[Nutrition and diet research progress series] Rodríguez, José Manuel Lorenzo_ Ruiz, Daniel Franco - Grape seeds_ nutrient content, antioxidant properties and health benefits (2016, Nova Publishers) - libgen.lc.pdf

NUTRITION AND DIET RESEARCH PROGRESS
GRAPE SEEDS
NUTRIENT CONTENT, ANTIOXIDANT
PROPERTIES AND HEALTH BENEFITS
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GRAPE SEEDS
NUTRIENT CONTENT, ANTIOXIDANT
PROPERTIES AND HEALTH BENEFITS
JOSÉ MANUEL LORENZO RODRÍGUEZ

AND
DANIEL FRANCO RUIZ
EDITORS
New York
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CONTENTS
Preface vii
Chapter 1 Extraction of Antioxidants from Grape Seeds 1
A. Moure, E. Falqué and H. Domínguez
Chapter 2 Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 27
L. M. Palade and V. S. Chedea
Chapter 3 Flavonoids Interaction with Gastrointestinal Tract:
Modulation of Enteroendocrine System, Intestinal
Permeability and Metabolic Endotoxemia 57
X. Terra, M. T. Blay, M. Pinent and A. Ardévol
Chapter 4 LC-ESI-FTICR-MS Analysis of Flavan-3-ols in Seeds
of Grape Pomace 85
I. Rockenbach, B. Santiago-Schübel, B. Thiele and R. Fett
Chapter 5 Methods to Evaluate Antioxidant Properties of Grape Seeds 101
M. L. González-SanJosé, M. D. Rivero-Pérez
and P. Muñiz-Rodríguez
Chapter 6 Modulation Effects of Polyphenolic Extracts in Experimental
Arterial Hypertension 125
M. Ciocoiu, M. Badescu and L. Badescu
Chapter 7 The Use of Grape Seed Extract on Meat Products: A Review 157
Daniel Franco, Javier Carballo and José M. Lorenzo
Chapter 8 The Science of Grape Seeds Applied to Health Food Development 181
C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
Chapter 9 Grape Seeds (Vitis Vinifera) and Their Nutritional Value 197
M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
Chapter 10 Antimicrobial and Antiviral Activities of Grape Seed Extracts 211
G. Pasqua and G. Simonetti
vi Contents
Chapter 11 Grape Extracts: Antioxidant Properties in Meat Product 225

G. Nieto and G. Ros
About the Editors 237
Index 239
PREFACE
The grape seed is a by-product obtained from wine industries, which represents a good
source of natural antioxidants, instead of synthetic antioxidants to produce foods with a
longer shelf life. In addition, grape seeds show health promoting benefits in relation to
cardiovascular protection, attenuation of oxidative stress, neuroprotective agent, and activities
on tumour, inflammation, ageing and other degenerative diseases, therefore study and
valorisation of their compounds has a great interest.
This book review and discusses the composition of grape seed, rich source of high-value
fatty oil, proteins and essential amino acids, vitamins, dietary fiber, as well as of macro and
micro elements, thus could use as a food supplement to improve the nutritive value of the
human diet. Indeed the search of a healthier diet has led to food technicians to develop new
products with wholesome characteristics and greater consumer acceptance. Sausages, yogurt,
milk, fruit juices and others can be supplemented with phenolic compounds from grape seed
without affecting the sensory properties demanded by consumers.
In other chapters the aim is focused in phenolic profiles (flavan 3-ol compounds,
catechin, epicatechin, epicatechin gallate, epigallocatechin and procyanidims) by their
recognised health benefits. As their composition varies greatly depending on origin, soil and
harvest conditions, storage conditions, variety, extraction methodologies and concentration
levels all this traits are considered in this work. Different extraction procedures (conventional
solvent extraction, enzyme, ultrasound and microwave assisted extraction, subcritical water
extraction and supercritical fluid extraction) are reported for extraction of phenolic
compounds are described as well as chromatographic assay that identified them.
Also the most important methods “in vitro” (DPPH, FRAP, ABTS) and “in vivo” to
evaluated antioxidant capacity are discussed in the book, considering that usually antioxidant
capacity or activity is measured in complex matrix where a lot of different antioxidants can be
present, hence this activity is not due to only one compound. As antioxidants can block
different oxidative agents, positions and reactions, each method is based in different reaction
mechanism, showing advantages and disadvantages respect others.
The book contains chapters dedicated to review the antioxidants properties of grape seed
extract over different meat product as: raw or cooked meat, dry-cured sausages and liver pates
of different species. Taking into account the results, numerous studies proposed grape seed
extract as preservatives in the food industry. In addition, antimicrobial antifungal and antiviral
activities of grape seed extract have been described. These activities together with lack of
toxicity-grape seed extract is generally recognized as safe, according to FDA- suggest that
viii José Manuel Lorenzo Rodríguez and Daniel Franco Ruiz
they could be used for the prevention and control of infection diseases without side effects
making greater potential for this extracts in the field of food and pharmaceutical application.
Finally, two chapters about modulation effects of polyphenolic extracts in arterial
hypertension and in the enteroendocrine system and metabolic endotoxemia are discussed in
the present book. In the first study it has been proved that natural polyphenol extracts will
have increased effectiveness in reducing blood pressure and limiting the side effects of the
major classes of antihypertensive agents used so far as monotherapy. The second research
shows that flavonoids could modulate the body homeostasis acting directly on the
gastrointestinal tract.
The book contains contributions from authors of international repute with great expertise
in their respective areas. Overall, it is a useful for graduates studying food science and
technology and for researchers, scientists, policy makers and professional from food and
biotechnology industries working grape seeds related to nutritional content, antioxidant
properties and health benefits.
In: Grape Seeds ISBN: 978-1-63484-578-6
Editors: J. M. Lorenzo Rodríguez and D. F. Ruiz © 2016 Nova Science Publishers, Inc.
Chapter 1
EXTRACTION OF ANTIOXIDANTS
FROM GRAPE SEEDS
A. Moure1,2,*, E. Falqué3 and H. Domínguez1,2

1
Departamento de Enxeñaría Química, Universidade de Vigo, Pontevedra, Spain
2
Centro de Investigación, Transferencia e Innovación (CITI), Ourense, Spain
3
Departamento de Química Analítica, Universidade de Vigo, Pontevedra, Spain
ABSTRACT
Grapes, grape products and by-products represent a widely distributed and studied
source of natural antioxidants, which also show health benefits in relation to
cardiovascular and degenerative diseases. Grape seeds are part of the wine industry by-
products and the valorization of their constituents has interest. The present chapter
reviews the composition of grape seeds, particularly the phenolic profile, the different
extraction methodologies reported for the extraction of phenolic compounds for
production of antioxidant extracts and the antioxidant capacities evaluated by different
protocols.
Keywords: grape seeds, phenolics, antioxidant activities, extraction
INTRODUCTION
Grape phenolics have a great number of potential bioactivities, including antioxidant,
cardioprotective, anticancer, anti-inflammation, antiaging and antimicrobial properties [1].
Grape products have been used for food applications for their antioxidant activities to delay
lipid oxidation and for their antimicrobial properties against aerobic mesophilic bacteria and
lactic acid bacteria [2]. The compounds showing higher and more diverse range of biological
*
Corresponding author: Andrés Moure, Departamento de Enxeñaría Química, Universidade de Vigo (Campus
Ourense), Edificio Politécnico, As Lagoas, Ourense, 32004, Spain, Email: amoure@uvigo.es.
2 A. Moure, E. Falqué and H. Domínguez
action have phenolic structures and among the different parts of the grape, the seed contains
higher amounts of phenolic compounds.
Grape pomace (GP) is one of the major by-product of the wine industry. This waste
obtained after pressing or after fermentation represents an ecological and economical problem
for this industry. At this moment, its major use is for tartaric acid and/or ethanol production,
the final waste being used as animal feed supplement [3, 4].
However, other valorization way is possible for this residue based on the functional
properties of its bioactive compounds. The GP after fermentation is a residual material mainly
composed of the skins, seeds and in lesser extent by the stems. Depending on the winemaking
procedure, the amount of polyphenols in GP is variable, but usually it still contains high
levels of polyphenols with good health-promoting effects and biological properties suitable
for food, cosmetic and/or pharmaceutical industries [5, 6]. The valorization of GP based on
the functional properties of its bioactive compounds is the most usual option. On this basis,
the grape seeds have an interesting potential as source of healthy products, since their oil has
been studied as a possible source of linoleic acid associated, with the promotion of
cardiovascular health [7]. As in the GP, the polyphenols are the major compounds found in
the seeds and skins of grape [8], being mainly flavan-3-ols (catechins and their polymers, the
proanthocyanidins).
The aim of this chapter is to review composition, antioxidant activities and the methods
reported to extract grape seed antioxidants.
GRAPE SEEDS COMPOSITION

The composition of grape seeds is basically (w/w) 40% fibre, 16% oil, which is rich in
essential fatty acids, 11% proteins, and 7% complex phenolic compounds including tannins,
in addition to sugars, mineral salts, etc. [9], tocopherols and -carotene, which are mainly
concentrated in grape seed oil.
Phenolic compounds play a vital role in plants, such as structural integrity, UV
photoprotection, plant reproduction and fertility, internal regulation of plant cell physiology
and signaling, protection against pathogen and herbivores, etc. and contribute to their color,
taste, and nutritional value.
In the particular case of grapes and grape seeds, phenolic compounds have shown
significant biological activities. Phenolic compounds are substances that have one or more
hydroxyl groups on the aromatic skeleton, and can be classified according to different
approaches: ‗simple‘ or ‗highly polymerized‘ compounds, or simple phenols and
polyphenols; ‗soluble‘ or ‗insoluble‘ phenolics [10]; phenolic acids, simple flavonoids, and
tannins and proanthocyanidins [11]. Phenolic compounds are generally classified into classes
and sub-classes based on the similarity of their chemical structures, that is, the types of
building blocks that appear as repeated units: the simplest are phenolics (C6) and their
derivatives such as phenolic acids and related compounds (C6-C1), acetophenones and
phenylacetic acids (C6-C2), derivatives of cinnamic acid (C6-C3), coumarins, isocoumarins
and chromones (C6-C3), chalcones, dihydrochalcones, aurones, flavans, flavones, flavanones,
flavanonols, leucoanthocyanidins, anthocyanidins and anthocyanins (C15, respectively C6-
C3-C6 also generally called ―flavonoids‖), biflavonyls (C30), benzophenones, xanthones and
Extraction of Antioxidants from Grape Seeds 3
stilbenes (C6-C1-C6, resp. C6-C2-C6), quinones (C6, C10, C14), lignans, neolignans and
tannins (dimers or oligomers, e.g., proanthocyanidinis, dimers or oligomers of flavan-3-ol)
and polymeric compounds such as lignin and phlobaphenes [12].
Flavonoids and stilbenes are synthetized along the general phenylpropanoid metabolic
pathway, in which the amino acid phenylalanine is transformed into 4-coumaroyl-CoA. This
can be combined with 3 molecules of malonyl-CoA to yield the stilbenes and the chalcones,
which contain two phenyl rings. The conjugate ring closure of chalcones results in the true
backbone of flavonoids, the three-ringed structure of a flavone. The metabolic pathway
continues through a series of enzymatic modifications to yield flavanones, dihydroflavonols,
and anthocyanins [13]. The chalcones also are the more direct precursors to other flavonoid
compounds such as flavones and isoflavones, flavonols, proanthocyanidins (tannins) and
other phenolics (Figure 1).
Phenolic acids, precursors of flavonoids, are phenols that possess one carboxylic acid
functional group and are divided into hydroxycinnamic acids and hydroxybenzoic acids. The
hydroxycinnamic acids are more common than hydroxybenzoic acids, and they mainly
include gallic, p-coumaric and caffeic acids.
The total concentration of phenolic compounds in grape were about 2178.8, 374.6, 23.8,
and 351.6 mg/g GAE (gallic acid equivalent) in seed, skin, flesh, and leaf, respectively [14].
The percentage of the total extractable polyphenols in grape tissues are: 60-70% in the seed,
10% or less in the pulp, and 28-35% in the skin. The polyphenolic content of the seed may
range from 5 to 8 wt % [15].
The phenolic compounds in grape seeds are essentially flavonoids, particularly, flavan-3-
ols (catechin, epicatechin and epicatechin-3-O-gallate monomers) and their polymers. The
most abundant flavan-3-ols monomers are (+)-catechin and (-)-epicatechin, and also (+)-
gallocatechin, (-)-epigallocatechin and their 3-O-gallic acid esters. Catechin is the isomer
with trans configuration and epicatechin is the one with cis configuration. Catechin and
epicatechin can form polymers, which are often referred to as proanthocyanidins because an
acid-catalyzed cleavage of the polymeric chains produces anthocyanidins. Other compounds
presents in the grape seeds are the phenolic acid precursors (gallic acid) [16] and stilbenes.
The degree of polymerization of the procyanidins may also determine the antioxidant activity.
The higher the degree of polymerization, the greater is the antioxidant activity [17].
Standardized grape seed extracts contain 74–78% oligomeric proanthocyanidins and less than
approximately 6% of free flavanol monomers on a dry weight basis. These can combine with
gallic acid to form gallate esters and ultimately glycosides [17].
The proanthocyanidins, also called procyanidins or condensed tannins, provide bitterness
and astringency to wine, especially to red wine, and are the oligomers of flavan-3-ol units,
especially (+)-catechin and (-)-epicatechin. Dimeric proanthocyanidins are formed by 4→8
(B1, B2, B3 and B4 are the most common) or 4→6 (B5, B6, B7 and B8) linked monomers
and sometimes esterified by gallic acid on the epicatechin moiety(ies). Trimers of
procyanidins have C1, C2 and C3 isomers. Levels of galloylated flavan-3-ols are more
important in seeds than in skins [18]. The degree of polymerization ranges from 2 to >15 and
an average molecular mass ranges from 578 to >5000 Da [19]. The average polymerization
degree of seed tannins is ~11, lower than the ~28 of skin tannins, but the levels of gallate in
the seeds (>30%) are higher than those in the skin and stems (5.16%) [11].
Figure 1. Schematic pathways of flavonoid biosynthesis and chemical structures of the main phenolic
compounds in grape seed.
The grape seeds structure can be divided into five zones: (i) cuticle and epidermis, (ii)
outer integument or soft seed coat, (iii) medium integument or hard seed coat, (iv) inner
integument and (v) endosperm and embryo. Polyphenols have been observed mainly in the
epidermis and in the outer integument [20]. During ripening of the grape, environmental
factors and endogenous enzymes promote modifications in the phenolic composition.
Visually, the seeds changed color from an initial green to a light buff, and finally to a dark
brown at harvest and some changes in polyphenols happened: gallic acid levels remained low
throughout the season, the flavan-3-ol monomers and procyanidins declined dramatically
(much of these decline occurred in the first month after véraison) with a 90% and 60%,
respectively. The fractional procyanidins composition of the individual subunits changed with
maturity: (-)-epicatechin-3-O-gallate decreased (38 ± 18%), (+)-catechin did not change (ca.
43%), and (-)-epicatechin increased (17 ± 41%). Procyanidins containing over 30 subunits
remained essentially constant (ca. 200 mg/berry). For the procyanidins that declined, those
containing 6 ± 10 subunits declined the least during fruit ripening (53.5%), followed by those
containing 2 ± 5 and 11 ± 15 subunits (60.4 and 62.9% decline, respectively). Procyanidins
containing 16 ± 20 and 21 ± 25 subunits declined the most (71.6 and 73.6% decline,
respectively) [21].
Resveratrol is another important non-flavonoid polyphenol found in grape seeds. The
trans-resveratrol content was found to be 1.42 ± 0.18 mg per 100 g dry mass in white grape
seeds [22].
The phenolic composition of white and red grape seeds is comparable, but catechin,
epicatechin and procyanidin B1 are higher in white grape seeds. Overall, grape seeds
contained lower amount of phenolic acid than grape skin, but are richer in catechins and
procyanidins. Pinelo et al. [11] and Teixeira et al. [6] reported general contents of the main
phenolics presents in seed grapes.
ANTIOXIDANT PROPERTIES OF GRAPE SEED PHENOLICS

The antioxidant properties of Vitis vinifera grape seed components have been evaluated
in different works. Most studies confirmed the reducing power, the ability to scavenge free
radicals and the potential to protect against lipid oxidation (Table 1).
Among the pomace fractions showing antioxidant properties, the seeds exhibited the
highest capacity [2]. Although grape skin anthocyanins present a high protective capacity
against lipid oxidation, grape seeds have higher levels of total phenolics per sample dry
weight when compared to grape skins [23]. The concentrations of catechin, epicatechin and
gallic acid, the major compounds in Chardonnay seeds and Merlot seeds, were lower in grape
skins than in seeds [18]. Seed extracts showed ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-
6-sulphonic acid)) scavenging activity, delayed the onset of lipid oxidation and the onset of
olive oil and pork lard oxidation using the Rancimat method [2]. The activity in other lipid
systems was observed. Grape seed extract dose-dependently retarded lipid oxidation of
sunflower oil under simulated frying conditions up to 240 min, being as efficient as butylated
hydroxytoluene (BHT) [24].
Table 1. Antioxidant properties of grape seeds and grape seed extracts
Product Antioxidant activity Reference

Commercial GSE RP, RS: AAPH [25]
Protection against lipid oxidation
Commercial GSE Prevents oxidative stress and reduced ROS in animals [43]
GSE RS: Superoxide, hydroxyl [40]
GSE Reduced postprandial oxidative stress [44]
Protection against LDL oxidation
GSE TRAP [41]
GSE RS: ORAC [18]
GSE RS: DPPH [32]
GSE RS: ORAC [54]
RS: Superoxide, hydroxyl, DPPH [38]
GSE RS: ABTS, DPPH [36]
GSE Reduced oxidative stress caused by high fat diet [42]
GSE FRAP, RS: DPPH [27]
GSE Inhibition of sunflower lipid oxidation [24]
GSE DPPH [29]
GSE RP, RS: ABTS, DPPH [34]
GSE RP; RS: DPPH, ABTS [31]
GSE RP, RS: AAPH, ABTS, DPPH [37]
Protection against lipid oxidation
GSE RS: DPPH, AAPH [35]
GSE RS: ABTS [23]
GSE procyanidins Protection against LDL oxidation [39]
PRGSO and extract FRAP, RS: ABTS [28]
PS RS: ABTS [2]
Oil and fat oxidation prevention
ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); AAPH: 2,2'-azobis(2-amidinopropane)
dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FRAP: ferric reducing antioxidant power;
GSE: grape seed extract; ORAC: oxygen radical antioxidant capacity; PRGSO: press residue from
grape seed oil production; PS: powdered seeds; RP: reducing power; ROS: reactive oxygen
species; RS: radical scavenging; TRAP: plasma total radical-trapping antioxidant parameter.
Schevey and Brewer [25] confirmed that grape seed extracts showed higher antioxidant
efficacy than synthetic antioxidants, such as α-tocopherol, ascorbic acid and gallic acid, in
model systems of commercial canola oil, lard and ground beef during oxidation, but were less
efficient than butylated hydroxyanisole (BHA). In that study, the influence of the antioxidant
was not noticeable on the intensities of the off-odour characteristics for samples containing an
antioxidant, suggesting that all of them could control the negative sensorial properties
conferred by oil oxidation. Procyanidins from grape seeds also protected low-density
lipoprotein (LDL) from in vitro oxidation induced by 2,2′-azobis-(2-amidinopropane)
dihydrochloride (AAPH) or by peroxynitrite [26].
The previous processing is an important factor affecting the properties of the extracts.
Both the antioxidant and antibacterial capacities of grape seeds aqueous extracts were lower
when the extracts were obtained from seeds after wine making [27]. Despite the highest
phenolic content in integral grape seeds than in the press residues after grape seed oil
production, this is still a rich source of polyphenols with strong reducing power and
antiradical activity. Total amount of phenolic compounds ranged from 4.8-19.1 g/kg defatted
dry matter for integral grape seeds, whereas their content in the press residues was in the
range 2.8-13.8 g/kg of defatted grape seeds [28]. The de-oiling method influenced the
ultrasound-assisted extraction (UAE) of phenolics from grape seed. The highest polyphenol
content (105.2 mg GAE/g) and antioxidant activity were found in grape seed extracts
obtained by a sequence of ultrasound and maceration [29].
The type of heating can influence the phenolic profile in a different manner depending on
the grape seed product, the effect of autoclave was more severe than furnace heat treatment
but no influence on the antioxidant activity occurred [30]. Furnace thermal treatment at 100
°C for 15, 30 and 60 min of grape seed extract (GSE) did not affect the total phenolic content,
tannin content, procyanidins and the ABTS and DPPH radical scavenging activity.
Autoclaving at the same temperature and time caused an extensive hydrolysis of
gallocatechin, catechin, epicatechin, procyanidin B1 and B2, but did not affect the antioxidant
activity of the extract. Differences among varieties and operational conditions are expected,
i.e., European V. vinifera grape seed extracts showed a higher total phenolics, tannins,
catechins and phenolic acids content than Japanese ones and also better radical scavenger
properties and stronger reducing power. The antioxidant capacity of the grape seed extracts
depends on the content of total polyphenol compounds [27] and many other studies have
shown a correlationship between the phenolic content and the antioxidant properties. Acetone
provided higher content of phenolic compounds and tannins and stronger potency than
methanol [31]. The ethyl acetate extracts of seeds from V. vinifera varieties cultivated in
Greece, contained different low molecular weight compounds: catechin was the most
abundant polyphenol, accounting for 49.8% of the total content, followed by epicatechin,
epicatechin gallate and procyanidin, whereas epigallocatechin and gallic acid were in lower
amounts. Procyanidin B1 may be one of the most important radical scavengers in grape seed
extracts [32]. Catechin was also the most abundant compound, followed by epicatechin,
procyanidin B2, gallic acid, gallocatechin and epicatechin gallate on the grape seed extracts
obtained with 50% ethanol from a variety cultivated in Eastern Croatia. The total
proanthocyanidins content of the extract had the highest positive correlation with the
antioxidant activity; also phenolic content and color of extracts were found to be correlated
[33].
Strong correlation between FRAP (ferric reducing antioxidant power) and total phenolics,
flavonoids and flavan-3-ols contents was observed for the seed extracts from red wine grape
cultivars of Pusa Navarang and Merlot, which contained catechin hydrate and epicatechin as
the major components [34]. Hot-water extracts from grape seeds showed the highest content
of polyphenols (43.9 mg/g) among different winery residues. These extracts showed
protective effects against oxidative stress induced by AAPH peroxyl radical in African
monkey kidney (MA 104) cells. The extracts containing the highest phenolic content also
showed the highest antioxidant activity measured by the DPPH test and the highest protective
effect on AAPH-induced cytotoxicity in MA104 cells, evaluated using the MTT assay. A
correlation was observed between antioxidant activities and phenolic contents of extracts
[35]. Total phenolic content showed a significant correlation with DPPH and ABTS values in
grape seed extracts obtained from different varieties grown in Turkey, extracted with 70%
acetone, and with the total phenolic content ranged from 33 to 58 mg GAE/100 g extract [36].
The polymeric fraction of grape seed extracts showed higher antioxidant capacity than
the monomeric and oligomeric fractions, according to ABTS, DPPH and FRAP, but ORAC
values (Oxygen radical absorbance capacity) showed the opposite trend [37]. These authors
found correlations between antioxidant properties and phenolic content but not with the
polymerization and galloylation degrees. Catechin, epicatechin and gallic acid were the major
phenolic constituents of grape seeds, but contributed less than 26% to the antioxidant capacity
measured as ORAC. Peroxyl radical scavenging activities of phenolics present in grape seeds
were, in decreasing order, resveratrol > catechin > epicatechin = gallocatechin > gallic acid =
ellagic acid. The results indicated that dimeric, trimeric, oligomeric, or polymeric
procyanidins accounted for most of the antioxidant capacity of grape seeds [18]. Spranger
et al. [38] reported that two highly pure procyanidin fractions, namely oligomers and
polymers isolated from grape seed methanolic extract showed a degree of polymerization
ranging from 2 to 17-18 and 12 to 32-37, respectively. Grape seed procyanidins presented
higher antioxidant activities than vitamin C. On the basis of molar concentration, polymeric
procyanidins were the most potent antioxidants, followed by oligomeric procyanidins;
catechins presented a lower antioxidant activity than its oligomers and polymers. The
antioxidant activities of grape seed procyanidins, measured as superoxide, DPPH and
hydroxyl radical scavenging, are positively related to their degree of polymerization.
The more active fractions from grapes seeds against in vitro oxidation of LDL were
components of the catechin family, the catechin oligomers and the procyanidin dimers (B2,
B3, B4, B6, B8) and trimers (C1, C2). The procyanidin dimers B2 and B8, and trimer C1, and
the monomers catechin, epicatechin and myricetin had the highest antioxidant activity [39].
Epicatechin 3-O-gallate and procyanidins from grape seeds were tested for their scavenging
capacity for superoxide radical and hydroxyl radical. All the compounds assayed are potent
scavengers of these radicals compared to Trolox. Gallic acid esterification increased the
antiradical capacity of the dimer procyanidins, the esterification position having importance
in the scavenging capacity [40].
Studies dealing on the dietary interventions with grape seed extracts confirmed the
in vivo antioxidant properties of these products. Supplementation with grape seed extract for
30 days had a marginal effect on the plasma antioxidant potential, measured by TRAP
(Plasma Total Radical-Trapping Antioxidant Parameter) and on the level of plasma α-
tocopherol. However, dietary procyanidins exerted their antioxidant protection in vivo by
sparing liposoluble vitamin E. A considerable reduction of oxidative DNA damage, and a
slight increase of polyunsaturated fatty acids in the phospholipids of red blood cells
membranes was observed [41]. A polyphenolic grape seed extract was provided for 12 weeks
to hamsters receiving a high-fat diet. Grape seed extract prevented in part the increase on
plasma glucose, triglycerides, insulin and insulin resistance, lowering insulinemia and
leptinemia. Oxidative stress, assessed by cardiac production of superoxide anion and
NAD(P)H oxidase expression, was reduced [42]. Grape seed proanthocyanidin rich extracts
dose and time dependently prevented tert-butylhydroperoxide-induced oxidative stress in two
intestinal cell types: the absorptive cell line Caco-2 and the enteroendocrine cell line STC-1.
The 1 h acute treatment with 1 g extract/kg body weight reduced the reactive oxygen species
(ROS) in fasted animals [43]. The effect of supplementing a meal with grape seed
proanthocyanidins on plasma lipid hydroperoxides (LPO) was evaluated to determine plasma
postprandial oxidative stress in healthy volunteers. The content of LPO was 1.5-fold higher
after the control meal than after the grape seed extract supplemented meal. The plasma
antioxidant capacity increased in the postprandial phase following the grape seed extract
supplemented meal. LDL isolated 3 h after the control meal tended to be more susceptible to
oxidative modification, whereas an opposite trend was observed following the grape seed
extract (GSE) supplemented meal [44].
EXTRACTION OF THE PHENOLIC FRACTION

The present section is focused on the availability of the traditional and new technologies
to valorize winemaking by-products. The quality and composition of the products obtained by
these technologies from winemaking wastes depend on the kind of waste (solid wastes,
effluents, lies…), grape varieties and maturity on harvest, the agro-climatic conditions of the
production area, vinification process and other treatments done on grapes. However, other
factors are also important as the solvent or combination of solvents chosen, the extraction
procedures and finally, additional factors such as the quality of material picked, its storage
conditions and pretreatment. For example, in the red winemaking the skins and seeds are in
contact with the fermenting broth for several days whereas in the white winemaking the skins
and seeds are removed from the broth.
To revalorize this fraction, the development of an extraction process of polyphenols
compounds is an important step, followed by the performance of isolation protocols and
compounds identification. The extraction process involves the following sequential
mechanisms: i) transport of solvent from the bulk solution to the external surface of the
matrix, ii) solvent penetration and diffusion in the solid matrix, iii) solubilization of the
components, iv) transport of the solute(s) through the solid matrix, and v) transport of the
solute(s) from the external surface of the solid to the bulk solution [45]. In literature there are
a wide set of extraction technologies applied to recover phenolic compounds from the
winemaking residues in general and from the grape seeds in particular. The most widespread
technique used is the conventional extractions with solvents (CSE), but owing to its
operational conditions, the use of organic solvents with their toxicology and environmental
problems led to researches towards the development of new processes. New technologies
namely, ultrasound assisted extractions (UAE), microwave assisted extractions (MAE),
enzyme assisted extraction (EAE), accelerated solvent extraction (ASE), hot water extraction
(HWE), subcritical water extraction (SWE), supercritical fluid extraction (SFE) and
supercritical antisolvent extraction (SAE) have been tried.
Conventional Solvent Extraction. Many factors have been described to be involved in
the efficiency of CSE, described as extraction yield and type of extracted solute. The type of
solvent, relative solvents proportion, solvent/sample ratio, particle size, temperature, and
extraction time are some of the most influential variables. The comparison of the ability that
distinct solvents and mixtures of them provide in the solubilization of the different classes of
phenolics present in grape seeds and their extraction yield are summarized in Table 2.
Methanol is a solvent widely employed in the extraction of grape seeds, however its
toxicity restricts its use to analytical procedures. In this regard, ethanol and ethanol-water
mixtures have been studied as a more environmentally friendly solvents, recognized as safe
according to the European Food Safety Authority (EFSA) and FAO/WHO Expert Committee
on Food Additives [46, 47]. The results obtained from literature studies do not allow choosing
an ideal extracting solvent, and different mixtures have been proposed. The mixtures based on
acetone/water or methanol/water/acetone have been suggested to give better results for the
extraction of flavonols and procyanidins [48, 49, 50]. Other researchers have found that the
extraction of catechins and procyanidins was more efficient with the use of an ethanol/water
mixture [51-53]. In contrast, methanol was reported for extracting catechins, epicatechins,
and epigallocatechins from grape seeds [54, 55]. On this matter, Agustín-Salazar et al. [56]
reported that a mixture of methanol/water (3:3, v/v) and methanol/water/acetone in a ratio
3:3.5:3.5 (v/v/v) were a good extractant agent for i) catechin and epicatechin, and ii) for total
phenolic compounds, total flavonoids, quercetin-3-rutinoside and myricetin, respectively.
Jayaprakasha et al. [57] extracted antioxidant compounds from grape seeds using various
solvents, such as acetone, ethyl acetate, methanol and mixtures of different solvents, such as
ethyl acetate and water in 9:1, 17:3 and 4:1 (v/v) ratios. Ethyl acetate demonstrated the
highest efficiency in material with bigger content in lipids owing to its low polarity.
Table 2. Extraction of antioxidants for grape seeds valorization using different

extraction technologies and operational conditions
Grape varieties Solvent /Extraction conditions Compounds Reference

Solvent; Liquid to solid ratio; extracted
Temperature; Stirring; pH; Time
Conventional Solvent Extraction (CSE)
Ada Karasi, 70% A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
Alphonso 50ºC; -; 2 h EGCG, EGC, PC
B1, PC B2, TP
Agiorgitiko Defatting: Hex; overnight C, ECG, GA, TA [52]
60% E (1 g CiA /L);1:20 (w;v); RT;
400 rpm; 2-6; 1-5 h
Aglianico 12% E (0.1 g SO2/L; 5 g TA /L); -; C, EC, ECG, EGC [97]
30ºC-; OpD; 3.2; 120 h
Albariño E:W (80:20, v:v); 1:8 (w:v); Low T, - ; C, EC, ECG, GA, [53]
3.5; 0.08 h PA-H, PC B2, PC
B3, PC B4
Albarossa 1st step: M (0.1 HCl), 1:16 (w:v); RT; An, PAn, TP [55]
CS; -; 2 h
2nd step: 12 h incubation at 4ºC
Bailey Alicante A M (1% HCl); -; -; 24000rpm; -; 0.016 h TAn, TF, TP [54]
Barbera 1st step: M (0.1% HCl), 1:16 (w:v); An, PAn, TP [54]
RT; CS; -; 2 h
Black queen 60% , 1:10 (w:v); RT; 180 rpm; -; 5 h C, EC, PC B2, TP [69]
Cabernet 1st step: M (0.1% HCl); 1:16 (w:v); An, PAn, TP [55]
Sauvignon RT; CS; -; 2 h
70% A or W; -; -; -; -; 18 h T [98]
E:W:HCl 0.12 M (70:29:1; v:v:v); -; -; C, EC, TP, R [99]
-; -; -
12% E (0.1 g/L SO2; 5 g/L TA); -; C, EC, ECG, EGC [97]
30ºC-; OpD; 3.2; 120 h
M:W:A (80:20:5); 1:50 (w:v); 4ºC; C, CA, EC, R [49]
MS; -; 1 h

70% A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
50ºC; -; 2 h EGCG, EGC, PC
B1, PC B2, TP
Campbell Early Defatting: Hex; 1:5 (w:v); -; CS; 1 h /- TP [100]
/60% E; -; -; -; 0.5 h
Canaiolo Nero E:W:HCl 0.12 M (70:29:1; v:v:v); -; -; C, EC, TP, R [99]
-; -; -
Carignan 70% A (2 g SO2/L); 1:3.3 (w:v); 4; -; - [101]
0.25 h
60% M or 70% M or 90% M or C, EC, ECG, Q, Q- [56]
M:W:A (3:3.5:3.5); 1:2 (w:v); G, R
25; -; -; -;
60% E or 70% E or 90% E; 1:2 (w:v);
25; -; -; -;
Chardonnay 1st step: M (0.1% HCl); 1:16 (w:v); An, PAn, TP [55]
RT; CS; -; 2 h
Cinsault 70% A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
B1, PC B2, TP
Colorino del E:W:HCl 0.12 M (70:29:1; v:v:v); -; -; C, EC, TP, R [99]
Valdarno -; -; 4 h
Dolcetto 1st step: M (0.1% HCl); 1:16 (w:v); An, PAn, TP [55]
RT; CS; -; 2 h
Ebizuru M (1% HCl); -; -; 24000 rpm; -; TAn, TF, TP [54]
0.016 h
Frankovka 50% E; 1:40 (w:v); 25-80ºC; 200 rpm; TP [102]
-; 0.08-3.3 h
Fernão Pires 80% M; 1:10 (w:v); RT; stirring; -; PAn, TP [103]
3 h + 75% A; 1:10 (w:v); RT; stirring
Isabel M:W:A (80:20:5); 1:50 (w:v); 4ºC; C, CA, EC, R [49]
MS; -; 1 h
Kadainou R-1 M (1% HCl); -; -; 24000 rpm; -; TAn, TF, TP [54]
0.016 h
Kalecik Karasi 70%A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
B1, PC B2, TP
Labruscana 80% E; 1:4 (w:v); RT; -; -; 168 h TP [35]
Malvar -; - ; - ; - ; - ; - C, EC, ECG, PC B1 [104]
Merlot E:W:HCl 0.12 M (70:29:1; v:v:v); -; -; C, EC, TP, R [99]
-; -; -
12% E (100 mg/L SO2; 5 g/L TA); - C, EC, ECG, EGC [97]
;30ºC-; OpD ; 3.2 ; 120 h
80% M; -; RT; shaking; -; overnight C, CA, EC, ECC, [34]
GA
70% A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
B1, PC B2, TP
Table 2. (Continued)

Monastrell MWS (0-15% E); 8.3 (w:v); RT; 2 PAn [60]
shaken/d; 3.6; 240 h
Montepulciano E:W:HCl 0.12 M (70:29:1; v:v:v); -; -; C, EC, TP, R [99]
-; -; -
Moscato Bianco 1st step: M (0.1% HCl); 1:16 (w:v); An, PAn, TP [55]
RT; CS; -; 2 h
Moschofilero Defatting: Hex; overnight C, ECG, GA, TA [52]
60% E (1 g CiA /L); 1:20 ( w:v);
RT; 400 rpm; 2-6; 1-5 h
Muller Thurgau 1st step: M (0.1 HCl); 1:16 (w:v); RT; An, PAn, TP [55]
CS; -; 2 h
Muscat Alexandria M (1% HCl); -; -; 24000 rpm; -; TAn, TF, TP [54]
0.016 h
Muscat Hamburg 70%A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
B1, PC B2, TP
Negro Amaro M:W:A (80:20:5); 1:50 (w:v); 4ºC; C, CA, EC, R [49]
MS; -; 1 h
Palomino Fino M (1% HCl); 1:2 (w:v), 0.5 h into Res [93]
ultrasonic bath
Papaz Karasi 70% A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
B1, PC B2, TP
Pinot Noir 1st step: M (0.1 HCl); 1:16(w:v); An, PAn, TP [55]
RT; CS; -; 2 h
Pinot Noir M:W:A (80:20:5); 1:50 (w:v); 4ºC; C, CA, EC, R [49]
MS; -; 1 h
Primitivo M:W:A (80:20:5); 1:50 (w:v); 4ºC; C, CA, EC, R
MS; -; 1 h
Pusa Navrang 80% M; -; RT; shaking; -; overnight C, CA, EC, ECC, [34]
GA
Ryukyuganebu M (1% HCl); -; -; 24000 rpm; -; TP, TAn, TF [54]
0.016 h
Sangiovese E:W:HCl 0.12 M (70:29:1; v:v:v); TP, C, EC, R [99]
Sangiovese M:W:A (80:20:5); 1:50 (w:v); 4ºC; C, CA, EC, R [49]
MS; -; 1 h
Savatiano Defatting: Hex; overnight C, ECG, GA, TA [52]
60% E (1 g CiA /L); 1:20 (w:v); RT;
400 rpm; 2-6; 1-5 h
Senso 70% A (0.5% AA); 1:15 (w:v); C, EC, ECG, [96]
B1, PC B2, TP
Shiohitashidudou M (1% HCl); -; -; 24000 rpm; -; TAn, TF, TP [54]
0.016 h
Shiragabudou
Solvent /Extraction conditions

Compounds
Grape varieties Solvent; Liquid to solid ratio; Reference
extracted
Shiraz 70% A (v:v); ; -; 23ºC; 200 rpm; 3.6; An [105]
72 h
15.2% E (v:v); -; 23ºC; 200 rpm; 3.6;
24 h
Unknown TB or TB (not Na2S2O5) or A:W TAn, TF [106]
(70:30; v:v); -; -; -; 3.2; 4 h
Unknown 50% E; 1:5 (w:v); 55ºC; 150 rpm; -; TP [107]
1.5 h
Vitis vinifera 50% E; 1:4 (w:v); RT; -; -; 1 h TP [15]
Vitis vinifera Maceration: 80% E or W; 8.3 (w:v); C, EC, GA [108]
80ºC; MS; -; 3 h
Vranac M/A/W/AA (30/42/27.5/0.5); 1:80 C, EC, PC [50]
(w:v); RT; 200 rpm; -; 0.5 h
White grapes 50% E; 1:10-40 (w:v); 25-80ºC; 20-s; 2 TP [58]
h
Yamabudou M (1% HCl); -; -; 24000 rpm; -; 0.016 TAn, TF, TP [54]
h
Aqueous two-phase extraction
Riesling Aqueous two phase system: PAn, TF, TP [109]
14-24% (w:w) A + 14-22% (w:w)
ammonium citrate
Enzyme-Assisted Extraction (EAE) Solvent; Enzyme; Liquid to solid ratio; Temperature;
Stirring; pH; Time
Cabernet 5 g/L TA in water or 5 g/L TA in 12% PAn [77]
Sauvignon E
Monastrell 0.25 unit Xyl/mL; 0.19 units Cell/mL;
0.06 units PGal/mL; 0.68 unit
PME/mL; 1:30 (w:v), RT, gentle; 3.6;
48 h
Muscadine defatting: Hex; 1:10 (w:v); RT; 24 h An, TP [75]
50% E; SA_B; Cell; Pect; Gluc; Mix;
1:8 (w:v); 50ºC; -; 4.8; 1-24 h
50% E 1:20 (w:v); RT; - ; -; 1 h
País grape Pect (E/S: 1%): AB; 1:100 (w:v); C, EC, ECG, TP [76]
25ºC; 150 rpm; 4; 1 h
Cell (E/S: 1%): AB; 1:100 (w:v);
37ºC; 150 rpm; 5; 1 h
Tn (E/S: 1%): AB; 1:100 (w:v); 30ºC;
150 rpm; 4.7; 1 h
Ultrasound-Assisted Extraction (UAE) Frequency; Power, Solvent; Liquid to solid ratio;
Temperature; Stirring; Time
Black Queen 40 kHz; 320 W; 60% E; 1:10 (w:v); C, EC, PC B2, TP [69]
RT; -; 0.5 h
Campbell Early 40 kHz; 250 W; 33-67% E; 1:50 (w:v); TA, Tp [62]
33-67ºC; -; 16-34 min
Mavrokountoura -; -; M (1% FA); 1:10 (w:v); -; VS (5 An, Tp [68]
(Mandilaria clone) min); 0.16 h

Raboso Piave 20 kHz; 50-150 W; Hex; 1:8 (w:v); Ice F, TAn, TP, TT [29]
bath; -; -; 0.5 h
Vranac 40 kHz; 500 W; M/A/W/AA C, EC, PAn [63]
(30/42/27.5/0.5); 1:40- 60 (w:v); 30-
50ºC; -; 0.08-0.25 h
Accelerated Solvent Extraction (ASE)
Aglianico Auto P at 25ºC; 5 g GS; 8 ext. cycles An, PAn, TP [79]
80% A + 4 cycles 60% M
Auto P at 25ºC; 5 g GS; 6 ext. cycles
60% M
Babić Auto P at 25ºC; 5 g GS; 8 ext. cycles An, PAn, TP [78]
80% A + 4 cycles 60% M
Auto P at 25ºC; 1 g GS; 6 ext. cycles
M (0.1% HCl)
Cabernet Auto P at 25ºC; 5 g GS; 8 ext. cycles An, PAn, TP [79]
Sauvignon 80% A + 4 cycles 60% M
Merlot Auto P at 25ºC; 5 g GS; 6 ext. cycles
Plavac mali 60% M
Microwave-assisted aqueous two-phase extraction
Riesling 650 W; aqueous two phase system: PAn, TF, TP [109]
14-24% (w:w) A + 14-22% (w:w)
ammonium citrate
Microwave-assisted extraction (MAE) Frequency; Power; Solvent; Liquid to solid ratio;
Temperature; Stirring; Time
Bangalore blue Defatting: Hex; 1:3 (w:v); 60ºC; 6 h TP [110]
2450 MHz; 100-200 W; 30-60% E;
1:10 (w:v); -; -; 0.033-0.1 h
Cabernet - ; 100-200 W; 10-90% E; 1:10-50 TP [111]
Sauvignon (w:v); 40-60ºC; -; 0.033-0.53 h
Chardonnay
Sauvignon Blanc
Shiraz
Subcritical water extraction (SWE) Solvent; Pressure; Temperature; Time; Flow rate
Pinot Nero SC-CO2 defatting seeds: 50ºC; 50 TP [82]
MPa; 8 g CO2/min
SWE: static mode: 10 MPa; 80-120ºC;
0.33 h; -dynamic mode: 10 MPa; 80-
120ºC; 2 h; 2-5 mL/min
Tempranillo 1st step: 70% E; 1.5 MPa; 90ºC; 1 h; 5 TP [81]
mL/min;
2nd step:
1) W or W:H2O2 (91:9, w:w); 1.5
MPa; 150-320ºC; 2.25 h; 5 mL/min
2) W or W:H2O2 (91:9, w:w); 1.5 MPa;
165-330ºC; 2.25 h; 5 mL/min
3) W or W:H2O2 (91:9, w:w); 1.5 MPa;
180-340ºC; 2.25 h; 5 mL/min

Hot Water Extraction (HWE) Solvent; Liquid to solid ratio; Temperature; Time
Labruscana W; 1:4 (w:v); BP; 1 h TP [35]
Supercritical fluid extraction (SFE) & Supercritical Antisolvent extraction (SAE)
Pressure; Temperature; Modifier; Time; Flow rate
Carignan 25-30 MPa; 30-50ºC; 5-20% E; 1 h; C, EC, ECG, EGC, [94]
5 g CO2/min EGCG, GA
Chardonnay -; 35-55ºC; 10-40% E or M; 1 mL C [91]
CO2/min
Palomino fino 10-40 MPa; 35-55ºC; 0-5% E; 3 h; Res [93]
0.8 g CO2/min
Syrah Defatting: Hex; 1:10 (w:v); RT; 24 h C, EC, GA, Res [95]
FS: SE; E; 4 h (5 min/cycle)
SAE: 10 mL FS (3% solids, w:w);
8-15 MPa; 35-60ºC; 2.38 kg CO2/h
Unknown 20-30 MPa; 40ºC; 2-15% E; 0.33 h; C, EC, GA, PC B2 [92]
0.5 mL CO2/min
A: acetone; AA: acetic acid; AB: acetate buffer, An: anthocyanins; BP: boiling point; C: catechin; CA:
chlorogenic acid; Cell: cellulase; CiA: citric acid; CS: continuous stirring; E: ethanol; EC:
epicatechin; ECG: epicatechin gallate; EGCG: epigallocatechin gallate; F: flavonols; FA: formic
acid; FS: feed solution; GA: Gallic acid; GS: grape seed; Hex: hexane; M: methanol; Mix:
mixture; MS: mechanical stirring; MWS: model wine solutions; OpD: flask rotating once per day;
P: pressure; PA-H: protocatechuic acid-O-hexoside; PAn: proantocyanidin; PC Bn: procyanidin Bn;
Pect: pectinase; PME: pectinmethylesterase; PGal: polygalacturonase; Q: quercetin; Q-G:
quercetin-3-O-glucoside; R rutin; Res: resveratrol; RT: room temperature; SA_B: saline acetate
buffer; SE: soxhlet extraction; T: tannin; TA: tartaric acid; TAn: total anthocyanins, TB: tartaric
buffer; TF: total flavonoids; Tn: tannase; TP: total polyphenols; TT: total tannins; VS: vigorous
shaking; Xyl: xylanase; W: water.
Buciš-Kojiš et al. [58] optimized the yield and kinetics of the phenolics extraction from
grape seeds using Peleg‘s mathematical model, definitely influenced by temperature, particle
size and solid–liquid ratio, with extraction time not being an important parameter after 40 min
of treatment. The effectiveness of the solvent not only depends on the physical properties of
the residue considered, but also on the target phenolic to be extracted. Thus, the extraction of
bioactive phenolics present in grape seeds using ethyl acetate as solvent, showed that this
solvent largely recovered flavonols, whereas methanol has been reported to be the best
solvent for extracting flavan-3-ols (catechin, epicatechin, and epigallocatechin). The use of
ethyl acetate or acetone with addition of water resulted in a significantly increased yield of
extracted proanthocyanidins. This is a consequence of a low permeability of the sample
tissues to only ethyl acetate or methanol, which is a non-polar aprotic solvent [59].
Hernández-Jiménez et al. [60] examined the extraction of seed proanthocyanidins in
model solutions with increasing concentrations of ethanol, from 0 to 15% (v/v). This study
shows that ethanol is not required for proanthocyanidin extraction, although its presence
increases the rate of extraction. However, operational variables such as time, temperature,
solid to solvent ratio influence the success of the extraction procedure but only affect to
solutes weakly linked to the cell wall structure and to those contained in vacuoles [45]. CSE
requires long extraction time by using certain grades of organic solvents, usually at higher
temperature. The long extraction time or higher temperature may cause the degradation of
phenolic compounds of grape seeds.
On the other hand, temperature, extraction time and the large volumes of solvent are
important factors to scale-up extraction process from laboratory to the industry [45, 61]. In
this sense, new technologies or modifications on the protocols of CSE are focusing towards
more environmentally friendly methods with shorter extraction times, better extraction yield
and therefore towards the quality improvement of extracts [62]. Various novel techniques
developed for the extraction of nutraceuticals from plants, including ultrasound-assisted
extraction, microwave-assisted extraction and supercritical fluid extraction, have been applied
to the extraction of antioxidants from grape seeds.
Ultrasound-Assisted Extraction has been developed to extract these high added-value
components in a shorter time and with less solvent requirements. This extraction technique is
recognized as inexpensive, simple and efficient [63]. UAE enhances the solute extraction
owing to its cavitation forces upon the propagation of the acoustic waves. Cavitation provides
a higher cell wall disruption, enhancing mass transfer and facilitating solvent access to the
cell content [6].
UAE was used to extract valuable compounds such as phenolic acids, tartaric acid and
anthocyanins from grapes and their by-products [62, 64, 65, 67]. These authors agree that
UAE must be applied carefully to avoid degradation of susceptible solutes.
When comparing the phenolic compounds extraction from grape seeds using UAE and
CSE procedures, the ultrasounds-assisted appears to be faster (Table 2). Time between 10 and
34 min were reported [29, 62, 63, 68, 69] for UAE whereas in CSE the extraction time ranged
between few hours and several days.
Microwave Assisted Extraction utilizes microwave energy to cause molecular
movements and rotation of polar solvents with permanent or induced dipoles [70]. This
technology is the simplest, fastest and most economical technique for extraction. It requires
less solvent and provides higher extraction yields and shorter extraction times compared to
CSE. These advantages are associated with the quick heating owing to the dipole rotation of
the solvent in the microwave field. MAE heats the material internally and externally without a
thermal gradient. Table 2 summarizes some examples of phenolic extraction from some grape
seeds varieties.
Enzyme-Assisted Extraction. Enzymes are an usual tool in winemaking applications such
as pressing and wine clarification [71] and in other juice industries [72]. Research has focused
on the application of cell wall hydrolyzing enzymes, such as cellulases, glucanases, and
pectinases, to release phenolics from grape fruit and pomace. Kammerer et al. [4] reported
that pectinases and cellulases could result in notably higher recovery rates of phenolics from
V. vinifera L. grape pomace. Nevertheless the enzymatic treatment may also hydrolyze the
polyphenols released into low molecular weight phenolics, which may increase the
availability and bioactivity of these phenolics [73, 74, 75, 76].
To our knowledge, there has been little information on enzyme hydrolysis for the release
of phenolic compounds from grape seeds (V. vinifera). Examples from those are summarized
in Table 2. Bautista-Ortin et al. [77] described the effect of different pure enzyme activities
(xylanase, cellulase, polygalacturonase and pectinmethylesterase) on the release of
proanthocyanidins from seeds of Monastrell and Cabernet Sauvignon grapes. Their results
showed that polygalacturonase and cellulase favour the degradation of seed cell walls
involved in the solubilization of proanthocyanidins.
País grape seeds were enzymatic treated with three enzymes (pectinase, cellulase and
tannase) and an enzymatic blend. The phenol concentrations increased and the degree of
polymerization of proanthocyanidin was reduced. The three enzymes efficiently increased the
phenolic extraction and their effects on the degree of polymerization were according to their
enzymatic activity [76].
However, the enzymatic pretreatment of Muscadine seeds led to a decrease in the total
phenolic yield compared with solvent (50% ethanol) alone [75]. Whereas the enzymatic
treatment shortens the extraction time, modified the galloylated form of polyphenols
enhancing its antioxidant activity. The use of carbohydrases favoured the release of
polyphenols and monosaccharides from grape by-products, improving the antioxidant
capacity and the nutritional value. The use of tannase in grape seed extract changed the
galloylated form of catechin to its free form, releasing gallic acid and increasing the
antioxidant activity. The relative concentrations of phenolic compounds in grape seed extract
changed during enzymatic treatment with tannase, the total polyphenolic content increased up
to 41%, gallic acid up to 6 times, epicatechin up to 22% and procyanidin B2 up to 42% and
decreased the concentrations of epigallocatechin gallate, gallocatechin gallate and epicatechin
gallate [74].
Accelerated Solvent Extraction is also known as pressurized fluid extraction or
pressurized liquid extraction. It is an efficient, innovative and environmentally clean
technique. ASE operates at high temperature and pressure to maintain the solvent in liquid
state and enhances the extraction of solutes from solid samples. The operational variables
employed allow the solvent to penetrate deeper into the material and increase the extraction of
target compounds. Scarce information about ASE process with grape seeds has been found.
The works found [78, 79] used the ASE technology as a tool to extract anthocyanins and
procyanidins from several grape seed varieties. In these works the variables influencing the
extraction by ASE were not studied. These works are related in Table 2.
Subcritical Water Extraction is an environmentally friendly technology. It has been
chosen for the extraction of bioactives since it is simple, rapid and can be scaled-up [80].
Moreover water is a clean, safe and environmentally benign solvent [81]. SWE is an
alternative technique for the extraction of both polar and non-polar compounds [82]. It is
defined as water at a temperature between its boiling and critical point where the pressure is
regulated in such a way that water remains in its liquid state [83]. This technology was used
to obtain high added value products from grape pomace [84-86] and seeds [81, 82, 87]
because under subcritical conditions the water‘s dielectric constant has a similar value to the
range of dielectric constants of methanol, ethanol and acetone at room temperature [82].
SWE from Pinot Nero defatted grape seeds was carried out at 10 MPa, at temperatures
ranges between 80 and 120ºC and two water flows. The total phenolic yield increased (from
44 to 124 mg/g) with the increase of temperature. Nevertheless, the total phenolic yield
decreased with flow rate at constant temperature and kinetic curves were successfully fitted
with a model on the base of mass transfer and partition coefficients.
Tempranillo grape seeds were fractionated with a combination of two processes: i)
solvothermal and ii) hydrothermal sequential extraction process [81]. At the first step a 4.46%
of total polyphenols were extracted with 70% ethanol at 90ºC.
Supercritical fluid extraction is based on the properties of a fluid under supercritical
conditions to facilitate the extraction and purification of natural compounds from solid
samples. A supercritical fluid is a substance above its critical temperature and pressure that
has a good solvating power (like liquid), high diffusivity, low viscosity, and marginal surface
tension.
SFE may observe the environmental, toxicological, and health regulations and may be
denote as a green technology. Besides SFE may be an environmentally sustainable alternative
to the conventional organic solvent extraction because it avoids the use of large amounts of
toxic solvents, being also rapid, automatable, and selective. Carbon dioxide shows favourable
properties, including environmental safety, high availability, purity, and suitability for heat-
labile, natural compounds. The physico-chemical properties of supercritical (SC) CO2 (high
diffusivity, low viscosity and low surface tension in comparison with conventional solvents)
facilitate mass transfer, enabling an environmentally friendly operation.
SFE basically consists of two major steps: (1) extraction of the soluble substances from
the solid matrix by the supercritical fluid solvent and (2) separation of extracted compounds
from the supercritical solvent after the expansion [5].
The use of modifiers enhances the solubility of some compounds and may increase the
extraction selectivity, allowing operation at a lower pressure. The SC-CO2 extraction can use
a moderate extraction temperature as low as 30°C, avoiding the degradation of susceptible
solutes and it has the advantage of being environmentally friendly to achieve extracts free of
residual toxic solvents.
Besides the modifier, the main important variables on the SFE are the previous
conditioning of the solid, pressure and temperature because these latest variables determine
the solubility equilibrium, whose knowledge is basic for process design.
Not many studies are found in literature with grape seeds and SFE, and mostly are
focused on the extraction of grape seed oil [88-90]. Only a few works are focusing on
antioxidant compounds extraction [91-94] from seeds nevertheless there are a bigger number
of studies using grape pomace as feedstock.
The selectivity of SC-CO2 was assayed to isolate proantocyanidins [92] and resveratrol
[93] from different varieties of grape seeds. Yilmaz et al. [94] reported different extraction
conditions to maximize the level of each target compound. So, epigallocatechin gallate
extraction was maximum at 300 bar, 50ºC and 20% ethanol, whereas the maximum amount of
catechin and epicatechin were obtained at 300 bar, 30ºC and 20% ethanol.
Resveratrol was extracted from the Palomino fino grape variety using supercritical
carbon dioxide extraction in wide operational conditions, pressure (100-400 bar), temperature
(35-55°C) with the addition of ethanol as modifier (5%, v/v). At high pressure, low
temperature and 5% ethanol, the optimal conditions of resveratrol extraction were reached.
Supercritical Antisolvent Extraction technology may be proposed to fractionate polar
compounds from an organic solution [5]. A continuous contact SC-CO2/feed solution in a
pressurized precipitation vessel was carried out. The solution feed was sprayed in the
supercritical fluid to enhance the mixing of the two fluids. The solution feed can be
fractionated because the compounds not soluble in SC-CO2 precipitate as solid powder in the
high pressure vessel. Marqués et al. [95] extracted and concentrated catechin, epicatechin,
resveratrol and gallic acid from Syrah grape seeds variety. Several experiments were carried
out using pressures and temperatures ranging from 8 to 15 MPa and from 35 to 60ºC. When
the operational variables chosen were 15 MPa and 40ºC, the extracts were enriched in
antioxidants by more than 150% with respect to the starting extracts.
CONCLUSION
Grape seeds are a worldwide available source of bioactive compounds, which show
antioxidant properties, confirmed both by in vitro assays and by in vivo experiments. Phenolic
compounds account for a significant portion of these bioactives and have been the focus of
interest in last years. Since the grape seeds are present in the discarded by-product from
wineries and therefore it is a low cost material, its valorization could offer environmental and
economic advantages to these industries. Different extraction technologies have been tried
and can be successfully used for the efficient extraction and concentration of the phenolic
fraction, including the extraction with conventional solvents, the use of pressurized solvents
or the utilization of enzymes, microwaves or ultrasound as technological aids.
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Chapter 2
ANTIOXIDANT/PRO-OXIDANT ACTION
OF POLYPHENOLS FROM GRAPE SEEDS
L. M. Palade and V. S. Chedea

National Institute for Research and Development in Animal Biology and Nutrition
Balotesti, Ilfov, Romania
ABSTRACT
Grape seeds, a by-product obtained through wine making techniques, represent a
good source of functional compounds such as polyphenols, which are exploited due to
their antioxidant properties. Grape seed extracts have been shown to possess health
promoting benefits such as: attenuation of oxidative stress, cardiovascular protection,
neuroprotective agent, antitumor activity, anti-inflammation activity, anti-aging activity.
The easiest and most used method to assess the presence of polyphenols from natural
matrices, including the grape seeds, is the Folin-Ciocalteu method of total polyphenols
determination. The antioxidant/pro-oxidant activity of polyphenols from grape seeds is
mainly determined using the following methods: DPPH, FRAP assay, ABTS assay. This
property of grape seed polyphenolic components is given by the total polyphenols content
and also by the compostion in antioxidant molecules. The determination of grape seed
polyphenolic composition is largely done by liquid chromatography combined with mass
spectrometry techniques. Grape seed extracts tested, considered as an antioxidant
nutritive supplement, might have prooxidant activity as well, depending on dose, duration
of administration and other dietary components. There are also synergistic/antagonistic
interactions between antioxidant polyphenolics and other prooxidant compounds (e.g.,
the oxidative lipoxygenase) that influence the antioxidant/prooxidant balance, either in
model solution or food matrices, either in vitro or in vivo.
Keywords: grape seed extract, antioxidant/pro-oxidant activity, LC-MS, intermolecular

interactions

Corresponding Author address: Veronica Sanda CHEDEA, National Institute for Research and Development in
Animal Biology and Nutrition, Calea Bucuresti nr. 1, Balotesti, Ilfov, 077015, Romania, Email:
chedea.veronica@ibna.ro.
28 L. M. Palade and V. S. Chedea
INTRODUCTION
Fruits and vegetables wastes and by-products, produced in great amounts during
industrial processing, represent a challenge for the food industry that they need to be managed
accordingly, as they have a big influence on the environment. On the other hand, their high
content of bioactive components, which have a beneficial effect on health, has attracted
interest regarding their exploitation in terms of antioxidants and other bioactive compounds
[1]. Grape (Vitis vinifera L.) seeds are generally considered to be by-products of grape
processing technologies like grape juice, grape pekmez and wine production [2]. They contain
important vitamins, minerals and polyphenols including flavonoids, proanthocyanidins and
procyanidins [3].
Grape Seed Extract (GSE) is chemoprotective [4], anti-inflammatory [5], anti-bacterial
[6], anticarcinogenic [7] and anti-diabetic [8]. Recent studies also indicate that polyphenols
from GSEs play an important part in the regulation of intestinal barrier as well as the
prevention intestinal inflammatory ailments [9, 10]. Many studies report the antioxidant
activity of GSE‘s polyphenols as being one of the most important health promoting properties
ascribed to them [11-13] by activating important detoxifying enzymes [14].
POLYPHENOLS FROM GRAPE SEEDS

The grape seeds are anatomically composed of: seed coat (outer layers), endosperm and
embryo. The chemical composition of the seeds expressed as percentage of the total mass is:
25-45% water, 34-36% sugars, 13-20% oils, 4-6% tannins and phenolics, 4-6.5% nitrogenous
compounds, 2-4% minerals and 1% fatty acids [15].
The composition of the endosperm is formed of a lipid fraction made of around 50%
linoleic acid, 30% oleic acid, 10% saturated fatty acids, and 1% unsaponifiable residue. In
general, the oil fraction is extracted through the pressing of the grape seeds, resulting in
around 500 mL of oil for every 100 L of wine [15]. The minerals present in the seeds take up
4% to 5% of their total weight and the distribution of cations differs from that of the other
parts of the grape. Calcium tends to be the most abundant followed by potassium,
magnesium, and sodium, and then much lower levels of iron, manganese, zinc, and copper, in
that order [15].
The outer layers of the seeds contain high amounts of tannins. Depending on the crop, the
content is between 22% and 56% of the total polyphenols of the grape. Here are included the
procyanidins (67% - 86%) and an important proportion of the total gallic and caffeic acid.
The woody part is surrounded by a thin film that has also substantial concentrations of tannins
[15-18].
Many authors have reported that grape seed present a higher phenolic content than that
contained in the peel and pomace, therefore grape seeds could represent a valuable source of
phenolics and antioxidants [19]. Monomeric phenolic compounds, such as (+)-catechin, (-)-
epicatechin and (-)-epicatechin-3-O-gallate and dimeric, trimeric and tetrameric procyanidins
are dominant phenolic substances in grape seeds [2, 14, 20, 21]. Moreover, they contain
higher concentration of monomeric, oligomeric and polymeric flavan-3- ols than those of
grape skins [22].
Antioxidant/Pro-Oxidant Action of Polyphenols from Grape Seeds 29
Flavonoids are primarily synthesized in the skins and seeds of the grapes, with up to 75 to
90% being located in the seeds. Flavonols and anthocyanins are mainly found in the skins,
and flavan-3-ols and their proanthocyanidin polymers are synthesized primarily in seeds and
stems (about 60 and 20%, respectively) [23].
Seed tannins are smaller, being less polymerized than those localised in the skins (mean
of 10 units vs. 30 for skin tannins). They show a richer content of epicatechin gallate – about
30 vs. 5% in skin tannins and 15% in stem tannins [23]. Flavanol moieties present in the seed
tannins are in a total up to 28, as compared to skin tannins - up to 74 units [23].
As the polyphenolic content and composition of grape seed extracts were not
systemically evaluated, their characterization is important not only for the quality control, but
also for the elucidation of the mechanisms involved in their biological activities [24]. For the
evaluation of the polyphenolic compounds extraction separation by liquid chromatography
and UV-Vis and MS spectral identification are some of the important steps.
EXTRACTION AND EVALUATION

