Content uploaded by Cristina Andres-Lacueva
Author content
All content in this area was uploaded by Cristina Andres-Lacueva
Content may be subject to copyright.
http://fst.sagepub.com/
International
Food Science and Technology
http://fst.sagepub.com/content/11/3/159
The online version of this article can be found at:
DOI: 10.1177/1082013205054498
2005 11: 159Food Science and Technology International
R. M. Lamuela-Raventós, A. I. Romero-Pérez, C. Andrés-Lacueva and A. Tornero
Review: Health Effects of Cocoa Flavonoids
Published by:
http://www.sagepublications.com
On behalf of:
Consejo Superior de Investigaciones Científicas (Spanish Council for Scientific Research)
can be found at:Food Science and Technology InternationalAdditional services and information for
http://fst.sagepub.com/cgi/alertsEmail Alerts:
http://fst.sagepub.com/subscriptionsSubscriptions:
http://www.sagepub.com/journalsReprints.navReprints:
http://www.sagepub.com/journalsPermissions.navPermissions:
http://fst.sagepub.com/content/11/3/159.refs.htmlCitations:
What is This?
- Jun 20, 2005Version of Record >>
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
Review: Health Effects of Cocoa Flavonoids
R.M. Lamuela-Raventós,
1,
* A.I. Romero-Pérez,
1
C. Andrés-Lacueva
1
and A. Tornero
2
1
Nutrició i Bromatologia, CÈRTA, Facultat de Farmàcia, Universitat de Barcelona, Avinguda Joan XXIII s/n,
08028 Barcelona, Spain
2
Faculty of Science, Engineering and Environment, School of Environment and Life Sciences, University of
Salford, Salford, Greater Manchester M5 4WT, UK
Flavonoids are phenolic substances widely found in fruits and vegetables. Many epidemiological studies
associate the ingestion of flavonoids with a reduced risk of cardiovascular disease and certain types of
cancer. These effects are due to the physiological activity of flavonoids in the reduction of oxidative
stress, inhibiting low-density lipoproteins (LDL) oxidation and platelet aggregation, acting as vasodila-
tors in blood vessels, inhibiting the adherence of monocytes to the vascular endothelium, promoting fib-
rinolysis, acting as immunomodulators and anti-inflammatory agents and as inhibitors in the different
phases of tumour process. Cocoa is an important source of polyphenols, which comprise 12–18% of its
total weight on dry basis; the major phenolic compounds are epicatechin, proanthocyanidins and cate-
chin. The levels of flavonoids contained are higher than the ones founds in apples, onions or wine, foods
known for their high amount of phenolic compounds. Cocoa and cocoa products are important sources
of flavonoids in our diet. In the Dutch population chocolate contributes up to 20% of the total flavonoid
intake in adults, and in children the percentage is even higher. The bioavailability of these compounds
depends on other food constituents, and their interaction with the food matrix. This article reviews
current evidence on the health effects of cocoa flavonoids in our diet. The compiled data supports the
premise that the consumption of cocoa flavonoids is beneficial to human health.
Key Words: cocoa, chocolate, flavonoids, flavan-3-ol, flavonol, flavone
INTRODUCTION
Cocoa beans and their derivatives such as cocoa
powder and chocolate, are important sources of
polyphenols (Waterhouse et al., 1996; Sanbongi et al.,
1998; Lamuela-Raventós et al., 2001; Wollgast and
Anklam, 2000a). Between the 1950s and 1970s the
research into cocoa phenolic compounds focused on
their relation with the formation and evolution of
chocolate flavour. Interest in the nutritional attributes
of vegetal phenols has significantly increased, particu-
larly in flavonoids, and even more so, now we have
been made aware of the possible relation between
their consumption, and the reduction of the risk factors
of cardiovascular disease and certain types of cancer.
This relation was established after observations were
pointed out in different epidemiological studies.
*To whom correspondence should be sent
(e-mail: lamuela@ub.edu).
Received 5 June 2004; revised 3 March 2004.
Food Sci Tech Int 2005; 11(3):159–176
© 2005 Sage Publications
ISSN: 1082-0132
DOI: 10.1177/1082013205054498
Three main roles have been detected; as antioxi-
dants, as cardiovascular protectors and with possible
preventive roles against tumoural processes. However,
the physiological effects of these compounds may
extend beyond the modulation of oxidative stress.
(Scalbert et al., 2005). Different mechanisms of activity
have been established within these main effects, both
for cocoa products and for the main isolated
flavonoids. The literature shows high biological activity
for these compounds, and it seems reasonable to say
that the effects observed after the consumption of
cocoa and its by-products result from a combination of
them all.
Literature on these compounds is extensive, since
these substances are well known for their widespread
distribution in fruits and vegetables, as well as for their
activity as active substances in medicinal plants or
because they have already been used in clinical train-
ing, e.g. quercetin for treatment of the early stages of
venous insufficiency.
The study of the individual activity of these
substances allows in depth examination of the poten-
tially beneficial effects of cocoa consumption, taking
into account their content in cocoa and cocoa products
and their bioavailability. All these factors let us
establish hypotheses with regard to the amounts that
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
should be ingested so that their effects can be
observed.
COMPOSITION OF COCOA
FLAVONOIDS
Phenolic compounds comprise 12–18% of the total
weight of dried cocoa nibs. Approximately 35% of the
total content of polyphenols in non-fermented cocoa
nibs belonging to the Forastero variety is epicatechin.
The epicatechin content in non-fermented cocoa nibs
of different varieties is in the 34.65 and 43.27mg/g
range (defatted sample). This amount decreases during
the process of fermentation and drying, depending on
the place of production the epicatechin content may
vary between 2.66 and 16.5mg/g of defatted sample
(Forsyth, 1955; Forsyth and Quesnel, 1963; Kim and
Keeney, 1984; Wollgast and Anklam 2000b), whilst the
amount of catechin increases (Porter et al., 1991).
As catechins are the major compounds in cocoa
nibs, most of the articles published about the analysis
of polyphenols in cocoa and its by-products have
focused on these compounds, finding great variability
in the levels depending on the type of product
analysed, as seen in Table 1, as has been pointed out
previously by Kris-Etherton and Keen (2002).
Apart from flavan-3-ols, catechin, epicatechin and
their dimers procyanidin B1 and procyanidin B2, which
are major compounds in cocoa, the following flavonols
have also been identified and quantified: quercetin,
quercetin-3-O-glucoside (isoquercitrin), quercetin-3-O-
galactoside (hyperoside), quercetin-3-O-arabinoside,
as well as the following flavones: apigenin, apigenin-8-
C-glucoside (vitexin), apigenin-6-C-glucoside (isovi-
texin), luteolin and luteolin-7-O-glucoside
(Sánchez-Rabaneda et al., 2003). Table 2 shows the
levels of these compounds in a sample of cocoa nibs
(Lamuela-Raventós et al., 2003), applying the method
described by Sanchez-Rabaneda et al. (2003).
160 R.M. LAMUELA-RAVENTÓS ET AL.
FLAVONOID IN DIET INGESTION
Most studies on the biological activity of cocoa
flavonoids briefed below, have been made in vitro in
isolated systems. Although the results obtained in such
studies cannot directly be extrapolated in vivo, they
can give us an idea on the plasmatic concentrations or
levels in target tissues that we should achieve to obtain
observable effects. Owing to the great amount of
numeric data that we can obtain from these studies, we
have carried out a global approach. In Table 3, the sub-
stances have been divided into groups and concentra-
tion ranges in which a particular activity has been seen
(the maximum and minimum concentrations that have
shown some effect in the studies have been included).
Most studies give concentrations in the 10 and 30M
range for the main activities in each group, except for
the antioxidant activity where these substances show
high activity at low concentrations. Furthermore, we
must bear in mind that these substances can act syner-
gically, so that inactive concentrations of each of them
acting by themselves could have a certain activity when
Table 1. Monomeric catechins content in different cocoa by-products.
Author Analysed product Amount of catechins
Bonvehi and Coll, 1997 Cocoa powder (100 g) 300 mg
Arts et al., 1999 Milk chocolate (100 g) 16 mg
Dark chocolate (100 g) 54 mg
Vinson et al., 1999 Milk chocolate (100 g) 15–16 mg
Dark chocolate (100 g) 48–137 mg
Cocoa powder (100 g) 296–327 mg
Hammerstone et al., 2000 Dark chocolate (100g) 108 mg
(445 mg total procyanidins)
Arts et al., 2000 Cocoa drink (1 L) 2–21 mg
Pascual-Teresa et al., 2000 Soluble cocoa (100 mL) 1.33 mg
Chocolate (100 g) 3.33mg
Table 2. Levels of flavonoids found in cocoa nibs
(Lamuela-Raventós et al., 2003).
Compound Cocoa nibs (mg/100 g)
Catechin 72.4
Epicatechin 328.1
Procyanidin B1 16.6
Procyanidin B2 92.1
Quercetin 2.5
Isoquercitrin 11.0
Hyperoside 9.0
Quercetin 3-O-arabinoside 16.5
Apigenin 0.5
Vitexin 0.4
Isovitexin 0.4
Luteolin 0.5
Luteolin-7-O-glucoside 1.2
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
combined, as Pignatelli et al. (2000) saw for catechin
and quercetin.
On the other hand, research has been carried out on
human volunteers that relate the ingested amounts of
some flavonoids from cocoa with their corresponding
plasmatic levels, and in some of them certain activities
have been observed. Table 4 summarises data from
these studies.
The high flavonoid content of cocoa in comparison
with other foods known to have high amounts of these
compounds is remarkable. Apart from the values that
we have found in cocoa beans (Table 2) and the cate-
chins values for cocoa and chocolate described in the
literature (Table 1), we have also included data
extracted from the US Department of Agriculture
database, in which flavonoid values in different foods
are compiled (Table 5). There are also mentioned
values for catechin, epicatechin, quercetin and the total
values of flavan-3-ols (catechins) and flavonols in dif-
ferent foods such as wine, apples, onions and different
kinds of tea. The values obtained for cocoa and choco-
late are, in some cases, higher than the ones found in
those foods. As for minor flavonoids in cocoa
(flavonols and flavones), we have observed that cate-
chins have a high biological activity and despite the
fact that the amount is very small in some cases, our
interest lies in the possibility of a synergetic action,
thus potentiating the activity shown by this group of
substances.
In spite of the high content of flavonoids high-
lighted, the assessment of these data must consider the
real contribution of cocoa and its by-products to the
diet. Arts et al. (1999) pointed out the importance of
chocolate as a catechin source in the diet, contributing
20% to the total intake of the Dutch population. This
percentage could be even higher in the younger age
groups, where chocolate is preferred to other popular
and widely consumed foods with a high content of cat-
echins such as tea or, in countries where the consump-
tion of this drink is not widespread, other foods or
beverages. This consumption implies that cocoa
flavonoids could significantly contribute to the effect
observed in epidemiological studies.
Apart from epidemiological observations, the only
available data were on the effects observed after acute
consumption of these substances in cocoa and by-prod-
ucts (Table 4). A study on multiple intakes has recently
been published, which allows us to confirm some of the
previous observations. In this blinded parallel-designed
study, 32 healthy subjects were assigned to consume a
tablet containing cocoa flavonoids (234mg of
monomeric catechins and procyanidins) or a placebo
Health Effects of Cocoa Flavonoids 161
Table 3. Concentration ranges with reported activity.
Catechins Flavonols Flavones
Antioxidant 224 nM–40 M 0.25–10 M 0.58–15 M
(Arteel and Sies, 1999; (Day et al., 2000; (Lin et al., 2002)
Zhu et al., 2002) Johnson and Loo, 2000)
Platelet antiaggregation 3–100 M 10–650 M No data
(Pignatelli et al., 2000; (Xie et al., 1996;
Rein et al., 2000c; Pignatelli et al., 2000)
Schewe et al., 2001)
Cardiovascular protection 1 nM–10 M 1 nM–100 M 4–10 M
(Waterhouse et al., 1996; (Kameda et al., 1987; (Zhang et al., 2000a)
Abou-Agag et al., 2001) Wang et al., 1996;
Kobuchi et al., 1999;
Zhao et al., 1999;
Abou-Agag et al., 2001)
Cancer protection No data 1–60 M 1–40 M
(Kang and Liang 1997; (Huang et al., 1996, 1999;
Xiao et al., 1997; Yin et al., 1999;
Rodgers and Grant 1998; Gupta et al., 2001;
Richter et al., 1999; Lindenmeyer et al., 2001;
Wang et al., 1999; Xagorari et al., 2001;
Ranelleti et al., 2000; Lautraite et al., 2002;)
Han et al., 2001;
Xagorari et al., 2001;
Lautraite et al., 2002;
Romero et al., 2002;
Soleas et al., 2002)
Neuronal protection 100–300 M 10–20 M No data
(Shimada et al., 2001) (Lee et al., 2001;
Chen and Ma 1999)
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
for 28 days. After this period, plasma concentrations of
epicatechin and catechin in the active group increased
by 81% and 28%, respectively, and significantly
decreased platelet aggregation induced by collagen or
adenosine diphosphate (ADP) (Murphy et al., 2003).
