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BLUEBERRIES
HARVESTING METHODS,
ANTIOXIDANT PROPERTIES
AND HEALTH EFFECTS
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NUTRITION AND DIET
RESEARCH PROGRESS
BLUEBERRIES
HARVESTING METHODS,
ANTIOXIDANT PROPERTIES
AND HEALTH EFFECTS
MALCOLM MARSH
EDITOR
New York
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Preface vii
Chapter 1 Blueberries: Market, Cultivars, Chemical
Composition and Antioxidant Capacity 1
Paula Becker Pertuzatti, Isidro Hermosín-Gutiérrez
and Helena Teixeira Godoy
Chapter 2 Bioactive Compounds, Color and Physicochemical
Parameters of Blueberries 31
Paula Becker Pertuzatti, Milene Teixeira Barcia,
Andressa Carolina Jacques and Rui Carlos Zambiazi
Chapter 3 Blueberries: Antioxidant Properties, Health and
Innovative Technologies 55
Guillermo Petzold, Jorge Moreno, Pamela Zúñiga,
Karla Mella and Patricio Orellana
Chapter 4 Blueberry Anti-Inflammatory Effects over Metabolic
Diseases Associated with Obesity 71
J. Soto-Covasich, M. Reyes-Farias, A. Ovalle-Marin,
C. Parra-Ruiz and D. F. Garcia-Diaz
Chapter 5 Blueberry Extracts Protect against Gross Mouse
Fetal Defects Induced by Alcohol Toxicity 89
Zach S. Gish, Sharang Penumetsa, Diana J. Valle
and Roman J. Miller
Index 111
PREFACE
researches have correlated the high antioxidant capacity with the phenolic
content in the fruit, especially flavonoids, because these kinds of compounds
are the major pigments found in these fruits. Among the phenolic compounds,
the blueberries are rich in flavonoids, namely anthocyanins, and flavanols, and
hidroxycinamic acids, which are associated in the literature with beneficial
health effects due to their ability to act as antioxidants, helping to protect the
body against free radicals and thus to avoid various types of cancer.
Roopchand et al. observed that the polyphenols present in blueberry may be
useful for the dietary management of diabetes, because lowered fasting blood
glucose levels, lowered serum cholesterol and reduced weight gain in mice.
Studies dealing with blueberries highlighted that, besides the presence of
phenolic compounds, other interesting compounds such as carotenoids,
tocopherols and ascorbic acid, also are present in this fruit, being sometimes
found in high levels. These compounds may also impact on the antioxidant
capacity of this fruit, once the antioxidant capacity of carotenoids has been
reported in the literature, as well as for tocopherols and ascorbic acid.
However, the composition in bioactive compounds of blueberries can be
highly variable, depending on cultivar, stage of maturation and harvesting and
storage conditions, usually because of its non climacteric nature with regard to
their production and responsiveness to ethylene. Therefore, this chapter aimed
to discuss about the blueberry producer and consumer market, the importance
of blueberry production in Brazil and in the world, which are the cultivars
produced in Brazil, in addition to its chemical and bioactive composition and
its influence in antioxidant capacity.
Chapter 2 - According to Food and Agriculture Organization (FAO)
statistics, from 1970 to 2011 the world production of blueberries increased
approximately 7 times (FAOSTAT, 2012). According to the National
Agricultural Statistics Service (NASS) of the United States Department of
Agriculture (USDA), the United States is the world leading producer of
blueberries, being the second most produced and commercialized small fruit in
the country after strawberry. There are three main groups of blueberries
commercialized and produced in the world: the lowbush, the highbush and the
Rabbiteye (RASEIRA e ANTUNES, 2004). The high amount of bioactive
compounds present in both the pulp and in the peel of the blueberries, makes it
a fresh fruit rich in natural antioxidants. Therefore, the identification and
quantification of the major bioactive compounds and some physicochemical
parameters in the peel, pulp and entire fruit of six blueberry cultivars
belonging to the Rabbiteye group are discussed in this chapter. Phenolic
compounds, anthocyanins, color, hydrolyzed and condensed tannins,
Preface ix
Chapter 1
ABSTRACT
The production of blueberry in Brazil began at the decade of 80’s
and its commercialization during the decade of 90’s of the XXth century.
Despite being a new crop in the country, it is observed that each day the
fruit has been gaining ground, which led to an increase in the number of
producers and cultivars marketed. The growing in blueberry cultivation in
Brazil is mainly attributable to the privileged climatic conditions with
temperate regions in four states, Rio Grande do Sul, Santa Catarina,
Paraná and São Paulo. Thus, the country has production potential over all
the year. In addition, another economically important aspect is that
Brazilian production of this fruit mainly occurs from December to
February, which happens between the harvest seasons in the United
* paulapertuzatti@yahoo.com.br.
2 P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy
States and that of the European Union that are the main consumer centers
[1, 2]. The health benefits of blueberries became widely accepted after
Prior [3] reported that blueberries had the highest antioxidant capacity of
more than forty fruits and vegetables evaluated. In addition, most
researches have correlated the high antioxidant capacity with the phenolic
content in the fruit, especially flavonoids, because these kinds of
compounds are the major pigments found in these fruits [4]. Among the
phenolic compounds, the blueberries are rich in flavonoids, namely
anthocyanins, and flavanols, and hidroxycinamic acids [5], which are
associated in the literature with beneficial health effects due to their
ability to act as antioxidants, helping to protect the body against free
radicals and thus to avoid various types of cancer [6]. Roopchand et al.
[7] observed that the polyphenols present in blueberry may be useful for
the dietary management of diabetes, because lowered fasting blood
glucose levels, lowered serum cholesterol and reduced weight gain in
mice. Studies dealing with blueberries highlighted that, besides the
presence of phenolic compounds, other interesting compounds such as
carotenoids, tocopherols and ascorbic acid, also are present in this fruit,
being sometimes found in high levels [2, 8]. These compounds may also
impact on the antioxidant capacity of this fruit, once the antioxidant
capacity of carotenoids has been reported in the literature [9, 10, 11], as
well as for tocopherols and ascorbic acid [12, 13]. However, the
composition in bioactive compounds of blueberries can be highly
variable, depending on cultivar, stage of maturation and harvesting and
storage conditions, usually because of its non climacteric nature with
regard to their production and responsiveness to ethylene [14]. Therefore,
this chapter aimed to discuss about the blueberry producer and consumer
market, the importance of blueberry production in Brazil and in the
world, which are the cultivars produced in Brazil, in addition to its
chemical and bioactive composition and its influence in antioxidant
capacity.
INTRODUCTION
Berry fruits, cultivated in temperate zones have increasingly attracted the
interest not only of consumers, due to its taste and high content of bioactive
compounds, but also the interest of producers, due to its high productivity,
good profitability and high demand. Because of this, the cultivation of
strawberry, blackberry, raspberry, cranberry and blueberry has been gaining
ground in several countries. According to data from Food and Agriculture
Market, Cultivars, Chemical Composition and Antioxidant Capacity 3
Organization of the United Nations [15] in 2013, more than 9 million tons of
berries were produced, contributing significantly to the increase availability of
berries.
Brazil has a privileged climatic condition for such cultivation, with
temperate regions in four states, including Rio Grande do Sul, Santa Catarina,
Paraná and São Paulo. Thus, the country has the potential to produce
blueberries throughout the year. In addition, another economically important
aspect is that Brazilian production of this fruit mainly occurs from December
to February and can extend until April, depending on the cultivar produced,
thus including the harvest season in the United States and that of the European
Union that are the main consumer centers [1, 2].
Berries are widely recognized as having a basic chemical composition that
accentuates its sweet taste, fruity aroma and beneficial health properties, which
are appreciated worldwide. This can be highly variable, depending on cultivar,
stage of maturation and harvesting and storage conditions, usually because of
its non climacteric nature with regard to its production and responsiveness to
ethylene [14].
Regarding blueberry, a small fruit native from North America, its
cultivation began in Brazil at 80’s, since then, the Brazilian Agricultural
Research Corporation (Embrapa) began developing researches in order to
verify the blueberry adaptability under the climatic conditions of the country,
in addition to organize workshops and other events, focused on small farmers
and scientific community, disseminating and encouraging the production of
this crop in the country, which contributed to increase in production and
marketing of this fruit in Brazil.
Worldwide, special attention began to be given to blueberry after Prior [3],
reported that blueberries had the highest antioxidant capacity of more than
forty fruits and vegetables. This finding sparked the interest of consumers and
researchers. Thereafter, several studies about antioxidant capacity of
blueberries began to be taken correlating this high value with the phenolic
content in the fruit, because this kind of compounds are their major pigments
[4].
Among the phenolic compounds, blueberries are rich in flavonoids,
namely anthocyanins and flavanols, and hydroxycinnamic acids [5], which are
associated in the literature with beneficial health effects due to their ability to
act as antioxidants, helping to protect the body against free radicals and thus to
avoid various types of cancer [6]. Roopchand et al. [7] observed that the
polyphenols presents in blueberries may be useful for the dietary management
4 P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy
CULTIVARS
Blueberries are from the family Ericaceae, subfamily Vaccinoideae and
genre Vaccinium, and their fruits may be classified in three main categories or
groups: Highbush, Lowbush and Rabbiteye [14, 26].
The Highbush group is originally from the west coast of North America
and within it the predominant specie is V. corymbosum L., although the
species V. australe and V. darrowi can be used for breeding [27], their plants
have a need for 650 to 850 h of cold (with temperatures lower or equal to
7.2°C) and the fruits have the best quality, in size and taste or with regard to
pruine content (waxy skin responsible for blue color of fruits) [28].