The extraction process is an important step in the recovery, isolation and identification of
phenolic compounds [25]. Polyphenolic compounds and their purity depend on the extraction
protocols: solid-phase extraction, shaking extraction [26], soxhlet extraction [27], microwave-
assisted extraction [28], ultrasound-assisted extraction [29] and supercritical fluid extraction
[30].
Extraction of polyphenols from grape seeds can be achieved by using alcoholic or
hydroalcoholic solutions, with or without mineral acids [31]. The use of solvents containing a
mineral acid permits the extraction of mainly all the phenolic classes, but the hydrolysis
reactions, that proanthocyanidins and acylated anthocyanins are subjected to, may produce
some artefacts. When solvents without acids are used, the extraction of phenolic compounds,
is regarded as not satisfactory. The possibility of other artefacts to occur during the extraction
technique are caused mainly due to the polymerization reactions of polyphenols, as well as
due to the action of polyphenol oxidases [32].
The manual solid-liquid extractions of polyphenols, after the grape seeds are
subsequently separated from the rest of the grape parts, are increasingly replaced with
techniques to reduce the time of extraction, solvents consumption, as well as to increase the
quality of the resulted extracts [33-36].
Different extraction solvents determined the extraction of different procyanidin
molecules besides the monomers catechin and epicatechin. Using hot water, procyanidin
oligomers with not a high degree of galloylation are extracted. Extracting the second time
with ethyl acetate, we get procyanidins with a higher galloylation degree which also
influences their antioxidant properties. We can conclude that besides the fact that due to the
environmental and each grape cultivar‘s genetic inheritance influence, a specific antioxidant
polyphenolic can be extracted, the mixture of different cultivars can also provide unique
recipes for polyphenolic GSE with desired antioxidant action through the valorization of
waste products of the winery and grape juice industry [43].
For the determination of total polyphenols, as a first step in polyphenol analysis in any
extract considered to contain phenolic compounds, different methods are used and none are
perfect [37]. The following methods are used ones: the Folin-Denis method, Folin-Ciocalteu
method, permanganate titration, colorimetry with iron salts, and ultraviolet absorbance [38].
From all these the Folin Ciocalteu is the one used the most and it relies on the transfer of
electrons in alkaline medium from phenolic compounds to phosphomolybdic/phosphotungstic
acid complexes to form blue complexes that are determined spectroscopically at
approximately 760 nm [37-39]. The results are expressed, mostly, as milligrams of
equivalents of galic acid (GAE) per kilogram or liter of extract, gallic acid being widely used
as the comparison standard. The major shortcoming of Folin Ciocalteu assay is the fact that it
measures besides the total polyphenols other oxidation substrates also. Singleton et al. [37]
discussed the effects of potential interference compounds and methods for correcting these
factors. However, despite these disadvantages, the Folin-Ciocalteu assay is simple and
reproducible and has been widely used for quantification of phenolic compounds in plant
materials and extracts [38] in general and in grape seed extracts in particular [43].
High performance liquid chromatography (HPLC) is the main technique used for the
separation of phenolic compounds [33]. A great number of papers have been published, using
HPLC to analyze the polyphenolic compounds in grape seed extracts [40]. A wide variety of
columns, supports, mobile phases and detectors are available for the characterization of
polyphenols [33]. In addition, various detection methods are used in tandem with LC for their
detection. These include UV-Vis (ultra violet visible), photodiode array (DAD), fluorescence
and mass spectrometry. The UV detection is generally the most highly used technique, due to
the natural absorbance of phenolic compounds in the UV region [32, 33, 40].
Mass spectrometry (MS) represents a valuable tool to obtain structural information for
the identification of the polyphenols extracted from grape seeds. MS is usually coupled with
liquid chromatography (LC). Thus, polyphenol structure as well as the mechanisms in which
they are involved is elucidated through the vast development of efficient LC-MS and multiple
mass spectrometry (MS/MS and MSn) systems [33].
Searching on Google Scholar Database for polyphenols identification from grape seeds,
only for the year 2015, there were retrieved 385 results. This shows the interest in scientific
research regarding the grape seed polyphenols (GSP) analysis by LC-MS. Table 1 and 2
present the characterization of grape seed compounds in both red and white varieties in terms
of extraction and separation conditions.
It has been reported that gallic acid is one of the major phenolic compounds found in
grape seeds and skins together with catechin and epicatechin [41]. Gallic acid was identified
in all grape seed samples (280 nm, m/z 169 – dissociated to form ions at m/z 125 through the
loss of CO2). It has been previously identified in Merlot, Cabernet Sauvignon, Bordeaux and
Isabel varieties, Merlot Recas [42], mixture of greek red grape seeds [43, 44]. Catechin and
epicatechin ([M−H]− at m/z 289) were also identified. They showed characteristic MS2
fragments at m/z 245 (loss of CO2), 205, and 203 (cleavage of the A-ring of flavan-3-ol) [45].
Using LC-MS, Ivanova et al. [46] detected flavan-3-ol monomers, (+)-catechin and (−)-
epicatechin, with [M–H]– at m/z 289 [46, 47] in the seeds of red and white grapes. Their
characteristic fragmentations in ESI+ mode (loss of one water, RDA, HRF and BFF for the
m/z 291* precursor ion, and loss of a -CH2-CHOH group or CO2, loss of C4H4O2 from the A-
ring and C6H6O2 from B-ring for the m/z 289) are in agreement with previously reported
findings [45, 48, 49].
Table 1. Compounds in grape seeds identified using liquid chromatography coupled with mass spectrometry (red varieties)
Compound Grape seed variety (Red) Applied technique UV-Vis m/z Year (sample Authors
(extraction and separation) λ max (nm) collection)
Gallic acid Vranec Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Merlot Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Pinot Noir, Negro Amaro, Cabernet Act/H2O (70/30, v/v), ESi-IT-MS, - - [50]
Sauvignon, Primitivo, Isabel, Sangiovese FTICR-MS
Feteasca Neagra H2O (°C), ESI+ - 171 - [47]
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 169 - [45]
HPLC-DAD-ESI-MSn
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 169 - [24]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 270 - [43]
Caffeic acid Feteasca Neagra H2O (°C), ESI+ - 181 - [47]
Ellagic acid Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 301 - [45]
HPLC-DAD-ESI-MSn
Hydroxybenzoic Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 298 339 - [43]
acid derivatives
Catechin Vranec Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
BRS Violeta MeOH/H2O (80/20, v/v), - 2012 [52]
ESI-MS/MS
Pinot Noir, Negro Amaro, Cabernet Act/H2O (70/30, v/v), - 289 - [50]
Sauvignon, Primitivo, Isabel, Sangiovese ESI-IT-MS, FTICR-MS
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 240, 278 291 - [43]
Epicatechin Vranec Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
BRS Violeta MeOH/H2O (80/20, v/v), ESI-MS/MS - 2012 [52]
Pinot Noir, Negro Amaro, Cabernet Sauvignon, Act/H2O (70/30, v/v), ESi-IT-MS, - 289 - [50]
Primitivo, Isabel, Sangiovese FTICR-MS
HPLC-DAD-ESI-MSn
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 289 - [24]
Epicatechin BRS Violeta MeOH/H2O (80/20, v/v), - 441 2012 [52]
gallate ESI-MS/MS
Pinot Noir, Negro Amaro, Primitivo, Isabel, Act/H2O (70/30, v/v), ESi-IT-MS, - - [50]
Sangiovese FTICR-MS
HPLC-DAD-ESI-MSn
Quercetin Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) - 447 - [45]
HPLC-DAD-ESI-MSn
Quercetin GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 463 - [24]
3-glucoside GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 477 - [24]
Proanthocyanidins Vranec, Act/H2O (80/20, v/v) – ESI-IT-MS 577 2007 [51]
BRS Violeta MeOH/H2O (80/20, v/v), - 577 2012 [52]
ESI-MS/MS
Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), - 577 - [50]
Cabernet Sauvignon, Primitivo, ESi-IT-MS, FTICR-MS
Isabel, Sangiovese
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) HPLC- - 577 - [45]
DAD-ESI-MSn
Proanthocyanidins GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 577 - [24]
Epicatechin Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) HPLC- - 593 - [45]
digallate DAD-ESI-MSn
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), - 729 - [50]
dimer monogallate Cabernet Sauvignon, Primitivo, ESi-IT-MS, FTICR-MS
Isabel, Sangiovese
Muscadine Act/H2O/AcOH (70/29.7/0.3, v/v) HPLC- - 729 - [45]
DAD-ESI-MSn
GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 729, - [24]
731
trimers Cabernet Sauvignon, Primitivo, ESi-IT-MS, FTICR-MS
Isabel, Sangiovese
867
dimer digallate Cabernet Sauvignon, Primitivo ESi-IT-MS, FTICR-MS
(A - type)
Proanthocyanidin Pinot Noir, Negro Amaro, Act/H2O (70/30, v/v), ESi-IT-MS, - 881 - [50]
dimer digallate Cabernet Sauvignon, Primitivo, FTICR-MS
(B - type) Isabel, Sangiovese
trimer Cabernet Sauvignon, Primitivo, FTICR-MS
monogallate Isabel, Sangiovese
tetramers Cabernet Sauvignon, Primitivo, FTICR-MS
Isabel, Sangiovese
1155
trimer digallate Cabernet Sauvignon, Primitivo, FTICR-MS
Isabel, Sangiovese
Proanthocyanidin GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 1441, - [24]
pentamers B-type 1443
Proanthocyanidin GSE (GravinolSuperTM) ESI-Q-TOF-MS/MS - 1729, - [24]
hexamers B-type 1731
Table 2. Compounds in grape seeds identified using liquid chromatography coupled with mass spectrometry (white varieties)
Compound Grape seed variety (White) Applied technique (extraction and separation) UV-Vis λ max m/z Year Authors
(nm) (sample
collection)
Gallic acid Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Chardonnay Act/H2O (80/20, v/v) – ESI-IT-MS 169 2007 [51]
Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 169 2010 [123]
Zalema EtOH, MeOH, ESI-IT-MS 272 2011 [124]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 271 2011 [125]
Feteasca Regala H2O (°C), ESI+ - 171 - [47]
Mixture of Greek red varieties Hot water, LC-DAD-ESI+-MS 270 - [43]
Caftaric acid Zalema EtOH, MeOH, ESI-IT-MS 297, 328 311 2011 [124]
Ellagic acid Malvar MeOH/H2O (80/20, v/v), Q-TOF 252,366 301 2011 [125]
Catechin Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Albariño EtOH/H2O (80:20, v/v), QqToF and QqQ - 2010 [123]
Fernão Pires MeOH/H2O (80/20, v/v), ESI-IT-MS - 2008 [126]
Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 240, 278 291 - [43]
Epicatechin Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 289 2007 [51]
Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 2010 [123]
Compound Grape seed variety (White) Applied technique (extraction and separation) UV-Vis λ m/z Year Authors
max (nm) (sample
collection)
Epicatechin gallate Malvar MeOH/H2O (80/20, v/v), Q-TOF 277 441 2011 [125]
Proanthocyanidins Smederevka Act/H2O (80/20, v/v) – ESI-IT-MS 577 2007 [51]
Chardonnay 1. Act/H2O (80/20, v/v) – ESI-IT-MS; - 577 2007 [51]
2. MeOH, ESI-IT-MS - 2007 [127]
Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 2010 [123]
Malvar MeOH/H2O (80/20, v/v), Q-TOF 278-280 2011 [125]
(epi)gallocatechin- Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 593 2010 [123]
(epi)catechin
Proanthocyanidin Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 729 2010 [123]
dimer monogallate Zalema EtOH, MeOH, ESI-IT-MS 278 2011 [124]
Proanthocyanidin Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 865 2010 [123]
trimers Zalema EtOH, MeOH, ESI-IT-MS 279 2011 [124]
Dimer digallate Albariño ethanol/water (80:20, v/v), QqToF and QqQ - 881 2010 [123]
Proanthocyanidin Zalema EtOH, MeOH, ESI-IT-MS 279 1153 2011 [124]
tetramers Mixture of Greek white varieties Hot water, LC-DAD-ESI+-MS 240, 278 1155 - [43]
Rockenbach et al. [50] and Caboni et al. [32] evaluated the profile of galloylated and
non-galloylated flavan-3-ols and the presence of enantiomers of catechin and epicatechin in
seeds of pomace from the vinification of different grape varieties with a view to their
exploitation as a source of natural antioxidants. The HPLC/DAD-MSn method employed
showed the presence of several galloylated and non-galloylated flavan-3-ol compounds and
the presence of condensed products of catechin with acetaldehyde.
Ivanova et al. [46] identified 4 procyanidin dimers of flavan-3-ols, B3, B1, B4 and B2 (in
the order of elution), with retention times of 18.8, 19.1, 21.6 and 24.4 min, respectively, with
molecular ions at m/z 577, and fragmentation ions at m/z: 559, 451, 425 and 289. Water
elimination gave the first fragment at m/z 559, and the one at m/z 451 was formed through the
loss of a phloroglucinol structure (m/z 126). The fragment ion at m/z 425 resulted from a
Retro Diels-Alder reaction (RDA) in the C-ring [46].
A procyanidin dimer consists of an extension unit and a terminal unit. The heterocyclic
ring of the flavan-3-ol units fragments through retro-Diels-Alder (RDA) and heterocyclic ring
fission (HRF) mechanisms. Singly charged negative ions with a [M−H]− at m/z 577
corresponded to the different procyanidin dimers [52]. [M−H]−ions at m/z 605 were
tentatively identified as (epi)catechinethyl dimers, condensed products of (epi)catechin with
acetaldehyde, corresponding to two (epi)catechin units linked by an ethyl-bridge [50].
Rockenbach et al. [50] also found [M−H]− ions at m/z 729 (monogallates of procyanidin
dimers) that generated MS2 ([M−H−152]−) fragment ions at m/z 577 corresponding to the
loss of a galloyl group, m/z 441 ([M−H−288]−) corresponding to the loss of an (epi)catechin
molecule, and m/z 603 ([M−H−126]−) corresponding to the elimination of a phloroglucinol
molecule (A-ring). Ions at m/z 407, 559, 425 and 451 suggest the characteristic fragmentation
pattern of the procyanidin dimers described. Compounds with [M−H]− ions at m/z 865 were
assigned to procyanidin trimers, known components of grape seeds [53, 42, 43].
Zhang and Zhu [24] compared polyphenolics from different grape extracts and managed
to characterize A-type proanthocyanidins. By the use of direct-infusion electrospray
ionization tandem mass spectrometry, they analysed red and white grape pomace extracts, as
well as grape seed extracts. The composition of the samples revealed glucosides of quercetin
and peonidin in both red and white grape pomace extracts, whereas quercetin, malvidin
derivatives and petunidin 3-p-coumaroylgluside were found only in the red ones.
(Epi)catechins, B-type PACs, A-type PAC dimers, and single A-type linked PAC trimers and
tetramers were detected in all the samples. On the other hand, singly and doubly charged
A-type PACs were only observed in the GSE [24]. Quercetin-3-glucoside and
quercetin-3-glucuronide were also detected in ESI-, with [M-H]- ions at m/z 463 and m/z 477,
respectively.
GSE revealed the presence of monogalloylated B-type epicatechin oligomers, with [M-
H]- ions at m/z 729, 1017 and 1305, attributed to monogalloylated B-type dimers, trimers and
tetramers, respectively. Moreover, they observed A-type proanthocyanidins. Under ESI+,
monogalloylated B-type dimers at m/z 731* and monogalloylated B-type trimers (m/z 1019*)
and tetramers (m/z 1307*) were also detected in GSE. Doubly charged A-type
proanthocyanidins were identified in the ESI- mode [24, 54].
ANTIOXIDANT/PRO-OXIDANT ACTIVITY OF
POLYPHENOLS FROM GRAPE SEEDS
A great deal of work has been carried out on the antioxidant properties of polyphenols
that can be recovered from abundant, inexpensive residual sources, such as wine industry by-
products. Polyphenol compounds and extracts that contain polyphenols from grape seeds and
grape seed pomace have been shown to exert protective effects regarding various foods [55-
58]. An important issue pertaining the use of said compounds, arises from their potential use
as protection agents for the food matrices, such as proteins and lipids. Foods are subjected to
oxidative reactions, which are considered to be the main cause of deterioration. They are
responsible for the losses of nutritional value, aroma, taste as well as degradation of texture
[1].
The antioxidant characteristics of polyphenols from grapes, notably from grape seeds,
have been widely investigated [59, 60]. Grapes and wines, containing considerable amounts
of polyphenolic compounds that have been studied for their beneficial effects on health. They
are considered as good agents in lowering the oxidative stress, modulating the inflammatory
cascade, reducing the oxidation of LDL-c and inducing protection against atherothrombotic
episodes (also myocardial ischemia and inhibition of platelet aggregation). The antioxidant
properties of phenolic compounds are generally considered to occur in several different ways
in biological systems (donation of hydrogen, free radical scavenging and metal chelating
activities, as well as their involvement in the cell signaling pathways and gene expression)
[61, 62].
The antioxidant activity of grape seeds is given by the dry matter content, and also by
their oil content [63]. Grape seed oil contains approximately 72% linoleic acid, thus being
regarded as having a highly beneficial nutritional importance [64]. The low solubility of low
molecular weight polyphenols present in the grape seed oils, in conjunction with their linkage
to the seed material, seem to explain the low contents of total polyphenols in grape seed oils
(cca. 100 times lower than in GSEs), as well as their low antioxidant capacity (10 to 100
times lower than that of GSEs) [65].
There is a series of methods which enable the evaluation of the antioxidant potential of
phenolic compounds extracted from grapes, grape seeds and the other parts of the grape, such
as the 1,1-diphenyl-2-picryhidrazyl (DPPH) method, oxygen radical absorbance capacity
(ORAC) assay, crocin bleaching assay (CBA), 2,2‘-azino-bis-(3- ethylbenzothiazoline-6-
sulfonic acid) (ABTS) assay, the thiobarbituric acid reactant substances (TBARS), Trolox
equivalent antioxidant capacity (TEAC) assay, the ferric reducing antioxidant power (FRAP)
assay [62], and the Rancimat test [43] (Table 3).
It was suggested that the antioxidant properties of phenolic compounds have a limit of
concentration at which the antioxidant capacity would not rise with the concentration [66].
Various studies revealed though inconsistency among the results, thus indicating that the
antioxidant activity of the analyzed phenolic compounds is influenced by more factors, not
only their concentration [67, 68].
Pinelo et al. [69] noted that the solvent used for the extraction of the antioxidant
influences its potency. For catechin and resveratrol, the antiradical activity of the extracts
decreased from ethanol, followed by methanol, and the minimum being in water [69].
Table 3. Polyphenolic content and antioxidant activity of grape seed extracts
Variety TP (total phenolics content) DPPH (1,1-diphenyl-2- ABTS (2,2‘-azino-bis-(3- FRAP (ferric reducing Year Source
picryhidrazyl) ethylbenzothiazoline-6-sulfonic acid) antioxidant power)
Cabernet 0.75-4.04 GAE/100g DM 3.35-11.8 mg/mL (EC50) 88.6-962.3 µM TE/g DM 2008 [128]
Sauvignon 4369.2 mg GAE/100g GSE 121.2 mg TEAC/g sample 120.2 mg TEAC/g sample [129]
869 mg/100 g of seeds 0.55 µg/µg DPPH (EC50) [130]
Grenache 41.2 mg GAE/g DW 56.4 TE/g DW 94.8 μM TE/g DW 114.6 μM Fe2+/g DW 2009 [61]
982 mg GAE/100g GSE 0.51 µg/µg DPPH (EC50) [130]
17.78 mg Chlorogenic acid 64.2% inhibition [131]
equivalents/100g DW
Merlot 1689 mg GAE/100g GSE 0.54 µg/µg DPPH (EC50) [130]
Counoise 42.7 mg GAE/g DW 39.6 TE/g DW 81.5 μM TE/g DW 43.8 μM Fe2+/g DW 2009 [61]
Grenache 88.7 mg GAE/g DW 41.4 TE/g DW 77.4 μM TE/g DW 43.11 μM Fe2+/g DW 2010 [61]
Syrah 72.8 mg GAE/g DW 35.5 TE/g DW 77.9 μM TE/g DW 41.3 μM Fe2+/g DW 2010 [61]
Carigan Noir 58.6 mg GAE/g DW 42.1 TE/g DW 85.0 μM TE/g DW 40.2 μM Fe2+/g DW 2010 [61]
Mourvèdre 59.6 mg GAE/g DW 48.6 TE/g DW 96.5 μM TE/g DW 51.6 μM Fe2+/g DW 2010 [61]
Alicante 76.4 mg GAE/g DW 73.0 TE/g DW 133.0 μM TE/g DW 88.9 μM Fe2+/g DW 2010 [61]
Bouchet
Feteasca 37.835 mg GAE/g 95.515% inhibition [47]
Neagra
Feteasca 27.065 mg GAE/g 94.585% inhibition [47]
Regala
Shiraz 116.73 g GAE/100g 0.47 µg/µg DPPH (EC50) 2008 [132]
Emir 75.5 mg GAE/g extract 428.1 µg/g IC50 [133]
Gamay 255.4 mg GAE/g extract 151.8 µg/g IC50 [133]
Romy 14.40 mg CAE 54.2% inhibition [131]
(chlorogenic acid)/100g DW
Mandilaria 628 mg GAE/g extract 6.7 µg/g estract (IC50) 129.0 µM [134]
FeSO4
Variety TP (total phenolics content) DPPH (1,1-diphenyl-2- ABTS (2,2‘-azino-bis- FRAP (ferric reducing Year Source
picryhidrazyl) (3- antioxidant power)
ethylbenzothiazoline-6-
sulfonic acid)
Mixture of Greek red 40.11 mg GAE/g seeds 0.99 EC50 (mM Trolox 21.36 [43]
varieties equivalents/mg GAE)
Mixture of Greek 38.05 mg GAE/g seeds 1.10 EC50 (mM Trolox 18.28 [43]
white varieties equivalents/mg GAE)
Black queen 28.24 mg GAE/g DM 160.63 µmol TE/g DM 313.49 µmol TE/g DM 152.64 µmol TE/g DM [25]
Pinot Noir 67.88 mg GAE/g DW 78.6 µl extract/µg DPPH [135]
Zalema 644.62 mg GAE/100 g 96.46 mmol TE/100 g 38.61 mmol TE/100 g [124]
GSE 7.01 µg/ml TE [136]
Montepulciano 8708 mg GAE/100g DM 2.68 IC50 (mg GAE/L) 2005 [91]
Tuscan (Colorino) 5033 mg GAE/100g DM 3.20 IC50 (mg GAE/L) 2005 [91]
Muscat 196 mg AAE/g 11 IC50 (µg/ml) [137]
(ascorbic acid equivalents)
Taking into account the metal-binding properties of polyphenols, Perron and Brumaghim
[70] concluded that polyphenol compounds containing metal binding catechol and galloyl
groups have very different activities depending on the metal ion. In systems involving iron
and H2O2, antioxidant potency is observed through several assays, including cell and
inhibition of DNA damage studies, while in the case of systems involving copper and H2O2,
the antioxidant activity is commonly highly lower. Also, in many instances, the interactions
between polyphenols and copper exert a pro-oxidant activity [70].
MOLECULAR INTERACTIONS AND MOLECULES INVOLVED

IN ANTIOXIDANT/PRO-OXIDANT ACTION – SYNERGISTIC
AND ANTAGONISTIC INTERACTIONS
Grape seed catechins exhibit both antioxidant and pro-oxidant activities. Their unique
structure is thought to contribute significantly to their beneficial health effects, provided by
the phenolic groups in their molecular structure. Pyrogallol and hydroxyl moieties show
superoxide anion scavenging potential, whereas the galloyl groups are responsible for
quenching hydroxyl radicals [71]. Yordi et al. [73] noted that the structural features that have
been associated with antioxidant activity are: a) a catechol group on the B-ring, which confers
high stability to the radical formed after the capture reaction of the free radical, b) the
2,3-double bond in conjugation with a 4-oxofunction of a carbonyl group in the C-ring and c)
the presence of hydroxyl groups at the 3 and 5 position (Figure 1) [72].
Currently, several authors have described that phenolic compounds extracted from plants
materials, have antioxidant properties in various model systems and in several foods [74, 75].
However, it has been also described that these compounds can exhibit pro-oxidant and
cytotoxic properties under certain conditions such as the presence of metals, pH, structural
characteristics and concentration [76]. On the other hand, it has been shown that mixtures of
different phenolic compounds can produce synergistic or antagonistic pro-oxidant effects
[77].
Figure 1. The relationship between the antioxidant activity and structure relationship of flavonoids. (a)
catechol moiety of the B-ring, (b) presence of hydroxyl groups at the 3 and 5 position, (c) 2,3-double
bond in conjugation with a 4-oxofunction of a carbonyl group in the C-ring [72].
Flavonoids from different classes are present together and are distributed in various
amounts in food systems [78]. The interactions between phytochemicals are highly important
because, in a mixture, they rarely act independently [79]. As food components, their
beneficial effects are influenced by many factors, including the in vitro activities, food
processing technique, and absorption and metabolism [80]. Catechins, known constituents of
green tea and grape seeds, may act both as antioxidants and pro-oxidants. The influencing
factors are the concentration and the exposure time on the cell culture, as Braicu et al. [81]
have shown.
Ostrowska and Skrzydlewska [82] reported that the antioxidative activity of catechins
may be altered through the esterification of the hydroxyl group in position 3 by gallic acid;
the esterification of the carboxylate group by gallic acid diminishes their antioxidant activity.
It is highly significant as the gallate acid residues induce lipid peroxidation by reacting with
lipid radicals.
Chedea et al. [14] reported that the pro-oxidant activity could be explained also through
the catechin-quinone redox system. Quinones are compounds that contain carbonyl groups,
and are capable of producing new compounds by means of coupling reactions that retain a
number of hydroxyl groups. O-quinones may act as pro-oxidants through redox-cycling, by
forming reactive oxygen species (ROS). By contrary, when they are reduced to semiquinones
by radicals, and then back to diphenols, they exert an antioxidant effect. When catechin o-
quinones are subjected to phenolic coupling reactions, they form dimers and oligomers,
retaining their original number of reactive hydroxyl groups. Thus, the antioxidant capacity is
enhanced up to a level where the oligomers precipitate; they become insoluble [71].
Mahmoud et al. [83] and Abou Samra et al. [77] studied the order of antioxidant tendency
of model antioxidants (ascorbic acid (AA), caffeic acid, quercetin, catechin (Cat), and
hesperetin). They observed that when catechin is mixed with ascorbic acid, catechin is a
stronger antioxidant than ascorbic acid is. The luminol chemiluminescence test was used to
investigate their antioxidant/pro-oxidant effects as well as their synergistic behavior. The
values indicate that when mixed, ascorbic acid becomes more antioxidant and catechin more
pro-oxidant, assuming that catechin regenerates ascorbic acid. Their results support the idea
of a significant antioxidant behavior because of catechin dimerization through oxidation,
which leads to procyanidin formation. Catechin dimerization was also previously proposed as
to affect the antioxidant/pro-oxidant balance in primary leucocyte culture [14]. Thus,
antioxidant activity is not only based on intramolecular stabilisation of formed radicals
through delocalisation, but also on intermolecular delocalisation to the extent where this one
is favoured [83].
The antioxidant and pro-oxidant activity was determined in model solutions of standard
compounds by a cyclic voltammetry assay using the conditions described by Simiš et al. [84].
They observed that compounds with low oxidation potentials (<0.45) possess antioxidant
activity, while compounds with high oxidation potential values (>0.45) act as pro-oxidants
[84].
It is suggested that the free radical scavenging activity and the electrochemical behavior
of phenolic compounds are highly influenced by their multiple OH substitution and
conjugation. It has been pointed out that compounds with low oxidation potentials act as
antioxidants, while compounds with high oxidation potentials act as pro-oxidants. This should
be taken into consideration as a trait for the antagonistic interaction between phenolic
compounds present in some extracts showing simultaneously antioxidant and pro-oxidant

activities [85].
Azam et al. [86] stated that: similar to several other classes of polyphenols, both
epicatechin (EC) and epigallocatechin gallate (EGCG) exhibit pro-oxidant activities like the
generation of superoxide anion and the hydroxyl radical. Both polyphenols induce oxidative
DNA breaking in the presence of copper ions; oxidation mediated by copper of EC and
EGCG may lead to the formation of polymerized polyphenols. It is indicated that copper
oxidized catechins are better pro-oxidants as compared to their unoxidized forms [86].
Through the oxidation of phenols in vivo, polyphenol oxidase produces free radical
intermediates that are extremely reactive. They generate polymeric products with different
stoichiometry through condensation, subsequent to being released from the enzyme. This
phenomenon is believed to be associated with the pro-oxidant activity of polyphenols. It is a
result of their predisposition to auto-oxidize, in tandem with the formation of ROS and H2O2
[87]. The active role of GSEs in oxidative stress or cell proliferation is supported by in vivo
and in vitro studies [14, 88].
The action of GSEs in vivo in was explored in yeast cells, which is very useful as a first
screening tool to investigate oxidative stress at the cellular level, despite the limitations
inherent to the use of such a simple single cell experimental model. This microorganism
conserves not only most enzymatic equipment and cellular structures, but also the redox
balance and the response to oxidative stress specific to higher eukaryotes. Our results clearly
emphasize the role of the GSEs supplementation in yeast cell growth and growth recovery
assays, which can vary from antioxidant to pro-oxidant depending on the cellular antioxidant
machinery deficiency [88].
Iacopini et al. [91] studied the capacity of phenolic compounds from red grapes to
scavenge free radicals. International and Italian red grape varieties of Native Tuscan, grown
in the same location, were analyzed for their free radical scavenging activity by using two
different models: the DPPH assay and the inhibition of the tyrosine nitration by peroxynitrite.
Considering the interactions among phenolic compounds as a possibility for the low
correlation between antioxidant activity of crude extracts and phenolic content, they
experimented with combinations of two, three and five compounds, in order to assess these
possible interactions. In the case of the DPPH test, all six combinations exerted antagonistic
interactions. The inhibition of DPPH activity was found to be actually lower than expected by
adding the antioxidant activities values of individual compounds [89, 90].
The combinations of rutin with trans-resveratrol and rutin with trans-resveratrol and with
quercetin showed synergistic interaction. The obtained inhibition was higher than the sum of
the individual effects. On the other hand, the combinations of quercetin with resveratrol and
of all five compounds together showed a lower activity than anticipated. Also, the mixtures of
catechin with epicatechin and quercetin with rutin gave activities that did not exceed the ones
expected by adding the individual values [91].
Phenolic compounds present in a mixture can interact and the interactions may interfere
with the total antioxidant capacity of that mixture. Moreover, using different modalities for
the assessment of the antioxidant capacity, the results are different. A possible explanation
could be the difference between the ways the polyphenols are scavenging free radicals, as in
the case of DPPH or peroxynitrite degeneration [92, 93].
For aqueous extracts of red and white mixtures of grape seeds, the total polyphenols and
different antioxidant parameters where measured and correlated [43]. Thus, the values of
FRAP, antiradical activity (AAR) determined by DPPH test and chemiluminescence, and
oxidative stability of sunflower oil (Rancimat), were plotted against the TP content and the
degrees of correlation between these results were calculated.
In this case, no correlation was found between TP and FRAP and the oxidative stability
of the purified sunflower oil containing GSE [43]. A better correlation was found for TP and
DPPH test, antiradical activity and even better with the oxidative stability of the unpurified
sunflower oil containing the antioxidants. The best was registered for antiradical activity
determined by chemiluminescence measurements and DPPH tests. No correlation is observed
between FRAP and the other antioxidant measurement methods used in this study. AAR might
be significantly associated with flavanols (catechin, epicatechin, proantocyanidins) [94].
Synergism and antagonism phenomena occurring between various polyphenol
compounds, as observed in real food substrates [95], may cause outcomes different from the
anticipated ones, regarding the antioxidant manifestations, due to the addition of specific
antioxidants. Such behaviours require a more complex assessment of the antioxidant activity,
not only the simple evaluation of the antioxidant status observed in a food after addition of
the antioxidant [96].
Thus, the assessment of the antioxidant efficiency of a mixture of antioxidants should not
envisage only the measurement of standard proportions, but also the manifestations of the
simultaneous variation of the content of the compounds in the mixture system. The
importance comes from the fact that resulted antioxidant effects might be caused by certain
ratio between the existing antioxidant compounds. Under certain conditions, an antioxidant
system might transform into a pro-oxidant one, thus, any deviation from the ratio could cause
unwanted results. In the case of food matrices, due to the occurring redox reactions, such
variations are real and the overall antioxidant efficiency should take into account the
consumption of one antioxidant at the expense of another [96-98].
The deactivation of oxidant species by antioxidant polyphenols is based, in the case of
food matrices that tend to deteriorate because of the action of peroxyl radicals [99, 100], on
the donation of hydrogen:
It was hypothesized [99, 100] that phenoxyl radicals generated through this reaction
could be stabilized by resonance and/or intra-molecular hydrogen bonding, or combine to
give dimers:
Brand-Williams et al. [101] and Bondet et al. [102] described the potency of an
antioxidant component to reduce R· as being highly dependent on its hydrogen donating
ability.
The effects of mixtures of polyphenolic antioxidants were postulated to include: a) a
synergistic effect if the less efficient antioxidant regenerates the more efficient one; b) an
antagonistic effect if the more efficient compound regenerates the less efficient one; c) no
mixture effect if both antioxidant molecules have the same efficiency. There is substantial
evidence that the regenerating ability of an antioxidant towards another depends on the
relative contents of the two components in the mixture [97, 98, 103, 104].
GRAPE SEED ANTIOXIDANTS INTERACTIONS WITH

THE OXIDANT LIPOXYGENASE
The intermolecular interaction between lipoxygenase and polyphenols is studied because

in terms of the structure and function, lipoxygenases (LOXs) are unique, due to their metal
cofactor is a single ion bound by the side chains of the surrounding amino acids and the
carboxylic group of the C-terminus, and their inhibitors bind to or near the Fe co-factor [105].
Lipoxygenases are inhibited by a large number of chemicals, some of which also serve as co-
substrates [106], polyphenols being some of them [107].
Lipoxygenase (LOX, EC 1.13.11.12) is a dioxygenase, found widely distributed in plants,
animals, and microorganisms. It catalyses the oxidation of polyunsaturated fatty acids to
hydroperoxides [108]. Peroxyl radical complexes have been reported to exist during the
catalytic cycle of LOX and can serve as sources of free radicals [109]. As previously noted,
some antioxidants, which act as free radical quenchers, are also considered to inhibit LOX
[110]. Schewe et al. [111], after studying the inhibitory effect of (−) epicatechin and of
related oligomers, procyanidins, towards mammalian lipoxygenase, hypothesized that the
potential for lipoxygenase inhibition by flavanols and procyanidins could have a beneficial
influence on the cardiovascular system in man [111].
Lipoxygenase activity implies responses that are unwanted. The reduction of these effects
can be accomplished through the use of phenolic compounds that are able to inhibit
lipoxygenase activity [112]. Depending on various factors, the mechanisms that have been
reported involve iron reduction at the active site of lipoxygenase [113]. Also, semi-quinones
and quinones, formed during the oxidation of polyphenols, might inhibit lipoxygenase by
linking to its sulfhydryl or amino structures [14, 118]. Another example is the inhibition
action of caffeic acid, catechin and quercetin against lipoxygenase from pea seeds [114, 115].
Babich et al. [116] pointed out that lipid peroxidation is a classic indicator of oxidative
stress, in which case electrons are pulled away from cell membranes by free radicals, thus
inducing damage. In order to quantify lipid peroxidation, malondialdehyde (MDA) is used,
the end product of lipid peroxidation. The reaction between MDA and thiobarbituric acid
(TBA), producing thiobarbituric acid reactive substances (TBARS), is further quantified
through visible or fluorescence spectrophotometry [116].
Chedea et al. [14] used a standard soybean lipoxygenase and a raw extract from soybean
containing the isoenzymes LOX-1 and LOX-3 as pro-oxidant inducers, in order to determine
the potential inhibitory action of GSE polyphenols against LOX enzymes [14]. To some
extent, the grape seed extract tested, considered as an antioxidant nutritive supplement, may
have pro-oxidant activity as well, depending on the dose, duration of administration, and
other dietary components [14].
As lipoxygenases contain an iron moiety at the active site, they are inhibited by
flavonoids, in particular those containing a catechol group. They are known to chelate iron
and other transition metal ions [117]. This possible chelation through a pro-
oxidant/antioxidant effect was evaluated by the TBARS (thiobarbituric acid reactive

substances) and MTT ((3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assays
on the leucocyte culture [118]. Short time periods of coincubation of cells with the GSE,
followed by the exposure to lipoxygenase standard or in extract, revealed a high level of lipid
peroxidation and a pro-oxidative effect. Longer coincubation and addition of lipoxygenase
resulted in the reversal of the pro-oxidant action either to antioxidant activity or to the control
level for. Lipid peroxidation was significantly reduced when cells were exposed to
polyphenols over a longer period and the lipoxygenase-GSE polyphenols complex formation
was found to be responsible for the observed effects [118].
Hu et al. [119] studied the effect of natural flavonoids on the lipoxygenase activity and
observed that they may exert anticarcinogenic and protective action by inhibiting the
lipoxygenase generated during oxidative activation. They observed that epigallocatechin-
gallate, quercetin and rutin inhibited the co-oxidation rate of guaiacol, benzidine,
paraphenylenediamine and dimethoxybenzidine by soybean lipoxygenase-1 [119].
Bors et al. [100] postulated that there are several ways in which flavonoids act as good
radical scavengers: (i) the presence of the catechol structure (o-dihydroxyl moiety in the B
ring), which plays the role of a target site for radicals, inducing electron delocalization and
stabilizing the phenoxy radical; (ii) the delocalization of an electron (unpaired) from the B
ring through the 2,3-double bond conjugated to the 4-oxo group; (iii) increased electron
delocalization along the entire flavonoid unit, by the use of the hydroxyl moieties at the
positions 3 and 5, thus improving radical scavenging activity. The inhibition potential was
explained as to be attributed to: a) the presence in the A or B ring of a catechol structure, and
b) the presence in the C ring of both a carbonyl structure and a 2,3-double bond [111].
Szymanowska et al. [114] proved that some phenolic compounds might efficiently inhibit
the activity of lipoxygenase from pea seeds. The conducted study showed that flavonoids
(catechin, quercetin) can significantly reduce the activity of lipoxygenase. Quercetin has been
reported to be among the inhibitors with the highest potency against lipoxygenases [120]. It
seems quercetin combines both inhibitory and radical-scavenging properties, being regarded
as a very powerful and efficient natural antioxidant [121].
Data obtained from studies pertaining inhibitory effect of epigallocatechin gallate on
lipoxygenases showed EGCG (epigallocatechin gallate) as the most potent inhibitor against
lipoxygenase from mackerel muscle [108]. Schewe et al. [111] reported that EGCG also had
the best inhibition potential against rabit 15-lipoxygenase-1, and epicatechin showed higher
inhibition in the case of recombinant human platelet 12-lipoxygenase.
Skrzypczak-Jankun et al. [122] observed the interaction between soybean lipoxygenase-3
and EGCG. The X-ray analysis reveals ring A close to the iron co-factor and linked to the C-
terminal end of the enzyme through a hydrogen bond. The hydroxyl moieties of ring B are
involved in the hydrogen bonds and the van der Waals interactions with the adjacent amino
acids and water molecules [122].
The interactions of these flavanols with LOX can be more complicated than simply
blocking the access to the enzyme‘s active site. Therefore, it warrants future endeavors to
thoroughly understand thus reliably predict interaction mechanism between the LOX proteins
and therapeutic agents at the molecular level. This will be important in fully understanding
the exact role of lipoxygenase inhibition by quercetin in therapy targeting and possibly
identifying new bioactive molecules which would be used as drugs [120].
ACKNOWLEDGMENTS
This work was supported by funds from the National Research Project PN-II-RU-TE-
2012-3-0048 granted by the UEFISCDI of Romanian Ministry of Education.
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Chapter 3
FLAVONOIDS INTERACTION WITH

GASTROINTESTINAL TRACT: MODULATION OF
ENTEROENDOCRINE SYSTEM, INTESTINAL
PERMEABILITY AND METABOLIC ENDOTOXEMIA
X. Terra, M. T. Blay, M. Pinent and A. Ardévol

MoBioFood Research Group. Departament de Bioquímica i Biotecnologia,
Universitat Rovira i Virgili, Tarragona, Spain
ABSTRACT
Obesity and related disorders, such as insulin resistance and diabetes, are among
major health problems. Flavonoids are plant bioactive compounds that have been
suggested to have several beneficial effects, including antiobesity and antidiabetic
action/effect agents; however the exact mechanisms to exert these effects remain to be
determined. Flavonoids might act within the body after being metabolized and absorbed,
but besides that flavonoids could also modulate the body homeostasis acting directly on
the gastrointestinal tract. Several mechanisms at this level could help to explain their
effects on obesity-related pathologies, such as modulation of the release of hormones by
enteroendocrine cells, regulation of the intestinal immune system and protection of the
intestinal barrier, and/or modification of the intestinal microbiota. In this chapter we
relate the existing evidence of flavonoid action in these processes.
Keywords: flavonoids, phenolic compounds, intestine, enterohormones, inflammation
INTRODUCTION
The gastrointestinal tract has, among its various functions, the responsibility to inform
and protect the body from entry and contact with the great diversity of chemical structures
that are ingested simultaneously with the nutrients required for energy and plastic functions of
the human beings [1]. Specifically, the intestinal epithelium acts as a selective filter of the
58 X. Terra, M. T. Blay, M. Pinent et al.
components of the intestinal lumen: it allows absorption of nutrients, electrolytes and water;
communicates what molecules we eat; and also discriminates between pathogenic and non-
pathogenic bacteria, and defends from toxins and food antigens. The epithelium, together
with the intestinal microbiota, has a conditioning effect on gut homeostasis, generating
paracrine and endocrine signals and regulating the mucosal immune response and metabolic
activity of the intestine and the whole organism. Thus the intestine has recently gained
attention for its identified new role in the pathophysiology of several metabolic diseases
including obesity, insulin resistance and diabetes [2, 3].
The currently existing knowledge about the molecular mechanisms that explain the
enteroendocrine and immunomodulatory functionality would support the possibility of
improving metabolic homeostasis or prevent its disruption through ingested food compounds.
Flavonoids, are a group of polyphenols found in high concentrations in fruits and vegetables.
They have been shown to have several beneficial effects on human health [4]:
cardioprotectors [5], hipolipidemics [6], antioxidants [7], antigenotoxics [8], antimicrobian
agents [9], anti-cancer/anti-proliferatives [10, 11], anti-inflamatories [12] and
antihyperglycaemics [13]. Many of the flavonoid structures have definite bioactive properties
for which there is not a clear available molecular explanation to define clear structure-
function-bioactivity, partly because of the difficulties in analyzing the bioavailability of the
great diversity of structures involved [14]. However there is evidence to suggest the
possibility that these molecules may explain part of their effects due to its action previous to
their absorption process, directly through interaction with members of the tract
gastrointestinal [15, 16].
In this chapter we analyze the effects of flavonoids in the gastrointestinal tract, as a
mechanism that could help to explain their effects on obesity-associated disorders described
elsewhere [17]. We focus on the effects of flavonoids on the enteroendocrine system, which
controls several digestive and metabolic processes such as food intake and glucose
homeostasis. On the other hand, we compile evidences of flavonoid effects on intestinal
inflammatory response, permeability alterations and changes in gut microbiota related to
obesity-associated complications and diet.
OVERVIEW OF FLAVONOID STRUCTURE AND METABOLISM

Flavonoid structures include from simple monomers, as (+)-catechin and its isomer (-)-
epicatechin, to complex structures that include the oligomeric and polymeric
proanthocyanidins, which are also known as condensed tannins. The monomeric forms can be
hydroxylated to form gallocatechins, and monomers can also undergo esterification with
gallic acid [18]. Catechin and epicatechin are the main flavanols in fruit, whereas
gallocatechin (GC), epigallocatechin (EGC), and epigallocatechingallate (EGCG) are found
in certain seeds of legumes, in grapes, and in tea. Although the exact flavonoid content is
difficult to determine due to the wide range of structures, they are major components in the
human diet due their widespread presence in fruits, berries, nuts, beans, some spices, cocoa-
based products, wine, and beer [19].
The biological properties of polyphenols depend on their bioavailability. The chemical
structure of polyphenols determines their rate, the extent of intestinal absorption and the
Flavonoids Interaction with Gastrointestinal Tract 59
nature of the metabolites that circulate in the plasma [20]. Moreover, the degree of
polymerization and galloylation of flavan-3-ols are factors that affect their bioavailability.
Monomeric flavan-3-ols are absorbed in the small intestine, where they are extensively
metabolized into glucuronide conjugates. Flavan-3-ols can also enter the liver, where they are
mainly sulfated and methylated [21]. Then, conjugated flavonoids enter into systemic
circulation or can be returned to the intestinal lumen via bile (entero-hepatic circulation) [22,
23]. Approximately 90-95% of the total consumed polyphenols cannot be absorbed by the
small intestine; as a result, they pass to the colon and, in addition to the compounds returned
by the entero-hepatic circulation, they are metabolized by the colonic microbiota [24]. The
microbial metabolites are absorbed by the colonocytes and arrive at the liver, where they are
subjected to glucuronidation, methylation and sulphatation. Then, they enter into systemic
circulation or to the kidneys, where they are excreted in the urine [21]. Flavonoids have been
detected in a wide range of tissues in mice and rats, including the brain, endothelial cells,
heart, kidney, spleen, pancreas, prostate, uterus, ovary, mammary gland, testes, bladder, bone,
and skin [25].
INTESTINAL ALTERATIONS IN OBESITY AND INSULIN RESISTANCE

Incretins and the Enteroendocrine System
The enteroendocrine system is one of the highest endocrine systems of the organism [26].
On the intestinal surface, there are absorptive enterocytes, bactericidal Paneth cells, mucus-
producing goblet cells and hormone-secreting enteroendocrine cells. These last cells are fully
differentiated cells that, together with the goblet and Paneth cells, constitute the secretory
lineages in the intestine, composing 10% of the epithelium [27]. The different
enteroendocrine cell types have been classified according to their epithelial localization: first,
the ―closed cells‖ that do not reach the gut lumen, and second, the ―open cells‖ that project a
tuft of apical microvilli into the intestinal lumen and extend to the basal lamina (lamina
propria) [28]. The open type cells are considered to be primary chemoreceptors, responding to
the luminal nutrients by releasing their secretory products, which activate neuronal pathways,
nearby cells or distant targets. Closed cells can be regulated by luminal content indirectly
through neural and humoral mechanisms [28]. Enteroendocrine cells have also been classified
into at least 10 types based on their morphology, principal hormone product(s) and
distribution along the intestinal tract [26]. The most studied enteroendocrine cells are I-, L-,
and K-cells due to their secreted products, which are cholecystokinin (CCK), glucagon-like
peptides (GLP), and glucose-dependent insulinotropic polypeptide (GIP), respectively [27].
Thus following meal intake, a complex set of physiological responses is activated,
providing neural and endocrine signals regulating the ingestion, absorption, and assimilation
of ingested nutrients [29]. Among these signals are found the incretins which must
accomplish specific criteria to be defined as it: they must be released in response to an oral
nutrient ingestion and reach physiological concentrations in vivo to provoke insulin release
[30]. Currently, two gut hormones are considered incretins: GLP-1 and GIP. These two
incretins reach their target cells and bind to specific G protein-coupled receptors (GPCRs) to
activate their signaling pathways. GIP receptor is expressed in pancreatic β-cells, adipose
tissue, bones and brain; while GLP-1 receptor is expressed in pancreatic α and β-cells, central
and peripheral nervous system, heart, kidney, lung and gastrointestinal tract [31]. This wide
distribution of receptors involves a wide range of biological activities for incretins. Both
hormones promote glucose-dependent insulin secretion and biosynthesis, enhance
proliferation and inhibit apoptosis of pancreatic β-cells. In addition to its insulinotropic
effects, GLP-1 inhibits gastric empting and reduces food intake and the rate of endogenous
glucose production [32]. On the other hand, GIP also regulates fat metabolism in adipocytes,
enhancing insulin-stimulated incorporation of fatty acids into triglycerides, increasing
lipoprotein lipase activity and stimulating fatty acid synthesis [33].
In obesity, a decrease on the capacity of adipose tissue to store lipids and trap fatty acids
is found, resulting in an increase of circulating fatty acids that leads to an ectopic storage of
fat in non-adipose tissues such as muscle, liver, pancreas and possibly other organs. This
increase on visceral fat causes several metabolic derangements associated with deterioration
on glucose tolerance and insulin resistance, leading to an increase on insulin secretion to
maintain glucose homeostasis [34, 35]. The important role of GLP-1 on glucose homeostasis
suggests that incretin impairment might be involved in the pathogenesis of obesity, but there
are only a few studies reporting that. In this way, the incretin effect was found to be decreased
in insulin resistant obese subjects compared to non-insulin resistant lean subjects [36].
Several authors have linked this impaired incretin effect to a decrease on GLP-1 secretion by
L-cells [37] and Dao et al. also reported a decreased intestinal GLP-1 production in rats fed a
high-fat diet [38]. However, other authors linked this reduced incretin effect to a decreased
incretin sensitivity on pancreatic β-cell [39, 40, 41], which has been suggested to represent a
very early stage in the pathophysiology of type 2 diabetes mellitus (T2DM). In T2DM
subjects an attenuated incretin effect was found [41], and associated to a decreased GLP-1
secretion. Besides, the decrease on incretin effect and GLP-1 secretion have been found to be
proportionally correlated with the degree of obesity [36], suggesting that incretin dysfunction
could be involved in the development of T2DM, but whether the decrease on GLP-1 secretion
is cause or consequence of these metabolic deregulations remains to be elucidated. Otherwise,
an increase on dipeptidyl peptidase IV (DPP4) activity, the enzyme that cleaves and
inactivates incretins, has also been linked to the reduced incretin effect, suggesting a higher
inactivation of incretin. Thereby, several authors reported an increase on circulating DPP4
activity in obese and T2D subjects [42]. However, a few studies showed decreased plasma
DPP4 activity in diabetic subjects [43] or unchanged activity in obese, diabetic and impaired-
glucose tolerance subjects [44, 45]. Taking into account these findings, it was concluded that
a severe degree of hyperglycemia is needed to induce an increase on circulating DPP4
activity, while mild hyperglycemia is not enough to increase it, which suggests that decreased
active GLP-1 levels in early phases of T2D might be due to a decreased hormone secretion
[44, 45]. The important role of GLP-1 in glucose homeostasis is supported by the fact that
GLP-1 analogues, GLP-1 receptor agonists and DPP4 inhibitors are currently considered
antidiabetic drugs [46].
Therapies involving not only GLP-1 but also other gut hormones such as Ghrelin, CCK-1
and PPY are also considered for their potential as antiobesity agents, and clinical trials are
ongoing [47, 48]. Therefore current understanding of gut hormones highlights the modulation
of the enteroendocrine system as a mechanism to combat obesity.
Obesity-Associated Intestinal Inflammation: The Link between High Fat

Diet, Changes in Gut Microbiota and Metabolic Endotoxemia
The gut microbiota represents an ensemble of microorganisms that resides in the

intestine, where it plays important roles in ensuring proper digestive functioning, in the
immune system and in performing a barrier effect. With respect to the major role that gut
microbiota plays in the normal functioning of the body and the different functions it
accomplishes, experts currently consider it an ―organ.‖ Furthermore, compelling evidence
supports the role of the intestinal microbiota in the regulation of adiposity and body weight
[49], and it has received increased attention from researchers worldwide [50-52].
The highest microbiota density in the human body is found in the colon. This
compartment is primarily composed of anaerobic bacteria, such as Bacteroides,
Porphyromonas, Bifidobacterium, Lactobacillus and Clostridium, that belong to the most
abundant phyla: Bacteroidetes, Actinobacteria, and Firmicutes [53]. The proportion of each
phyla varies between individuals and depends on age, diet, stress, geographical location, and
other factors [54-56]. In addition, certain bacteria tend to adhere to the surface of the
intestinal mucosa, while others inhabit the lumen. Whereas the bacteria of the mucosal
surface interact with the host immune system, the microorganisms residing in the lumen may
be more relevant to metabolic interactions with food or other digestion derivatives [57]. Data
in humans and rodents revealed that 90% of the normal gut microbiota consists of the
Bacteroidetes and Firmicutes phyla, while obesity is linked to changes in their proportions
[58-60]. In this sense, it has been suggested that changes in the composition of the gut
microbiota and epithelial functions may play a role in obesity-associated inflammation [61].
However, due to the high microorganism diversity found between subjects, it has been
difficult to obtain clear conclusions about the predominant phyla present in metabolic
diseases [62].
Components that originated from gut microbiota, such as lipopolysaccharides (LPS),
lipoteichoic acid, peptidoglycan, flagellin and bacterial DNA, can cause immune system
activation. Among them, LPS is thought to be a major inducer of the inflammatory response
[63, 64]. LPS are large glycolipids that consist of lipid and polysaccharide fractions joined by
a covalent bond. They are found in the outer membrane of Gram-negative bacteria, act as
endotoxins, and can elicit strong immune responses. The ability of LPS to promote low-grade
inflammation and metabolic disturbances may differ, primarily because of the chemistry of its
components due to the existing variations between strains [65].
The stomach and the duodenum harbor very low numbers of microorganisms, typically
less than 103 microbes/mL of the luminal content. The number progressively increases along
the jejunum and ileum, up to 1011-1012 microbes/ml of the luminal content in the distal tract
[65, 66]. Consequently, the gut microbiota is a very large reservoir of LPS. Under normal
conditions, the presence of this endotoxin in the intestinal lumen does not cause negative
health effects. However, some factors can favor the transfer of LPS into the circulatory
system and cause metabolic endotoxemia [66]. In this respect, recent studies have shown that
rodents fed a HF diet display alterations in the gut microbiota that increase intestinal
permeability through gut barrier dysfunction [50]. Indeed, LPS has been reported to be able to
diffuse from the gut to the bloodstream during high fat (HF) diet consumption, either by
direct diffusion through altered intestinal paracellular permeability or through absorption by
enterocytes during chylomicron secretion [67-69]. Because LPS is able to transfer into the
circulatory system and to induce the inflammatory response, a possible association between
intestinal LPS and metabolic diseases has been suggested [63, 64].
Animal research indicated that germ-free mice fed a HF diet do not gain weight, exhibit
adiposity, or display other metabolic effects, such as insulin resistance. When the microbiota
was transplanted from lean mice or genetically or diet-induced obese mice into germ-free
mice, the recapitulation of the original phenotype was observed, showing an increase in body
fat [70]. In genetically obese mice and obese patients, there is a significant change in the
composition of the gut microbiota compared with lean controls [60, 71], and in rodents, these
modifications can be induced by ingestion of a HF diet [72]. It has been hypothesized that the
body weight gain is associated with an increase in the capacity of the microbiota to extract
nutrients from the diet [63]. However, other mechanisms, such as changes in gut function,
have not been fully explored [73]. It has been suggested that the type of diet consumed,
particularly HF diets, can contribute to metabolic endotoxemia [74]. In this respect, it has
been observed that a HF meal promotes the translocation of intestinal endotoxins into the
circulation in human subjects [69, 75] and in mice [67, 76]. Plasma levels of LPS were also
shown to increase in response to a 4-week HF diet, by genetically induced hyperphagia [76],
or in the blood of mice orally gavaged with LPS [77]. Moreover, a link was revealed between
HF diet, inflammation, and the occurrence of pro-inflammatory products from gut microbiota
Gram-negative bacteria in plasma [78, 79]. Therefore, an important question is how dietary
fat promotes intestinal LPS absorption. One possibility is that dietary fat promotes
paracellular leakage of LPS across the intestinal epithelium. This possibility is supported by
observations that intestinal-epithelial tight-junction integrity is compromised in obese mice
[50] and by studies demonstrating that experimental exposure of the intestinal lumen to some
fatty acids can cause small-intestinal epithelial damage [80]. An alternative possibility could
be that LPS enters the bloodstream by transcellular transport through intestinal epithelial
cells. This process could occur through the so-called intestinal-epithelial microfold cells (M-
cells), which are permeable to bacteria and macromolecules and facilitate sampling of gut
antigens by the underlying lymphoid tissue [67].
Taken together, these data suggest that HF diet-induced changes in the intestinal
microbiota could be responsible for metabolic endotoxemia and for the onset of the
corresponding diseases, although the causative link between intestinal bacteria populations,
endotoxemia, and metabolic disease needs further assessment [81].
Furthermore, despite the origin of metabolic endotoxemia is still unclear, it is suggested
that the consumption of a HF diet induces changes in the gut microbiota, leading to increased
activation of inflammatory pathways, as nuclear factor (NF)-kB or mitogen activated protein
kinases (MAPK), and impaired insulin signaling [82]. This alteration seems to interfere with
intestinal permeability and increases the absorption of LPS. LPS then reaches the circulation
and initiates the activation of Toll-like receptor (TLR)-2 and 4 and LPS receptor CD14,
leading to increased activation of inflammatory pathways through cytokine release. An
impairment of insulin signaling is observed with the activation of these pathways, along with
decreased phosphorylation of the insulin receptor, insulin receptor substrate (IRS) and protein
kinase B or Akt, as well as increased inhibitory serine phosphorylation of IRS-1 [82].
In terms of human insulin resistance, it has been found that LPS is present at higher
levels in the blood of subjects with T2DM or insulin resistance, compared to healthy subjects
[83-85]. Circulating LPS was also shown to correlate with insulin levels, glucose and with a
homeostasis model assessment of insulin resistance [83-85]. Interventional studies suggest
that high levels of LPS may play a direct role in the progression of insulin resistance, because
a bolus injection of LPS into healthy human volunteers was shown to cause a 35% decrease in
insulin sensitivity [86]. Other authors showed that altering the gut microbiota composition by
antibiotic treatment reduced the antigenic load and hence the inflammatory reaction that had
led to potent pancreatic β-cell destruction in autoimmune diabetes development [87].
Furthermore, it has been proposed that antibiotic treatment partially protects against T2DM
[88, 89]. These data indicate that the microbial ecology in humans could be an important
factor affecting glucose homeostasis [61].
More studies are needed to precisely define how intestinal inflammation favors obesity
and insulin resistance, but the mechanisms likely include altered epithelial permeability,
translocation of bacterial products, up-regulation of pro-inflammatory cytokines and
hormones produced from gut endocrine cells and the modulation of neural signaling between
the gut and brain that impacts appetite or satiety [90].
FLAVONOID’S EFFECTS AT THE GASTROINTESTINAL TRACT

Modulation of Enterohormone Release by Flavonoids
Due to its key role in the regulation of glucose homeostasis, as well as in food intake,
GLP-1 is the enterohormone that has been most studied concerning its modulation by
flavonoids. The modulation of incretins by polymeric flavonoids, i.e., proanthocyanidins, has
been partly evaluated. In healthy rats, an acute oral dose of grape seed extract (1 g/kg bw) has
been shown to increase GLP-1 levels after an oral glucose load. The mechanisms that exert
this effect could arise from their capacity to inhibit the incretin-degrading enzyme DPP4 [91],
their ability to modulate GLP-1 secretion from L-cells [92], as shown in the enteroendocrine
cell line STC-1, and/or by altering the number of enteroendocrine cells in the intestine [93].
Similarly, a dose of 10 g/kg bw of the procyanidin tetramer cinnamtannin A2 also increases
plasma active GLP-1 when it is acutely administered to fasted mice [94]. Furthermore,
Torronen et al. working with healthy humans, showed that a single administration of a berry
purée (800 mg polyphenols including anthocyanins, flavonols, phenolic acids,
proanthocyanidins, and ellagitannins) administered together with sucrose tended to increase
GLP-1 [95]. Not only acute doses, but also a preventive dose of 25 mg/kg bw of the grape
seed procyanidin extract, for 12 weeks, prevented the cafeteria-induced decrease in colon
GLP-1 producing cells [93].
Other monomeric polyphenols have also been shown to modulate GLP-1 levels.
Chlorogenic acid, which is a major phenol found in coffee, was shown to improve plasma
GLP-1 levels [96] and increase GLP-1 secretion and production in STC-1 cells, a murine
enteroendocrine cell line [97]. Berberine, which is a major active constituent of
Rhizomacoptidis, has been reported to increase portal active GLP-1 levels in healthy and
streptozotocin-induced diabetic rats (STZ) and to enhance GLP-1 secretion and biosynthesis
in NCI-H716 cells, a human enteroendocrine cell line [98, 99]. Genistein and daidzein are
flavonoids, which are derived from soybean fermentation, have been reported to increase
GLP-1 secretion from NCI-H716 cells [100]; glyceollins and phytoalexins that are derived
from daidzein in soybean with a fungi infection showed the same effect in vitro [101].
Resveratrol, a polyphenolic compound produced by fruits such as red grapes or berries, was
found to increase portal active GLP-1 levels and intestinal biosynthesis in HF diet-fed rats
[38]. Finally, curcumin, a phenolic compound that is isolated from the rhizomes of Curcuma
longa L., can increase GLP-1 secretion in the murine enteroendocrine cell line, GLUTag
[102].
Another studied enterohormone is ghrelin, produced by X-cells of the stomach, and
whose main role is highly related to the regulation of food intake. Ghrelin‘s modulation by
isoflavones has been evaluated, while there is less information regarding other types of
flavonoids. Soy isoflavones decreased plasma ghrelin, increased CCK, and increased,
although not significantly, peptide YY (PYY) when administered to ovariectomized rats that
were fed a high-fat diet for 4 weeks. These changes were found at 3 doses of isoflavones: low
(26 mg/kg bw), medium (74 mg/kg bw), and high (206 mg/kg bw). However, the body weight
was increased at the lower dose and was reduced at the other two doses. At the higher doses,
the energy intake was reduced [103]. A similar effect was found in female mice by
Ryokkynen. In this case, 8 mg /kg bw/day of the isoflavone genistein administered to mice
for 8 weeks reduced plasmatic ghrelin in females, while it had no effect in males. In these
mice, food consumption was reduced at weeks 1 and 5 but not at the end of the experiment
because these animals had pups that were in the lactation period [104]. Thus, animal studies
suggest that some isoflavones can modify the levels of ghrelin, and it appears that such a
modification precedes changes in body weight. Similar studies in humans do not clearly show
this effect, but these results could be due to the different doses that were administered. In
healthy postmenopausal women, 80 or 120 mg (i.e., 1.19 or 1.79 mg/kg bw) of soy
isoflavones for 12 months did not modify the fasting levels of appetitive hormones (ghrelin,
insulin, leptin, and adiponectin). In the same sense, the body composition was not affected by
soy isoflavones. Food intake was not assessed [105]. Similarly, in another study in healthy
postmenopausal women, 50 mg/day of isoflavones neither affected preprandial ghrelin
plasma levels, nor insulin, glucose body weight or energy intake. Instead, PYY was increased
by isoflavones, and the authors concluded that this hormone level is not a major factor in the
regulation of body weight [106]. In a smaller study [107], isolated isoflavonoids (114
mg/day) for three months inhibited the age-dependent rise of fasting plasma ghrelin in
postmenopausal women with a history of breast cancer, although this finding was not
accompanied by modifications in the lipid profile or insulin sensitivity, and the body
composition and food intake were not assessed.
An extract of Citrus grandis that is rich in naringenin was administered for 12 weeks at
different concentrations (300, 600, 1200 mg/kg bw) in Zucker fatty rats that were fed a high
fat/high cholesterol diet and did not induce significant changes in the body weight nor in the
food intake, although the authors suggested that there was a tendency to reduce the body
weight accompanied by an increased energy intake. The hormones were analyzed, and the
extract appeared to counteract the HF diet-induced decrease in ghrelin. The extract also
decreased the plasma GLP-1 (which was not affected by the diet), while it did not change the
insulin, PYY, leptin nor amylin [108]. In type 2 diabetic humans, a decaffeinated green tea
extract (11.12 mg EGCG/Kg bw) for 16 weeks did not show any difference in plasma ghrelin
or leptin compared to the placebo group. Treatment also did not modify the body weight or
plasmatic parameters (insulin, glucose, HOMA-IR) [109]. Finally, in healthy humans, carob
pulp reduced the acylated but not the total ghrelin. The effects on ghrelin might account for
the observed reduction in non-esterified fatty acids and triglycerides and the change in the
substrate utilization toward lipid oxidation (decrease in respiratory quoficient (RQ)) [110].
The effects on plasma acylated ghrelin and fat oxidation after a meal were maintained 24-
hour after carob pulp intake [111]. Because the treatment was acute; no report on hunger or
energy intake was made. Moreover, carob pulp is rich in insoluble dietary fiber and
polyphenols, mainly gallic acid, gallotannins and flavonol glycosides, and from these
experiments, it cannot be deciphered whether the effects were due to the fiber or the
polyphenols [110].
There are very few studies that evaluate flavonoid effects on PYY; these studies were
cited above. Raasmaja et al. showed no effects of an extract that was rich in naringenin gived
simultaneously with a HFD in Zucker fatty rats [108]. Zhang et al. reported that an isoflavone
treatment of ovarectomitzed rats increased PYY [112]. Finally, Weickert al. described that
soy isoflavone supplementation for eight weeks did not significantly reduce the energy intake
or body weight, even though plasma PYY increased during the isoflavone treatment [106].
The relation of CCK with flavonoids is indeed less analyzed. The scarce existing studies
are already indicated above.
Taking these studies together, flavonoids have been shown to modulate GLP-1 levels.
Other polyphenols also modulate GLP-1 and ghrelin. There is very little data that concerns
the effects on other enterohormones. Some of the studies point out to the relationship between
modulation of enterohomones (mostly GLP-1) levels and beneficial effects on insulin
resistance. Instead, whether such modulation involves effects on food intake, a key point in
the development of obesity, has not been assessed.
Table 1. Summary of flavonoids acting on enteroendocrine system
Flavonoid type Enteroendocrine hormone Ref.