Despite the great amount of data published, it is
very difficult to make any kind of extrapolation, due to
the complexity of the metabolism of these compounds
and the possibility of synergism between them, in addi-
tion to the interaction phenomenon with other com-
ponents from foods.
BIOAVAILABILITY OF COCOA
FLAVONOIDS
Biological properties of cocoa flavonoids are condi-
tioned by their bioavailability. Different studies have
proven the absorption of catechin, epicatechin and
dimeric procyanidins as a result of an increase in their
plasmatic concentrations after the intake of different
cocoa by-products by animals and humans (Richelle et
al., 1999; Rein et al., 2000a; Baba et al., 2000b and
2001a; Holt et al., 2002b; Steinberg et al., 2002; Zhu et
al., 2002; Keen et al., 2005). Likewise, after ingesting
onions, an increase in the plasmatic concentrations of
quercetin derivatives has been observed in humans
(Hollman and Katan, 1999; Day et al., 2001; Graefe et
al., 2001; Wittig et al., 2001).
The absorption of these flavonoids across the
human intestinal epithelial membrane presents distinc-
tive aspects for aglycons, glycosides or polymeric
forms. Studies with rats revealed that quercetin
aglycon can be partially absorbed in the stomach, in
opposition to its glycoside isoquercitrin (Crespy et al.,
2002). However, in contrast to what was initially
thought, neither glycosides nor dimeric or polymeric
forms (procyanidins) are hydrolysed at a gastric level,
thus arriving intact to the small intestine (Ríos et al.,
2002). The aglycons, using the Caco-2 cellular model of
162 R.M. LAMUELA-RAVENTÓS ET AL.
Table 4. Relation between ingestion, plasmatic levels and observed effects (acute studies).
Ingestion Plasmatic level Observed effect Reference
557 mg total procyanidins, which 137 mg 257 66nM Increases plasmatic antioxidant capacity Rein et al., 2000a
are epicatechin (as 80 g of chocolate) (epicatechin)
897 mg epicatechin and procyanidins No data Diminishes platelet aggregation Rein et al., 2000b
(as 300 mL of cocoa drink containing
18.75 g cocoa powder)
280 mg total procyanidins, which 69 mg are 258 29 nM Increases plasmatic antioxidant capacity Wang et al., 2000
epicatechin (as 53 g of chocolate) (epicatechin)
220 mg catechins and procyanidins 427 249nM Diminishes platelet aggregation and Holt et al., 2002a
(as 25 g of semisweet chocolate chips) increases prostacyclin/leukotriene ratio
70 mg catechins and 106 mg procyanidins No data Increases plasmatic nitric oxide Heiss et al., 2003
(as 100 mL of cocoa drink)
26 g of cocoa 41 4nM (proc. B2) No data Holt et al., 2002b
5.92 0.60M (epicat.)
0.16 0.03M (catechin)
Table 5. Flavonoid content of some foods. Data are from US Department of Agriculture, Agricultural Research
Service. 2004. USDA Database from the Flavonoid Content of Selected Foods. Nutrient Data Laboratory Home
Page, http://www.nal.usda.gov/fnic/foodcomp.
Flavan-3-ols (mg) Flavonols (mg)
Food (100 g) Catechin and epicatechin Total Quercetin Total
Red wine 11.9 11.9 0.84 1.64
Apple, with skin 9.09 9.09 4.42 4.42
Onions, red, raw – – 19.93 37.87
Tea, black, ready-to-drink 0.49 27.72 0.74 2.27
Tea, black, brewed, prepared with tap water 3.85 133.79 2.07 3.86
Tea, green, ready-to-drink 1.98 11.9 0.21 1.56
Tea, green, brewed 11.2 133.28 2.69 5.21
Cocoa, dry powder – – 20.13 20.13
Chocolate, dark 53.49 53.49 – –
Chocolate, milk 13.35 13.35 – –
Lack of data in the database does not mean the compound is not present in a particular food.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
human intestinal absorption, go across the intestinal
cells by means of a multispecific organic anion trans-
porter (Vaidyanathan and Walle, 2001), although Day
et al. (2003) also observed their absorption in an iso-
lated rat small intestine preparation by means of
passive diffusion. Procyanidin absorption depends on
its molecular weight. Dimers and probably trimers are
absorbed in the small intestine but less efficiently
(under 0.5%) than epicatechin and catechin
monomers, whose absorption level is in the 22–55%
range (Baba et al., 2001a, 2001b; Gonthier et al., 2003).
However, epicatechin is much better absorbed than
catechin (Holt et al., 2002b; Steinberg et al., 2002) pos-
sibly due to sterochemical differences which results in
differences in hydrophobicity (Keen et al., 2005). On
the other hand, polymeric procyanidins, given their
high molecular weight, are scarcely absorbed.
However, we must carefully consider the results from
the activity of cocoa procyanidins in the in vitro assays,
especially the data obtained with long-chain oligomers,
because their potential activity in vivo is conditioned
by their poor bioavailability.
Some glycosides interact with a sodium-dependent
glucose transporter in the intestine (Gee et al., 1998,
2000; Ader et al., 2001; Wolffram et al., 2002; Manach
and Donovan, 2004), which increases their absorption
compared with aglycons (Morand et al., 2000; Olthof et
al., 2000). Once inside the intestinal cell, they are
hydrolysed by means of cytostolic -glucosidases
(Nemeth et al., 2003; Manach and Donovan, 2004). A
second hypothesis on the absorption of these glyco-
sides has been proposed. Lactase phlorizin hydrolase
(an enzyme located in the luminal side of enterocytes
in the small intestine membrane) may also act by
hydrolysing the glycoside allowing the released aglycon
to be absorbed by passive diffusion. The position of the
glucose molecule in the aromatic ring could determine
the contribution of each one of these mechanisms to
the absorption process (Day et al., 2003; Sesink et al.,
2003). The inter-individual variability observed in the
activity of these two enzymes may be one of the factors
that explains the high variability in the percentages of
flavonoid absorption that have been published. So,
some individuals could have better absorption than
others, possibly because of particular polymorphisms
(Manach et al., 2005).
The free aglycons resulting from the deglycosilation
process, together with the rest of the aglycons, are
extensively metabolised during their movement across
the epithelial cells of the small intestine, resulting in
the appearance of glucuronide conjugates (mainly),
sulphate conjugates, sulphoglucuronide conjugates and
methylated conjugates of corresponding aglycons
(Shimoi et al., 1998; Baba et al., 2000b, 2001b; Murota
et al., 2000; Day et al., 2001; Graefe et al., 2001; Wittig
et al., 2001). These metabolites could be responsible, at
least in part, for the effects observed on the central
nervous system after cocoa and chocolate consump-
tion. Specifically, this has been seen in laboratory
animals where epicatechin metabolites (glucuronide
and 3-O-methylglucuronide) can cross the blood-brain
barrier and act at a cerebral level (Abd El Mohsen et
al., 2002).
As for hepatic metabolites in humans, it has been
pointed out that quercetin in vitro is methylated at a
hepatic level and is quickly eliminated, mainly due to
oxidative degradation, leading to the formation of car-
boxylic acids like protocatechuic acid. These metabolic
changes could also occur in vivo, which would con-
tribute to the multiple biological actions reported for
quercetin (Boulton et al., 1999). Furthermore, the
existence of interactions in the hepatic metabolism is
possible as quercetin is a potent inhibitor of hepatic
phenolsulphotransferase (De Santi et al., 2002).
Data on oral absorption percentages of these com-
pounds are very limited. According to Donovan et al.
(2001) approximately 35% of catechin is absorbed in
rats. Hollman and Katan (1999) state that 24% of
quercetin and 52% of its glycosides can be absorbed in
humans. In a more recent study carried out by Walle et
al. (2001), healthy volunteers were given radio-labelled
quercetin both orally and intravenously and the per-
centages of oral absorption were high, ranging from
36.4 to 53.0%. Added to this great interindividual vari-
ability, diet constituents that are present containing
polyphenols and their interaction in the food matrix
can also have a relevant effect on their oral bioavail-
ability. Milk is usually consumed with cocoa and by-
products. The studies made on the effect of milk on
polyphenolic compounds show contradictory effects.
The results obtained in previous studies suggest that
phenol bioavailability is not affected by the matrix.
Hollman et al. (2001) proved that adding milk to tea
does not affect the absorption of flavonols in humans.
On the contrary, in a recent study published by Serafini
et al. (2003) point out that milk, either added to choco-
late during its manufacture or consumed with it, dimin-
ishes the efficiency of chocolate polyphenols
absorption. Perhaps in this case, the differences found
in both studies might be due to the different amounts
of milk added to these foods, smaller in the case of tea
(15mL of milk in 135ml of tea, whilst 200mL of milk
with 100g of chocolate).
Consumption of foods rich in carbohydrates com-
bined with chocolate could increase the absorption of
these compounds, whilst foods rich in lipids and pro-
teins or treatment with famotidine, an antagonist of
histamine H
2
receptors with antiacid effect, seem not to
have a significant effect on that absorption (Schramm
et al., 2003). Tuck et al. (2001) also observed that, in
the case of olive oil polyphenols, the lipophilic or
hydrophilic character of the matrix remarkably con-
dition their absorption, being much higher in the lipidic
moiety. The addition of oil in the diet or the presence
Health Effects of Cocoa Flavonoids 163
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
of an emulsifier like lecithin also notably increased the
absorption of quercetin and their glycosides when
onions have been consumed (Azuma et al., 2003).
The flavonoids that are not absorbed through the
gut barrier (mainly long chain procyanidins) or that are
excreted in the bile, reach the colon where they can
interact with the existing microbiota. Gut microflora
catalyses the breaking of these compounds into more
simple molecules, producing phenolic acids of low mol-
ecular weight, which are more bioavailable, and might
well be absorbed through the colon (Scalbert and
Williamson, 2000) and could be excreted in urine.
Thus, it has been observed that consumption of pro-
cyanidin-rich chocolate increases the urinary excretion
of m-hydroxyphenylpropionic acid, ferulic acid, 3,4-
dihydroxyphenylacetic acid, m-hydroxyphenylacetic
acid, vanillic acid and m-hydroxybenzoic acid (Ríos et
al., 2003).
This metabolisation had already been detected in
vitro (Déprez et al., 2000). In respect of the organisms
responsible for this conversion, Schneider et al. (1999)
identified 2 types of bacteria in human faeces capable
of metabolising these compounds, Enterococcus cas-
seliflavus that would only react with the sugar molecule
and Eubacterium ramulus which is capable of degrad-
ing the aromatic ring, thus transforming flavonoids into
lower molecular weight phenols, like hydroxypheny-
lacetic acids (Aura et al., 2002). Later, it was observed
that the human faecal bacteria Clostridium orbi-
scindens is capable of degrading quercetin, apigenin
and luteolin into more simple compounds (Schoefer et
al., 2003). This implies that monomeric flavonoids that
have not been absorbed in the small intestine and
reach the colon intact could also suffer this metabolisa-
tion. All these phenolic acids of low molecular weight,
with antioxidant activity, could also have biological
activity and have a local effect in the colon or be
absorbed, thus contributing to the biological effects
observed after the consumption of cocoa and its
derivatives.
EFFECTS OF FLAVONOIDS
CONSUMPTION
Antioxidant Activity
A main key in the prevention of cardiovascular,
tumoural and degenerative diseases is maintaining the
balance between prooxidant and antioxidant sub-
stances in the organism. Oxidative stress is involved in
mutagenesis, carcinogenesis, lipid peroxidation, alter-
ations of the correct membrane function and oxidation
and fragmentation of proteins. As an example, the
intake of flavonoid rich-foods could be useful in pro-
tecting the skin from solar radiation damage or from
the damage of the erythrocyte membranes caused by
tobacco (Erden-Inal and Kahraman, 2000; Begum and
Terao, 2002; Basu-Modak et al., 2003; Jeon et al.,
2003). The high antioxidant activity of cocoa flavonoids
has been shown both in vitro and in vivo.