Blueberries of Highbush group, can be divided into Northern Highbush,
which is the most commonly planted in the world, with a cold requirement of
over 800 h and Southern Highbush, developed to allow blueberry production
in regions with mild winters or warmer (200 to 300 h of cold) [29]. According
to Galletta and Ballington [30], Southern Highbush blueberry prefers plateau
areas, soil rich in organic matter, is not bothered by many pest problems and
produce great fruits with excellent quality. These fruits have a very early
production compared to the other blueberry groups. They are grown in the less
cold regions of the United States, Chile and predominantly in Argentina and
6 P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy
CHEMICAL COMPOSITION
According to Moraes et al. [23], blueberry has carbohydrate content about
15%. The main sugars found are sucrose, glucose and fructose, occurring in
blueberry in concentrations of 0.12-1.14%, 3.28-3.87% and 3.34-3.88%
respectively, the high amount of fructose, usually the major sugar in
blueberries, making this fruit a good option for diabetics [6, 35]. Wang et al.
[36] support this statement saying that the main sugars found in blueberries are
fructose and glucose, while sucrose is found in lower amounts, probably due to
high activity of invertase during the final stage of maturation [37]. The
concentration of these sugars is important to fruit quality because fructose is
1.8 times sweeter than sucrose, while glucose presents 60% of fructose
sweetness. The cultivation system also influences the sugar concentration,
because blueberries grown under organic cultivation system have higher
amounts of sugar (fructose and glucose) than blueberries grown under
conventional system [36].
Among polysaccharides found in ripe blueberries cellulose, hemicellulose,
pectin and lignin, which are mainly found in the cell wall, stand out. Besides
contributing to the nutritional value, sugars and organic acids are also
responsible for texture and flavor of fruits [38, 39].
Sugar content in blueberries is compensated by the presence of organic
acids and also phenolic acids, which can give an additional bitter or astringent
flavor. The completion of organic acids with phenolic acids is responsible for
titratable acidity that is commonly measured as a global index of fruit quality.
Organic acids also help to stabilize ascorbic acid and are fundamental in fruit
color, stabilizing anthocyanins and extending the shelf life of fresh and
processed fruits [35]. The organic acids found in blueberries are citric acid and
malic acid. Talcott [35] reports only the presence of malic acid in blueberry,
ranging from 0.06 to 1.10% while Milivojevic et al. [37] found both organic
acids, with citric acid, 0.1–0.23% being the major organic acid while malic
acid had amounts of 0.05 – 0.12%.
Volatile compounds are typically esters, alcohols, acids, aldehydes and
ketones [38]. In blueberries, Simon et al. [40] demonstrated that the emission
levels of volatile compounds are very low compared with other fruits such as
strawberry, only butyl-acetate could be observed. However, according to Su
Market, Cultivars, Chemical Composition and Antioxidant Capacity 9
Phenolic Compounds
UV radiation and consequently less rain during the maturation period, higher
is the phenolic content in fruits.
As can be seen in Table 2 the reported levels of total phenolic compounds
in Highbush, Lowbush, Rabbiteye and Southern Highbush blueberries ranged
between 118 and 461 mg/100 g of fresh fruit, from 299 to 374 mg/100 g of
fresh fruit, from 175 to 592 mg/100 g of fresh fruit, and between 116 and
586 mg/100 g of fresh fruit, respectively. According to Prior et al. [52], the
medium content of total phenolic in blueberry, in dry weight, is about
2500 mg/100g being one of the highest among fruits and vegetables.
Figure 1. Structure of major flavonoids found in blueberry. (a) quercetin, (b) (+)-
catechin, (c) myricetin, (d) (‒)-epicatechin, (e) kaempferol, (f) anthocyanidin.
12 P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy
Phenolic Acids
The predominant phenolic acids in berries are hydroxybenzoic and
hydroxycinnamic acids. Among the phenolic compounds analyzed by
Castrejón et al. [52] the hydroxycinnamic acids were the major group found in
blueberries in all maturity stages. This class of phenolic acids is found in all
parts of blueberry and bilberry plants [61].
These acids occur rarely as free acids, they are commonly found in the
conjugated form as esters and glucosides. In blueberries, hydroxybenzoic
acids: gentisic, gallic, protocatechuic, salicylic, syringic and vanillic, are
present in free, ester and glucoside forms, with ester and glucoside form of
predominant salicylic acid. The hydroxycinnamic acids, namely caffeic, m-
coumaric, o-coumaric, p-coumaric and ferulic acids, are also present in the
free, ester and glucoside forms, with sinapic acid and 3-4-dimethoxycinnamic
(veratric) acid present in ester and glucoside forms and dihydroxycinnamic
acid in ester form [48, 62]. In Table 3, are shown phenolic acids found in
blueberries.
Flavonols
Flavonols most commonly found in blueberries are quercetin, myricetin
and kaempferol, usually these compounds are present in fruits in glucoside
forms, with a sugar linked in C3 position. Glucose and galactose are the most
commonly sugar bonded, even though rutinose, xylose, arabinose and
rhamnose are also found [48]. According to Howard and Hager [48] fourteen
quercetin derivatives, including acylated compounds (caffeic and acetic acid),
together with four myricetin derivatives and two kaempferol derivatives were
found in blueberries.
Blueberry contains higher levels of flavonols than strawberry and
raspberry, quercetin levels ranged between 1.7 and 4.7 mg/100g of fresh fruit,
while myricetin shows a variation between 0.8 and 1.8 mg/100g of fresh fruit
[48]. Quercetin derivatives are the predominant flavonols in blueberry, being
quercetin 3-galactoside the major compound, however, some genotypes
contain appreciable amounts of quercetin 3-rhamnoside [70, 71]. According to
Johnson and Arjmandi [72], the potential of blueberry and apple juice blends,
rich in quercetin, in protection against induction of oxidative DNA damage in
human volunteers have been examined, in one of these studies [73] during four
weeks, volunteers consumed one liter of juice per day, that provided 97 mg of
Market, Cultivars, Chemical Composition and Antioxidant Capacity 15
Flavonol References
Myricetin 3-arabinoside 36, 75
Myricetin 3-galactoside 71, 76
Myricetin 3-glucoside 71
Myricetin 3 rhamnoside 71
Quercetin 3-galactoside 36, 62, 71, 75, 76
Quercetin 3-glucoside 36, 62, 70, 71, 75, 76
Quercetin-diglucoside 76
Quercetin 3-rutinoside (Rutin) 70, 71, 76
Quercetin 3-rhamnoside 62
Quercetin 3-arabinoside 70, 76
Quercetin 3-acetylglucoside 76
Quercetin 3-acetylrhamnoside 71
Kaempferol 3-glucuronide 75
Kaempferol 3-glucoside 62, 75, 76
Laricitrin 3-glucoside 70
Syringetin 3-glucoside 70
16 P. Becker Pertuzatti, I. Hermosín-Gutiérrez and H. Teixeira Godoy
Anthocyanins
Anthocyanins are the subgroup of flavonoids most widely distributed in
nature and especially abundant in red, purple and blue small fruits, among
them blueberry, which are the main phenolic compounds found [6]. These
pigments are present in both peel and pulp of bilberry and just in the peel of
other species of blueberry, because of this the anthocyanin content of bilberry
is superior to the others, which makes it to be considered one of the best
sources of anthocyanins [61]. Chromatographic profiles have shown that
anthocyanins represent 35–74% of phenolic compounds in blueberry [70].
Studies indicate that blueberries of Rabbiteye group have higher levels of
anthocyanins than Highbush, Southern Highbush and Lowbush blueberries
[48].
According to Table 2 the total anthocyanin content in Highbush,
Lowbush, Rabbiteye and Southern Highbush blueberries ranges between 74
and 278 mg/100 g of fresh fruit, 91 to 255 mg/100 g of fresh fruit, 62 to 242
mg/100 g of fresh fruit, and 73 to 220 mg/100 g of fresh fruit, respectively.
Size is another factor related to the amount of anthocyanins in fruit, several
studies reported that smaller the berry size higher the amount of anthocyanins
is [51, 77].
Anthocyanidins are very unstable and rarely found in free form among
plant tissues [48, 78]. Anthocyanidins occur mainly in glycosylated form
(anthocyanins), because the substitution of sugar increases its stability and
solubility [78]. Due to the diversity of monoglucosides and acylation with
aliphatic acids such as malonic and acetic acid, many anthocyanins have been
identified in different blueberry groups (Table 5). In Highbush blueberry
extracts, 15 non-acylated anthocyanins and 5 acylated anthocyanins were
identified [65, 79], however in Rabbiteye blueberries none acylated
anthocyanin was found and 14 non-acylated anthocyanins were found [80].
Regarding Lowbush blueberries cultivars, 25 anthocyanins (11 acylated) were
reported [77].
According to Cho et al. [71] the percentage of monomeric anthocyanin
distribution in five genotypes of blueberry belonging to Highbush, Southern
Highbush and Rabbiteye groups are based on the following anthocyanidin
structures: delphinidin (27% to 40%), malvidin (22% to 33%), petunidin (19%
to 26%), cyanidin (6% to 14%) and peonidin (1% to 5%); with regard to the
percentage of glycosylated anthocyanin distribution, predominate galactosides,
60 to 67%, followed by arabinosides (26 to 32%) and glucosides (2 to 29%).
