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Review: Health Effects of Cocoa Flavonoids


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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 vasodilators in blood vessels, inhibiting the adherence of monocytes to the vascular endothelium, promoting fibrinolysis, 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.
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Food Science and Technology
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
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Review: Health Effects of Cocoa Flavonoids
R.M. Lamuela-Raventós,
* A.I. Romero-Pérez,
C. Andrés-Lacueva
and A. Tornero
Nutrició i Bromatologia, CÈRTA, Facultat de Farmàcia, Universitat de Barcelona, Avinguda Joan XXIII s/n,
08028 Barcelona, Spain
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
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
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
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should be ingested so that their effects can be
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).
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
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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
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)
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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.
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
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
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.
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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
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
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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
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,
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
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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
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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.
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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
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
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Activity in Phases of Tumour Progression and
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.
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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).
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... Quercetin is one of the polyphenolic compounds isolated from PCP and widely studied for antiinflammatory and antioxidant compound. Quercetin prevents alcohol-induced hepatotoxicity [52], has scavenging properties [53], decreased the cytotoxicity [54], inhibits neurotoxicity [55], and possesses several medicinal benefits. Herbal antioxidants are major supporting agents in the battle of diseases and infections. ...
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Traditional Chinese medicines (TCM) play an important role in the control and treatment of several animal diseases. Penthorum chinense Pursh (PCP) is a famous plant for its use in traditional medication practice and therapeutic effects in numerous pathological conditions. In China, PCP is utilized for both food and medication due to numerous bioactivities. PCP is widely administered in prevention and treatment of traumatic injury, edema, and liver diseases with functions of reducing swelling, support diuresis, blood stasis, and mitigation symptoms of excessive alcohol intake. Recently, PCP highlighted for research trials in various fields including pharmacology, pharmacognosy, cosmeceuticals, nutraceuticals, and pharmaceuticals due to medicinal significance with less toxicity and an effective ethnomedicine in veterinary practice. PCP contains diverse important ingredients such as flavonoids, organic acids, coumarins, lignans, polyphenols, and sterols that are important bioactive constituents of PCP exerting the therapeutic benefits and organ-protecting effects. In veterinary, PCP extract, compound, and phytochemicals/biomolecules significantly reversed the liver and kidney injuries, via antioxidation, oxidative stress, apoptosis, mitochondrial signaling pathways, and related genes. PCP water extract and compounds also proved in animal and humans’ clinical trial for their hepatoprotective, antiaging, nephroprotective, anti-inflammatory, antidiabetic, antibacterial, antiapoptotic, immune regulation, and antioxidative stress pathways. This updated review spotlighted the current information on efficiency and application of PCP by compiling and reviewing recent publications on animal research. In addition, this review discussed the toxicology, traditional use, comparative, and clinical application of PCP in veterinary practices to authenticate and find out new perspectives on the research and development of this herbal medicine.
... [33,34] These compounds' content comprises catechin, epicatechin, and procyanidin B2 dimer. [19,35] Thus, the higher the flavonoids, especially in the number of monomers and dimers in cocoa products, the greater the health benefits obtained. [19] Antioxidant activity The IC₅₀ of the green coffee bean extract was 18.48 ± 0.40 ppm, and that of the encapsulated extract was 73.20 ± 0.73 ppm. ...
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Cocoa drinks are made from powdered cocoa beans that have been processed. Cocoa powder has antioxidants, but processing may lower the antioxidant activity. Therefore, adding powerful antioxidants, such as green coffee extract, is essential. The extract needs to be encapsulated to preserve its stability and properties. This study investigated the antioxidant activity and characteristics of a cocoa drink incorporated with encapsulated green coffee extract. The percentage of encapsulated coffee extract in cocoa drinks was designated as C1 (0%), C2 (2%), C3 (4%), C4 (6%), C5 (8%) and C6 (10%). The results showed that incorporating a higher percentage of the encapsulated extract increased the number of total polyphenols and flavonoids and antioxidant activity. Additionally, before brewing, the powdered form showed a significant increase in hygroscopicity and total color difference, along with an increase in the percentage of encapsulated extract in the cocoa drink formulation. However, the moisture content of the powder decreased significantly. After brewing the pH value, dissolution time and sedimentation decreased while the viscosity, total soluble solids, and solubility increased as the encapsulated extract percentage increased. According to the sensory evaluation, all sensory attributes of the new cocoa drink formulation were acceptable. Therefore, cocoa drink can be used as a ready-for-consumption drink rich in healthy functional compounds.