In Vitro
Extracts of different cocoa by-products have shown
a powerful antioxidant activity proportional to their
flavonoid content (Azizah et al., 1999; Lee et al., 2003).
This effect is shown in different experimental systems
based on linoleic acid, erythrocytes or hepatic micro-
somes (Sanbongi et al., 1998; Zhu et al., 2002).
The antioxidant effect has also been proved with
isolated cocoa flavonoids, including the main com-
pounds; catechin, epicatechin, procyanidins (Adamson
et al., 1999; Zhu et al. 2002) and its metabolites
(Spencer et al., 2001). One of the main metabolites of
epicatechin, the 3-O-methylepicatechin, has shown
protective effects against cellular death caused by
oxidative stress (Spencer et al., 2001) and both epicate-
chin and this metabolite have demonstrated their
ability to protect against cellular damage caused by
UVA-radiation exposure in a culture of human fibrob-
lasts (Basu-Modak et al., 2003).
Quercetin scavenges reactive oxygen species (Saija
et al., 1995; Miller, 1996), inhibits xanthine oxidase
(Chang et al., 1993) and inhibits lipid peroxidation in
vitro (Chen et al., 1990). Quercetin, by itself or acting
with ascorbic acid, reduces the incidence of oxidative
injury in skin neurovasculare structures, it inhibits
neural damage caused by a decrease of glutation
(Skaper et al., 1997) and protects DNA from oxidative
damage caused by hydrogen peroxide (Duthie and
Dobson, 1999). On the other hand, hyperoxide can act
as an antiradical, neutralising the hydroxyl radical
(Chen et al., 2002b). This effect has been seen in a
preparation of human erythrocytes treated with an
aqueous extract of tobacco tar. Quercetin and, to a
lesser degree, its metabolite 3-O--D-glucuronide
inhibited loss of deformability and lipid peroxidation
of the erythrocyte membrane (Begum and Terao,
2002).
In Vivo
The consumption of cocoa powder or chocolate with
a high procyanidin and epicatechin content enhances
the antioxidant capacity of plasma and decreases the
content of lipid oxidation products in human plasma
(Wang et al., 2000) and in rats (Baba et al., 2000a). The
isolated compounds have also shown their antioxidant
properties in vivo. Cocoa flavanols and procyanidin
oligomers increased the antioxidant capacity of blood
plasma and the resistance of erythrocyte haemolysis
induced by oxidant agents in rats (Zhu et al., 2002).
Catechin has also proved to be an active element in the
164 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
protection of epidermic cells against damage caused by
UVB-radiation, when added to the diet of mice (Jeon
et al., 2003). Quercetin also reveals antioxidant effects
when added to the diet of rats, increasing the antioxi-
dant capacity of plasma (Morand et al., 1998), dimin-
ishing or preventing oxidative stress caused by
exposure to UV rays (Erden-Inal and Kahraman,
2000) and preventing the hepatotoxicity and nefrotoxi-
city caused by oxidative damage (Peres et al., 2000;
Satyanarayana et al., 2001).
Cardiovascular Protective Activity
Epidemiological Evidence
In 1993, Hertog et al. established an inverse relation
between flavonoid consumption and mortality due to
coronary disease after a study of 805 men aged
between 65 and 84 over 5 years. Other trials carried
out afterwards confirmed this relation. In 1996, Keli et
al. made a prospective cohort study of 552 men aged
between 50 and 69, who were observed for 15 years.
During this period, they detected an inverse relation
between the consumption of flavonoids in their diet
and the incidence of strokes. Arai et al. (2000)
observed that the consumption of flavonoids in Japan-
ese women was inversely related to low levels of plas-
matic cholesterol (total cholesterol as well as LDL
cholesterol), which could contribute to the low inci-
dence of cardiovascular disease in comparison with
women from other countries. Arts et al. published two
studies in 2001 in which the protective cardiovascular
activity of catechin and epicatechin was stated. In the
first, data was extracted from the Iowa Women’s Study
(a prospective study carried out between 1986 and
1998 that included more than 34,000 women aged
between 55 and 69), a decrease in the risk of death due
to cardiovascular disease was found, related to catechin
and epicatechin consumption (Arts et al., 2001a). In
the second one, the association between the intake of
catechin and the incidence of ischemic heart disease
and strokes was evaluated, taking into account mortal-
ity due to these causes. For that purpose, the data from
the Zutphen Elderly Study were used, a prospective
cohort study where 806 men aged between 65 and 84
were included and an inverse relation between cate-
chin consumption and mortality due to ischemic heart
disease, but not strokes, was found (Arts et al., 2001b).
In 2002, Geleijnse et al. used data from the Rotterdam
Study to prove the association between the consump-
tion of flavonoids and the incidence of myocardial
infarction in the Dutch population. In this study, 4,807
subjects aged 55 or older were monitored for 5 years.
Flavonoid consumption was then associated with a
reduction of risk of fatal myocardial infarction. A later
study carried out in Finland with 10,054 subjects who
were observed for a longer period of time (28 years)
confirmed this inverse association between flavonoid
intake and mortality due to ischemic heart disease, the
incidence of cerebrovascular disease or the incidence
of certain types of cancer (Knekt et al., 2002).
The cardiovascular protection shown by flavonoids
in these epidemiological studies would be stimulated
by the multiple action mechanisms of flavonoids also
present in cocoa, as they produce a modification of risk
factors for these pathologies, e.g. reduction of LDL
oxidative susceptibility, inhibition of platelet aggrega-
tion or induction of vasodilation. In other studies
where these substances were isolated, other types of
mechanisms were also observed, such as the inhibition
of the adhesion of monocytes to endothelial cells or the
promotion of fybrinolysis.
LDL Oxidation Inhibition
LDLs are the main carriers of cholesterol in circula-
tion and play key roles in cholesterol transfer and
metabolism. LDLs are susceptible to oxidation and
oxidative modification of LDL has been related to the
pathogenesis of atherosclerosis.
In Vitro Activity
The first effect observed in vitro when investigating
the activity of cocoa extract was the inhibition of
human LDL oxidation (Kondo et al., 1996; Water-
house et al., 1996). Later on, Hirano et al. (2000)
showed that cocoa liquor polyphenols inhibit LDL oxi-
dation in a dose-dependent manner, this effect is
evident in the tested range 0.1mg/dL–0.5mg/dL. Like-
wise, isolated flavonoids have shown a great inhibition
capacity. Catechin inhibits LDL oxidation up to 87%
(Waterhouse et al., 1996). This activity has also been
shown by cocoa epicatechin and procyanidins after
LDL treatment with different oxidant agents (Lotito et
al., 2000; Osakabe et al., 2002; Steinberg et al., 2002,
2003). LDL protection in vitro tends to increase with
procyanidin chain length. However when the inhibition
is based on equivalent amounts of monomeric units is
similar (Steinberg et al., 2002). Da Silva et al., (2000)
could even observe a greater activity of epicatechin in
the inhibition of LDL oxidation catalysed by 15-lipoox-
igenase than the one shown by ascorbic acid or -toco-
pherol in similar concentrations. Quercetin also
inhibits LDL cholesterol oxidation, either by directly
inhibiting such oxidation or protecting vitamin E in
LDL from oxidation or by regenerating oxidised
vitamin E (De Whalley et al., 1990). Conjugated
metabolites of quercetin inhibit LDL and VLDL oxi-
dation catalysed by the copper cation, and are four
times more powerful than trolox at inhibiting the oxi-
dation of LDL (Morand et al., 1998; Da Silva et al.,
1998). Likewise, metabolite 3-O-methylated (isorham-
netin) is a powerful inhibitor of lipidic peroxidation in
Health Effects of Cocoa Flavonoids 165
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
the mythocondrial membrane of rats and on LDL oxi-
dation (Manach et al., 1998; Santos et al., 1998). This
antioxidant effect shown by quercetin metabolites is
lower than that shown by the aglycon, reaching in
some instances 50% (Manach et al., 1998).
In Vivo Activity
Osakabe et al. (2000) studied the effects of cocoa
liquor polyphenols on the susceptibility of LDL oxida-
tion in hypercholesterolemic rabbits. They observed
that by supplementing the diet of these animals with
1% cocoa liquor polyphenols, an increase in the resis-
tance to LDL oxidation induced by 2–2-azobis(4-
methoxy-2,4-dimethylvaleronitrile was produced. This
increase was evaluated by measuring the production of
conjugated dienes and thiobarbituric acid reactive sub-
stances (TBARS). When applied to humans, the
results are very similar. Rein et al. (2000a) observed
that after a procyanidins-rich chocolate intake, the
plasma antioxidant capacity measured using the total
antioxidant potential (TRAP) assay increased by 31%,
at the same time there was a significant decrease in the
concentration of plasma oxidation products (TBARS).
Osakabe et al. (2001) observed an increase in the resis-
tance to oxidation of LDL induced by 2–2-azobis(4-
methoxy-2,4-dimethylvaleronitrile or by copper ion.
After consuming 36g of cocoa powder a day for 2
weeks, the lag time to conjugated diene production was
significantly prolonged. Wan et al. (2001) assessed the
effect of a diet enriched with 22g of cocoa powder and
16g of dark chocolate for 4 weeks, revealing an
increase in the LDL oxidation lag time and a slight
increase in the antioxidant capacity of plasma meas-
ured by oxygen radical absorbance capacity.
Inhibition of Platelet Aggregation
The increase in platelet activation and function is
related to the development and progress of atheromas,
and therefore, the thrombotic processes. Cocoa
flavonoids have been shown to be potent inhibitors of
platelet aggregation both in vitro and in vivo.
In Vitro Activity
Catechin inhibits platelet aggregation induced by
collagen and the platelet adhesion to collagen (Pig-
natelli et al., 2000). Furthermore, cocoa procyanidins,
added to human blood samples, inhibit the platelet
activation in response to epinephrine (Rein et al.,
2000c). This effect could be caused by the modulation
in the eicosanoid synthesis produced by cocoa pro-
cyanidins (Chang and Hsu, 1989; Schramm et al.,
2001), this effect is also shown by epicatechin (Schewe
et al., 2001). The cardiovascular effects of quercetin are
mainly due to their antioxidant and anti-inflammatory
activity and to their capacity to inhibit platelet aggrega-
tion (Pace-Asciak et al., 1995). Xie et al. (1996)
observed this inhibitor effect of quercetin in platelet
aggregation induced by ADP as well as by reactive
oxygen species. This effect on platelet aggregation can
be stimulated by the concomitant presence of several
types of flavonoids. Pignatelli et al. (2000) showed that
quercetin produces a synergic effect with catechin in
the inhibition of platelet aggregation induced by colla-
gen and in the platelet adhesion to collagen since a
combination of both, in doses that proved to be inact-
ive separately, produced a significant inhibitory effect.
In Vivo Activity
After the consumption of a cocoa drink by healthy
volunteers, Rein et al. (2000b) observed inhibition on
platelet activity and reactivity stimulated by ADP or
epinephrine, resulting in an aspirin-like effect on
primary haemostasis. Schramm et al. (2001) postulated
that these inhibitory effects on platelet activation may
be caused by changes in the plasma leukotriene/prosta-
cyclin ratio. These authors carried out a short-term
clinical trial with a randomised, blinded and crossover
design, in order to determine and compare the ability
of cocoa procyanidins to modify the synthesis of
eicosanoids in vivo in humans and in cultured human
aortic endothelial cells. Ten volunteers were included
in the study. They consumed two types of chocolate,
one rich in procyanidins and another with a low
content. There was a 1-week wash-out period between
both consumptions. After treatment, a decrease of
52% in the plasma leukotriene/prostacyclin ratio in
vivo and 58% in the test in vitro were observed.
Bearing in mind that prostacyclins of vascular endothe-
lium act promoting vasodilation and inhibiting platelet
aggregation, conversely, leukotrienes are vasoconstric-
tors and can increase platelet aggregation, a regular
intake of the active components of cocoa could help to
diminish the risk of thrombosis. Innes et al. (2003)
reached the same conclusion in a recent study carried
out on 30 volunteers, randomised in three groups to
take 100g of dark chocolate, white chocolate or milk
chocolate. It was pointed out that dark chocolate sig-
nificantly inhibited platelet aggregation induced by col-
lagen whereas white chocolate, where flavonoid
content is minimal, had no significant effect.