Market, Cultivars, Chemical Composition and Antioxidant Capacity 17
Anthocyanin References
Delphinidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Delphinidin 3-glucoside 36, 50, 71, 76, 77, 79, 81
Delphinidin 3-arabinoside 36, 50, 71, 75, 76, 77, 79, 81
Cyanidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Cyanidin 3-glucoside 50, 71, 77, 79, 81
Cyanidin 3-arabinoside 71, 76, 77, 79, 81
Petunidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Petunidin 3-glucoside 36, 50, 71, 75, 77, 79, 81
Petunidin 3-arabinoside 36, 71, 75, 76, 77, 79, 81
Peonidin 3-galactoside 50, 71, 76, 77, 79, 81
Peonidin 3-glucoside 79
Peonidin 3-arabinoside 71, 76, 77, 79
Malvidin 3-galactoside 36, 50, 71, 75, 76, 77, 79, 81
Malvidin 3-glucoside 36, 50, 71, 75, 76, 77, 79, 81
Malvidin 3-arabinoside 36, 50, 71, 75, 76, 77, 79, 81
Delphinidin 3-(acetyl)-galactoside 50, 77
Delphinidin 3-(6”-acetyl)-glucoside 50, 71, 76, 77
Delphinidin 3-(malonyl)-glucoside 81
Cyanidin 3-(6”-acetyl)-galactoside 77, 81
Cyanidin 3-(6”-acetyl)-glucoside 77, 81
Cyanidin 3-(acetyl)-arabinoside 77
Cyanidin 3-(malonyl)-glucoside 81
Petunidin 3-(acetyl)-galactoside 77
Petunidin 3-(6”-acetyl)-glucoside 71, 76, 77, 81
Petunidin + pentose 81
Peonidin 3-(6”-acetyl)-galactoside 77, 81
Peonidin 3-(6”-acetyl)-glucoside 77, 81
Malvidin 3-(6”-acetyl)-galactoside 50, 77, 81
Malvidin 3-(6”-acetyl)-glucoside 50, 71, 76, 77, 81
Malvidin 3-(malonyl)-glucoside 81
Malvidin + acetyl + hexose 81
Carotenoids
fresh fruit [87]. Although these concentrations are low compared with other
fruits rich in carotenoids, its presence indicates the diversity of phytochemicals
in this fruit as well as contributing as an aroma precursor, when converted in
norisoprenoids, compounds identified as responsible for grape aroma [35, 88].
In the literature data on carotenoid content in berries is limited. Heinonen
et al. [87] found lutein+zeaxanthin, β-cryptoxanthin, α-carotene and β-carotene
in blueberry and Marinova and Ribarova [89] found lutein, zeaxanthin, β-
cryptoxanthin and β-carotene, and found that there was no presence of α-
carotene because during the fruit ripening occurs hydroxylation of this
carotenoid and then it is converted into lutein, which together with β-
cryptoxanthin and β-carotene are major carotenoids in blueberry [2, 89, 90].
Regarding to bilberry, six carotenoids were found, namely, neoxanthin,
violaxanthin, anteraxanthin, lutein, zeaxanthin and β-carotene [91].
Antioxidant Capacity
from 18.5 to 161.4 µmol of Fe2+/g of fresh fruit [51, 56], this wide range of
results occurs due to different factors such as, cultivar, maturity degree, season
and storage conditions [76].
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In: Blueberries ISBN: 978-1-63484-885-5
Editor: Malcolm Marsh © 2016 Nova Science Publishers, Inc.
Chapter 2
ABSTRACT
According to Food and Agriculture Organization (FAO) statistics,
from 1970 to 2011 the world production of blueberries increased
approximately 7 times (FAOSTAT, 2012). According to the National
Agricultural Statistics Service (NASS) of the United States Department
of Agriculture (USDA), the United States is the world leading producer
of blueberries, being the second most produced and commercialized small
fruit in the country after strawberry. There are three main groups of
blueberries commercialized and produced in the world: the lowbush, the
highbush and the Rabbiteye (RASEIRA e ANTUNES, 2004). The high
Corresponding author: Paula Becker Pertuzatti. Universidade Federal de Mato Grosso (UFMT),
Av.Senador Valdon Varjão 6390, Barra do Garças - MT, 78600-000, Brazil. E-mail:
paulapertuzatti@yahoo.com.br.
32 P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.
amount of bioactive compounds present in both the pulp and in the peel
of the blueberries, makes it a fresh fruit rich in natural antioxidants.
Therefore, the identification and quantification of the major bioactive
compounds and some physicochemical parameters in the peel, pulp and
entire fruit of six blueberry cultivars belonging to the Rabbiteye group are
discussed in this chapter. Phenolic compounds, anthocyanins, color,
hydrolyzed and condensed tannins, carotenoids and physicochemical
analyses were done. There were evaluated Powderblue, Briteblue,
Bluebelle, Climax, Delite and Woodard cultivars. The blueberry fruits
showed as rich sources of phenolic compounds and anthocyanins, besides
to have considerable amounts carotenoids and tannins. All
phytochemicals analyzed were found at the highest levels in the peels of
blueberry cultivars tested.
INTRODUCTION
Statistics data from 1970 to 2011 relate that the world production of
blueberries increased approximately 7 times [1]. The United States follows as
the world leading producer of this fruit. The increase in blueberry production
can be related with the special attention given to blueberry after that Prior et
al. [2] found its high antioxidant capacity, related with the large amount of
bioactive constituents such as phenolic compounds, vitamins and carotenoids.
Some quality factors influence the fresh-market value and the suitability
of the berries for processing. Color, related to the anthocyanins content, and
taste, related to the physicochemical composition are some of these. Studies by
Bargmann, Wu and Powers [3] have established a significant relation between
low titratable acidity/high pH and the acceptability testing of a few varieties.
Furthermore, varieties that showed higher absorbance, due to the darker color,
were judged superior in appearance.
Therefore, this chapter aimed to discuss about the identification and
quantification of the major bioactive compounds in the peel, pulp and entire
fruit of six blueberry cultivars belonging to the group Rabbiteye (Powderblue,
Climax, Briteblue, Bluebelle, Delite and Woodard), from the 2007/2008
harvest, from the city of Pelotas, RS, Brazil. The material was donated by
Embrapa Temperate Climate (Brazil).
Bioactive Compounds, Color and Physicochemical Parameters … 33
BIOACTIVE COMPOUNDS
Bioactive compounds from plants may be nutritive or non-nutritive
substances which ones have important role when are ingested, due to the
biological activity such as antioxidant, anticarcinogenic, antimicrobial and
anti-inflammatory effects. Depending on the biological activity attributed to
the bioactive compounds, diets with high ingestion of fruits and vegetables has
been correlated with decrease of incidence of degenerative diseases.
Phenolic Compounds
Anthocyanins
Tannins
Among the bioactive compounds, tannins are also highlighted, which, like
the other phenolic compounds, are derived from the secondary metabolism of
plants. They are present in most plants and can vary in concentration
depending on the age and size of the plant part collected, the time or even the
Bioactive Compounds, Color and Physicochemical Parameters … 37
collection site. The tannins can be categorized into hydrolyzable, and non-
hydrolyzable or condensed tannins [16, 17].
The hydrolyzable tannins are esters of phenolic acids (gallic, caffeic,
ellagic acids) linked to simple sugars. These compounds have smaller
molecular chains than condensed tannins and can be hydrolyzed more easily,
just by the action of dilute acids [18, 19]. Typically, hydrolysable tannins are
classified in gallotannins which produce gallic acid after hydrolysis and
ellagitannins which produce ellagic acid [20]. However, unlike other berries
such as raspberry, strawberry and blackberries, blueberries do not contain
ellagitannins nor other derivatives of ellagic acid. The ellagic acid content in
blueberry is lower than 5 mg.100 g-1 fresh fruit [21] after acid hydrolysis.
For the determination of hydrolyzable tannins in blueberry, the method
adapted from Brune et al. [22], which consists of the extraction of tannin with
methyl alcohol, was used. The methanol extract was then mixed with a
reaction solution of ferric ammonium sulfate, consisting of 89% urea buffer:
acetate, 10% arabic gum solution 1% in deionized water and 1% ferric
ammonium sulfate solution 5% in hydrochloric acid 1 mol.L-1. The absorbance
was read at 578 nm in a spectrophotometer (Ultrospec 2000). The
determination of the content of hydrolysable tannins was performed through a
gallic acid standard curve and results were expressed in mg of gallic acid.100
g-1 sample.
The non-hydrolyzable or condensed tannins, also known as
proanthocyanidins, are compounds formed by the polymerization of flavonoid
units, predominantly catechin and are present in a wide variety of foods, and
can be divided into two main classes that include procyanidins, mixtures of
oligomers and polymers composed of units of (+) -catechin and/or (-)-
epicatechins, and propelargonidins composed exclusively of epiafzelechin
units [23]. However, blueberries exclusively have procyanidins, which are
considered one of the major phenolic compounds present in the fruit pulp [15,
19, 21, 24]. These compounds are ranked according to their degree of
polymerization (DP) where DP = 1 indicates a monomer, while DP = 2-10 and
DP > 10 refer to oligomers and polymers, respectively [25].
The monomer units of procyanidins are connected via a C4-C8 or C4-C6
(type B) bond, and can coexist with a C2-O-C7 bond or, less frequently, with
C2-O-C5 bond (type A) [23]. Hwang et al. [26] found 300 mg procyanidin
B1.100 g-1 fresh fruit in blueberry extracts, well below the contents found for
black chokeberries (Aronia melanocarpa) of 2.5 g.100 g-1 fresh fruit, which is
known for its high astringency.
38 P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.
Carotenoids
COLOR
Color is a primary indicator of food quality, because it has great
importance in evaluating the degree of maturity and freshness of fruits, the
storage conditions, postharvest handling and transportation. Therefore, color is
characterized as a decisive factor utilized at the time of choice and acceptance
of a product, especially blueberry which has as its remarkable characteristic
blue color.
Since color is a parameter used to describe quality, its determination is
useful to correlate with the concentration of the pigments present in the fruit.
Anthocyanin quantification methods and color indices have been
established and used in industrial control applications [35]. The data from
instrumental color evaluation in this study were performed with a colorimeter
(Minolta CR-300) for six blueberry fruit cultivars and are shown in Table 4.