... Comparatively, the higher TFC obtained in this work could be attributed to the coconut oil additions. The importance of these compounds is important in the health sector because it reduces blood pressure and oxidative stress, acting as anti-inflammatory agents, promoting fibrinolysis and inhibiting tumor formation (Bauer, Ding, & Smit, 2011;Lamuela-Raventós, Romero-Pérez, Andrés-Lacueva, & Tornero, 2005;Peluso, Miglio, Morabito, Ioannone, & Serafini, 2015;Socci, Tempesta, Desideri, De Gennaro, & Ferrara, 2017;Övet, 2015). These findings go to suggest that enhancing TPC and TFC content in chocolate has additional advantages. ...
To appreciate and explore the potential use of coconut oil as substitute in chocolate this research was conducted. In achieving this, three different chocolates were formulated using varied amount of coconut oil (5–15%) as cocoa butter substitute. The formulated chocolate samples and a control were stored for 21 days to evaluate the kinetic reaction of oxidative stability and phenolic and flavonoid content, color and sensory properties. Results revealed that characterizations of oxidative reactions were dependent on coconut oil which had a positive correlation with free fatty acids, saponification and peroxide. Based on statistical tools such as coefficient of correlation (0.9879–0.9976), root mean square error (0.0176–0.3620) and reduced chi-square (1.5 × 10–4–0.1310), second-order polynomial model accurately predicted kinetic evolution of oxidative stability during storage. In conclusion, coconut oil could be used as cocoa butter replacer in chocolate formulation, however only 5% is recommended based on better mouthfeel, appearance and taste and enhanced polyphenolic content
... This polyphenol content includes cinnamic acid, pyrogallol, tannin, resorcinol, quercetin, and epicatechin-3-galat [4]. The main compounds found from cocoa shell are (-)-epicatechin and phidroxybenzoic acid [5], these compounds both have functions as antimicrobials [6] [7]. ...
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Cocoa has become the leading export commodity from the plantation sector. The Increasing of cocoa production results in the increase of the cocoa shell waste amount. Cocoa shell utilization is still very limited. The benefit of cocoa shell is that this waste contains secondary metabolites that can be used as antimicrobial agents. One of the products that can be produced from cocoa shell waste is liquid smoke. This study was focused in determining the antimicrobial activity and the minimum kill concentration from liquid smoke on the growth of the Lasiodiplodia theobremae fungi from different amounts of raw materials moisture content and pyrolysis temperatures. This research indicated that cocoa shells liquid smoke from different amounts of water content and temperature of pyrolysis had antimicrobial activity and the application of different concentrations in the minimum kill concentration test can inhibit Lasiodiplodia theobremae fungi growth. Liquid smoke antimicrobial activity at 10% water content and temperatures at 200, 300, and 400°C obtained inhibition zone values of 10.40; 16.75 and 17.80 mm. At the 15% moisture content and temperatures of 200, 300and 400°C, the inhibition zone value is 10.15; 15.70 and 16.15 mm. At 20% water content and temperatures of 200, 300and 400°C, it obtained inhibition zone values of 4.25; 11,45 and 12.30 mm. At 25% water content and temperatures of 200, 300, and 400°C, it obtained inhibition zone values of 3.70; 7.65 and 8.65 mm. The value of minimum kill concentration and minimum inhibitory concentration of liquid smoke at 10, 15, 20, and 25 % water content at temperatures of 200, 300, and 400°C obtained the values of 1% and 9%.
... Cocoa is rich in polyphenols, basically catechins (flavan-3-ols) and proanthocyanidins. Phenolic substances contain 12-18% of the total weight of dried cocoa nibs (Lamuela Raventos et al., 2005). Phenolic compounds play a crucial role in contributing to the sensory attributes of cocoa and cocoa products. ...