Grape seed proanthocyanidin Increase active GLP-1 [92]
Cinnamtannin A2 Increase active GLP-1 [94]
Polyphenols Increase GLP-1 [95]
Chlorogenic acid Increase GLP-1 secretion [97]
Berberine Increase GLP-1 [98, 99]
Genistein and daidzein Increase GLP-1 secretion [100]
Glyceollins and phytoalexins Increase GLP-1 secretion [101]
Resveratrol Increase active GLP-1 [38]
Curcumin Increase GLP-1 secretion [102]
Narigenin Decreased plasma GLP-1 [108]
Soy isoflavones Decreased plasma ghrelin [103, 104]
Carob pulp Reduced the acylated ghrelin [110]
Naringin Activation of ghrelin receptor [160]
Soy isoflavones Increased CCK [103]
Isoflavones Increased PYY [106, 112]
Naringenin No effect on PYY [108]
Flavonoid Effects on Intestinal Inflammation
NF-κB plays a key role in the intestinal inflammatory response [113]; therefore, the
compounds that could modulate this inflammatory pathway are an interesting field of
investigation. Flavonoid-mediated modulation of the inflammatory response has been
extensively studied in several in vivo and in vitro models [12, 114]; however, there are fewer
studies regarding its effects on intestinal inflammation. They point out that in the intestine,
flavonoids might also modulate this pathway, including effects at several levels as signal
reception, NF-kB transcriptional activity and/or cytokine release.
Figure 1. Schematic view of the anti-inflammatory mechanisms of flavonoids on intestinal

inflammation. The mechanisms underlying the anti-inflammatory effects of flavonoids involve, among
others, the production and secretion of inflammatory mediators, protection of TJ cytokine-induced
damage and the modulation of the MAPK and NF-kB pathways. AA, arachidonic acid; DSS, dextran
sulfate sodium; MyD88, myeloid differentiation primary response gene 88; NIK, NF-kB-inducing
kinase.
The initial step in the activation of the NF-kB pathway by endotoxins is LPS binding to
its receptor TLR4. Dou et al. [115] studied the effect of naringenin on flavonoid modulation
of TLR4 expression in colonic inflammation using female C57BL/6 mice. Naringenin is a
flavanone present in citrus fruits that plays an important role as anti-inflammatory and
antioxidant agent [116]. In this report, colonic inflammation was produced using dextran
sulfate sodium (DSS), one of the most widely utilized chemical compounds for inducing an
intestinal inflammatory model [117]. After a 6-day DSS treatment, both the mRNA and
protein expression of TRL4 was significantly increased. Naringenin treatment inhibited its
expression, thus demonstrating that naringenin protects mice from DSS-induced colitis by
suppressing of TLR4/NF-κB signaling pathway [115].
Luteolin is another flavonoid that has been related to NF-κB inhibition; it is a flavone that
is abundant in carrots, peppers, celery, olive oil, peppermint, thyme, rosemary, and oregano.
Luteolin has been shown to produce various beneficial health effects, including antioxidant,
anti-inflammatory, antimicrobial and anti-cancer activities [118]. Once the membrane
receptor is activated by e.g., LPS, the classical pathway of NF-kB activation leads to the
phosphorylation of inhibitory protein kB (IkB) kinase (IKK). Kim et al. [119] observed that
IKK activity was suppressed by pretreating IEC-18 cells (a rat non-transformed small
intestinal cell line) with luteolin followed by LPS stimulation. This effect resulted in an
inhibition of NF-κB signaling and the consequent pro-inflammatory gene expression in these
intestinal epithelial cells.
Ruiz et al. [120] found that treatment with functionally diverse flavonoids, such as 3'-
hydroxy-flavone and luteolin, followed by tumor necrosis factor (TNF)-α stimulation,
inhibited NF-κB signaling by targeting different points of the pathway. They observed that 3'-
hydroxy-flavone was able to inhibit IKK activity and that luteolin inhibited NF-κB RelA
transcriptional activity in Mode-K cells, a murine intestinal epithelial cell line.
Some authors have demonstrated that flavonoids are able to inhibit the NF-κB
translocation to the nucleus, preventing pro-inflammatory gene transcription. This effect can
be explained by the protective role that some flavonoids exert over IκB degradation. Nunes
et al. [121] found that treatment with a red wine extract rich in procyanidins and
anthocyanidins significantly inhibited IκB degradation. These results were observed in HT-29
cells (human epithelial colorectal adenocarcinoma cells) stimulated with TNF-α, interleukin
(IL)-1β and interferon (INF)-γ. Some in vitro and in vivo studies have proven the effect of
flavonoids on IκB degradation. An in vitro study showed that Opuntia ficus-indica juice, also
known as cactus pear juice, acted as an antioxidant and anti-inflammatory agent in Caco-2
cells [122]. The extract constituents were flavonoids, such as isorhamnetin and some of its
derivates. Pre-treatment with Opuntia extract followed by stimulation with TNF-α, IL-1β and
LPS slightly prevented IκB depletion. Moreover, the co-incubation of the extract with these
inflammatory inducers led to a more significant effect, showing higher levels of IκB. Other
authors [115] also showed similar effects of flavonoids on NF-kB translocation. Specifically,
naringenin significantly blocked the NF-κB signaling pathway in DSS-induced colitis by
suppressing IκBα phosphorylation/degradation, blocking NF-κB p65 nuclear translocation
and inhibiting NF-κB-mediated transcriptional activity [115].
Upon activation, NF-κB regulates the transcriptional activation of many genes involved
in the immune and inflammatory responses, such as pro-inflammatory cytokines (TNF-α, IL-
1β, and IL-6) and enzymes [123]. The beneficial effect of flavonoids on intestinal
inflammation has directly been related to the suppression of pro-inflammatory enzyme
expression, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2).
Nunes et al. observed that pre-treatment with a red wine extract rich in catechins, oligomeric
procyanidins and anthocyanidins inhibited COX2 and iNOS cytokine-induced expression and
it also suppressed IL-8 overproduction in HT-29 cells [121]. In another study, also in HT-29
cells, pre-treatment with pomegranate juice, which is rich in anthocyanidins and catechins,
reduced TNF-α-induced COX2 expression [124]. Some authors suggested that this finding
may be related to the inhibition of phosphatidylinositide 3-kinase (PI3K) and protein kinase
B, both necessary for NF-κB pathway activation [125]. Another hypothesis is that flavonoids
inhibit COX2 activation by modulating the MAPK pathway [126, 127].
Other authors found that the pre-treatment of Caco-2 cells with a Sardinian red wine
extract, rich in flavanols, flavolons and anthocyanidins, prevents IL-6 and IL-8 expression
and synthesis after being challenged with an oxysterol mixture [128]. In addition, During
et al. [129] studied the effects of chrysin, a flavone found in some plants, such as passion
flowers or chamomile. They concluded that o-methylated chrysin was able to modulate
intestinal inflammation in Caco-2 cells. The cells were pre-treated with both the o-methylated
and the non-methylated forms of chrysin and then stimulated with IL-1β. The results
indicated that the o-methylated form was able to reduce IL-6 and IL-8 secretion and COX2
activity more effectively than the non-methylated form, indicating a structure-related effect. It
has also been reported that naringenin is able to down-regulate the expression of adhesion
molecules (ICAM-1), chemokines (MCP-1), iNOS, COX2, TNF-α and IL-6 [115] in a model
of DSS-induced colitis using female C57BL/6 mice. Furthermore, in a rat model of
spontaneous inflammatory bowel disease, Castagnini et al. found that Marie Ménard
lyophilized apples, which are rich in flavonols and flavan-3-ols, reduced myeloperoxidase
(MPO) activity and COX2 and iNOS gene expression [130].
Very recently, Mascaraque et al. [131] tested the intestinal anti-inflammatory activity of
apigenin K, a soluble form of apigenin, in two models of rat colitis, namely, the
trinitrobenzenesulfonic acid model and the DSS model. Apigenin K pre-treatment
ameliorated the morphological signs and biochemical markers in both models. Specifically,
Apigenin K pre-treatment tended to normalize the expression of a number of colonic
inflammatory markers (e.g., TNF-α, transforming growth factor-β, IL-6, intercellular
adhesion molecule 1 or chemokine ligand 2) and to reduce colonic MPO and alkaline
phosphatase activities. Finally, some authors have suggested that flavonoid metabolites
derived from the intestinal microbial metabolism may also have anti-inflammatory effects
[132]; however, flavonoid metabolites have only been tested in few intestinal cell models. For
example, Larrosa et al. [132] concluded that some polyphenol-derived metabolites from the
colon microbiota inhibit DSS-induced colitis lipid peroxidation and DNA damage in the
colon mucosa and down-regulate the fundamental cytokines involved in the inflammatory
process (TNF-a, IL-1b, and IL-8). Together, the literature suggests that flavonoids reduce the
inflammatory processes driven by NF-κB activation by inhibiting cytokine expression and
synthesis and down-regulating TLR-4/NF-κB pathway in intestinal cell models.
Flavonoid Effects on Intestinal Mucosa Barrier Integrity
The paracellular and transcellular pathways are the two major pathways mediating
transmembrane transfer of intestinal bacterial substances. Both mechanisms may be involved
in intestinal mucosal barrier damage and bacterial translocation. The paracellular pathway is
integrated by tight junctions (TJs), consisting of zonulin/zonula occludens (ZO)-1, occludin,
claudins, and actin-myosin cytoskeletal proteins. Previous studies have shown that
inflammatory cytokines and bacterial antigens can affect the expression level and assembly of
these elements, thereby exerting an influence on TJ functions [133]. Because the integrity of
the intestinal barrier has been compromised in several intestinal pathologies [134, 135], the
potential protective effects of naturally occurring bioactive compounds have been evaluated
in some in vitro and in vivo models.
Quercetin is a flavonoid that has been proposed to exert beneficial effects over the
intestinal barrier [136]. It is the most common flavonoid in nature and can be found in fruits
and vegetables, including onions, kale and apples [137]. Amasheh et al. tested the effect of
quercetin on cytokine-induced intestinal barrier damage both in HT-29 cells and in the distal
colon from male Wistar rats ex vivo [138]. In vitro, quercetin was added on both sides of the
culture insert and TNF-α was added only to the basolateral side, which produced a decrease in
transepithelial electrical resistance (TEER). Interestingly, quercetin treatment partially
inhibited this effect. In this study, the expression of claudin-2 was also evaluated. Claudin-2
forms cation-selective channels, and consequently, its upregulation could contribute to the
altered barrier function by allowing the massive transit of cations and water to the lumen
[139]. In this context, the authors found that quercetin exerts a protective effect on the
intestinal barrier by down-regulating claudin-2. The analysis of intestinal permeability in rat
colon ex vivo revealed that the application of TNF-α and INF-γ reduced the total resistance of
the intestinal barrier, which was partially inhibited by quercetin.
Carrasco-Pozo et al. [140] tested the effect of quercetin and EGCG against the
indomethacin-induced disruption of epithelial barrier integrity in Caco-2 cells [140].
Indomethacin is a non-steroidal anti-inflammatory drug that causes mitochondrial
dysfunction, oxidative stress and apoptosis in chronic administration [141, 142]. The results
showed that quercetin and EGCG completely protected against the indomethacin-induced
decrease in TEER. The same results were obtained when the permeability was assessed by
measuring fluorescein isothiocyanate-labeled dextran (FD-4) transport across the Caco-2 cell
monolayer [140]. Finally, they evaluated the protective effect of quercetin on ZO-1 and
occludin in Caco-2 cells treated with indomethacin and rotenone (an environmental toxin).
Immunofluorescence analysis revealed that either indomethacin or rotenone, both inhibitors
of mitochondrial complex I, caused TJ disruption through ZO-1 delocalization. Treatment
with quercetin protected ZO-1 delocalization and also prevented the decrease in ZO-1 and
occludin expression. The authors hypothesized that quercetin‘s effects may be due to its
mitochondrial-protecting property.
The effect of naringenin was evaluated in a murine model of chronic intestinal
inflammation [143]. To induce intestinal damage, male BALB/c mice were fed with 2%
(wt:v) DSS. The colonic permeability was studied by measuring FD-4 paracellular transport.
The authors found that the animals fed with DSS exhibited higher permeability than the
control group. In contrast, the DSS + naringenin group did not differ from the control group.
Furthermore, the expression of the occludin, junctional adhesion molecule (JAM)-A, claudin-
3 and claudin-1 proteins was decreased in the DSS group. However, the level of these
proteins was equivalent to the control group after treatment with naringenin. Taken together,
all of these findings suggested that naringenin was able to protect TJ by suppressing DSS-
induced damage in the intestinal epithelial cells.
Puerarin, an isoflavone extracted from a Chinese medicinal herb, can modulate TJ
expression in the altered intestinal barrier in vivo [144]. Male Sprague-Dawley rats were fed
an ethanol (EtOH) liquid diet producing intestinal barrier dysfunction [145]. In this study,
ZO-1 protein expression was significantly down-regulated by EtOH intake, whereas the
groups treated with puerarin exhibited an upregulation of this protein. The authors concluded
that the expression of ZO-1 in the EtOH diet rats was indicative of injury to the intestinal
barrier function and that puerarin mitigated such intestinal alterations.
The molecular mechanisms of genistein, quercetin, myricetin and EGCG in protecting the
intestinal barrier have been extensively reviewed by Suzuki et al. [146]. These molecules
exerted protective and promoting effects on intestinal TJ barrier function. In particular,
genistein and quercetin interact with intracellular signaling molecules, such as tyrosine
kinases, resulting in the regulation of TJ protein expression and assembly. More specifically,
it has been demonstrated that oxidative stress-induced TJ dysfunction is related to the tyrosine
phosphorylation of occludin, ZO-1 and E-cadherin in Caco-2 cells [147]. It has been
hypothesized that genistein acts against the oxidative stress in the intestinal barrier by
suppressing c-Src kinase (a tyrosine kinase) activation, which inactivates tyrosine
phosphorylation of the TJ. Furthermore, EGCG‘s effects on INF-γ-induced intestinal barrier
dysfunction were evaluated in T84 human colonic cells [148]. The results showed that EGCG
restored the decreased TEER values caused by INF-γ. The authors suggested that the ability
of EGCG to limit the IFN-γ-induced increases in epithelial permeability is likely a component
of the anti-inflammatory nature of this polyphenol.
Flavonoid-Microbitoa Interaction: Modulation of Gut Microbiota

Composition
The human body is an excellent culture medium for the growth of many varieties of
microorganisms, comprising the human microbiota [149]. In mammals, the microbiota is
involved in the maintenance and development of the immune system, in the regulation of
several metabolic pathways, and in general body homeostasis [150, 151]. Several authors
have suggested that both dietary flavonoids, which are the substrates of intestinal bacteria,
and the metabolites produced during flavonoid degradation in the colon may modulate and
induce oscillations in the composition of the microbiota populations by means of prebiotic
and antimicrobial effects against gut pathogenic microorganisms [152-155]. However, the
mechanisms involved are still poorly understood. In the following section, we summarize the
effects of flavonoids on the gut microbiota composition.
Etxeberria et al. [156] assessed the potential of quercetin to reverse alterations of the gut
microbial composition associated with diet-induced obesity in Wistar rats. All of the animals
were fed a high-fat sucrose diet, containing 17% of the energy as sucrose, for six weeks, and
the treated group was also supplemented with quercetin at 30 mg/kg bw/day during the
experiment. According to the results, quercetin generated a significant impact on different
taxonomic grades of the gut microbiota composition. At the phylum level, quercetin
administration attenuated the Firmicutes/Bacteroidetes ratio, decreasing Firmicutes levels by
34.2%. Furthermore, the quercetin-treated group showed a statistically significant inhibition
in the growth of some bacterial species previously associated with diet-induced obesity
(Erysipelotrichaceae, Bacillus, and Eubacterium cylindroides). Overall, quercetin
administration effectively reduced the high-fat sucrose diet-induced gut microbiota dysbiosis.
Similar results were obtained in a randomized, double-blind, crossover intervention study
that included twenty-two human volunteers who were provided with a dietary supplement of
494 mg of cocoa flavanols/day. After four weeks, the consumption of flavanols induced a
significantly increase in the growth of Lactobacillus and Bifidobacterium but significantly
decreased the Clostridia counts. These microbial changes were correlated with reductions in
plasma C-reactive protein (CRP) concentrations, which is considered to be a blood marker of
inflammation and a hallmark of the acute phase response [153]. Queipo-Ortuó et al.
performed a randomized, crossover, controlled intervention study, in which 10 adult men
participated. The results showed that a daily consumption of 272 mL of red wine, which is
mainly rich in flavan-3-ols, anthocyanins, flavonols, and other flavonoids, decreased the
plasma levels of triglycerides and high-density lipoprotein cholesterol, and these significant
reductions may be partly due to the flavonoid-induced increase in the growth of Bacteroidetes
phyla [154]. Other authors noted a significant reduction in the plasma concentration of CRP
after red wine treatment, which was related to an increase in the number of Bifidobacterium
[157].
In obese individuals, the proportion of Bacteroidetes to Firmicutes is decreased, which
produces signals that control gene expression in epithelial intestinal cells. A daily intake of
some flavonoid-rich fruits and drinks, including apples, pears, grapefruit and green tea, has
been implicated in a significant decrease in body weight in obese subjects [158]. The
metabolism of flavonoids by gut microbiota includes the cleavage of glycosidic linkages,
which generates different products, such as glycans, that are necessary for the survival of the
intestinal microbiota. The Firmicutes family possesses fewer glycan-degrading enzymes than
Bacteroidetes and is more repressed by antimicrobial effects of flavonoid compounds than the
Bacteroidetes family. Instead, the Bacteroidetes family prevails following dietary flavonoid
intake, and the flavonoids are fermented to phenolic compounds due to the presence of more
glycan-degrading enzymes. This has been described as a possible mechanism by which
dietary flavonoids exert their weight lowering effect [158].
Although there are few studies regarding the effects of flavonoid consumption on the gut
microbiota composition, it has been suggested that the effects of flavonoids in human health
depend on their transformation by the gut microbiota [159]. Moreover, flavonoids and their
metabolites contribute to the maintenance of gut health, inducing the growth of beneficial
bacteria and inhibiting the growth of pathogen species [152]. However, the mechanisms
involved in this two-way relationship remain to be elucidated.
CONCLUSION
Flavonoids are a large and diverse group of natural compounds of which only a few have
been evaluated regarding their effect at the level of gastrointestinal tract. The strongest
conclusion that can be drawn from the revision of the current literature is that some
flavonoids are able to reduce the intestinal inflammatory processes targeting the TLR4/NF-kB
pathway. Most of the studies performed to date on the effects of flavonoids both in vitro and
in vivo address the issue using models of cytokine, endotoxin or chemically induced intestinal
inflammation. Furthermore, the specific effects of flavonoids on obesity-associated intestinal
alterations have not yet been evaluated. Severe intestinal inflammation, including
inflammatory bowel disease, and obesity-associated intestinal inflammation, show different
degrees of severity, but both pathologies share common pathways and mechanisms.
Therefore, it can be hypothesized that the effects of flavonoids on obesity-associated

pathologies can be partially explained through the flavonoid-mediated anti-inflammatory
effects on intestinal inflammation. Although there are few studies regarding the flavonoid
effects on intestinal permeability, most of them point out that flavonoids are able to protect
barrier integrity by primarily acting on TJ stability. The review of the literature on the effects
of flavonoid consumption on the gut microbiota populations suggests that flavonoids may
modulate the microbiota composition by means of prebiotic and antimicrobial properties.
However, the mechanisms involved are still poorly understood. It also remains unclear
whether the documented modulation of enterohormone release by different flavonoids is
linked to their effects on food intake and insulin resistance.
Future investigations are thus required to elucidate the precise mechanisms underlying
these flavonoid-mediated effects on the gastrointestinal tract, and to determine the influence
that they have on obesity-related disorders.
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Chapter 4
LC-ESI-FTICR-MS ANALYSIS OF
FLAVAN-3-OLS IN SEEDS OF GRAPE POMACE
I. Rockenbach1,*, B. Santiago-Schübel2,
B. Thiele3 and R. Fett4
1
Department of Food Technology,
Federal University of Paraíba, Paraíba, Brazil
2
ZCH/BioSpec, Forschungszentrum Jülich GmbH, Jülich, Germany
3
Institute of Bio- and Geosciences (IBG-2),
Research Centre Jülich, Jülich, Germany
4
Department of Food Science and Technology,
Federal University of Santa Catarina, Florianópolis, Brazil
ABSTRACT
Negative-ion electrospray ionization Fourier transform ion cyclotron resonance (FT-
ICR) mass spectrometry has been used for analysis of seeds of pomace from the
vinification of five different grape varieties with a view to their exploitation as a source
of natural antioxidants. We were able to assign elemental compositions to 138 (Isabel
variety), 184 (Negro Amaro), 180 (Pinot Noir), 120 (Primitivo) and 150 (Sangiovese)
different flavan-3-ol compounds, including isomers of 25 different molecular classes. The
number of total B-type isomers was higher than that of total A-type isomers.
Furthermore, elemental compositions were assigned to isomers of condensed products of
(epi)catechin with acetaldehyde up to tetramers. Data presented in this chapter can be
very useful in the analysis of food ingredients and dietary supplements made of grape
seeds.
Keywords: LC-ESI-FTICR-MS, grape seeds, flavan-3-ols, procyanidins
* Corresponding Author address: Av. dos Escoteiros, s/nº, Mangabeira VII, 58055-000, João Pessoa, PB, Brazil.
Email: ismael.rockenbach@gmail.com.
86 I. Rockenbach, B. Santiago-Schübel, B. Thiele et al.
INTRODUCTION
In the wine industry, large amounts of grape pomace are produced, with every 100 kg of
grape-vine producing approximately 20-25 kg of grape waste. Grape pomace has a significant
impact on the environment due to the high phenols content considerably increasing both the
chemical and biochemical oxygen demands, making its disposal a serious environmental
problem [1]. The conversion of this by-product into value added ingredient is of considerable
interest to the wine industry since around 13-14 million ton are generated per year [2].
The seeds constitute a considerable proportion of the grape pomace, amounting to 38-
52% on a dry matter basis [3]. The composition of grape seeds is basically (w/w) 40% fiber,
16% essential oil, 11% protein, 7% complex phenolic compounds like tannins, and other
substances like sugars and minerals [4]. The phenolic compounds in grape seeds are
essentially all flavonoids. The presence of flavan-3-ol monomers, dimers, and trimmers has
been extensively reported. The most abundant phenolics isolated from grape seeds are
catechins and their polymers [5]. Vitis vinifera grape seed procyanidins are particularly
interesting because of their unlimited structural diversity based on the combination of only
three elemental units, i.e., catechin, epicatechin, and epigallocatechin. This is due to the
stereochemistry of the asymmetric carbons C2 and C3 of the flavan skeleton, the type of
interflavan bond (C4-C8 and C4-C6, B-type procyanidins), the length of the polymer chain
(degree of polymerization), the degree of galloylation, and the position of the gallic acid ester
[6]. Several studies reported antioxidant activity of the grape pomace extracts, suggesting the
winery by-products for production of natural phenolic antioxidants [7-9].
Different strategies have been explored for the analysis of procyanidins. Reversed-phase
high performance liquid chromatography (HPLC) coupled to diode array detection (DAD)
and/or mass spectrometry (MS) are the usually employed techniques for analysis of these
phenolic compounds. The former enables the distinction of the various classes of phenolic
compounds on the basis of their characteristic UV-visible spectrum, whereas the latter gives
access to intact molecular ions and fragment ions from which both the molecular weight of
the compound and information on its structural features can be established [10, 11]. However,
the complete resolution of all the possible components present in the complex profiles of
grape seed procyanidins is quite difficult to achieve, even using very lengthy gradient elutions
(from 60 to 120 min) [6].
Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized molecules
to separate one from another. This requires first that the various molecular species of interest
have been charged (often by loss or addition of protons) and transferred into the gas phase,
and that they are then separated as a function of their m/z values. These two steps are
achieved by the mass spectrometer source and analyser, respectively. Analysers that have
been used to analyse phenolic compounds are quadrupole (Q), magnetic sector (B), ion-trap
(IT), time-of-flight (TOF), and Fourier-transform ion cyclotron resonance (FTICR) that
differ, among other factors, by the available mass range and resolution. FTICR provides the
highest mass resolution and most accurate mass determination, making it theoretically
possible to assign molecular formula unambiguously for smaller oligomers [11, 12]. These
high specifications mean that FTICR is ideal for analysing complex mixtures, as
demonstrated by prior electrospray ionization (ESI) FTICR-MS analysis of black tea samples
containing thousands of chemically distinct constituents [13].
LC-ESI-FTICR-MS Analysis of Flavan-3-ols in Seeds of Grape Pomace 87
In this chapter, we applied LC-ESI-FTICR-MS analysis to elucidate the complex

composition of flavan-3-ol isomeric compounds in seeds of pomace from different grape
varieties widely produced in Brazil to wine production as a view to their exploitation as a
cheap source of natural antioxidants.
MATERIALS AND METHODS

Samples
Three 2 kg samples of each red grape pomace were obtained from the Experimental
Research Station plantation located in Santa Catarina state, Brazil. The varieties were as
follows: ‗Primitivo,‘ ‗Sangiovese,‘ ‗Pinot Noir,‘ ‗Negro Amaro‘ (Vitis vinifera) and ‗Isabel‘
(Vitis labrusca). The samples studied were by-products of the winemaking process, obtained
after fermentation. The seeds were manually separated and stored at -80ºC before analysis.
After lyophilization, grape seeds were ground with a ball mill (MM2000, Retsch, Haan,
Germany) under cooling with liquid nitrogen to obtain a fine powder.
Extraction of Polyphenols from Solid Samples
Pressurized liquid extraction (PLE) was carried out using an Accelerated Solvent
Extractor. One gram of diatomaceous earth at the bottom and 2 g of the sample material
mixed with 2 g of diatomaceous earth on the top were packed into 11 mL stainless steel
extraction cells after insertion of two cellulose filters. The PLE parameters were as follows:
solvent acetone plus water, 70 + 30 (v/v) [14], temperature 25ºC, time 10 min, two cycles,
100% flush volume. The extracts were submitted to solid-phase extraction (SPE) before
analysis.
Solid-Phase Extraction (SPE)
SPE was performed using a Gilson ASPEC XL system according to a method described
previously [15, 16]. Polyamide SPE cartridges were conditioned with 10 ml of distilled water
for 10 min and washed with 5 mL of water. The extract was diluted with water to contain
20% (v/v) of the organic solvent prior to loading onto the cartridge. The cartridge was washed
with 15 mL of water to remove matrix interferences, and the phenolic compounds bound to
the polyamide were eluted with dimethylsulfoxide plus formic acid, 99.9 + 0.1 (v/v). While
eluting, the initial 1.5 mL was discarded because it did not contain compounds of interest, and
the next 3 mL was collected, containing all of the compounds. These elution samples were
then subjected to analysis.
LC-ESI-FTICR-MS Analysis
HPLC analysis was carried out with an Agilent HPLC system equipped with a diode
array detector. The analytical column (Aqua 3 µm C18, 150 mm, 2 mm i.d.) was equipped
with a guard column (Security Guard, C18, 4 mm, 2 mm i.d.; both Phenomenex,
Aschaffenburg, Germany). The mobile phase consisted of water +1% formic acid (A) and
acetonitrile +1% formic acid (B). The following gradient was used at a flow rate of 0.3
mL/min: 100% A for 4 min, linear gradient to 92% A over 60 min, to 85% A for another 10
min, to 60% A over 60 min, to 100% B within 1 min holding for 10 min and then back to
100% A within 1 min which was held for 14 min. The injection volume was 10 µL for each
sample. The column temperature was 35°C.
Mass spectrometry was performed using a hybrid linear ion trap FTICR mass
spectrometer LTQ-FT Ultra equipped with a 7 T supra-conducting magnet. The electrospray
ionisation (ESI) source was operated in the negative mode with an ion spray voltage of 3.3
kV. Nitrogen was employed as sheath gas (40 arb). The transfer capillary temperature was set
to 300ºC. Voltages for capillary and tube lens were set to -137 and -218 V, respectively. Mass
spectra were recorded in full scan from 200 to 2000 Da and from 150 to 1500 Da with a
resolution of 100,000 at m/z 400. The automatic gain control was set to 5E5 for the FTMS
full scan. Spectra were recorded in the profile mode. Mass measurements are based on the
―monoisotopic‖ ion (i.e., the species in which all carbons are 12C, all oxygens are 16O, etc.).
All data were processed using the Xcalibur software version 2.0.
RESULTS AND DISCUSSION

LC-ESI-FTICR-MS Analysis
The profile of isomers for each of the five grape varieties is shown in Tables 1-5. Table 6
summarizes the number of isomers of each compound identified in the seeds of pomace from
the different grape varieties.
We were able to assign elemental compositions to 138 (Isabel), 184 (Negro Amaro), 180
(Pinot Noir), 120 (Primitivo) and 150 (Sangiovese) different flavan-3-ol compounds,
including isomers of 25 different molecular classes. The maximal tolerated mass accuracy
error was 2 ppm. Elemental compositions were assigned to flavan-3-ol procyanidins up to
pentamers. Additionally, elemental compositions were also assigned to mono and
digalloylated flavan-3-ols, in both cases up to tetramers. A-type procyanidins were detected
for all corresponding B-type galloylated flavan-3-ols up to tetramer monogallates and for
non-galloylated compounds up to pentamers, including compounds with two A-type bonds in
its structure in the case of non-galloylated procyanidin trimers. Furthermore, elemental
compositions were assigned to condensed products of catechin with acetaldehyde.
(epi)Catechin
The negative-ion mass spectrum of all five samples (Isabel, Negro Amaro, Pinot Noir,
Primitivo and Sangiovese) contained two ions at m/z 289.0718. Taking into account the
results in Rockenbach et al. [17], in which it was indicated that the fermentation of
winemaking process does not give rise to enantiomers not commonly found in grape seeds, it
is feasible to assign an elemental composition of C15H14O6 to these isomers, corresponding to
(+)-catechin and (−)-epicatechin.
Table 1. Elemental compositions assigned to peaks in the negative-ion ESI-FTICR mass

spectrum of seeds of pomace from Isabel grape
Calculated Retention Time (min) Elemental Major error

m/z [M ‒ H]‒ composition a (ppm)
289.0718 41.88, 61.98 C15H14O6 ‒0.28
441.0827 43.27, 58.75, 76.40, 82.51 C22H18O10 0.95
575.1195 81.61, 82.88, 85.27, 87.29, 93.66 C30H24O12 0.85
577.1351 39.88, 42.65, 51.53, 57.97, 70.41, C30H26O12 0.35
73.27, 80.14, 81.46
605.1664 76.31, 80.73, 81.67, 83.73, 87.83, C32H30O12 0.68
90.02, 91.04, 91.61, 93.36, 93.86,
96.10, 98.44, 98.88, 102.25
727.1305 63.67, 75.55, 76.13, 79.76, 80.73, C37H28O16 0.84
86.80, 87.59, 88.74, 92.01
729.1461 67.85, 69.65, 71.15, 72.03, 80.11, C37H30O16 ‒0.56
83.51, 86.83
861.1672 92.62, 94.59, 99.93, 104.24, 105.62 C45H34O18 1.00
863.1829 68.11, 70.90, 71.72, 73.33, 75.63, C45H36O18 1.01
78.65, 80.43, 85.76
865.1985 23.04, 35.22, 44.87, 53.09, 57.95, C45H38O18 0.78
63.44, 72.16, 75.02, 78.76
879.1414 84.89, 86.98 C44H32O20 1.12
881.1571 77.48, 89.40 C44H34O20 0.79
893.2298 74.31, 76.72, 78.59, 79.31, 79.94, C47H42O18 1.41
80.82, 81.49, 82.56, 83.67, 84.53,
85.21, 86.38, 87.51, 88.41, 89.06, 90.35
1015.1938 73.00, 75.90, 77.80, 81.49, 86.26, 88.12 C52H40O22 1.11
1017.2095 50.49, 67.29, 69.09, 72.09, 73.49, C52H42O22 0.97
73.93, 75.50, 78.64, 79.91, 84.15
1151.2463 70.26, 72.52, 73.32, 73.96, 75.03, C60H48O24 1.98
76.98, 79.61, 81.01, 81.61, 82.95, 87.30
1153.2619 45.30, 52.04, 57.97, 67.50, 74.21, C60H50O24 0.78
77.09, 78.14
1169.2205 73.61, 79.72, 80.73, 85.36 C59H46O26 1.43
1181.2932 93.78 C62H54O24 1.28
1305.2729 71.87, 74.13, 76.78 C67H54O28 ‒0.37
1439.3097 72.52 C75H60O30 0.25
1441.3253 73.00, 67.04, 77.09, 82.57 C75H62O30 ‒0.70
a
Each listed elemental composition refers to the neutral molecule, not the deprotonated ion observed by
mass spectrometry.

spectrum of seeds of pomace from Negro Amaro grape
Calculated Elemental Major error

Retention Time (min)
289.0718 41.90, 61.81 C15H14O6 ‒0.35
441.0827 76.44 C22H18O10 0.11
577.1351 40.32, 42.69, 51.50, 57.91, 59.28, 73.32, C30H26O12 0.28
80.13
605.1664 76.35, 80.71, 81.65, 83.69, 87.75, 89.97, C32H30O12 ‒0.84
90.97, 91.60, 93.34, 93.78, 95.97, 98.36,
98.71, 102.24
727.1305 47.17, 60.42, 69.54, 72.06, 86.66, 88.72, C37H28O16 0.98
92.04
729.1461 67.90, 69.51, 71.17, 71.96, 80.03, 86.72 C37H30O16 0.35
861.1672 60.66, 68.26, 70.41, 74.37, 77.00, 79.56, C45H34O18 1.34
83.69, 84.55, 94.56
863.1829 32.92, 46.57, 54.87, 59.00, 60.71, 68.17, C45H36O18 0.99
71.53, 75.67, 80.41, 81.92
865.1985 21.22, 22.98, 34.54, 44.92, 53.24, 56.31, C45H38O18 ‒0.88
63.28, 71.69, 72.25, 74.99, 78.80
879.1414 68.20, 73.29, 76.88, 82.43, 84.90, 89.31, C44H32O20 ‒1.59
94.27, 96.92
881.1571 77.50, 89.34 C44H34O20 ‒0.31
893.2298 74.64, 75.82, 76.67, 78.59, 79.35, 79.89, C47H42O18 0.82
82.76, 85.23, 85.82, 86.37, 86.90, 87.44,
88.40, 88.97, 89.29, 90.23, 92.17, 95.59
1015.1938 73.03, 76.05, 79.69, 81.43, 85.49, 86.20 C52H40O22 1.39
1017.2095 50.47, 66.11, 67.29, 69.07, 72.20, 73.55, C52H42O22 1.11
73.97, 74.90, 75.43, 77.11, 78.73, 79.29,
83.87, 85.23
1151.2463 63.88, 70.27, 72.62, 74.08, 77.01, 81.49, C60H48O24 1.88
83.40
1153.2619 40.09, 45.23, 49.44, 52.48, 54.94, 60.91, C60H50O24 1.08
62.25, 67.51, 71.12, 73.78, 76.97, 78.12
1167.2048 83.22, 86.49, 88.61, 92.95 C59H44O26 1.81
1169.2205 74.56, 77.32, 79.71, 80.65, 85.40, 88.15 C59H46O26 1.55
1303.2572 66.64, 74.47, 76.18, 76.44, 78.03, 79.74, C67H52O28 1.11
80.47, 83.05, 86.69, 87.26, 88.67
1305.2729 56.96, 67.08, 71.49, 74.17, 76.76, 79.85, C67H54O28 1.06
83.60, 91.13
1439.3097 60.86, 68.27, 72.61, 75.20, 76.05 C75H60O30 1.62
1441.3253 54.29, 56.91, 61.47, 63.83, 68.81, 70.84, C75H62O30 ‒0.69
73.65, 74.41, 75.14, 76.08, 77.23, 80.36
1457.2838 78.88, 79.29, 82.04, 83.14 C74H58O32 ‒1.40
a
mass spectrometry.

spectrum of seeds of pomace from Pinot Noir grape
Calculated Retention Time (min) Elemental Major

m/z [M ‒ H]‒ composition a error
(ppm)
289.0718 42.07, 62.13 C15H14O6 ‒0.39
441.0827 76.16 C22H18O10 ‒0.02
575.1195 82.83, 85.08, 87.24, 93.67 C30H24O12 ‒0.69
577.1351 36.76, 38.79, 40.44, 42.62, 49.04, 52.12, 54.95, C30H26O12 ‒0.31
57.49
605.1664 75.90, 76.02, 76.39, 80.33, 80.73, 80.77, 87.63, C32H30O12 ‒1.50
89.68, 89.78, 89.83, 89.89, 89.97, 90.06, 90.17,
90.93, 91.02, 91.10, 91.13, 91.22, 91.53, 91.63,
91.68, 91.77, 91.80, 91.91, 93.04, 93.13, 93.21,
93.24, 93.41, 93.50, 93.58, 93.69, 93.75, 98.87,
99.02, 102.19, 102.26, 102.35, 102.55
727.1305 50.48, 60.65, 63.65, 67.98, 75.67, 80.49, 83.50, C37H28O16 ‒0.99
86.73, 88.73, 92.00
729.1461 67.97, 69.74, 71.24, 72.13, 80.05, 86.84 C37H30O16 0.15
861.1672 92.46, 94.66, 95.95, 97.87, 100.05, 104.36, C45H34O18 1.00
105.79
863.1829 71.01, 71.77, 74.89, 75.18, 75.79, 80.18 C45H36O18 0.87
865.1985 45.07, 48.82, 50.59, 53.31, 54.57, 56.51, 63.13, C45H38O18 0.63
71.06, 72.25, 74.33, 78.49
879.1414 84.87, 87.01, 94.10, 96.61 C44H32O20 0.78
881.1571 77.56, 89.37 C44H34O20 0.29
893.2298 75.85, 78.31, 82.28, 84.47, 85.33, 88.53, 90.43, C47H42O18 0.86
91.99, 95.57
1015.1938 73.15, 75.55, 76.05, 78.46, 81.47, 85.88, 87.08, C52H40O22 1.05
88.15
1017.2095 50.52, 64.14, 67.35, 69.11, 72.51, 72.87, 73.56, C52H42O22 1.56
74.10, 74.92, 78.82, 79.98, 83.55, 85.30
1151.2463 70.29, 71.44, 72.96, 73.38, 74.12, 76.55, 77.07, C60H48O24 1.45
81.58, 82.69, 85.19, 87.31
1153.2619 45.51, 49.35, 52.45, 55.30, 64.56, 65.26, 67.73, C60H50O24 0.88
71.58, 73.78, 76.63, 77.36, 78.17
1167.2048 82.24, 86.64, 87.92, 93.00 C59H44O26 1.01
1169.2205 73.72, 76.48, 77.42, 78.90, 79.82, 84.55, 85.53, C59H46O26 1.45
88.26
1181.2932 84.29, 93.72 C62H54O24 ‒0.31
1303.2572 73.46, 75.84, 80.09, 82.71 C67H52O28 ‒0.62
1305.2729 70.89, 72.02, 73.38, 74.18, 76.03, 80.09, 82.84, C67H54O28 0.82
91.19
a
mass spectrometry.
(epi)Catechin Monogallate
Four isomers in Isabel sample, two isomers in Primitivo and a single isomer in Negro
Amaro, Pinot Noir and Sangiovese each sample presented m/z 441.0827, corresponding to a
monomer with one galloyl group linked to its structure. These isomers were associated to an
elemental composition of C22H18O10.
Procyanidin Dimer
An elemental composition of C30H26O12 was assigned to eight different isomers of B-type

procyanidin dimers (m/z 577.1351) detected in the grape seeds of Isabel, Pinot Noir and
Sangiovese varieties, probably corresponding to the typical isomers B1-B8. Negro Amaro
sample presented seven of these isomers, while Primitivo sample presented only six of them.
In addition to the B-type procyanidin dimers, different isomers of A-type procyanidin dimers
(m/z 575.1195) were also detected in a number of five isomers in Isabel sample and four
isomers in Pinot Noir and Sangiovese each sample, corresponding to an elemental
composition of C30H24O12. A-type procyanidin dimers were not detected in grape seeds of
Negro Amaro and Primitivo samples.
(epi)Catechin-Ethyl Dimer
Numerous isomers at m/z 605.1664 were found in all grape seeds analysed (Isabel and
Negro Amaro, 14; Pinot Noir, 40; Primitivo, 8; Sangiovese, 12). Their accurate masses were
presumably assigned to (epi)catechin-ethyl dimers, condensed products of (epi)catechin with
acetaldehyde, corresponding to two (epi)catechin units linked by an ethyl-bridge (Figure 1).
These isomers were associated to an elemental composition of C32H30O12.
Procyanidin Dimer Monogallate
We were able to assign an elemental composition of C37H30O16 to procyanidin dimer

monogallates at m/z 729.1461, corresponding to seven isomers in the Isabel grape seeds and
six isomers in each of the other four samples of grape seeds. A-type structures corresponding
to an elemental composition of C37H28O16 (m/z 727.1305) were also detected in all samples.
In this case, the larger number of isomers was detected in the seeds of the pomace from Pinot
Noir sample (10), followed by Isabel (9), Negro Amaro (7), Sangiovese (6) and Primitivo (3).
Procyanidin Trimer
Accurate masses of B-type procyanidin trimers (m/z 865.1985) were detected,

corresponding to an elemental composition of C45H38O18, and with a number of isomers
ranging from seven (Primitive) to twelve (Sangiovese). Procyanidin trimers with one A-type
bond in their structures (m/z 863.1829), corresponding to an elemental composition of

C45H36O18 were found in all samples, with a number of isomers ranging from six (Pinot Noir)
to ten (Negro Amaro). Furthermore, the presence of two A-type bonds in the structure of
procyanidin trimers (m/z 861.1672) was also observed, corresponding to an elemental
composition of C45H34O18, and the number of isomers ranging from four (Sangiovese) to nine
(Negro Amaro).
Figure 1. Elemental structure of possible dimers and trimers resulting from (epi)catechin-acetaldehyde
condensation.
Procyanidin Dimer Digallate
The presence of two galloyl groups was observed in B-type structures of procyanidin
dimers, corresponding to an elemental composition of C44H34O20 (m/z 881.1571). Two
isomers of procyanidin dimer digallates were identified in each of the samples analyzed. In
addition, a variable number of isomers identified in samples (Isabel, 2; Sangiovese, 3; Pinot
Noir, 4; Primitivo, 7; Negro Amaro, 8) was associated to structures of A-type procyanidin
dimer digallates, corresponding to an elemental composition of C44H32O20 (m/z 879.1414).
(epi)Catechin-Ethyl Trimer
An elemental composition of C47H42O18 was assigned to (epi)catechin-ethyl trimers (m/z

893.2298), corresponding to three (epi)catechin units with one ethyl-bridge (Figure 1). The
number of isomers ranged from seven (Primitivo) to eighteen (Negro Amaro).
Procyanidin Trimer Monogallate
B-type structures of procyanidin trimer monogallates at m/z 1017.2095 were detected in

all samples, corresponding to an elemental composition of C52H42O22. The number of isomers
in the seeds of the different grape varieties was as follows: Primitivo, 9; Isabel, 10; Pinot
Noir, 13; Negro Amaro and Sangiovese, 14. At the same time, procyanidin trimer
monogallates with one A-type bond in their structures (elemental composition of C52H40O22,
m/z 1015.1938) were also detected in all samples, however with a lower number of isomers
compared to that of the corresponding B-type structures for each of the grape varieties
(Sangiovese, 5; Primitivo, Isabel and Negro Amaro, 6; Pinot Noir, 8).

spectrum of seeds of pomace from Primitivo grape

289.0718 41.86, 62.04 C15H14O6 ‒0.39
441.0827 76.45, 78.88 C22H18O10 0.14
577.1351 40.32, 42.72, 51.91, 58.09, 73.39, C30H26O12 0.26
80.18
605.1664 80.64, 87.76, 89.95, 90.91, 93.37, C32H30O12 0.76
96.06, 98.83, 102.29
727.1305 86.70, 88.71, 92.11 C37H28O16 0.79
729.1461 68.03, 69.44, 71.60, 72.04, 80.14, C37H30O16 0.40
86.82
861.1672 73.94, 79.26, 83.49, 84.60, 86.99 C45H34O18 1.79
863.1829 66.58, 68.18, 70.92, 71.77, 73.13, C45H36O18 0.86
75.68, 80.42, 81.95
865.1985 22.57, 43.08, 45.01, 56.04, 63.38, C45H38O18 0.73
71.60, 78.70
879.1414 68.34, 73.27, 82.48, 84.86, 86.94, C44H32O20 ‒1.55
94.21, 96.95
881.1571 77.52, 89.34 C44H34O20 0.19
893.2298 84.33, 85.30, 86.19, 88.35, 89.03, C47H42O18 0.84
95.73, 97.30
1015.1938 73.07, 75.98, 78.52, 79.08, 86.25, C52H40O22 1.32
93.29
1017.2095 50.52, 63.99, 66.24, 67.36, 69.09, C52H42O22 0.94
73.35, 74.94, 78.78, 83.62
1151.2463 69.17, 70.30, 72.59, 73.50, 74.10, C60H48O24 1.44
77.05, 81.03, 83.26
1153.2619 45.67, 49.67, 51.01, 52.55, 54.93, C60H50O24 1.06
66.25, 67.55, 68.08, 71.24, 74.17,
77.31, 78.11
1167.2048 83.26, 86.42, 88.66, 92.94 C59H44O26 1.74
1169.2205 73.12, 74.38, 76.90, 79.25, 80.83, C59H46O26 0.94
85.53
1303.2572 74.15, 76.06, 79.84, 82.96, 88.69 C67H52O28 1.79
1305.2729 76.81, 79.74, 83.55, 85.68 C67H54O28 ‒1.30
1441.3253 55.07, 73.15, 75.87 C75H62O30 0.36
a
mass spectrometry.

spectrum of seeds of pomace from Sangiovese grape

289.0718 41.32, 61.84 C15H14O6 ‒0.39
441.0827 76.42 C22H18O10 0.18
575.1195 81.39, 85.13, 87.09, 93.53 C30H24O12 ‒0.59
577.1351 35.06, 37.59, 39.70, 42.10, 51.74, C30H26O12 0.57
55.46, 57.75, 79.52
605.1664 75.65, 76.37, 80.06, 80.57, 83.66, C32H30O12 ‒0.30
87.74, 89.50, 91.07, 93.24, 95.97,
98.34, 102.23
727.1305 43.76, 45.34, 46.79, 79.44, 85.78, C37H28O16 0.47
86.71
729.1461 67.84, 69.62, 70.69, 72.03, 86.08, C37H30O16 0.33
86.76
861.1672 72.99, 94.65, 99.80, 104.24 C45H34O18 1.47
863.1829 32.02, 65.73, 68.09, 70.68, 71.69, C45H36O18 1.43
74.81, 79.35
865.1985 20.89, 42.71, 44.56, 48.34, 50.44, C45H38O18 0.71
50.89, 52.87, 54.46, 71.25, 72.03,
75.00, 78.55
879.1414 73.21, 81.68, 84.82 C44H32O20 1.36
881.1571 77.51, 88.50 C44H34O20 0.58
893.2298 75.75, 78.93, 81.75, 84.46, 85.19, C47H42O18 0.78
87.53, 88.50, 91.88, 95.50
1015.1938 75.49, 78.70, 81.28, 85.52, 86.14 C52H40O22 1.06
1017.2095 45.19, 61.14, 63.54, 65.47, 67.08, C52H42O22 0.95
68.95, 71.85, 72.62, 73.47, 75.43,
78.66, 79.29, 83.29, 85.22
1151.2463 63.59, 68.74, 70.21, 72.53, 73.68, C60H48O24 1.42
77.01, 81.54
1153.2619 40.51, 42.99, 45.22, 48.97, 51.20, C60H50O24 0.78
52.15, 54.67, 64.96, 67.45, 69.01,
73.02, 73.74, 76.51
1169.2205 76.77, 77.36, 79.17, 79.74, 80.65, C59H46O26 1.31
81.31, 85.23, 88.20
1181.2932 81.04, 84.32, 87.18, 93.41 C62H54O24 0.99
1303.2572 75.78, 79.62, 87.67 C67H52O28 ‒1.65
1305.2729 52.23, 54.32, 56.48, 66.92, 70.44, C67H54O28 0.78
76.02, 76.72, 78.97, 82.63, 90.86
1439.3097 63.45, 66.08, 68.15, 71.73, 72.46, C75H60O30 0.74
73.67, 74.57
1441.3253 63.60, 73.51, 77.04 C75H62O30 0.35
a
mass spectrometry.
Table 6. Number of isomers of each compound identified in the seeds of pomace from
different grape varieties*
Calculated m/z Elemental Compound Number of isomers

[M ‒ H]‒ composition a I NA PN P S
289.0718 C15H14O6 (epi)Catechin 2 2 2 2 2
441.0827 C22H18O10 (epi)Catechin 4 1 1 2 1
monogallate
575.1195 C30H24O12 Procyanidin dimer 5 - 4 - 4
(A-type)
577.1351 C30H26O12 Procyanidin dimer 8 7 8 6 8
605.1664 C32H30O12 (epi)Catechin-ethyl 14 14 40 8 12
dimer
monogallate
(A-type)
monogallate
861.1672 C45H34O18 Procyanidin trimer 5 9 7 5 4
(two A-type bonds)
(A-type)
digallate
(A-type)
digallate
893.2298 C47H42O18 (epi)Catechin-ethyl 16 18 9 7 9
trimer
monogallate
(A-type)
monogallate
1151.2463 C60H48O24 Procyanidin 11 7 11 8 7
tetramer (A-type)
1153.2619 C60H50O24 Procyanidin 7 12 12 12 13
tetramer
1167.2048 C59H44O26 Procyanidin trimer - 4 4 4 -
digallate
(A-type)
digallate
1181.2932 C62H54O24 (epi)Catechin-ethyl 1 - 2 - 4
tetramer
Calculated m/z Elemental Compound Number of isomers

[M ‒ H]‒ composition a I NA PN P S
1303.2572 C67H52O28 Procyanidin - 11 4 5 3
tetramer
monogallate
(A-type)
1305.2729 C67H54O28 Procyanidin 3 8 8 4 10
tetramer
monogallate
1439.3097 C75H60O30 Procyanidin 1 5 - - 7
pentamer (A-type)
1441.3253 C75H62O30 Procyanidin 4 12 - 3 3
pentamer
1457.2838 C74H58O32 Procyanidin - 4 - - -
tetramer digallate
Total isomers 13 184 180 12 150
8 0
Total galloylated 47 77 68 54 58
isomers
Total non- 91 107 112 66 92
galloylated isomers
Total A-type 47 67 58 46 46
isomers
Total B-type 54 82 68 55 76
b
isomers
a
mass spectrometry. b (epi)catechin, (epi)catechin monogallate and condensed products of catechin
with acetaldehyde were not considered.
* I = Isabel, NA = Negro Amaro, PN = Pinot Noir, P = Primitivo, S = Sangiovese.
Procyanidin Tetramer
Procyanidins with four monomeric units, corresponding to m/z 1153.2619 were

associated to an elemental composition of C60H50O24. In the case of these structures, the
number of isomers ranged from seven (Isabel) to thirteen (Sangiovese). Procyanidin tetramers
with one A-type bond in their structures, corresponding to an elemental composition of
C60H48O24 (m/z 1151.2463) were detected in a number of isomers as follows: Negro Amaro
and Sangiovese, 7; Primitivo, 8; Isabel and Pinot Noir, 11.
Procyanidin Trimer Digallate
Four isomers in Isabel sample, six isomers in Negro Amaro and Primitivo and eight
isomers in Pinot Noir and Sangiovese samples presented m/z 1169.2205, corresponding to a
trimer with two galloyl groups linked to its structure. These isomers were associated to an
elemental composition of C59H46O26. Negro Amaro, Pinot Noir and Primitivo samples also
presented A-type structures of procyanidin trimer digallates (m/z 1167.2048, elemental

composition of C59H44O26), in a number of four isomers for each sample.
(epi)Catechin-Ethyl Tetramer
An elemental composition of C62H54O24 was assigned to (epi)catechin-ethyl tetramers

(m/z 1181.2932), corresponding to four (epi)catechin units with one ethyl-bridge. The number
of isomers was of one in Isabel sample, two in Pinot Noir sample, and four in Sangiovese
sample.
Procyanidin Tetramer Monogallate
Accurate masses of B-type procyanidin tetramer monogallates (m/z 1305.2729) were

detected in all samples, corresponding to an elemental composition of C67H54O28, and with a
number of isomers ranging from three (Isabel) to ten (Sangiovese). Except for Isabel sample,
procyanidin tetramer monogallates with one A-type bond in their structures (m/z 1303.2572),
corresponding to an elemental composition of C67H52O28 were also found in the samples. In
this case, the number of isomers was as follows: Sangiovese, 3; Pinot Noir, 4; Primitivo, 5;
Negro Amaro, 11.
Procyanidin Pentamer
Isomers of B-type procyanidin pentamers (m/z 1441.3253, elemental composition of

C75H62O30) were detected in four of the five grape seed samples. A number of three isomers
were found in Primitivo and Sangiovese samples, while four isomeric structures were found
in Isabel sample and twelve in Negro Amaro. Procyanidin pentamers with one A-type bond in
their structures, corresponding to an elemental composition of C75H60O30 (m/z 1439.3097)
were detected in a number of isomers as follows: Isabel, 1; Negro Amaro, 5; Sangiovese, 7.
Procyanidin Tetramer Digallate
The Negro Amaro sample presented four isomeric structures of procyanidin tetramer
digallates (m/z 1457.2838), corresponding to an elemental composition of C74H58O32.
Procyanidin tetramer digallates with A-type bonds in their structure were not detected.
CONCLUSION
The complex composition of isomers from flavan-3-ol compounds in seeds of pomace
from different grape varieties was characterized in details up to pentamers by the high
resolution and mass accuracy of Fourier transform ion cyclotron mass spectrometry. The
highest number of isomers was detected in seeds of Negro Amaro sample, followed by the
seeds of Pinot Noir sample. In all samples, the number of total B-type isomers was higher
than that of total A-type isomers. Furthermore, elemental compositions were assigned to
isomers of condensed products of (epi)catechin with acetaldehyde up to tetramers. Data
presented in this chapter can be useful in the analysis of food ingredients and dietary
supplements made of grape seeds.
ACKNOWLEDGMENTS
This work was supported by CNPq (National Council for Scientific and Technological
Development - Brazil). The authors are grateful to EPAGRI – Videira (Agricultural Research
Governmental Company of Santa Catarina state) for help in obtaining samples from industrial
producers.
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Chapter 5
METHODS TO EVALUATE ANTIOXIDANT