The samples (about 3 g, equivalent to the average weight of a fruit) were
placed in Petri dishes of 5 cm diameter and 2 cm in height. The measured
color parameters were: L*, a* and b*, where L* indicates brightness (0 =
black and 100 = white) and a* and b* represent chromaticity coordinates (+ a*
= red, - a* = green, + b* = yellow, - b* = blue). The color parameters were
converted to color angle H° = tan-1 b/a, indicating the angle Hue (H°) of the
sample (0° or 360° = red; 90° = yellow; 180° = green; 270° = blue) [36].
42 P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.
The hue is an attribute by which the colors are identified where positive
values of a* indicate red, while negative values represent green colors.
Similarly, positive b* values relate to yellow and negative values express the
blue colors. The luminosity or brightness (L*) is presented as an attribute that
describes a gray scale of the measure, characterizing the color as lighter or
darker (between black and white) in a range of measurement ranging from 0-
100. The meeting of the three values sets the color of the product [37]. Thus, it
can be seen by the data of Table 4, Climax cultivar showed lower values of b*
and L, meaning that the peel of this fruit has a darker shade of blue, which is
very favorable commercially.
With respect to the values of Hue angle, it can be seen that all the peels
had values near blue (h° = 360), which is also very favorable for blueberry,
considering that consumers prefer blue fruits with intense shades. The fruit
pulp presented values between red and yellow, where the values varied
between 11.86-63.77.
When color is compared with the determination of anthocyanins and
phenolic compounds, there was a strong negative correlation, according to the
Pearson correlation coefficient between the b* value and the determination of
phenolic compounds (- 0,8). This means that as the concentration of these
phytochemicals increases the b* values reduce, i.e., the fruit gets bluer. With
the values of h°, the opposite occurred where there was a Strong positive
correlation with the content of phenolic compounds (0,8); Therefore, as the
amount of these compounds increases, the Hue angle also increases. The h°
angle is also positively correlated with the pH, this is due to the presence of
anthocyanins in the fruit, because as the pH increases, anthocyanins become
blue due to the deprotonation of the structure resulting in an increase in the h°,
thus being closest to the value for the blue color. However, as the pH
decreases anthocyanins are mostly in the form of flavylium cation and
acquires a red color, and Hue angle decreases, getting closer to this color.
Figure 1 is a graphical representation of the values of L, a* and b* in the
CIELAB scale obtained in a Minolta CR-300 colorimeter, for the peel and
pulp of blueberry cultivars.
By observing Figure 1 (b) one realizes that all blueberry pulps were
arranged in the first quadrant of the circle, however the display of color in a
graphical representation of two dimensions for this part of the fruit is not
enough because it does not take into account the luminosity values. Thus, it is
noticed (Figure 1 (a)) that since the values of L* are higher in the pulp, they
characterize the pulp as whitish, while the peel, which has smaller values of
L*, has a blue tone, tending to purple, characteristic of anthocyanins.
44 P. Becker Pertuzatti, M. Teixeira Barcia, A. Carolina Jacques et al.
From Figure 1(b), it can also be observed that only the climax cultivar
presents difference in coloration, which was also statistically observed (Table
4). However, the location of the colors of the samples in the colorimetric
space, or even the statistic results of the color information are not sufficient to
express whether the color differences are possible to be distinguished visually,
but these differences may be calculated by the distances between two points in
three dimensional space (ΔE) defined by the parameters a*, b* and L*, and
these values can be compared with the classification used by the paint industry
for the perception of the human eye. In general, color differences of two
overlapping samples can be distinguished in ΔE values above 0.2-0.5 [37].
Mathematically, the colorimetric parameter ΔE is described by Equation 1 and
the values for the analyzed blueberry cultivars are shown in Table 5.
pH of the fruit increases and the acidity decreases as the fruit ripens due to
degradation of organic acids with the evolution of maturation becoming acidic
salts [39].
The soluble solids (SS) are also indicators of the degree of maturation of
the fruit, because as the fruit ripens the soluble solids content increases, by
increasing the content of sugars. However, this measure is related to all solids
dissolved in water, including salts, acids, proteins and other soluble
compounds in addition to sugars.
The pH values of the peel and the whole fruit of blueberry cultivar did not
differ significantly from each other, staying in the range of 2.87-3.57.
However, when the pH values of the different parts of the fruit were observed
between the different cultivars, it was observed that there were differences in
the comparisons. It is also observed that for all cultivars, except the Delite, the
pH value of the pulp (2.38) was lower than that of the other parts of the fruit.
Comparing the values of this study with pH values found by Moraes et al.
[40], who worked with of Delite, Bluebelle and Woodard blueberry cultivars
(pH 2.67; 2.59 and 2.62, respectively), the pH of the fruits of this study
showed higher values, however when compared with the data obtained by
Perkins-Veazie et al. [41] in a study of blueberry (Vaccinium corymbosum) of
Collins and Bluecrop cultivars, the results are similar (3.5 and 3.3
respectively). As well as those found by Souza et al. [11], who reported a
3.64 pH for blueberry fruit.
Observing the pH values found in the pulp, inferior and significantly
different values are noted for the peel, for all cultivars, showing that there is a
higher concentration of dissociable organic acids in this part of the fruit, since
this determination quantifies only the total of ionizable hydrogen atoms
present in the fruit. This same statistical difference occurs with acidity content
of the peel and pulp, for all cultivars, with higher acidity in the peel, showing
that unlike the pH, non-dissociated organic acids are present in greater
amounts in the peel. The blueberry cultivars showed significant differences in
the whole fruit acidity levels, with Woodard (0.09%) and Powderblue (0.07%)
being the cultivars with higher acidity values. The acid content in blueberry is
mainly represented by the presence of malic acid which is the major organic
acid in small fruits.
The soluble solids content in the peel of blueberry cultivars showed higher
content than the content in the pulp (Table 6). Similar results were also
observed by an Embrapa Grape and wine [42] research, where the authors also
claim that in years of higher solar radiation intensity, grapes are produced with
higher soluble solids.
Among the analyzed fruits, the Woodard and Delite cultivars were the
ones that showed soluble solids similar to those of Raseira and Antunes [10],
Woodard cultivar, 12-13.9 °Brix and Delite cultivar 10.8-12.5 °Brix. The other
cultivars analyzed showed high soluble solids content (11.87-17.87 °Brix)
both when compared to the cultivars analyzed by Moraes et al. [40] (12-13.2
° Brix) as well as those analyzed by Perkins-Veazie et al. [41] Collins cultivar,
10.9 °Brix and Bluecrop cultivar 12 °Brix. These data demonstrate that the
Bioactive Compounds, Color and Physicochemical Parameters … 49
fruits analyzed in the present study may have been collected with a greater
degree of ripeness.
Several factors can influence the content of soluble solids. Junior et al.
[43], when working with maturation curves and soluble solids in grapes,
describe climate as one of the factors that influence the accumulation of sugars
most. Volpe et al. [44] state that the temperature and the rains of months prior
to harvesting decisively influence the concentration of soluble solids of orange
juice. The soluble solids content of the blueberries in the present study showed
a positive correlation with pH (r = 0.7), which is justified by the increase in
value as the fruit ripens. This relationship was also observed by Perkins-
Veazie et al. [41], who found a coefficient of moderate Pearson correlation
(0.52) between pH and content of soluble solids in Highbush blueberry.
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In: Blueberries ISBN: 978-1-63484-885-5
Editor: Malcolm Marsh © 2016 Nova Science Publishers, Inc.
Chapter 3
ABSTRACT
Blueberries are a soft and small fruit native to North America with an
attractive blue color. In addition, blueberries are very popular because
they have low calories, high nutritional value and important antioxidant
properties. Blueberries have an interesting content of phenolic
compounds with high antioxidant capacity against free radicals and
reactive species, such that blueberry consumption may have a potential
beneficial effect on human health. Innovative technologies in the food
industry are new technologies based to develop more efficient process or
products, reduction of energy and water. Innovative technologies, such as
freeze concentration, osmotic dehydration and vacuum impregnation at
mild temperatures, are considered minimal processing techniques because
they preserve the fresh characteristics of fruits such as blueberries. Freeze
concentration is an innovative technology for producing a blueberry
concentrate juice in a process at low temperatures where no vapor/liquid
*
Emerging technologies and Bioactive Compounds in Food (TECBAL). Department of Food
Engineering, Universidad del Bío-Bío, Chillán, Chile. Email:gpetzold@ubiobio.cl.
56 Guillermo Petzold, Jorge Moreno, Pamela Zúñiga et al.
In the last years, there has been a growing interest in food that can provide
beneficial effects to human health. It is widely known that a diet rich in fruits
and vegetables has beneficial effects due to the high amounts of antioxidants
and bioactive compounds in these foods, which have an essential role in
prevent certain diseases and reduce the risk of some health problems including
cardiovascular disease, neurodegenerative diseases, stroke and cancer
(Giampieri et al., 2014, Seeram et al., 2006; Neto, 2007; Kalt et al., 2008).
Accordingly, many native fruits have been studied for their potential as a
functional food. Recently, investigations have focused to improve the
nutritional value of fruits with emphasis in bioactive compounds (Scalzo et al.,
2005). Studies have also reported that specific berries, i.e., blueberries, have
antidiabetic effects. For instance, a study performed in mice (DeFuria et al.,
2009) found that supplementation with whole blueberries reduced the blood
glucose (Martineau et al., 2006; Vuong et al., 2007).
Fresh fruits of blueberry typically contain 1-3% seeds by weight, the
highly unsaturated seed oils of V. myrtillus (21-37%) is an excellent source of
essential fatty acids and oleic acid (Johansson et al., 1997).
Compounds capable of protecting against the effects of reactive oxygen
and nitrogen species (ROS and RNS) are known as antioxidants (Karadag et
al., 2009), they interact with unstable molecules such as free radicals and may
prevent the oxidative damage caused by the free radicals (Wang et al., 2012).