Top trends in the food industry are mainly related with improvement of functionality of the products. Food manufacturers and researchers mainly focus on this subject and it is aimed to produce the functional products with quality characteristics similar to conventional ones. In the present chapter, the ways to improve functional characteristics of the chocolate products were reviewed. Calorie reduction in chocolates with using sugar and cocoa butter alternatives and its effect on chocolate quality was mentioned. In addition, the influence of processes applied for cocoa and chocolate productions on polyphenol composition and antioxidant activity was also addressed. Studies related with dietary fiber usage as a prebiotics and their effects on chocolate quality and probiotic chocolates and bioaccessibility of probiotics were also summarized in the chapter. It can be highlighted that functional characteristics of the chocolate or derived products can be improved by enrichment and adjusting production process parameters. Chocolate is a good functional compound carrier since it is lovely consumed by people of all age throughout the world.KeywordsChocolateCocoaFunctional foodSugar-freePolyphenolsProbiotics
... Cocoa is rich in polyphenols, basically catechins (flavan-3-ols) and proanthocyanidins. Phenolic substances contain 12-18% of the total weight of dried cocoa nibs (Lamuela Raventos et al., 2005). Phenolic compounds play a crucial role in contributing to the sensory attributes of cocoa and cocoa products. ...
Top trends in the food industry are mainly related with improvement of functionality of the products. Food manufacturers and researchers mainly focus on this subject and it is aimed to produce the functional products with quality characteristics similar to conventional ones. In the present chapter, the ways to improve functional characteristics of the chocolate products were reviewed. Calorie reduction in chocolates with using sugar and cocoa butter alternatives and its effect on chocolate quality was mentioned. In addition, the influence of processes applied for cocoa and chocolate productions on polyphenol composition and antioxidant activity was also addressed. Studies related with dietary fiber usage as a prebiotics and their effects on chocolate quality and probiotic chocolates and bioaccessibility of probiotics were also summarized in the chapter. It can be highlighted that functional characteristics of the chocolate or derived products can be improved by enrichment and adjusting production process parameters. Chocolate is a good functional compound carrier since it is lovely consumed by people of all age throughout the world.
... 1 Nutritional interest in phytochemicals has increased due to the relation between their consumption and health benefits, such as the reduction of the risk factors of cardiovascular disease and certain types of cancer. 2,3 The combined peak capacity of liquid chromatography ion mobility (LC-IM-MS) and CCS can be used to produce routine unequivocal identification of phytochemicals. Importantly, IM separation can often resolve LC coeluting and m/z coincident isomeric species, which makes it a powerful tool in complex mixtures analysis. ...
Conference Paper
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Ion mobility (IM) provides an added dimension of separation to that of LC (hydrophobicity) and MS (m/z), in addition to CCS (collision cross‐section) values which provide a complimentary physiochemical descriptor. The combined peak capacity of liquid chromatography (LC) ion mobility and CCS can be used to produce routine unequivocal identification of phytochemicals. The approach has been utilised to profile phytochemical makeup, using non-targeted screening of “off the shelf” cocoa powder food commodities (including vegan, organic, alkalized, low fat, supermarket home-brand, high-end brands, natural cocoa powder and unsweetened). Nutritional interest, particularly in flavonoids has increased due to the relation between their consumption and the reduction of the risk factors of cardiovascular disease and certain types of cancer. Sixty-seven cocoa powder food commodities have been analysed using a non-targeted screening strategy to determine the influence of manufacturing processing and production methods upon phytochemical make-up. LC linear TWIM data were acquired for the cocoa extracts. Ion mobility separated single component precursor ion/product ion spectra were generated for the [M+H]+ and [M-H]- components detected. Data were screened against natural product libraries incorporating positive and negative ion mass spectrometry