PROPERTIES OF GRAPE SEEDS
M. L. González-SanJosé*, M. D. Rivero-Pérez
and P. Muñiz-Rodríguez
Department of Biotechnology and Food Science,
University of Burgos, Burgos, Spain
ABSTRACT
Products and compounds are considered antioxidant when they show capacity to
delay, retard or prevent oxidation being in low concentrations relative to the substrate to
be oxidized; and when the oxidised resulting products are stable against further oxidation.
The capacity of one compound to block and inhibit, totally or partially, diverse
oxidative reactions is known as the antioxidant capacity or activity (AOC) of this
compound. Usually AOC is measured in complex matrices, where a lot of different
antioxidants can be present, in these cases it is usual the use of the term ―total antioxidant
capacity‖ (TAC), according to the fact that AOC is not due only to one compound.
Antioxidants can block different oxidative agents and oxidative reactions; so
different methodologies using different reactions or different substrates can be used to
measure qualitatively and quantitatively the antioxidant capacity of compounds and
products. There are multiple free radicals and oxidant agents; and both oxidants and
antioxidants have different chemical and physical characteristics. Due to these facts, a lot
of different methods have been developed to evaluate the AOC of products and
compounds, being especially variable the methods applied to assess AOC of foods,
botanicals, nutraceuticals, and other dietary supplements. Each AOC method is based in
different reaction mechanisms, showing advantages and disadvantages in relation to
others. The antioxidant properties of grape seed products have been evaluated in both ―in
vitro‖ and ―in vivo‖ assays and a large number of different assays have been used.
Keywords: grape seeds, antioxidant capacity, antioxidant assays
*
Corresponding author address: Department of Biotechnology and Food Science, University of Burgos, Spain,
Email: marglez@ubu.es.
102 M. L. González-SanJosé, M. D. Rivero-Pérez and P. Muñiz-Rodríguez
INTRODUCTION
World life is mainly conduced in presence of an oxygen rich medium. Under this
condition, oxidative reactions are continuously occurring in nature, including flora and fauna
which require oxygen for generating metabolic energy, while also produce reactive oxygenic
species (ROS) [1-3]. By extension, oxidative reactions also occur during human manipulation
of natural resources, including food industrial processes.
Oxidative reactions are those chemical reactions that produce an increase of the oxidation
state and those that involve losses of electrons. Oxidation reactions can produce free radicals
which, due to their high reactivity, can start oxidative chain reactions, producing very
negative consequences to life and industrial processes and products. Products or compounds
that produce oxidative reactions are oxidant compounds and those that inhibit oxidative
reactions are antioxidants. Antioxidants can block oxidative reactions by different
mechanisms [4] with their consequent oxidation while other compounds or products are
reduced, so antioxidants are usually reducing agents. Accordingly, antioxidant reactions
usually involve transference of electrons or protons.
Products and compounds are considered antioxidant when they show capacity to delay,
retard or prevent oxidation being in low concentrations relative to the substrate to be
oxidized; and when the oxidised resulting products are stable against further oxidation. The
capacity of one compound to block and inhibit, totally or partially, diverse oxidative reactions
is known as the antioxidant capacity or activity (AOC) of this compound. When AOC is
measured in a complex, where a lot of different antioxidants can be present, the term Total
Antioxidant Capacity (TAC) is generally used, in agreement with the fact that antioxidant
capacity of the matrix is not due only to one compound [5-7].
Due to the capacity of antioxidants to prevent the negative effect produced by oxidative
reaction, this type of compounds has a large number of preservative applications not only in
food industries [8], but also in others as the cosmetic or pharmaceutical industries. Besides,
when antioxidants are used the duality anti-oxidant/pro-oxidant effect should be always
considered.
There are natural and synthetic antioxidants, however the cosmetic and food industry
tend to use, more and more, natural antioxidants. There are a lot of natural antioxidants, as
polyphenols, ascorbic acid, tocopherols, selenium, etc., which are mainly present in
vegetables, arriving to animals through the diet. Animals have different systems to protect
themselves of the possible oxidative damages inherent to life, and diet antioxidants contribute
to their endogenous antioxidant/oxidant balance. Different types of antioxidants play an
essential role in this sense. Beside exogenous antioxidants (vitamins, minerals, etc.),
endogenous antioxidant mechanisms include metabolites such as glutathione or enzymes such
as peroxidases, catalase, superoxide dismutase, and others, who work simultaneously to
inhibit oxidative damages to cellular components as lipid, proteins and DNA [9-10].
Antioxidants try to prevent the formation of reactive oxygen species (ROS) or remove them
partially keeping the ideal ROS level, due to the fact that ROS have also necessary functions
as for example redox signalling [1, 9, 11]. Insufficient antioxidants or deficient action of
antioxidant mechanisms produce oxidative stress and the corresponding risk of oxidative
cellular damages including dead. Oxidative stress (OS) has been correlated with many animal
and human diseases. It is clear the role of OS in cardiovascular disease [12-13] and with a
Methods to Evaluate Antioxidant Properties of Grape Seeds 103
wide range of pathologies caused by diabetes and neurodegenerative diseases [14-15], and
diverse types of cancers [16]. In this sense, the use of antioxidants with therapeutic effect has
been intensively studied by the pharmacology industry but also by medical institutions and
researchers. Controversial results have been published about antioxidant supplements,
however nowadays nobody discusses the importance of a balanced diet which includes a
sufficient quantity of natural antioxidants in order to extend both the length and the quality of
life [17-19]. This fact has lead to the extensive use of different natural antioxidants as dietary
supplements, nutraceuticals and their use as ingredients of ―functional foods.‖ However, it is
important to have in mind that the food industry has used traditionally antioxidants to protect
foods, and nowadays the tendency is towards the use of natural antioxidants, answering the
expectative of modern consumers who demand foods free from synthetic ingredients.
Antioxidants are used in food industries to inhibit oxidative deterioration of foods, which
can occur also under refrigeration and freezing conditions. The oxidation of unsaturated fat
producing rancidity [20] is probably the most important oxidative degradation of foods. In
fact, the antioxidants mainly used in the food industry are those that prevent oxidation of fats.
To obtain satisfactory food industry applications, the solubility of antioxidants is a
determinant factor; so lipophilic antioxidants are probably the most demanded, although
hydrophilic antioxidants also have an important role to prevent oxidations conduced in water
medium such as fruit, meat, juice, body fluids and tissues.
The antioxidant capacity exerted by one antioxidant is dose dependent, being possible
that it can also act as pro-oxidant depending on its concentration in the medium. Furthermore,
other factors, as its reactivity towards a particular oxidant [10] and the presence of other
antioxidants with which it can interact making possible synergistic and interdependent effects
[21], have to be considered.
METHODS TO EVALUATE ANTIOXIDANT PROPERTIES

Antioxidants can block several oxidative agents and oxidative reactions, so various
methodologies using different reactions or substrates can be used to measure qualitatively and
quantitatively the antioxidant capacity of compounds and products. Some examples of this
diversity are the assays of antioxidant capacity carried within biological systems, in which
different antioxidant defences can be considered as enzymes (i.e., superoxide dismutase), or
small molecules as glutathione, among others. Furthermore, individual antioxidants may, in
some cases, act by multiple mechanisms in a single system [22] or by a different single
mechanism depending on the reaction system; and antioxidants may respond differently to
varied radical or oxidant sources. Furthermore, there are multiple free radicals and oxidant
agents and both oxidants and antioxidants have different chemical and physical
characteristics. Due to these facts, a lot of different methods have been developed to evaluate
the antioxidant capacity of compounds and products, and those applied to assess AOC of
foods, botanicals, nutraceuticals, and other dietary supplements are especially variable. Each
method is based in different reaction mechanisms, showing advantages and disadvantages
with respect to others [5].
It must be appreciated that there is no simple universal method by which AOC can be
measured accurately and quantitatively, because multiple reaction characteristics and
mechanisms as well as different phase localizations are usually involved during antioxidant
actions [5], so the study of AOC, specially of foodstuffs, requires the use of diverse methods
which provide a wide picture of their multiple antioxidant effects. Thus, total antioxidant
capacity (TAC) can only be properly valuated when multiple assays are used in order to build
up the antioxidant profile of the product under study.
The two major mechanisms of antioxidants to deactivate radicals are Hydrogen Atom
Transfer (HAT) and Single Electron Transfer (SET). The HAT methods most commonly used
to evaluate the AOC of foodstuffs are: ORAC (Oxygen Radical Absorbance Capacity), TRAP
(Total Radical-trapping Antioxidant Parameter), Chemiluminescence (CL), and Low-Density
Lipoprotein (LDL), while the most commonly used SET method is FRAP (Ferric Reducing
Antioxidant Power).
ORAC [23-26] measures antioxidant inhibition of peroxyl radical induced oxidations
and, thus, reflects classical radical chain-breaking antioxidant activity by H atom transfer
[26]; TRAP [23, 27-29] monitors the ability of antioxidant compounds to interfere with the
reaction between peroxyl radicals and a target probe; Chemiluminescence [30-31] is a high-
sensitivity modification of TRAP based on the reaction of radical oxidants with marker
compounds to produce excited state species that emit chemiluminescence; LDL [32-33] is a
method based on the inhibition of LDL oxidation, This is a method adapted to assess
antioxidant capacity in physiologically relevant systems. FRAP [34-35] measures the
reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) to a coloured product, detecting
antioxidants with redox potential lower than 0,7V. This method was initially developed to
measure reducing power in plasma, and was subsequently adapted to assay antioxidant
capacity of botanical products [7, 36-37]. This is a method with reasonable screen for
measuring the redox status in cells and tissues, and it is an index of the reductive ability of an
antioxidant [38].
Other methods as TEAC (Trolox equivalent antioxidant capacity) and other ABTS
assays, such as DPPH assay, have been classified as (SET) mechanisms. However, both
radicals (ABTS and DPPH) may be neutralised by SET and HAT mechanisms making
particularly difficult to know which mechanism is actually involved without knowing the
exact antioxidant in action [39]. The reaction mechanism may shift with pH, so, for example,
in the case of ABTS assay, electron transfer is facilitated at acid pH [40].
TEAC was first reported by Miller and Rice-Evans [41] and it is based on the scavenging
ability of antioxidants the long-life radical ABTS (2,2‘-Azinobis 3-ethylbenzothiazoline-6-
sulfonic acid) which is an intensively coloured compound. AOC is measured as the ability to
decrease the colour, expressing the results as relative to Trolox. Different posterior protocols
were proposed to avoid overestimations [42-43] and to improve reaction time [44]. Besides
this modification, other modifications in terms of wavelengths that are used to monitor the
reaction and to quantify calibration curves were also made by different researchers. This fact
led to a large number of similar methods that actually share the same reaction mechanism and
use the same radical (ABTS) [5]. Variation has been adapted also to measure selectively
hydrophilic and lipophilic antioxidants by running the assay in buffered media and organic
solvents [45]. DPPH assay (2,2-Diphenyl-1-picrylhydrazyl) [46-47] is based on the
measurement of the reducing ability of antioxidants toward DPPH, one of the few stable
organic nitrogen radicals, which bears a deep purple colour. The widely used decolouration
assay was first reported by Brand-Williams and co-workers [44]. The DPPH assay has been
considered as mainly based on SET mechanisms being HAT a marginal pathway (2). DPPH
and ABTS have some similarities as both radicals show similar bi-phase kinetic reactions
with many antioxidants, but they also show some important differences in their solubility.
ABTS can be dissolved in aqueous and organic media, being able to measure the antioxidant
activity due to the hydrophilic and lipophilic antioxidant, while DPPH can only be dissolved
in organic media, especially in ethanol, showing an important limitation to interpret the role
of hydrophilic antioxidants.
Considering that Total Polyphenols (TPP) assay, which has been applied for many years
as a measure of the total phenolic content in natural products, is based on an oxidation-
reduction reaction, this method can be considered another useful TAC method [5] and it is
simple, sensitive, and precise. However, under original conditions the reaction was slow and
it lacked specificity. Singleton and Rossi [48] improved the original method to increase the
phenols selectivity but the reaction of Folin is not actually specific to phenol compounds. TPP
assay is also a SET method. A similar method, Phosphomolybdate assay [49], has been
recently developed, being the main difference that ascorbic acid instead of gallic acid is used
as standard.
AOC methods described previously are in vitro methods which can also be carried out
―ex-vivo,‖ as it has been commented about LDL and FRAP. It is generally recognized that
effects of foodstuffs on the redox balance in vivo cannot be merely extrapolated from their
activities measured in vitro, with the possible exception of their antioxidant action at the
gastrointestinal tract [50-51]. However, the determination of the in vitro TAC by simple and
relatively fast chemical methodologies could be a useful tool of approximation to the healthy
potential of antioxidants and antioxidant-rich products. Furthermore, in vitro AOC assays
give technologically outstanding information to the food industry that needs the use of
antioxidants to increase shelf life of products. In this sense, other antioxidant assays, mainly
based on the measurement of products resulting from fat oxidation, are also of interest and
have been extensively applied. The most commonly used methods are the inhibition of
TBARS (Thiobarbituric acid reactive substances) and hexanal formation, which are an index
of the fat oxidation, as well as RANCIMAT, which is based on oxidation, under accelerated
conditions, of a fat placed on a closed container [20].
In order to approximate in vitro measures of AOC to the possible antioxidant activities
that antioxidants can exert in vivo, different biologically relevant methods have been also
used [52]. Some of these methods are based on the evaluation of scavenger capacity towards
ROS, being mainly focused on some of the ROS generated in the organism itself due to the
aerobic metabolism. Some of these methods are SRSC (superoxide radical scavenger
capacity) and HRSC (hydroxyl radical scavenger capacity). The hydroxyl radical is
particularly unstable, reacts rapidly and non-specifically with most biological molecules and
can damage cells by starting chemical chain reactions such as lipid peroxidation, or by
oxidizing DNA or proteins [9]. Damage to DNA could cause cancer [16], while damage to
proteins could produce, among others, enzyme inhibition [53], with the corresponding
deficient expressions. Furthermore, and considering the fact that oxidative stress in biological
systems arises from an imbalance between oxygen species and antioxidants, the study of
biomarkers of oxidative stress are also recognised as biologically relevant methods [54]. The
measurement of lipid peroxidation inhibition (LPI) and the study of DNA and protein
damages are some of the most applied methods of this group [55-58]. The main product
formed during lipid peroxidation is malondialdehyde, a powerful genotoxic and carcinogenic
compound. Oxidative modifications of proteins often lead to the modification of certain
amino acid residues forming carbonyl derivates, which is linked to losses in physiological
functions under pathological processes or during aging [59].
SRSC is based on the capacity of superoxide radical to react with NBT
(4-nitroblue tetrazolium chloride) to generate a coloured compound with absorbance at 560
nm [60] being the antioxidant capacity directly correlated with the produced decoloration.
The results are expressed as percentage of inhibition in relation to a control (test without
antioxidant). HRSC is based on the oxidation of desoxyribose (2-desoxy-D-ribose) by
hydroxyl radicals generated by Fenton reaction [61]. Malondialdehyde (MDA) formed from
the decay of the desoxyribose is evaluated by reaction with TBA (tiobarbituric acid) giving a
coloured compound with absorbance at 532 nm. The result is expressed as a percentage of
inhibition in relation to a control.
Other interesting biologically relevant methods are those based on lipid peroxidation,
DNA damages and protein oxidation. Lipid peroxidation inhibition (LPI) can be measured
with different biological systems in which lipid peroxidation is induced by different oxidant
agents. One of the methods frequently used is based on the lipid peroxidation of microsomes
isolated from liver of Wistar rat, using ABAP (2,2‘-azobis-(2-amidinopropane)-
dihydrochloride) as oxidative agent [62]. Lipid peroxidation is evaluated indirectly by
quantification of MDA levels. Absorbance measured at 532 nm was proportional to the
quantity of peroxyl radicals generated and the results are expressed as a percentage of
inhibition in relation to a control. Damage to DNA is usually evaluated using DNA calf
thymus [61, 63], which is exposed to hydroxyl radicals generated by Fenton reaction, and
oxidative damages are evaluated considering the DNA fragments separated by electrophoresis
in agarose gel. The results are expressed as DNA fragments, calculated using standard
molecular weight markers. Oxidative DNA damage can be also measured by quantification of
the levels of the modified base 8-hydroxy-2-deoxiguanosine [64]. Oxidative damage to
proteins can be evaluated quantifying the levels of carbonyl groups or evaluating the levels of
tyrosine oxidation or nitration [64].
All the commented methods show the limitation that they only measure the activity of
soluble compounds present in liquid products or in extracts, being the extraction procedure a
critical step [65-66]. This reason motivated the development of the QUENCHER (QUick,
Easy, New, CHEap, and Reproducible) assays to measure the antioxidant activity of food
materials [67]. These methods are a very interesting approach that avoids time and solvent
consuming extraction steps involved in the classical protocols. The basis of QUENCHER
(Q-) protocols is to place in direct contact solid powdered foodstuffs and the reagent
solutions. Thus, the soluble antioxidants of the sample quench the radicals present in the
reaction medium according to usual liquid-liquid reactions, whereas the antioxidants bound to
the insoluble particle matter exert their antioxidant activity taking advantage from surface
reactions occurring at the solid-liquid interface [67]. Several of the frequently used TAC
methodologies (ABTS, DPPH, ORAC, FRAP, and FC) have recently been adapted to
QUENCHER approach. Up to now, these assays have been successfully applied to some
foods in which the antioxidant activity is largely dependent on the insoluble part, like cereals
and bakery products, seeds, nuts, pulses, and dietary fibers [68-70]. Despite of the advantages
and previous applications of the QUENCHER methods, these assays are not yet extensively
used, probably due to a lack of validation studies of these methodologies. Recently, some of
the cited methods have been validated and new QUENCHER methodologies targeted on
biologically relevant radicals such as SRSC and HRSC, as well as with other biomarkers of
oxidative stress as LPI, have been developed [71].
Biological in vitro assays, as it was previously indicated, are only an approximation to the
antioxidant effect occurring in the organism, because these methods do not consider
bioavailability aspects (absorption, metabolic transformation and organ distribution). Besides,
biological in vitro assays have been recognised as an invaluable tool for clinical studies if
they are combined with bioavailability data [72]. The ability to act of antioxidants depends on
their absorption, transport, distribution and their properly retention in biological fluids, cells
and tissues. So, bioavailability of antioxidants (concentration and duration effect) has been
studied in biological fluids (plasma, urine) and in different tissues of animals and humans [50,
73]. Furthermore, bioavailability studies have been complemented with data of oxidation of
lipids and proteins among other parameters [64, 74]. Moreover metabolic biotransformation
of antioxidants, such as enzymatic conjugation sulfations, methylations or glucuronidation,
has been also studied and their effect on antioxidant activity has been evaluated.
Bioaccessibility and bioavailability studies are complex and expensive and cannot always
be developed in animals or humans, which is the optimum option. Fortunately, nowadays,
there are the adequate methodologies to reproduce or mimic diverse biological process,
allowing the study of bioaccessibility and bioavailability more easily. Some examples are the
useful methods to study the bioactivity under gastrointestinal conditions and colonic
fermentation. These methods reproduce, with different digestion models, the gastrointestinal
digestion and the bacterial colonic fermentation under laboratory scale and conditions [75-
77]. Differences between initial levels of antioxidants and those remaining under diverse
digestion steps allow the estimation of bioavailability of the antioxidants in small and large
intestine [78-79].
Other methodologies applied to evaluate antioxidant properties have been carried out
using cell cultures. The advantage of using cell cultures is that they can be used as model
systems for specific diseases. These methods are used to assess cell viability, proliferation,
radical free generation, oxidative damage to biomolecules, changes in intracellular
antioxidant, etc. [80]. Therefore, the use of cell cultures is a model to indirectly assess the
antioxidant capacity of compounds, but results have to be interpreted with precaution.
Cell, animal and clinical studies can use some of the methods previously described to
evaluate the effects produced by the supplemented antioxidants. TEAC and other methods as
FRAP have been used to evaluate the antioxidant stage of cells [81-82]. However, clinical and
animals studies usually use biological antioxidant markers, as glutathione, diverse enzymatic
systems as catalase, oxidative damage biomarkers as DNA and also changes in the mRNA
expression. This fact is due to the capacity of exogenous antioxidant to act on or interacting
with diverse endogenous antioxidant defence mechanisms [83].
Some of the endogenous antioxidant systems most usually valuated in animals and
human studies are probably Glutathione system (GSH/GSSG) and Superoxide dismutases
(SOD). Glutathione system includes glutathione, glutathione reductase, glutathione
peroxidase and glutathione S-transferases [84]. Glutathione (GSH) is a peptide with
antioxidant properties due to the presence of cysteine thiol group, having the possibility of be
reversibly oxyded (GSSG) and reduced (GSH). Glutathione reductase maintains glutathione
in the reduced form, and then maintains its antioxidant potential. Superoxide dismutases [85]
(SODs) usually acts together with other closely enzymes as catalases and peroxidises. SODs
catalyze superoxide conversion into hydrogen peroxide and oxygen, and then hydrogen
peroxide is removed by action of catalases, which convert hydrogen peroxide in water and
oxygen [86-87].
ANTIOXIDANT CAPACITY OF GRAPE SEED PRODUCTS

Grape seed is a complex matrix containing approximately 40% fiber, 16% oil, 11%
proteins, and 7% complex phenols including tannins. Other minor constituents are sugars and
mineral salts, etc. [88]. The polyphenol composition of grape seeds varies depending on the
varieties of grapes and is influenced by the growth medium, climate, maturity and the time of
fermentation in the case of by-products of the wine industry [89]. Besides, grape seeds have
been generally appreciated because of their content of phenolic compounds such as gallic
acid, catechin and epicatechin, and a wide variety of condensed tannins [90].
Proanthocyanidins of grape seeds are de most important group of flavonoids, which are
known to possess broad pharmacological activities and therapeutic potential [91]. The
proanthocyanidins identified in grape seeds can range in size from monomers to long-chain
polymers, then including monomers as epicatechin and cathechin, diverse oligomers from
dimmers to tetramers, and polymers until a polymerization degree of about 30 units [92]. The
dimmers are the most numerous (around twenty) and they are the most widely distributed in
grape seeds, and dimmers, together with trimmers, are the most active proanthocyanidins
[93]. The dimmers are often referred to as B-series, and the trimmers as C-series [94].
Proanthocyanidins of grape seeds can be galloylated, so monogalloylated oligomers, with the
degree of polymerisation of 2–5, and digalloylated oligomers, with the degree of
polymerisation of 2–3, among others, have been described [95]. The chemical properties of
proanthocyanidins in terms of the availability to donate electrons and phenolic hydrogen
atoms determine their antioxidant activity, mainly as hydrogen donating radical scavengers
and singlet oxygen quenchers [96]. The presence of hydroxyl functional groups and their
position on the rings of the flavonoid structure influences notably these properties. Hydroxyl
groups enhance the antioxidant activity, while substitution by –OCH3 group decreases the
antioxidant activity. Furthermore, the antioxidant activity of proantho-cyanidins also depends
on their degree of polymerization [97].
In addition to phenolic compounds, grape seeds also contain non-phenolic antioxidants
such as tocopherols and β-carotene; both of them are potent antioxidants and are critical to
human health. Tocopherols and β-carotene are mainly concentrated in grape seed oil [98],
where also sterols are present [89]. Grape seeds contain certain amount of phytosterols,
compounds with interesting healthy effects as anti-arteriosclerotic activity, and which
contribute to the antioxidant capacity of grape seed oils.
Grape Seed Products
The products most commonly derived from grape seed are the extracts, which are
obtained by different extraction and purification processes. Extracts could be commercialized
in liquid formats or as powdered products obtained after drying processes. All of them are
products rich in polyphenols, with intense antioxidant and other interesting properties as
antimicrobial and anti-inflammatory [97].
The technique most widely used to obtain proantocyanidin extracts from grape seeds is
the extraction with organic solvents, such as ethanol [99], ethyl acetate [100], methanol [101]
and mixtures of different solvents [102]. Solvent mixtures are used to enhance the solubility
of proanthocyanidins of different solubility, and to improve the antioxidant activity of the
obtained extracts. In this way, it has been described that extracts obtained with mixtures of
ethyl acetate and water, at different concentrations, exhibited more antioxidant activity than
other extracts obtained with other solvents [102].
Environmentally friendly extractive methodologies, using water instead of organic
solvent, which is also healthier (no risk of solvent residues) have been studied and proposed
as alternative possibilities [103]. The obtained results showed that this technology allowed to
obtain grape seed extracts with important in vitro and in vivo antioxidant activities.
Nowadays, there are different commercial available grape seed proanthocyanidin extracts
(GSPE). GSPE were declared as potent antioxidants, rich in bioavailable free radical
scavengers, safe and novel products, possessing a broad spectrum of health benefits [96].
Moreover, the cardioprotective ability of GSPE was observed in animals and humans. [104]
According to these results, the utilization of extracts of grape seeds, as functional ingredients,
was announced as a promising field.
Recent research has focused on delivering new approaches to improve the performance of
conventional extractions, and to modify the composition of extracts to obtain more active
products. Diverse enzymatic treatments have been studied for these purposes. The use of
specific enzymes can help to increase the liberation of proanthocyanidins from seed matrices
and, simultaneously, reduce the average molecular weight of extracted oligomers and
polymers. The reduction of size of proanthocyanin would be desirable because the
bioavailability of proanthocyanidins is largely influenced by their degree of polymerisation
and galloylation. Although monomeric flavan-3-ols are readily absorbed in the small
intestine, oligomeric and polymeric forms pass intact through the gastrointestinal tract and
reach the colon, where they must be transformed by the intestinal microbiota before
absorption [105].
Significant changes of polyphenol and polysaccharide contents of grape seed extracts
were obtained using different enzymes [106]. Pectinase, cellulose, tannase and different
sequential blends were used to increase the phenol concentration and to reduce the
proanthocyanidins molecular size of grape seed extracts [107]. The three enzymes were
effective, increasing the phenolic extraction around 1.3 times, with respect to the controls.
However, - different results were obtained depending on the grape variety [108-109], on the
terroir [110] and on the degree of grape maturation [111].
The processing of grape seeds is usually necessary to improve extraction processes. In
this sense, there is a significant number of reports that describe the variables that influence the
antioxidant capacity and the characteristics of the resulting grape seeds extracts. Some recent
investigations have focused on delivering the effects of heating and grinding treatments. Heat
treatment of grape seeds liberated phenolic compounds, and thus increased the amounts of
active compounds in extracts. Therefore a simple heating process can be used as a tool to
increase antioxidant activity of grape seed extract [112]. This work also showed that
antioxidant activity of grape seed extract was affected by the heating conditions and by the
physical conditions of grape seeds at the time of heat treatments. Particle size of seeds is the
physical parameter that most affects extraction processes. The extraction yields increase with
smaller particles, but size should remain large enough to prevent clogging phenomena.
Previous studies showed that simple heat treatment converted insoluble phenolic compounds
into soluble phenolics [113]. However, the heating treatment was not able to cleave
covalently bound phenolic compounds [114]. Furthermore, slight differences between the
kinds of phenolic compounds detected in non-heated and heated seeds (at 150ºC for 40 min)
were noted. Several low molecular weight phenolic compounds such as 3,4- dihydroxy
benzoic acid and o-cinnamic acid were newly found. Besides, other authors described no
changes in the phenolic composition of grape seeds treated to 90ºC [115].
Although the extracts are probably the products that are becoming more popular in recent
years, other authors have proposed the use of wine by-products, as grape seeds, without any
prior extraction of the phenolic compounds; this approach presents clear economic and
environmental advantages [115-116]. Different powdered products have been developed and
some of them have been patented. They show different characteristic and technological
applications, some of them directly correlated with their antioxidant properties, which derived
from the grape seed antioxidants, which are also present in the powdered products. In these
cases, probably the particle size of the products is one of the main critical points. A direct use
of grape seeds requires their downsizing, reaching the right size for each of the final
applications. Downsizing facilitates the incorporation of grape seeds into a final matrix (food,
cosmetics, etc.) but also enhances the antioxidant activity that can be exerted by grape seeds.
The smaller the particle size of the seeds is, the greater their capacity to release extractable
compounds and the higher their antioxidant potential. Furthermore, recently superfine
grinding technology has been applied to produce grape pomace powders (seeds, skins and
stems) in the submicron range. This technology uses a mini-type airflow pulverization
system, [117], and it was observed that superfine grinding caused an increase in ABTS,
FRAP, and TPP values, although a reduction in DPPH radical scavenging activity, of the
obtained products, was also detected.
Antioxidant Capacity of Grape Seed Products
The antioxidant properties of grape seed products has been evaluated in both ―in vitro‖
and ―in vivo‖ assays, and a lot papers attribute these properties mainly to the potent free
radical scavenging abilities of grape seed proanthocyanidins.
AOC of Grape Seed Products Measured by “In Vitro” Methods

The antioxidant properties of grape seeds can be evaluated through very different
methodologies based on their different capacities to block oxidative reactions. Among these
capacities probably the most used is the ability of scavenging different free radicals. Different
methods as ABTS (1,1-diphenyl-2-picrylhydrazyl), DPPH (2,2‘-azino-bis-3-ethyl-benzo-
thiazoline-6-sulfonic acid), superoxide and hydroxyl scavenging capacities, among others,
have been used to evaluate the antioxidant properties of grape seed extracts and products.
Apart from the scavenging properties, other properties as the capacity of metal chelating and
the reducing antioxidant power have also been used [118-120]. Positive results respect the
antioxidant properties of grape seeds and derived products were always noted. Grape seeds
showed the highest antioxidant capacity compared with the skin and flesh [121]. The extracts
of defatted grape seeds expressed less antioxidant capacity than whole grape seeds, which is
correlated with the losses and degradation of different antioxidant compounds during oil
extraction processes [122].
Grape seed proanthocyanidins showed free radical scavenging abilities against
biochemically generated superoxide and hydroxyl radicals, which were measured using
cytochrome c reduction and chemiluminescence assays [123]. Grape seed proanthocyanidins
demonstrated superior free radical scavenging abilities than vitamin C, and superior peroxyl
radical scavenging abilities than Trolox. Both total polyphenol and total flavonoid contents
were positively correlated with radical scavenging activity (R = 0.9), and these results were
considered to provide basic information about health-beneficial effects of grape seeds [124].
Regarding to their reducing power some grape seed extracts showed marked antioxidant
activity using the potassium ferricyanide reduction method [102]. Among the diverse
antioxidant actions, the grape seed flavonoids, mainly proanthocyanidins, may act in a similar
fashion as reductive compounds, by donating electrons, and as radical scavengers, by reacting
with free-radicals to convert them into more stable products ending the free-radical chain
reaction. Grape seed extracts were also able to prevent different oxidative damage induced by
H2O2. [104] Oxidative damage to lipids (TBARS assay) and proteins (evaluation of carbonyl
group), as well as enzymatic (catalase activity) and non-enzymatic (protein sulfhydryl assay)
defences were used to evaluate the protection against H2O2 damages.
The relation between lipid peroxidation and diseases, its modulation by antioxidants and
other contexts, are being extensively studied. The antioxidant capacity of tannin fractions
isolated from grape seeds was measured using the conjugated autoxidizable triene (CAT)
procedure. This assay is based upon the high sensitivity to oxidation of the α-eleostearic acid,
which is contained in the triacylglycerols of stripped Tung (Vernicia fordii) oil [125]. Upon
oxidation, the degradation of the conjugated triene system into a conjugated diene is
accompanied by a decrease in the signal at 273 nm. Tannins from grape seeds showed good
antioxidant activity in oil in water emulsion measured by CAT assay. Their antioxidant
properties come from their hydroxyl groups and more specifically from their catechol
moieties (i.e., –OH groups on ortho positions on aromatic rings).
The capacity to inhibit lipid peroxidation of microsomes isolated from rat and mouse
livers has been also applied to evaluate the antioxidant activity of grape seed extracts [126].
Data from an aniline hydroxylation assay showed inhibitor effect on grape seed extract-
exposed livers, and inhibition was concentration dependent.
Similarly to previously described biological assays, the formation of lipid hydroperoxides
and thiobarbituric acid was also applied to determine the antioxidant effect of grape seed
extracts in raw and cooked ground muscle during refrigerated and frozen storage [127]. The
obtained results showed that a grape seed extract, at concentrations as low as 0.1%, was a
very effective inhibitor of primary and secondary oxidation reactions, showing interesting
potential as a natural antioxidant to raw and cooked meat systems.
Antioxidant capacity of grape seeds was also analyzed through oil oxidative stability, an
important parameter to evaluate possible applications in food industry. The method Cd 12-
b92 (AOCS, 2009), which uses a Rancimat instrument, has been utilized in different studies.
Rancimat values showed high antioxidant stability of oils extracted from grape seeds [128];
and this method also showed the protective effect of grape seed extract on sunflower oil
oxidation [129] and the protective effect of a powdered grape seed product on olive oil and
pork lard lipid oxidation [115].
AOC of Grape Seed Products Measured by “In Vivo” Methods

The study of the healthy properties of grape seed and derived products that are associated
to its antioxidant properties has been carried out by in vivo and ex vivo methodologies and
usually non- invasive instrumental methods have been selected.
Studies about the bioaccessibility and bioavailability of grape seed, carried out in vitro
simulated digestion and colonic fermentation, have showed that during digestion antioxidant
properties of seed products were modified due, at least in part, to the qualitative and
quantitative variations of the polyphenol content [77, 130-131]. These results point out that to
correlate antioxidant properties of grape seed components and their healthy properties, and it
is very important to consider the modification of these components after they are ingested.
Possible antioxidant properties of grape seed products have also been studied using cell
cultures. To understand the mechanistic pathways of cytoprotection of grape seed products, it
has been necessary to analyze different parameters directly correlated with the oxidative
stress of cells. In this sense, ROS levels, lipid oxidation, protein oxidation, DNA damage, and
cell death have been evaluated [91, 132-133]. Furthermore, diverse cellular lines have been
employed, being probably the Caco line the most used. The gastrointestinal tract is constantly
exposed to damage produced for ROS present in food and beverages. Probably, for that
reason, intestinal cell models are very usually employed; furthermore these studies provide
significant information for human health. Grape seed products showed capacity to prevent
oxidative damages caused by tert-butylhydroperoxide-induced oxidative stress in Caco cells,
and the observed protection was dose and time dependent [134].
The anticancer potential of grape seed products has been studied in different cancer cell
lines such as colorectal, prostate, breast, lung, skin, gastric, etc. [132, 135-141]. In general,
results showed that the effect of the grape seed products on cell cytotoxicity is concentration
dependent. Treatment of cancerous cells with grape seed products produced changes in the
intracellular redox potential. High concentrations of grape seed products (25-400 mg/mL)
induced cytotoxicity and antiproliferative effect and produced a high oxidative stress in terms
of generation of ROS. These results were observed in different cell lines as bladder, breast,
lung, mouth, colon and head cancer cells, as well as in neck squamous carcinoma cells. These
studies also showed that low concentrations (1-10 mg/mL) of grape seed products inhibits the
generation of ROS in cultures of lymphocytes exerting protective antioxidant effect.
Furthermore, it was observed that the concentration of 25 and 50 mg/L of a grape seed extract
produced a cytotoxic effect in different human cancer cells, whiles enhancing the growth and
viability of the standard cells [132]. Based on the positive obtained results, different
preparations of grape seed have been used as dietary supplements to the chemoprevention of
cancer.
Some studies have showed the preventive effect of grape seed products, this conclusion
has been derived from the diverse results obtained depending on the moment in which the
seed products are added into the cells. The pretreatment with grape seed extract (50 µg/mL)
and ulterior treatment with tert-butyric acid (oxidant agent) showed capacity to prevent
oxidative stress, whiles grape seed extract was not able to reduce ROS levels when the cells
were not pretreated [134]. This study also showed that low doses of GSPE (grape seed
proanthocyanidin extracts) (0.01 µg/mL) were sufficient to prevent oxidative stress.
Diverse studies showed also the capacity of grape seed and derived products to
ameliorate chemotherapy toxicity. Thus, a grape seed product decreased the inhibitory cell
growth effect produced by chemotherapeutic drugs such as idarubicin, doxorubicin, among
others, indicating its potential to ameliorate the toxic effects associated with
chemotherapeutic agents [134, 142].
Grape seed products showed an antioxidant function by influencing the antioxidant
activity and the antioxidant gene expression of diverse enzymatic and not enzymatic cell
antioxidants. In this sense, the effect of grape seed on the antioxidant and redox state of the
cell is very interesting. Cells treated with H2O2 in presence of different concentrations of
GSPE enhanced the cell viability and activity together with the antioxidant mRNA expression
levels of the catalase, superoxide dismutase and glutathione peroxidase enzymes, and
improved the redox stage of the cells increasing TAC values [82].
The healthy effects of grape seeds on oxidative stress have been evaluated in humans and
experimental animals under normal conditions and under oxidative stress, generated in
clinical intervention trials and in animal models, for specific diseases. The bioavailability of
bioactive compounds of grape seed products has been evaluated in vivo through
pharmacokinetic studies analyzing and quantifying the related metabolites (glucorinated or
methylated) in plasma, at different times after oral administration. A rapid absorption of
different compounds of grape seed extracts was observed by different authors, detecting
maximal concentration of phenolic compounds in plasma between one and two hours after
administration of the extract [143-144]. Furthermore, it was observed that the repeated oral
administration (10 days) of a grape seed extract (300-400 nM) led to accumulations of
catechin, epicatechin and gallic acid, and also of their metabolites in blood, as well as of
catechin and epicatechin in the brain [145-146]. Other authors noted that grape seed extract
compounds were also detected in tissues such as heart and liver [144, 147].
One characteristic of the intake of the compounds with antioxidant capacity is that the
effect is dependent of the concentration [134] being possible pro-oxidant effects. In this
sense, it is also proper to consider that phenolic compounds act as antioxidants in the reduced
forms whiles phenoxyl radicals (oxidised forms) may exert cytotoxic and pro-oxidant activity
when the lifetime of the radicals is prolonged by effectors of spin-stabilization [148]. This
occurs also with other natural antioxidants like vitamin C, vitamin E and carotenoids or others
[149]. Different studies have showed that GSPE does not have toxicity effects unless in high
doses of ingestion (5g/kg) [150-154]; therefore, it is possible to assert that no significant pro-
oxidant activities have been described for these products.
Animal studies suggest that grape seed extracts have beneficial effects on different
diseases. In this sense, a protective effect of a defatted milled grape seed was observed in
hepatocytes of rat treated with the oxidant agent adryamicin. A supplemented diet with grape
seeds produced significant decrease of the high levels of ATP and GSH induced by the
adryamicin treatment, so the toxicity was restored to control value and protein and lipid
oxidation was also reduced [155]. Other authors did not find similar results [156], no
significant increase of TAC neither in plasma nor in the liver was observed, and the
antioxidant defense system (catalase, glutathione reductase, and superoxide dismutase) did
not show any remarkable change, with the exception of a notable increase in the glutathione
peroxidase activity. However, it is important to consider that in this study, rats were
supplemented with grapes not with seed grape products so the results are not directly
comparable.
The protective effect of GSPE on diet-induced hypercholesterolemic rats was described
by Thiruchenduran et al. [157]. In this study, the authors used cholesterol colic acid diet to
induce the increase of lipid peroxidation, cholesterol and triglycerides accumulation, the
decrease of the concentration of high density lipoproteins, and the induced alteration of the
activity of creatine kinase, in both serum and heart; all of them accompanied by a decrease of
non-enzymatic and enzymatic antioxidant defense systems in the heart, and with the increase
of the expression of cytochrome c and caspase-3. These negative changes were partially
restored to normal values in rats treated with grape seed proanthocyanidins. Furthermore, an
extract of grape seeds also showed protective effect on oxidative stress in streptozotocin-
induced diabetic rats, acting through the inhibition of lipid peroxidation, restoring endothelial
function and reducing the risk of cardiovascular disease [158].
Grape seed products can also ameliorate oxidative stress and prevent gastrointestinal
diseases before being absorbed. In this sense, a study, where the oxidative stress was
generated, at the level of the duodenum in an experimentation animal study, with cafeteria
diet, showed that the administration of 1g of GPSE/kg produced a significant decrease of the
ROS levels evaluated in the stomach, being the effect dose and time dependent [134]. Studies
in humans have reported that grape seeds possess a broad spectrum of healthy properties,
such as the capacity to prevent cardiovascular diseases [91, 158-159], and to reduce blood
pressure in individuals with hypertension or metabolic syndrome [160-161] and
inflammation-associated bone destruction [162], as well as the delay of Alzheimer
propagation [154]. All of these effects have been associated to their antioxidant properties.
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Chapter 6
MODULATION EFFECTS OF POLYPHENOLIC

EXTRACTS IN EXPERIMENTAL
ARTERIAL HYPERTENSION
M. Ciocoiu1, M. Badescu and L. Badescu2

1
Department of Pathophysiology
2
Department of Cell and Molecular Biology
University of Medicine and Pharmacy ―Gr. T. Popa‖ Iasi, Romania
ABSTRACT
Hypertension is a significant cardiovascular risk factor, associated to endothelial
dysfunction and oxidative stress. The proposed areas of study provide experimental basis
for a possible trial study in which we combine two different classes of substances: renin
inhibitors and natural polyphenol extracts. The aim of this study would be to identify if
this combination can have increased effectiveness in reducing blood pressure and
reducing the side effects of the major classes of antihypertensive agents used so far as
monotherapy. Polyphenolic extracts from Aronia melanocarpa and Sambucus nigra are
used in the following studies on an L-NAME induced experimental model of arterial
hypertension. HPLC/DAD coupled with ElectroSpray Ionization-Mass Spectrometry
allowed identification of five phenolic compounds in berries ethanolic extract:
chlorogenic acid, kuromanin, rutin, hyperoside and quercetin. The serous activity of
glutathione-peroxidase (GSH-Px) has significantly lower values in the hypertensive
(AHT) group as compared to the group protected by polyphenols (AHT+P). The total
antioxidant capacity (TAC) values are significantly higher in the AHT+P group
compared to AHT group. All the measured blood pressure components revealed a bio-
statistically significant blood pressure drop between the AHT group and the AHT+P
group. The results reveal the normalization of the reduced glutathion (GSH)
concentration, as well as a considerable reduction in the MDA serum concentration in the
AHT+P group. The polyphenols are extracted from isolated and purified vegetable
material represented by the mature fruit of the Aronia melanocarpa. The combination

Corresponding Author address: Magda Bădescu, University of Medicine and Pharmacy Iași, Universității St.16,
700115, Iași, Romania Email: magda.badescu@gmail.com.
126 M. Ciocoiu, M. Badescu and L. Badescu
between the Aliskiren and polyphenolic extract produce superior hypolipidemic and
antioxidant effects than in the case of separate administration within experimental AHT.
Polyphenolic extract from Aronia melanocarpa reduce systolic and diastolic arterial
tension values in rats with drug-induced hypertension, a phenomenon more pronounced
when polyphenols are associated with Aliskiren. The histological modifications at the
level of the aortic arch, identified in the hypertensive group, consist in the alteration of
the endothelium and the lysis of the intima, whereas in the group AHT+P, the Sambucus
nigra extract provided protection, ensuring the integrity of the vascular endothelium and
keeping within normal limits the elastic elements from the media, as well as the internal
elastic limiting membrane. The myocardial alterations in the hypertensive rats which
received Sambucus nigra extract are considerably diminished. The polyphenolic extract
has cardio-protective effects and could be used as a nutritional supplement in chronic
cardiovascular and metabolic diseases.
Keywords: polyphenols, oxidative stress, arterial hypertension
IMPORTANCE AND RELEVANCE OF THE SCIENTIFIC CONTENT

In the world, at least one in four adults is diagnosed with hypertension that is more than
one billion persons in the world, according to the AHA Association Estimates [1].
International guides for the management of arterial hypertension are improvable, and the
current tendency is to try to identify new therapeutic targets for the development of optimal
strategies of cardiovascular disease prevention in general and of arterial hypertension in
particular [2]. From epidemiological and dietary intervention studies, it appears, however,
that exogenous antioxidants at physiologic (nutritional) doses play an important role in the
maintenance or re-establishment of redox homeostasis, an essential state in maintaining
healthy biological systems [3, 4]. Polyphenols can also interact with phospholipid head
groups, particularly with those containing hydroxyl groups, so they can also decrease the
fluidity in the polar surface of phospholipid bilayer [5]. Their localization in the membrane
interior and their influence on fluidity of membrane components can hinder diffusion of free
radicals and thereby decrease the kinetics of free radical reactions [6].
Bioavailability of each and every polyphenol differs however there is no relation between
the quantity of polyphenols in food and their bioavailability in human body. A better
knowledge of some variables of polyphenol bioavailability, such as the kinetics of absorption
[7], accumulation and elimination, will facilitate the design of such studies. Importantly it is
the chemical structure of polyphenols and not its concentration that determines the rate and
extent of absorption and the nature of the metabolites circulating in the plasma [8]. To ensure
the safety of consuming dietary polyphenols and until sufficient data are collected to establish
a dietary reference intake for each class of compounds [9, 10], the development of sustainable
functional foods should only consider the same level of polyphenols as the best dietary source
can deliver at dietary doses [11].
The increase of oxidative stress, not related to blood pressure values, is accompanied by a
reduction in the most important antioxidant mechanisms and by the accumulation of ROS
byproducts, not only from lipid peroxidation but also from oxidized genomic and
mitochondrial DNA [12]. The impact of the released ROS products from the peripheral
mononuclear cells may contribute to the endothelial dysfunction, if antioxidant systems are
Modulation Effects of Polyphenolic Extracts … 127
not efficient in counterbalancing the effects. Antioxidant systems include certain antioxidant
enzymes [superoxide-dismutase (SOD), catalase (CAT), glutathion-peroxidase (GSH-Px)], as
well as components with an antioxidant action such as the vitamins E and C [13].
The results linked to the antioxidant capacity have determined us to use a new class of
natural compounds for the following studies, respectively polyphenolic extracts from
Sambucus nigra and Aronia melanocarpa in an arterial hypertension experimental model. The
chemical characterization of extracts/extractive fractions of Sambucus nigra and Aronia
melanocarpa will enable us to have insight into the components with high antioxidant
potential and to achieve phytopreparations as adjuvanttherapy or prevention therapy in
cardiovascular diseases or chronic metabolic diseases. Phytopreparations offer many
advantages:
 due to the fact that they have a complex composition, they develop qualitative effects
which are superior to the ones of the preparations which condition the synthesis and
semisynthesis substances;
 they do not generate secondary effects;
 they completely lack toxicity and they are cheap.
The flavonoids are one of the most important groups of natural polyphenols due to their
considerable genetic potential and their extremely wide spreading [14]. The main flavonoidic
structures are: catechina, flavones, flavonones, anthocyans and proantocyanidolic oligomers
[15]. The natural polyphenols were characterized by different chromatographic techniques
HPLC and techniques of spectral analysis as RMN spectroscopy, mass spectroscopy,
ionization-dessorbtion in the field – mass spectroscopy, UV-VIZ spectroscopy.
The activity at a vascular level, especially the capillary-protective activity, is the best
known and consequently the most used therapeutic property of flavonoids. The capillary-
protective flavonoids increase the resistance of the blood capillary walls and diminish
capillary permeability [16]. Some flavonoids (diosmina, hesperidina, proantocyanidolic
oligomers) also act at a venous level: they are venotonic, antiinflammatory and antiedematous
[17]. The therapeutic efficacy in venous actions is also explained by diminishing venous
distension and volume, as well as vasoprotective properties. A connection was established
between the spasmolitic action of flavonoids and their activity of phosphodiesterase‘s
inhibitors (spasmolysis on the isolated rat‘s aorta). At the level of the big circulation, the
anthocyans have a hypotensive and coronary-dilatating action [18].
The hypocholesterol/hypolipemia action. The flavonoids, especially the flavonic dimers,
diminish the degradation speed of collagen. The degradation of the collagen from the
capillary endothelium determines the fixation of cholesterol [19]. The fact that the flavonoids
stimulate the activity of the ascorbic acid has as an effect the decrease of the cholesterol‘s
seric level.
The anti-atherosclerotic activity of flavonoids is a consequence of several effects, among
which the most important ones are the following: hypocholesterol/hypolipemia action,
platelet aggregate action, antioxidant action, angioprotective action [20]. A connection was
established between the percent of inhibition belonging to LDL oxidation and the total
content of polyphenols expressed in galic acids equivalents. The flavonoids are capable of
saving vitamin E, a major catcher of free radicals, an endogenous antioxidant of LDL and it
inhibits the lipooxygenases [21].
The dynamics of the scientific research on international level in the field of arterial
hypertension (AHT) treatment and its complications can be seen in the creation of some
products with a wide spectrum of activity and variable secondary effects, which need to be
evaluated. These products aim to have a complete activity, as much as possible, both in their
main activity and in their pleiotropic effects.
The renin-angiotensin system is one of the most well -known parts of the APUD system,
having major roles in the hydroelectrolytic and cardiovascular regulation, namely in the
regulation of arterial tension. The final product of the renin-angiotensin system is angiotensin
II, a biologically active octapeptide, which is formed under the influence of the conversion
enzyme (a carboxypeptidase dipeptide) from angiotensin I. The circulating angiotensin II is
considered to be a true hormone of the hydroelectrolytic homeostasis, interfering in the
regulation of the water and electrolyte income and outcome [22]. Its effects are due to the
activity at the level of the vascular smooth muscles, including renal, as well as at the level of
the functional relationship of the renin-angiotensin system with corticoadrenal, adrenal
medulla, the central nervous structures, hypophysis and epiphysis.
The inhibition of this system by the currently available medication [23] is accomplished
by: decreasing the renin secretion (the beta-adrenergic blockers), the inhibition of the
conversion enzyme of the angiotensin (with captopril, enalapril), the antagonization of
angiotensin II at the level of its specific receptors (with losartan, candesartan, valsartan) [24].
The inhibitors of the conversion enzyme of angiotensin (IECA) form a group of
anthypertensives whose effect is mainly due to their stopping the angiotensin I in turning into
active angiotensin II, as a consequence of the enzyme inhibition which catalyses this
biochemical reaction. The angiotensin II is formed under the influence of the conversion
enzymes, a carboxypeptidase dipeptide, which takes out the last two amino acids – histidine
and leucine – from the components of angiotensin I.
IECA activates on the renin-angiotensin system, of endocrine type, represented by the
renin which is segregated by the kidney and by the plasmatic angiotensin, as well as on the
renin-angiotensin systems, local or tissular, of autocrine or paracrine type, from the level of
the myocard or other structures. As a consequence of the conversion enzyme inhibition, the
quantity of angiotensin II is decreased, further diminishing the phenomena for which it is
responsible – vasoconstriction in the arterial area (direct or secondary to the sympatic
stimulation), the increase in the secretion of aldosteron. Apart from the function of
vasoconstriction, there is also influenced their proliferative function, consequently improving
the structural changes produced by hypertension. The deficit of angiotensin II determines the
increase of renin secretion, by a retro-afferent mechanism, forming increased quantities of
angiotensin I. For the treatment in short periods of time, there is a good correlation between
the anti-hypertensive effect and the level of activity of the conversion enzyme in the plasma.
For the chronic treatment, the tension decrease is not rigorously correlated with the decrease
in the activity of the conversion enzyme or with the level of angiotensin II. In this situation, it
is possible that the excess of angiotensin I might surpass the inhibiting capacity of the
conversion enzyme.
The fear of the secondary effects of IECA, as well as the necessity to interfere on other
levels as well, determined the discovery and practical use of the receptor blockers for the type
I angiotensin (BRA) [25]. As a mechanism, the BRA effect is due to the blocking of the AT1
type angiotensin II receptors. Their activity stimulates the C phospholipase, activating the
secondary messenger inositol-triphosphate-diacylglycerol system and increasing the calcium
ions available, phenomena which are responsible for the stimulation of the vascular smooth
muscles by the angiotensin II. There is also caused an inhibition of the adenylaticyclase, with
the decrease of the AMP cyclical quantity. Thus, the stimulation of the AT1 receptors leads to
an enzymatic avalanche which will produce (depending on the local conditions)
vasoconstriction, the increase of atherogenity, inflammation increase and proliferation and/or
coagulation. These processes are crucial in different stages of the atherosclerosis and they are
also important in hypertension, left ventricular hypertrophy, chronic heart failure and atrial
fibrillation [26].
The antagonists of angiotensin, blocking mainly the AT1 receptors, unlike IECA,
focusses as well on angiotension II produced in an alternative way. The specific blocking of
the AT1 receptors no longer determine the increase in the level of vasoactive kinine, thus
avoiding the negative reactions given by IECA. Also, the blocking of the AT1 receptors
allows the renin and angiotensins to increase the stimulation on the AT2 receptors, which can
become therapeutically important. The medication used in the AHT treatment, IECA, BRA,
statine type has net effects on AHT and its complications, undertaking cardio-, reno- and
neuroprotective actions [27].
Since 2007 there has been accepted in Europe the introduction in therapy of a fourth
group of medication which reduces the blood pression by blocking the renin-angiotensin
system at its origin [28, 29]. We are talking about the renin inhibitors, which have Aliskiren
as prototype (Rasilez), considered to be the first in the group of oral inhibitors directly from
renin [2, 30]. If IECA and the blockers of the angiotensine receptors interfere at the end of the
enzymatic avalanche, the renin inhibitors interfere at the beginning of the molecular events
which lead to the release of angiotensin, which is involved in increasing the blood pressure
and the heart diseases. Aliskiren is specific to this enzyme and even renin is specific to this
reaction.
In the experimental studies which have been made, the effects obtained after the
administration of Aliskiren as a monotherapy or in combination with a diuretic [31], with a
blocker of the calcium channels, with an inhibitor of the conversion enzyme of angiotensin or
with a blocker of the angiotensin receptors [32, 33], have proved a better control on the blood
pressure comparative to the singular administration of these medicines.
The renin inhibitors could be an alternative of the conversion enzyme inhibitors of
angiotensin (ACEI) or the blockers of the angiotesin receptors (ARB), in treating
hypertension and it could also accomplish a multi-protection of the organs (heart, kidney,
brain) [34]. Since ACEI and ARB increase the level of plasma renin, Aliskiren combination
can be beneficial because it reduces plasma renin activity. It is now clear that the blockade of
the renin-angiotensin-aldosterone system not only reduces the blood pressure but also
decreases the risk of cardiovascular events and slows the progression of renal disease in
different types of patients [35]. Renin-angiotensin-aldosterone system blockade via direct
renin inhibition has the potential to provide organ protection independent of BP reductions.
The models of experimental arterial hypertensionrepresent one way of understanding the
cellular and molecular mechanisms modified throughout the cardiovascular disturbance and
of looking for means of preventing, slowing down and improving them.
THE ANTIOXIDANT, HYPOCOLESTEROLEMIANT AND HYPOTENSIVE

EFFECTS OF SAMBUCUS NIGRA EXTRACT
Polyphenols are organic compounds synthesized by plants, including tannins, lignans and
flavonoids. Isoflavones are flavonoid compounds with both antioxidant and estrogenic
properties, such as the soybean isoflavones genistein and daidzein which can behave as
estrogen mimics [36]. The effects of polyphenols, therapeutically relevant for the biological
systems, are: they reduce the scavenger properties for oxygen free radicals, they have the
capacity of interacting with the system, leading to the release of NO from the endothelium,
and they have an antiatherogenic effect [37].
They show high affinity for different structures and may therefore be able to decrease
oxidative damage mainly at such particular sites [38]. On the other hand, since polyphenols
are redox active compounds they may also cause increased radical formation if they uncouple
electron pathways in the body or if they chelate transition metals in such a way that they
become more reactive like in the experimental Fenton oxidation systems. More evidence for a
protective role of polyphenols against cardiovascular diseases arose from a number of clinical
trials [39], experiments on animal models and mechanistic studies [40]. Various
epidemiological studies have shown an inverse association between the consumption of
polyphenols or polyphenol-rich foods and the risk of cardiovascular diseases [41, 42].
Our research [43] emphasizes the effects of the polyphenolic extract from vegetable
material represented by the mature fruit of the Sambucus nigra on biochemical parameters
and blood pressure modifications. The experiment was performed on the arterial hypertension
model.
Dried berries of Sambucus nigra Michx. (Elderberry) (100 g) were chopped into small
pieces and extracted with 3 x 700 ml ethanol using a magnetic stirrer (FALC F30ST), each
time for 3 h. The combined extracts were taken to dryness by evaporation under reduced
pressure (BÜCHI R-210 rotavapor, BÜCHI V-850 vacuum controller, BÜCHI V-700 vacuum
pump). The total phenolic content was expressed as mg gallic acid equivalents/g extract.
Models of experimental hypertension are one of the ways to decipher the cellular and
molecular mechanisms of cardiovascular dysfunction and to search means to prevent, slow or
improve them [44]. The research was performed on Wistar white rats, with an average weight
of 250-280 g, which were divided into 4 groups of 12, namely:
 Group W -control, normal animals, that didn‘t receive natural polyphenols;

 Group AHT- animals which were administered L-NAME 40 mg/kg body/day, i p., at
every 2 days, for 8 weeks;
 Group P – animals that were administered polyphenols under the form of solution,
from the extract obtained from the Sambucus nigrafruit, with a dosage of 0.045 g/Kg
body, p.o., (by tube feeding), at every 2 days, for 8 weeks;
 Group AHT+P – animals which were administered polyphenols in the dosage
mentioned p.o., at every 2 days, concomitantly with L-NAME, for 8 weeks.
Reduced glutathione (GSH) was also determined by the Beutler method, through the use
of 5,5‘ ditio-bisnitro-benzoic acid (DTNB) and was expressed in g GSH/mg protein or g Hb
in erythrocyte. The malondialdehide(MDA) concentration– the index of lipid peroxidation –

was determined by the Ohkawa method using the tiobarbituric acid (TBARS) [45].
The exploration of the lipid profile included the measurement by photocolorimetry, in the
serum obtained after separation, of the concentration of total cholesterol (Ch-T), of
triglycerides (TG), of total lipids (LT), [the method with sulfovaniline], of high-density
lipoproteins (HDL) [46], of low-density lipoproteins (LDL) [according to the Friedewald
formula] for all the animals included in the experiment.
The rat blood pressure values were recorded using a CODATM system which uses a non-
invasive blood pressure measuring method. The actual experiment consists of performing at
least 6 blood pressure measurements in each laboratory animal, data collection by means of
the CODATM software and subsequent data processing.
Statistical data interpretation. All the data are shown as mean value ± standard error of
the mean (SEM). In order to assess the normal distribution of the groups, Shapiro-Wilk test
was performed. Additionally, Levene test was performed to confirm the homoscedasticity of
the groups, followed by ANOVA and paired or unpaired t-test to reveal the pairs of groups
that differ biostatistical significantly in term of means. Statistical data interpretation
considered the corresponding differences for a given significance threshold: P > 0.05
statistically insignificant; P < 0.05 statistically significant; P < 0.01 strong
statisticalsignificance; P < 0.001 very strong statistical significance.
Oxidative stress generates free radicals and oxidants that play a role in increasing lipid
peroxidation, as confirmed by the high levels of MDA, of serum lipids and fractions of
membrane lipids. MDA has been proposed as an indicator of lipid peroxidation because this
molecule is one of the end products of this oxidative process [47].
There are highly significant values (P < 0.01) for group P when compared with group W
and for group AHT +P when compared with group AHT and extremely significant values
(P < 0.001) for group AHT when compared with group W, as shown by the statistical
analysis of the MDA values (Table I).
Malondialdehide, the most abundant among the reactive aldehydes derived from lipid
peroxidation, was significantly increased in blood as well as in peripheral mononuclear cells.
These aldehydes have been implicated as causative agents in cytotoxic processes, and it is
reasonable to suppose that releases from cell membranes may diffuse, interact, and induce
oxidative modifications in other cells and in LDL molecules, thereby increasing the risk of
cardiovascular damage. The significant lipid peroxide diminution in the serum contained by
the AHT+P group compared to the P group is a result of a considerable reduction in the MDA
serum concentration.
Reduced levels of GSH have been related to an extensive number of metabolic and gene
expression disturbances, since the tripeptide is not only an efficient antioxidant but also an
important regulatory substance in biological systems. Whether the low GSH levels and
activity of the antioxidant enzymes is the cause or the consequence of the increased oxidative
status needs further evaluation, but the fact that the low activity included several systems
points to the reduction being more a consequence than a cause.
The tripeptide γ-glutamyl-cysteinylglycine or GSH is the major nonenzymatic regulator
of intracellular redox homeostasis, ubiquitously present in all cell types at millimolar
concentration. This cysteine-containing tripeptide exists either in reduced (GSH) or oxidized
(GSSG) form, better referred to as glutathione disulfide, and participates in redox reactions by
the reversible oxidation of its active thiol. Reactive oxygen species oxidized GSH to GSSG,
leading to a decrease in GSH and an increase in GSSG concentrations. Moreover, even

though the increment in ROS may upregulate the antioxidant enzymes under higher amounts
of pure oxygen or related species, consumption by ROS can overcome the increased
production, leading to the low activity observed.
Since polyphenols may modulate eNOS via O2.−mediated activation of src kinase [48], it
seems relevant to further investigate the source(s) and role(s) of O2.−−and other ROS in soy
isoflavone mediated activation of eNOS and antioxidant genes. Under conditions of oxidative
stress, upregulation of Hsp90 expression and increased intracellular Ca2+ will promote
turnover and proteosomal degradation of proteins such as calmodulin and eNOS [49] and
thereby affect NO bioavailability. The ability of dietary polyphenols to generate both NO and
ROS in endothelial cells and activate ARE/EpRE (Antioxidant response element/Electrophile
response element) mediated gene expression underlies their cardioprotective properties [50].
Dietary polyphenols may counteract oxidative stress in vascular and inflammatory
diseases by modulating key redox sensitive gene transcription via NF-κB [51] and Nrf2/ARE
[52] signaling pathways. The balance between antioxidant and pro-oxidant characteristics of
polyphenols have been attributed not only to their structural features, but also to the
concentration, suggesting induction of antioxidant defence metabolism by low concentrations
and ROS production at high concentrations [53]. Dietary polyphenols may offer an indirect
protection by activating endogenous defense systems and by modulating cellular signaling
processes such as NF-kB activation, glutathione biosynthesis, MAPK proteins, and PI3-
kinase/Akt pathway. The higher GSH levels in the heart of animals subjected to experimental
arterial hypertension is an adaptive reaction triggered by the activation of the non-enzymatic
antioxidant systems. GSH may be covalently bound to proteins through a process called
glutathionylation and acts as a coenzyme of numerous enzymes involved in cell defense. The
antioxidant capacity of the serum is significantly improved (P < 0.001) in the AHT+P rats, as
well as the GSH concentration being normalized (Table 1).
It should also be noted that the total cholesterol and triglycerides-lowering activity of
Sambucus nigraextracts was found in the case of rats fed with standard [54], non-
hypercholesterolemic diet supplemented with high doses of chokeberry anthocyanins for 4
weeks. When comparing total cholesterol and LDL-col levels, the results show that these are
significantly higher in the AHT group than in the W group. There are significant
improvements taking place against the dislipidemia occuring in arterial hypertention as a
result of the administration of polyphenols extracted from Sambucus nigra fruit. The serum
LDL levels in the AHT+P group were kept within normal limits by the polyphenolic
protection (Table 2). From the viewpoint of the variability coefficient (%), the mean values
obtained are typical of the series considered. Research comparing the AHT+P and AHT
groups shows that the HDL-chol is significantly higher in the first group.
The systolic and diastolic blood pressures, as well as their calculated mean, were
measured. The Shapiro-Wilk test was positive, which supports sample normality, and the
descriptive statistics and box-and-whisker plots are shown in Figure 1 and 2.
The Levene test confirmed group homoscedasticity, whereas the ANOVA test revealed a
significant difference between the means of the 4 groups, as concerns systolic and diastolic
blood pressure. All the measured blood pressure components revealed a bio-statistically
significant (P < 0.05) blood pressure drop between the AHT and the AHT+P groups.
Table 1. GSH and MDA modifications in the studied groups
W P AHT AHT + P
GSH (moli/mL) 7.42  0.19 7.86  0.28* 4.91  0.58*** 6.88  0.28##
MDA (nmol/ml) 0 0 8.73 x 10-2*** 6.92 x 10-2##
Values are mean ± SEM. Statistical analyses *- P < 0.05;**- P < 0.01;***- P < 0.001, vs. W group.
#
- P < 0.05; ## - P < 0.01;###- P < 0.001 vs. AHT group.
Table 2. Lipid profile in the studied groups
Groups W P AHT AHT+P

Ch-T (mg/dL) 73.41  4.56 66.22  1.72 95.3  6.74*** 70.42  3.77###
TG (mg/dL) 86.53  6.62 71.54  6.66* 144  15.38*** 95.77  22.85###
HDL-col (mg/dL) 34.21  4.36 33.16  3.62 21.74  4.88*** 28.27  3.42##
LDL-col (mg/dL) 23.41  5.32 20.88  2.79 42.83  5.41*** 27.12  7.36###
Values are mean ± SEM. Statistical analyses *- P < 0.05;**- P < 0.01;***- P < 0.001 vs. W group.
#
- P < 0.05; ## - P < 0.01;###- P < 0.001 vs. AHT group.
Figure 1. The box-and-whisker plot of systolic blood pressure.