Vasodilation Activity
Flavonoids may cause dilation of the blood vessels
by relaxing their muscular walls. This provokes an
increase in blood flow, but a decrease in systemic vas-
cular resistance.
166 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
In Vitro Activity
Epicatechin induces vascular relaxation acting on
the endothelium and increasing the nitric oxide level
(Chen et al., 2002a). Quercetin and its metabolites
isorhamnetin and tamarixetin produce vasodilation by
means of endothelium dependent and non-dependent
mechanisms (Kobuchi et al., 1999; Zhao et al., 1999;
Pérez-Vizcaíno et al., 2002)
In Vivo Activity
In patients with some cardiovascular risk factor,
Heiss et al. (2003) observed that the consumption of
100mL of a cocoa drink with a high content in cate-
chins (70mg of catechin plus epicatechin and 106mg of
procyanidins) increased the plasmatic levels of nitric
oxide producing a positive effect on vascular endothe-
lium-dependent dilation (arterial vasorelaxing effect).
This effect was not observed with the consumption of
100mL of another cocoa drink with a catechin content
less than 10mg. Isolated compounds have also shown
this effect (Duarte et al., 2001).
Inhibition of Monocyte Adhesion to Endothelial Cells
The adherence of monocytes to the vascular
endothelium is an important early event in atherogene-
sis. Flavonoids have shown an inhibitory activity in
vitro. Koga and Meydani (2001) studied the effect of
catechin and its metabolites (glucuronide and sulphate
conjugates and methylated forms) on the monocyte
U937 adhesion to human aortic endothelial cells and on
the production of reactive oxygen species in these cells
after stimulation with interleukin-1 or hydrogen per-
oxide. Pre-treatment of human aortic endothelial cells
with catechin metabolites inhibited monocyte adhesion,
whilst catechin did not show this effect. The production
of reactive oxygen species induced by hydrogen perox-
ide was inhibited by catechin and its metabolites, whilst
the one induced by interleukin-1 was only inhibited by
catechin metabolites. According to these results,
flavonoid metabolites, rather than their intact forms,
could contribute to the effects observed in cardiovascu-
lar protection by means of this mechanism.
In Vivo Activity
Data from humans who consumed cocoa, showed an
increase in NO activity (Sies et al., 2005) causing vascu-
lar endothelium relaxation and decrease in blood pres-
sure (Taubert et al., 2003).
Promotion of Fibrinolysis
Fibrin forms the essential portion of the blood clot
and of the thrombus. Fibrinolytic compounds are
useful in the prevention and treatment of thrombotic
disease.
According to Abou-Agag et al. (2001), cardiopro-
tective effects of flavonoids may also be attributed, at
least in part, to the capacity shown by catechin and epi-
catechin to increase fibrinolytic activity, which they
produce by means of stimulation of the genetic tran-
scription of fibrinolytic proteins t-PA (tissue-type plas-
minogen activator) and u-PA (urokinase-type PA).
Protective Activity against Cancer
The mechanisms involved in this protective effect
appear at different stages of the carcinogenesis process,
both in phases of tumour initiation and promotion and
in tumour progress and metastasis.
Epidemiological Evidence
In Spain, García-Closas et al. (1999) carried out a
case-control study to see the possible relation between
the incidence of gastric cancer and flavonoid consump-
tion. A total of 354 cases and 354 controls were
included, and a dietary history questionnaire record
was kept. Once the statistical analysis of the data was
made, an inverse relation between flavonoid consump-
tion and gastric cancer could be determined. Arts et al.
(2002), taking data from the Iowa Women’s Study,
established an inverse epidemiological relation
between catechin consumption and the incidence of
rectal cancer in post-menopausal women. In a recent
clinical trial carried out in Greece with cases (820) and
controls (1,548) a significant inverse association was
found between flavone consumption and the incidence
of breast cancer (Peterson et al., 2003).
Activity in Phases of Tumour Initiation and Promotion
Cocoa liquor polyphenols have shown antimutagenic
properties in the tests normally carried out in the initial
phases to find out the mutagenic behaviour of a sub-
stance, such as the Ames test and the micronucleus test
in mice. In the former, an inhibition of the mutagenic
effect of the heterocyclic amines in the bacterial culture
(Yamagishi et al., 2000) was observed, whilst in the
latter, the cocoa liquor polyphenols administered orally
reduced the occurrence of micronucleated cells after
the injection of mitomycin C (Yamagishi et al., 2001).
Weyant et al. (2001) observed that catechin, added
to a diet in concentrations of 0.1 and 1%, diminishes
the formation of intestine tumours in a mouse model
genetically modified to express a similar defect to the
one primarily expressed in the majority of human col-
orectal cancers. This chemopreventive effect has also
been observed in cocoa liquor procyanidins (Yamag-
ishi et al., 2003).
Health Effects of Cocoa Flavonoids 167
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
Activity in Phases of Tumour Progression and
Metastasis
Carnesecchi et al. (2002) recently studied the effect
of cocoa powder extract on tumour progression. In the
mentioned study, the treatment of human colonic
cancer cells with procyanidin-enriched cocoa powder
extracts reduced 70% of cellular growth, an effect that
may have been caused by the inhibition of polyamine
biosynthesis by cocoa polyphenols.
In opposition to this, the early studies carried out
with quercetin showed a mutagenic activity, which
made researchers hesitant about the safety of this com-
pound. Nevertheless, we must point out that when a
mutagenic activity appears in early studies in vitro, this
does not necessarily involve carcinogenic activity. In
this case, anticarcinogenic activity was later seen in
studies carried out on rats, mice and hamsters (Stavric
1994; Hertog and Hollman, 1996; De et al., 2000; Soleas
et al., 2002) and in cultures of human cancer cell lines
from different locations, including different types of
breast cancer cells (Scambia et al., 1993; Singhal et al.,
1995; Rodgers and Grant, 1998; Choi et al., 2001; Han
et al., 2001), leukaemic cells (Larocca et al., 1991, 1995;
Kang and Liang, 1997; Xiao et al., 1997; Yamashita and
Kawanishi, 2000), colonic cancer cells (Pereira et al.,
1996; Richter et al., 1999), ovarian cancer cells
(Scambia et al., 1990, 1992), squamous cancer cells
(Castillo et al., 1989), endometrial cancer cells (Scambia
et al., 1992), gastric cancer cells (Yoshida et al., 1990),
lung cancer cells (Caltagirone et al., 1997), hepatic
cancer cells (Kang et al., 1999) and prostatic cancer
cells (Asea et al., 2001; Romero et al., 2002). Further-
more, quercetin has proved to be very active, increasing
the efficacy of different chemotherapeutic agents
(Scambia et al., 1990, 1992). The anticarcinogenic activ-
ity of quercetin seems to be due to its action in different
stages of tumour development, either reducing cellular
proliferation by inhibiting different protein-kinases
(Kang and Liang, 1997), inhibiting the enzymatic acitiv-
ity of tumour cells (Rodgers and Grant, 1998), inhibit-
ing angiogenesis (Igura et al., 2001), inducing apoptosis
(Xiao et al., 1997; Richter et al., 1999), or inhibiting the
expression of oncogenes, as seen with rats oncogene in
colon cancer cell lines and in primary colorectal
tumours (Ranelletti et al., 2000). It even seems to have
an antimetastatic activity, as observed in vitro when
inhibiting the invasion and mobility of murine
melanoma cells (Zhang et al., 2000b).
Other Activities Observed
Immunomodulatory Activity
Cocoa, as a potential modulator of immune
response, may be beneficial in alterations involving an
anomalous activation of the immune system. The phe-
nolic extract of cocoa liquor modulates the cellular
immune response in vitro, thus inhibiting the produc-
tion of oxygen reactive species (hydrogen peroxide and
superoxide anion) by lymphocytes and activated gran-
ulocytes. Furthermore, cocoa liquor phenols inhibit the
proliferation of lymphocytes and the production of
immunoglobins, an effect partly caused by modulation
in the cytokine transcription and secretion by periph-
eral blood mononuclear cells (Sanbongi et al., 1997;
Mao et al. 2000a).
Epicatechin has shown a modulator effect on the
immune function in vitro, by inhibiting in a dose-depen-
dent manner the superoxide anion production generated
by granulocytes and the proliferation of T cells (San-
bongi et al., 1997). On the other hand, Mao et al. (2000b)
observed a modulation of genetic transcription and
secretion of interleukin-1 beta (IL-1 beta), IL-2 and IL-3
pro-inflammatory cytokines by peripheral blood
mononuclear cells when treated with cocoa procyanidins.
Anti-inflammatory Activity
Peroxynitrite is a high pro-inflammatory mediator.
Epicatechin and cocoa procyanidins have proved to be
efficacious in vitro in cellular protection against perox-
ynitrite, inhibiting oxidation and nitration reactions
mediated by this molecule (Arteel and Sies, 1999;
Schroeter et al., 2001) and inhibiting the activity of
human 5-lipooxygenase against araquidonic acid
(Schewe et al., 2002). The anti-inflammatory activity of
quercetin seems to be due to its antioxidant properties
and to its inhibitory effect on the enzymes involved in
the inflammatory process (cyclooxygenase and lipooxy-
genase) with the subsequent inhibition of inflammation
mediators, including leukotrienes and prostaglandins
(Della Logia et al., 1988; Kim et al., 1998). The
inhibitory effect on the release of histamine by masto-
cytes and basophils also contributes to this anti-inflam-
matory activity (Bronner and Landry, 1985; Fox et al.,
1988). Conversely, high amounts of nitric oxide and
tumour necrosis factor-alpha by activated macrophages
lead to pathophysiological conditions during acute and
chronic inflammation processes. These effects can be
minimised by quercetin, inhibiting the overproduction
of these substances by macrophages (Manjeet and
Ghosh, 1999).
Neuronal Protective Activity
Commenges et al. (2000) investigated if flavonoid
intake could be associated with a minor incidence of
dementia. For that purpose, they used a group of 1,367
individuals aged over 65, between 1991 and 1996. After
statistical analysis of the data, it was observed that the
relative risk of suffering dementia was significantly
lower in those individuals having consumed the larger
amount of flavonoids.
168 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
Oxidative stress has been associated with neuronal
loss in neurodegenerative diseases and during age-
related cognitive decline. Epicatechin and its 3-O-
methylepicatechin metabolite inhibit neuronal toxicity
in vitro induced by oxidised low density lipoproteins,
thus inhibiting the activation of protein-kinases and the
caspase-3-like protease activity in neurons. Therefore,
according to Schroeter et al. (2001), they can be used
as protective agents against neuronal apoptosis caused
by oxidative stress.
At the same time, Shimada et al. (2001) observed
that both catechin and epicatechin and B1 and B2 pro-
cyanidins protect from neuronal death induced by glu-
tamate in cultured rat cerebellar granule cells, by
means of the inhibition of the calcium ion influx.
Potential Toxicity
It is necessary to have more information to evaluate
the adverse effects of these compounds. Procyanidins
have been considered antinutritional compounds
because they can interact with proteins and inhibit
certain enzymes (Mennen et al., 2005). This binding
depends on the degree of polymerization, the larger
molecules tend to bind more efficiently (Santos-Buelga
and Scalbert, 2000). However at the dose present in
cocoa no adverse effect has been observed.
On the other hand, it has been observed that popu-
lations at lower risk of heart disease and certain types
of cancers are Asians and vegetarians. Based on the
average daily intake of flavonols (68mg) and
isoflavones (20–240mg) in Asian populations, dietary
exposures at these doses are not likely to cause adverse
health effects. In addition, the level of flavonoids
required to induce mutations and cytotoxicity may not
be physiologically achievable through dietary sources;
however the use of flavonoid supplements could result
in exposure to potential toxic levels (Skibola and
Smith, 2000).
REFERENCES
Abd El Mohsen M.M., Kuhnle G., Rechner A.R.,
Schroeter H., Rose S.M., Jenner P. and Rice-Evans
C.A. (2002). Uptake and metabolism of epicatechin
and its access to the brain after oral ingestion. Free
Radical Biology and Medicine 33: 1693–1702.