detection results for precursors ion, ion mobility product ion, retention time and TWCCSN2 values. We report initial findings. Using positive ion mode, abundant phytochemicals identified across the majority of cocoa powder food commodities analysed include: Methylxanthine alkaloids which have been identified based on accurate mass and CCS: caffeine (ref TWCCSN2 136.8 Å2, Δ TWCCSN2 0.1 %); theobromine (ref 130.4Å2, Δ TWCCSN2 0.4 %) and theophylline (ref CCS 132.2 Å2, Δ TWCCSN2 -1.5 %). In negative ion mode, abundant phytochemicals identified across the majority of cocoa powder food commodities analysed include: Flavanols: (−)-Epicatechin (Δ TWCCSN2 0.7 %) and (+)-catechin (Δ TWCCSN2 0.9%). Also, quercetin, kaempferol, quercetin-3-glucoside and isoquercitrin. Other minor polyphenolic compounds identified include flavonoids (vitexin, isovitexin, isoorientin, apigenin and apigenin-7- O glucoside), flavones (luteolin), flavanones (naringenin and eriodyctiol) and polyphenols (caffeic acid and ferulic acid). Additionally in negative ion mode: procyanidin B1 (TWCCSN2 221.9 Å2), procyanidin B2 (TWCCSN2 223.4 Å2) and procyanidin C1 (TWCCSN2 275.2 Å2) TWCCSN2 values have been determined, from identification within the analysed samples. In the case of procyanidins, initial reference CCS values were determined using CCS prediction, to facilitate identification. Using LC-IM, a combination of accurate mass measurement, retention time and CCS values has facilitated identification of both abundant and trace level phytochemicals of processed cocoa products, enabling impact of manufacturing processes upon food commodity nutritional value to be assessed.
One of the most abundant flavonoids present in cacao is (-)-epicatechin (Epi) and this flavanol has been linked to the cardiovascular health promoting actions of cocoa products. We previously demonstrated that Epi reduces infarct size in rodent models of ischemia/reperfusion and permanent coronary occlusion. Reduced infarct size was associated with decreased left ventricular (LV) oxidative stress (OS) and indicators of inflammation factors, which foster myocardial fibrosis. In this study, we examine the antifibrotic actions of Epi in an aging female rat model of pre-heart failure with preserved ejection fraction (pre-HFpEF) as well as its potential to mitigate plasma levels of OS, proinflammatory/profibrotic cytokines, and improve passive and active LV function. Epi treatment [1 mg/(kg·d)] was provided daily by gavage from 21 to 22 months of age, whereas controls received water. A Millar catheter was used to assess hemodynamic function. Subsequently, hearts were arrested in diastole, a balloon inserted into the LV and passive pressure-volume curves generated. Fixed LV sections were processed for collagen area fraction quantification using Sirius Red staining. Treatment with Epi did not lead to detectable changes in LV contractile function. However, passive LV pressure volume curves were significantly right shifted with Epi. Collagen area fraction values indicated that Epi treatment significantly reduces LV fibrosis. Epi also significantly reduced plasma OS markers and levels of profibrotic and proinflammatory cytokines. In conclusion, Epi reduces cardiac fibrosis in an aged, female rat model of pre-HFpEF, which correlates with significant reductions in OS and cytokine levels in the absence of changes in LV contractile function.
Bioactive ingredients, whether derived from various botanical or animal sources, have acquired important relevance during the last few decades due to a large number of potential applications, which could be useful in food products. An important part of this potential resides in the composition and the concentration of the bioactive constituents. These two aspects are deeply related to the techniques applied to recover, characterize, and the way to stabilize them in the final matrix. First, the extraction and purification of bioactive compounds from the natural sources are key points to procure enriched extract in bioactive compounds. In addition, in the characterization step, it is necessary to identify the composition of the attained extracts. Finally, the protection and stabilization of the compounds allow improving their incorporation in the food matrix to get products with great organoleptic properties. This chapter reviews different bioactive compounds present in natural sources and the aspects which are to be considered in the selection of techniques and tools for recovering, characterizing, and stabilizing suitable potential applications considering the nature of bioactive compounds under study.