Figure 2. The box-and-whisker plot of diastolic blood pressure.
In conclusion, in the arterial hypertensive model the cardio-protective effects of the

polyphenolic extract from Sambucus nigra are represented by the antioxidant,
hypocolesterolemiant intervention. The results prove that oxidative stress is considerably
lower, statistically speaking, in rats with hypertension but also provided with natural
polyphenolic protection from Sambucus nigra fruits than in the rats belonging to the control
group. In addition to the demonstrated antioxidant effects, natural polyphenols also have other
biological properties that might contribute to the cardioprotective effects.
MORPHOLOGICAL ASPECTS RELATED WITH

SAMBUCUS NIGRA EXTRACT ADMINISTRATION AT
THE MYOCARDIUM LEVEL IN ARTERIAL HYPERTENSION
This study uses usual (hematoxylin-eozyne) and special (PAS) stains. The sections were
examined at the optical microscope with a x10, 20 lens. However, the histological lesions are
localized in the myocardium (Figure 3) in the group that is not protected by the polyphenols
(group AHT). Thus, in the animals from group AHT, the myocardial fibers have an interstitial
edema that keeps them dissociated. In Figure 4 one may notice an artery like vessel dilated in
the interstice, associated with perivascular fibrosis in the same group. One may also notice an
interstitial edema and PAS positive deposits in the interstice in the myocardial fibers from the
group AHT, which is not protected with Sambucus nigra extract at the PAS stain (Figure 5).
Figure 3. Longitudinal section through the miocardial fiber, AHT group (HE, X20).
Figure 4. Cross section through the miocardial fiber, AHT group (HE, X10).
The histological modifications at the level of the aortic arch, identified in the
hypertensive group, consist in the alteration of the endothelium and the lysis of the intima,
whereas in the group AHT+P, the Sambucus nigra extract provided protection, ensuring the
integrity of the vascular endothelium and keeping within normal limits the elastic elements
from the media, as well as the internal elastic limiting membrane. In the group of
hypertensive rats which were protected by polyphenols, the myocardial modifications are
significantly more diminished when compared with the group AHT, the myocardial fiber with
nuclei suggesting the normalization of the histological aspect (Figure 6).
The ability of Sambucus nigra extract to provide antioxidant protection via inhibition of
LDL-oxidation and scavenging of free radicals makes it a potentially valuable tool in the
treatment of disease resulting from oxidative stress. The selective activation of those
metabolic pathways conducting to the synthesis of more stable or bioactive final products of
polyphenols will open a promising perspective for the design of functional foods or new
foods with health related claims.
Figure 5. Cross section through the miocardial fiber, AHT group (PAS, X20).
Figure 6. Cross section through myocardial fibre containing nuclei, AHT+P group (HE, X10).
In conclusion, the myocardial alterations in the hypertensive rats which received

polyphenolic protection are considerably diminished. The polyphenolic extract from
Sambucus nigra has cardio-protective effects and could be used as a nutritional supplement in
cardiovascular and metabolic diseases.
POLYPHENOLIC EXTRACT FROM ARONIA MELANOCARPA

FRUITS IN ARTERIAL HYPERTENSION
Dietary polyphenols are mostly derivatives and/or isomers of flavones, isoflavones,
flavonols, catechins, and phenolic acids. Aronia melanocarpa Elliot (Rosaceae, black
chokeberry) is a shrub native to North America [55]. Its berries, which are rich in
polyphenols, have been used by native Indians both as a remedy and as a food [56].
Anthocyanins (cyanidin glycosides), flavonoids (quercetin glycosides) [57], chlorogenic acids
and proanthocyanidins are the main polyphenols identified in Aronia berries [58]. Several
reports indicated that extracts from Aronia berries exhibited different biological effects both
in vitro and in vivo (antioxidant, gastroprotective, hepatoprotective, antiproliferative activities
via antioxidant pathways, but also via impacting signal transduction/intracellular signalling
cascades, impacting apoptosis, etc.) [59].
(a) detection at 280 nm
(b) detection at 515 nm
Figure 7. HPLC-DAD chromatograms of ethanolic extract of black chokeberry fruits (1 - chlorogenic

acid, 2 - kuromanin, 3 - rutin+unknown compound, 4 - hyperoside, 5 - quercetin).
Results of our study [60] focused on determining the content of Aronia melanocarpa
extract and also to estimate the influence of polyphenolic compounds contained in
chokeberries on oxidative stress, on an L-NAME induced experimental model of arterial
hypertension.
Preparation of Extract and Chemical Determinations
Chemicals. Folin-Ciocalteu‘s phenol reagent, chlorogenic acid and rutin trihydrate were
purchased from Merck (Darmstadt, Germany). Quercetin dihydrate, hyperoside and
kuromanin chloride were purchased from Carl Roth (Karlsruhe, Germany). Gallic acid,
caffeic acid and (+)-catechin hydrate were purchased from Sigma-Aldrich (Steinheim,
Germany). Except for HPLC grade solvents, all other solvents and reagents were of analytical
grade. Ultrapure water was obtained using a SG Water Ultra Clear TWF water purification
system (Barsbüttel, Germany).
Plant material. Ripe berries of Aronia melanocarpa Elliott (Rosaceae, black chokeberry)
were sampled in Botanical Garden, Iasi, Romania. The berries were shade-dried at room
temperature for one week. Extraction. Dried berries (100 g) were chopped into small pieces
and extracted with 3 x 700 mL ethanol using a magnetic stirrer (FALC F30ST), each time for
3 h. The combined extracts were taken to dryness by evaporation under reduced pressure
(BÜCHI R-210 rotavapor, BÜCHI V-850 vacuum controller, BÜCHI V-700 vacuum pump).
Total phenolic content. Total phenolics quantification was performed by Folin-Ciocalteu
method [61]. Briefly, berries extract was mixed with 3.16 mL of water and 0.2 mL of Folin-
Ciocalteu‘s phenol reagent. After 5 min., 0.6 mL of 20% sodium carbonate were added. The
absorbance was measured at 765 nm after 2 h of incubation at room temperature. A
calibration curve was plotted using gallic acid as standard. The total phenolic content was
expressed as mg gallic acid equivalents/g extract. Sample was assayed in triplicate and the
results were given as the mean ± SD.
Total anthocyanin content. Anthocyanins quantification was performed according to a
described procedure [62]. Berries extract was mixed with methanol - hydrochloric acid (99:1,
v:v) and kept at room temperature, in dark for 2 h followed by centrifugation (1000 xg, 15
min). Anthocyanin content in supernatant was measured both at 530 nm and 657 nm.
Absorbance values were converted into anthocyanin concentration using an extinction
coefficient of 31.6 M-1 cm-1. The results were expressed as µmole anthocyanin/g extract.
Sample was assayed in triplicate and the results were given as the mean ± SD.
HPLC/DAD/ESI-MS analysis(High-Performance Liquid Chromatography coupled with
Diode Array Detection and ElectroSpray Ionization-Mass Spectrometry) was conducted on an
Agilent 1200 Series HPLC system with a diode array detector coupled to an Agilent 6520
Accurate-Mass Q-TOF LC/MS system (Quadrupole Time-of Flight Liquid
Chromatography/Mass Spectrometry) equipped with an ESI source. Separations were done on
a Zorbax Eclipse XDB-C18 column (150 x 4.6 mm, i.d. 5 µm). The mobile phase consisted of
(A) water and acetic acid (99:1, v/v) and (B) acetonitrile and acetic acid (99:1, v/v). The
compounds were monitored at 254, 280 and 320 nm; anthocyanins were monitored at 515
nm. Mass spectrometric detection was performed in the negative ion mode for non-
anthocyanin polyphenols and in the positive mode for anthocyanins. The mass spectrometric
conditions for negative and positive ion mode were: drying gas (N2) flow rate 7.0 L/min;
drying gas temperature 220ºC; nebuliser pressure 15 psig. In negative ion mode the capillary
voltage was set to −4.2 kV and the skimmer voltage to -60 V. In positive ion mode the
capillary voltage was set to 4.2 kV and the skimmer voltage to 60 V. A fragmentor voltage of
200 V was used in both modes. The full-scan mass spectra of the investigated compounds
were acquired in the range m/z 50–2000 [63]. Data were collected and processed using a
MassHunter Workstation software.
Extraction. Ethanol extraction of black chokeberry fruits yielded 47.17 g extract. The
extract was stored at -20ºC until used. Phenolic contents. Berries ethanolic extract contained
24.87 ± 0.54 mg total phenolics/g and 4.46 ± 0.06 µmole anthocyanin/g. HPLC/DAD/ESI-MS
analysis. In our study, the phenolic profile of berries ethanolic extract was characterized by
HPLC/DAD/ESI-MS (Figure 7).
HPLC/DAD/ESI-MS allowed identification of chlorogenic acids, quercetin and cyanidin
glycosides, proanthocyanidins as major polyphenols in black chokeberry fruits [64]. (+)-
Catechin hydrate, chlorogenic acid, caffeic acid, rutin trihydrate, hyperoside, quercetin
dihydrate and kuromanin chloride were used as standards. Main phenolic constituents were
identified by comparison of their retention times and mass spectral data to those of authentic
standards. Five phenolic compounds have been detected in berries ethanolic extract:
chlorogenic acid, kuromanin, rutin, hyperoside and quercetin; their retention times and mass
spectral data are given in Table 3.
Table 3. Retention time and mass spectral data of polyphenolic compounds detected in
ethanolic extract of black chokeberry fruits
Non-anthocyanin polyphenols
Peak Rt Mass spectral data Peak assignment
No. (min) Deprotonated Fragment ions (m/z)
molecule [M-H]- (m/z)
1. 32.9 352.90 190.93 chlorogenic acid (5-O-
[M-H-Caffeoyl] caffeoyl quinic acid)
3. 61.9 608.87 - rutin* (quercetin-3-O-
glucorhamnoside)
4. 63.2 462.86 - Hyperoside (quercetin-3-
O-galactoside)
5. 87.9 300.86 - quercetin
Anthocyanins
Peak Rt Mass spectral data Peak assignment
No. (min) Molecular ion Fragment ions
[M]+ (m/z) (m/z)
2. 39.3 449.21 287.13 Kuromanin
[M-Glucose] (cyanidin-3-O-glucoside)
* rutin co-eluted with a compound (m/z 462.86), possible another quercetin glycoside.
Rt, retention time.
THE EFFECTS OF ARONIA MELANOCARPA

POLYPHENOLIC EXTRACT ON OXIDATIVE STRESS
The experiment used Aronia melanocarpaactive therapeutic doses, well-determined
fractions of DL50 on an experimental model of arterial hypertension [60]. Fractions of DL50
are doses representing 1/5, 1/10, 1/20, 1/40 of DL50. The dose representing 1/20 of DL50
was chosen, as it is the smallest dose that determined the pharmacodynamic effect that is
being researched, without producing significant toxic effects.
The research was performed on Wistar white rats, with an average weight of 250-280 g,
which were divided into 4 groups of 12, namely:
 Group W – control, normal animals, that didn‘t receive natural polyphenols;

 Group AHT–animals which were administered L-NAME 40 mg/kg body/day, i.p., at
every 2 days, for 8 weeks;
 Group P – animals that were administered polyphenols under the form of solution,
from the extract obtained from the Aronia melanocarpa fruit 0.050 g/Kg body every
two days, at every 2 days, for 8 weeks;
 Group AHT+P – animals which were administered polyphenols in the dosage
mentioned p.o. at every 2 days, concomitantly with L-NAME, for 8 weeks.
The blood samples necessary to the biochemical determinations were drawn from the
retrorbital venous sinus. The malondialdehyde (MDA) concentration– the index of lipid
peroxidation – was determined by the Ohkawa method using the tiobarbituric acid [45]. The
MDA concentration was expressed in nmol/mL. Glutathione peroxidase (GSH-Px) (H2O2:
GSH oxidoreductase) was determined by the Gross and Beutler method; the GSH-Px activity
was expressed in µM oxidized GSH per minute/g Hb or mg protein. Reduced glutathione
(GSH) was also determined by the Beutler method, through the use of 5,5‘ ditio-bisnitro-
benzoic acid (DTNB) and was expressed in g GSH/mg protein or g Hb in erythrocyte.
For the extracellular response the total antioxidant capacity (TAC) was determined by using
a RANDOX kit for manual use by Randox Laboratories Ltd. The major advantage of this test
is to measure the antioxidant capacity of all antioxidants in a biological sample and not just
the antioxidant capacity of a single compound. The method is based on formation of the
ABTS•+ cation [2,2‘-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] and its scavenging by
antioxidant sample constituents (e.g., serum or food) measured by spectrophotometry (decay
of green/blue chromophore absorbance is inversely associated with antioxidant sample
content and the control antioxidant is Trolox, a hydrophilic vitamin E analog).
The GSH-Px serum activity in hypertensive rats (AHT) has significantly low values (P <
0.001) when compared to the rats in groups W and AHT+P, which is a consequence of
oxidative stress increase (Table 4). As a consequence of oxidative stress increase in
hypertensive rats (AHT), the reduced glutathione (GSH) values are significantly low (P <
0.001) as compared to the rats in the W and AHT+P groups. The group of hypertensive rats
protected by the administration of polyphenolic extract showed significantly lower (P < 0.01)
values of GSH serum concentration compared to the hypertensive group. The results achieved
reveal a significant serum antioxidant capacity improvement (P < 0.001) in the AHT+P rats,
the normalization of the GSH concentration, as well as a considerable reduction in the MDA
serum concentration, causing a significant lipid peroxide diminution in the serum. Depending
on the significance threshold values (P), the statistical analysis of the MDA values reveals
significant differences (P < 0.01) between the AHT+P and the AHT groups, respectively, and
highly significant differences (P < 0.001) between the AHT and the W groups.
The TAC levels were significantly decreased in AHT group (P < 0.001) as compared to
the rats in the W and AHT+P groups. As expected, severe oxidative stress disturbs the
antioxidant balance by generating reactive species and decreasing the total antioxidant
capacity in the extracellular space. There are similar TAC values in group W and group P and
the differences between the AHT and AHT+P groups are statistically significant (P < 0.01).
Hypertension is a significant cardiovascular risk factor, associated to endothelial
dysfunction and oxidative stress. The oxidative process participates in increasing systemic
arterial pressure, reducing NO availability and vasodilation [65]. Oxidative stress is involved
in remodelling of myocardial microvascular architecture and subsequent development of left
ventricular hypertrophy [66]. Assessment of antioxidant activities and lipid peroxidation
byproducts in hypertensive subjects indicates an excessive amount of ROS and a reduction of
antioxidant mechanism activity in both blood as well as in several other cellular systems,
including not only vascular wall cells but also those found in circulating blood [67].
Polyphenols act as free radicals scavengers by donating hydrogen atoms or electrons
from phenolic hydroxyls. This is the main mechanism by which polyphenols scavenge many
ROS (superoxide anion radical, hydroxyl radical). During arterial hypertension, due to the high
oxygen consumption, the reactive oxygen species acting chiefly on unsaturated lipids, belonging
to the membrane, with the formation of certain peroxidation products, which generate MDA.
Since the most reactive radicals are short-lived they might be expected to react close to the
site where they are formed. Polyphenols vary strongly in their absorption and distribution.
They show high affinity for different structures and may therefore be able to decrease
oxidative damage mainly at such particular sites.
A clear idea about the antioxidant potentials and the dependence of antioxidant activities
on the quality and the quantity of phenolic substances can be obtained by comparison of the
antioxidant activities of different phenolic extracts at equal total phenol concentrations.
Recent studies have revealed a total phenolic content of 20-90 mg/kg fresh wt (or mg/L) in
strawberry, of 20-40 mg/kg fresh wt (or mg/L) in apple and of 15-40 in black grape [68]. The
results of this study show that the total phenolic content in the black chokeberry extract is
similar to that found in fresh strawberries, apples and black grapes.
In normal conditions, the intrinsic antioxidant systems counteract the effects of oxidative
stress. Therefore, the polyphenolic extract used contributes to actively maintain the effects of
these systems (this can be construed as an explanation to why the MDA values in the animals
from P group are less lowered than the ones in AHT+P group). Free radicals scavenging
activity and metal chelation partially explain polyphenols inhibitory effects on lipid
peroxidation and LDL oxidation [69]. In addition, some polyphenols increase the activity of
several endogenous antioxidant defense systems (GSH-Px, SOD, CAT) and induce a
significant increase in GSH level [70].
Table 4. GSH- Px, GSH and TAC modifications in the studied groups
W P AHT AHT+P
MDA(nmol/mL) 0 0 8.76 x 10-2*** 6.43 x 10-2##
GSH-Px (moli 2.53  0.19 2.37  0.49* 1.17  0.20*** 1.56  0.21##
GSSG/min/mL)
GSH(moli/mL) 7.29  0.21 7.53  0.40* 5.10  0.49*** 6.710.35##
TAC (mmol/L) 1.55  0.29 1.58  0.31 1.31  0.16** 1.53  0.27##
Values are mean ± SEM. (n = 12 animals). Statistical analyses:
*- P < 0.05;**- P < 0.01;***- P < 0.001, vs. W group.
#
- P < 0.05;## - P < 0.01;###- P < 0.001 vs. AHT group.
Phenolic compounds detected in ethanolic extract of black chokeberry fruits have shown
antioxidant effects [71]. Chlorogenic acid, quercetin and cyanidin are effective radical
scavengers and iron chelators; glycosylated derivatives of quercetin and cyanidin have a
lower scavenging activity in comparison to quercetin and cyanidin, respectively [72].
However, it may still be true that specific antioxidants are preventive, and since polyphenols
are both potent antioxidants and abundant in plant foods they are likely candidates for
fullfilment of the antioxidant hypothesis.
The current study revealed an important reduction in the antioxidant mechanisms, both in
GSH levels and antioxidant enzymatic activities in the hypertensive group. Reduced levels of
GSH have been related to an extensive number of metabolic and gene expression
disturbances, since the tripeptide is not only an efficient antioxidant but also an important
regulatory substance in biological systems. The low activity of GSH is a consequence and not
a cause of the increase in the oxidative status. Reactive oxygen species oxidized GSH to
GSSG, leading to a decrease in GSH and an increase in GSSG concentrations. Long-time
oxidative stress can consume antioxidants, and reduce SOD, CAT and GSH-Px levels in
cardiovascular diseases in general, and especially in arterial hypertension.
A wide range of evidence suggests that the Keap1 (Kelch ECH associating protein) -
Nrf2 (Nuclear factor erythroid 2-related factor) complex constitutes a sensor of oxidative
stress involved in triggering antioxidant response element (ARE) mediated gene expression to
restore the cellular redox status [73]. Under basal conditions, Nrf2 interacts with a cytosolic
repressor protein Keap1 limiting Nrf2 mediated gene expression [74]. In cells exposed to
oxidative stress, Nrf2 is released from Keap1 and translocates to the nucleus, where it
activates ARE dependent transcription of phase II and antioxidant defense enzymes, such as
NAD(P)H:quinone oxidoreductase, glutathione-S transferase, glutathione peroxidase and
heme oxygenase-1 [75]. Polyphenols may modify the capability of Keap1 in sequestering
Nrf2 and/or activate MAPK proteins (ERK, JNK and p38), probably involved in Nrf2
stabilization [76]. The role of antioxidant nutrients in fighting against oxidative stress is well-
established in several diseases including cardiovascular and neurological pathologies [77]. In
this sense, researchers had materially proved that following consumption of diets rich in fruits
and vegetables there was an increase in serum TAC [78]. The decline of MDA levels may be
caused by increased antioxidant status.
The TAC levels were significantly decreased in the hypertensive group. In this
experimental model, all groups were put on the same diet. The reduction in antioxidant
mechanisms can be neutralized through natural polyphenol compounds of Aronia
melanocarpa, which will maintain TAC to levels capable of neutralizing ROS effects. The
decay in TAC, as significant as it was, was not as severe as the changes observed in
intracellular enzyme activity. The TAC evaluation, used with other oxidative stress and
antioxidant defense biomarkers, constitutes the first step in search for a healthy body status.
In order to form strategies for the intervention and prevention of cardiovascular diseases, an
understanding of the basic molecular mechanisms by prophylactic agents is required.
The study demonstrated that an ethanolic extract from fruits of Aronia melanocarpa
Elliott is able to reduce endothelial dysfunction and improve total antioxidant capacity in
early arterial hypertension. Evidence shows that polyphenols can increase the antioxidative
capacity of plasma and that this effect is directly related to the plasma concentration and the
intrinsic antioxidant capacity of the compounds. Evidence that the intake of catechins and
related procyanidins are linked to the increased antioxidant capacity of plasma is necessary in
order to assess more acurately whether there is a relationship between polyphenol intakes and
health risks mediated by antioxidation.
THE EFFECTS OF ARONIA MELANOCARPA POLYPHENOLIC EXTRACT

ON SYSTOLIC AND DIASTOLIC ARTERIAL PRESSURE
The rat blood pressure values were recorded using a CODATM Non-invasive Blood
Pressure System which uses a non-invasive blood pressure measuring method. The
experiment consists in performing at least 6 blood pressure measurements in each laboratory
animal, data collection by means of the CODATM.
that differ biostatistical significantly in term of means.
The systolic and diastolic blood pressures were measured in AHT group, and AHT+P
group, respectively. The Shapiro-Wilk test was positive, which supports sample normality,
and the descriptive statistics and box-and-whisker plots are shown in Figure 8.
The blood pressure decrease effect of polyphenols could be due to particular actions, non-
mediated by estrogenic receptors, of nitric oxide or superoxide anion bio-availability
modulation by polyphenols [79, 80]. The significant blood pressure value drop in the
hypertension group protected by polyphenols could be related to their ability to decrease
in vivo reactive oxygen species production. In our study, polyphenols intake is associated
with blood pressure decrease, not through the lowering of heart rate, but through the
antioxidant mechanism especially.
A good way to raise the intake of antioxidants from Aronia melanocarpa fruits is to
increase the proportion of consumption, and another effective way is to substitute the fruit
and vegetables that have low antioxidant capacity with antioxidant-rich extract from Aronia
fruits. In addition, a colorful variety of all fruit and vegetables, healthfully prepared, makes a
significant contribution to a diet that promotes good health.
There are potential clinical benefits in using the polyphenolic extract coupled with the
antihypertensive drugs with therapeutic purposes. This would lead to using a smaller dose of
antihypertensive drugs and thus diminishing the secondary effects they produce.
In conclusion, ethanolic extract of black chokeberry fruits has a potential value as
prophylactic agent, but also may function as a nutritional supplement in the therapy of arterial
hypertension. The role of the polyphenolic extract of Aronia melanocarpa is to prevent the
total antioxidant capacity decrease and also to reduce the oxidative stress.
(a) The box-and-whisker plot of systolic blood pressure
(b) The box-and-whisker plot of diastolic blood pressure
Figure. 8. Systolic and diastolic blood pressure at studied groups.
POLYPHENOLIC EXTRACT ASSOCIATION WITH RENIN INHIBITORS

IN EXPERIMENTAL ARTERIAL HYPERTENSION
Polyphenols and other food phenolics are the subject of increasing scientific interest
because of their possible beneficial effects on human health. The fruit of the Aronia
melanocarpa, a shrub of the rosaceous family, has a dark purple peel and contains high levels
of flavonoids and phenolic acids. Main flavonoid subgroups in chokeberry are
proanthocyanins, anthocyanins, flavonols and catechins. Aronia melanocarpa extract may

involve inhibition of nitric oxide, prostaglandin (E2), and tumor necrosis factor-α production,
resulting from suppressed expression of inducible nitric oxide synthase and cyclooxygenase-2
enzymes [58].
More evidence for a protective role of polyphenols against cardiovascular diseases arose
from a number of clinical trials [39], experiments on animal models and mechanistic studies
[40]. Various epidemiological studies have shown an inverse association between the
consumption of polyphenols or polyphenol-rich foods and the risk of cardiovascular diseases
[81, 82]. Since polyphenols are redox active compounds they may also cause increased
radical formation if they uncouple electron pathways in the body or if they chelate transition
metals in such a way that they become more reactive like in the experimental Fenton
oxidation systems [83, 84]. A substantial body of epidemiological and experimental evidence
suggests the significance of serum uric acid as an important and independent risk factor of
cardiovascular [85, 86, 87] and renal diseases especially in patients with hypertension [88,
89].
Our study [90] focused on determining the influence of the association between the renin
inhibitor and the polyphenolic extract on biochemical parameters and systolic and diastolic
blood pressure. The research was performed on Wistar white rats, with an average weight of
250-280 g, which were divided into 6 groups of 12, namely: - Group W -control, normal
animals, that didn‘t receive natural polyphenols; - Group AHT- animals which were
administered L-NAME 40 mg/kg body/day, i.p., at every 2 days, for 8 weeks; - Group PA –
animals that were administered polyphenols under the form of solution, from the extract
obtained from the Aronia melanocarpafruit, with a dosage of 0.045 g/Kg body, p.o. (by tube
feeding), at every 2 days, for 8 weeks; - Group AHT+PA – animals which were administered
polyphenols in the dosage mentioned p.o. at every 2 days, concomitantly with L-NAME, for
8 weeks; Group AHT+Alisk - animals with AHT induced by L-NAME, that were given
Aliskiren 30 mg/kg body/day, administered s.c. for 8 weeks; Group AHT+Alisk+PA -
animals with AHT that were given Aliskiren and polyphenols in the above mentioned dosage,
for 8 weeks. Between experiments, the rats were kept in individual cages and were given food
and water. A 12 hours light/12 hour darkness cycles was employed.
Ceruloplasmin determination was performed on an EOS BRAVO automatic analyzer.
Uric acid determination was carried out by means of Siedel‘s modified colorimetric method.
The exploration of the lipid profile included the measurement by photocolorimetry, in the
serum obtained after separation, of the concentration of total cholesterol (Ch-T), of total lipids
(LT), [the method with sulfovaniline], of high-density lipoproteins (HDL) (46), of low-
density lipoproteins (LDL) for all the animals included in the experiment.
The rat blood pressure values were recorded using a CODATM system. The actual
experiment consists of performing at least 6 blood pressure measurements in each laboratory
animal, data collection by means of the CODATM software and subsequent data processing.
that differ biostatistical significantly in term of means.
Figure 9. Correlation of the cerulo-plasmin individual values with the cardiac frequency (FC) and
HDL-col.
Figure 10. The box-and-whisker plot of uric acid.

The lowest average values of the ceruloplasmin have been recorded in the AHT group.
The highest average values of the ceruloplasmin have been recorded in the witness group and
in the groups treated with Aronia melanocarpa. Note that all the groups present higher
average values of the ceruloplasmin compared to the hypertensive group, which suggests the
antiradical beneficial effect of the ceruloplasmin. The most efficient administration proved to
be Aronia melanocarpa, which determines average values of the ceruloplasmin close to those
recorded in witness group (Figure 9). The ceruloplasmin and uric acid were determined as
nonspecific oxidative stress markers. Statistically speaking, the uric acid values remain
significantly high both in the AHT group, and in the AHT+PA group in comparison with
control group (Figure 10).
It should also be noted that the total cholesterol and triglycerides-lowering activity of
Aronia melanocarpa extracts was found in the case of rats fed with standard, non-
hypercholesterolemic diet supplemented with high doses of chokeberry anthocyanins for 4
weeks [18].
When comparing total cholesterol and LDL-col levels, the results show that these are
significantly higher in the AHT group than in the W group. There are significant
improvements taking place against the dislipidemia ocurring in arterial hypertention as a
result of the administration of polyphenols extracted from Aronia melanocarpa fruit. The
serum LDL-col levels in the AHT+PA group were kept within normal limits by the
polyphenolic protection. The highest average values of the total cholesterol have been
recorded in the AHT group, significantly higher than in the other investigated groups. The
lowest average values of the total cholesterol have been recorded in the AHT+Alisk+PA
group, significantly lower than those recorded in AHT and W groups. From the viewpoint of
the variability coefficient (%), the mean values obtained are typical of the series considered
(Figure 11).
Research comparing the AHT+PA, AHT+Alisk+PA and AHT groups shows that the
HDL-col is significantly higher in the first and second group (Figure 12). The individual
values of the total cholesterol have presented direct correlations with the systolic blood
pressure, moderated as intensity, for the PA (r= +0,58) and AHT + PA (r= +0,76) groups.
In a combined therapy, chokeberry extracts were given as supplements with the diet of
patients after myocardial infarction, as an addition to the statin treatment. Compared to the
control group, treated only with statins, patients receiving additional Aronia extract for 6
weeks had significantly lower LDL-col oxidation status as well as reduced levels of serum 8-
isoprostanes and increased adiponectin levels, which indicate diminished oxidative stress and
reduced endothelial inflammation [91].
The positive anti free radical effect of polyphenols has been largely proven by the high
SOD values in the treated groups. It is noteworthy thatthe mean SOD values of the
hypertension groups which received dietary supplement such as Aroniamelanocarpa and/or
Aliskiren were close to the values recorded in the control group. The SOD values of the
hypertension group were lower and associated with high cholesterol, triglyceride and LDL-
cholesterol values, as well as with low HDL-cholesterol levels. The low SOD values of the
hypertension group were also associated with low ceruloplasmin values and high uric acid
values.
The lowest mean GSH values were recorded in the hypertension group, and they were
significantly lower than the ones recorded in the other groups under survey. One should note
that the mean GSH values of the groups which received dietary supplement such as
Aroniamelanocarpa and/or Aliskiren were close to the ones of the control group.
Figure 11. The box-and-whisker plot of total cholesterol.
Figure 12. The box-and-whisker plot of HDL-cholesterol.

Figure 13. The box-and-whisker plot of systolic blood pressure.
Figure 14. The box-and-whisker plot of diastolic blood pressure.
The systolic and diastolic blood pressures, as well as their calculated mean, were
measured. The Shapiro-Wilk test was positive, which supports sample normality, and the
descriptive statistics and box-and-whisker plots are shown in Figure 13 and 14.
Polyphenolic extract from Aronia melanocarpa reduce systolic and diastolic arterial
tension values in rats with drug-induced hypertension, a phenomenon more pronounced when
polyphenols are associated with Aliskiren. Associating a renin inhibitor with natural
polyphenols might be a therapeutic modality that reduces the side effects of renin inhibitors in
the long-term treatment of essential AHT and would lead to usinga smaller dose of
antihypertensive drugs.
In conclusion, the arterial hypertensive model the cardio-protective effects of the
polyphenolic extract from Aronia melanocarpa are represented by the antioxidant,
hypocolesterolemiant intervention. The combination between the Aliskiren and polyphenolic
extract produce superior hypolipidemic and antioxidant effects than in the case of separate
administration within experimental arterial hypertension induced in the murine model.
The pleiotropic effects of polyphenols are able to offer a better understanding in
approaching personalized therapies in the future. The knowledge and understanding of the
mechanisms involved in the production of cardiovascular disease is a starting point in
primary, secondary and tertiary prevention with a high prevalence of these diseases.
Prudently transfering to humans the results obtained on animals, the conclusions of
our biomedical research will have a special impact on the individuals ‘ state of health –
an essential condition for the social and economic parameters of their integration.
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194, e179-e184.
Chapter 7
THE USE OF GRAPE SEED EXTRACT ON

MEAT PRODUCTS: A REVIEW
Daniel Franco1,*, Javier Carballo2 and José M. Lorenzo1

1
Centro Tecnológico de la Carne de Galicia,
San Cibrao das Viñas, Ourense, Spain
2
Area de Tecnologia de los Alimentos,
Facultad de Ciencias de Ourense, Universidad de Vigo,
Vigo, Spain
ABSTRACT
Oxidative processes in meat and meat products during storage lead to the
degradation of color pigments, lipids and proteins that, in turn, can contribute to the
deterioration in flavour, texture, color and nutritional value of the meat. For fresh meat,
microbial growth, lipid oxidation and color are important factors to shelf-life and
consequently for consumer acceptance. Antioxidants are used to minimize the oxidative
changes in meat and meat products. Many synthetic antioxidants such as butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone
(TBHQ), and propyl gallate (PG) are typically used to protect foods from spoilage,
although their use is restricted due to possible carcinogenic effects. Therefore, there has
been increasing interest in alternative additives from natural sources, which has gradually
provided impetus to eliminating synthetic preservatives in food. In response to recent
demand for natural products and consumers' willingness to pay significant premiums for
natural foods, the meat industry is actively seeking natural solutions to minimize
oxidative rancidity and increase the shelf-life of products. Grape seed is a waste by-
product of the food industry and due to its high phenolic compound content grape seed
extract (GSE), it provides a good alternative to conventional antioxidants. The present
chapter reviews how in meat products (raw, cooked and dry-cured) the use of GSE
decrease the amount of primary and secondary lipid oxidation products, reducing the
rancid flavor development and therefore increasing shelf life and quality.
Keywords: grape seeds, meat products, lipid oxidation, color
*
E-mail: danielfranco@ceteca.net.
158 Daniel Franco, Javier Carballo and José M. Lorenzo
1. INTRODUCTION: ANTIOXIDANTS AND MEAT SHELF LIFE

Meat is composed mainly of water, proteins, lipids, minerals and a small amount of
carbohydrates. Due to their rich nutritional composition, meat and meat products are prone to
quality deterioration caused by chemical and microbial changes. The most common forms of
chemical degradation are the oxidation of lipids and proteins. In fact, oxidation of these
fractions has been demonstrated as the main, non-microbial cause of quality deterioration,
especially during processing, and currently is one of the biggest economic problems in the
meat industry. It compromises the nutritional quality, limits shelf-life, increases toxicity and
decreases the market value of meat and meat products [1].
Lipid oxidation is a complex mechanism that depends on several factors such as chemical
composition of meat, light exposition, oxygen presence, and storage temperature [2].
Oxidation of lipids is a radical chain reaction consisting of three steps (initiation, propagation,
and termination) with the production of free radicals (Figure 1).
Protein oxidation is less studied, in fact it is one of the most innovative issues in meat
quality evaluation currently in special due to advancement of proteomic techniques.
According to Shacter [3] and Lund et al. [4] protein oxidation is described as the covalent
modification of a protein induced by reactive oxygen substances to form a protein which is
consequently converted into a radical in the presence of oxygen. Similarly to lipid oxidation
in animal tissues, protein oxidation occurs through a chain reaction of free radicals [4].
Figure 1. Scheme of chemical reactions of lipid oxidation in meat products.

The Use of Grape Seed Extract on Meat Products 159
In addition both oxidation mechanisms are closely connected, because protein oxidation
also occurs due to the interaction between proteins, especially in nitrogen or sulphur positions
of reactive amino acid residues, and lipid hydroperoxides or secondary lipid oxidation
products, such as aldehydes or reducing sugars [5]. At the end of oxidation processes, several
compounds which have negative effects on the sensory characteristics of meat products
(colour, texture and flavour) and on nutritional or safety aspects [6] (potentially toxic
compounds such as fatty acid peroxides, cholesterol hydroperoxide, and peroxy radicals) are
produced [7]. Free oxygenated radicals are particularly involved in these oxidative processes
and lipid oxidation, particularly of polyunsaturated fatty acids, (PUFA) is one of the primary
causes for deterioration of flavour. As indicated by Arabshahi et al. [8] oxidation in
polyunsaturated fatty acids leads to secondary oxidative compounds such as aldehydes
(hexanal, pentanal, heptanal and octanal) that are responsible not only of quality deterioration,
but represent health risks, including carcinogenesis. The most widely used methods for
determination of primary products of oxidation (hydroperoxides and conjugated dienes) and
secondary products as aldehydes and ketones are peroxide and TBARs values or index, so
values referred to these methods appear on this chapter mainly.
Both types of oxidation can be diminished, using antioxidants in meat products and thus
improving the product quality and shelf-life. Antioxidants can reduce oxidation of lipids and
proteins oxidation employing different strategies such as: scavenging initial radicals, breaking
chain reactions, decomposing peroxides, decreasing oxygen levels and binding by chelation
of initiating catalysts, such as metal ions [9]. The use of antioxidants in food products is
controlled by regulatory laws of each country or by international standards from organizations
such as FDA, FAO/WHO, UE or Codex Alimentarius [10].
Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) have been
widely used as antioxidants in meat and poultry products [11, 12], but these compounds have
been questioned due to potential toxicological and carcinogenic effects [13, 14] and thus
counteract any beneficial effect for the consumer [15]. Their use is questionable because they
are suspected to act as tumour promoters [16]. In this situation, current research tends for
their replacement by natural antioxidants obtained from natural sources to be applied in the
food area [14]. In this sense there are an important number of raw materials (plants, herbs,
fruits) that have been reported to possess compounds having antioxidant activity (see review
of Shah et al. [17]) and whose antioxidant effect has been studied in different meat products.
Overall, this fact has led the meat industry to seek inexpensive and effective natural
antioxidants that might replace synthetic antioxidants without adversely effect on quality of
meat products and consumer perceptions [10].
The shelf life of meat comprises the time that it remains in the exhibitor of sale until its
rejection for the consumer or withdrawn by expiry date, both facts not always being
coincident. As we commented above, the oxidative damage is the most important factor in the
meat deterioration, together with the microbial spoilage. The increase of rancidity and loss of
color is produced by fat and myoglobin oxidation, respectively. The duration of shelf life is
also conditioned by the protein oxidation that provokes conformational changes and
fragmentation of peptide chains, liberating amino acids, which can produce unpleasant smells
and flavours [18]. The use of antioxidants allows increasing preservation time, so increasing
the possibilities of sale and decreasing economic losses. Nowadays, meat industry needs to
incorporate new solutions in order to be more competitive, since current manufacturing
processes are high-scale voluminous and points of sale are increasingly far. Overall, meat
shelf-life can be extended with the addition of antioxidant and/or antimicrobial compounds
[19, 20].
2. GRAPE SEED EXTRACTS

Agro-industries generate numerous waste materials resulting in environmental problems
and therefore the achievement of a sustainable agriculture implies the reduction/elimination
of waste. The possibility of using these wastes as natural antioxidants in the food industry
could represent a significant step towards maintaining environmental balance. For instance in
wineries, wastes composed by seeds and peels account for approximately 30% of the total
volume of grapes used representing serious storage, processing or disposal problems. These
by-products, are rich in phenolic compounds, which are known to have high antioxidant
activity [21, 22].
In addition, the use of antioxidant extracts from grape in meat products has gathered
interest from consumers and scientific community because of their health promoting
properties. The benefits of these extracts have been attributed to their high phenolic
compound content, mainly quinones, flavanols, flavonoids alkaloid and lectins [14]. In Figure
2 the chemical structure of the most common flavonoids is shown. Specifically, numerous
studies have been conducted on the antioxidant potential of grape and grape seed extract in
meat as cooked beef [19], pork patties [23] and poultry products as turkey [24]. We will
discuss them in deep in the next sections. Technically according to Lau and King [24] grape
seed extract (GSE) is a by-product derived from the grape seeds (Vitis vinifera) obtained in
the process of production of wine. Seeds are extracted and extract is next purified and dried to
obtain a polyphenolic compound rich preparation. GSE is sold commercially as dietary
supplement listed on the ―Everything Added to Food in the United States (EAFUS)‖ and it
also has the GRAS (Generally Recognized as Safe) status approved by the Food and Drug
Administration (FDA). Indeed, apart from the extracts specifically prepared for research in
trials, there are also available commercial grape extracts such as ActiVin™ (Inter Health,
Benicia, CA, USA) or Gravinol Super™ (Kikkoman, Tokyo, Japan) which have been tested
in several studies. The most abundant phenolics isolated from GSE are catechins,
procyanidins and their polymers [25, 26]. GSE are known for their high contents of
polyphenolic, oligomeric procyanidins [27] and proanthocyanidin compounds [28].
Indeed, standardized grape seed extracts contain 74-78% oligomeric proanthocyanidins
and less than approximately 6% of free flavanol monomers on a dry weight basis [29]. GSR
are rich in proanthocyanidins in the form of monomeric phenolic compounds, such as
catechin, epicatechin and epicatechin-3-O-gallate, and in dimeric, trimeric and tetrameric
procyanidin forms. These can combine with gallic acid to form gallate esters and ultimately
glycosides [30, 31]. The red color and astringency taste of the GSE can be attributed to
polyphenol rich compounds especially proanthocyanidins. This fact could affect color and
sensory characteristics of the product when used at higher concentrations [31, 32]. Recent
literature has evidenced antioxidant properties of GSE ―in vivo‖ and ―in vitro‖ assessed by
different antioxidant methods, such as DPPH, TEAC, FRAP or ORAC, and mainly due to the
flavonoid ability to scavenge free radicals [27, 33-35].
Flavonoid Subclass Compound

Flavonols
R1
OH
OH O
Quercetin (R1=OH, R2=H)

Kaempferol (R1=R2=H)
OH O Myricetin (R1=R2=OH)
Flavones
R1
OH
OH O
R2
OH
OH O
Catechins Apigenin (R1=H)
OH Luteolin (R1=OH)
Catechin (R1=H, R2=OH)
OH
Epicatechin (R1=R2=H)
Epigallocatechin (R1=OH, R2=OH)
OH O
R1 Epicatechingallate (R1=H, R2=OC-
Ph(OH)3)
Epigallocatechingallate (R1=OH, R2= OC-
R2
Ph(OH)3)
OH Gallocatechin (R1= OC-Ph(OH)3, R2=H)
Flavanones
R1
R2
Hesperetin (R1=OH, R2=OMe
OH O Naringenin (R1=H, R2=OH)
H
OH O
Figure 2. (Continued).
Anthocyanidins
R2
OH
Delphinidin (R1=R2=OH)
OH O+ Cyanidin (R1=OH, R2=H)
R1 Pelargonidin ((R1=R2=H)
Petunidin (R1=OMe, R2=OH)
OH
Peonidin (R1=OMe, R2=H)
OH Malvidin (R1=R2=OMe)
Procyanidin B-type dimers
OH
OH
OH O
R3
R4
OH B1: R1=OH, R2=H, R3=H, R4=OH
OH B2: R1=OH, R2=H, R3=OH, R4=H
OH
B3: R1=H, R2=OH, R3=H, R4=OH
H B4: R1=H, R2=OH, R3=OH, R4=H
OH O
R2
R1
OH
Figure 2. Chemical structure of the most common flavonoids in GSE.
Also, the resveratrol is a well-known free radical scavenger present in GSE, so the high
antioxidant activity could be due to their content [36]. The higher the level of polymerization
of the procyanidins, the higher the antioxidant activity of these molecules [37].
Extracts from grape seed possess an antioxidant potential 20 and 50 times higher than
vitamins E and C, respectively [38, 39]. The polyphenol content in grapeseed vary from 35
mg GAE/g grape seed and 85 mg GAE/g grape seed [22, 30] to 373 mg GAE/g grape seed
[40]. Moreover, different authors have reported EC50 values obtained for the scavenging
effects of extracts from red grape varieties (V. vinifera L.) on DPPH, ranging from 2.71 and
4.62 μg/mL for Turkish [41] and Italian [42] grape seed extracts, respectively, to values of
160 μg/mL for Spanish grape seed extracts [40].
In addition, many positive effects on human health have been described for polyphenols
including anti-inflammatory, anti-carcinogenic, cardioprotective and vasodilatory properties
[43]. Specifically, several studies have shown that grape extracts can play a role as potential
chemoprotective anticancer factor [44] and cardio protective [45]. As the GSE are composed
of polymers of flavan-3-ols such as catechin and epicatechin, they have antimicrobial
properties due to the galloyl moiety present in the structures of these compounds. This
activity is based on the interaction with bacterium cell membranes. Membranes are composed
of a double layer of phospholipids and proteins that are the main interaction site with the
molecules possessing antimicrobial activity.
The rupture of the cell membrane leads to the death of the bacteria by physical disruption
[46]. GSE have shown antimicrobial activities in meat [19, 47], specifically against Listeria
monocytogenes [48-50]. Also antimicrobial activity of GSE extracts has been reported against
Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa [47].
These authors suggested that high concentration of flavonoids and other phenolic
compounds were responsible for antimicrobial activity. Gallic acid has been identified as the
active compound against Escherichia coli and Salmonella enteritidis in methanolic GSE [51].
However, Rhodes et al. [50] did not find a significant antimicrobial activity against other
microorganisms such as Bacillus cereus, Salmonella menston, Escherichia coli,
Staphylococcus aureus or Yersinia enterocolitica.
3. USE OF GRAPE EXTRACTS AND GRAPE SEED EXTRACTS ON

MEAT PRODUCTS
3.1. Food Models
In different studies and using various raw food models composed by meat of pork and
chicken, it was verified that GSE (Gravinol Super™) at concentrations as low as 0.1% was a
very effective inhibitor of the formation of primary (hydroperoxides) and secondary (TBARs
values) oxidation products [52]. In addition, the antioxidant ability of GSE was later
confirmed in cooked ground muscle during refrigerated or frozen storage [52].
In another study, employing a meat model system sausage [53], GSE (ActiVin™) was
added at different levels (100 to 500 ppm). Sausages were made from beef, cooked at 70ºC
and stored for four months at -18ºC. The results showed that after 4 months of storage
samples containing GSE showed rancidity odour and flavour scores lower (P < 0.05) than
control samples. While TBARs value of the control samples increased (P < 0.05), samples
with GSE did not modify significantly (P > 0.05) the values of this parameter over the storage
period. TBARs values of samples manufactured with 100 and 300 ppm of GSE remained
constant or slightly decreased during the storage period with values below 0.2 mg MDA/kg
sausage.
3.2. Raw Meat
GSE (GMBH and Co., Cadolzburg, Germany) at level of 300 ppm was evaluated as
antioxidant in raw beef patties. The results showed the possibility of using GSE in
combination with 100 mg of sulphite to increase shelf life of raw beef patties. There was a
reduction of the loss of red index and in the lipid oxidation level, thus increasing the shelf life
in three days respect to control samples [54].
In another study carried out with beef steaks packed in modified protective atmosphere
(MAP) and vacuum conditions it has been reported than mean luminosity values of samples
treated with GSE at level of 40 ppm (Grape-Maxi™) were significantly higher than those for
control, thus enhancing the luminosity of meat [55]. In addition, these authors found a
positive correlation (P < 0.01) between lightness and yellowness with TBARs values, in
agreement with Insausti et al. [56] that indicated that color parameters were positively
correlated with several traits that reflect the degradation of beef quality such as sensory
degradation of color and odour, fat oxidation, and increase in microbial spoilage. Concerning
lipid oxidation of intramuscular fat from steaks, a positive effect on the decrease of TBARs
values of meat with the addition of GSE to beef steaks compared to samples without
antioxidant extract was found. This positive effect has been reported for cooked beef and
pâtés [39, 52, 57]. Regarding antimicrobial activity, Franco et al. [55] indicated a moderate
anti-bacterial activity of GSE in agreement with results previously reported [58, 59].
In other work carried out with raw pork patties packed in MAP in refrigerated conditions
[60], the samples suffered discoloration (mainly defined by loss of redness) as already other
authors point out in raw pork during chilled storage [61-63], but the addition of GSE (1000
ppm of a non-commercial extract of GSE, obtained from Vitis vinifera ―Albari o‖ variety and
a Vitis labrusca hybrid and well characterized from the antioxidant activity point of view)
resulted in higher redness values compared to control samples. Pork patties including GSE
were brighter, redder and more yellow than control ones. Preservation of raw pork patties
towards the desirable red color, has influence on consumers purchase decision. This
stabilising effect on color has also been observed in studies with red grape (pomace from Vitis
vinifera var. Monastrell) at a dose of 600 ppm in pork burger packed under aerobic conditions
and stored at 4°C for 6 days [64]. In this sense, one might think that the red pigments such as
anthocyanins from red grape could contribute to show a desirable mixture of color on meat,
but redness values did not modify in samples with red grape extracts [64]. Indeed, redness
value and myoglobin content are closely associated parameters [65] and positively correlated
between them. The discoloration that occurs during storage of red meat cuts is generally
explained by the oxidation of the iron atom within the heme group in red oxymyoglobin,
transforming it to brownish metmyoglobin [66], and by the oxidative degradation of certain
nitrosopigments in pork [62], and beef [67]. The accumulation of metmyoglobin in the meat
surface and subsequent discoloration largely depends on the presence of reducing systems and
on lipid oxidation [61, 66]. GSE has an effect in the inhibition of lipid oxidation in pork
patties, showing that GSE was the most effective against TBARs formation, even better than
the commercial antioxidant BHT in agreement with information reported by Garrido et al.
[64]. Lorenzo et al. [60] indicated results in good agreement with above statements, because
redness was negatively correlated to TBARs values (r = − 0.495, P < 0.01), indicating this
correlation between lipid and myoglobin oxidation. Also, protein radicals and other protein
oxidation products are known to induce lipid oxidation [68] and hence, heme pigments in raw
meat could also be affected by their pro-oxidant action, but this is less reported in the
literature. According to Georgantelis et al. [69] rancid flavour is detected in meat products
with TBARs values higher than 0.6 mg MDA/kg. TBARs values of GSE pork patties
remained below than this value during the whole storage (20 days), whereas control samples
reached values higher than this indicated threshold. Concerning antimicrobial activity,
Lorenzo et al. [60] observed a significant (P<0.001) decrease in total viable counts (TVC) in
samples containing GSE in their formulation. Similar results were reported by Jayaprakasha
et al. [47] who observed in vitro antibacterial effect of GSE. Lactic acid bacteria constitute a
substantial part of the natural microbiota of MAP meats [70] and Pseudomonas have been
characterized as the microorganisms responsible for the spoilage of aerobically stored fresh
meat [71]. Pork patties treated with GSE showed a reduced in LAB and Pseudomonas
population during storage [60]. Finally, all pork patties showed a significant (P<0.05)
increase in psychotropic aerobic bacterial counts during refrigerated storage, but at the end of
shelf-life, patties with GSE presented lower psychotropic aerobic bacterial counts (6.5 log10
CFU/g) than control patties (7.7 log10 CFU/g) [60].
The GSE also has been tested successfully in raw beef and pork patties, vacuum
packaged and frozen stored (-18ºC) for 4 months, providing protection against oxidation
without significant alteration of the sensory perception of oxidation and color [72]. In another
work from the same research group, where the frozen storage period was extended to 6
months, authors showed that GSE had higher antioxidant activity than synthetic antioxidants
such as BHA and BHT, and only PG provided slightly better results than GSE based on
TBARs values (0.21 and 0.23 MDA/kg for PG and GSE, respectively). Also, once again GSE
had no negative effect on redness or yellowness [73].
In other species less studied such as buffalo, results with GSE (Mega Natural Inc.,
Madera, CA, USA) were positive as indicated Tajik et al. [74] in raw buffalo patties during
storage at 8°C for 9 days with GSE added at 0.1 and 0.2%. TBARs values of both treated
GSE samples remained less than 1.0 for the entire 9 days of storage time. The TBARs values
higher than 1.0 are usually associated with rancid flavour/odour by sensory panelist and it is
considered as the beginning of organoleptic perception of lipid oxidation [75].
Rababah et al. [76] studied the effect of commercial GSE (Mega Natural Inc.) at different
levels (3000 and 6000 ppm) on lipid peroxidation and redness of goat meat stored at 5°C for 9
days. Addition of GSE significantly decreased the lipid oxidation of goat meats. Further,
higher level of addition of antioxidants was more effective in minimizing lipid oxidation. In
agreement with the last statement, Murcia et al. [77] also found that reduction of lipid
oxidation and peroxidation of meat products by GSE takes place in a concentration-dependent
manner. Regarding, color parameters GSE increased the redness index significantly in goat
meat samples [76].
3.3. Cooked Meat
Numerous studies concluded that GSE is an effective antioxidant in cooked meat. For
instance, Ahn et al. [19] informed that GSE (ActiVin™) at dose of 0.1% improved
significantly the oxidative stability compared with control samples of cooked beef after 3
days of refrigerated storage. During this period TBARs values, hexanal content, and warmed-
over flavor were reduced. In a later study in cooked beef from the same research group, when
the dose of ActiVin™ was increased until 1%, a 92% of inhibition percentage in TBARs
index is achieved with respect to control samples, after 9 days of storage [57]. In addition, the
hexanal content was significantly lowered throughout the storage period. The color of cooked
beef treated with ActiVin™ had less luminosity, more redness and less yellowness than that
treated with a mixture of synthetic antioxidants BHA/BHT.
In another study, GSE at a concentration of 300 ppm was evaluated as antioxidant in
cooked beef patties. The results showed that samples with GSE delayed the onset of rancid
flavours in cooked patties [54]. In agreement with this work, Rojas and Brewer [78]
confirmed the effectiveness of GSE at 0.02% on oxidative processes and color stability of
cooked beef and pork patties (71ºC) stored at refrigerated conditions (4ºC) for 8 days.
Samples with GSE exhibited less oxidation based on TBARs values and off-odors associated
with lipid oxidation such as rancidity and grassy. In addition, the addition of extract did not
modify meat cooked color and also decreased the visual green discoloration in beef patties.
Color changes are an important factor influencing the quality and acceptability of meat and
meat products. In fact, color tends to be used as an indicator of quality and freshness of meat
as indicated Carpenter et al. [79] that point out a close relationship between color preference
and the decision to purchase. Specifically in some meat and poultry products, consumers may
perceive an increase in red color as a negative effect because a fully cooked product may
appear to be undercooked. As it mentioned above, the color of meat products depends of
many factors such as concentration of haem pigments and particularly of myoglobin, the
physical characteristics of the meat, essentially pH, and the chemical state of these pigments.
With time or thermal process discoloration results from conversion of oxymyoglobin to
metmyoglobin which gives an unattractive brown color [80]. Also, previous reports have
shown that meat color stability is muscle-dependent [81], hence comparisons between studies
is difficult. In this sense, Ahn et al. [19] by means of a panel sensorial test found that low
concentrations of GSE (0.1%) did not modify the sensory characteristics as a consequence of
the color of the extract, whereas when extract concentration was increased (1%) [57], the
redness of final product increased by 50% and the index of yellow decreased by 20%, when
compared with control samples. In agreement with Ahn et al. [19], other authors, who used
other commercial GSE (Gravinol SuperTM) at low dose (0.02%) did not find differences for
index of red between treated and control samples of cooked pork and beef [78]. In the same
line, Kulkarni et al. [53] using GSE (ActiVin, San Joaquin Valley Concentrates, Fresno, CA,
USA) at concentrations of 100, 300, and 500 ppm in precooked beef sausages cooked at 70°C
and stored for 4 months (−18°C), they reported that samples with GSE had lower rancid
odour and flavour scores (P < 0.05) and retained their fresh cooked beef odour and flavour
longer (P < 0.05) than the control samples. In other cooked meat product as frankfurters, the
effect of GSE added at different levels (0.01 to 0.5%) was also evaluated over quality
properties. The pH, water and fat content and color parameters were significantly affected.
The results indicated that there was a decrease in TBARs values as the GSE concentration
increases, without affecting the sensory properties at the beginning of storage according to the
overall acceptability results [82].
GSE (Dakshin Laboratories, Hyderabad, India) was also tested in cooked restructured
mutton slices, packed in aerobic and vacuum conditions and stored at refrigerated
temperatures. Slices with GSE had significantly lower TBARs values and free fatty acids
compared to control and BHA samples. In addition, samples with GSE showed significantly
higher scores for color, flavour, juiciness and overall palatability than control and BHA
treated restructured mutton slices [83]. In goat meat, GSE minimized lipid oxidation in
cooked goat meat. The results showed that GSE addition significantly (P < 0.05) increased
the redness [76].
The antioxidant efficacy of extracts of grape skin (200 ppm) in precooked, vacuum
packaged pork patties stored in refrigerated conditions (<5ºC) was also assessed. TBARs
value of the cooked pork patty manufactured with the extract was 15.8 μmol MDA/kg,
whereas it was 30.0 μmol MDA/kg in the control. Hexanal contents were 13.2 and 21.6 ppm
in skin grape extract-containing cooked pork patties and control, respectively [23].
Table 1. Summary of the main research exposed in this chapter on the use of
commercial and non-commercial GSE in different meat products
Commercially extracts Concentration Meat product Reference