Abou-Agag L.H., Aikens M.L., Tabengwa E.M., Benza
R.L., Shows S.R., Grenett H.E. and Booyse F.M.
(2001). Polyphenolics increase t-PA and u-PA gene
transcription in cultured human endothelial cells. Alco-
holism: Clinical and Experimental Research 25:
155–162.
Adamson G.E., Lazarus S.A., Mitchell A.E., Prior R.L.,
Cao G., Jacobs P.H., Kremers B.G., Hammerstone J.F.,
Rucker R.B., Ritter K.S. and Schmitz H.H. (1999).
HPLC method for the quantification of procyanidins in
cocoa and chocolate samples and correlation to total
antioxidant capacity. Journal of Agricultural and Food
Chemistry 47: 4184–4188.
Ader P., Block M., Pietzsch S. and Wolffram S. (2001).
Interaction of quercetin glucosides with the intestinal
sodium/glucose co-transporter (SGLT-1). Cancer
Letters 162: 175–180.
Arai Y., Watanabe S., Kimira M., Shimoi K., Mochizuki
R. and Kinae N. (2000). Dietary intakes of flavonols,
flavones and isoflavones by Japanese women and the
inverse correlation between quercetin intake and
plasma LDL cholesterol concentration. The Journal of
Nutrition 130: 2243–2250.
Arteel G.E. and Sies H. (1999). Protection against perox-
ynitrite by cocoa polyphenol oligomers. FEBS Letters
462: 167–170.
Arts I.C.W., Hollman P.C.H. and Kromhout D. (1999).
Chocolate as a source of tea flavonoids. The Lancet
354: 488.
Arts I.C.W., Van de Putte B. and Hollman P.C.H. (2000).
Catechin contents of foods commonly consumed in The
Netherlands. 2. Tea, wine, fruit juices, and chocolate
milk. Journal of Agricultural and Food Chemistry 48:
1752–1757.
Arts I.C., Jacobs Jr D.R., Harnack L.J., Gross M. and
Folsom A.R. (2001a). Dietary catechins in relation to
coronary heart disease death among postmenopausal
women. Epidemiology 12: 668–675.
Arts I.C., Hollman P.C., Feskens E.J., Bueno de
Mesquita, H.B. and Kromhout D. (2001b). Catechin
intake might explain the inverse relation between tea
consumption and ischemic heart disease: the Zutphen
Elderly Study. The American Journal of Clinical Nutri-
tion 74: 227–232.
Arts I.C., Jacobs Jr D.R., Gross M., Harnack L.J. and
Folsom A.R. (2002). Dietary catechins and cancer inci-
dence among postmenopausal women: the Iowa
Women’s Health Study. Cancer Causes and Control 13:
373–382.
Asea A., Ara G., Teicher B.A., Stevenson M.A. and
Calderwood S.K. (2001). Effects of the flavonoid drug
quercetin on the response of human prostate tumours
to hyperthermia in vitro and in vivo. International
Journal of Hyperthermia 17: 347–356.
Aura A.M., O’Leary K.A., Williamson G., Ojala M.,
Bailey M., Puupponen-Pimia R., Nuutila A.M.,
Oksman-Caldentey K.M. and Poutanen K. (2002).
Quercetin derivatives are deconjugated and converted
to hydroxyphenylacetic acids but not methylated by
human fecal flora in vitro. Journal of Agricultural and
Food Chemistry 50: 1725–1730.
Azizah A.H., Ruslawati N.M.N. and Tee T.S. (1999).
Extraction and characterization of antioxidant from
cocoa by-products. Food Chemistry 64: 199–202.
Azuma K., Ippoushi K., Ito H., Horie H. and Terao J.
(2003). Enhancing effect of lipids and emulsifiers on
the accumulation of quercetin metabolites in blood
plasma after the short-term ingestion of onions by rats.
Health Effects of Cocoa Flavonoids 169
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
Bioscience, Biotechnology, and Biochemistry 67:
2548–2555.
Baba S., Osakabe N., Natsume M., Yasuda A., Takizawa
T., Nakamura T. and Terao J. (2000a). Cocoa powder
enhances the level of antioxidative activity in rat
plasma. British Journal of Nutrition 84: 673–680.
Baba S., Osakabe N., Yasuda A., Natsume M., Takizawa
T., Nakamura T. and Terao J. (2000b). Bioavailability
of ()-epicatechin upon intake of chocolate and cocoa
in human volunteers. Free Radical Research 33:
635–641.
Baba S., Osakabe N., Natsume M., Muto Y., Takizawa T.
and Terao J. (2001a). Absorption and urinary excretion
of ()-epicatechin after administration of different
levels of cocoa powder or ()-epicatechin in rats.
Journal of Agricultural and Food Chemistry 49:
6050–6056.
Baba S., Osakabe N., Natsume M., Muto Y., Takizawa T.
and Terao J. (2001b). In vivo comparison of the
bioavailability of ()-catechin, ()-epicatechin, and
their mixture in orally administered rats. The Journal
of Nutrition 131: 2885–2891.
Basu-Modak S., Gordon M.J., Dobson L.H., Spencer J.P.,
Rice-Evans C. and Tyrrell R.M. (2003). Epicatechin
and its methylated metabolite attenuate UVA-induced
oxidative damage to human skin fibroblasts. Free
Radical Biology and Medicine 35: 910–921.
Begum A.N. and Terao J. (2002). Protective effect of
quercetin against cigarette tar extract-induced impair-
ment of erythrocyte deformability. The Journal of
Nutritional Biochemistry 13: 265–272.
Bonvehi J.S. and Coll F.V. (1997). Evaluation of the bit-
terness and astringency of polyphenolic compounds in
cocoa powder. Food Chemistry 60: 365–370.
Boulton D.W., Walle U.K. and Walle T. (1999). Fate of
the flavonoid quercetin in human cell lines: chemical
instability and metabolism. Journal of Pharmacy and
Pharmacology 51: 353–359.
Bronner C. and Landry Y. (1985). Kinetics of the
inhibitory effect of flavonoids on histamine secretion
from mast cells. Agents Actions 16: 147–151.
Caltagirone S., Ranelletti F.O., Rinelli A., Maggiano N.,
Colasante A., Musiani P., Aiello F.B. and Piantelli M.
(1997). Interaction with type II estrogen binding sites
and antiproliferative activity of tamoxifen and quercetin
in human non-small-cell lung cancer. American Journal
of Respiratory Cell and Molecular Biology 17: 51–59.
Carnesecchi S., Schneider Y., Lazarus S.A., Coehlo D.,
Gosse F. and Raul F. (2002). Flavanols and procyani-
dins of cocoa and chocolate inhibit growth and
polyamine biosynthesis of human colonic cancer cells.
Cancer Letters 175: 147–155.
Castillo M.H., Perkins E. and Campbell J.H. (1989). The
effects of the bioflavonoid quercetin on squamous cell
carcinoma of head and neck origin. The American
Journal of Surgery 158: 351–355.
Commenges D., Scotet V., Renaud S., Jacqmin-Gadda H.,
Barberger-Gateau P. and Dartigues J.F. (2000). Intake
of flavonoids and risk of dementia. European Journal
of Epidemiology 16: 357–363.
Chang W.C. and Hsu F.L. (1989). Inhibition of platelet
aggregation and arachidonate metabolism in platelets
by procyanidins. Prostaglandins, Leukotrienes and
Essential Fatty Acids 38: 181–188.
Chang W.S., Lee Y.J., Lu F.J. and Chiang H.C. (1993).
Inhibitory effects of flavonoids on xanthine oxidase.
Anticancer Research 13: 2165–2170.
Chen Y.T., Zheng R.L., Jia Z.J. and Ju Y. (1990).
Flavonoids as superoxide scavengers and antioxidants.
Free Radical Biology and Medicine 9: 19–21.
Chen Z.W. and Ma C.G. (1999). Effects of hyperin on
free intracellular calcium in dissociated neonatal rat
brain cells. Zhongguo Yao Li Xue Bao 20: 27–30.
Chen Z.W., Yao X.Q., Chan F.L., Lau C.W. and Huang
Y. (2002a). ()-Epicatechin induces and modulates
endothelium-dependent relaxation in isolated rat
mesenteric artery rings. Acta Pharmacologica Sinica 23:
1188–1192.
Chen J.W., Zhu Z.Q., Hu T.X. and Zhu D.Y. (2002b).
Structure-activity relationship of natural flavonoids in
hydroxyl radical-scavenging effects. Acta Pharmacolog-
ica Sinica 23: 667–672.
Choi J.A., Kim J.Y., Lee J.Y., Kang C.M., Kwon H.J.,
Yoo Y.D., Kim T.W., Lee Y.S. and Lee S.J. (2001).
Induction of cell cycle arrest and apoptosis in human
breast cancer cells by quercetin. International Journal
of Oncology 19: 837–844.
Crespy V., Morand C., Besson C., Manach C., Demigne
C. and Remesy C. (2002). Quercetin, but not its glyco-
sides, is absorbed from the rat stomach. Journal of
Agricultural and Food Chemistry 50: 618–621.
Da Silva E.L., Piskula M.K., Yamamoto N., Moon J.H.
and Terao J. (1998). Quercetin metabolites inhibit
copper ion-induced lipid peroxidation in rat plasma.
FEBS Letters 430: 405–408.
Da Silva E.L., Abdalla D.S. and Terao J. (2000).
Inhibitory effect of flavonoids on low-density lipopro-
tein peroxidation catalyzed by mammalian 15-lipoxyge-
nase. IUMB Life 49: 289–295.
Day A.J., Bao Y., Morgan M.R. and Williamson G.
(2000). Conjugation position of quercetin glucuronides
and effect on biological activity. Free Radical Biology
and Medicine 29: 1234–1243.
Day A.J., Mellon F., Barron D., Sarrazin G., Morgan
M.R. and Williamson G. (2001). Human metabolism of
dietary flavonoids: identification of plasma metabolites
of quercetin. Free Radical Research 35: 941–952.
Day A.J., Gee J.M., DuPont M.S., Johnson I.T. and
Williamson G. (2003). Absorption of quercetin-3-glu-
coside and quercetin-4-glucoside in the rat small intes-
tine: the role of lactase phlorizin hydrolase and the
sodium-dependent glucose transporter. Biochemical
Pharmacology 65: 1199–1206.
Della Loggia R., Ragazzi E., Tubaro A., Fassina G. and
Vertua R. (1988). Anti-inflammatory activity of ben-
zopyrones that are inhibitors of cyclo- and lipo-oxyge-
170 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
nase. Pharmacological Research Communications 20:
S91–S94.
Déprez S., Brezillon C., Rabot S., Philippe C., Mila I.,
Lapierre C. and Scalbert A. (2000). Polymeric proan-
thocyanidins are catabolized by human colonic
microflora into low-molecular-weight phenolic acids.
The Journal of Nutrition 130: 2733–2738.
De Santi C., Pietrabissa A., Mosca F., Rane A. and Paci-
fici G.M. (2002). Inhibition of phenol sulfotransferase
(SULT1A1) by quercetin in human adult and foetal
livers. Xenobiotica 32: 363–368.
De S., Chakraborty J., Chakraborty R.N. and Das S.
(2000). Chemopreventive activity of quercetin during
carcinogenesis in cervix uteri in mice. Phytotherapy
Research 14: 347–351.
De Whalley C.V., Rankin S.M., Hoult J.R., Jessup W. and
Leake D.S. (1990). Flavonoids inhibit the oxidative
modification of low density lipoproteins. Biochemical
Pharmacology 39: 1743–1749.
Donovan J.L., Crespy V., Manach C., Morand C., Besson
C., Scalbert A. and Remesy C. (2001). Catechin is
metabolized by both the small intestine and liver of
rats. The Journal of Nutrition 131: 1753–1757.
Duarte J., Perez-Palencia R., Vargas F., Ocete M.A.,
Perez-Vizcaino F., Zarzuelo A. and Tamargo J. (2001).
Antihypertensive effects of the flavonoid quercetin in
spontaneously hypertensive rats. British Journal of
Pharmacology 133: 117–124.
Duthie S.J. and Dobson V.L. (1999). Dietary flavonoids
protect human colonocyte DNA from oxidative attack
in vitro. European Journal of Nutrition 38: 28–34.
Erden Inal M. and Kahraman A. (2000). The protective
effect of flavonol quercetin against ultraviolet A
induced oxidative stress in rats. Toxicology 154: 21–29.
