Cacao and Human Health: from Head to Foot — A Review

Abstract

The cacao, as part of the wonderful nature, provides the mankind a wide variety of valuable food products and health benefits. The most known and universally relished product derived from this fruit is chocolate, an amazing and unique food for the human nutrition with records of consumption of similar products dating to 1000 years B.C. In fact, the cocoa is a complex food that includes over 300 different components. This review is designed to inform scientists, technicians, academicians, farmers, and interested communities of numerous studies that have been conducted worldwide to investigate the properties of various cocoa constituents, their relations to human health, and their potential role in the prevention and treatment of many medical conditions. The general population, for example in Brazil, despite being one of the major producers of cocoa, is poorly informed of the significant and beneficial properties of cocoa. The present review covers important topics linking cocoa to human health and show the state of the art of effect of cocoa in different systems that comprise the human body. The paper is organized based on the main human organ system and includes: Cardiovascular / Circulatory, Neurological / Nervous, Oral health, Endocrine, Lymphatic and Immunological, Respiratory, Reproductive, Dermatological systems. Scientific findings tend to confirm the historic designation of cacao as “food of the Gods”.

Full text

DOI:10.1080/10408398.2012.657921
1
Cacao and Human Health: from Head to Foot – A Review
QUINTINO REIS DE ARAUJO
1,2
, JAMES NASCIMENTO GATTWARD
2
, SUZANA
ALMOOSAWI
3
, MARIA DAS GRAÇAS CONCEIÇÃO PARADA COSTA SILVA
1
,
PAULO ALFREDO DE SANTANA DANTAS
2
and QUINTINO REIS DE ARAUJO
JÚNIOR
4
¹ CEPLAC - Executive Commission for the Cocoa Farming Plan / Cocoa Research Centre, Road Ilhéus-Itabuna, km 22,
45600-970, Itabuna, Bahia, Brazil; ² State University of Santa Cruz, Road Ilhéus-Itabuna, km 16, 45662-900, Ilhéus,
Bahia, Brazil; ³ Queen Margaret University / Department of Dietetics, Nutrition and Biological Sciences, Queen
Margaret Drive, Musselburgh EH21 6UU, UK;
4
UniJorge, Luis Viana Anenue / Paralela, 6775, 41745-130, Salvador,
Bahia, Brazil.
Corresponding Author:
Quintino Reis de Araujo
quintino@cepec.gov.br
CEPLAC / CEPEC,
Road Ilhéus-Itabuna, km22.
CP.07.
CEP 45600-970, Itabuna, Bahia, Brazil.
Tel. +55 73 3214 3256
Fax. +55 73 3214 3204
(Short Title: Cacao and Health)

DOI:10.1080/10408398.2012.657921
2
Cacao and Human Health: from Head to Foot – A Review
The cacao, as part of the wonderful nature, provides the mankind a wide variety of valuable food
products and health benefits. The most known and universally relished product derived from this
fruit is chocolate, an amazing and unique food for the human nutrition with records of consumption
of similar products dating to 1000 years B.C. In fact, the cocoa is a complex food that includes over
300 different components. This review is designed to inform scientists, technicians, academicians,
farmers, and interested communities of numerous studies that have been conducted worldwide to
investigate the properties of various cocoa constituents, their relations to human health, and their
potential role in the prevention and treatment of many medical conditions. The general population,
for example in Brazil, despite being one of the major producers of cocoa, is poorly informed of the
significant and beneficial properties of cocoa. The present review covers important topics linking
cocoa to human health and show the state of the art of effect of cocoa in different systems that
comprise the human body. The paper is organized based on the main human organ system and
includes: Cardiovascular / Circulatory, Neurological / Nervous, Oral health, Endocrine, Lymphatic
and Immunological, Respiratory, Reproductive, Dermatological systems. Scientific findings tend to
confirm the historic designation of cacao as "food of the Gods".
Key Words: Theobroma cacao L., food, antioxidant, heart, neurology, immunology.