Gravinol-S Kikkoman International 0.1-1% Raw pork and chicken [52]
Gravinol-S Kikkoman International 0.1-1% Cooked pork and chicken [52]
ActiVin 100-500 ppm Cooked beef sausages [53]
GMBH and Co. 300 ppm Raw beef patties [54]
GMBH and Co. 300 ppm Cooked beef patties [54]
Grape-Maxi™ soft gels 40 ppm Raw beef steaks [55]
Mega Natural Inc. 0.1-0.2% Raw buffalo patties [74]
Mega Natural Inc. 3000-6000 ppm Raw goat meat [76]
Mega Natural Inc. 3000-6000 ppm Cooked goat meat [76]
ActiVinTM InterHealth 0.02% Cooked ground beef [19]
ActiVinTM Natural Health Science 1.0% Cooked ground beef [57]
Gravinol SuperTM 0.01-0.02% Cooked beef patties [78]
Gravinol SuperTM 0.01-0.02% Cooked pork patties [78]
Local food supplements company 0.01-0.5% Frankfurts [82]
Dakshin Laboratories 0.1% Restructured mutton [83]
slices
Gravinol-S Kikkoman International 0.1% Raw chicken meat [102]
Gravinol-S Kikkoman International 0.1% Cooked chicken meat [102]
Grapines-Nature‘s Sunshine Products, Inc. 0.4-1.6 g/kg Cooked Turkey breast [103]
Guinness Chemical 50-1000 µg/g Raw pork patties [39]
Guinness Chemical 50-1000 µg/g Cooked pork patties [39]
Gravinol SuperTMKikkoman 0.01-0.02% Frozen raw beef patties [72]
Gravinol SuperTMKikkoman 0.01-0.02% Frozen raw pork patties [72]
Gravinol SuperTMKikkoman 0.02% Frozen cooked pork [73]
patties
Gravinol SuperTMKikkoman 200 ppm Frozen cooked beef [104]
patties
Non commercially extracts
Vitis vinifera var. Albariño and 1000 ppm Raw pork patties [60]
Vitis labrusca hybrid
Pomace from Vitis vinifera var. 600 ppm Pork burgers [64]
Monastrell
Vitis vinifera var. Albariño and 1000 ppm Liver pate [85]
Vitis vinifera var. Albariño and 50-1000 ppm Liver pate [95]
Skin grape from (Unilever, Vlaardingen, 200 ppm Pork patties [23]
Holland)
Vitis vinifera L., from Australian local 200 ppm Raw pork [105]
market
Vitis vinifera var. Albariño and 1000 ppm ―Chorizo‖ [40]
Vitis vinifera var. Albariño and 50-1000 ppm ―Chorizo‖ [112]
Another interesting meat product is the liver pâté, a traditional cooked meat product that
consist of minced liver, fat and meat mixed with water and different additives, packed in glass
or can, and finally subjected to a thermal process. The high content of fat and the exposure to
high temperatures (>70ºC) make this type of meat products highly susceptible of onset of
oxidative reactions after cooking [84]. Other concern about this type of product is that nitrites
have been widely used in cooked meat products but the potential health risks related to
residual nitrite levels and the formation of harmful N-nitrosamines in meat products led to
demanding a significant decrease in the use of sodium nitrite.
In a study of liver pâté manufactured using liver from Celta pig breed and GSE at level of
1000 ppm [85] stored in the dark at 4ºC for 24 weeks, the mere fact of introducing the
antioxidant extract in the formulation modifies the pH of the final product, due to the acidity
of the GSE (pH 4.29). This fact could have consequences on color characteristics of final
product. Indeed, luminosity, redness and yellowness values were lower in pâtés with GSE
than in control samples or in samples manufactured with BHT [85]. Obviously, apart from pH
modifications, these color changes could be related to oxidation phenomena and to the
presence of antioxidants as noted in cooked pork sausages [86]. According to Tarladgis [87]
the compound responsible for brownish grey colors in cooked meats is the ferric-porphyrin of
the molecule denatured, globin. The modification of this structure causes the degradation of
heme molecule and the release of iron, which could explain the color modification of pâtés.
On the contrary, Morrissey et al. [88] linked the variation of luminosity and of redness with
nitrosopigment degradation in ostrich liver pâté. Despite of the initial modification of color in
samples containing GSE, authors noted that liver pâté manufactured with GSE showed lower
color modifications (0 to 24 weeks) than control and BHT samples, so it is indicating a
protective effect on color features of the product [85].
In meat products where high temperatures are applied, the formation and destruction of
peroxides begin during the manufacturing and thermal treatment. In pâté the first heat
treatment consists in scalding of fat and liver [89]. In the study of Pateiro et al. [85] samples
with BHT retarded the beginning of lipid oxidation because the inhibition of the formation of
peroxides during the manufacture of pâté. At the end of storage period (24 weeks) samples
with GSE showed peroxide index values significantly lower than control pâtés, with values of
3.80 and 1.50 meqO2/kg (P < 0.05) for control and GSE pâtés, respectively. For secondary
products of lipid oxidation, at the end of storage time the lowest TBARs values were obtained
in batches with GSE with values of 1.62 and 2.92 mg MDA/kg (P < 0.05) for GSE and
control, respectively. According to TBARs values, in this product and conditions, GSE
improved the results obtained by BHT in agreement with previously published studies [84,
90] that reported higher effectiveness of natural antioxidants compared to synthetic
antioxidants and suggested the possibility of using these extracts as replacers of commercial
compounds. In liver pâtés, the determination of volatile compounds provides an indication of
the chemical processes that occur during manufacture and storage [91, 92]. Also, these
compounds allow knowing the oxidative stability and the aroma characteristics of this product
[93]. Specially, the aldehydes are the main compounds and probably the most interesting
volatile compounds [94] because they can produce a wide range of flavors and odors. Liver
pâtés that contained GSE showed a decrease in the amount of lipid-derived volatile
compounds (hexanal and heptanal) with respect to control samples [85]. Similar results were
reported in other pork pâté samples employing essential oils of sage and rosemary as
antioxidants in the formulation [84].
The main disadvantages of the use of natural antioxidants are that they are usually more
expensive than the synthetic ones, and they may impart color or taste to the product.
Therefore trials studying the dose effect are necessary to achieve a better extract
characterization. In this sense Pateiro et al. [95] studied the effect of the addition of increasing
levels of GSE (50, 200 and 1000 ppm) on oxidative stability of refrigerated stored pig pâtés
with respect to control samples. There were significant differences in luminosity of pâtés by
dose effect (luminosity values of 70.03 and 64.40 for 50 and 1000 ppm of GSE) in agreement
with results reported by Zhang et al. [96] who observed lower luminosity values in pâté
samples with higher amount of sage extracts. As occur with luminosity, increasing amounts
of GSE added to the pâté determined decreasing values of redness and yellowness in the
sample. At low dose (50 ppm) similar luminosity, redness and yellowness values were found
between control and samples treated with GSE. The higher dose (1000 ppm) modifies the
initial color of pâté, but it has a stronger effect on color stabilization during the storage
period. A similar trend was found in lipid oxidation, because the higher the amount of GSE
added to the pâté, the lower was the TBARs value of the sample. As in the study of Pateiro et
al. [95], many works reported that the improving effects of extracts were concentration-
dependent [39, 97] descending the production of TBARs as the levels of antioxidants
increased. Apart from peroxide values, TBARs values or volatiles compounds, the amount of
polyunsaturated fatty acids (PUFA) can be used as a measure of the oxidative deterioration of
meats due to the fact that PUFA contain double bonds, the preferred substrates in oxidative
reactions [98], and are destroyed in the oxidation processes. Pateiro et al. [95] reported that
the oxidative degradation of PUFA mainly occurred after the fourth week of storage, in
agreement with Reddy et al. [84]. This phenomenon may be attributed to the gradual
degradation of endogenous antioxidants and the release of iron from the heme molecule [99].
It can be inferred that GSE addition would protect slightly the pâtés from oxidative
degradation between week 4 and 8, taking into account the higher amount of PUFA found in
GSE samples compared to control samples (10.51 vs. 11.66 vs. 10.68% for control and GSE
at level of 50 and 1000 ppm, respectively). Within volatile compounds, the predominant
aldehyde was hexanal that is mainly generated as a result of the oxidative decomposition of
PUFAs and it has been related to rancid flavors [100].
Indeed, hexanal content, as happened with the TBARs values, has been frequently used
as marker for lipid oxidation [101]. In fact Pateiro et al. [95] found a strong correlation
between these two parameters (r = 0.65, P < 0.01). Also, Pateiro et al. [95] reported that the
greater the amount of GSE added to the pâté, the lower was the concentration of aldehydes in
the sample. Particularly, the addition of GSE had a significant (P < 0.05) effect on the
generation of most of the volatiles with relation to control samples, even improving the
results obtained using BHT as antioxidant.
GSE has also demonstrated antioxidant activity in cooked poultry meat as chicken [102]
and turkey [103], or in meat products frozen after cooking process composed by beef or pork
[72, 73, 104], by decreasing the concentration of primary and secondary lipid oxidation
compounds (hydroperoxides, hexanal and thiobarbituric acid reactive substances) and
therefore reducing rancid flavour development.
Despite the fact that in heat treated meat products the microbial spoilage is less
determinant than in raw meats, antimicrobial properties of GSE have been also tested in
cooked meats. GSE added at 1% level showed antimicrobial activity against Gram-negative
bacteria such as Escherichia coli O157:H7 and Salmonella typhimurium in cooked ground
beef [19].
3.4. Dry-Cured Products
Dry-cured meat products are above all fermented sausages that undergo drying-ripening
processes before consumption. During the manufacture of dry-cured sausages, apart from the
microbial evolution some important chemical modifications occur. In particular dehydration,
fermentation of carbohydrates and acidification, development of color, lipolysis and fat
autooxidation and proteolysis takes place [106]. Specifically, lipid oxidation is one of the
main deteriorative chemical changes that occur during the sausage ripening process as we
commented in the introduction section. ―Chorizo‖ is a typical dry fermented sausage from
Spain. To our knowledge, few studies regarding the effect of natural antioxidants on the
oxidation stability of dry ripened sausage ―Chorizo‖ were found in the literature perhaps
because it is a well-designed product to achieve long shelf life (low moisture content and low
water activity values, use of natural spices as garlic and paprika, and modern packed based on
vacuum conditions). However, in a study performed by Lorenzo et al. [40] it can be noted
how the use of GSE (non-commercial extract) at level of 1000 ppm in ―Chorizo‖ modified the
moisture content of the dry-cured sausage, showing the batches with GSE higher water
contents than control batches (27.99 vs. 20.58%) at the end of the ripening process. This fact
has effect on color parameters, indeed the luminosity of ―Chorizo‖ manufactured with GSE
was affected (P < 0.001) they presenting higher luminosity, redness and yellowness values
than control ones at the end of process. Clearly, the addition of GSE affects color parameters,
in disagreement with Karabacak and Bozkurt [107] and Bozkurt [108] who observed that
CIELAB color parameters were not affected (P > 0.05) by addition of natural antioxidants
other than GSE in Turkish sausages. As occurred with other meat products previously
exposed in this chapter, color is one of the most important quality attributes of sausages that
attract the preference of the consumers. These products are most attractive when a desired
brick-red color is formed during the ripening period and therefore the addition of natural
antioxidants provide more attractive sausages.
Besides the consequences on color parameters, modification of moisture content has
influence on textural parameters (confirmed by significant Pearson correlation (P < 0.01)
between moisture and textural profile parameters). Indeed, the addition of GSE decreased
(P < 0.001) hardness, gumminess and chewiness, measured by instrumental devices and
confirmed by sensorial panel (the textural acceptability was significantly (P < 0.001) higher
for Chorizo with GSE than for the control batch). Although this effect was not expected, the
presence of GSE is very interesting from a technological point of view. The explanation is out
of the aim of this chapter but some researches have shown that polyphenolic compounds are
able to react with thiol groups in meat proteins to form covalent thiol-quinone adducts [109].
Specifically, it has been hypothesized that polyphenolic compounds from green tea extract
can alter the textural properties of Bologna type sausages [110], so especially at high
concentrations phenolic compounds could interact with the protein thiols to modify water
holding capacity and other textural parameters. Statistical analysis showed that TBARs values
decreased (P < 0.001) with the addition of GSE when compared with control batches or even
with batches manufactured with synthetic antioxidant as indicated by Bozkurt [111] who
worked with a Turkish dry-fermented sausage formulated with extract from sesame and
Thymbra spicata oil. Hexanal, a typical indicator from linoleic acid oxidation was higher in
control sausages than in GSE ―Chorizo‖ (16.8 vs. 2.27 x 106 area units) [40]. In this sense,
sausages with GSE had similar hexanal values than samples treated with BHT (2.27 vs.
2.19 x 106 area units), but ―Chorizo‖ manufactured with GSE showed the lowest values for
total volatile compounds, so this indicates that the addition of GSE effectively inhibited the
lipid oxidation. GSE showed no antimicrobial activity in sausages, because no differences in
total microbial counts were detected between control and batches treated with GSE. Pateiro et
al. [112] completed the work of Lorenzo et al. [40] using other levels of GSE (50 and 200
ppm). At the end of the ripening process, the samples that contained GSE 200 ppm in their
formulation were those that showed the highest values for luminosity, even improving the
results obtained using BHT as antioxidant. Samples manufactured with GSE at 200 ppm
presented higher redness values compared to control batch. Regarding dose effect, significant
differences (P < 0.05) were found for redness in samples that contained GSE in their
composition. Similar results were reported by O´Grady et al. [97] because the improving
effects of extracts were concentration-dependent. Also, Jayawardana et al. [113] showed the
capacity of adzuki bean extract to hold the color on pork cured sausages. Samples with GSE
in their composition showed values below 0.4 mg MDA/kg, reducing lipid oxidation of the
dry-cured sausage. The levels reported after the storage period were significantly (P < 0.05)
lower than the limit (2.0 mg MDA/kg) which is accepted as deterioration level [114]. Within
dose effect, no significant differences (P < 0.05) were found among samples treated with 50,
200 and 1000 ppm of GSE, so that the lowest concentration of GSE would be sufficient to
improve the results obtained in control samples. When the antimicrobial activity of GSE was
assessed in Chorizo, Pateiro et al. [112] noted that regarding dose effect, significant
differences (P < 0.001) were found for the different GSE batches studied. An increase in the
level of GSE decreased the TVC count values and samples manufactured with GSE at 1000
ppm showed lower values than those made with GSE at 50 ppm and 200 ppm, with counts of
7.91, 8.14, 8.15 and 8.55 log10 CFU/g, for 1000, 200, and 50 ppm GSE an control,
respectively). Also GSE provoked a decrease in the population of LAB, being this decrease
more pronounced as the GSE concentration increased, obtaining LAB counts of 7.70, 7.93,
and 7.96 log10 CFU/g for GSE at 1000, 50 and 200 ppm, respectively.
CONCLUSION
From the information reviewed in this chapter, it can be concluded that overall the
addition of GSE to meat products improves the characteristics related with shelf life such as
color and oxidative status, and in certain cases (such as in raw meats) microbial spoilage. In
most of the papers revised, control (without antioxidants) and samples with added synthetic
antioxidants are manufactured and assessed, hence results are consistent. Overall, the addition
of GSE shows a preservative effect over raw, cooked and dry-cured meat products (raw
patties, cooked patties, frankfurters, liver pâté and dry-cured sausage) during storage normally
under refrigeration conditions, improving in certain researches the results obtained using
synthetic antioxidants; so, the addition of GSE would be advisable to replace them.
Further research on the extraction conditions, mode of addition to the product
(microencapsulated), how the addition of GSE affects the sensory properties and volatile
compounds, relationships between GSE and lipid, and protein oxidation and meat quality
would be of interest to obtain more extendable and attractive products for the consumers. In
addition, GSEs are obtained from by-products, so the valorization of agricultural waste
products to produce natural antioxidants, with potential application in processed meat

products, would be attractive for concerned consumers.
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Chapter 8
THE SCIENCE OF GRAPE SEEDS APPLIED

TO HEALTH FOOD DEVELOPMENT
C. J. Contreras-Castillo1,*, J. P. Cruz-Tirado2
and L. Din Shirahigue1
1
Universidade de São Paulo, Escola Superior de Agricultura Luiz de Queiroz,
Departamento de Agroindústria, Alimentos e Nutrição, São Paulo, Brazil
2
Universidad Nacional de Trujillo, Facultad de Ciencias Agropecuarias,
Escuela de Ingeniería Agroindustrial, Trujillo, Peru
ABSTRACT
The search for a healthier diet has led food science and technology to develop new
products with healthier characteristics and greater consumer acceptance. Consumer
products such as sausages, yogurt, milk, fruit juices, and others can be supplemented with
phenolic compounds derived from fruits (namely quince, apple, barriers, and grapes) or
waste generated by industries in order to improve taste and aroma of these products.
Grape seed extract reduced oxidation and provided higher microbial stability in foods,
without significantly affecting the sensory characteristics demanded the consumer.
Keywords: grape seeds extract, procyanidins, antioxidant, food development
INTRODUCTION
Food production has evolved alongside the needs of consumer satisfaction. Today, this
satisfaction includes the consumption of products which incorporate diet compounds that are
favorable to the body, without losing the specific traditional taste sought in the product. There
are many food products in the world which contain functional compounds. Grape seeds, as a
residual source from the wine industry, represent a striking source of these compounds,
having a high percentage content of compounds such as catechins, proanthocyanidins,
*
E-mail: ccastillo@usp.br.
182 C. J. Contreras-Castillo, J. P. Cruz-Tirado and L. Din Shirahigue
anthocyanins, and phenolic acids, among others. These phenolic compounds are antioxidants,
and therefore they not only favor the development of health food products, but also preserve
the microbiological quality of food and retard chemical reactions that oxidize the main
components of the product. The ability to develop these properties in products depends on the
number of these phenolic compounds that are adsorbed in the food. Therefore, food science is
working on the design of processes and technologies that enable maximum efficiency in the
development of new products which contain these antioxidant compounds.
GRAPE SEEDS
The production of Vitis vinifera L. is widespread worldwide, exceeding 69 million tons
per year [1]. Its seeds comprise about 5% of the weight of the fruit, which means that more
than 3 million tons of grape seeds are discarded annually worldwide. This byproduct is a
valuable raw material that is scarcely used in food. The seeds‘ composition is basically (w/w)
40% fiber, 16% essential oil, 11% protein and 7% complex phenolic compounds. It is known
that the protein and oil quality of grape seeds is similar to many other sources currently used
in foods.
Moreover, phenolic compounds of grape seeds have many applications in cosmetics,
pharmaceuticals and several foods. Precisely, in the latter, it presents advantages over other
sources of phenolic compounds because grape seeds are considered industrial waste.
HOW TO TAKE ADVANTAGE OF GRAPE SEEDS?

In recent years, numerous plant sources rich in different compounds have been placed in
the market as ingredients that complement and promote the nutritional, microbiological and
chemical quality of different food products, without affecting their sensory acceptance. One
of the plant sources that has recently been more widely exploited is grape seeds. Among the
compounds of interest extracted from grape seeds are: protein extract, phenolic extracts and
oil, all of great interest to the food industry.
POLYPHENOLIC EXTRACT
Phenolic compounds are antioxidant compounds, that is, they delay, prevent or eliminate
the oxidative damage caused by excess free radicals. Among the numerous phenolic
compound extracts, grape seed extract (GSE) stands out for it is a powerful exterminator of
free radicals, and may have a better effect than vitamin E – in equal weight doses – in the
protection of tissues from oxidative damage [2, 3]. The GSE may contain from 74 to 78%
oligomeric proanthocyanidins and less than about 6% free flavonoid monomers on a dry base.
Proanthocyanidins, in the monomeric phenolic compound form, such as catechin, epicatechin
and epicatechin-3-O-gallate, and in forms of procyanidin, dimerics, trimerics and tetramerics,
are abundant in GSE. These, when combined with gallic acid, form gallate esters and finally
glycosides [4]. This extract contains the necessary antioxidants to slow the deterioration of
The Science of Grape Seeds Applied to Health Food Development 183
food from oxidation and prevent microbiological contamination (in association with other
additives). Moreover, its consumption in the diet has been suggested by the WHO/FAO as a
key element to promote smooth functioning of the metabolism and reduce cell apoptosis.
How to Extract the Phenolic Extracts?
The first step in the use of phenolic compounds from a natural source is the extraction. It
is important to note that these compounds are susceptible to temperature variations and one
should always try to obtain the highest extraction efficiency. For this propose, food process
engineering has developed several technologies that optimize this process and best take
advantage of the content of phenolic compounds from their natural source.
Currently the most commonly used method for extracting polyphenols and other
constituents from the seeds of various products is extraction using solvents. This particular
method often requires large volumes of solvent and long periods of extraction [5]. Various
methods have been developed to reduce extraction time, which usually implies a high cost for
obtaining polyphenols.
Ultrasound Assisted Extraction Using a Solvent
Assisted extraction of phenolic compounds with ultrasound has proven beneficial. The
technology is based on the fact that ultrasound favors mass transfer, a better penetration of the
solvent into the plant tissue and capillary effects [6]. The collapse of cavitation bubbles near
the cell wall allows more efficient penetration of the solvent, which results in less time and
lower solvent consumption in the extraction of a significant fraction of polyphenols.
Microwave Assisted Extraction (MAE) Using a Solvent
This technology is based on the fact that microwave assisted extraction (MAE) using a
solvent, heats up the matrix internally and externally without a thermal gradient and because
of this, the functional compounds can be extracted efficiently and safely. MAE evaporates the
water within the cells due to the microwave effect, raising the pressure on the cell wall and
eventually breaking it. This facilitates the leaching of components in the solvent surrounding
the cells and improves extraction efficiency. The benefits of MAE are many, including
shorter extraction times and reduced solvent volume [5]. Interestingly, the three different
methods for extracting phenolic compounds (microwave assisted extraction - MAE,
ultrasound assisted extraction - UAE, and solvent extraction - SE), used similar levels of
temperature and ethanol content, but differed dramatically in terms of the time required
(MAE was 4.6 min, USE 29.4 min and 200 min). When tested (since the seeds used in the
different extractions were from different types of grapes) MAE proved much faster than the
others. In addition to being fast, MAE is relatively inexpensive and it does not significantly
pollute the environment, reaching an efficiency of over 90% extraction of polyphenols
available in seeds in a matter of minutes.
Supercritical Fluid Extraction
Although the above techniques result in better extract yields with shorter extraction
times, conventional solvents are still used (ethanol, methanol, n-hexane, etc.), resulting in
environmental pollution. Extraction with supercritical water, also called pressure water or low
polarity water, is seen as an interesting option for the extraction of polar and nonpolar
compounds [7, 8, 9]. When we speak of supercritical water, we refer to water at a temperature
between boiling point and critical point, without phase change [10].
How does it work? In a supercritical state, the dielectric constant of water is adjusted to
temperature. For example, at standard temperature and pressure (25º C and 101 kPa) the
dielectric constant of water is about 80; whereas when the temperature varies from 200 to
350° C, the dielectric constant lowers to around 20-30, similar to that of the solvents used in
traditional extraction methods, such as methanol, ethanol and acetone, when they are at room
temperature [9]. Although the solubility of organic compounds in the water depends on
several factors, an increase in temperature leads to a reduction in strength of the hydrogen
bonds, which makes water a low-polarity solvent, increasing the solubility of organic
compounds. Polyphenolic substances represent a wide range of organic compounds, thus to
obtain an optimal performance you basically need a good management system and good
operating conditions. Duba et al. [9], found that the extraction efficiency of phenolic
compounds from grape seeds increases as the temperature is raised, however, it decreases
when the flow is higher, which is a curious behavior, though it represents a viable and
efficient option for producing GSE.
PROTEIN EXTRACT
From the total residual grape seeds produced in the winemaking process a small part is
used for the extraction of oil and natural antioxidants. However, some researchers have
recently proposed the use of a protein extract from grape seeds as a fining agent [11]. The
nutritional quality of the proteins of grape seeds has long been considered similar to that of
other oil seeds or cereals. The major amino acids present in grape seeds are glycine 13.4%,
glutamic acid 26.7% and aspartic acid 9%, similar to the content of these same amino acids in
soy protein. The essential amino acids isoleucine, leucine, lysine, methionine, valine,
phenylalanine and threonine make up approximately 24.1% of the protein content of grape
seeds. Amino acids isoleucine (42.9 mg/g protein), leucine (75.6 mg/g protein), phenylalanine
+ tyrosine (84.7 mg/g protein) and valine (53.9 mg/g protein) are within the provisions of the
FAO/WHO [12].
Gazzola et al. [11] fractionated endosperm proteins of seeds of different grape varieties
according to their solubility and characterized them by electrophoresis in terms of polypeptide
composition, to later identify the major electrophoresis bands by mass spectrophotometry.
They found that the endosperm contained approximately 40% protein, from which the
albumin and globulin content represented 58.4% of the total extracted. The identification of
polypeptides was similar in all varieties tested, homologous to the 11S globulin protein stored
in grape seeds. However, researchers also generated new information about a 43 kDa
polypeptide having homology to the 7S globulins from other legume seeds.
This composition of amino acids present in grape seeds is very similar to that of cereals
and other oil seeds, therefore a treated seed may replace these food products, facilitating the
industrial use of this residue.
OIL
One of the useful components from grape seeds is the oil. Seeds contain 8 to 20% of oil
(dry basis) in their composition, which is made up mainly of linoleic fatty acids (58-78%,
C18: 2n-6) and oleic acid (10-20%, C18: 1n-9). Saturated fatty acids (C16: 0, C18: 0, C20: 0)
account for 10% of total fatty acids [13].
The fatty acid composition of this oil may be considered for edible purposes, since it
contains bioactive tocopherols and phenolic compounds in its unrefined form that may be
beneficial to the diet.
For the industrial extraction of grape seed oil, seeds are separated from the grape pulp (by
drying and screening), then the oil is extracted by continuous mechanical pressing (screw
pressing) and/or a solvent process. Currently, considering that heat treatment may degrade the
quality of oil, cold-extraction methods have been designed to obtain oil with higher
nutritional quality, since it not only prevents oxidation of the oil, but it preserves a good
percentage of its minor components, such as the antioxidants. Although the yield is lower
than when using a solvent, there is no concern about solvent waste in the oil, making it a safer
product [14]. Grape seed oil can be extracted using supercritical fluids such as CO2. This
extraction technique can achieve higher yields than the conventional solid-liquid extraction
performed with a solvent (n-hexane). Furthermore CO2 is nontoxic, nonflammable and is
cheap, aside from preventing traces of solvent in the oil; and although the initial investment of
this technology is expensive, the operating cost is low if the process is carried out in optimal
operating conditions [15-17].
FROM GRAPE SEEDS TO FOOD

Food processes have always been a challenge for food technology engineers. Eating
habits have changed over time and food processing has become increasingly complex. One of
the most important characteristics of food is its shelf-life, which is influenced by the
microbiological, physical and chemical quality of the food. The use of natural preservatives is
being tested to try to increase the shelf-life of many products, without perceptibly affecting
their sensory characteristics. Phenolic extracts from grape seeds are capable (alone or in
combination with other products) to significantly slow down the oxidation of fats, proteins
and sugars in food, as well as microbial spoilage, thus favoring a longer shelf-life.
MEAT TECHNOLOGY
Meat is one of the most widely consumed foods in the world. In its different forms (raw
or processed), it tends to have a short shelf-life, which is why the industry uses different
additives to preserve these products for a longer time periods, including sulphite. However,
the use of sulphite in foods has been questioned due to its association with certain health
problems, including allergies, lack of thiamine absorption and disruption of carbohydrate
metabolism. Using phenolic grape seed extract (GSE) can influence the shelf-life of raw meat
and its processed forms, due to its antioxidant and antimicrobial properties.
Microbiological Preservation
The protein and lipid composition and the high moisture content of the meat make it
susceptible to microbial contamination. Due to this, several research studies have been
conducted to evaluate the effect of GSE on microbial preservation, being that gallic acid, the
main active component, is effective against gram-positive bacteria. Moreover, as GSE is a
rich source of polymers of flavan-3-ols, such as (+) - catechin and (-) -epicatechin, its
antimicrobial properties can also be attributed to the general mechanisms of its phenolic
compounds. Phenolic compounds extracted from defatted grape seed extract, demonstrated
inhibitory effects on S. aureus and E. coli [18]. The minimum inhibitory GSE concentration
to have antilisterial activity is 0.26 mg GAE/L [19]. Bañón et al. [20] evaluated the effect of
the partial substitution of sulphite (SO2) by GSE for microbial conservation of raw burgers,
which have a shelf-life of about seven days in refrigeration and aerobiosis depending on
conditions of hygiene and conservation. The authors of this research described the effects of
adding GSE and the storage time of total viable counts (TVC) and total coliforms (TCC) in
raw meat patties. The study found that the partial substitution of sulphite by GSE,
significantly affects microbial count (P < 0.05), with lower counts of TVC and TCC
compared with the control on days 6 and 9. The study also demonstrated that it is possible to
reduce the use of sulphite (100 mg SO2/kg) by using GSE to retard the growth of total aerobic
and total coliform bacteria in raw meat hamburger patties, packaged in air and stored up to 9
days under conditions of minimal exposure; thus increasing their shelf-life up to three days
without affecting the pH of the food.
Similar results were found in restructured sliced lamb, refrigerated at 4°C, where the GSE
reduced the psychrophilic count under aerobic (14 days) and vacuum-packed (28 days)
conditions, being that in the latter, the total psychrophilic count was reduced from 3.51 to
3.23 log CFU/g, showing no microbial spoilage on all 28 days (4.42 log CFU/g) [21].
Therefore, it is possible to design food of good microbiological quality that also contributes to
the diet with antioxidant compounds.
Antioxidant Capacity
Lipid oxidation is one of the main reasons for the deterioration of many foods during
processing and storage. Various synthetic antioxidants such as butylated hydroxytoluene
(BHT), butylated hydroxyanisole (BHA), propyl gallate (PG) and tert-butylhydroquinone
(TBHQ) [21, 22] are currently used to retard this deterioration and prolong the shelf-life of
foods containing fat. However, it is well known that some of these compounds at
concentrations greater than those indicated could have negative effects on consumer health
and for these and other reasons new sources of natural antioxidants have been sought.
Phenolic compounds have been reported to have efficient protective action against oxidation
of lipids in meat products – especially sausages with high lipid content – and over long
periods of storage [23, 24].
The antioxidant capacity of GSE is mainly due to the flavonoids which can perform a
wiping action on free radicals (superoxide, hydroxyl, and 1,1-diphenyl-2-picrylhydrazyl
(DPPH)), on chelating properties of metals, on hydroperoxide formation reduction and,
consequently, on its effects on cell signaling pathways and gene expression [4]. Therefore,
GSE is an advantageous source of polyphenols which slows lipid oxidation when applied on
various meat products. Studies on raw burgers show that GSE together with a small amount
of sulphite (100 mg/kg), can delay lipid oxidation (TBARS) significantly (P<0.05), when
compared to a control and a sample treated merely with sulphite (0.69 ± 0.33, 3.86 ± 0.51 and
3.31 ± 0.86 mg malonaldehyde/kg sample, respectively at 9 days of storage), thus prolonging
the shelf-life of the product.
In Brazil, we used of grape seed and peel extract, from Isabel (Figure 1) and Niagara
varieties, as a natural antioxidant showed satisfactory results on the lipid oxidation retardation
of processed and cooked chicken meat stored for 14 days under refrigeration, with results
similar to the synthetic antioxidant butyl hydroxytoluene (BHT) [25]. According to these
authors, the use of grape extracts combined with vacuum packaging has demonstrated to be a
good technique to increase the lipid stability on cooked chicken meat.
Utilizing grape pomace from the same varieties (Isabel and Niagara), Selani et al. [26]
showed that grape seed and peel extracts from both varieties (Figure 2) as effective as
synthetic antioxidants BHT and sodium erythorbate on lipid oxidation prevention of
processed raw and cooked (Figure 3) chicken products stored frozen for nine months. The use
of vacuum packaging and seed and peel extracts can be considered and effective method to
retard lipid oxidation.
The use of grape seed and grape peel extract, from Isabel and Niagara varieties, as a
natural antioxidant showed satisfactory results on the lipid oxidation retardation of processed
and cooked chicken meat stored for 14 days under refrigeration, with results similar to the
synthetic antioxidant BHT [25]. According to these authors, the use of grape extracts
combined with vacuum packaging has demonstrated to be a good technique to increase the
lipid stability on cooked chicken meat.
Furthermore, the combination of different technologies for food processing has often
proved beneficial not only in terms of quality but also economically. Mielnik et al. [29],
validated the antioxidant effect of GSE on cooked turkey meat stored under refrigeration.
They showed that the antioxidant effect of GSE is dependent on the concentration (the higher
the concentration of GSE, the greater the antioxidant effect) and that when samples are
vacuum packed the effect is potentiated, remaining quite stable for 13 storage days at 4°C.
Similar results were observed in vacuum packed lamb slices [21].
Carpenter et al. [30] also showed that GSE applied to raw pork meat hamburgers, packed
under a modified atmosphere (75% O2: 25% CO2) at 4°C, had a greater positive antioxidant
effect than if left alone without the GSE; and that when the concentration was of 1000 mg/g,
it had an effect that was 4 times greater than when 50 mg/g were applied, without causing a
pro-oxidant effect. The above mentioned is important since plant extracts at low
concentrations may have pro-oxidant effects, while they cause antioxidant activity at higher
concentrations [31]. The β-carotene showed a pro-oxidant effect on chicken meat when the
concentration was 50 µg/g [32].
Source: Melo [27].
Figure 1. Grape pomace residue (Isabel variety) showed peel and seeds.
Source: Selani [28].
Figure 2. Aqueous extracts of Isabel (red) and Niagara Grape (yellow).

Source: Selani [28].
Figure 3. Raw (left photo) and cooked (right photo) burgers with four types of antioxidants (The first
figure to the left each photo: Control without antioxidant, Butyl hydroxytoluene, Isabel variety grape
seed and peel extract, Niagara variety grape seed and peel extract, and Commercial mixture of sodium
erythorbate, citric acid and sugar).
However not all extracts have the same behavior, some show antioxidant activity at low
concentrations and pro-oxidant at higher concentrations [33]. In a number of studies and in
various concentrations, GSE did not show a pro-oxidant effect, perhaps because its
composition is different from other extracts and because these are stable over time (Figure 3),
that is, no secondary compounds are generated.
When tested on fish oil to prevent oxidation, grape seed oil suppressed lipid peroxidation
in general, but it also protected the eicosapentaenoic acid (EPA), docosahexaenoic acid
(DHA) and the n-3 bioactive fatty acids from peroxidation [14] – delaying lipid and protein
oxidation in Silver carp fillets stored at 4°C [34].
Improvement of Nutritional Composition
Meat products are an important source of high bioavailable protein, fat, vitamins and
minerals in the diet of human beings, so the improvement of their overall nutritional balance
would be attractive from a nutritional point of view [35]. Therefore, the use of plant extracts
rich in phenolic compounds are being studied not only to improve product quality but also to
improve their composition.
In the area of meat technology the use of grape seed extract is very beneficial to
supplement the nutritional content in the meat. The benefits of using GSE are not only its
antioxidant and antimicrobial capacities; the phenolic compounds contained in the extract,
when absorbed in the product are conserved in it, allowing also their ingestion when the meat
product is consumed. Ribas-Agusti et al. [24] evaluated the residual concentration of the
phenolic compounds of GSE in two types of dry fermented cured meats, "salchichón" and
―Fuet,‖ at the end of the maturation process (when the product is ready for consumption). He
found that the main phenolic compounds showed a reduction at the end of the maturation
process, but that their level did not decrease significantly, at least until the end of the
commercial shelf-life. Catechin and epicatechin were at 54-61% from their initial
concentration, while higher residual percentages were found for gallic acid (81% of initial
content), oligomeric flavan-3-ols (procyanidins) (72-95% of initial content) and Epicatechin
gallate (61% of initial content).
One of the most popular ways of consuming meat is to fry it, especially chicken.
Although nowadays there are a lot of negative impressions about these food products, they
remain popular to consumers, creating a considerable challenge for food technologists. In this
regard, the nutritional quality of a piece of fried chicken depends on it containing the least
amount of oil possible, without compromising its sensory characteristics. Applying a bath of
grape seed powder and whey, may improve different characteristics of fried products,
including oil absorption. Some studies found that the moisture content in chicken nuggets
after frying varies from 65.32 ± 0.34 and 70.54 ± 1.90%, and showed a tendency to increase
in moisture with higher quantities of grape seed powder (GSP), that is, higher moisture
content and lower oil content. The author states that the higher moisture content of the
samples containing a higher amount of GSP may have led to an increase in surface tension of
the coating, thus reducing the amount of oil absorption. This means that a sufficient amount
of GSP provided a protective coating over the chicken nugget, preventing excessive
evaporation of water during the frying process [36] and generating, therefore, a healthier
product with less oil. Similarly, Özvural and Vural [23] showed that sausages made with
grape seed flour improve protein quality and dietary fiber, as well as their capacity to retain
water, which from a health viewpoint is very beneficial.
DAIRY PRODUCT TECHNOLOGY

Cheese is among the most traditional and preferred dairy derivatives in the world. For
this reason, it may be utilized as a vehicle to carry the polyphenols from a particular plant
source to the consumer‘s metabolism. Polyphenolic extracts from grape seeds can be added to
milk to produce cheese with high concentrations of these active compounds. Felix da Silva
et al. [37] evaluated the influence of adding commercial grape seed extract to milk at
concentrations of 0, 0.1, 0.2, and 0.3% w/w, for the manufacture of cheese. They determined
that the radicals scavenging was directly proportional to the phenolic compound content
contained in the extract, and that the concentration added to the milk was the main factor
influencing coagulation properties. Higher concentrations of GSE decrease syneresis of milk
gels, resulting in cheeses with higher moisture levels; also slightly increasing the recovery of
proteins in the cheese. GSE shows a good recovery ratio of polyphenols (0.87), at any
concentration; however, the author recommends a concentration range of 0.1 to 0.3% w/w to
produce cheese with potential benefits for health, without negative effect on cheese yield.
Aside from cheese, another widely consumed and growing dairy product is yogurt. Why?
Yogurt has been promoted as a nutritional and delicious product. The health benefits offered
by yogurt are related to the presence of living microorganisms, such as lactic acid bacteria,
streptococci, bifidobacteria or combinations [38]. When yogurt is complemented with
antioxidants compounds, it not only potentiates its nutritional content, but it also allows new
sensory sensations that could be attractive to the consumer. Various studies have been
conducted to complement yogurts with antioxidant compounds, such as extracts from wine,
grape extract, apple polyphenols and tea. One may think that as GSE has bactericidal
properties, it should not be used to supplement yogurt. However, Chouchouli et al. [38]
successfully used GSE for phenolic strengthening of yogurt, being that at concentrations of 5-
10 mg GAE/100 g neither the pH nor the viability of lactic acid bacteria are affected; and
also, it does not cause defects in the consistency, flavor and color, favoring oxidative stability
in storage.
TECHNOLOGY OF JUICE
Another application of GSE is related to the microbiological preservation of juices.
Alicyclobacillus spp. is a gram-positive bacteria that is non-pathogenic, aerobic, elongated,
thermophilic and acidophilic, and bacterial spore maker [39], which has been identified in
apple, tomato, white grape, grapefruit, orange and pineapple juices [40]. Among the
Alicyclobacillus species, Alicyclobacillus acidoterrestris is capable of generating compounds
that alter the taste to guaiacol and halophenols in fruit juices, something which represents a
problem in the industry. The main active component of GSE is gallic acid, which has a
destructive effect on gram-positive bacteria and has previously shown inhibitory effects on E.
coli and Salmonella enteritidis [41] – enabling its use in the elimination of A. acidoterrestris.
The effect of GSE on the elimination of vegetative cells may be due to the breakdown of the
cell wall of A. acidoterrestris; however, Molva and Baysal [40] report that more research is
needed to determine the effect of GSE on the elimination of bacterial spores, and the
variability of the sensitivity of these to GSE in terms of sporulation. It is generally accepted
that a higher concentration of GSE has a greater destructive effect on bacterial cells, by
destroying their cell wall.
However, antimicrobial activity depends on GSE concentration. A concentration between
340-390 mg GAE L-1 inhibits Gram (+) bacteria such as Bacillus and Staphylococcus, while a
concentration of 475-575 mg GAE L-1 inhibits Gram (-) bacteria such as P. aeruginosa and
E. coli [42].
NEW HORIZONS IN THE SENSORY ASPECT OF THE PRODUCT

It is not enough that a product is complete from a nutritional point of view, or that it has a
long shelf-life in terms of quality, it must taste good. Almost always, the addition of a new
ingredient to the formulation of a food modifies its chemical, physical, microbiological and
sensory properties (sometimes perceptibly, sometimes not). Here lies the challenge to mask
these changes!
In most of the examples shown in this chapter, a sensory analysis was developed along
with the main objective of the research. The authors understand that their efforts may not be
valid if the product they design shows significant variations that are perceptible by the
consumer, or worse if they do not accept them.
For example, the color red and the astringent taste of GSE can be attributed in particular
to the proanthocyanidins, which can affect the color and sensory characteristics of the product
when used in higher concentrations [4]. However, the degree of polymerization of
proanthocyanidins can also determine the antioxidant activity, since the higher the degree of
polymerization, the higher the antioxidant activity [43].
When a marinade containing grape seed flour was used to fry chicken nuggets, the
sensory analysis revealed that samples containing high amounts of GSP had lower scores of
acceptance, color and sharpness, suggesting that a level of 5% represents a viable option to
fry chicken, without significantly altering the sensory characteristics of the original product
[36]. In the sensory test of raw burgers, panelists did not detect significant differences in color
and smell among the hamburgers that had been treated with GSE and those which had not
been treated with extracts, representing a viable option for use [44]. Reddy [21] reported the
effect of GSE on the sensory attributes of slices of restructured lamb, where samples with
GSE have general acceptability scores comparable to those of the control sample and of the
sample treated with the synthetic antioxidant BHA.
Not only meat products have benefited from the addition of GSE in their formulation, but
also yogurts, cheese and even products from the wine industry. Recently, it has been used as
an additive to influence the structure and organoleptic properties of wine [5].
Used in the right amount, GSE is beneficial to the microbiological quality, oxidative
stability and nutritional composition of food products, without adversely affecting consumer
acceptance, which is the cornerstone of product development.
FOODS THAT NOURISH HEALTH

The most direct way to take these important compounds to the human metabolism is
through food. Unbalanced diets have been associated with some chronic diseases, such as
obesity, cancer and heart disease [45]. This is why many foods are being designed to include
compounds that nourish and protect the metabolism. Phenolic compounds are important in the
diet because they help prevent undesirable effects on consumer health related to high
consumption of fatty foods, such as the absorption of malonaldehyde [46] or some alterations
of lipid metabolism [47]. However, the food universe is very large and different technologies
are required to incorporate these compounds into the food.
Grape seeds (Vitis vinifera L.) are a rich source of oil with high-value of fatty acids,
proteins, phenolic compounds and a wide variety of procyanidins, which generate many
health benefits. GSE has a wide range of biological activities, such as: antioxidant properties
and radioprotective effects; prevention of cataracts; antihyperglycemic effects; improvement
of postprandial lipemia; improvement of insulin sensitivity; prevention of
hypertriglyceridemia; protection effects against oxidative damage in mouse brain cells and
anti-inflammatory effects [12].
One area where the antioxidant benefits of GSE has been proven, is the area of meat
technology, which has been able to slow lipid oxidation and improve nutritional and
microbiological quality. Since FAO estimates that in 2050 the per capita consumption of meat
will be 50 kg/person/year, the challenge of technologists, engineers and researchers is to
develop ―healthier‖ meat products by applying the knowledge generated in the research: to
improve its oil content, balance the generation of unwanted fatty acids and protein content,
and generate complete products for consumer health. How? This chapter described the
various ways in which grape seeds may be exploited and the importance of their components.
For example, it is possible to formulate marinates and sausages using grape seed flour, which
helps to reduce oil absorption, contributes to protein composition and retards oxidation.
Furthermore, the widespread use of GSE would help avoid the use of synthetic antioxidants
such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA).
The oil extracted from grape seeds has also earned a prominent place among the other
oils, since in addition to being a rich source of linoleic acid – which is associated with
promoting cardiovascular health by down-regulating the production of low density lipoprotein
cholesterol (LDL-C) [48, 49] – it also contains phenolic compounds which retard oxidation
and can be incorporated into the organism in various forms of consumption.
Other products have also taken advantage of the benefits of GSE, such as cheese and
yogurt. These are already considered nutritious products, but a supplement of GSE phenolic
compounds potentiates their metabolic function. When consumers consume yogurt, they not
only incorporate beneficial bacteria to the organism, but also the GSE metabolites which in
the circulatory system exert modulatory effects on cells through selective actions, on different
components of intracellular signaling cascades; this in turn is vital for cell functions such as
growth, proliferation and apoptosis.
It is therefore advantageous for humans to consume products containing grape seed
extract, whether in the form of polyphenol, protein or oil, to improve and potentiate the best
functioning of the metabolism.
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Chapter 9
GRAPE SEEDS (VITIS VINIFERA)

AND THEIR NUTRITIONAL VALUE
M. Rubilar1,2,3, C. Burgos-Díaz3 and J. M. Lorenzo4

1
Department of Chemical Engineering,
Universidad de La Frontera, Temuco, Chile
2
Scientific and Technological Bioresource Nucleus,
BIOREN, Universidad de La Frontera, Temuco, Chile
3
Agriaquaculture Nutritional Genomic Center, CGNA,
Technology and Processes Unit, Temuco, Chile
4
Meat Technology Center of Galicia, Ourense, Spain
ABSTRACT
Grape (Vitis vinifera L.) seeds, being a rich source of high-value fatty oil, proteins,
phenolic compounds and a wide variety of procyanidins, have received increasing
attentions because of their recognised health benefits. Grape seeds are a good source of
oil (5.8-16.8%) and protein (7.5-16.7%). Grape seed oil is characterized by lower
saturated fatty acid content and higher levels of monounsaturated fatty acids and
polyunsaturated fatty acids. Regarding fatty acid profile, linoleic acid (C18:2) is the
predominant fatty acid followed by oleic acid (C18:1) and palmitic acid (C16:0). Grape
seed oil is also rich in tocopherols, α, β and γ-tocopherols and α and γ-tocotrienols, which
are the most powerful natural fat soluble antioxidants, and are invaluable antioxidant
sources for human nutrition and healthy diets. Grape seed also presents higher essential
amino acid contens (ranged from 19.8 mg/100 g to 48.7 mg/100 g). The results of the
mineral analysis show that the grape seeds contained a considerable number of macro and
microelements. Thus, grape seeds could be used as a food supplement to improve the
nutritive value of the human diet.
Keywords: fatty acid profile, amino acid composition, mineral content, chemical composition

Corresponding author: Mónica Rubilar, Department of Chemical Engineering, Universidad de La Frontera,
Temuco, Chile. E-mail: monica.rubilar@ufrontera.cl.
198 M. Rubilar, C. Burgos-Díaz and J. M. Lorenzo
INTRODUCTION
Grapes belong to the family Vitaceae, and Vitis is a genus grouped into two sections, with
11 series and more than 60 species [1]. Vitis vinifera L., by far the most widely cultivated
species of Vitis genus, is grown throughout the temperate and tropical regions of the world for
fresh fruit, raisins, juice, and wine [2, 3]. It is therefore an extremely important fruit, and
according to the FAO over 77 million metric tons were produced in 2013 [4].
Management and disposal of large amounts of wastes produced by the food-processing
industry is a serious environmental problem. New processes for the controlled disposal of
wastes are currently being sought, aiming at converting the waste materials into other added-
value bio-products [5, 6]. These processes would cope with the environmental problems while
creating new sources of revenue that would increase the profit of the main industrial process.
During of production of water of grape, large quantities of pomace are produced as a
byproduct. In general, the pomace is not economically utilized except locally. However, the
use of pomace in the food industry can create some opportunities to lower production costs
and to create a new food source for human consumption.
About 80% of the grape production is used in juice-making, and also in this process seeds
and grape skins remained as by-product which was formulated for an animal feed [7]. Wine
by-products containing some valuable substances such as fatty acids, tocopherols and
polyphenols with potential application in the food industry mostly go to waste. Pomace,
consisting of seeds, skins and stems, is an important by-product of winemaking and the
production of traditional foods such as molasses and vinegar. An estimated 13% by weight of
the grapes processed by the wine industry ends up as by-products after pressing [8]. So, the
use of pomace in the food industry can create some opportunities to lower production costs
and to create a new food source for human consumption. Depending on the grape variety, the
seed is between 2% to 6% of the weight of the grapes and 1% to 4% of the weight of the
grape. The average yield of a seed is between 20-25% of the weight of the marc; marc dry
seeds contain 40-65% and have an oil content of 12-22% [9]. Nowadays the investigation on
grape seed has been increasing since its positive effects on human health [10, 11].
The chemical composition of grape seed is shown in Table 1. The grape seed shows a
moisture content range 5.02% and 7.16% for Narince and Aligoté varieties, respectively [12,
13]. The grape seeds are a good source of oil and protein [12]. The oil content varies
depending on the grape variety, the degree of ripeness of the grapes, soil, climate, etc.
Usually, oil content range from 5.8% to 16.8% of dry matter [12-18]. Oil of grape seed has
some unique attributes as compare with other oils and it has no effect on main flavor of food
moreover has a little flavor of butter in food; therefore it is admired by everyone [19].
Regarding protein content, grape seed shows values range from 7.5% to 16.7% [12]. In line
with this, Cotea [20] showed that the grape seed contains: water (28-40% of its weight),
cellulose (28%), nitrogenous (0.8-1.2%), tannins (4-6%), oil (10-25%) and mineral
substances (2-4%).
In addition, Licev et al. [21] also observed that grape seeds contain: water (25-45%), oil
(34-36%), tannin (4-6%), phenolic substances (2-4%), nitrogenous (4-6%), mineral
substances (2-4%), cellulose (10-11%), pentozane (8-10%) and lignin (25-28%).
Grape Seeds (Vitis Vinifera) and Their Nutritional Value 199
Table 1. Proximate compositions of grape seeds
Grape varieties Moisture (%) Oil (%) Protein (%) Ash (%) Reference
Razaki 6.54 14.29 7.48 8.0
Alphonse Lavallée 5.45 11.13 9.24 17.81
Muscat of Hamburg 5.29 10.45 10.27 19.56
Öküzgözü 5.30 11.42 11.78 18.61
Horoz karasi 5.21 11.65 11.67 20.12 [12]
Narince 5.02 13.10 12.05 19.06
Alicnate Bouchet 5.07 13.05 10.34 19.10
Cosmo 2 5.81 14.46 14.89 16.22
Salt creek 4.95 16.73 16.71 8.49
Mixtures of white varieties 5.56 6.26 9.0 3.18
Aligoté 7.16 7.21 9.78 3.35
Hybrid variety Nohan white 6.48 8.08 8.68 2.14 [13]
Hybrid variety Frăguţă a 6.98 8.15 8.75 3.20
Hybrid variety Frăguţă b 6.96 9.01 9.0 8.28
Grape seed oil is also rich in tocopherols, α, β and γ-tocopherols and α and γ-tocotrienols,
which are the most powerful natural fat-soluble antioxidants, and are invaluable antioxidant
sources for human nutrition and healthy diets [22]. According to Sabir et al. [23] grape seeds
contain primarily α-tocopherol, which possesses the highest antioxidant activity among the
tocopherols existing in functional foods. Therefore, grape seed appears to have unique
potential for human health as a great source of vitamin E. The antioxidant activity in these
compounds help to protect the oil against oxidative deterioration, and the biological activity
reduces oxidative stress on cells. Phytosterols have also been established as a prominent
feature of the unsaponifiable fraction, which can inhibit intestinal cholesterol absorption,
thereby lowering total plasma and low-density lipoprotein (LDL) cholesterol levels [24, 25].
In light of its premium antioxidant properties, the food, pharmaceutical and cosmetic
industries have shown great interest in grape seed oil.
On the other hand, grape seed is an interesting source of dietary fiber (about 35%, dried
seed) because it contains large amounts of lignin, polysaccharides and polyphenols [24, 26].
Dietary fiber (non-digestible polymers such as non-starch polysaccharides and lignin) is of
particular interest due to its benefits for human health. According to Touriño et al. [26], fibers
have laxative properties and some have been shown to reduce blood cholesterol and glucose.
Moreover, when dietary fiber reaches the colon, the polysaccharides and other non-digestible
dietary compounds (e.g., resistant starches and proteins, and high molecular weight
polyphenols) are fermented to some extent by the colonic microbiota. Since these compounds
help to stabilize the number of colonic bacteria, they are called prebiotics and may contribute
to the health benefits of fiber. The polyphenolic components of some fibers may add their
antioxidant potential to the presumed benefits of the whole preparation. Polyphenols are
powerful free-radical scavengers with antioxidant activity and antitumor effects [27]. The
consumption of polyphenol-rich food and beverages has been associated with the prevention
of diseases, particularly different types of cancer and coronary heart conditions [28].
Therefore, due to these unit properties of grape seed oil, it‘s commercialized as a food
ingredient in food industry, pharmaceutical applications, medical purposes and for cosmetic
[29].
FATTY ACID COMPOSITION OF GRAPE SEED

The fatty acid profile of grape seeds oil is presented in Table 2 and Table 3. The fatty
acid composition of grape seed and pomace are similar to the oils of safflower, sunflower,
soybean, maize, cotton seed, popy and tobacco, which belong to the linoleic type [18].
However, some authors have reported higher or lower values, depending on seed origin and
method of oil extraction. It has also been shown that extraction method influences the fatty
acid profiles of grape seed oils, as demonstrated by the comparison between oils extracted
with supercritical carbon dioxide and petroleum ether [15].
Table 2. Fatty acid composition of grape seed oil
Fatty acid (%)

Grape varieties Reference
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
Ruby red nd 7.1 nd 4.2 21.9 66.0 0.3
Muscadine nd 7.7 nd 4.7 15.4 70.2 1.1
[30]
Concord nd 7.1 nd 2.5 13.9 75.3 0.4
Chardonnay nd 7.8 nd 3.6 19.3 68.8 0.3
Kalecik Karas nd 10.2 nd 4.0 21.6 63.3 0.4
Narince nd 7.4 nd 2.9 17.7 71.4 0.1
[18]
Hasandede nd 8.7 nd 4.7 16.2 69.7 0.3
Emir nd 8.9 nd 4.1 19.8 66.2 0.3
Ghara
0.1 6.7 0.6 4.4 0.2 72.3 nd
Ghandoma
Goye Maleki 0.2 10.2 5.8 0.1 78.2 nd
[31]
Rasha 0.3 7.9 0.3 4.7 3.3 66.4 nd
Rezghi 0.2 11.5 3.6 nd 72.4 nd
Shirazi nd 9.9 0.7 5.2 nd 81.6 nd
Saperavi 6.3 8.1 nd 4.5 21.1 51.4 nd [32]
Egyptian
0.4 12.8 0.2 7.9 28.9 49.0 nd [33]
vineyards
Perlette 1.3 7.7 nd 3.2 18.2 71.7 0.9
Anib-eshahi 0.3 7.3 nd 4.8 16.7 73.3 0.8
Madess Field 0.2 8.9 nd 3.5 17.1 76.6 1.1
[34]
Black Hobbage 1.5 10.3 nd 4.3 19.8 73.2 1.5
South Columbia 0.6 11.1 nd 15.0 17.2 76.6 0.8
Autumn 0.4 9.8 nd 3.9 18.7 74.1 0.9
Lal Shahrodi 0.1 8.9 0.4 4.2 23.2 63.2 0.8
[29]
Asgari Shahrodi 0.1 9.0 0.4 4.3 25.1 65.4 0.8
Monastrell 0.1 7.9 nd 5.8 18.4 67.1 0.4
Garnacha
0.1 8.5 nd 4.6 24.9 60.9 0.5
Tintorera [35]
Petit Verdot 0.1 9.2 nd 4.7 16.1 69.2 0.5
Syrah 0.1 8.0 nd 4.4 21.8 64.5 0.6
Red Shahrodi 1.0 9.7 0.3 3.7 22.3 60.2 0.6
[19]
Fakhri Shahrodi 0.1 7.4 0.2 4.7 20.7 64.9 0.4
Fatty acid (%)

Grape varieties Reference
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
Razaki 0.1 9.5 0.2 4.4 19.1 66.4 0.4
Alphonse
0.1 8.7 0.2 3.6 19.8 67.3 0.3
Lavallée
Muscat of
0.1 8.0 nd 4.3 18.1 69.2 0.3
Hamburg
Öküzgözü 0.1 9.8 0.1 4.4 19.3 65.8 0.2
[12]
Horoz karasi 0.1 10.0 0.2 5.0 20.1 64.3 0.3
Narince 0.1 9.6 0.2 3.1 20.5 66.1 0.3
Alicnate
0.1 8.5 0.3 4.0 22.9 62.5 0.4
Bouchet
Cosmo 2 nd 8.8 nd 4.0 18.3 67.5 0.2
Salt creek nd 8.6 nd 3.4 19.5 67.6 0.2
Alicante
nd 8.3 nd 5.1 25.5 60.1 0.5
Bouschet
Bogazkere nd 7.1 nd 5.1 23.9 62.9 0.4
Clairette nd 8.1 nd 4.2 20.7 66.2 0.4
Emir nd 8.6 nd 4.7 17.8 68.1 0.5
Hasandede nd 8.6 nd 4.5 18.7 67.5 0.4
Muscat of
nd 6.5 nd 4.3 18.3 70.1 0.3 [14]
Hamburg
Kalecik karasi nd 9.0 nd 4.0 22.2 62.9 0.9
Karasakiz nd 7.9 nd 5.4 19.2 66.7 0.4
Narince nd 8.4 nd 3.5 18.7 68.8 0.5
Öküzgözü nd 8.6 nd 4.3 20.1 66.3 0.4
Pinot noir nd 9.0 nd 3.5 22.6 64.9 nd
Riesling nd 9.0 nd 4.5 22.2 63.4 0.4
nd: not detected.
The fatty acid of grape seed oils range from 9.6% to 26.7% saturated fatty acids (SFAs),
from 13.9% to 29.1% monounsatured fatty acids (MUFAs) and from 49.0% to 81.6%
polyunsatured fatty acids (PUFAs). Among the identified SFAs, palmitic acid (C16:0) is the
predominant fatty acid (between 6.7% for Ghara Ghandoma [31] and 12.8% for Egyptian
vineyards [33]) followed by stearic acid (C18:0) ranged from 2.5% for Concord [30] to 15.0%
for South Columbia [34]. The dietary guidelines have recommended avoiding saturated fat in
order to prevent cardiovascular disease [36]. The mechanisms through which SFA exert
pejorative effects in cardiovascular and general metabolic health are diverse. In this line,
Kennedy et al. [37] have proposed that an excessive consumption of SFA could promote
white adipose tissue expansion and hypertrophy leading to apoptosis. These phenomena
would promote the release of inflammatory proteins like cytokines and chemokines inducing
inflammation and insulin resistance, thus increasing the risk of cardiovascular disease and
metabolic syndrome [38, 39]. Regarding MUFA content, the oleic acid (C18:1) is the main
fatty acid range from 0.1% for Goye Maleki [31] to 28.9% for Egyptian vineyards [33]. Oleic
acid, a monounsaturated fatty acid, also has great importance in terms of their nutritional
implication and the effect on oxidative stability of oils [40].
Table 3. Nutritional index of grape seed oil
Grape varieties SFA (%) MUFA (%) PUFA (%) PUFA/SFA Reference
Ruby red 11.5 22.2 66.3 5.87 [30]
Muscadine 12.6 16.0 71.4 5.75
Concord 9.7 14.5 75.8 7.89
Chardonnay 11.5 19.5 69.0 6.06
Kalecik Karas 14.2 21.6 63.7 4.49 [18]
Narince 10.3 17.7 71.5 6.94
Hasandede 13.4 16.2 70 5.22
Emir 13 19.8 66.5 5.12
Ghara Ghandoma 11.2 16.5 72.3 6.46 [31]
Goye Maleki 16.2 5.6 78.2 4.83
Rasha 12.9 20.7 66.4 5.15
Rezghi 15.3 12.3 72.4 4.73
Shirazi 15.1 3.3 81.6 5.40
Saperavi 18.9 21.1 51.4 2.72 [32]
Egyptian vineyards 21.1 29.1 49.8 2.32 [33]
Perlette 12.2 18.2 72.6 5.95 [34]
Anib-eshahi 12.4 16.7 74.1 5.98
Madess Field 12.6 17.1 77.7 6.17
Black Hobbage 16.1 19.8 74.7 4.64
South Columbia 26.7 17.2 77.4 2.90
Autumn 14.1 18.7 75 5.32
Lal Shahrodi 13.2 23.6 64 4.85 [29]
Asgari Shahrodi 13.4 25.5 66.2 4.94
Monastrell 13.8 18.4 67.5 4.89 [35]
Garnacha Tintorera 13.2 24.9 61.4 4.65
Petit Verdot 14 16.1 69.7 4.98
Syrah 12.5 21.8 65.1 5.21
Red Shahrodi 14.4 22.6 60.8 4.22 [19]
Fakhri Shahrodi 12.2 20.9 65.3 5.35
Razaki 14 19.3 66.8 4.77 [12]
Alphonse Lavallée 12.4 20 67.6 5.45
Öküzgözü 14.3 19.4 66 4.62
Horoz karasi 15.1 20.3 64.6 4.28
Narince 12.8 20.7 66.4 5.19
Cosmo 2 12.8 18.3 67.7 5.29
Salt creek 12 19.5 67.8 5.65
Alicante Bouschet 13.4 25.5 60.6 4.52 [14]
Bogazkere 12.2 23.9 63.3 5.19
Clairette 12.3 20.7 66.6 5.41
Emir 13.3 17.8 68.6 5.16
Hasandede 13.1 18.7 67.9 5.18
Kalecik karasi 13 22.2 63.8 4.91
Karasakiz 13.3 19.2 67.1 5.05
Narince 11.9 18.7 69.3 5.82
Öküzgözü 12.9 20.1 66.7 5.17
Pinot noir 12.5 22.6 64.9 5.19
Riesling 13.5 22.2 63.8 4.73
On the other hand, the major fatty acid identified as PUFA is linoleic acid (C18:2)
accounting for 49.0% and 78.2% of total PUFAs in Egyptian vineyards [33] and Goye Maleki
[31], respectively. Compared with other vegetable oils, grape seed oil is high in linoleic acid,
only equaled by safflower seed oil [41]; however the ratio between linoleic and linolenic
acids is much lower than the usual (about 6) normally considered as optimal in the human diet
[42]. Grape seed oil is rather poor in linolenic acid (C18:3) (below 1.6%; Table 3).
Low levels of linolenic acid are desired in edible oils, because high levels of this fatty
acid can produce an unfavourable odour and taste in oil. Furthermore, since linolenic acid is
oxidised due to having three double bonds on its hydrocarbon chain, the stability or shelf-life
of oil rich in linolenic acid would be short [43, 44]. Compared with other vegetable oils,
grape seed oil has low values for this fatty acid which raises oxidative stability [45]. So, grape
seed and pomace oils having low quantities of linolenic acid, can be an advantage in terms of
human consumption and the shelf-life of the oil [18].
It was also observed that the proportions of fatty acids change among the genotypes. The
fatty acid profile of grape seeds generally exhibits a dominance of the two classes, MUFAs
and PUFAs (Table 3). Among grape seeds Egyptian vineyards have the highest level of
MUFAs (29.1%) and Goye Maleki give the highest PUFAs (78.2%). The PUFA/SFA ratio is
one of the main parameters used to assess the nutritional quality of the lipid fractions of
foods. The British Department of Health [46] recommends a PUFA/SFA ratio between 0.4
and 0.5. However, all grape seeds present PUFA/SFA ratio above 2. From the nutritional and
therapeutic point of view, grape seed oil has a high linoleic acid content (important for
prostaglandin synthesis, which has an influence on platelet aggregation and inflammatory
processes), high vitamin E content (helps to reduce the risk of suffering from arteriosclerosis)
and low values of cholesterol, and therefore it is recognized that its intake may be beneficial
to prevent heart and circulatory problems [45, 47, 48]. Although grape seed is also rich in
phenolic compounds, mainly proanthocyanidins, natural antioxidants with strong antiradical
activity, the amounts of these compounds that are recovered with the oil are very limited, due
to the polar nature of their chemical structure [49, 50].
AMINO ACID COMPOSITION OF GRAPE SEED

The amino acid composition and properties of grape seed proteins are very similar to
other cereals and oilseeds, and they could be used as food additives [51]. Moreover, grape
seed proteins show interesting functional properties such as good solubility and emulsifying
activity [52]. The amino acid content of grape seed proteins is shown in Table 4. Arginine
was included between the essential amino acids, as was done by Hoffman et al. [53], because
arginine is considered a conditionally essential amino acid [54]. The content on essential
amino acid of grape seed range from 19.8 mg/100 g to 48.7 mg/100 g, while the non essential
fraction range from 9.5 mg/100 g to 45.5 mg/100 g.
In the essential fraction, the major amino acid is tryptophan (18.7 mg/100 g) followed by
threonine (9.8 mg/100 g) and phenylalanine (8.8 mg/100 g) [32]. However, Zhou et al. [52]
observed that leucine (4.8 mg/100 g) was the most abundant amino acid followed by arginine
(4.2 mg/100 g) and valine (3.8 mg/100 g). In contrary, El-Shami et al. [33] and Kamel et al.
[55] found that arginine (9.8 mg/100 g and 5.9 mg/100 g, respectively) was the main essential
amino acid. Grape seed has higher levels of glycine and arginine in comparison to those
soybean proteins [52]. Like other legume proteins, grape seed protein lacks the sulfur-
containing amino acids methionine and cysteine (Table 4). Regarding non essential amino
acids, glutamic acid presented the higest contents (above 16 mg/100 g; [52, 55]) followed by
glycine, aspartic acid and alanime. However, El-Shami et al. [33] found that tyrosine (5.3
mg/100 g) was the most abundant, while Mchedluri et al. [32] observed that aspartic acid
(11.7 mg/100 g) was the main non essential amino acid.
Table 4. Amino acids composition (mg/100 g) of grape seed proteins
Amino acid Reference