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The main dietary sources of polyphenols are reviewed, and the daily intake is calculated for a given diet containing some common fruits, vegetables and beverages. Phenolic acids account for about one third of the total intake and flavonoids account for the remaining two thirds. The most abundant flavonoids in the diet are flavanols (catechins plus proanthocyanidins), anthocyanins and their oxidation products. The main polyphenol dietary sources are fruit and beverages (fruit juice, wine, tea, coffee, chocolate and beer) and, to a lesser extent vegetables, dry legumes and cereals. The total intake is ∼1 g/d. Large uncertainties remain due to the lack of comprehensive data on the content of some of the main polyphenol classes in food. Bioavailability studies in humans are discussed. The maximum concentration in plasma rarely exceeds 1 μM after the consumption of 10–100 mg of a single phenolic compound. However, the total plasma phenol concentration is probably higher due to the presence of metabolites formed in the body's tissues or by the colonic microflora. These metabolites are still largely unknown and not accounted for. Both chemical and biochemical factors that affect the absorption and metabolism of polyphenols are reviewed, with particular emphasis on flavonoid glycosides. A better understanding of these factors is essential to explain the large variations in bioavailability observed among polyphenols and among individuals.
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Flavonoids occur in most plant species, and account for a significant percentage of the chemical constituents of some; e.g. dried green tea leaves contain approximately 30% flavonoids by weight. Flavonoids have been shown to have antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, and vasodilatory activity. The potent antioxidant activity of flavonoids-their ability to scavenge hydroxyl radicals, Superoxide anions, and lipid peroxy radicals-may be the most important function of flavonoids, and underlies many of the above actions in the body. Qxidative damage is implicated in most disease processes, and epidemiological, clinical, and laboratory research on flavonoids and other antioxidants suggest their ; use in the prevention and treatment of a number of these. Catechin and its derivatives, oligomeric proanthocyanidins, quercetin and quercetin chalcone, Ginkgo flavone glycosldes, silymarin, and others can be utilized in preventative and treatment protocols for cardiovascular disease, cancer, inflammatory conditions, asthma, periodontal disI ease, liver disease, cataracts and macular degeneration.
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Flavonoids apigenin, acacetin, tilianin, kaempferol and kaempferol-3-O-L-rhamnoside have been isolated from the herb Saussurae stella Maxim and superoxide anion scavanging and lipid peroxidation inhibitory activities of these flavonoids are reported.
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Background: Flavonoids are polyphenolic compounds of plant origin with antioxidant effects. Flavonoids inhibit LDL oxidation and reduce thrombotic tendency in vitro. Little is known about how cocoa powder and dark chocolate, rich sources of polyphenols, affect these cardiovascular disease risk factors. Objective: We evaluated the effects of a diet high in cocoa powder and dark chocolate (CP-DC diet) on LDL oxidative susceptibility, serum total antioxidant capacity, and urinary prostaglandin concentrations. Design: We conducted a randomized, 2-period, crossover study in 23 healthy subjects fed 2 diets: an average American diet (AAD) controlled for fiber, caffeine, and theobromine and an AAD supplemented with 22 g cocoa powder and 16 g dark chocolate (CP-DC diet), providing ≈466 mg procyanidins/d. Results: LDL oxidation lag time was ≈8% greater (P = 0.01) after the CP-DC diet than after the AAD. Serum total antioxidant capacity measured by oxygen radical absorbance capacity was ≈4% greater (P = 0.04) after the CP-DC diet than after the AAD and was positively correlated with LDL oxidation lag time (r = 0.32, P = 0.03). HDL cholesterol was 4% greater after the CP-DC diet (P = 0.02) than after the AAD; however, LDL-HDL ratios were not significantly different. Twenty-four–hour urinary excretion of thromboxane B2 and 6-keto-prostaglandin F1α and the ratio of the 2 compounds were not significantly different between the 2 diets. Conclusion: Cocoa powder and dark chocolate may favorably affect cardiovascular disease risk status by modestly reducing LDL oxidation susceptibility, increasing serum total antioxidant capacity and HDL-cholesterol concentrations, and not adversely affecting prostaglandins.