The high antioxidant activities in fruits are attributed to phenolic compounds,
such as anthocyanins, and other flavonoid compounds. However, the
antioxidant activity depends on their structure and content in berries.
Blueberries are a good sources of phenolic compounds, including
anthocyanins, flavonols, chlorogenic acid and procyanidins, that have high
antioxidant activity (Cho et al., 2004; Howard et al., 2003; Wang et al., 2012).
Antioxidant Properties, Health and Innovative Technologies 57
INNOVATIVE TECHNOLOGIES
Freeze Concentration
Osmotic Dehydration
Blueberries OD Application
Blueberries are fruits available in the growing season and show a limited
shelf-life due they are highly perishable commodities and sensitive to bacterial
and fungal contamination; hence, preservation and processing methods are
usually applied to extend berry commercial life and consumption occasions,
usually by either freezing or drying technologies.
Within existing methods available for drying blueberries, freeze-drying
and vacuum-drying obtain the highest-quality end product, considering the
losses in total anthocyanins and phenolics (Lohachoomplo et al., 2004).
However, these drying methods require a high input of energy and their cost
are elevated (Ratti, 2001).
On the other hand, osmotic dehydration is a simple and cheaper
dehydration method that has been proposed as an alternative for preserving
food products with high nutritional value, which has been successfully applied
to vegetables and fruits.
However, most berries (such as blueberries) have an impermeable
epidermis which behaves as a barrier that impedes mass transfer during
osmotic dehydration, slowing down the process (Ketata et al., 2013). This
epidermis is very thick and contains, among others, waxes and pectines which
are often necessary to pretreatment before processing the raw material.
To enhance skin permeability in blueberries, chemical, mechanical and
thermal pretreatments have been used to decrease the hydrophobicity of the
skin and promote moisture diffusion during drying of whole berries, which are
briefly described in Figure 3.
Nevertheless, the steam-blanching has been proving successful results
(using 85°C steam for 3 min) increasing mass transfer phenomena during the
osmodehydration treatments and reducing the loss of phenolic compounds,
62 Guillermo Petzold, Jorge Moreno, Pamela Zúñiga et al.
Vacuum Impregnation
ACKNOWLEDGMENTS
Author Guillermo Petzold is grateful for the financial support provided by
CONICYT through FONDECYT Project No. 11140747.
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thawing in maple sap cryoconcentration technology. Journal of Food
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impregnation. Journal of Food Engineering, 56(2–3), 273-277.
Bonilla-Zavaleta, E., Vernon-Carter, E. J. and Beristain, C. I. (2006).
Thermophysical properties of freeze-concentrated pineapple juice. Italian
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Antioxidant Properties, Health and Innovative Technologies 65
Cho, M., Howard, L. R., Prior, R. L., Clark, J. R. (2004). Flavonoid glycosides
and antioxidant capacity of various blackberry, blueberry, and red grape
genotypes determined by high-performance liquid chromatography/mass
spectrometry. Journal of Science and Food Agriculture, 84, 1771-1782.
Cho, M., Howard, L., Prior, R., Clark, J. (2005). Flavonol glycosides and
antioxidant capacity of various blackberry and blueberry genotypes
determined by high-performance liquid chromatography/mass
spectrometry. Journal of Science and Food Agriculture, 85, 2149-2158.
Cortés, M., Osorio, A. and García, E. (2007). Manzana deshidratada
fortificada con vitamina E utilizando la ingeniería de matrices. Vitae, 14,
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DeFuria, J., Bennett, G., Strissel, KJ., Perfield, JW. II, Milbury, PE.,
Greenberg, AS., Obin, MS. (2009). Dietary blueberry attenuates whole-
body insulin resistance in high fat-fed mice by reducing adipocyte death
and its inflammatory sequelae. Journal of Nutrition, 139 (8), 1510–1516.
Deshpande, S.S., Bolin, H.R. and Salunkhe, D.K. (1982). Freeze concentration
of fruit juices. Food Technology, 36(5), 68-82.
Falade, K. O. and Igbeka, J. C. (2007). Osmotic dehydration of tropical fruits
and vegetables. Food Reviews International, 23(4), 373-405.
Gao, L., Sun, J., Zhang, M., Majumdar, A. S. and An, J. (2011). Effect of pre-
drying and vacuum impregnation with nano-calcium carbonate solution on
stawberry, Carrot, corn, and blueberry. European Drying Conference-
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Giampieri, F., Alvarez-Suarez, JM., Battino, M. (2014). Strawberry and
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Agricultural and Food Chemistry, 62 (18), 3867-3876.
Giovanelli, G., Brambilla, A. and Sinelli, N. (2013). Effects of osmo-air
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Giovanelli, G., Brambilla, A., Rizzolo, A. and Sinelli, N. (2012). Effects of
blanching pre-treatment and sugar composition of the osmotic solution on
physico-chemical, morphological and antioxidant characteristics of
osmodehydrated blueberries (Vaccinium corymbosum L.). Food Research
International, 49(1), 263-271.
Howard, L. R., Clark, J. R., Brownmiller, C. (2003). Antioxidant capacity and
phenolic content in blueberries as affected by genotype and growing
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Wang, S., Chen, H., Camp, M., Ehlenfeldt, M. (2012). Genotype and growing
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In: Blueberries ISBN: 978-1-63484-885-5
Editor: Malcolm Marsh © 2016 Nova Science Publishers, Inc.
Chapter 4
BLUEBERRY ANTI-INFLAMMATORY
EFFECTS OVER METABOLIC DISEASES
ASSOCIATED WITH OBESITY
ABSTRACT
Inflammation is a natural defense mechanism triggered as a response
to an alteration of the physiological functions of the organism. This
process is responsible for the secretion of mediators crucial for tissues
repair, integrating different signalling pathways between distinct cells and
organs. Likewise, it has been observed that in metabolic diseases some
classic mediators present during short-term inflammation are involved,
although the features of its actions differ from the classic pathways. Thus
it is considered as a subclass of inflammation often referred as meta-
inflammation. In the case of obesity for example, this response is
exacerbated and, at the long term, a chronic inflammatory state associated
with cardiovascular diseases, insulin resistance and type-2 diabetes
development is established. Since obesity-associated inflammation is
known to be a key feature of the etiology of non-communicable diseases,
Authors contribute equally.
72 J. Soto-Covasich, M. Reyes-Farias, A. Ovalle-Marin et al.
several efforts have been made for identifying novel agents with anti-
inflammatory properties capable of ameliorate its negative long-term
effects. In this regard, blueberry consumption has been described to
induce important health benefits through anti-inflammatory and
antioxidant features. Therefore, in the present chapter, we will discuss the
impact on the low-grade inflammatory status associated to metabolic
diseases provided by a blueberry treatment or diet, previously described
in the literature. In this context, will be addressed: a) in vitro studies over
inflammation in macrophages and changes in adipogenesis; b) in vivo
studies over pro-oxidant and inflammatory status, related to amelioration
of insulin resistance, hyperglycemia, dyslipidemia, hyperphagia and
weight gain induced by a high fat feeding, and improvement of blood
pressure, renal function and beta cell function; and c) human clinical
evidence, over antioxidant defense mechanisms and inflammation,
influencing blood pressure and insulin sensitivity in susceptible subjects.
In this sense, recent findings supports that a blueberries-rich diet has been
able to modulate the inflammatory status in a positive manner, likewise
exerting its effects in different crucial stages of metabolic alterations
development and hence contributing to the prevention and reduction of
obesity-associated comorbidities. It is still pending to deepen into the
cellular and molecular mechanisms in order to take advantage from a
commercially-available fruit for improve human life quality.
INTRODUCTION
Obesity is a Global Pandemic
One of the aspects that influence the most on each individual’s day-by-day
wellbeing is the body weight fluctuation. Despite the fact that humans require
the presence of adipose tissue in the organism, when this tissue develops
excessively several harmful consequences occur (Bray et al., 2004). Indeed, it
is well known that an excessive body fat accumulation is a masterpiece for
several associated clinical manifestations such as type 2 diabetes (T2D),
cardiovascular diseases (CVD), among others (Guh et al., 2009). Obesity
prevalence has been doubled from 6.4% in 1980 to 12.0% in 2008 in the entire
world. Half of this rise occurred from 2000 to 2008 (Stevens et al., 2012).
Obesity is defined as the pathogenic increment of the organism fat
content, accompanied by a total body weight augment, due to a positive
balance in the equation comprising both energy intake and energy expenditure
(Hartroft et al., 1960). This augmentation has been related specially with an
increase in white adipose tissue (WAT) content. WAT it is considered an
Blueberry Anti-Inflammatory Effects over Metabolic Diseases … 73
stress arise in WAT (Furukawa et al., 2004; Hosogai et al., 2007; Ozcan et al.,
2004). These stresses induce the release of inflammatory signals, which
initiate the infiltration of monocyte into this tissue (Suganami et al., 2010).
Adipose tissue inflammation is triggered by this macrophages infiltration; the
subsequent crosstalk between these cells and resident adipocytes highlights as
a key factor for the development of associated co-morbidities (Weisberg et al.,
2003; Xu et al., 2003), especially insulin-resistance (IR) (Weisberg et al.,
2003). In this sense, several inflammatory products produced by this
interaction, such as TNF-α, MCP-1 and NO, correlates with increased body
adiposity (Ferrante et al., 2007) and appear to participate in the induction and
maintenance of the chronic inflammatory state associated with obesity
(Shoelson et al., 2000). In addition, WAT overgrowth leads to downregulation
of several anti-inflammatory products, e.g., adiponectin (Maeda et al., 2002).
In this context, it has been observed that macrophages contribute to the
development of IR in obese patients, while weight loss reduces macrophage
infiltration and the expression of inflammation-related factors in adipose
tissue, being related with insulin-sensitivity improvement (Cancello et al.,
2005; Clement et al., 2004).