[52] [33] [32] [55]
Leucine 4.80 nd 5.34 4.88
Valine 3.82 8.51 nd 3.44
Isoleucine 2.72 4.85 nd 2.48
Phenylalanine 2.20 6.40 8.76 2.30
Threonine 1.23 3.57 9.77 2.30
Arginine 4.15 9.84 nd 5.94
Lysine 0.65 8.78 nd 2.11
Methionine 0.19 2.21 6.15 0.86
Tryptophan nd nd 18.67 nd
Essential 19.76 44.16 48.69 24.30
Glutamic acid 17.30 nd 8.05 16.81
Glycine 8.68 nd 7.49 6.80
Aspartic acid 5.83 nd 11.65 2.87
Alanine 4.54 nd 7.48 3.35
Proline 2.59 nd 4.80 2.01
Serine 3.37 nd nd 3.16
Tyrosine 1.88 5.29 nd 1.05
Histidine 0.39 4.23 nd nd
Cysteine 0.39 nd nd 0.86
Non Essential 44.97 9.52 45.47 36.90
nd: not detected.
Table 5. Nutritional quality of protein from grape seed protein
Amino acids IOM/FNB FAO/WHO References

[52] [33] [55]
Histidine 18 15 6.0 17.2 17.8
Isoleucine 25 30 42.9 19.7 35.7
Leucine 55 59 75.6 nd 70.2
Lysine 51 45 10.0 35.7 30.3
Methionine 17 16 2.9 9.0 12.4
Phe + Tyr 47 38 84.7 47.5 48.1
Threonine 27 23 18.9 14.5 33.0
Valine 32 39 53.9 34.6 49.6
nd: not determined.
The nutrional quality of protein from grape seed and the FAO/WHO pattern [56] for
humans (children of more than one year old and adults) is shown in Table 5. The profile of
the Institute of Medicine, Food and Nutrition (FNB) [57] is also presented for comparative
purposes. The percentage of some essential amino acids of the grape seed protein, for
example, phenylalanine + tyrosine, leucine and valine fulfilled or exceeded their respective
contents fixed in the FAO⁄WHO pattern [56].
However, the overall quality of the grape seed protein is slightly compromised by the low
level of lysine and methionine, which accounted for only 22-79% and 18-77% of lysine and
methionine requirements, respectively, as recommended by the FAO⁄WHO [56] (Table 3).
The limiting levels of essential amino acids may require supplementation with
complementary proteins to reach the minimal nutritional requirements of these nutrients.
Nevertheless, it should be noted that grape seed proteins showed higher levels of glycine and
arginine compared to those of soy protein isolate [52]. Soybean protein-based formulas have
been used as the source of nutrition for infants due to their good amino acid composition [58].
Therefore, proteins from grape seeds may have a similar nutritional function to soy protein
isolate.
MINERAL CONTENT OF GRAPE SEED

Grape seeds also contain other micronutrients with interesting properties and health
benefits, such as minerals (2-4%). Minerals are very important nutrients for the human body
because they play an important role in the metabolism. They are an essential part of many
important enzymes, and they also act as catalysts and antioxidants. The content of macro and
micro elements of grape seeds is presented in Table 6 and 7. Within macro elements, calcium
is the most abundant range from 34 mg/kg to 436 mg/kg [13, 59]. The second macro mineral
is potassium with values range from 11 mg/kg to 190 mg/kg [13, 59]. Potassium is the
principal intracellular caution and with sodium helps to regulate osmotic pressure and pH
equilibrium. It also is involved with cellular enzyme function. Potassium is essential for life
but rarely is limiting even in the most meager diets [60]. On the other hand, phosphorous
presents values range 29 mg/kg to179 mg/kg [12, 13], while magnesium shows values range
from 7.1 mg/kg to 17.0 mg/kg [12, 59]. Magnesium is required in the plasma and extra
cellular fluid, where it helps to maintain osmotic equilibrium. It is required in many enzyme
catalyzed reactions, especially those in which nucleotides participate where the reactive
species is the magnesium salt [61].
Regarding micro elements, iron is the most abundant with mean values of 21.1 mg/kg
followed by manganese (16.9 mg/kg) and zinc (14.9 mg/kg) [12]. Iron is a trace mineral that
is essential for our health. It gives blood the dark red color and helps transport oxygen to our
cells ant it is also important for muscle protein and traces of it can be found in liver, spleen,
bone marrow and in our muscles [62]. However, Mironeasa et al. [13] noticed that manganese
was the main micro mineral of grape seed with mean values of 1 mg/kg followed by zinc (0.4
mg/kg) and copper (0.3 mg/kg). Manganese plays a number of essential roles in cellular
function and human metabolism. At the biochemical level, manganese can function both as a
constituent of metallo-enzymes and as an enzyme activator [61]. In addition, zinc for its part
is a multi functional nutrient involved in glucose and lipid metabolism, hormone function and
wound healing [63] and is also associated with proper hair growth [58]. Therefore, mineral
nutrients from grape seeds constitute an excellent source of several essential bioelements.
Table 6. Macrominerals content (mg/kg) of grape seeds
Grape varieties P K Mg Ca Reference

Razaki 29.0 50.0 13.0 48.0 [12]
Öküzgözü 32.0 33.0 17.0 74.0
Horoz karasi 39.0 44.0 14.0 74.0
Narince 37.0 33.0 13.0 73.0
Cosmo 2 38.0 45.0 17.0 79.0
Salt creek 44.0 41.0 14.0 64.0
Mixtures of white 48.8 84.1 nd 177.3 [13]
varieties
Aligoté 55.7 91.8 nd 179.1
Hybrid variety Nohan 40.3 50.6 nd 117.8
white
Hybrid variety Frăguţă a 68.0 76.5 nd 166.9
Hybrid variety Frăguţă b 179.5 190.9 nd 436.3
Cabernet Sauvignon nd 29.5 15.2 47.8 [59]
Merlot nd 27.5 13.2 66.5
Chardonnay nd 37.9 19.5 48.4
Seibel nd 33.9 11.4 53.6
Steuben nd 11.0 10.5 57.2
Kyoho nd 30.5 14.7 69.8
Campbell Early nd 12.9 7.1 33.8
nd: not determined.
Table 7. Micromineral content (mg/kg) of grape seeds
Grape varieties Zn Fe Mn Cu Reference

Razaki 12.3 17.3 11.1 9.3 [12]
Öküzgözü 12.6 23.0 15.3 8.8
Horoz karasi 14.8 18.6 13.8 7.3
Narince 12.3 17.7 15.7 7.5
Cosmo 2 18.9 19.3 21.5 8.6
Salt creek 19.0 21.2 23.9 12.4
Mixtures of white varieties 0.4 nd 0.8 0.2 [13]
Aligoté 0.5 nd 1.1 0.3
Hybrid variety Nohan white 0.1 nd nd 0.1
Hybrid variety Frăguţă a 0.3 nd 0.8 0.3
Hybrid variety Frăguţă b 0.8 nd 1.4 0.6
nd: not determined.
CONCLUSION
Utilisation of agro-industrial waste materials is an excellent way of adding value to crop
production and processing industries, with the added advantage of reducing waste disposal
problems. Wine and juice manufacturing produces tons of fruit seeds as by-products. Grape
seeds are only about 2-3% of the whole grape and yet, in light of their powerful natural
antioxidants, minerals, proteins, fiber and oil with high vitamin E content and essential fatty
acids, they definitely merit attention. The fatty acid profile shows that linoleic and oleic acid,
which are essential fatty acids for body and less volume of saturation acid rather other oils,
are the main fatty acid of grape seed oil. Grape seed also is an interesting source of dietary
fiber (about 35%, dried seed) because it contains large amounts of lignin, polysaccharides and
polyphenols. The amino acid composition and properties of grape seed proteins are very
similar to other cereals and oilseeds, and they could be used as food additives.
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Chapter 10
ANTIMICROBIAL AND ANTIVIRAL ACTIVITIES

OF GRAPE SEED EXTRACTS
G. Pasqua1, and G. Simonetti2

1
Department of Environmental Biology,
Sapienza Università di Roma, Rome, Italy
2
Department of Public Health and Infectious Diseases,
Sapienza Università di Roma, Rome, Italy
ABSTRACT
Grape seed extract (GSE) is a rich source of polyphenols. The polyphenols are
important secondary metabolites which play multiple essential roles in plant physiology
and which show a broad range of bioactive properties in human organism, mainly as
antioxidant, anti-inflammatory, anticancer, cardioprotection, and antiaging. GSE is
recognized as a complex mixture of monomeric, oligomeric, and polymeric flavan-3-ols.
The principal monomers identified are (+)-catechin, (−)-epicatechin, (−)-epicatechin
gallate, (−)-epigallocatechin, and (−)-epigallocatechin gallate. The content of flavan-3-ols
in seed grapes is influenced by several factors mainly cultivar, irrigation, nitrogen
fertilization, delayed harvest, and storage conditions. Antimicrobial and antiviral
activities of GSE have been described. Moreover, some researchers showed that seed
extracts were more effectively antimicrobial than other parts of grapes. The decrease
order of the antimicrobial activity is seed, skin and flesh grape extracts.
We demonstrated, for the first time, a significant correlation between the content of
the flavan-3-ols in GSEs, with a polymerization degree ≥4, and antifungal activity.
Recently, we also demonstrated a significant inhibition of Candida albicans, in an
experimental murine model of vaginal candidiasis, using GSE with high content of
polymeric flavan-3-ols. Technologies to deliver GSEs for an effective inhibition of
pathogens have been reported. GSE is Generally Recognized as Safe (GRAS), approved
by Food and Drug Administration (FDA) and also sold as dietary supplement.
Antimicrobial activity together with lack of toxicity suggests that GSE could be used for

Corresponding Author address: Gabriella Pasqua, Department of Environmental Biology, Sapienza Università di
Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy. Email: gabriella.pasqua@uniroma1.it.
212 G. Pasqua and G. Simonetti
the prevention and control of infection diseases without side effects, making greater
potential for grape in the field of food and pharmaceutical application.
Keywords: Vitis vinifera, grape seed extract, proanthocyanidins, antifungal activity,

antibacterial activity, antiviral activity
INTRODUCTION
Phytomedicine, which has historically been an important aspect of traditional medicine in
non-industrialized countries, is now becoming an integral part of healthcare in industrialized
countries. Plants are the source of thousands of new phytochemicals, and different strategies
can be applied to improve the yields of bioactive metabolites in the plant and to obtain
chemically standardized extracts [1, 2]. Along with conventional methods, numerous new
methods have been established but till now no single method is regarded as standard for
extracting bioactive compounds from plants. The efficiencies of conventional and non-
conventional extraction methods mostly depend on the critical input parameters;
understanding the nature of plant matrix and chemistry of bioactive compounds [3].
Grapes is the second largest fruit crop after orange in the world, cultivated especially in
Mediterranean area [4]. As reported by the wide literature, grapes is a rich source of
polyphenols, which are important secondary metabolites produced by the higher plants and
which play multiple essential roles in plant physiology. Polyphenols from grapes show
healthy properties in human organism, mainly as antioxidant, antiallergic, anti-inflammatory,
anticancer, antihypertensive, renoprotective, and antimicrobial agents [5-7]. Vitis vinifera
seeds contain lipids, proteins, carbohydrates, and 5–8% polyphenols, depending on the
cultivar of Vitis vinifera [8]. Standardized grape seed extracts contain from 74 to 78%
oligomeric proanthocyanidins and less than approximately 6% of free flavanol monomers on
a dry weight. The content of flavan-3-ols is influenced by several factors mainly cultivar,
irrigation, nitrogen fertilization, delayed harvest, and storage conditions [9, 10]. Moreover,
the application of an extraction process suitable to efficiently recover the target metabolites
and an appropriate analytical method for an accurate qualitative and quantitative
determination of extract components are required [11, 12].
Grape seed extract (GSE), rich in proanthocyanidins, shows potential antimicrobial
activities in preventing pathogen contamination of food [6, 13, 14].
The phenolic compounds from different parts of grapes displayed different antimicrobial
effects [6]. The decrease order of the antimicrobial activity is seed, skin and flesh grape
extracts. Anastasiadi et al. [15] suggested that high concentrations of flavonoids and their
derivatives by grape seeds, and flavonoids, stilbenes, and phenolic acids by grape stems, were
responsible for the antimicrobial activity. Ferrazzano et al. [16] reported that red grape seeds,
with high polyphenol content, had anti-cariogenic properties against Streptococcus mutans.
Moreover, GSEs inhibit the growth of anaerobic bacteria, such as Porphyromonas gingivalis
and Fusobacterium nucleatum, associated with periodontal diseases.
Rhodes et al. [17] demonstrated that polymeric phenolic fractions produced the highest
inhibition activity for all Listeria species, but not for other bacteria, such as Bacillus cereus,
Salmonella menston, Escherichia coli, Staphylococcus aureus or Yersinia enterocolitica.
Antimicrobial and Antiviral Activities of Grape Seed Extracts 213
Recently, high antifungal activity of GSE, rich in polymeric flavan-3-ols, against broad
panel of human fungal pathogens has been demonstrated against Candida spp., Cryptococcus
neoformans, and dermatophytes [10, 18]. The increase and spread of antimicrobial resistance
and the infections caused by opportunistic pathogens are the most serious public health
problems. Antimicrobial agents for the treatment of infections have become limited, leading
frequently to recurrent infections, treatment failure and increase of morbidity and mortality.
The decreased effectiveness and toxicity of current antibiotics led to search of alternative
antimicrobial substances. Plants are recognized as a source of unexplored chemical structures
with high therapeutic potential, including antimicrobial activity against clinically important
microorganisms. Main classes of phytochemicals with antimicrobial properties and their
mode of action have been studied [6, 19].
In this review, an overview of the activity of GSE against human microbial pathogens is
reported. Moreover, novel techniques of GSE are described.
GSE ACTIVE CONSTITUENTS

Grape is a phenol-rich plant and these molecules are mainly distributed in the skin, stem,
leaf and seed. In the grape berry, anthocyanins, flavonols and simple flavan-3-ols are
localized in the pericarp (skin), while flavan-3-ols are the most abundant class in the seed.
GSEs contain from 74 to 78% oligomeric proanthocyanidins and less than approximately 6%
of free flavanol monomers with respect to dry weight [20].
Catechins and proanthocyanidins are located essentially in the seeds, then in the skins and
in traces in the pulp [21]. Proanthocyanidins vary in size, ranging from dimers to polymers
with more than 40 units. GSE is recognized as a complex mixture of monomeric, oligomeric,
and polymeric flavan-3-ols. The principal monomers identified are (+)-catechin, (−)-
epicatechin, (−) epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-
epigallocatechingallate (EGCg). Prieur et al. [22] found that 55% of the procyanidins
extracted from grape seeds consisted of more than five monomer units and determined that
their mean degree of polymerization ranged from 2 to 16. Mattivi et al. [23] demonstrated that
upper and extension units of polymeric proanthocyanidins are constituted mainly of
epicatechin units, with the co-presence of catechin and epicatechin gallate. Recently we
published GSEs HPLC profiles in terms of monomers, oligomers, and polymers from
different V. vinifera cultivars. Optimizing the chromatographic method, also with the help of
an RP 18 Poroshell column, several monomer and oligomer compounds have been separated
and quantified in all the fifteen extracts. Moreover, two groups of polymeric procyanidins
(Pol 1 and Pol 2) with a polymerization degree ≥4 have been separated and determined by
their mass spectra in negative ionization mode [10]. GSE extract can be obtained from
viticultural and winemaking supply chain's waste matter, such as seeds and/or pomace and or
green seeds. Storage, delayed harvesting and the different kinds of water supply are the
variables mostly affecting grape polyphenol content. Polyphenol content diminished by more
than 50% after 6 weeks if the grapes was stored in a refrigerator [9]. It is known that to obtain
reproducible plant extracts during the years it is of fundamental importance to have a raw
material obtained in agronomic controlled conditions [24]. In GSE extracts, a high amount of
total phenols and, above all, their optimal distribution between oligomers, polymers and
gallate forms within the extract is obtained by growing the grapevines under moderate hydric
and nitrogen stress [10, 18].
Wine grape pomace (WGP), containing both seeds and skins, is a rich source of
polyphenols. Dehydration of wet pomace is a first step before developing further applications.
However, polyphenolics are sensitive to heat and oxygen. Several studies have evaluated the
effects of different drying methods on the biochemical changes of fruit pomace [25, 26].
The minimum loss of bioactive compounds was found at drying temperature not higher
than 50°C [27]. Physiochemical properties and chemical composition of dried pomace of
Pinot Noir and Merlot, subjected to different drying methods followed by storage at 15 ± 2ºC,
were evaluated by Tseng and Zhao [26]. Overall, 40ºC oven and ambient air dry are highly
acceptable by considering the amount of retention of most measured bioactive compounds
and their much less cost compared with freeze dry, thus may be employed in commercial
application of drying large quantity of wine processing by products.
ANTIBACTERIAL ACTIVITY
Gram-Positive Bacteria
Some authors reported that GSE was more effective in inhibiting Gram-positive than
Gram-negative bacteria.
GSE of V. vinifera var. Ribier black table grapes was found to be highly inhibitory
towards Listeria monocytogenes. L. monocytogenes is ubiquitous in the environment and
causes the disease listeriosis, which, although rare, has a 20–30% mortality rate. Fractionation
of the extracts showed that the antilisterial activity was strongest using the polymeric
phenolic fractions [17].
GSE from New Zealand subjected to extraction process using three different solvents
(50% aqueous acetone, 50% aqueous ethanol or 50% aqueous methanol) were tested against
Staphylococcus aureus NCTC 6571. Methanol/water seed extracts exhibited higher
antimicrobial activity against S. aureus than the other extracts. New Zealand Pinot noir grapes
was more effective against S. aureus bacteria than extracts from Pinot Meunier grapes,
suggesting that extraction solvent, fraction, and grape variety significantly influence
antimicrobial properties. The relationship between total phenolic content and antimicrobial
activities against S. aureus was found to be positively significant [28]. Baydar [29] reported
that seed extracts at 2.5% concentration had a bactericidal effect on S. aureus during the 24 h.
Both growth and biofilm formation of S. mutans UA159 were inhibited by GSE at 4
mg/mL [30]. Cueva et al. [31] reported that extracts from grape seeds were active against
Enterococcus faecalis V583, S. aureus ATCC 25923 and Streptococcus pneumonia. Furiga
[32] investigated the preventive effects of GSE on dental plaque formation. CLSM
observations of biofilms incubated with 2000 μg/ml of GSE revealed a decrease in the
number of microcolonies and thickness of biofilms. These observations suggest the need to
evaluate the germ-killing efficacy of grape compounds in oral rinses and chewing gums.
Gram-Negative Bacteria
GSE extracts prepared from Vitis rotundifolia, (cultivar Ison and from cultivar Carlos)
exhibited strong antimicrobial activity against Escherichia coli O157:H7. E. coli O157:H7 is
an enteropathogen responsible for hemorrhagic colitis, bloody diarrhea, and hemolytic uremic
syndrome. Kim et al. [33] reported that heat treatment of both extracts increased antibacterial
activity and total phenolic content.
Qui ones et al. [34] reported that commercial GSEs inhibited Shiga toxin1 and Shiga
toxin 2 produced by E. coli O157:H7. Chardonnay seed flour extracts at 165 μg seed flour
equivalents/mL exhibited bactericidal activity against E. coli [35].
Ethanol extracts of grape seeds were found to be effective in inhibiting Klebsiella
pneumoniae with MIC value of 40 µg/mL [36]. Two grape extracts currently sold as
nutritional supplements inhibited cholera toxin and E. coli heat-labile toxin activity against
cultured cells and intestinal loops by blocking toxin binding to the cell surface [37].
Cholera toxin (CT), produced by Vibrio cholera, is an AB5 toxin responsible for the
profuse, life-threatening diarrhea of cholera. The MIC of GSE, against Vibrio vulnificus was
10 mg/mL. Treatment with 500 mg/mL GSE reduced the initial inherent microbiota in fresh
shucked to below the detection level [38].
Anti-Helicobacter pylori activity of muscadine (Vitis rotundifolia) seed extract was
determined. H. pylori is considered the etiological agent of peptic ulcer and gastritis. The
MIC results indicated that the GSE had significant effects against growth of H. pylori strains
(MIC range from 256 to 1,024 μg/mL) [39].
Silván et al. [40] examined GSE activity against different Campylobacter strains
demonstrating the capacity of the GSE to inhibit Campylobacter growth in the range from
5.08 to 6.97 log CFU/mL. The analysis of the antibacterial activity against C. jejuni of the
collected fractions showed that phenolic acids, catechins and proanthocyanidins were the
main responsible of the behavior observed.
ANTIFUNGAL ACTIVITY
The highly fatal fungal systemic infections, associated with immunosuppression, are
supported mainly by Candida species, Cryptococcus neoformans and Aspergillus spp.
Superficial mucosal and cutaneous infections are mainly caused by Candida spp.,
dermatophytes and Malassezia spp. The increase in fungal infections caused an increase in
the prescription of antifungal drugs, with a resulting increase of costs. In addition, for some
fungal infections, mainly the superficial ones (for example, onychomycosis caused by
dermatophytes), the timing of the therapeutical treatment is long and often is not resolving
and subsequently they become chronic and recurrent and the remarkable side effects make
even more dramatic the already poor health conditions of the individuals who are being
treated. Many antifungal agents currently in use bring about undesired effects, they are often
ineffective toward new or recurrent agents of opportunistic infections and resistance to
antifungal therapy continue to increase [41].
The number of therapeutic options for the treatment of fungal infections is quite limited
when compared with those available to treat bacterial infections. Indeed, only three classes of
molecules are currently used in clinical practice and only one new class of antifungal drugs
has been developed in the last 30 years [42].
We reported, for the first time, anti-fungal activity and chemical analysis of GSEs
obtained from different wine and table cultivars of V. vinifera L., grown in different
agronomic conditions against a broad panel of human fungal pathogen [18].
Cheng et al. [28] have demonstred that extracts obtained from Pinot noir and Pinot
meunier seeds showed anti-Candida activity with MIC values of 0.39 and 50 mg/mL for
Pinot noir and Pinot meunier, respectively.
Recently, we compared the anti-Candida activity with respect of phenolic content in GSE
obtained by several V. vinifera cultivars. We demonstrated a significant negative correlation
coefficient of total flavan-3-ols contained in the different extracts and MIC values
(𝑟 = −0.648, 𝑃 = 0.00896). Moreover, we demonstrated, for the first time, that the antifungal
activity (MIC) of GSEs is attributable mostly to the polymeric flavan-3-ols (with a
polymerization degree ≥4) (Figure 1), with a significative negative correlation coefficient
(𝑟 = −0.6974, 𝑃 = 0.0038). Differently, the content of gallate monomers and oligomers did
not seem to be correlated to antifungal activity (𝑟 = −0.4334, 𝑃 = 0.1065) [10].
Figure 1. Flavan-3-ols with degree of polymerization ≥4 in grape seed extracts obtained by different
cultivars of Vitis vinifera L. and geometric mean MIC against Candida albicans reference strains
(ATCC90028, ATCC3153, ATCC10261, ATCC10231 and ATCC24433).
It is important to emphasize that the typical catechin of green tea, EGCg, known to be
responsible of growth-inhibitory effect on clinical isolates of Candida spp. [43], was absent in
our samples [10].
ANTIVIRAL ACTIVITY
Several authors reported antiviral activities of resveratrol, as active component in skin
grape [44-46]. The effect of a polyphenol-based grape extract (NE) obtained from Portuguese
white-winemaking by-products, and resveratrol in pure form, on adenovirus type 5 infection
has been evaluated. The NE and resveratrol reduced 4.5 and 6.5 log (TCID50/mL) on total
infectious Ad-5 production, respectively [47].
Few papers report antiviral activity of GSE. Hala [48] found that GSE was active upon
inhibiting the hepatitis C virus (HCV) replication into HepG2 cells. Feline calicivirus, FCV-
F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain
HM175) were treated with commercial GSE (Gravinol-S). At high titers (∼7 log10 PFU/ml),
FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log10 PFU/ml; MNV-1 by 0.82,
1.35, and 1.73 log10 PFU/ml; MS2 by 1.13, 1.43, and 1.60 log10 PFU/ml; and HAV by 1.81,
2.66, and 3.20 log10 PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE,
respectively in a dose-dependent manner [49]. GSE at 1 mg/ml in apple juice reduced MNV-1
to undetectable levels after 1 h and by 1 log in milk after 24 h. GSE at 1 and 2 mg/ml in AJ
reduced HAV to undetectable levels after 1 h, while 2 and 4 mg/ml GSE in milk caused ∼1
log reduction after 24 h. GSE at 2 mg/mL in intestinal fluid reduced FCV-F9, MNV-1 and
HAV to undetectable levels after 6 h [50].
Nair et al. [51] found that GSE seemed to exert antiviral effects by inducing Th1-derived
cytokine γ interferon (IFN-γ) by peripheral mononuclear cells, suggesting that the beneficial
immunostimulatory effect of GSE may be mediated through induction of IFN-γ. The
enhancing effect of GSE on IFN-γ expression was further supported by a concomitant
increase in the number of cells with intracytoplasmic IFN-γ as well as the synthesis and
secretion of IFN-γ.
ANTIMICROBIAL ACTIVITY OF GSE IN DIFFERENT

DELIVERY SYSTEMS
Over the past several years, great advances have been made on development of novel
drug delivery systems for plant extracts. The variety of novel herbal formulations like
polymeric nanoparticles, nanocapsules and liposomes, has been reported using bioactive plant
extracts. The novel formulations are reported to have remarkable advantages over
conventional formulations of plant extracts which include enhancement of solubility,
bioavailability, protection from toxicity, enhancement of pharmacological activity,
enhancement of stability, sustained delivery, and protection from physical and chemical
degradation [52].
There has been an increasing trend in the application of electrostatic spray in various
agricultural and biological systems as it has great potential in distributing the antimicrobial
compounds when applied [53]. Electrostatic spraying is a novel technology that can be used
for fine coating of antimicrobials on a biotic surface so that it can provide greater retention
and efficient distribution required to interact with pathogens [54]. Ganesh et al. [55] reported
that electrostatic spraying (in comparison to conventional treatments) of spinach with GSE
and malic acid showed 2.6 and 3.3 log reductions of L. monocytogenes and Salmonella
Typhimurium on days 7 and 14 respectively.
A novel technique of delivering employs nanoparticles that show high interactions in
biological systems [56]. Application of nanotechnology has been extensively explored in
several bio-medical areas, especially drug delivery and also has demonstrated huge potential
in nutraceuticals and functional foods for delivering bioactive compounds [57, 58]. Recently,
we demonstrated the ability of polylactic-co-glycolic acid nanoparticles (PLGA NPs) to cross
the plant cell wall and membrane of V. vinifera cell cultures and grapevine-pathogenic fungi
[59]. In grapevine plants PLGA NPs can be absorbed by the roots and transported to the
leaves through the vascular tissues. Moreover, PLGA NPs can enter in leaf tissues through
stomata openings [59].
Ravichandran et al. [60, 61] have investigated the inhibitory effects of polylactic-co-
glycolic acid nanoparticle-encapsulated GSE and MA (1% GSE + 1% MA) against L.
monocytogenes, S. Typhimurium, E. coli O157:H7 and C. jejuni. GSE-MA nanoparticles
showed effective inhibitory action against L. monocytogenes with a reduction of 5.5 log
CFU/mL and against E. coli O157:H7 and S. Typhimurium with a reduction of 5.2 log
CFU/mL and 4.9 log CFU/mL, respectively.
Edible films containing natural antimicrobials can minimize post packaging pathogen
contamination. GSE incorporated in pea starch film inhibited the growth of S. aureus, E.
faecalis, E. faecium, and L. monocytogenes [62].
Soy protein isolates (SPI) are widely used to prepare edible films [63, 64]. Antimicrobial
and physical properties of soy protein films with various natural antimicrobials have been
demonstrated [65, 66]. SPI films containing grape seed extract (GSE 1% w/w), nisin (10,000
IU/g), ethylenediaminetetraacetic acid (EDTA 0.16% w/w), inhibited the growth of L.
monocytogenes (reduction of 2.9 log CFU/mL), E. coli (1.8 log reduction) and S.
Typhimurium (0.6 log reduction) [64].
CONCLUSION
GSE has the potential to provide inexpensive antimicrobial agent for use against a broad
range infectious diseases and to protect food contamination. In phytotherapy it is crucial to
obtain extracts with reproducible chemical composition, as often the pharmacological activity
shows itself thanks to the concurrent presence of molecules, often belonging to different
chemical classes that in this way concur to define the phytocomplex. The activity of
phytoterapic products is generally performed by the synergic action of different components,
comprising the less concentrated ones, which contribute to the modulation of the product's
activity. The main industrial interest is to obtain a phytocomplex with a standardized and
reproducible content in characterized active principles, starting from low cost and safe plant
matrices and by using simple and reproducible extraction procedures. In spite of interesting
antimicrobial activity, up to date GSE hasn't been developed as antimicrobial product for
human and animal infections. Besides pharmaceutical application, the recent literature shows
positive results on the possibility to use GSE as antimicrobial agent in food conservation
systems.
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Chapter 11
GRAPE EXTRACTS: ANTIOXIDANT PROPERTIES

IN MEAT PRODUCT
G. Nieto and G. Ros

Department of Food Technology, Nutrition and Food Science,
Veterinary Faculty University of Murcia, Campus de Espinardo, Murcia, Spain
ABSTRACT
Oxidative stress, the consequence of an imbalance of prooxidants and antioxidants in
the organism, has rapidly gained recognition as a key phenomenon in chronic diseases:
cardiovascular disease, hypertension, diabetes mellitus or cancer. In addition, oxidation is
a well-known non-microbial cause of quality loss in meat. The lack of synthetic
antioxidants to combat multiple health risks associated with this stress and maintenance
of functional integrity of oxidised meat hitherto remains a challenge to the meat industry.
The harmful effects of oxidative processes in living organisms can be reduced by the
dietary intake of phenolic compounds, which are secondary plant metabolites that play a
key role in the sensory and nutritional quality of fruits, vegetables and other plants.
A search for a viable alternative amidst the unexploited novel sources of natural
antioxidants stands as a sustainable option for preserving the meat quality. Consequently,
there is a need to explore a suitable alternative from natural sources, such as plant-
derived antioxidants, to combat the challenges of oxidative instability of lipids and
protein in meat. For example, phenolic extracts from grape and grape derivatives have
been shown to be useful preservatives in fruit juices, chicken, turkey, pork meat and
horse mackerel. Among phenolic compounds in grapes, the most abundant are catechin,
epicatechin, and procyanidins (as dimers and trimers). Catechin, gallic acid and
anthocyanin have demonstrated anticancer activities, and flavanols may contribute
molecules to anti-inflammation activities. Catechin and gallic acid act as free radical
scavengers, and epicatechin has also demonstrated antibacterial activity and protective
effects against membrane oxidation. Taking into account, the properties of grape, its
extracts have been proposed as new preservatives in the food industry due to their

Corresponding Author address: Gama Nieto, Department of Food Technology, Nutrition and Food Science,
Veterinary Faculty University of Murcia, Campus de Espinardo, 30100 Espinardo, Murcia, Spain.
Email: gnieto@um.es.
226 G. Nieto and G. Ros
antioxidant properties. In this chapter, the potential use of bioactive compounds in grape
seed extract is reviewed as remedy against lipid oxidation of meat products.
Keywords: functional meat product, lipid oxidation, antioxidant, grape
INTRODUCTION
Meat and meat products are essential components in the diets of developed countries.
Health promoting functional foods, prepared on basis of meat is becoming more and more
popular. Modern consumers require nutritional food with functional properties. Among the
strategies used, meat and meat products can be modified by adding ingredients considered
beneficial for health. These strategies have been emphasized in the alteration of nutritional
profiles, for example increasing the content of polyunsaturated fatty acid (PUFA), or improve
the oxidative stability, with addition of natural antioxidants to minimize protein and lipid
oxidation in meat. The beneficial effect of producing meat products containing medicinal
plant extracts would be to combat different health related problems that have been associated
with consumption of meat over the years. Duthie et al. [1] found that including vegetable
powder in the formulation of processed turkey meat patties increase the antioxidant content,
and this may contribute to the prevention of meat related diseases. It has also been
demonstrated that the consumption of meat rich in natural antioxidant can reinforce the
activity of the endogenous antioxidants against degenerative diseases linked to oxidative
stress and ROS-related tissue damage [2].
The use of natural preservatives to increase the shelf life of meat products is a promising
technology since many vegetal substances have antioxidant and antimicrobial properties.
The quality attributes of meat products deteriorate due to the lipid oxidation during
processing and storage. Lipid oxidation is responsible for development of primary and
secondary oxidation products, reduction in nutritional quality, as well as changes in flavor [3],
which can precipitate economic losses in terms of inferior product quality [4]. Lipid oxidation
is a rather complex process whereby the unsaturated fatty acid fraction of membrane
phospholipids is oxidized, and hydroperoxides are formed which are further susceptible to
oxidation or decomposition to secondary oxidation products, such as short-chain aldehydes
and ketones, that may adversely affect the overall quality and acceptability of meat and meat
products.
The major strategies for preventing lipid oxidation are the use of antioxidants and
restricting the access to oxygen during storage vacuum-packaging. The antioxidant additives
are added to fresh and further processed meats to prevent oxidative rancidity, retard
development of off-flavors, and improve colour stability [5]. Plants‘ extracts rich in
polyphenols are good candidates, since they are easily obtained from natural sources and they
efficiently prevent lipid oxidation in food products.
Synthetic antioxidants, such as butylated hydroxytoluene (BHT), have been used to
prevent the lipid oxidation by scavenging chain-carrying peroxyl radicals or suppressing the
formation of free radicals. However, because of the concern over the safety of these synthetic
compounds, extensive work is being carried out to find novel and naturally occurring
compounds to delay the oxidative degradation of lipids, improve quality, and maintain the
Grape Extracts 227
nutritional value of foods. Thus, natural antioxidants have greater application potential in the
meat industry because of the consumers‘ acceptability over the synthetic antioxidants.
Much interest has been developed during the last few years for naturally occurring
antioxidants because of the adverse attention received by synthetic antioxidants, and also
because of the worldwide trend to avoid or minimize the use of synthetic food additives.
The research for natural antioxidants has also increased in recent years; these antioxidants
may be found in herbs, spices, grains, fruits, nuts, seeds, leaves and roots. The majority of
natural antioxidants are phenolic compounds, and the most important are the tocopherols,
flavonoids, and phenolic acids. All are generally common to all plant sources. They are added
to an extensive variety of foods, in order to prevent or retard lipid oxidation.
The use of natural preservatives to increase the shelf life of meat products is a promising
technology since many plant derived substances show antioxidant and antimicrobial
properties. In this sensse, grape seed extract has been evaluated for its antioxidative effect on
a few meat types and has been reported to improve the oxidative stability of cooked beef [6],
turkey patties and cooked stored turkey meat [7, 8].
The addition of grape extracts as antioxidant in meat, is related with these effect
associated with the presence of nutrients and phytochemical compounds: phenolic
compounds. Among phenolic compounds in grapes, the most abundant are catechin,
epicatechin, and procyanidins (as dimers and trimers). Catechin is usually the most important
individual flavanol in both grape skins and seeds, although epicatechin is also usually well
represented. Procyanidin B1 has been reported to be the main oligomer in skins, whereas all
C4–C8 procyanidin dimers (i.e., B1–B4) are usually found in seeds, of which procyanidin B2
is normally the most abundant. Levels of galloyl flavan-3-ols are more important in seeds
than in skins.
In this chapter, attempts were made to address these issues and appraise the potential use
of natural bioactive compounds from grape to ameliorate oxidative stress in meat, to prevent
lipid oxidation and improve oxidative stability in meat and meat products.
THE PHENOMENON OF OXIDATION IN MEAT

Oxidation is one of the major causes of quality deterioration in meat. Meat becomes
susceptible to oxidative deterioration due to high concentrations of unsaturated lipids, heme
pigments, metal catalysts and a range of oxidizing agents in the muscle tissue [9].
Oxidative deterioration in meat manifests in form development of off flavour, formation of
toxic compounds, nutrient and drip losses [10]. Also in form of discoloration, because the
exposure of meat to oxygen, light and temperature, as well as preservative and processing
techniques, such as chilling, freezing, additives (salt, nitrate and spices), cooking, irradiation,
high pressure and packaging, could influence the extent of oxidation and as a consecuence the
deterioration of color. Color is the most important factors influencing the consumers decision
to purchase. Consumers rely primarily on muscle colour at the point of purchase as an
indicator of freshness and anticipated palatability.
In addition, it‘s important to indicate that under normal physiologic conditions, the
molecular oxygen undergoes a series of reactions that leads to the generation of free radicals.
A small portion (about 2–5%) of the oxygen consumed during the metabolic reaction is
converted to free radicals in the form of reactive oxygen species (ROS) and these radicals
play key regulatory roles in several homeostatic processes by interacting with proteins, fatty
acids and nucleic acids and also act as intermediate agents in essential oxidation–reduction
reactions [11].
Therefore, it‘s really important to understand the activity of free radicals in meat, since
high levels of the ROS in meat could reduce its sensory quality [12] and cause loss of protein
functionality [13].
The presence of elevated levels of molecular oxygen (or triplet state oxygen) in
packaging atmospheres holds the risk for increased oxidation. However, molecular oxygen
requires energy (as from exposure to light or other irradiation) or ocurrence of reactive
species (as reactive radicals, peroxidases, metal ions) to become activated and induce
damage. The nature of protein and lipid oxidation products formed is highly dependen on
how oxidation is initiated. In general, the more reactive the formed radicals are, the less
selective reactions are initiated [14].
Currently, lipid and protein oxidation is one of the biggest economic problems in the
meat industry. It compromises the nutritional quality, limits shelf life, increases toxicity and
decreases the market value of meat and meat products [15].
Between the consequences of oxidation of cooked meat: it‘s importnat to remark the
damages in both odor/flavor of cooked meat, resulting in the development of warmed over
flavor (WOF) [16]. WOF includes odors and flavors commonly described as ―stale,‖
―cardboard-like‖ or ―rancid.‖ It starts with meat lipid oxidation, particularly the
polyunsaturated fatty acids. Once oxidized, the fatty acids usually break apart into smaller
molecules, such as pentanal, hexanal, and 2,4- decadienal, which have the off-odors and
flavors recognized as ―warmed over.‖ These substances are extremely volatile and are
perceptible in very low concentrations. Heat (cooking) is one of the primary initiators of
oxidation. Once oxidation is initiated, it is autocatalytic, increasing over time. Oxidation
proceeds rapidly in cooked meats because iron released from denatured myoglobin catalyzes
the reaction. Antioxidants which protect meat lipids, pigments or both may be effective in
reducing/delaying oxidation.
To avoid the lipid oxidation in meat, synthetic antioxidants such as butylated hydroxyl
toluene (BHT) and butylated hydroxyl anisole (BHA) have been extensively used in the food
industry. However, in recent years, consumer's pressure to reduce synthetic additives use in
foods have led to attempts to increase meat stability by natural antioxidants.
NATURAL ANTIOXIDANTS IN MEAT AND MEAT PRODUCTS

In recent years, special attention has been paid to a number of medicinal plants and
extracts that could be used as potential sources of antioxidants for muscle food preservation
and nutritional quality improvement.
The most effective antioxidants contain aromatic or phenolic rings capable of donating
H• to the free radical formed during lipid oxidation. Synthetic phenolic antioxidants
effectively inhibit WOF [17]. However, consumer concern regarding the safety of these
synthetics has motivated food manufacturers to investigate the benefits of natural antioxidants
as replacements [17].
Grape Extracts 229
Various plant materials containing polyphenolic compounds are effective antioxidants

and can retard the development of WOF in meat systems [18]. In the interest of a ―clean
label,‖ naturally occurring substances with antioxidative activity similar to the synthetics have
been of particular interest to the meat industry. Grape seed extract (GSE) is listed on the
Everything Added to Food in the United States (EAFUS) database that the Food and Drug
Administration (FDA) approved as food additives or affirmed as Generally Recognized As
Safe (GRAS). The list can be accessed on the FDA Center for Food Safety and Applied
Nutrition (CFSAN) website at http://www.cfsan.fda.gov/~dms/eafus.html.
Natural plant extract such as grape, rosemary, thyme, sage and green tea have been
shown to be effective antioxidant [19-21]. These plant extracts with antioxidant properties are
very interesting for the meat industry, and give the opportunity to develop innovative meat
products with improved nutritional and health benefits, better shelf-life and quality.
GRAPE SEED EXTRACT

Byproducts of wine/grape juice processing provide an abundant source of flavonoid
compounds. After grapes are pressed and the juice is collected, the remaining material is
known as pomace. This material contains grape seeds, skins, and/or stems. Grape seeds (rich
in proanthocyanidins) from grape juice and wine processing can be separated, extracted,
dried, and purified into GSE, which contains phenolic compounds [22].
The phenolic composition varies greatly due to grape variety, environmental and climate
conditions, soil type, degree of ripeness, and winemaking process. Touns et al. [23] reported
wide variations in the contents of total phenols (122 to 441 mg GAE/g), flavonoids 17 to 48
mc epicatechin [EC]/g), and tannins (15 to 37 mc EC/g) in the methanolic extracts from seeds
of 3 varieties of grapes. The phenolic content of grape seeds defatted with hexane then
extracted with methanol and dried under vacuum has been reported to be about 5 mg/100 g,
while the anthocyanin content is between 0.14 and 0.68 g/100 g [24].
Phenolic compounds in grape seeds and skins include catechins, epicatechins,
epicatechin-3-O-gallate, phenolic acids, caffeic acid, quercetin, myricetin, proanthocyanidins,
and resveratrol [25]. Many have strong antiradical activity. Resveratrol, quercetin, and rutin
are generally found in grape skin extracts, while catechin and epicatechin are found in the
seeds. Resveratrol inhibits peroxidation in a concentration-dependent manner. It does not
scavenge hydroxyl radical nor does it react with peroxide of nitrogen, making it an inefficient
catalyst of subsequent oxidation [26].
In addition, grape is rich in procyanidins in the form of monomers, oligomers and
polymers of polyhydroxy flavan-3-ols such as (þ)-catechin and (-)-epi-catechin, which can
couple with gallic acid to form gallate esters or sugar molecules to form glycosides [27].
The multiple mechanisms of the antioxidative activity of GSE are expressed in its ability
of radical scavenging, metal chelation, and synergism with other antioxidants [28]. Many
in vitro and in vivo studies have been conducted to examine the antioxidative properties of
GSE. GSE showed to have antioxidant activity when fed to animals. Grape seed tannins or
proanthocyanidins have been shown to have a hypocholesterolemic, antiatherosclerotic, and
antioxidant effect in vivo when fed to rats receiving diets with cholesterol [29]. Fasted rats
were administered GSE via intragastric intubation, and plasma was collected and incubated
with oxidants [30]. Results suggest that GSE protects blood plasma from oxidative stresses
[30].
In addition, grape extracts inhibited conjugated diene and hexanal formation in lecithin
liposomes [31]. Total phenolic content was highly correlated with relative percent inhibition
of conjugated diene and hexanal. Another study showed that fresh grape extracts inhibited
human LDL oxidation in vitro [32]. Moreover, antioxidative activity of grape seed extract has
been confirmed by b-carotene linoleate and linoleic acid peroxidation methods [33] as well as
by DPPH and phosphomolybdenum complex methods [34].
PREVENTION OF OXIDATIONI MEAT USING GRAPE EXTRACTS