Forsyth W.G.C. (1955). Cacao polyphenolic substances. 3.
Separation and estimation on paper chromatograms.
Biochemical Journal 60: 1108.
Forsyth W.G.C. and Quesnel V.C. (1963). The mechanism
of cacao curing. Advances in Enzymology and Related
Subjects of Biochemistry 25: 457–492.
Fox C.C., Wolf E.J., Kagey-Sobotka A. and Lichtenstein
L.M. (1988). Comparison of human lung and intestinal
mast cells. Journal of Allergy and Clinical Immunology
81: 89–94.
García-Closas R., González C.A., Agudo A., Riboli E.
(1999). Intake of specific carotenoids and flavonoids
and the risk of gastric cancer in Spain. Cancer Causes
and Control 10: 71–75.
Gee J.M., DuPont M.S., Rhodes M.J., Johnson I.T.
(1998). Quercetin glucosides interact with the intestinal
glucose transport pathway. Free Radical Biology and
Medicine 25: 19–25.
Gee J.M., DuPont M.S., Day A.J., Plumb G.W.,
Williamson G. and Johnson I.T. (2000). Intestinal
transport of quercetin glycosides in rats involves both
deglycosylation and interaction with the hexose trans-
port pathway. The Journal of Nutrition 130: 2765–2771.
Geleijnse J.M., Launer L.J., Van der Kuip D.A., Hofman
A. and Witteman J.C. (2002). Inverse association of tea
and flavonoid intakes with incident myocardial infarc-
tion: the Rotterdam Study. The American Journal of
Clinical Nutrition 75: 880–886.
Gonthier M.P., Donova J.L., Texier O., Felgines C.,
Remesy C. and Scalbert A. (2003). Metabolism of
dietary procyanidins in rats. Free Radical Biology and
Medicine 35: 837–844.
Graefe E.U., Wittig J., Mueller S., Riethling A.K.,
Uehleke B., Drewelow B., Pforte H., Jacobasch G.,
Derendorf H. and Veit M. (2001). Pharmacokinetics
and bioavailability of quercetin glycosides in humans.
The Journal of Clinical Pharmacology 41: 492–499.
Gupta S., Afaq F. and Mukhtar H. (2001). Selective
growth-inhibitory, cell-cycle deregulatory and apop-
totic response of apigenin in normal versus human
prostate carcinoma cells. Biochemical and Biophysical
Research Communications 287: 914–920.
Han D., Tachibana H. and Yamada K. (2001). Inhibition of
environmental estrogen-induced proliferation of human
breast carcinoma MCF-7 cells by flavonoids. In Vitro Cel-
lular and Developmental Biology - Animal 37: 275–282.
Hammerstone J.F., Lazarus S.A. and Schmitz H. (2000).
Procyanidin content and variation in some commonly
consumed foods. The Journal of Nutrition 130:
2086S–2092S.
Heiss C., Dejam A., Kleinbongard P., Schewe T., Sies H.
and Kelm M. (2003). Vascular effects of cocoa rich in
flavan-3-ols. The Journal of the American Medical
Association 290: 1030–1031.
Hertog M.G., Feskens E.J., Hollman P.C., Katan M.B.
and Kromhout D. (1993). Dietary antioxidant
flavonoids and risk of coronary heart disease: the
Zutphen Elderly Study. The Lancet 342: 1007–1011.
Hertog M.G.L. and Hollman P.C.H. (1996). Potential
health effects of the dietary flavonol quercetin. Euro-
pean Journal of Clinical Nutrition 50: 63–71.
Hirano R., Osakabe N., Iwamoto A., Matsumoto A.,
Natsume M., Takizawa T., Igarashi O., Itakura H. and
Kondo K. (2000). Antioxidant effects of polyphenols in
chocolate on low-density lipoprotein both in vitro and
ex vivo. Journal of Nutritional Science and Vitaminol-
ogy 46: 199–204.
Hollman P.C. and Katan M.B. (1999). Health effects and
bioavailability of dietary flavonols. Free Radical
Research 31: S75–S80.
Hollman P.C., Van Het Hof K.H., Tijburg L.B. and Katan
M.B. (2001). Addition of milk does not affect the
absorption of flavonols from tea in man. Free Radical
Research 34: 297–300.
Holt R.R., Schramm D.D., Keen C.L., Lazarus S.A. and
Schmitz H.H. (2002a). Chocolate consumption and
platelet function. The Journal of the American Medical
Association 287: 2212–2213.
Holt R.R., Lazarus S.A., Sullards M.C., Zhu Q.Y.,
Schramm D.D., Hammerstone J.F., Fraga C.G.,
Schmitz H.H. and Keen C.L. (2002b). Procyanidin
dimer B2 [epicatechin-(4-beta-8)-epicatechin] in
human plasma after the consumption of a flavanol-rich
Health Effects of Cocoa Flavonoids 171
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
cocoa. The American Journal of Clinical Nutrition 76:
798–804.
Huang Y.T., Hwang J.J., Lee P.P., Ke F.C., Huang J.H.,
Huang C.J., Kandaswami C. and Middleton E. (1999).
Effects of luteolin and quercetin, inhibitors of tyrosine
kinase, on cell growth and metastasis-associated prop-
erties in A431 cells overexpressing epidermal growth
factor receptor. British Journal of Pharmacology 128:
999–1010.
Huang Y.T., Kuo M.L., Liu J.Y., Huang S.Y. and Lin J.K.
(1996). Inhibitions of protein kinase C and proto-
oncogene expressions in NIH 3T3 cells by apigenin.
European Journal of Cancer 32: 146–151.
Igura K., Ohta T., Kuroda Y. and Kaji K. (2001). Resvera-
trol and quercetin inhibit angiogenesis in vitro. Cancer
Letters 171: 11–16.
Innes A.J., Kennedy G., McLaren M., Bancroft A.J. and
Belch J.J.F. (2003). Dark chocolate inhibits platelet
aggregation in healthy volunteers. Platelets 14: 325–327.
Jeon S.E., Choi-Kwon S., Park K.A., Lee H.J., Park M.S.,
Lee J.H., Kwon S.B. and Park K.C. (2003). Dietary
supplementation of ()-catechin protects against
UVB-induced skin damage by modulating antioxidant
enzyme activities. Photodermatology, Photoimmunol-
ogy and Photomedicine 19: 235–241.
Johnson M.K. and Loo G. (2000). Effects of epigallocate-
chin gallate and quercetin on oxidative damage to cel-
lular DNA. Mutation Research 459: 211–218.
Kameda K., Takaku T., Okuda H., Kimura Y., Okuda T.,
Hatano T., Agata I, and Arichi S. (1987). Inhibitory
effects of various flavonoids isolated from leaves of
persimmon on angiotensin-converting enzyme activity.
Journal of Natural Products 50: 680–683.
Kang T.B. and Liang N.C. (1997). Studies on the
inhibitory effects of quercetin on the growth of HL-60
leukemia cells. Biochemical Pharmacology 54:
1013–1018.
Kang Z.C., Tsai S.J. and Lee H. (1999). Quercetin inhibits
benzo[a]pyrene-induced DNA adducts in human Hep
G2 cells by altering cytochrome P-450 1A1gene expres-
sion. Nutrition and Cancer 35: 175–179.
Keen C.L., Holt R.R., Oteiza P.I., Fraga C. and Schimitz
H.H. (2005). Cocoa antioxidants and cardiovascular
health. The American Journal of Clinical Nutrition 81:
298S–303S.
Keli S.O., Hertog M.G., Feskens E.J. and Kromhout D.
(1996). Dietary flavonoids, antioxidant vitamins, and
incidence of stroke: the Zutphen Study. Archives of
Internal Medicine 156: 637–642.
Kim H. and Keeney, P.G. (1984). ()-Epicatechin
content in fermented and unfermented cocoa beans.
Journal of Food Science 49: 1090–1092.
Kim H.P., Mani I., Ziboh V.A. (1998). Effects of natu-
rally-occurring flavonoids and bioflavonoids on epider-
mal cyclooxygenase from guinea pigs. Prostaglandins,
Leukotrienes and Essential Fatty Acids 58: 17–24.
Knekt P., Kumpulainen J., Järvinen R., Rissanen H.,
Heliövaara M., Reunanen A., Hakulinen T. and
Aromaa A. (2002). Flavonoid intake and risk of
chronic diseases. The American Journal of Clinical
Nutrition 76: 560–568.
Kobuchi H., Roy S., Sen C.K., Nguyen H.G. and Packer
L. (1999). Quercetin inhibits inducible ICAM-1 expres-
sion in human endothelial cells through the JNK
pathway. American Journal of Physiology 277:
C403–C411.
Koga T. and Meydani M. (2001). Effect of plasma
metabolites of ()-catechin and quercetin on mono-
cyte adhesion to human aortic endothelial cells. The
American Journal of Clinical Nutrition 73: 941–948.
Kondo K., Hirano R., Matsumoto A., Igarashi O. and
Itakura H. (1996). Inhibition of LDL oxidation by
cocoa. The Lancet 348: 1514.
Kris-Etherton PM and Keen C.L. (2002). Evidence that
the antioxidant flavonoids in tea and cocoa are benefi-
cial for cardiovascular health. Current Opinion in Lipi-
dology 13: 41–49.
Lamuela-Raventós R.M., Andrés-Lacueva C., Permanyer
J. and Izquierdo-Pulido M. (2001). More antioxidants
in cocoa. The Journal of Nutrition 131: 834.
Lamuela-Raventós R.M., Olga J., Ibern-Gómez M., Pons-
Raga J.V. and Andrés-Lacueva C. (2003). Cocoa
extract is a rich ingredient in flavanols and flavonols
compounds. Oxidants and Antioxidants. Book of
Abstracts 150.
Larocca L.M., Teofili L., Leone G., Sica S., Pierelli L.,
Menichella G., Scambia G., Benedetti Panici P., Ricci
R., Piantelli M. et al. (1991). Antiproliferative activity of
quercetin on normal bone marrow and leukaemic prog-
enitors. British Journal of Haematology 79: 562–566.
Larocca L.M., Teofili L., Sica S., Piantelli M., Maggiano
N., Leone G. and Ranelletti F.O. (1995). Quercetin
inhibits the growth of leukemic progenitors and
induces the expression of transforming growth factor-
B1 in these cells. Blood 85: 3654–3661.
Lautraite S., Musonda A.C., Doehmer J., Edwards G.O.
and Chipman J.K. (2002). Flavonoids inhibit genetic
toxicity produced by carcinogens in cells expressing
CYP1A2 and CYP1A1. Mutagenesis 17: 45–53.
Lee K.W., Kim Y.J., Lee H.J. and Lee C.Y. (2003). Cocoa
has more phenolic phytochemicals and a higher antiox-
idant capacity than teas and red wine. Journal of Agri-
cultural and Food Chemistry 51: 7292–7295.
Lee M.H., Lin R.D., Shen L.Y., Yang L.L., Yen K.Y. and
Hou W.C. (2001). Monoamine oxidase B and free
radical scavenging activities of natural flavonoids in
Melastoma candidum D. Don. Journal of Agricultural
and Food Chemistry 49: 5551–5555.
Lin C.M., Chen C.T., Lee H.H. and Lin J.K. (2002). Pre-
vention of cellular ROS damage by isovitexin and
related flavonoids. Planta Medica 68: 365–367.
Lindenmeyer F., Li H., Menashi S., Soria C. and Lu H.
(2001). Apigenin acts on the tumor cell invasion
process and regulates protease production. Nutrition
and Cancer 39: 139–147.
Lotito S.B., Actis-Goretta L., Renart M.L., Caligiuri M.,
Rein D., Schmitz H.H., Steinberg F.M., Keen C.L. and
Fraga C.G. (2000). Influence of oligomer chain length
172 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
on the antioxidant activity of procyanidins. Biochemi-
cal and Biophysical Research Communications 276:
945–951.
Manach C., Morand C., Crespy V., Demigne C., Texier
O., Regerat F. and Remesy C. (1998). Quercetin is
recovered in human plasma as conjugated derivatives
which retain antioxidant properties. FEBS Letters 24:
331–336.
Manach C. and Donovan J. (2004). Pharmacokinetics and
metabolism of dietary flavonoids in humans. Free
Radical Research 38:771–785.