Polyphenols are abundant micronutrients in our diet, and evidence for their role in the prevention of degenerative diseases is emerging. Bioavailability differs greatly from one polyphenol to another, so that the most abundant polyphenols in our diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues. Mean values for the maximal plasma concentration, the time to reach the maximal plasma concentration, the area under the plasma concentration-time curve, the elimination half-life, and the relative urinary excretion were calculated for 18 major polyphenols. We used data from 97 studies that investigated the kinetics and extent of polyphenol absorption among adults, after ingestion of a single dose of polyphenol provided as pure compound, plant extract, or whole food/beverage. The metabolites present in blood, resulting from digestive and hepatic activity, usually differ from the native compounds. The nature of the known metabolites is described when data are available. The plasma concentrations of total metabolites ranged from 0 to 4 mumol/L with an intake of 50 mg aglycone equivalents, and the relative urinary excretion ranged from 0.3% to 43% of the ingested dose, depending on the polyphenol. Gallic acid and isoflavones are the most well-absorbed polyphenols, followed by catechins, flavanones, and quercetin glucosides, but with different kinetics. The least well-absorbed polyphenols are the proanthocyanidins, the galloylated tea catechins, and the anthocyanins. Data are still too limited for assessment of hydroxycinnamic acids and other polyphenols. These data may be useful for the design and interpretation of intervention studies investigating the health effects of polyphenols.
The flavonoid quercetin is metabolized into isorhamnetin, tamarixetin, and kaempferol, the vascular effects of which are unknown. In the present study, the effects of quercetin and its metabolites were analyzed on isometric tension in isolated rat thoracic and abdominal aorta, in isolated intact and β-escin-permeabilized iliac arteries, and on perfusion pressure in the isolated mesenteric resistance vascular bed. In noradrenaline-precontracted vessels, the four flavonoids produced a vasodilator effect, which was inversely correlated with the diameter of the vessel studied; i.e., quercetin, isorhamnetin, tamarixetin, and kaempferol were 5-, 25-, 4-, and 6-fold, respectively, more potent in the resistance mesenteric bed (−log IC50 = 5.35 ± 0.15, 5.89 ± 0.11, 5.34 ± 0.10, and 5.66 ± 0.06, respectively) than in the thoracic aorta (−log IC50 = 4.68 ± 0.08, 4.61 ± 0.08, 4.73 ± 0.11, and 4.81 ± 0.13, respectively; n = 4–6). The vasodilator responses of quercetin and isorhamnetin were not significantly modified after removal of the endothelium in the thoracic aorta or in the mesenteric bed. Furthermore, the guanylate cyclase inhibitor ODQ (1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxalin-1-one; 10−6 M), the adenylate cyclase inhibitor SQ22536 [9-(tetrahydro-2-furanyl)-9 H -purin-6-amine; 10−6 M], KCl (40 mM), or ouabain (10−3 M) had no effect on isorhamnetin-induced vasodilation in the mesenteric bed. In permeabilized iliac arteries stimulated with Ca2+(pCa of 5.9), isorhamnetin was also significantly more potent (−log IC50 = 5.27 ± 0.15) than quercetin (−log IC50 = 4.56 ± 0.15). In conclusion, quercetin and its metabolites showed vasodilator effects with selectivity toward the resistance vessels. These effects are not due to or modulated by endothelial factors and are unrelated to changes in cytosolic Ca2+.
Flavonols are efficient antioxidants with the potential to protect biological macromolecules from oxidative damage in vivo, and if absorbed into the circulation they may protect against cardiovascular disease. Although flavonol aglycones are present in foods at low concentrations, their glycosides are abundant in onions, apples, beans and tea, and are thought to be stable under the conditions of the human stomach and small bowel. There is, however, recent evidence to suggest that intact glycosides of quercetin may be absorbed from the small intestine by a mechanism involving the glucose transport pathway. In the present study we tested this hypothesis by measuring the effect of quercetin glycosides on the rate of efflux of galactose from the jejunal mucosa. Everted sacs of rat jejunum preloaded with 14C-galactose were exposed to quercetin glycosides isolated from onions. Quercetin mono- and diglucosides were shown to accelerate the carrier-mediated efflux of galactose via a sodium-dependent pathway. HPLC analysis confirmed the stability of the glycosides under conditions simulating those in the upper alimentary tract. These studies suggest that purified quercetin glucosides are capable of interacting with the sodium dependent glucose transport receptors in the mucosal epithelium and may therefore be absorbed by the small intestine in vivo.