At the molecular level, the adipose tissue at enlargement presents
activation of some mitogen-activated protein kinases (MAPKs), such as ERK,
p38 MAPK, and JNK (Johnson et al., 2002; Bost et al., 2005; Hirosumi et al.,
2002). Once activated by upstream kinases, these enzymes are rapidly
inactivated by MKP-1 (Farooq et al., 2004). During the course of adipocyte
hypertrophy the expression of this protein is downregulated, which leads to an
increased secretion of MCP-1 (Ito et al., 2007). MCP-1 plays a crucial role in
the inflammatory response in obesity by enhancing monocyte migration and
activation of macrophages (Yu et al., 2006). Once inside the adipose tissue,
macrophages participate in the activation of inflammatory pathways mainly
through TNF-α secretion. TNF-α activates the hypertrophied adipocytes
through their TNF-α receptor, inducing pro-inflammatory cytokines
production by NFκB-dependent mechanisms and lipolysis by NFκB-
independent (MAPK-dependent) mechanisms (Suganami et al., 2005). On the
other hand, free fatty acids (FFA), which are consequently liberated by
adipocytes, bind TLR4 complex, which is essential for the activation of NFkB
signaling by lipopolysaccharides (LPS) stimulation in macrophages (Suganami
et al., 2007), establishing a vicious cycle (Suganami et al., 2010). TNF-α and
FFA both can induce disruption of the molecular signaling pathway of insulin
action mainly by aberrant key-proteins phosphorylation (e.g., on IRS1, Akt),
declining GLUT4 translocation to membrane (Hotamisligil et al., 1999; Boden
Blueberry Anti-Inflammatory Effects over Metabolic Diseases … 75
et al., 1999). This phenomenon is first observed locally (in WAT), and then at
the systemic level, the latter when advanced stages of obesity development are
reached.
Regarding other diseases, e.g., cardiovasculares diseases (CVD), which
includes heart and vascular disease, and atherosclerosis, are characterized also
as a chronic inflammatory condition. This condition relies majorly over the
secreted factors from the inflamed adipose tissue, that is, adipokines. These
molecules present contrasting actions for the cardiovascular system. In this
sense: adiponectin, apelin and omentin preserve normal cardiovascular
function, whereas leptin, resistin and visfatin contribute to inflammation and
endothelial dysfunction (Mattu and Randeva, 2013). It has been described that
an increase in proinflammatory state cause: alterations of vascular tone and
flow, increased expression of adhesion molecules (VCAM-1, ICAM-1),
increased vascular permeability (increase of VEGF), less fibrinolysis (increase
of PAI-1), increased cytokines and C-reactive protein (PCR) (Rajendran et al.,
2012). On the other hand, insulin-resistance itself (as a results of adipose tissue
local inflammation) it is known to contribute to CVD development (Li et al.,
2011). Therefore, adipose tissue inflammation, directly and indirectly, is
recognized as a key ignitor factor for cardiac tissue injury.
As it mentioned previously, obesity co-morbilities such as, insulin
resistance, T2D and cardiovascular disease have been reconsidered as
inflammatory diseases. In this regard, the treatment with anti-inflammatory
agents, such as polyphenolic compounds, could be an effective therapeutic
strategy. In this sense, blueberry (Vaccinium corymbosum, L.) is a fruit
worldwide known as a rich source of anthocyanins, one of the main
polyphenolic compounds. In this chapter, we will explore the anti-
inflammatory effect of blueberry over metabolic diseases associated with
obesity and its preventive effects of in reducing theses co-morbilities.
IN VITRO EVIDENCE
Effects of Blueberries Extracts on Inflammation Induced
In Vitro
Obesity is the main risk factor for the development of other diseases, such
as insulin resistance, T2D, CVD, fatty liver disease, cancer and other
pathologies. The main works that describe beneficial effects of blueberry
treatment in this regards are described as follows.
adipogenesis and cell proliferation. Other study has been demonstrated that the
treatment of 3T3-L1 preadipocytes with blueberry peel extract (BPE) results in
an important adipogenesis reduction. This finding is supported by the gradual
reduction in the number of lipid droplets in a dose-dependent manner (ranged
from 50 to 300 ug/ml of BP extract) and the diminished level of triglyceride
content (by 37, 7%) after seven days of treatment of differentiating cells with
BPE (200 ug/ml). Furthermore, the mRNA and protein levels of C/EBPb,
C/EBPa and PPAR also exhibit a reduction after concomitant treatment of
differentiating 3T3-L1 cells with increasing concentrations of BP extracts
(Song et al., 2013). This same study revealed that the phosphorylation of Akt
and its downstream substrate, phospho-GSK3 was also reduced by the
treatment of BPE in 3T3-L1 cells. Hence, the evidence shows that BP extracts
has the potential to modulate the adipogenic activity via PI3K/Akt/GSK3
pathway. However, further studies are required to assure the therapeutic
potential of blueberry to adipogenesis and obesity.
Cardiovascular Disease
Sakaida et al. (2007) observed a potent inhibitory effect of blueberry leaf
extract on angiotensin converting enzyme activity, which was stronger
compared to other leaves of Ericaceae plants and Camellia sinensis L. (green
tea). Additionally, spontaneously hypertensive rats were fed a diet
supplemented with 3% of freeze-dried blueberry leaf and showed a decrease in
systolic blood pressure from the second week as compared to rats without
supplementation. These in vitro and in vivo results demonstrated the
antihypertensive property of blueberry leaf. Along with this, Louis et al.,
(2014) investigated the cardioprotective action of an aqueous blueberry extract
and five polyphenolic fractions (Phe: phenolic fraction; Flv: flavonoid
fraction; Acn: anthocyanins-enriched fraction; Hep: Heteropolymers-enriched
fraction; and Pac: proanthocyanidins-enriched fraction) on an in vitro model of
heart disease. Adult Sprague Dawley rats cardiomyocytes were isolated and
cultured, performing a pretreatment with extract and fractions and a further
treatment with norepinephrine (NE). Regarding hypertrophy and cell death of
cardiomyocyte, the Phe, Flv, Acn and Hep fractions presented a preventing
effect on cardiomyocyte injury by NE. Pretreatment of cardiomyocytes upon
Phe fraction prevented an increase of apoptosis, increase of oxidative stress,
decrease of superoxide dismutase and catalase activities, increase of calpain
activity and decrease of contractile function induced by NE treatment
compared to condition without pretreatment. As a first approach, these results
of in vitro model suggest a protective effect of blueberry over cardiovascular
diseases.
IN VIVO EVIDENCE
Anorexic Effect of Blueberry Supplementation
mice fed with high fat diet plus 4% freeze-dried blueberry powder show
improved glucose tolerance, similar to low-fat-diet group (DeFuria et al.,
2009). Moreover, fasting glucose values and HOMA-BCF score (β cell
function) were diminishes to normal levels in animals supplemented with
blueberry anthocyanins (Prior et al., 2010).
Finally, it has been described that blueberry protects renal system. Obese
male Zucker rats treated with 2% lyophilized blueberry in water (BB) for 15-
weeks presented an improvement in glucose tolerance compared with
untreated group. When the kidney function was assessed, BB supplementation
decreased mean arterial pressure and renal vascular resistance, nevertheless it
improved glomerular filtration rate and renal blood flow. In addition BB
enriched diet attenuates the expression ACE and AT1 and improved the
expression levels of ACE2 and AT2 genes in renal tissue, exerting a protective
role. Kidneys examination showed severe glomerular adhesions, cortical and
medullary tubular lesions and interstitial nephritis in obese animal, however
BB supplementation exhibit a reduction in those pathological changes. At the
end, BB enriched diet increases SOD and catalase levels in kidneys and
Blueberry Anti-Inflammatory Effects over Metabolic Diseases … 83
regular consumption of a wild blueberries drink for 6 weeks reduced the levels
of oxidized DNA bases and increased the resistance to oxidatively induced
DNA damage in subjects with risk factors for cardiovascular disease. In
addition, blueberries improve endothelial function in adults with metabolic
syndrome. Subjects received a blueberry or placebo smoothie twice daily for
six weeks. The group that consumed blueberry showed an increase in
endothelial function versus the placebo group (Stul et al., 2015). Moreover,
another study revealed that blueberry consumption improves blood pressure in
postmenopausal women with pre-and stage 1-hypertension (Johnson et al.,
2015). These studies reveal the protective effect of blueberry over
cardiovascular disease. However, further studies are required to assure the
therapeutic potential of blueberry.
CONCLUSION
All the listed evidence reveals important beneficial effects of the treatment
and/or consumption of blueberries. The low number of human trials that have
been performed in this sense, must lead future clinical trials in order to
confirm definitively the major potential of this fruit on human health, specially
on obesity and the metabolic diseases associated with.
REFERENCES
Ahmet, I., Spangler, E., Shukitt-Hale, B., Joseph, J. A., Ingram, D. K. &
Talan, M. (2009a). Survival and cardioprotective benefits of long-term
blueberry enriched diet in dilated cardiomyopathy following myocardial
infarction in rats. PLoS One, 4, e7975.
Ahmet, I., Spangler, E., Shukitt-Hale, B., Juhaszova, M., Sollott, S. J., Joseph,
J. A., Ingram, D. K. & Talan, M. (2009b). Blueberry-enriched diet
protects rat heart from ischemic damage. PLoS One, 4, e5954.
Basu, A., Du, M., Leyva, M. J., Sanchez, K., Betts, N. M., Wu, M., Aston, C.
E. & Lyons, T. J. (2010). Blueberries decrease cardiovascular risk factors
in obese men and women with metabolic syndrome. J. Nutr., vol. 140(9)
pp. 1582-7.