Manach C., Williamson G., Morand C., Scalbert A. and
Rémésy C. (2005). Bioavailability and bioefficacy of
polyphenols in humans. I. Review of 97 bioavailability
studies. The American Journal of Clinical Nutrition 81:
230S–242S.
Manjeet K.R. and Ghosh B. (1999). Quercetin inhibits
LPS-induced nitric oxide and tumor necrosis factor-
alpha production in murine macrophages. International
Journal of Immunopharmacology 21: 435–443.
Mao T., Van de Water J., Keen C.L., Schmitz H.H. and
Gershwin M.E. (2000a). Cocoa procyanidins and
human cytokine transcription and secretion. The
Journal of Nutrition 130: 2093S–2099S.
Mao T.K., Powell J., Van de Water J., Keen C.L., Schmitz
H.H., Hammerstone J.F. and Gershwin M.E. (2000b).
The effect of cocoa procyanidins on the transcription
and secretion of interleukin 1 beta in peripheral blood
mononuclear cells. Life Sciences 66: 1377–1386.
Mennen L.I., Walker R., Bennetau-Pelissero C. and Scal-
bert (2005). A. Risk and safety of polyphenols con-
sumption. The American Journal of Clinical Nutrition
81: 226S–229S.
Miller A.L. (1996). Antioxidant flavonoids: structure,
function and clinical usage. Alternative Medicine
Review 1: 103–111.
Morand C., Crespy V., Manach C., Besson C., Demigne
C. and Remesy C. (1998). Plasma metabolites of
quercetin and their antioxidant properties. American
Journal of Physiology 275: R212–R219.
Morand C., Manach C., Crespy V. and Remesy C. (2000).
Quercetin 3-O-beta-glucoside is better absorbed than
other quercetin forms and is not present in rat plasma.
Free Radical Research 33: 667–676.
Murota K., Shimizu S., Chujo H., Moon J.H. and Terao J.
(2000). Efficiency of absorption and metabolic conver-
sion of quercetin and its glucosides in human intestinal
cell line Caco-2. Archives of Biochemistry and Bio-
physics 384: 391–397.
Murphy K.J., Chronopoulos A.K., Singh I., Francis M.A.,
Moriarty H., Pike M.J., Turner A.H., Mann N.J. and
Sinclair A.J. (2003). Dietary flavanols and procyanidin
oligomers from cocoa (Theobroma cacao) inhibit
platelet function. The American Journal of Clinical
Nutrition 77: 1466–1473.
Nemeth K., Plumb G.W., Berrin J.G., Juge N., Jacob R.,
Naim H.Y., Williamson G., Swallow D.M. and Kroon
P.A. (2003). Deglycosylation by small intestinal epithe-
lial cell beta-glucosidases is a critical step in the absorp-
tion and metabolism of dietary flavonoid glycosides in
humans. European Journal of Clinical Nutrition 42:
29–42.
Olthof M.R., Hollman P.C., Vree T.B. and Katan M.B.
(2000). Bioavailabilities of quercetin-3-glucoside and
quercetin-4-glucoside do not differ in humans. The
Journal of Nutrition 130: 1200–1203.
Osakabe N., Natsume M., Adachi T., Yamagishi M.,
Hirano R., Takizawa T., Itakura H. and Kondo K.
(2000). Effects of cacao liquor polyphenols on the sus-
ceptibility of low-density lipoprotein to oxidation in
hypercholesterolemic rabbits. Journal of Atherosclero-
sis and Thrombosis 7: 164–168.
Osakabe N., Baba S., Yasuda A., Iwamoto T., Kamiyama
M., Takizawa T., Itakura H. and Kondo K. (2001).
Daily cocoa intake reduces the susceptibility of low-
density lipoprotein to oxidation as demonstrated in
healthy human volunteers. Free Radical Research 34:
93–99.
Osakabe N., Yasuda A., Natsume M., Takizawa T., Terao
J. and Kondo K. (2002). Catechins and their oligomers
linked by C4→C8 bonds are major cacao polyphenols
and protect low-density lipoprotein from oxidation in
vitro. Experimental Biology and Medicine 227: 51–56.
Pace-Asciak C.R., Hahn S., Diamandis E.P., Soleas G.
and Goldberg D.M. (1995). The red wine phenolics
trans-resveratrol and quercetin block human platelet
aggregation and eicosanoid synthesis: implications for
protection against coronary heart disease. Clinica
Chimica Acta 235: 207–219.
Pascual-Teresa S., Santos-Buelga C. and Rivas-Gonzalo J.
(2000). Quantitative analysis of flavan-3-ols in Spanish
foodstuffs and beverages. Journal of Agricultural and
Food Chemistry 48: 5331–5337.
Pereira M.A., Grubbs C.J., Barnes L.H., Li H., Olson
G.R., Eto I., Juliana M., Whitaker L.M., Kelloff G.J.,
Steele V.E. and Lubet R.A. (1996). Effects of the phy-
tochemicals, curcumin and quercetin, upon
azoxymethane-induced colon cancer and 7,12-
dimethylbenzyl[a]anthracene-induced mammary
cancer in rats. Carcinogenesis 17: 1305–1311.
Pérez-Vizcaíno F., Ibarra M., Cogolludo A.L., Duarte J.,
Zaragoza-Arnaez F., Moreno L., Lopez-Lopez G. and
Tamargo J. (2002). Endothelium-independent vasodila-
tor effects of the flavonoid quercetin and its methylated
metabolites in rat conductance and resistance arteries.
The Journal of Pharmacology and Experimental Thera-
peutics 302: 66–72.
Peres W., Tunon M.J., Collado P.S., Herrmann S.,
Marroni N. and Gonzalez-Gallego J. (2000). The
flavonoid quercetin ameliorates liver damage in rats
with biliary obstruction. Journal of Hepatology 33:
742–750.
Peterson J., Lagiou P., Samoli E., Lagiou A., Katsouyanni
K., La Vecchia C., Dwyer J. and Trichopoulos D.
(2003). Flavonoid intake and breast cancer risk: a case-
control study in Greece. British Journal of Cancer 89:
1255–1259.
Pignatelli P., Pulcinelli F.M., Celestini A., Lenti L., Ghis-
elli A., Gazzaniga P.P. and Violi F. (2000). The
Health Effects of Cocoa Flavonoids 173
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
flavonoids quercetin and catechin synergistically inhibit
platelet function by antagonizing the intracellular pro-
duction of hydrogen peroxide. The American Journal
of Clinical Nutrition 72: 1150–1155.
Porter L.J., Ma Z. and Chan B.G. (1991). Cacao procyani-
dins: major flavanoids and identification of some minor
metabolites. Phytochemistry 30: 1657–1663.
Ranelletti F.O., Maggiano N., Serra F.G., Ricci R.,
Larocca L.M., Lanza P., Scambia G., Fattorossi A.,
Capelli A. and Piantelli M. (2000). Quercetin inhibits
p21-RAS expression in human colon cancer cell lines
and in primary colorectal tumors. International Journal
of Cancer 85: 438–445.
Rein D., Lotito S., Holt R.R., Keen C.L., Schmitz H.H.
and Fraga C.G. (2000a). Epicatechin in human plasma:
in vivo determination and effect of chocolate consump-
tion on plasma oxidation status. The Journal of Nutriti-
ton 130: 2109S–2114S.
Rein D., Paglieroni T.G., Wun T., Pearson D.A., Schmitz
H.H., Gosselin R. and Keen C.L. (2000b). Cocoa
inhibits platelet activation and function. The American
Journal of Clinical Nutrition 72: 30–35.
Rein D., Paglieroni T.G., Pearson D.A., Wun T., Schmitz
H.H., Gosselin R. and Keen C.L. (2000c). Cocoa and
wine polyphenols modulate platelet activation and
function. The Journal of Nutrition 130: 2120S–2126S.
Richelle M., Tavazzi I., Enslen M. and Offord E.A. (1999)
Plasma kinetics in man of epicatechin from black
chocolate. European Journal of Clinical Nutrition 53:
22–26.
Richter M., Ebermann R. and Marian B. (1999).
Quercetin-induced apoptosis in colorectal tumor cells:
possible role of EGF receptor signaling. Nutrition and
Cancer 34: 88–99.
Ríos L.Y., Bennett R.N., Lazarus S.A., Remesy C., Scal-
bert A. and Williamson G. (2002). Cocoa procyanidins
are stable during gastric transit in humans. The Ameri-
can Journal of Clinical Nutrition 76: 1106–1110.
Ríos L.Y., Gonthier M.P., Rémésy C., Mila I., Lapierre
C., Lazarus S.A., Williamson, G. and Scalbert A.
(2003). Chocolate intake increases urinary excretion of
polyphenol-derived phenolic acids in healthy human
subjects. The American Journal of Clinical Nutrition
77: 912–918.
Rodgers E.H. and Grant M.H. (1998). The effect of the
flavonoids, quercetin, myricetin and epicatechin on the
growth and enzyme activities of MCF7 human breast
cancer cells. Chemico-Biological Interactions 116:
213–228.
Romero I., Paez A., Ferruelo A., Lujan M. and Berenguer
A. (2002). Polyphenols in red wine inhibit the prolifer-
ation and induce apoptosis of LNCaP cells. BJU Inter-
national 89: 950–954.
Saija A., Scalese M., Lanza M., Marzullo D., Bonina F.
and Castelli F. (1995). Flavonoids as antioxidant
agents: importance of their interaction with biomem-
branes. Free Radical Biology and Medicine 19: 481–486.
Sanbongi C., Suzuki N. and Sakane T. (1997). Polyphe-
nols in chocolate, which have antioxidant activity, mod-
ulate immune functions in humans in vitro. Cellular
Immunology 177: 129–136.
Sanbongi C., Osakabe N., Natsume M., Takizawa T.,
Gomi S. and Osawa T. (1998). Antioxidative polyphe-
nols isolated from Theobroma cacao. Journal of Agri-
cultural and Food Chemistry 46: 454–457.
Sánchez-Rabaneda F., Jáuregui O., Casals I., Andrés-
Lacueva C., Izquierdo-Pulido M. and Lamuela-Raven-
tós R.M. (2003). Liquid chromatographic/electrospray
ionization tandem mass spectrometric study of the phe-
nolic composition of cocoa (Theobroma cacao).
Journal of Mass Spectrometry 38: 35–42.
Santos A.C., Uyemura S.A., Lopes J.L., Bazon J.N., Min-
gatto F.E. and Curti C. (1998). Effect of naturally
occurring flavonoids on lipid peroxidation and mem-
brane permeability transition in mitochondria. Free
Radical Biology and Medicine 24: 1455–1461.
Santos-Buelga C. and Scalbert A. (2000). Proanthocyani-
dins and tannin-like compounds – nature, occurrence,
dietary intake and effect on nutrition and health.
Journal of the Science of Food and Agriculture 80:
1118–1125.
Satyanarayana P.S., Singh D. and Chopra K. (2001).
Quercetin, a bioflavonoid, protects against oxidative
stress-related renal dysfunction by cyclosporine in rats.
Methods and Findings in Experimental and Clinical
Pharmacology 23: 175–181.
Scalbert A. and Williamson G. (2000). Dietary intake and
bioavailability of polyphenols. The Journal of Nutrition
130: 2073S–2085S.
Scalbert A., Johnson I.T. and Saltmarsh M. (2005).
Polyphenols: antioxidants and beyond. The American
Journal of Clinical Nutrition 81: 215S–217S.
Scambia G., Raneletti F.O., Benedetti Panici P., Bonanno
G., DeVincenzo R.; Piantelli M. and Mancuso S.
(1990). Synergistic antiproliferative activity of
quercetin and cisplatin on ovarian cancer cell growth.
Anti-Cancer Drugs 1: 45–48.
Scambia G., Raneletti F.O., Benedetti Panici P., Piantelli
M., Bonanno G., DeVincenzo R., Gerrandina G., Mag-
giano N., Capelli A. and Mancuso S. (1992). Inhibitory
effect of quercetin on primary ovarian and endometrial
cancers and synergistic activity with cis-
diamminedichloroplatinum (II). Gynecologic Oncology
45: 13–19.
Scambia G., Raneletti F.O., Panici P.B., Piantelli M.,
DeVincenzo R., Ferrandina G., Bonanno G., Capelli
A. and Mancuso S. (1993). Quercetin induces type-II
estrogen-binding sites in estrogen-receptor-negative
(MDA-MB231) and estrogen-receptor-positive (MCF-
7) human breast-cancer cell lines. International Journal
of Cancer 54: 462–466.