In this section, an overview of different methods for the applications of grape in meat and
meat products is shown. GSE has been evaluated for its antioxidative effect on different meat
types and has been reported to improve the oxidative stability of meat, including raw and
cooked meat of different species:
In 2000, Nissen et al. [35] evaluated grape seed extract compared with other antioxidants:
coffee, rosemary and greep tea for its antioxidative effect in food. Nissen et al. [35] tested the
oxidative stability of four natural antioxidants in dehydrated chicken meat that was
mechanically deboned. The polyphenol content (millimoles per gram) of the antioxidants in
grape skin (1.60), coffee (1.23), rosemary (0.92), and green tea (0.57) was determined.
Analyses performed to determine the extent of lipid oxidation include electron spin
resonance, hexanal, thiobarbituric acid reactive substances (TBARS), sensory evaluation, and
conjugated dienes. The investigators found that the order of efficiency in inhibiting lipid
oxidation was rosemary ∼ synthetic antioxidants > coffee ∼ tea > grape skin > control.
Although grape skin was least efficient in decreasing lipid oxidation, it was considerably
more effective in retarding it than the control sample (no antioxidants).
In 2001, Kanner and Lapidot [36] evaluated catechin in a simulated stomach model
system, and the inhibiting lipid oxidation of meat during a gastric digestion. These authors
investigated reactions that could occur in the acidic pH of the stomach and accelerated the
generation of lipid hydroperoxides and co-oxidation of dietary constituents. The ability of
dietary polyphenols (catechin or red wine polyphenols) to invert catalysis from pro-oxidation
to antioxidation was examined. In the presence of catechin or red wine polyphenols,
metmyoglobin catalyzed the breakdown of hydroperoxides to zero, totally preventing lipid
peroxidation and β-carotene cooxidation. The results indicated the potentially harmful effects
of oxidized fats intake in the presence of endogenous catalysts found in foods, and the major
benefit of including in the meal plant dietary antioxidants.
In 2003, Lau and King [37] studied the addition of grape seed extract added at 10 and 20
g/kg to turkey thigh meat. As result of the addition of grape TBARS decreased values nearly
ten-fold as compared to the control. The authors suggested that the optimal level of grape
seed extract supplemented to poultry meat was between 1 and 10g/kg meat.
In 2007, Ahn et al. [38] evaluated the antixidant properties of grape extracts in cooked
beef. In this study the effects of butylated hydroxyanisole/butylated hydroxytoluene
(BHA/BHT), GSE, pine bark extract and oleoresin rosemary on color change, and lipid
oxidation were investigated in cooked, ground beef. The color of cooked beef treated with
Grape Extracts 231
grape seed extract showed less light (L*), more red (a*), and less yellow (b*) than those
treated with BHA/BHT, pine bark extract, and rosemary. Therefore, GSE effectively retained
the redness in cooked beef during storage. The control showed significantly higher
thiobarbituric acid reactive substances (TBARS) and hexanal content over storage. GSE
retarded the formation of TBARS by 92% after 9 days, and significantly lowered the hexanal
content throughout the storage period. GSE, in particular, has the potential to reduce lipid
oxidation and WOF in cooked ground beef when added at between 0.1% and 1%. Results of
this work show that GSE is a promising additive for maintaining the quality and safety of
cooked beef.
In 2006, Mielnik et al. [39] studied the efficiency of four concentrations of GSE (0.0, 0.4,
0.8, and 1.6 g/kg) in retarding oxidative rancidity (TBARS and volatile compound formation)
in cooked turkey breast meat stored during 13 days of refrigerated storage. Supplementation
of GSE prior to cooking significantly improved oxidative stability of minced turkey meat
during heat treatment and storage. The ability of GSE to prevent lipid oxidation was
concentration-dependent. The authors concluded that GSE could be very effective in
inhibiting lipid oxidation of cooked turkey meat during chill-storage.
In 2008, Brannan [40] studied the effect of GSE (0.1%) in ground chicken thigh meat at
59%, 76% and 88%, elaborated with a 1% NaCl level, during refrigerated storage.
The addition of GSE inhibited the formation of TBARS and altered the prooxidant effects of
NaCl without affecting the moisture content and pH of the product during storage, when
compared with the untreated control.
In 2011, Garrido et al. [41] studied the antioxidant effect of red grape pomace extracts at
0.06 g/100 g of final product concentration obtained by different extraction systems
(Extraction 1: methanolic extraction + high- low instantaneous pressure, and Extraction 2:
methanolic extraction) in pork burgers packed under aerobic conditions (4 °C) at 0, 3, and 6
days. At day zero, TBARS values in control burgers were double that of the burgers with the
grape extraction 1 (1.07 compared with 0.54); and on day 6 the TBARS values for the burgers
containing extract was 2 and 7 times lower than in the grape extraction and control burgers,
respectively, which showed the intense antioxidant effect of the red grape pomace extract in
pork burgers.
Also, in 2011, the study of Kulkarni et al. [42] was realized in pre-cooked, frozen and
reheated beef sausage. The objective was to compare three levels of GSE to commonly used
antioxidants (ascorbic acid and propyl gallate). For that sausage was manufactured from lean
beef (70%), pork fat (28%), and salt (2%). Antioxidants added for comparison with control
included grape seed extract (100, 300, and 500 ppm), ascorbic acid (AA, 100 ppm of fat) and
propyl gallate (PG, 100 ppm of fat). Product was formed into rolls, frozen, sliced into patties,
cooked on a flat griddle to 70°C, overwrapped in PVC, and then frozen at −18°C for 4
months. GSE- and PG-containing samples retained their fresh cooked beef odor and flavor
longer than controls during storage. Rancid odor and flavor scores of GSE-containing
samples were lower (P < 0.05) than those of controls after 4 months of storage. GSE-
containing samples were darker at the initiation of the study, however L* values (lightness) of
all samples increased during the 4 month storage period. TBARS of GSE containing samples
remained constant or decreased during storage while those of the control and ascorbic acid-
containing samples increased starting during the first month of storage continuing through the
4th month. GSE at the lower concentrations (100 and 300 ppm) protected these samples
against oxidation better than PE. In conclusión, based on sensory characteristics, instrumental
color (L*, a*, b* values, and hue angle) and TBARS values, GSE at the lower concentrations
(100 ppm and 300 ppm) generally performed as well as PG in maintaining product quality
throughout the 4-month storage period.
In 2014, a previous experiment that was realized by our reseach group [43] evaluated the
antioxidant properties of grape seed extract in meat. In this study he amount of GSE to
depress oxidation in turkey and pork emulsions make with mechanically separated meat
(MSM) could be estimated by means of Antioxidant capacity (AC: ORACFL) and lipid
oxidation (TBARS and lipid peroxides) before and after in vitro gastric digestion. To verify
this hypothesis and explore the efficiency of GSE in retarding of oxidation development in
processed turkey and pork meat, the new attempt was undertaken. The aim of the present
contribution was to study the influence of GSE and a simulated gastric digestion on the
antioxidant capacity of turkey and pork emulsions elaborated with mechanically separated
meat. For that, GSE was added as antioxidant in turkey and pork emulsions make with
mechanically separated meat (MSM) [43]. The Antioxidant capacity (AC: ORACFL) and
lipid oxidation (TBARS and lipid peroxides) were measured on control and emulsion added
0.5% GSE before and after in vitro gastric digestion. Results showed that gastric digestion
increased the AC of meat by 8-11-fold. Based on the data obtained, the incorporation of GSE
at 0.5% to meat emulsions has shown to be adequate to prevent lipid oxidation and improve
the AC of the emulsions; this effect was retained after simulated gastric digestion of the
samples.
FUTURE PERSPECTIVES
The use of grape seed plays an anti-oxidative and preservative role in meat during
processing and storage. In some cases, supplementing meat with medicinal plants rich-
antioxidants can act as functional or nutraceutical food to promote consumers' health and
wellness compared to the use of synthetic antioxidants. Grape seed extract have been
identified to function in this capacity, and their application in meat can provide functional or
nutraceutical meat or meat products. The beneficial effect of producing meat products
containing grape seed extracts would be to combat different health related problems that have
been associated with consumption of meat over the years. Further research will be needed to
determine the amount of natural antioxidants that is required to produce functional meat.
CONCLUSION
The use of bioactive compounds in grape seed extract as natural antioxidants has a great
antioxidant potential to preserve meat from oxidative deterioration. However, since the effect
of oxidative stress on meat quality has not been adequately investigated, there is a need to
explore this area to respond the challenges of quality losses due to oxidation. Threfore, Itis
important to investigate the efficient use of medicinal-plant-rich antioxidants as grape seed
extract to preserve the functionality of meat.
Grape Extracts 233
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ABOUT THE EDITORS
Daniel Franco has a Master in Chemical Engineering and Ph.D in Chemist Science from
university of Santiago of Compostela in 2002. During the doctorate stage he acquired
extensive knowledge of solid-liquid extraction from food matrix, as well as about near-
infrared spectroscopy and liquid chromatography. From 2003 to 2005, he worked as a
technologist in the Marine Research Institute of Vigo. During this time he acquired skills
focused on the management of R&D and technology transfer for SMEs. From 2005 to 2009
he obtained a post-doctoral position on Department of Animal Production of Agricultural
Research Center of Mabegondo. During this time he obtained experience in the field of
animal production, mainly focused on cattle and methodological aspects of meat quality such
as determination of texture parameters, sensorial and nutritional analysis (fatty acid and
amino acid profiles) by GC-FID and HPLC-FLD. From 2009 to today he joined to Meat
Technology Centre where he is head of physicochemical lab. During this time he has been
Senior Researcher of several projects of R & D and innovation related to meat science and
technology. Published more than 70 scientific articles in indexed international journals (SCI)
related to meat science and technology and published more than 110 communications to
congresses, mostly international. Email: danielfranco@ceteca.net.
José Manuel Lorenzo is Head of Research at the Meat Technology Centre of Galicia,
Ourense, Spain. He received his B.S. and M.S. in Food Science and Technology at the
University of Vigo. He obtained his Ph.D. in Food Science and Technology from University
of Vigo in 2006. He has pursued his scientific career in the Department of Food Science and
Technology at the University of Vigo, first as researcher scholarship, then, since April 2006,
as academic Researcher. In 2006-2005 from October to March, he completed a stage period
for his research project at the Stazione Sperimentale per L´Industria delle Conserve
Alimentaria, Parma, Italy.
This activity has allowed the publication of scientific paper and the share to national and
international Conferences, Seminars and Workshop. In some manifestations has contributed
with oral relations or poster. He has authored of more than 100 scientific works, more than
half published on the most accredited national and international Food Science journals (Food
Chemistry, Meat Science, Food Control, LWT Food Science and Technology, Journal of
Food Science, Journal of the Science and Food Agriculture, Journal of Food Science and
Technology, Grasas y Aceites, Journal of Integrative Agriculture, European Food Research
and Technology) and Animal Science journals (Poultry Science, Animal, Animal Production
Science, Animal Science, Spanish Journal of Agricultural Research; Archivos de Zootecnia).
Email: jmlorenzo@ceteca.net.
INDEX
albumin, 184
A aldehydes, 131, 159, 168, 169, 226
aldosterone, 129
abuse, 176
alkaline phosphatase, 68
access, 16, 46, 86, 226
alters, 76
accessibility, 122
ambient air, 214
accounting, 7, 203
American Heart Association, 209
acetaldehyde, 37, 85, 88, 92, 97, 99
amine(s), 196, 221
acetic acid, 15, 138
amino, vii, 3, 45, 46, 106, 128, 155, 159, 173, 184,
acetone, 7, 10, 15, 17, 87, 184, 214
185, 197, 203, 205, 207, 210, 237
acetonitrile, 88, 138
amino acid(s), vii, 3, 45, 46, 106, 128, 159, 173, 184,
acetylcholine, 154
185, 197, 203, 204, 205, 207, 210, 237
acid, 2, 3, 5, 6, 7, 8, 15, 16, 17, 18, 20, 28, 29, 30,
ammonium, 13, 14
31, 35, 38, 39, 40, 42, 45, 46, 49, 51, 52, 53, 54,
amylase, 22
58, 60, 61, 63, 65, 66, 68, 78, 81, 86, 87, 88, 104,
anaerobic bacteria, 61, 212
105, 106, 108, 110, 111, 112, 113, 116, 117, 123,
angiotensin II, 128, 129, 152
125, 127, 130, 137, 138, 139, 140, 142, 145,
aniline, 111, 121
147,153, 156, 159, 160, 163, 164, 169, 173, 174,
ANOVA, 131, 132, 143, 145, 150
178, 182, 185, 186, 189, 190, 191, 194, 196, 197,
antagonism, 44, 53
200, 201, 203, 204, 205, 207, 208, 209, 210, 218,
anthocyanin, 23, 52, 120, 138, 139, 153, 176, 225,
222, 225, 226, 229, 230, 231, 233, 235, 237
229
acidic, 230
antibacterial activity, 20, 56, 212, 214, 215, 220,
acidity, 168
221, 225
active compound, 109, 130, 145, 163, 190
antibiotic, 63, 78
active site, 45, 46
anti-cancer, 58, 67
adaptation, 75, 77
antifungal activity, 211, 212, 213, 215, 216, 221
additives, 157, 167, 183, 186, 227, 228
antihypertensive agents, viii, 125
adenovirus, 217
antihypertensive drugs, 143, 150
adhesion, 68, 69
antioxidant activity, 3, 6, 7, 8, 17, 21, 22, 23, 24, 25,
adiponectin, 64, 147
28, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51,
adipose, 59, 60, 76, 77, 201, 209
53, 55, 86, 99, 100, 104, 105, 106, 107, 108, 109,
adipose tissue, 60, 76, 77, 201, 209
110, 111, 113, 116, 117, 118, 119, 120, 121, 122,
adiposity, 61, 62, 74, 79
151, 154, 155, 159, 160, 162, 164, 165, 169, 172,
adults, 126, 205, 210
173, 187, 189, 191, 199, 220, 229, 234, 235
advancement, 158
antioxidant additives, 226
aerobic bacteria, 165
antioxidant/pro-oxidant activity, 27, 38
aflatoxin, 173
antioxidative activity, 42, 173, 229, 230
age, 61, 64, 152
antioxidative potential, 234
agriculture, 160
antitumor, 27, 199
240 Index
antiviral activity, 212, 217 benefits, vii, viii, 1, 27, 109, 143, 153, 160, 183, 189,
AOC, 101, 102, 103, 104, 105, 110, 112 190, 192, 193, 197, 199, 205, 210, 219, 228, 229,
aorta, 127 233
apoptosis, 60, 69, 73, 122, 137, 183, 193, 201, 219 benign, 17
appetite, 63 benzene, 54
apples, 24, 68, 69, 71, 80, 141 beverages, 112, 114, 199
aqueous solutions, 50 bile, 59
arginine, 203, 205 bioavailability, 58, 72, 73, 107, 109, 112, 113, 126,
aromatic rings, 111 132, 151, 156, 217
Aronia melanocarpa, 125, 127, 136, 137, 138, 139, biochemistry, 20, 209
140, 142, 143, 144, 145, 147, 150, 151, 154, 155 biodiesel, 207
arrest, 122 biological activities, 2, 29, 52, 60, 80, 151, 192
arterial hypertension, v, viii, 125, 126, 127, 128, 129, biological activity, 199
130, 132, 134, 136, 137, 139, 141, 142, 143, 144, biological fluids, 107, 116
150, 153, 154, 156 biological samples, 172
arteriosclerosis, 203 biological systems, 38, 103, 105, 106, 126, 130, 131,
artery, 134 142, 154, 217, 218
arthritis, 77, 124 biomarkers, 82, 105, 107, 117, 142
ascorbic acid, 6, 40, 42, 53, 102, 105, 116, 127, 173, biomaterials, 220
176, 231 biomolecules, 107
Asia, 153, 220 biosynthesis, 4, 20, 60, 63, 132
aspartic acid, 184, 204 biotechnological applications, 19, 219
assessment, 43, 44, 62 biotechnology, viii, 20
assimilation, 59 biotic, 218
astringent, 191 black tea, 86, 100
atherosclerosis, 77, 129, 234 bladder cancer, 122
atmosphere, 163, 172, 173, 175, 176, 177, 187 bleaching, 38
atoms, 108 blends, 109
ATP, 113 blood, viii, 62, 71, 82, 113, 114, 124, 125, 126, 127,
atrial fibrillation, 129 129, 130, 131, 132, 140, 141, 143, 145, 150, 151,
autooxidation, 170 152, 154, 155, 156, 199, 205, 230
blood plasma, 230
blood pressure, viii, 114, 124, 125, 126, 129, 130,
B 131, 132, 133, 134, 143, 144, 145, 147, 149, 150,
151, 152, 154, 155, 156
Bacillus subtilis, 163
bloodstream, 61, 62
bacteria, 1, 58, 61, 62, 70, 71, 75, 163, 164, 169,
body composition, 64
186, 190, 191, 193, 199, 212, 214, 221, 222
body fat, 62, 75
bacterial cells, 191
body fluid, 103
bacterial infection, 215
body weight, 8, 61, 62, 64, 65, 71, 79
bacteriophage, 217
bonding, 44
bacteriostatic, 175
bonds, 88, 93, 96, 98
bacterium, 162
bone(s), 59, 60, 80, 82, 114, 124, 205
barriers, 181
bone marrow, 205
basal lamina, 59
brain, 59, 60, 63, 113, 123, 129, 192
base, 17, 106, 115, 182
Brazil, 49, 85, 87, 99, 100, 181, 187
beef, 6, 50, 160, 163, 164, 165, 167, 169, 172, 173,
breakdown, 191, 230
175, 176, 177, 178, 194, 196, 227, 230, 231, 233,
breast cancer, 64, 122
234, 235
breast carcinoma, 73
beer, 58
buffalo, 165, 167, 176
beneficial effect, 28, 38, 42, 57, 58, 65, 67, 69, 111,
building blocks, 2
113, 144, 147, 150, 159, 226, 232
Index 241
by-product(s), vii, 1, 2, 9, 16, 17, 19, 21, 22, 23, 27, cell signaling, 38, 80, 115, 187
28, 38, 50, 55, 86, 87, 108, 110, 157, 160, 171, cell surface, 215
176, 198, 207, 210, 217, 234 cellulose, 87, 109, 198
ceruloplasmin, 147
challenges, 221, 225, 232
C cheese, 190, 192, 193, 195
chemical(s), 2, 4, 18, 25, 26, 28, 45, 54, 57, 58, 67,
Ca2+, 78, 132
86, 101, 102, 103, 105, 108, 117, 118, 123, 126,
CAE, 39
127, 158, 160, 166, 168, 170, 173, 174, 182, 185,
caffeine, 78
191, 194, 197, 198, 203, 208, 209, 213, 214, 216,
calcium, 117, 129, 205
217, 218, 223
calibration, 104, 138
chemical degradation, 158, 217
cancer, 47, 48, 55, 80, 105, 112, 115, 117, 121, 122,
chemical properties, 18, 108, 208, 223
192, 199, 222, 225
chemical reactions, 102, 158, 182
cancer cells, 112, 122
chemical structures, 2, 4, 57, 213
cancerous cells, 112
chemiluminescence, 42, 44, 52, 104, 111, 116
candidates, 142, 226
chemokines, 68, 201
candidiasis, 211
chemoprevention, 47, 48, 112, 121
capillary, 88, 127, 138, 183
chemoreceptors, 59
carbohydrate(s), 24, 158, 170, 186, 210, 212
chemotherapeutic agent, 113, 122
carbohydrate metabolism, 186
chemotherapy, 112
carbon dioxide, 18, 25, 49, 196, 200, 208
chicken, 50, 163, 167, 169, 176, 178, 187, 189, 191,
carbonyl groups, 42, 106
194, 195, 222, 223, 225, 230, 231, 233, 235
carboxylic acid, 3
children, 205
carcinogenesis, 54, 159
Chile, 197
carcinoma, 47, 112, 122
chitosan, 176
cardiovascular disease(s), 72, 82, 102, 114, 115, 126,
cholera, 215, 221
127, 130, 142, 145, 150, 151, 152, 153, 201, 209,
cholesterol, 64, 71, 79, 113, 123, 127, 132, 147, 148,
225
151, 159, 193, 199, 203, 210, 229, 234
cardiovascular morbidity, 152
cholic acid, 123
cardiovascular risk, 76, 125, 141, 153, 156, 209
chromatograms, 137
cardiovascular system, 45
chromatographic technique, 127
cargoes, 22
chromatography, 120, 208, 209
caries, 221
chromium, 124
carob, 64, 79
chronic diseases, 155, 192, 194, 225
carotene, 2, 108, 187, 195, 230
chronic heart failure, 129
carotenoids, 113, 123
cimetidine, 152
cascades, 137, 193
circulation, 59, 62, 127
catalysis, 230
classes, viii, 2, 9, 19, 29, 42, 43, 85, 86, 88, 125, 203,
catalyst, 229
213, 215, 218
catechin(s), vii, 2, 3, 5, 7, 8, 10, 15, 17, 18, 20, 25,
cleavage, 3, 30, 71
28, 29, 30, 31, 35, 36, 37, 38, 41, 42, 43, 44, 45,
climate, 108, 198, 220, 229
46, 47, 49, 51, 52, 53, 58, 67, 85, 86, 88, 89, 92,
clinical trials, 60, 130, 145
93, 96, 97, 98, 99, 108, 113, 123, 136, 138, 139,
clone, 13
142, 145, 160, 161, 162, 174, 181, 182, 186, 189,
closure, 3
211, 213, 215, 217, 221, 225, 227, 229, 230, 234
CO2, 14, 15, 18, 30, 119, 185, 187, 194
cation, 69, 116, 140
coatings, 223
cattle, 175, 237
cocoa, 54, 58, 71, 82, 194
cecum, 118
coenzyme, 132
cell biology, 20, 221
coffee, 63, 230
cell culture, 42, 107, 112, 218
colic, 113
cell death, 112
colitis, 47, 67, 68, 79, 81, 215
cell line(s), 8, 47, 63, 67, 112
collaboration, 150
cell membranes, 45, 131, 162
242 Index
collagen, 77, 127, 210 Croatia, 7, 24, 48, 120, 172, 233
colon, 47, 59, 61, 63, 68, 69, 70, 73, 80, 81, 109, crop, 28, 207, 212
112, 199 crop production, 207
colon cancer, 80 CRP, 71
color, 2, 5, 7, 157, 159, 160, 164, 165, 166, 168, 170, cultivars, 7, 24, 29, 53, 55, 120, 121, 194, 209, 213,
171, 175, 177, 190, 191, 205, 227, 230, 232, 235 216, 219, 234
colorectal adenocarcinoma, 67 culture, 42, 46, 69, 70, 176
colour stability, 176, 226 culture medium, 70
combined effect, 176 curcumin, 55, 64, 77
commercial, 6, 109, 160, 164, 165, 166, 167, 168, cuticle, 5
170, 173, 189, 190, 195, 214, 215, 217, 234 cycles, 14, 87, 145
community, 160 cycling, 42
complement, 182, 190 cyclooxygenase, 67, 145
complications, 58, 128, 129 cysteine, 107, 131, 204
composition, vii, 1, 2, 5, 9, 19, 21, 22, 23, 25, 27, 28, cytochrome, 111, 114, 121
29, 37, 50, 51, 55, 61, 62, 63, 64, 70, 71, 72, 75, cytokines, 63, 67, 68, 69, 80, 81, 201
76, 77, 82, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, cytomegalovirus, 222
97, 98, 100, 108, 109, 110, 119, 127, 154, 158, cytoskeleton, 82
171, 172, 174, 178, 182, 184, 185, 186, 189, 192, cytotoxicity, 7, 52, 81, 112, 122
194, 197, 198, 200, 203, 204, 205, 207, 208, 209,
210, 214, 218, 229
condensation, 43, 93 D
conditionally essential, 203
damages, 102, 105, 111, 112, 228
conditioning, 18, 58
data collection, 131, 143, 145
configuration, 3
data processing, 131, 145
conjugated dienes, 159, 230
database, 229
conjugation, 41, 42, 107
decay, 106, 140, 142
consensus, 150
decomposition, 169, 226
conservation, 186, 219
defects, 81, 190
constituents, 1, 8, 21, 42, 51, 53, 55, 67, 80, 86, 108,
defence, 107, 122, 132
118, 120, 139, 140, 173, 179, 183, 208, 209, 230,
deficiency, 43
234
deficit, 26, 128
consumers, vii, 103, 157, 160, 164, 166, 170, 171,
degenerative diseases, vii, 1, 19, 226
190, 193, 226, 227, 232
degradation, 16, 18, 38, 67, 70, 103, 111, 114, 127,
consumption, 29, 44, 61, 62, 64, 71, 72, 73, 79, 130,
132, 153, 157, 164, 168, 169, 226
132, 142, 143, 145, 150, 151, 155, 170, 181, 183,
dehydration, 170
189, 192, 193, 198, 199, 201, 203, 226, 232
dendritic cell, 80, 82
contamination, 183, 186, 212, 218
dental plaque, 214
control group, 69, 134, 147, 148
dentin, 50
controversial, 75
deoxyribose, 114, 117
cooking, 168, 169, 227, 228, 231
deposition, 123
cooling, 87
deposits, 134
copper, 28, 41, 43, 205
depression, 233
correlation(s), 7, 8, 43, 44, 128, 147, 164, 169, 170,
derivatives, 2, 31, 37, 61, 116, 117, 136, 142, 190,
211, 216
212, 225
correlation coefficient, 216
desoxyribose, 106
cosmetic(s), 2, 102, 110, 182, 199
destruction, 63, 114, 124, 168
cost, 19, 183, 185, 214, 218
detection, 23, 30, 86, 99, 137, 138, 195, 215
cotton, 200
developed countries, 226
coumarins, 2
deviation, 44
covalent bond, 61
diabetes, 57, 58, 63, 72, 74, 75, 77, 103, 115, 124,
c-reactive protein, 82
154, 209, 225
creatine, 114
diabetic patients, 74
Index 243
diacylglycerol, 129
diarrhea, 215
E
diastolic blood pressure, 132, 134, 143, 144, 145,
E. coli, 186, 191, 215, 218, 222
149, 150
EAE, 9, 13
dielectric constant, 17, 184
E-cadherin, 70, 82
diet, vii, 6, 8, 22, 47, 58, 60, 61, 62, 64, 69, 70, 75,
ecology, 63, 76, 82
76, 77, 78, 79, 82, 102, 113, 114, 118, 123, 132,
economic evaluation, 25, 194
142, 143, 147, 151, 175, 181, 183, 185, 186, 189,
economic losses, 159, 226
192, 193, 196, 197, 203
economic problem, 158, 228
diet composition, 76
edema, 134
dietary fat, 62
effluents, 9
dietary fiber, vii, 65, 79, 106, 118, 121, 123, 190,
eicosapentaenoic acid, 189
199, 207, 208
electrical resistance, 69
dietary intake, 72, 115, 225
electrochemical behavior, 42
dietary supplements, 85, 99, 101, 103, 112, 114, 122
electrolyte, 128
diffusion, 9, 61, 126
electron(s), 30, 45, 46, 102, 104, 108, 111, 130, 141,
diffusivity, 18
145, 230, 235
digestion, 61, 107, 112, 118, 122, 230, 232, 235
electrophoresis, 106, 184
dilation, 153
elemental compositions, 85, 88, 89, 90, 91, 94, 95,
dimerization, 42
99
dimethylsulfoxide, 87
elucidation, 29, 100
dipeptides, 173
emulsions, 232, 235
dipoles, 16
enamel, 221
discharges, 99
enantiomers, 37, 89
diseases, vii, viii, 1, 19, 58, 61, 62, 76, 102, 107,
encapsulation, 99
111, 113, 114, 115, 126, 127, 129, 130, 136, 142,
endocrine, 58, 59, 63, 128
145, 150, 151, 199, 208, 212, 218, 226
endocrine system, 59
dissociation, 82
endosperm, 5, 28, 184, 194
distilled water, 87
endothelial cells, 59, 132
distribution, 20, 28, 48, 59, 60, 73, 107, 141, 213,
endothelial dysfunction, 125, 126, 141, 142, 151
218
endothelium, 126, 127, 130, 135, 153, 154
diuretic, 129
endotoxemia, viii, 61, 62, 76, 77
diversity, 54, 57, 58, 61, 75, 80, 86, 103
endotoxins, 61, 62, 66
DNA, 8, 41, 43, 61, 68, 102, 105, 106, 107, 112,
energy, 16, 57, 64, 65, 70, 75, 76, 102, 210, 228
114, 117, 121, 122, 126
engineering, 183
DNA damage, 8, 41, 68, 106, 112, 117, 122
enteroendocrine system, v, viii, 57, 58, 59, 60, 65
DNA repair, 121
environment(s), 28, 86, 183, 214, 220
docosahexaenoic acid, 189
environmental factors, 5
Doha, 55
environmental stress(es), 155
dominance, 203
enzymatic activity, 17
dosage, 130, 140, 145
enzymes, vii, 5, 9, 16, 17, 19, 24, 28, 43, 45, 46, 53,
dosing, 123
60, 63, 67, 71, 72, 102, 103, 105, 107, 109, 113,
double bonds, 169, 203
115, 119, 123, 127, 128, 129, 131, 132, 142, 145,
double-blind trial, 152
154, 177, 205, 219
downsizing, 110
enzyme inhibitors, 129
drug delivery, 217, 218, 222
EPA, 189
drug resistance, 221
epicatechin, vii, 3, 5, 7, 8, 10, 15, 17, 18, 20, 25, 28,
drugs, 46, 60, 112, 143, 215, 216
29, 30, 32, 33, 35, 36, 37, 43, 44, 45, 46, 49, 52,
dry matter, 7, 38, 86, 198
53, 58, 86, 89, 108, 113, 160, 161, 162, 174, 182,
drying, 108, 138, 170, 185, 214, 220
186, 189, 211, 213, 225, 227, 229, 234
DSM, 195
epicatechin gallate (ECG), vii, 7, 10, 11, 12, 13, 15,
duodenum, 61, 114
17, 29, 32, 36, 189, 211, 213
dyslipidemia, 196
epidemiology, 221
244 Index
epidermis, 5 fiber(s), 25, 65, 86, 108, 118, 121, 123, 134, 135,
epigallocatechin, vii, 3, 7, 15, 17, 18, 43, 46, 52, 58, 136, 182, 199, 207
82, 86, 161, 211, 213 fibrillation, 129
epiphysis, 128 fibrosis, 134
epithelial cells, 62, 67, 69, 80, 82 films, 218, 223
epithelium, 57, 59, 62, 81 filters, 87
equilibrium, 18, 194, 205 fish, 50, 172, 189, 221, 233
equipment, 43 fish oil, 189, 221
ESI, v, 30, 31, 32, 33, 34, 35, 36, 37, 48, 50, 85, 86, fission, 37
87, 88, 89, 90, 91, 94, 95, 100, 119, 138, 139, fixation, 127
154, 175 flavan-3-ol compounds, vii, 37, 85, 88, 98, 120
essential fatty acids, 2, 207 flavonoids, v, viii, 2, 3, 7, 10, 15, 20, 23, 26, 28, 29,
ester, 86, 116 41, 42, 45, 46, 49, 52, 54, 57, 58, 59, 63, 64, 65,
estrogen, 130 66, 67, 68, 70, 71, 79, 80, 81, 82, 86, 108, 111,
ethanol, 2, 7, 9, 15, 17, 18, 23, 35, 36, 38, 48, 69, 82, 115, 123, 127, 130, 137, 144, 151, 153, 154, 155,
105, 109, 130, 138, 183, 184, 214 156, 160, 162, 163, 174, 187, 208, 212, 227, 229
ethyl acetate, 7, 10, 15, 29, 109 flavonol, 22, 65, 207
Europe, 129 flavor, 157, 165, 173, 178, 190, 198, 220, 226, 228,
evaporation, 130, 138, 190 231
evidence, 45, 51, 57, 58, 61, 77, 81, 120, 130, 142, flavour, 24, 157, 159, 163, 164, 165, 166, 169, 172,
145, 153 178, 227
evil, 115 flight, 22, 55, 86, 208
evolution, 119, 170 flora, 82, 102
excretion, 50 flora and fauna, 102
experimental design, 219 flour, 190, 191, 192, 194, 215
expertise, viii flowers, 68
exploitation, 28, 37, 85, 87 fluid, vii, 9, 15, 16, 17, 18, 25, 29, 48, 194, 205, 207,
exposure, 42, 46, 62, 168, 186, 227, 228 217, 219, 235
extinction, 138 fluid extract, vii, 9, 15, 16, 17, 25, 29, 48, 194, 207,
extraction, vii, 1, 7, 9, 10, 13, 14, 15, 16, 17, 18, 19, 219
20, 21, 22, 23, 24, 25, 26, 29, 30, 31, 32, 33, 34, fluorescence, 30, 45, 115
35, 36, 38, 48, 49, 51, 76, 87, 99, 100, 106, 108, food, vii, viii, 1, 2, 19, 20, 27, 28, 38, 42, 44, 47, 52,
109, 110, 111, 114, 118, 119, 120, 139, 171, 176, 58, 60, 61, 63, 64, 65, 72, 79, 85, 99, 102, 103,
183, 184, 185, 193, 194, 200, 207, 212, 214, 218, 105, 106, 110, 111, 112, 118, 119, 121, 123, 126,
219, 220, 231, 235, 237 137, 140, 144, 145, 151, 153, 157, 159, 160, 163,
extraction procedures, vii, 9, 218 167, 172, 173, 174, 177, 181, 182, 183, 185, 186,
187, 189, 191, 192, 193, 197, 198, 199, 203, 207,
208, 212, 218, 220, 222, 225, 226, 227, 228, 229,
F 230, 232, 233, 234, 237
food additive(s), 177, 203, 207, 227, 229, 234
fasting, 64, 74
Food and Drug Administration (FDA), vii, 159, 160,
fat, 6, 8, 21, 47, 60, 62, 64, 65, 70, 75, 76, 77, 82,
211, 229
103, 105, 159, 164, 166, 167, 168, 170, 177, 178,
food industry, vii, 19, 28, 102, 103, 105, 111, 121,
186, 189, 196, 197, 199, 201, 207, 210, 231
157, 160, 182, 198, 199, 225, 228
fat soluble, 197
food ingredients, 85, 99
fatty acids, 28, 60, 62, 64, 159, 166, 185, 189, 192,
food intake, 58, 60, 63, 64, 65, 72, 79
198, 201, 203, 207, 209, 210, 228
food products, 159, 181, 182, 185, 189, 192, 226
fear, 128
food safety, 20, 119, 173, 193, 220, 233
feces, 82
formation, 42, 43, 46, 102, 105, 111, 130, 140, 141,
feedstock, 18
163, 164, 168, 178, 187, 196, 214, 221, 226, 227,
fermentation, 2, 63, 87, 89, 107, 108, 112, 118, 170,
230, 231
178
formula, 86, 131
fertility, 2
fertilization, 211, 212
Index 245
Fourier transform ion cyclotron resonance (FTICR), 48, 49, 50, 52, 54, 56, 63, 109, 110, 111, 112,
v, 31, 32, 33, 34, 50, 85, 86, 87, 88, 89, 90, 91, 113, 119, 120, 121, 122, 123, 124, 157, 160, 162,
94, 95, 100 163, 171, 173, 174, 175, 176, 177, 178, 181, 182,
fragments, 30, 37, 106 186, 189, 190, 193, 194, 195, 210, 211, 212, 213,
France, 207 215, 216, 218, 220, 221, 222, 223, 226, 227, 229,
free radicals, 5, 43, 45, 101, 102, 103, 110, 114, 126, 230, 231, 232, 233, 235
128, 130, 131, 135, 141, 154, 158, 160, 182, 187, grape seed procyanidins, 8, 72, 86, 99, 124
226, 227, 228 grape seeds, v, vii, viii, 1, 2, 3, 5, 6, 7, 8, 9, 10, 15,
freezing, 103, 227 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
fructose, 78 29, 30, 31, 35, 37, 38, 42, 43, 47, 49, 50, 55, 85,
fruits, 58, 64, 67, 69, 71, 79, 81, 134, 137, 139, 142, 86, 87, 89, 92, 99, 101, 108, 109, 110, 111, 113,
143, 156, 159, 177, 181, 210, 225, 227 114, 118, 119, 120, 121, 123, 157, 160, 173, 174,
functional food, 51, 103, 126, 135, 155, 195, 199, 181, 182, 184, 185, 190, 192, 193, 196, 197, 198,
218, 226, 233 199, 200, 203, 205, 206, 207, 208, 209, 212, 213,
funds, 47 214, 215, 219, 220, 229, 234
fungal infection, 215 grape varieties, 9, 10, 11, 12, 13, 14, 15, 20, 21, 37,
fungi, 63, 218, 222 43, 48, 50, 51, 52, 85, 87, 88, 94, 96, 98, 120,
162, 184, 199, 200, 201, 202, 206, 208, 209, 210,
220
G GRAS, 160, 211, 229
Greece, 7, 53
galloylation, 8, 29, 59, 86, 109
growth, 43, 47, 70, 71, 72, 108, 112, 122, 157, 173,
gastritis, 215
175, 186, 193, 206, 212, 214, 215, 217, 218, 235
gastrointestinal tract, viii, 57, 58, 60, 71, 72, 73, 105,
guidelines, 201, 209
109, 112
GC-FID, 237
gel, 106 H
gene expression, 38, 67, 68, 71, 80, 82, 113, 131,
132, 142, 153, 187 H. pylori, 215
genes, 67, 119, 132, 196 hair, 206
genetics, 20 hardness, 170
genomics, 75 harmful effects, 225, 230
genotype, 154 harvesting, 213
genus, 198 health, vii, viii, 1, 2, 18, 19, 27, 28, 38, 41, 57, 61,
Germany, 85, 87, 88, 138, 163 67, 71, 72, 73, 75, 82, 108, 109, 111, 112, 115,
gland, 59 119, 120, 135, 143, 150, 151, 153, 154, 159, 160,
glucagon, 59, 74, 78 168, 182, 186, 190, 192, 196, 197, 199, 201, 205,
glucose, 8, 58, 59, 60, 62, 63, 64, 73, 74, 75, 76, 78, 209, 210, 215, 219, 225, 226, 229, 232, 233, 234
199, 205 health condition, 215
glucose tolerance, 60, 74, 75, 78 health effects, 41, 61, 67, 154, 234
glucoside, 15, 32, 37, 139 health problems, 57, 186
glutamic acid, 184, 204 health risks, 143, 159, 168, 225
glutathion (GSH), 107, 113, 125, 127, 130, 131, 132, heart disease, 129, 192
133, 140, 141, 142, 147 heart failure, 129
glutathione, 102, 103, 107, 113, 125, 130, 131, 132, heart rate, 143
140, 142, 153, 154 Helicobacter pylori, 215, 221
glycans, 71 heme, 142, 164, 168, 169, 178, 227
glycine, 184, 204, 205 heme oxygenase, 142
glycoside, 139 hemolytic uremic syndrome, 215
goblet cells, 59 hepatitis, 217, 222
Google, 30 hepatocytes, 113, 123
grades, 15, 70 herpes simplex, 221
grape seed extract(s) (GSEs), v, vii, 3, 5, 6, 7, 8, 17, hexane, 15, 184, 185, 229
20, 21, 24, 27, 28, 29, 30, 37, 38, 39, 43, 45, 47, high density lipoprotein, 114
246 Index
high fat, 6, 61, 64, 76, 79 immunity, 76, 81

histidine, 128 immunomodulation, 75
history, 64 immunomodulatory, 58
HLA-B27, 80 immunostimulatory, 217
homeostasis, viii, 57, 58, 60, 62, 63, 70, 126, 128, immunosuppression, 215
131 improvements, 132, 147
hormone(s), 57, 59, 60, 63, 64, 65, 74, 78, 79, 128, in vitro, vii, 6, 8, 19, 21, 23, 25, 27, 42, 43, 47, 52,
205 53, 63, 66, 67, 69, 71, 78, 81, 101, 105, 107, 109,
host, 61 110, 112, 114, 117, 118, 119, 120, 122, 123, 137,
hue, 232 155, 160, 164, 207, 221, 222, 229, 230, 232, 235
human, vii, 21, 46, 47, 52, 53, 54, 57, 58, 61, 62, 63, in vivo, vii, 8, 19, 27, 43, 47, 52, 59, 66, 67, 69, 71,
67, 70, 71, 74, 75, 76, 77, 80, 82, 102, 107, 108, 78, 83, 101, 105, 109, 110, 112, 113, 117, 120,
112, 115, 118, 119, 122, 126, 144, 150, 151, 155, 122, 123, 137, 143, 155, 160, 219, 229
162, 189, 192, 197, 198, 199, 203, 205, 210, 211, incidence, 74, 156
212, 213, 216, 219, 222, 230 income, 128
human body, 61, 70, 126, 205 India, 166
human health, 58, 71, 108, 112, 119, 144, 162, 198, Indians, 137
199 individuals, 61, 71, 114, 150, 151, 155, 215
human subjects, 62 inducer, 61
humidity, 235 induction, 122, 132, 217, 219
hybrid, 22, 50, 55, 88, 164, 167 industrial processing, 28
hydrogen, 38, 44, 46, 107, 108, 141, 184 industrialized countries, 212
hydrogen atoms, 108, 141 industries, vii, viii, 2, 16, 19, 102, 103, 160, 181,
hydrogen bonds, 46, 184 199, 207
hydrogen peroxide, 107 industry, 1, 2, 16, 22, 29, 38, 50, 86, 103, 108, 111,
hydrolysis, 7, 16, 25, 29 121, 157, 158, 159, 172, 181, 185, 191, 192, 194,
hydroperoxides, 8, 45, 111, 159, 163, 169, 226, 230 195, 198, 207, 225, 227, 228, 229, 233
hydroquinone, 173 INF, 67, 69, 70
hydroxyl, 2, 6, 8, 41, 42, 43, 46, 105, 106, 108, 110, infants, 205
111, 117, 126, 141, 187, 228, 229 infection, viii, 63, 212, 217
hydroxyl groups, 2, 41, 42, 111, 126 inflammasome, 81
hygiene, 186 inflammation, vii, 1, 27, 57, 61, 62, 63, 66, 67, 68,
hypercholesterolemia, 123, 151 69, 71, 73, 76, 77, 78, 80, 81, 114, 123, 129, 147,
hyperglycemia, 60 153, 201, 209, 225
hypertension, viii, 114, 124, 125, 126, 127, 128, 129, inflammatory bowel disease, 47, 68, 71, 79, 81
130, 132, 134, 137, 139, 141, 142, 143, 145, 147, inflammatory disease, 132
150, 151, 152, 153, 154, 155, 156, 225 inflammatory mediators, 66
hypertriglyceridemia, 192 inflammatory responses, 67
hypertrophy, 129, 141, 152, 201 infrared spectroscopy, 237
hypotensive, 127 ingestion, 50, 59, 62, 74, 113, 189
hypothesis, 68, 142, 232 ingredients, 85, 99, 103, 109, 173, 182, 220, 226
inheritance, 29
inhibition, 38, 39, 41, 43, 45, 46, 53, 67, 68, 70, 80,
I 81, 104, 105, 106, 111, 114, 127, 128, 129, 135,
145, 150, 164, 165, 168, 177, 211, 212, 221, 230
ICAM, 68
inhibitor, 46, 111, 129, 145, 150, 152, 163, 174
ideal, 10, 86, 102
initiation, 158, 231
identification, 9, 23, 25, 26, 29, 30, 125, 139, 173,
injury, 70, 72, 81, 153
175, 184, 194, 196
innate immunity, 76
IFN, 70, 82, 217
inositol, 129
IL-8, 68
insertion, 87
ileum, 61
institutions, 103
immune response, 58, 61
immune system, 57, 61, 70, 77
Index 247
insulin, 8, 47, 57, 58, 59, 60, 62, 63, 64, 65, 72, 74,
77, 78, 79, 154, 192, 201, 209
L
insulin resistance, 8, 57, 58, 60, 62, 63, 65, 72, 77,
lactation, 64
78, 79, 201, 209
lactic acid, 1, 190
insulin sensitivity, 63, 64, 78, 79, 192
Lactobacillus, 61, 71
insulin signaling, 62
lactoferrin, 210
integration, 150
laws, 159
integrity, 2, 62, 69, 72, 126, 135, 225
LC-MS, 27, 30, 178
integument, 5
leaching, 183
intercellular adhesion molecule, 68
leakage, 62, 81
interface, 106
lecithin, 230, 234
interference, 30
legume, 184, 204
interferon, 67, 81, 217
lens, 88, 134
interferon gamma, 81
leptin, 64
intermolecular interactions, 27
lesions, 134
international standards, 159
leucine, 128, 184, 203, 205
intervention, 70, 82, 113, 117, 126, 134, 142, 150
leucocyte, 42, 46
intestinal tract, 59
liberation, 109
intestine, 57, 58, 59, 61, 63, 66, 81, 107, 109
LIFE, 152
intima, 126, 135
lifetime, 113
intoxication, 221
ligand, 68
investment, 185
light, 5, 145, 158, 199, 207, 222, 227, 228, 231
ionization, 37, 85, 86, 100, 127, 208, 213
lignans, 3, 130
ions, 30, 37, 43, 86, 88, 117, 129, 139
lignin, 3, 24, 198, 199, 207
iron, 28, 30, 41, 45, 46, 51, 121, 142, 164, 168, 169,
linoleic acid, 2, 28, 38, 54, 170, 192, 197, 203, 230
175, 178, 205, 228
lipemia, 192
irradiation, 227, 228
lipid metabolism, 192, 205
irrigation, 211, 212, 219
lipid oxidation, 1, 5, 6, 20, 65, 79, 111, 112, 113,
IRS, 62
121, 122, 157, 158, 159, 163, 164, 165, 166, 168,
isoflavone, 64, 65, 69, 79, 132
169, 170, 171, 172, 175, 176, 187, 192, 194, 221,
isoflavonoids, 64, 79
226, 227, 228, 230, 231, 232, 233, 235
isolation, 9, 29, 195
lipid peroxidation, 42, 45, 46, 68, 105, 106, 111, 113,
isoleucine, 184
126, 131, 140, 141, 165, 189, 196, 230
isomeric compounds, 87
lipid peroxides, 153, 232
isomers, 3, 85, 88, 89, 92, 93, 96, 97, 98, 136
lipids, 10, 26, 38, 60, 76, 79, 107, 111, 131, 141,
issues, 158, 227
145, 155, 157, 158, 159, 187, 212, 225, 226, 227,
Italy, 114, 211, 237
228, 233
lipolysis, 74, 170
J lipoproteins, 79, 114, 116, 131, 145, 153
liposomes, 217, 230
Japan, 22, 160, 196 liquid chromatography, 23, 27, 29, 30, 31, 35, 86, 99,
jejunum, 61 100, 120, 208, 234, 237
Jordan, 234 Listeria monocytogenes, 72, 163, 175, 214, 222, 223
liver, vii, 59, 60, 77, 81, 106, 113, 121, 123, 167,
168, 171, 177, 178, 196, 205
K liver disease, 77
local conditions, 129
ketones, 159, 226 localization, 59, 126
kidney(s), 7, 59, 60, 128, 129 lovastatin, 152
kinase activity, 80, 82 low-density lipoprotein (LDL), 6, 8, 9, 20, 21, 38,
kinetic curves, 17 52, 53, 104, 105, 116, 127, 131, 132, 133, 135,
kinetics, 15, 23, 26, 49, 126, 194 141, 145, 147, 155, 174, 193, 199, 230
Korea, 210 low-grade inflammation, 47, 61, 75, 76
248 Index
lumen, 58, 59, 61, 62, 69 medulla, 128

luminosity, 163, 165, 168, 169, 170, 171 mellitus, 60, 75, 77, 115, 154, 209, 225
lung cancer, 122 melon, 210
Luo, 54, 79, 81, 195, 234 membranes, 8
lymphocytes, 112 metabolic, v, 57, 61, 77
lymphoid tissue, 62 metabolic disturbances, 61
lysine, 184, 205 metabolic pathways, 70, 135
lysis, 126, 135 metabolic syndrome, 76, 77, 79, 114, 124, 155, 201,
209
metabolism, 42, 50, 60, 68, 71, 72, 73, 74, 76, 105,
M 132, 151, 153, 183, 190, 192, 193, 205, 209
metabolites, 59, 68, 70, 71, 73, 81, 82, 102, 113, 118,
Macedonia, 50
123, 126, 193, 211, 212, 225
machinery, 43
metabolized, 57, 59
macromolecules, 62
metal ion(s), 41, 159, 228
macrophages, 80, 155
metals, 41, 52, 123, 130, 187
magnesium, 28, 205
metastasis, 73
magnet, 88
methanol, 7, 10, 15, 17, 38, 109, 138, 184, 214, 229
majority, 227
methodology, 48, 53
MALDI, 173
methylation, 59, 80
mammals, 70
mice, 47, 59, 62, 63, 64, 66, 68, 69, 74, 76, 77, 78,
management, 126, 184, 237
79, 80, 81, 152
manganese, 28, 205
microbiota, 57, 58, 59, 61, 62, 63, 68, 70, 71, 72, 73,
manipulation, 102
75, 76, 77, 78, 81, 82, 109, 164, 199, 215
manufacturing, 159, 168, 179, 207
microcirculation, 154
marrow, 80, 82
micronutrients, 205
mass, 5, 16, 17, 18, 22, 27, 28, 30, 31, 35, 37, 50, 55,
microorganism(s), 43, 45, 61, 70, 163, 165, 190, 213
85, 86, 88, 89, 90, 91, 94, 95, 97, 98, 99, 100,
microscope, 134
127, 138, 139, 173, 183, 184, 194, 208, 209, 213
microsomes, 106, 111
mass resolution, 86
microwave heating, 20
mass spectrometry, 22, 23, 27, 30, 31, 35, 37, 50, 55,
microwaves, 19
85, 86, 88, 89, 90, 91, 94, 95, 97, 98, 99, 100,
migration, 122
125, 138, 173, 208, 209
milligrams, 30
materials, 30, 41, 106, 160, 198, 207, 219, 222, 229
Ministry of Education, 47
matrix, vii, 9, 87, 102, 108, 110, 123, 183, 212, 237
mitochondrial damage, 81
matter, 10, 106, 183, 213
mitochondrial DNA, 126
maturation process, 189
mitogen, 62
McGillicuddy, 77
mixing, 18
MCP, 68
model system, 6, 20, 41, 53, 54, 107, 121, 163, 175,
MCP-1, 68
230, 235
MDA, 45, 106, 122, 125, 131, 133, 140, 141, 142,
modelling, 24, 25
163, 164, 165, 166, 168, 171
models, 21, 23, 43, 54, 66, 68, 69, 71, 73, 80, 81,
measurement(s), 44, 55, 88, 104, 105, 115, 116, 117,
107, 112, 113, 118, 120, 123, 129, 130, 145, 163,
118, 131, 143, 145
194, 235
meat, vii, 50, 103, 111, 157, 158, 159, 160, 163, 164,
modifications, 3, 5, 16, 62, 64, 104, 105, 126, 130,
165, 166, 167, 168, 169, 170, 171, 172, 173, 175,
131, 133, 135, 141, 168, 170
176, 177, 178, 186, 187, 189, 192, 194, 195, 207,
moisture, 170, 186, 190, 198, 231
210, 222, 223, 225, 226, 227, 228, 229, 230, 231,
moisture content, 170, 186, 190, 198, 231
232, 233, 234, 235, 237
molasses, 198
media, 104, 126, 135
molecular formula, 86
medical, 103, 199, 218
molecular mass, 3
medication, 128, 129
molecular oxygen, 227, 228
medicine, 53, 151, 212, 222
molecular structure, 41
Mediterranean, 177, 212
Index 249
molecular weight, 7, 16, 20, 21, 26, 38, 49, 55, 86, NIR, 20
106, 109, 110, 120, 173, 199 nitric oxide, 67, 143, 145, 155
molecules, 3, 27, 29, 45, 46, 58, 68, 70, 86, 103, 105, nitric oxide synthase, 67, 145
131, 162, 163, 213, 216, 218, 225, 228, 229 nitrite, 168
monolayer, 69 nitrogen, 87, 104, 159, 211, 212, 214, 219, 229
monomers, 3, 5, 8, 29, 30, 58, 86, 108, 160, 182, nitrosamines, 168
211, 212, 213, 216, 229 non-polar, 15, 17
monotherapy, viii, 125, 129 norepinephrine, 156
monounsaturated fatty acids, 197 normal distribution, 131, 143, 145
morbidity, 213 North America, 136
morphology, 59 Nrf2, 132, 142, 155
mortality, 152, 156, 213, 214 nuclei, 135, 136
mortality rate, 214 nucleic acid, 228
mRNA, 67, 107, 113 nucleotides, 205
mucosa, 22, 61, 68, 73, 81, 122 nucleus, 67, 142
mucus, 59 nutraceutical, 232
muscles, 128, 129, 173, 205 nutrient(s), 57, 59, 62, 74, 75, 78, 142, 205, 206, 227
myocardial infarction, 147, 156 nutrition, 194, 197, 199, 205, 210
myocardial ischemia, 38
myocardium, 134
myoglobin, 159, 164, 166, 177, 210, 228 O
myosin, 68
obesity, 22, 57, 58, 60, 61, 63, 65, 70, 71, 72, 73, 74,
75, 76, 77, 78, 115, 192
N oil, vii, 2, 5, 6, 7, 18, 19, 20, 21, 24, 25, 26, 28, 38,
44, 48, 50, 51, 53, 86, 99, 108, 111, 119, 121,
NaCl, 231 170, 174, 176, 178, 179, 182, 184, 185, 189, 190,
NAD, 8, 142 192, 193, 194, 197, 198, 199, 200, 202, 203, 207,
NADH, 100 208, 209, 210
nanoparticles, 217, 218, 222 oil production, 6, 7, 21, 48, 121, 174
nanotechnology, 218, 222 oleic acid, 28, 185, 197, 201, 207
National Academy of Sciences, 76 oligomers, 3, 8, 29, 37, 42, 45, 86, 108, 109, 119,
natural antioxidants, vii, 1, 20, 22, 37, 49, 50, 52, 53, 127, 213, 216, 229
85, 87, 99, 102, 113, 123, 159, 160, 168, 170, olive oil, 5, 67, 111, 209
172, 173, 176, 177, 178, 179, 184, 186, 195, 203, onychomycosis, 215
207, 225, 226, 227, 228, 230, 232, 233, 234, 235 opportunities, 198
natural compound, 17, 18, 71, 127 optimal performance, 184
natural food, 157, 177 organ(s), 60, 61, 107, 129, 151
natural phenolic antioxidants, 86 organic compounds, 130, 184, 193
natural resources, 102 organic solvents, 9, 15, 104, 109
NCTC, 214 organism, 58, 59, 105, 107, 193, 211, 212, 225
negative consequences, 102 osmotic pressure, 205
negative effects, 159, 186 overproduction, 68
neolignans, 3 overweight, 154
neonates, 116 oxidation, 5, 6, 8, 20, 21, 30, 38, 42, 43, 45, 46, 49,
nervous system, 75 53, 54, 65, 101, 102, 103, 104, 105, 106, 107,
Netherlands, 81 111, 112, 113, 116, 117, 121, 122, 127, 130, 131,
neurodegenerative diseases, 103 135, 141, 145, 147, 157, 158, 159, 163, 164, 165,
neuronal cells, 115 166, 168, 169, 170, 172, 174, 175, 176, 177, 181,
neuroprotective agent, vii, 27 183, 185, 186, 187, 189, 192, 193, 225, 226, 227,
neutral, 89, 90, 91, 94, 95, 97 228, 229, 230, 231, 232, 233
New Zealand, 214 oxidation products, 157, 163, 226
NHANES, 156 oxidation rate, 46
niacin, 124 oxidative agents, vii, 101, 103
250 Index
oxidative damage, 52, 102, 106, 107, 111, 112, 115, 183, 184, 185, 186, 187, 189, 192, 193, 194, 197,
117, 118, 123, 130, 141, 159, 182, 192, 221 203, 212, 219, 220, 221, 222, 225, 227, 229
oxidative reaction, 38, 101, 102, 103, 110, 168, 169, phenotype, 62
177 phenoxyl radicals, 44, 113
oxidative stress, vii, 6, 7, 8, 21, 22, 27, 38, 43, 45, phenylalanine, 3, 184, 203, 205
69, 70, 72, 81, 82, 102, 105, 107, 112, 113, 114, phospholipids, 8, 163, 226
115, 117, 119, 122, 125, 126, 132, 134, 135, 137, phosphorous, 205
140, 141, 142, 143, 147, 151, 153, 154, 155, 199, phosphorylation, 62, 67, 70, 82
226, 227, 230, 232 phylum, 70
oxygen, 6, 38, 86, 102, 105, 107, 108, 114, 115, 116, physical characteristics, 101, 103, 166
122, 130, 131, 141, 142, 154, 158, 159, 205, 214, physical properties, 15, 218
226, 227, 228, 233 physicochemical properties, 176, 235
oxygen consumption, 141 physiology, 2, 211, 212
oysters, 221 phytosterols, 108
ozone, 222 phytotherapy, 218
pilot study, 124
placebo, 64, 79, 152
P plants, 2, 16, 41, 45, 52, 68, 114, 123, 130, 151, 159,
208, 212, 218, 225, 228, 232, 233
PACs, 37
plasma levels, 64, 71
pancreas, 59, 60
plasma proteins, 115
partition, 17
platelet aggregation, 38, 203
pathogenesis, 60, 76, 77, 81
platelets, 154
pathogens, 211, 213, 218, 221
pneumonia, 214
pathophysiological, 74
polar, 16, 17, 18, 126, 184, 203
pathophysiology, 58, 60
polarity, 10, 184
pathway(s), 3, 4, 38, 59, 62, 66, 67, 68, 71, 72, 80,
policy makers, viii
104, 112, 130, 132, 137, 145, 155, 187
pollution, 184
pea starch, 218, 223
polymer chain(s), 3, 86
peptic ulcer, 215
polymeric products, 43
peptidase, 60, 74, 75, 78
polymerization, 3, 8, 17, 29, 59, 86, 108, 162, 191,
peptide(s), 59, 64, 74, 78, 79, 107, 159
211, 213, 216
peptide chain, 159
polymers, 2, 3, 8, 29, 86, 108, 109, 119, 160, 162,
periodontal disease, 212
186, 199, 213, 229
peripheral nervous system, 60
polymorphisms, 82
permeability, 15, 58, 61, 62, 63, 69, 70, 72, 76, 77,
polypeptide(s), 59, 74, 75, 184
78, 81, 127
polyphenol extracts, viii, 125
peroxidation, 23, 45, 46, 54, 105, 106, 111, 114, 115,
polyphenolic extracts, viii, 19, 24, 99, 120, 125, 127,
120, 126, 131, 141, 151, 165, 189, 229, 230, 235
190
peroxide, 108, 117, 131, 140, 159, 168, 169, 229
polyphenols, 2, 3, 5, 7, 9, 15, 16, 17, 19, 20, 21, 22,
peroxynitrite, 6, 43, 121, 175
23, 24, 26, 27, 28, 29, 30, 38, 41, 43, 44, 45, 46,
Peru, 181
48, 49, 51, 52, 53, 54, 55, 58, 63, 65, 72, 73, 79,
petroleum, 196, 200, 208
80, 81, 82, 100, 102, 109, 117, 118, 121, 122,
pH, 10, 11, 12, 13, 14, 15, 41, 104, 116, 166, 168,
123, 125, 126, 127, 130, 132, 134, 135, 136, 138,
172, 176, 186, 190, 205, 230, 231
139, 140, 141, 142, 143, 145, 147, 150, 151, 153,
pharmaceutical(s), viii, 2, 102, 182, 199, 212, 219
155, 162, 173, 174, 183, 187, 190, 193, 196, 198,
pharmacology, 103, 155
199, 207, 208, 211, 212, 214, 219, 220, 221, 226,
phenol, 17, 20, 63, 105, 109, 138, 141, 213
230
phenolic compounds, vii, 1, 2, 3, 4, 7, 9, 10, 16, 17,
polysaccharide(s), 24, 61, 109, 117, 120, 199, 207
19, 20, 21, 22, 23, 26, 28, 29, 30, 38, 41, 42, 43,
polyunsaturated fatty acids, 8, 45, 159, 169, 197, 228
45, 46, 48, 49, 50, 51, 52, 54, 55, 57, 71, 73, 80,
population, 165, 171
86, 87, 99, 100, 108, 109, 110, 113, 125, 139,
positive correlation, 7, 164
142, 160, 163, 170, 173, 174, 175, 176, 181, 182,
potassium, 28, 111, 205
Index 251
potential benefits, 190

poultry, 159, 160, 166, 169, 172, 173, 230, 233, 235
Q
precipitation, 18
quality control, 29
preparation, 79, 160, 199
quality improvement, 16, 228
preservation, 159, 186, 191, 228
quality of life, 103
preservative, 102, 171, 227, 232
quantification, 30, 106, 138
prevention, viii, 6, 28, 47, 80, 115, 117, 119, 126,
quercetin, 10, 15, 25, 37, 42, 43, 45, 46, 52, 53, 54,
127, 142, 150, 151, 155, 172, 187, 192, 194, 196,
69, 70, 81, 82, 117, 125, 137, 139, 142, 154, 229
199, 208, 212, 226
quinone(s), 3, 42, 45, 142, 160, 170, 178
primary products, 159
principles, 218
proanthocyanidins, 2, 3, 7, 8, 15, 16, 22, 23, 24, 25, R
26, 28, 29, 33, 36, 37, 47, 48, 50, 58, 63, 72, 73,
100, 108, 109, 110, 111, 114, 119, 120, 123, 137, radiation, 121, 222
139, 160, 173, 181, 182, 191, 193, 196, 203, 210, radical formation, 130, 145
212, 213, 215, 229, 234 radical reactions, 126
probe, 104, 115 radicals, 8, 41, 42, 44, 46, 104, 106, 110, 111, 113,
probiotic, 82 114, 115, 117, 119, 126, 128, 130, 141, 150, 159,
producers, 99 164, 182, 190, 226, 228
production costs, 198 rancid, 157, 164, 165, 169, 228
professionals, 209 rape, 1, 5, 7, 26, 86, 108, 112, 178, 212, 232
profit, 198 rape seed, 1, 5, 26, 86, 108, 112, 212, 232
pro-inflammatory, 62, 63, 67 raw materials, 159
project, 59, 237 reactant, 38
proliferation, 43, 60, 107, 129, 193 reaction mechanism, vii, 101, 103, 104
pro-oxidant activity, 27, 41, 42, 43, 45, 113, 117, reaction medium, 106
195 reaction time, 104
propagation, 16, 114, 158 reactions, vii, 29, 42, 44, 101, 102, 103, 105, 106,
prophylactic, 80, 142, 143 110, 111, 117, 126, 129, 131, 159, 205, 227, 228,
prophylactic agents, 142 230
prostaglandin, 145, 203 reactive oxygen, 6, 8, 42, 102, 122, 141, 143, 158,
proteasome, 153 228
protection, vii, 2, 8, 27, 38, 57, 66, 72, 111, 112, 115, reactivity, 24, 102, 103
119, 126, 129, 132, 134, 135, 136, 147, 151, 153, reagents, 117, 138
165, 182, 192, 217 reception, 66
protective coating, 190 receptor(s), 47, 59, 60, 62, 65, 66, 67, 74, 77, 79, 81,
protective role, 67, 130, 145 83, 128, 129, 143, 152, 210
protein kinases, 62 recognition, 225
protein oxidation, 106, 112, 158, 159, 164, 171, 172, recovery, 16, 19, 29, 43, 190
176, 178, 179, 189, 195, 228, 234 recycling, 121
proteins, vii, 2, 24, 38, 46, 68, 69, 102, 105, 106, red blood cells, 8
107, 108, 111, 115, 117, 132, 142, 153, 157, 158, red wine, 3, 7, 9, 51, 67, 68, 71, 80, 82, 100, 117,
159, 163, 170, 178, 184, 185, 190, 192, 194, 197, 196, 230
199, 201, 203, 204, 205, 207, 210, 212, 228, 233 reducing sugars, 159
proteolysis, 170, 210 regions of the world, 198
protons, 86, 102 regulations, 18
prototype, 129 rejection, 159
Pseudomonas aeruginosa, 163 relaxation, 153
public health, 213 relevance, 24, 193
pulp, 3, 22, 50, 55, 64, 65, 79, 185, 213 remodelling, 141
purification, 17, 108 renin, 125, 128, 129, 145, 150, 152, 156
purity, 18, 29 renin inhibitors, 125, 129, 144, 150, 156
PVC, 231 repair, 121
252 Index
replication, 217, 222 shelf life, vii, 105, 157, 159, 163, 170, 171, 175, 178,
repressor, 142 194, 226, 227, 228
reproduction, 2, 79 showing, vii, 1, 5, 43, 62, 67, 101, 103, 105, 111,
requirements, 16, 205, 210 164, 170, 186
researchers, viii, 10, 61, 103, 104, 142, 184, 192, 211 side chain, 45
residue(s), 2, 6, 7, 9, 15, 28, 42, 50, 106, 109, 159, side effects, viii, 125, 150, 212, 215
173, 185, 188, 194, 195, 220, 222 signal transduction, 137
resistance, 62, 69, 76, 127, 213, 215, 220 signaling pathway, 59, 67, 80, 132
resolution, 54, 86, 88, 98, 208 signalling, 79, 80, 82, 102, 137
response, 43, 48, 53, 58, 59, 61, 62, 66, 71, 77, 132, signals, 58, 59, 71
140, 142, 157, 172 signs, 68, 74
resveratrol, 5, 8, 15, 18, 25, 38, 43, 49, 51, 53, 82, silver, 195, 234
162, 217, 222, 229, 234 skeleton, 2, 86
retardation, 187 skin, 3, 5, 22, 24, 26, 29, 48, 50, 55, 56, 59, 80, 99,
revenue, 198 110, 112, 120, 123, 166, 173, 211, 212, 213, 217,
ribonucleotide reductase, 174 220, 221, 229, 230
ribose, 106 small intestine, 59, 109
rings, 3, 108, 111, 228 smooth muscle, 52, 128, 129
risk, 82, 102, 109, 114, 125, 129, 130, 131, 145, 201, sodium, 28, 47, 66, 67, 81, 138, 168, 187, 189, 205
203, 228 software, 88, 131, 138, 145
RMN spectroscopy, 127 soil type, 229
rodents, 61, 62 solid matrix, 9, 18
Romania, 27, 125, 138 solid waste, 9, 207
room temperature, 15, 17, 138, 184 solubility, 18, 38, 103, 105, 109, 184, 203, 217
roots, 218, 227 solution, 9, 15, 18, 27, 54, 130, 140, 145
Royal Society, 72 solvents, 9, 16, 18, 19, 22, 29, 109, 138, 183, 184,
214
South Africa, 55
S soybeans, 78
Spain, 1, 57, 101, 157, 170, 197, 225, 237
safety, 18, 119, 123, 126, 159, 178, 226, 228, 231
species, vii, 6, 8, 42, 44, 70, 71, 86, 88, 102, 104,
Salmonella, 72, 163, 169, 191, 212, 218, 222, 223
105, 115, 121, 122, 131, 140, 141, 142, 143, 154,
salts, 2, 30, 108
165, 172, 175, 191, 198, 205, 212, 215, 228, 230
Sambucus nigra, 125, 127, 130, 132, 134, 135, 136,
specifications, 86
153
spectrophotometric method, 116
saturated fat, 28, 197, 201
spectrophotometry, 45, 140, 184
saturated fatty acids, 28, 201
spectroscopy, 20, 54, 127
saturation, 207, 210
spin, 113, 230, 235
scavengers, 7, 8, 46, 108, 109, 111, 141, 142, 199,
spleen, 59, 205
225
spore, 191
scholarship, 237
Sprague-Dawley rats, 69
science, viii, 181, 182, 237
squamous cell carcinoma, 122
secretion, 60, 61, 63, 65, 66, 68, 74, 78, 128, 217
stability, 19, 20, 24, 41, 44, 49, 50, 51, 72, 111, 165,
selectivity, 18, 105
166, 168, 169, 170, 172, 175, 176, 177, 178, 181,
selenium, 102
187, 190, 192, 194, 195, 201, 203, 209, 217, 226,
sensations, 190
227, 228, 230, 231, 233, 235
sensing, 73
stabilization, 113, 142, 169
sensitivity, 60, 104, 111, 191
standard error, 131, 143, 145
sensor, 142
starch polysaccharides, 199
sequencing, 50
state(s), 17, 87, 99, 102, 104, 113, 126, 150, 166,
serine, 62
184, 190, 228
serum, 114, 125, 131, 132, 140, 142, 145, 147, 156
statin, 147, 156
shade, 138
statistics, 132, 143, 149
Index 253
steel, 87 techniques, 16, 20, 23, 27, 29, 55, 86, 120, 127, 158,
sterols, 108 184, 213, 227
stimulation, 67, 128, 129 technologies, 9, 10, 16, 19, 28, 182, 183, 187, 192
stoichiometry, 43 technology, viii, 16, 17, 18, 109, 110, 181, 183, 185,
stomach, 61, 64, 114, 230, 235 189, 192, 218, 226, 227, 237
stomata, 218 technology transfer, 237
storage, vii, 9, 60, 111, 121, 157, 158, 160, 163, 164, temperature, 7, 9, 15, 16, 17, 18, 21, 23, 26, 87, 88,
165, 166, 168, 169, 171, 172, 175, 176, 177, 178, 138, 158, 172, 176, 183, 184, 214, 227
179, 186, 187, 190, 195, 211, 212, 214, 219, 220, tension, 126, 128, 150
226, 231, 232, 234, 235 texture, 38, 157, 159, 237
streptococci, 190 Thailand, 55
stress, 8, 19, 43, 61, 70, 78, 102, 105, 112, 113, 114, therapeutic agents, 46, 121
115, 117, 119, 122, 125, 126, 131, 140, 141, 142, therapeutic effect, 103
151, 155, 214, 225, 233 therapeutic targets, 126
structural changes, 128 therapeutics, 73
structural characteristics, 41, 208 therapy, 46, 47, 53, 75, 115, 127, 129, 143, 147, 152,
structure, 3, 5, 15, 30, 37, 41, 45, 46, 48, 51, 52, 54, 156, 215
58, 68, 81, 88, 92, 93, 97, 98, 100, 108, 119, 126, thermal degradation, 100
160, 162, 168, 192, 203, 209, 220 thermal treatment, 7, 19, 21, 51, 168
style, 153, 178 threonine, 184, 203
subgroups, 144 thymus, 106
substitution, 42, 108, 186 time periods, 46, 186
substrate(s), 30, 44, 45, 49, 62, 65, 70, 76, 101, 102, tissue, 183, 226, 227
103, 169 TLR, 62, 68
sucrose, 63, 70, 76, 78, 82 TLR4, 66, 71, 81
sulfate, 66, 67 TNF-α, 67, 68, 69
sulfur, 204 tobacco, 200
sulphur, 26, 159 tocopherols, 2, 50, 102, 108, 185, 197, 198, 199,
Sun, 21, 23, 26, 55, 79, 100, 119, 173, 174, 176, 196 208, 227
supplementation, 21, 43, 65, 77, 79, 124, 205 toluene, 228
supply chain, 213 total cholesterol, 131, 132, 145, 147, 148
suppression, 67, 79 toxic effect, 113, 139
surface reaction(s), 106 toxicity, vii, 9, 112, 113, 123, 127, 158, 211, 213,
surface tension, 18, 190 217, 228
survival, 71, 155 toxicology, 9
Switzerland, 22 toxin, 69, 215, 221
symptoms, 47 traits, vii, 164, 175
syndrome, 114, 124 transcription, 67, 132, 142
synergistic effect, 44 transesterification, 207
synthesis, 60, 68, 127, 135, 203, 217 transference, 102
synthetic antioxidants, vii, 6, 102, 157, 159, 165, transformation, 71, 107
168, 171, 174, 176, 177, 186, 187, 192, 225, 226, transforming growth factor, 68
227, 228, 230, 232 transition metal, 45, 130, 145
systolic blood pressure, 133, 144, 147, 149 transition metal ions, 45
translation, 79
translocation, 62, 63, 67, 68
T transport, 9, 62, 69, 107, 205
treatment, 7, 8, 15, 16, 17, 24, 63, 65, 67, 68, 69, 71,
tannins, 2, 3, 7, 15, 25, 28, 29, 48, 58, 86, 108, 121,
74, 80, 109, 112, 113, 120, 123, 128, 129, 135,
130, 193, 198, 220, 229, 234
147, 150, 152, 168, 185, 213, 215, 217, 221, 222,
target, 15, 17, 18, 46, 47, 59, 80, 104, 151, 212
231
Task Force, 150
trial, 79, 125
TBA, 45, 106
triglycerides, 8, 60, 64, 71, 113, 131, 132, 147
TCC, 186
254 Index
Trinidad, 79 vitamins, vii, 28, 102, 127, 151, 162, 189

tryptophan, 203 Vitis labrusca, 22, 49, 87, 99, 100, 164, 167, 173,
tumor, 67, 72, 81, 145 219
tumor necrosis factor (TNF), 67, 68, 69, 81, 145 Vitis vinifera, v, 5, 13, 19, 20, 21, 22, 23, 24, 25, 26,
tumorigenesis, 80 28, 47, 48, 49, 51, 52, 53, 55, 56, 86, 87, 99, 100,
Turkey, 7, 21, 25, 167, 174 119, 120, 121, 122, 160, 164, 167, 173, 174, 175,
turnover, 132, 155 182, 192, 193, 194, 195, 196, 197, 198, 207, 209,
type 2 diabetes, 60, 72, 74, 77 212, 216, 219, 220, 221, 222, 234, 235
tyrosine, 43, 47, 70, 82, 106, 184, 204, 205 VLDL, 196
U W
ultrasound, vii, 7, 9, 16, 19, 21, 23, 29, 48, 51, 183, Washington, 210
193 waste, 2, 9, 29, 86, 121, 157, 160, 171, 181, 182,
United States (USA), 76, 160, 165, 166, 173, 220, 185, 198, 207, 213, 220
223, 229 waste disposal, 207
universe, 192 water, vii, 7, 9, 13, 14, 15, 17, 24, 25, 26, 28, 29, 30,
uric acid, 145, 146, 147, 156 31, 32, 33, 34, 35, 36, 38, 46, 53, 58, 69, 79, 87,
urine, 59, 107 88, 103, 108, 109, 111, 115, 128, 138, 145, 158,
uterus, 59 166, 167, 170, 183, 184, 190, 193, 198, 213, 214,
221, 222, 234
water purification, 138
V wavelengths, 104
weight gain, 156
vacuum, 50, 130, 138, 163, 165, 166, 170, 176, 186,
weight loss, 75, 76, 82
187, 226, 229
wellness, 232
Valencia, 152
WHO, 9, 22, 159, 183, 184, 194, 204, 205, 210
validation, 106, 115, 118, 176
wine industries, vii
valine, 184, 203, 205
winemaking process, 87, 89, 184, 229
valorization, 1, 2, 10, 19, 29, 171
winery by-products, 19, 86
valuation, 21, 51, 55, 111, 123
workers, 104
vancomycin, 74
workplace, 156
variables, 9, 15, 17, 18, 109, 126, 213
worldwide, 19, 61, 182, 227
variations, 44, 61, 112, 183, 191, 229
wound healing, 206
varieties, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20,
21, 23, 25, 30, 31, 32, 33, 34, 35, 36, 37, 40, 43,
48, 50, 51, 52, 70, 85, 87, 88, 92, 94, 96, 98, 108, X
120, 162, 174, 184, 187, 198, 199, 200, 201, 202,
206, 208, 209, 210, 220, 229, 234 xanthones, 2
vascular wall, 141 X-ray analysis, 46
vasoconstriction, 128, 129
vasodilation, 141
vegetable oil, 203 Y
vegetables, 28, 58, 69, 81, 102, 116, 142, 143, 155,
yeast, 43
225
yield, 3, 9, 15, 16, 17, 26, 185, 190, 194, 198
virus replication, 221
viruses, 222
viscosity, 18 Z
vitamin C, 8, 111, 113, 123, 177
vitamin E, 8, 113, 123, 128, 140, 177, 182, 193, 199, zinc, 28, 205
203, 207

[Nutrition and diet research progress series] Rodríguez, José Manuel Lorenzo_ Ruiz, Daniel Franco - Grape seeds_ nutrient content, antioxidant properties and health benefits (2016, Nova Publishers) - libgen.lc.pdf

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NUTRITION AND DIET RESEARCH PROGRESS

Additional books in this series can be found on Nova‘s website

Additional e-books in this series can be found on Nova‘s website

JOSÉ MANUEL LORENZO RODRÍGUEZ

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

ISBN:  (eBook)

Library of Congress Control Number: 2015960806

Published by Nova Science Publishers, Inc. † New York

Chapter 11 Grape Extracts: Antioxidant Properties in Meat Product 225

A. Moure1,2,*, E. Falqué3 and H. Domínguez1,2

Keywords: grape seeds, phenolics, antioxidant activities, extraction

GRAPE SEEDS COMPOSITION

ANTIOXIDANT PROPERTIES OF GRAPE SEED PHENOLICS

Table 1. Antioxidant properties of grape seeds and grape seed extracts

Product Antioxidant activity Reference

EXTRACTION OF THE PHENOLIC FRACTION

Table 2. Extraction of antioxidants for grape seeds valorization using different

Grape varieties Solvent /Extraction conditions Compounds Reference

Grape varieties Solvent /Extraction conditions Compounds Reference

Grape varieties Solvent /Extraction conditions Compounds Reference

Solvent /Extraction conditions

Grape varieties Solvent /Extraction conditions Compounds Reference

Grape varieties Solvent /Extraction conditions Compounds Reference

diet-induced obesity in hamsters by improving adipokine imbalance and oxidative

L. M. Palade and V. S. Chedea

Keywords: grape seed extract, antioxidant/pro-oxidant activity, LC-MS, intermolecular

POLYPHENOLS FROM GRAPE SEEDS

EXTRACTION AND EVALUATION

MOLECULAR INTERACTIONS AND MOLECULES INVOLVED

compounds present in some extracts showing simultaneously antioxidant and pro-oxidant

GRAPE SEED ANTIOXIDANTS INTERACTIONS WITH

The intermolecular interaction between lipoxygenase and polyphenols is studied because

oxidant/antioxidant effect was evaluated by the TBARS (thiobarbituric acid reactive

FLAVONOIDS INTERACTION WITH

X. Terra, M. T. Blay, M. Pinent and A. Ardévol

Keywords: flavonoids, phenolic compounds, intestine, enterohormones, inflammation

OVERVIEW OF FLAVONOID STRUCTURE AND METABOLISM

INTESTINAL ALTERATIONS IN OBESITY AND INSULIN RESISTANCE

Obesity-Associated Intestinal Inflammation: The Link between High Fat

The gut microbiota represents an ensemble of microorganisms that resides in the

FLAVONOID’S EFFECTS AT THE GASTROINTESTINAL TRACT

Table 1. Summary of flavonoids acting on enteroendocrine system

Flavonoid type Enteroendocrine hormone Ref.

Flavonoid Effects on Intestinal Inflammation

Figure 1. Schematic view of the anti-inflammatory mechanisms of flavonoids on intestinal

Flavonoid Effects on Intestinal Mucosa Barrier Integrity

Flavonoid-Microbitoa Interaction: Modulation of Gut Microbiota

Therefore, it can be hypothesized that the effects of flavonoids on obesity-associated

glucose intolerance in LDLR-/- mice--role of intestinal permeability and macrophage

Keywords: LC-ESI-FTICR-MS, grape seeds, flavan-3-ols, procyanidins

In this chapter, we applied LC-ESI-FTICR-MS analysis to elucidate the complex

MATERIALS AND METHODS

Extraction of Polyphenols from Solid Samples

Solid-Phase Extraction (SPE)

RESULTS AND DISCUSSION

Table 1. Elemental compositions assigned to peaks in the negative-ion ESI-FTICR mass

Calculated Retention Time (min) Elemental Major error

Table 2. Elemental compositions assigned to peaks in the negative-ion ESI-FTICR mass

Calculated Elemental Major error

Table 3. Elemental compositions assigned to peaks in the negative-ion ESI-FTICR mass

Calculated Retention Time (min) Elemental Major

An elemental composition of C30H26O12 was assigned to eight different isomers of B-type

Procyanidin Dimer Monogallate

ISBN: (eBook)