Boden, G. (1999). Proc Assoc Am Physicians, 111(3), 241-8
Bost, F., et al. (2005). Biochimie, 87(1), 51-6.
Blueberry Anti-Inflammatory Effects over Metabolic Diseases … 85
Prior, R. L., S, E. W., T, R. R., Khanal, R. C., Wu, X. & Howard, L. R. (2010).
Purified blueberry anthocyanins and blueberry juice alter development of
obesity in mice fed an obesogenic high-fat diet. J Agric Food Chem, 58,
3970-3976.
Prior, R. L., Wu, X., Gu, L., Hager, T., Hager, A., Wilkes, S. & Howard, L.
(2009). Purified berry anthocyanins but not whole berries normalize lipid
parameters in mice fed an obesogenic high fat diet. Mol Nutr Food Res,
53, 1406-1418.
Rajendran, K., Devarajan, N., Ganesan, M. & Ragunathan, M. (2012).
Obesity, Inflammation and Acute Myocardial Infarction - Expression of
leptin, IL-6 and high sensitivity-CRP in Chennai based population.
Thromb J., 10(1), 13.
Sakaida, H., et al., (2007). Effect of Vaccinium ashei reade leaves on
angiotensin converting enzyme activity in vitro and on systolic blood
pressure of spontaneously hypertensive rats in vivo. Biosci Biotechnol
Biochem, 71(9), p. 2335-7.
Salcedo, R., Ponce, M. L., Young, H. A., Wasserman, K., Ward, J. M.,
Kleinman, H. K., Oppenheim, J. J. & Murphy, W. J. (2000). Blood, 96,
34-40.
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polyphenols in a whole diet. Food Chemistry, 101, p. 492–501.
Suganami, T., et al. (2010). J Leukoc Biol, 88(1), 33-9.
Seymour, E. M., Tanone, II., Urcuyo-Llanes, D. E., Lewis, S. K., Kirakosyan,
A., Kondoleon, M. G., Kaufman, P. B. & Bolling, S. F. (2011). Blueberry
intake alters skeletal muscle and adipose tissue peroxisome proliferator-
activated receptor activity and reduces insulin resistance in obese rats. J
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Seeram, N. P. (2008). Berry fruits: compositional elements, biochemical
activities, and the impact of their intake on human health, performance,
and disease. J. Agric. Food Chem., vol. 56(3) pp. 627-9.
Shoelson, S. E., et al. (2006). J Clin Invest, 116(7), 1793-801.
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Stull, A. J., Cash, K. C., Johnson, W. D., Champagne, C. M. & Cefalu, W. T.
(2010). Bioactives in blueberries improve insulin sensitivity in obese,
insulin-resistant men and women. J Nutr., 140(10), 1764-8.
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88 J. Soto-Covasich, M. Reyes-Farias, A. Ovalle-Marin et al.
Chapter 5
ABSTRACT
Alcohol is a powerful teratogen, systematically affecting prenatal
development as well as postnatal functioning in humans and other
mammals. Using a mouse model, this study explored the potential effects
of anthocyanins from blueberry extracts in protecting against alcohol-
induced prenatal developmental deficiencies.
Swiss mice were assigned to three experimental groups: control
(CO), binge alcohol (BA) and alcohol-anthocyanin (AA). CO mice were
administered normal saline (0.03 ml/g maternal body weight), while BA
and AA mice received alcohol (25% v/v of absolute ethanol in normal
saline at 0.03 ml/g maternal body weight), through intraperitoneal
injections on days 5 and 7 following impregnation. Supplemental
anthocyanins via blueberry extracts (0.03 mg/g maternal body weight)
Corresponding author: millerrj@emu.edu.
90 Zach S. Gish, Sharang Penumetsa, Diana J. Valle et al.
INTRODUCTION
Alcohol and Development
Many consumers are aware of the health benefits that fresh blueberries
provide [23]. These include basic nutrient properties, antioxidant activity, [24,
25] anti-aging properties, [26] cancer prevention, [27, 28] protection against
age-related neurological defects, [29] urinary tract health, protection against
diabetes, [30] and cardiovascular health [31]. The polyphenolics and
anthocyanins, found in ripened blueberries, are the primary health promoters
and protective antioxidant agents [32] In comparison to many other fruits,
blueberries contain higher levels of protective anthocyanins. These benefits are
based on various studies, many of them animal studies where the findings have
been superimposed on humans. This list has also been a clarion marketing call
and elicited many consumers to choose blueberries for consumption rather
than other fruits which have lower levels of antioxidants.
What if the health benefit list of blueberries or blueberry anthocyanins
could be expanded? This chapter, based on preliminary research in our
laboratory, documents that blueberry anthocyanins in the form of blueberry
extract can alleviate some of the teratogenic influences of maternally ingested
alcohol on embryonic/fetal development. That claim, if further verified, has
huge implications. The idea that anthocyanins protect against some of alcohol
teratogenic influences has been noted by several other investigators in other
biological systems; [33, 34] however to date, except for this model project,
that connection has not been demonstrated in an in vivo mammalian
developmental system.
MODEL PROJECT
Experimental Objectives
The teratogenic effects that binge alcohol alone can have on a developing
embryo or fetus are well documented in various model systems. However, the
potential protection of anthocyanins against this alcohol toxicity has not been
examined in developmental model systems. Consequently our project
approach used an in vivo mouse development model to inspect the extent of
the protection that anthocyanins provide in combating the life-threatening
effects of oxidative stress on embryos and fetuses from alcohol induction
exposure during gestation. To investigate the extent of gross anatomical
malformations, three experimental groups of pregnant mice were used: control
(CO), binge alcohol (BA), and binge alcohol supplemented with anthocyanins
(AA). The goal for this experiment was to clearly demonstrate the protective
role of anthocyanins against the teratogenic influences of alcohol as shown in
gross anatomic parameters – whole body, forebrain, and liver in the mouse
fetuses.
Blueberry Extracts Protect against Gross Mouse Fetal Defects … 95
Measurements/Stereological Data
Random representative fetuses from each group (N = 12) were used for
morphometric and stereological data collection. Morphometric data consisted
of determining gross liver and telencephalon area with direct measurements
using a Nikon SMZ 74ST microscope and NIS Elements BR 3.2 software. In
each fetus the telencephalon, a part of the forebrain area (prosencephalon), and
the liver area were determined via specific somatic landmarks. These organ
areas were circumscribed and their surface areas estimated in mm2 using the
calibrated software program from the image camera. Subsequently, these areas
were compared with the total fetus body surface area.
To obtain primary stereological data, a coherent Weibel grid imprinted on
an acetate sheet was superimposed on photomicrographs of the fetuses.
Following an established protocol, [45, 46] simple point counts, based on the
number of Weibel grid points falling on the image of the parameter of interest,
e.g., liver vs total body area or telencephalon area vs. total body area, were
converted into volume density determinations. Each measured fetus was
contained within one counting field of view and represented an “n” of one.
Statistical Analysis
Means and standard errors were calculated as group statistics for measured
parameters: fetal weight, crown-rump length, and gross tissue measurements
(organ areas and organ volume density measures). Significant differences
Blueberry Extracts Protect against Gross Mouse Fetal Defects … 97
Percent Increase in
Average Number
Fetuses / Mother
Fertility Percent
Total Fetuses
Body Weight
Mothers
(g)
(g)
(g)
Although the project was initiated with mating 15 female mice in the CO
group and 12 female mice in the BA and AA groups, the number of resultant
pregnant females in the BA and the AA groups was less than in the CO group:
CO = 12 pregnancies (80% fertility); BA = 7 pregnancies (58% fertility);
AA = 9 pregnancies (75% fertility). While the average number of fetuses per
pregnant mother varied slightly in the different groups with an average of 8.94
fetuses/mother, these differences were not statistically significant (See Table
1). Upon conclusion of the project, it was determined that one of the males
used was sterile, since each of the females this male mated throughout the
project did not produce offspring.
All females receiving binge alcohol injections exhibited similar patterns of
behavior following injections that included staggering and losing
consciousness within the span of a few minutes. But then later these females
revived and resumed normal activity after a period of time. Control females
with saline injections did not exhibit these behavioral patterns. Instead they
displayed mild agitation following injections.
The average food intake per day by each group generally reflected a
steady increase in consumption throughout the period of pregnancy. However
food intake did fluctuate as a consequence of alcohol injections (see Figure 1).
Figure 1. Daily food consumption by experimental group mothers over the course of
experiment. Control (CO) saline treated mice (N = 15), Binge Alcohol (BA) treated
mice (N = 12), Alcohol–Anthocyanin (AA) treated mice (N = 12). Feeding data began
after males were removed from female cages on day 4. Arrows denote days injections
were performed (large=ethanol) (narrow = anthocyanin). Values are expressed as
means ± standard error.
Blueberry Extracts Protect against Gross Mouse Fetal Defects … 99
Following necropsy, the average weight of the 117 CO fetuses was 196.7
mg (Figure 2) and the average crown to rump length was 11.19 mm (Figure 3).
The BA group of 58 fetuses averaged 90.4 mg for fetal weight at time of
collection and a length of 9.07 mm. The AA group of 79 fetuses averaged a
weight of 161.8 mg and a length of 10.71 mm. Both fetal body weight and
crown-rump length were not significantly different between the CO and AA
groups. However, significant differences were found between the CO and BA
groups as well as between AA and BA groups. Representative samples from
each experimental group demonstrating the developmental differences
pictorially are illustrated in Figure 4. Strong definition and detail differences in
the fetal mice can be more clearly observed in the CO (Figure 4A) and AA
(Figure 4C) mice while gross detail was far less distinct in the BA (Figure 4B)
mice. Especially in the BA group, the fetuses were frequently ill-formed, but
less so in the AA group when compared to the CO group.