Schewe T., Sadik C., Klotz L.O., Yoshimoto T., Kuhn H.
and Sies H. (2001). Polyphenols of cocoa: inhibition of
mammalian 15-lipoxygenase. Biological Chemistry 382:
1687–1696.
Schewe T., Kuhn H. and Sies H. (2002). Flavonoids of
cocoa inhibit recombinant human 5-lipoxygenase. The
Journal of Nutrition 132: 1825–1829.
174 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
Schneider H., Schwiertz A., Collins M.D. and Blaut M.
(1999). Anaerobic transformation of quercetin-3-gluco-
side by bacteria from the human intestinal tract.
Archives of Microbiology 171: 81–91.
Schoefer L., Mohan R., Schwiertz A., Braune A. and
Blaut M. (2003). Anaerobic degradation of flavonoids
by Clostridium orbiscindens. Applied and Environ-
mental Microbiology 69: 5849–5854.
Schramm D.D., Wang J.F., Holt R.R., Ensunsa J.L., Gon-
salves J.L., Lazarus S.A., Schmitz H.H., German J.B.
and Keen C.L. (2001). Chocolate procyanidins
decrease the leukotriene-prostacyclin ratio in humans
and human aortic endothelial cells. The American
Journal of Clinical Nutrition 73: 36–40.
Schramm D.D., Karim M., Schrader H.R., Holt R.R.,
Kirkpatrick N.J., Polagruto J.A., Ensunsa J.L., Schmitz
H.H. and Keen C.L. (2003). Food effects on the
absorption and pharmacokinetics of cocoa flavanols.
Life Sciences 73: 857–869.
Schroeter H., Spencer J.P., Rice-Evans C. and Williams
R.J. (2001). Flavonoids protect neurons from oxidized
low-density-lipoprotein-induced apoptosis involving c-
Jun N-terminal kinase (JNK), c-Jun and caspase-3. The
Biochemical Journal 358: 547–557.
Serafini M., Bugianesi R., Maiani G., Valtuena S., De
Santis S. and Crozier A. (2003). Plasma antioxidants
from chocolate. Nature 424: 1013.
Sesink A.L., Arts I.C., Faassen-Peters M. and Hollman
P.C. (2003). Intestinal uptake of quercetin-3-glucoside
in rats involves hydrolysis by lactase phlorizin hydro-
lase. The Journal of Nutrition 133: 773–776.
Shimada Y., Goto H., Kogure T., Shibahara N., Sakak-
ibara I., Sasaki H. and Terasawa K. (2001). Protective
effect of phenolic compounds isolated from the hooks
and stems of Uncaria sinensis on glutamate-induced
neuronal death. The American Journal of Chinese Med-
icine 29: 173–180.
Shimoi K., Okada H., Furugori M., Goda T., Takase S.,
Suzuki M., Hara Y., Yamamoto H. and Kinae N.
(1998). Intestinal absorption of luteolin and luteolin 7-
O-beta-glucoside in rats and humans. FEBS Letters
438: 220–224.
Sies H., Schewe T., Heiss C. and Kelm M. (2005). Cocoa
polyphenols and inflammatory mediators. The Ameri-
can Journal of Clinical Nutrition 81: 304S–312S.
Singhal R.L., Yeh Y.A., Prajda N., Olah E., Sledge G.W.
and Weber G. (1995). Quercetin down-regulates signal
transduction in human breast carcinoma cells. Bio-
chemical and Biophysical Research Communications
208: 425–431.
Skaper S.D., Fabris M., Ferrari V., Dalle Carbonare M.
and Leon A. (1997). Quercetin protects cutaneous
tissue-associated cell types including sensory neurons
from oxidative stress induced by glutathione depletion:
cooperative effects of ascorbic acid. Free Radical
Biology and Medicine 22: 669–678.
Skibola C.F. and Smith M. (2000). Potential health
impacts of excessive flavonoid intake. Free Radical
Biology and Medicine 29: 375–383.
Soleas G.J., Grass L., Josephy P.D., Goldberg D.M. and
Diamandis E.P. (2002). A comparison of the anticar-
cinogenic properties of four red wine polyphenols.
Clinical Biochemistry 35: 119–124.
Spencer J.P., Schroeter H., Rechner A.R. and Rice-Evans
C. (2001). Bioavailability of flavan-3-ols and procyani-
dins: gastrointestinal tract influences and their rele-
vance to bioactive forms in vivo. Antioxidants and
Redox Signalling 3: 1023–1039.
Stavric B. (1994). Quercetin in our diet: from potent
mutagen to probable anticarcinogen. Clinical Biochem-
istry 27: 245–248.
Steinberg F.M., Holt R.R., Schmitz H.H. and Keen C.L.
(2002). Cocoa procyanidin chain length does not deter-
mine ability to protect LDL from oxidation when
monomer units are controlled. The Journal of Nutri-
tional Biochemistry 13: 645–652.
Steinberg F.M., Bearden M.M. and Keen C.L. (2003).
Cocoa and chocolate flavonoids: implications for car-
diovascular health. Journal of the American Dietetic
Association 103: 215–223.
Taubert D., Berkels R., Roesen R. and Klaus W. (2003).
Chocolate and blood pressure in elderly individuals
with isolated systolic hypertension. The Journal of the
American Medical Association 290:1029–1030.
Tuck K.L., Freeman M.P., Hayball P.J., Stretch G.L. and
Stupans I. (2001) The in vivo fate of hydroxytyrosol
and tyrosol, antioxidant phenolic constituents of olive
oil, after intravenous and oral dosing of labeled com-
pounds to rats. The Journal of Nutrition 131:
1993–1996.
Vaidyanathan J.B. and Walle T. (2001). Transport and
metabolism of the tea flavonoid ()-epicatechin by the
human intestinal cell line Caco-2. Pharmaceutical
Research 18: 1420–1425.
Vinson J.A., Proch J. and Zubik L. (1999). Phenol antioxi-
dant quantity and quality in foods: cocoa, dark choco-
late, and milk chocolate. Journal of Agricultural and
Food Chemistry 47: 4821–4824.
Walle T., Walle U.K. and Halushka P.V. (2001). Carbon
dioxide is the major metabolite of quercetin in humans.
The Journal of Nutrition 131: 2648–2652.
Wan Y., Vinson J.A., Etherton T.D., Proch J., Lazarus
S.A. and Kris-Etherton P.M. (2001) Effects of cocoa
powder and dark chocolate on LDL oxidative suscepti-
bility and prostaglandin concentrations in humans. The
American Journal of Clinical Nutrition 74: 596–602.
Wang I.K., Lin-Shiau S.Y. and Lin J.K. (1999). Induction
of apoptosis by apigenin and related flavonoids through
cytochrome c release and activation of caspase-9 and
caspase-3 in leukaemia HL-60 cells. European Journal
of Cancer 35: 1517–1525.
Wang J.F., Schramm D.D., Holt R.R., Ensunsa J.L., Fraga
C.G., Schmitz H.H. and Keen C.L. (2000). A dose-
response effect from chocolate consumption on plasma
epicatechin and oxidative damage. The Journal of
Nutrition 130: 2115S–2119S.
Wang W.Q., Ma C.G. and Xu S.Y. (1996). Protective
effect of hyperin against myocardial ischemia and
Health Effects of Cocoa Flavonoids 175
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
reperfusion injury. Zhongguo Yao Li Xue Bao 17:
341–344.
Waterhouse A.L., Shirley J.R. and Donovan J.L. (1996).
Antioxidants in chocolate. The Lancet 348: 834.
Weyant M.J., Carothers A.M., Dannenberg A.J. and
Bertagnolli M.M. (2001). ()-Catechin inhibits intesti-
nal tumor formation and suppresses focal adhesion
kinase activation in the min/ mouse. Cancer Research
61: 118–125.
Wittig J., Herderich M., Graefe E.U. and Veit M. (2001).
Identification of quercetin glucuronides in human
plasma by high-performance liquid chromatography-
tandem mass spectrometry. Journal of Chromatogra-
phy B, Biomedical Sciences and Applications 753:
237–243.
Wolffram S., Block M. and Ader P. (2002). Quercetin-3-
glucoside is transported by the glucose carrier SGLT1
across the brush border membrane of rat small intes-
tine. The Journal of Nutrition 132: 630–635.
Wollgast J. and Anklam E. (2000a). Polyphenols in choco-
late: is there a contribution to human health Food
Research International 33: 449–459.
Wollgast J. and Anklam E. (2000b). Review on polyphe-
nols in Theobroma cacao: changes in composition
during the manufacture of chocolate and methodology
for identification and quantification. Food Research
International 33: 423–447.
Xagorari A., Papapetropoulos A., Mauromatis A.,
Economou M., Fotsis T. and Roussos C. (2001). Lute-
olin inhibits an endotoxin-stimulated phosphorylation
cascade and proinflammatory cytokine production in
macrophanges. The Journal of Pharmacology and
Experimental Therapeutics 296: 181–187.
Xiao D., Zhu S.P. and Gu Z.L. (1997). Quercetin induced
apoptosis in human leukemia HL-60 cells. Zhongguo
Yao Li Xue Bao 18: 280–283.
Xie M.L., Lu Q. and Gu Z.L. (1996). Effect of quercetin
on platelet aggregation induced by oxyradicals. Zhong-
guo Yao Li Xue Bao 17: 334–336.
Yamagishi M., Natsume M., Nagaki A., Adachi T.,
Osakabe N., Takizawa T., Kumon H. and Osawa T.
(2000). Antimutagenic activity of cacao: inhibitory
effect of cacao liquor polyphenols on the mutagenic
action of heterocyclic amines. Journal of Agricultural
and Food Chemistry 48: 5074–5078.
Yamagishi M., Osakabe N., Natsume M., Adachi T., Tak-
izawa T., Kumon H. and Osawa T. (2001). Anticlasto-
genic activity of cacao: inhibitory effect of cacao liquor
polyphenols against mitomycin C-induced DNA
damage. Food and Chemical Toxicology 39: 1279–1283.
Yamagishi M., Natsume M., Osakabe N., Okazaki K.,
Furukawa F., Imazawa T., Nishikawa A. and Hirose M.
(2003). Chemoprevention of lung carcinogenesis by
cacao liquor proanthocyanidins in a male rat multi-
organ carcinogenesis model. Cancer Letters 191: 49–57.
Yamashita N. and Kawanishi S. (2000). Distinct mechan-
ism of DNA damage in apoptosis induced by quercetin
and luteolin. Free Radical Research 33: 623–633.
Yin F., Giuliano A.E. and Van Herle A.J. (1999). Growth
inhibitory effects of flavonoids in human thyroid cancer
cell lines. Thyroid: Official Journal of the American
Thyroid Association 9: 369–376.
Yoshida M., Sakai T., Hosokawa N., Marui N., Mat-
sumoto K., Fujioka A., Nishino H. and Aoike A.
(1990). The effect of quercetin on cell cycle progression
and growth of human gastric cancer cells. FEBS Letters
260: 10–13.
Zhang Y.H., Park Y.S., Kim T.J., Fang L.H., Ahn H.Y.,
Hong J., Kim Y., Lee C.K. and Yun Y.P. (2000a).
Endothelium-dependent vasorelaxant and antiprolifer-
ative effects of apigenin. General Pharmacology 35:
341–347.
Zhang X., Xu Q. and Saiki I. (2000b). Quercetin inhibits
the invasion and mobility of murine melanoma B16-
BL6 cells through inducing apoptosis via decreasing
Bcl-2 expression. Clinical and Experimental Metastasis
18: 415–421.
Zhao X., Gu Z., Attele A.S. and Yuan C.S. (1999). Effects
of quercetin on the release of endothelin, prostacyclin
and tissue plasminogen activator form human endothe-
lial cells in culture. Journal of Ethnopharmacology 67:
279–285.
Zhu Q.Y., Holt R.R., Lazarus S.A., Orozco T.J. and Keen
C.L. (2002). Inhibitory effects of cocoa flavanols and
procyanidin oligomers on free radical-induced erythro-
cyte haemolysis. Experimental Biology and Medicine
227: 321–329.
176 R.M. LAMUELA-RAVENTÓS ET AL.
at UPC (CSIC) on December 10, 2013fst.sagepub.comDownloaded from