In concordance with previous work, perigestational ethanol exposure does
retard gross fetal size, [47] however proactive administration of anthocyanins
appears to partially neutralize the deficits in fetal size. These results clearly
demonstrate that the anthocyanin dosages mitigate the detrimental effects of
the alcohol at the very least on a gross fetal body scale.
In comparing telencephalon size with the rest of the fetal body (Figure 5),
BA fetuses demonstrated a trend of reduced telencephalon size (5%) compared
with those of CO (7%) and AA (7%). A similar trend was seen in another
study [48] that showed improper telencephalon division and development in
ethanol induced mouse embryos.
100 Zach S. Gish, Sharang Penumetsa, Diana J. Valle et al.
Figure 5. Fetus body composition comparing telencephalon size to relative body size,
using a Weibel grid to determine volume density measures. Control (CO) saline treated
fetuses (N = 12), Binge Alcohol (BA) treated fetuses (N = 11), Alcohol-Anthocyanin
(AA) treated fetuses (N = 11). Values are expressed as means. Circle diameters reflect
relative differences in experimental group body sizes.
Figure 7. Fetus body composition comparing liver size to relative body size, using a
Weibel grid to determine volume density measures. Control (CO) saline treated fetuses
(N = 12), Binge Alcohol (BA) treated fetuses (N = 11), Alcohol-Anthocyanin (AA)
treated fetuses (N = 11). Values are expressed as means. Circle diameters reflect
relative differences in experimental group body sizes.
Blueberry Extracts Protect against Gross Mouse Fetal Defects … 103
Figure 8. Morphometric liver analysis detailing average absolute liver area in each
experimental group. Control (CO) saline treated fetuses (N = 12), Binge Alcohol (BA)
treated fetuses (N = 11), Alcohol-Anthocyanin (AA) treated fetuses (N = 11). Values
are expressed as means ± standard errors.
CONCLUSION
Our research demonstrates that anthocyanin supplementation (in the form
of blueberry extracts) given to developing embryos/fetuses in the mouse
development system mitigates some of the detrimental effects of concomitant
perigestational exposure of alcohol. This mitigating response is seen following
two binge alcohol exposures during the early period of gestation when
accompanied with four applications of anthocyanin supplementation given
before, during, and after the exposure to alcohol.
Initial gross fetal body assessments in comparison to control fetuses show
that binge alcohol exposure reduces average fetal body weight by 54% while
binge alcohol with anthocyanin supplementation reduces average fetal body
weight by 18%. When considering fetal size as determined by crown-rump
length, binge alcohol reduces size by 19% and binge alcohol supplemented
with anthocyanins reduces size by 4% when compared with control fetuses.
In looking at the size of two organs – telencephalon and liver – a similar
outcome is observed. Based on surface area, fetuses in the control group have
telencephalon and liver surface areas that represent 7% and 12% respectively
of their total body surface area. In contrast binge alcohol fetuses have
telencephalon and liver surface areas that represent 5% and 7% respectively of
their total body surface area. However, fetuses from the binge alcohol and
anthocyanin supplemented group have telencephalon and liver surfaces that
represent 7% and 11% respectively of their total body surface area paralleling
the control group data. Inspection of these data shows that anthocyanin
supplementation has a beneficial effect in reducing the influence of alcohol
toxicity.
The specific mechanism for the protective role of anthocyanins against
alcohol toxicity in the developing mouse system is not yet determined.
Subsequent studies extending this research are currently focusing on
Blueberry Extracts Protect against Gross Mouse Fetal Defects … 105
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INDEX
crystals, 58, 59
C cultivars, vii, viii, 1, 5, 7, 9, 10, 16, 18, 21,
23, 24, 26, 27, 28, 32, 33, 34, 35, 36, 38,
Ca2+, 64
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 52
CAE, 39
cultivation, vii, 1, 2, 3, 4, 5, 8, 10, 27
calcium carbonate, 65
CVD, 75, 77, 83
cancer, viii, 2, 3, 17, 28, 29, 38, 56, 67, 68,
cyclooxygenase, 76
77, 83, 93, 108
cytokines, 73, 74, 75
cancer cells, 17, 28, 29, 38, 68
carbohydrates, 8, 10
cardiovascular diseases (CVD), ix, 56, 71, D
72, 75, 79, 84, 86
cardiovascular function, 75 dehydration, ix, 55, 60, 61, 62, 63, 64, 65,
cardiovascular risk, 84 66, 68, 69
cardiovascular system, 75 Delite, ix, 6, 12, 32, 33, 34, 36, 39, 40, 41,
carotene, 8, 19, 21, 22, 40 42, 46, 47, 48
carotenoids, viii, ix, 2, 4, 18, 19, 29, 32, 40, dendritic cell, 73
41, 52 Department of Agriculture, vii, viii, 4, 31
cell death, 79, 91, 102, 105, 109 derivatives, 14, 22, 37, 57
cell lines, 18, 76 detection system, 19
cell membranes, 61 developing brain, 108, 109
cellulose, 8 developmental change, 106, 109
central nervous system (CNS), 90, 92, 101 developmental process, 90, 101
cerebral cortex, 91, 105 diabetes, viii, ix, 2, 4, 18, 71, 73, 85, 93,
chemical, viii, 2, 3, 50, 51, 61, 65, 68, 73 108
chemical properties, 68 dialysis, 68
chemokines, 73 diastolic pressure, 82
Chile, 4, 5, 6, 7, 55, 71 diet, x, 18, 56, 72, 79, 80, 81, 82, 84, 87, 88,
Chitosan, 68 95, 107
chlorophyll, 10 dietary management, viii, 2, 4, 18
cholesterol, viii, 2, 4, 18, 21, 81 dietary supplementation, 83
Climax, ix, 6, 12, 32, 34, 35, 36, 39, 41, 42, diffusion, 61, 62
43, 46, 47 digestive enzymes, 107
clinical symptoms, 73 dilated cardiomyopathy, 84
clinical trials, 83, 84 diseases, ix, 8, 33, 56, 71, 73, 75, 76, 77, 83,
coenzyme, 92 84
colon, 18, 29 distillation, 58
colon cancer, 18, 29 distribution, 16, 26, 28, 50
consumption, ix, 5, 18, 20, 28, 55, 57, 61, diversity, 16, 19
72, 77, 83, 84, 85, 93, 95, 98, 99, 105 DNA, 14, 27, 84, 93, 108
contamination, 61 DNA damage, 14, 27, 84, 93, 108
control group, 77, 93, 104 double bonds, 40
copper, 10 drinking water, 81
c-reactive protein, 75 dry matter, 63, 66
crop, vii, 1, 3, 22 dyslipidemia, x, 72
crystallization, 59
Index 113
E F
elaboration, 14 farmers, 3
Elam, 85 fasting, viii, 2, 4, 18, 80
electric field, 63 fasting glucose, 81
electron, 34 fat, 9, 72, 80, 81, 83, 87, 88
electrons, 4 fat soluble, 9
electrophoresis, 68 fatty acids, 74
embryology, 105, 108 fermentation, 50
emission, 8, 24 fertility, 98
endocrine, 72 fetal alcohol syndrome, 106, 109
endothelial dysfunction, 75 fetal development, 93
energy, ix, 10, 55, 61, 63, 72, 83 fetus, 90, 94, 96, 101
energy expenditure, 72 filtration, 68, 82
energy transfer, 10 flavanols, viii, 2, 3, 57
engineering, 67 flavonoids, viii, 2, 3, 10, 11, 16, 29, 57, 85,
enlargement, 74 107
environment, 63 flavor, 6, 8, 58, 59, 62
enzyme, 57, 58, 68, 78 folate, 9
enzymes, 9, 58, 63, 74 food, ix, 10, 24, 41, 51, 52, 55, 56, 58, 59,
epidermis, 61, 63 60, 61, 63, 64, 67, 95, 98, 99
epithelial cells, 83 Food and Agriculture Organization (FAO),
equilibrium, 61 vii, viii, 3, 4, 22, 31, 49
equipment, 59 food industry, ix, 55, 64
ESI, 27 food intake, 98
essential fatty acids, 56 food products, 52, 61
ester, 13, 14 forebrain, x, 90, 94, 96
ethanol, x, 35, 78, 89, 91, 92, 93, 95, 98, 99, formation, 20, 22, 92
101, 102, 103, 105, 106, 107, 108, 109 free radicals, viii, ix, 2, 3, 4, 55, 56, 93, 94,
ethyl acetate, 77, 79 107
ethylene, viii, 2, 3 freeze concentration, ix, 55, 58, 59, 60, 65,
etiology, ix, 71 66, 67
Europe, 4, 7, 18 freezing, 58, 59, 60, 61, 66, 68
European Union, vii, 2, 3 fructose, 8, 80, 86
evaporation, 58 fruits, vii, ix, 2, 3, 5, 7, 8, 9, 10, 11, 14, 15,
evidence, x, 67, 72, 76, 78, 84, 105 16, 19, 20, 21, 24, 25, 29, 32, 33, 34, 38,
evolution, 10, 46 40, 41, 43, 45, 48, 50, 55, 56, 60, 61, 62,
exercise, 22 63, 64, 65, 66, 76, 87, 88, 93, 107
exposure, x, xi, 9, 10, 63, 90, 91, 92, 93, 94, functional food, 52, 56
99, 101, 103, 104, 105, 107, 109
extinction, 35, 40
extraction, 15, 37, 38 G
extracts, x, xi, 9, 16, 18, 20, 22, 27, 28, 29,
GABA, 109
33, 37, 38, 50, 51, 68, 76, 77, 78, 79, 83,
gastrointestinal tract, 77
88, 89, 90, 103, 104
114 Index
yield, 7
U
ultrasound, 63, 68 Z
United Nations, 3, 22, 49
United States, vii, viii, 2, 3, 4, 5, 6, 19, 31, zinc, 10
32