DOI:10.1080/10408398.2012.657921
3
Introduction
The nature has a huge, effective and wonderful collection of plant species that offer several
beneficial properties to human nutrition and health. These plant species represent important sources
of raw materials essential for maintaining human existence and improving its quality of life.
The cocoa tree, which forms the basis for one of the world’s most popular food products-
chocolate, has a rich history involving many cultures and carrying important economical and social
implications to millions of peoples worldwide. In recognition of its multiple health benefits, the
Maya gave cocoa its ancient name “kakawa” which translates into ‘‘Food of the Gods”. The Maya
of Central America are credited for being the first people to consume cocoa. The health-promoting
properties of cocoa have also been celebrated by the ancient Mesoamerican society with historical
records revealing more than 150 applications of cocoa for medicinal purposes. Christopher
Columbus was the first European to encounter cocoa in around 1502 A.D. But it was not until a
quarter of a century later that the great Spanish conqueror Hernán Cortéz introduced cocoa into
Europe. By the mid-1600s, cocoa was used in Europe as a medicine that promotes health, and as a
cure for all manner of aliments (Pucciarelli and Grivetti, 2008). Cocoa was particularly valued for
its ability to treat upper respiratory tract conditions such as colds and coughs, enhance mental well-
being and to protect against nutritional deficiencies (Pucciarelli and Grivetti, 2008; Selmi et al.,
2008; Tomaru et al., 2007).
In 2005, a meeting was set at Lucerne, Switzerland, where scientists and medical experts
gathered to discuss the latest evidence on the potential health properties of cocoa. Since then,
numerous studies have appeared in the literature indicating that the value of cocoa goes beyond its
nutritional properties to potentially disease prevention. Chocolate have for several years been
studied for its possible beneficial health effects (Cooper et al., 2008; Sarmadi et al., 2011).

DOI:10.1080/10408398.2012.657921
4
Cocoa is a complex plant product that contains over 300 different constituents (Fung, 2011),
even in roasted beans (Keeney, 1972). Its major components include: cocoa butter (oleic, stearic,
and palmitic fatty acids), minerals (magnesium, potassium, iron and zinc), methylxanithines
(theobromine and caffeine), polyphenols in addition to other compounds such as tyramine,
tryptophan, and serotonin. Polyphenols are a large group of compounds found in fruits and
vegetables. Recently attention has been drawn to these compounds because of their ability to
improve health and prevent numerous diseases owing to their antioxidant properties and their
potential anti-inflammatory and cardio-protective properties (Wollgast and Anklam, 2000). For
these compounds from chocolate were directed the cause of significantly improved Chalder Fatigue
Scale score, a measurement of symptoms for subjects with chronic fatigue syndrome (Sathyapalan,
2010). One class of polyphenols is the flavonoids which is present in high concentration in cocoa
(Stoclet et al., 2011; Tzounis et al., 2011; Jalil and Ismail, 2008; Mehrinfar and Frishman, 2008),
and because this, cacao seeds was recently called as “Super Fruit” (Crozier, 2011). The fiber of
cocoa can be considered as an excellent source of dietary fiber, and therefore could be used as an
ingredient in functional foods rich in fiber. Besides this, fiber cocoa would provide protection
against oxidative damage through its content in phenolic compounds (epicatechin) (Lecumberri et
al., 2007). The pharmacokinetics of cocoa flavanols are rapidly absorbed in the intestine and
metabolized (Spencer et al., 2001).
The present reviews deals with some of the literature concerning the health properties of
cocoa and its related products and constituents. It is aimed at researchers, academics, farmers, and
general public who would like to gain a broad knowledge of the most recent evidence. The structure
of the review spans round the main systems in the human organ and include:
1. Cardiovascular / Circulatory system
2. Neurological / Nervous system
3. Endocrine system

DOI:10.1080/10408398.2012.657921
5
4. Oral health
5. Lymphatic and Immunological system
6. Dermatological system
7. Respiratory system
8. Reproductive system
Cardiovascular / Circulatory
The cradioprotective effects of cocoa can be summarized to include effects on blood
pressure, endothelium function, lipid profile and platelet function (Beckett, 2008)
Blood pressure
The interest in the effects of cocoa intake on the cardiovascular system was initially
triggered by the observation that the indigenous Kuna Indians, a population living in an island off
the coast of Panama, have a very low incidence of ischemic heart disease, stroke and hypertension
(Bayard et al., 2007; Hollenberg et al., 1997). These Kuna Indians consumed over 5 cups of
flavanol-rich cocoa containing 900mg of polyphenols per day (Hollenberg et al., 2006). These
observations are supported by several European epidemiological studies. In the Zutphen Elderly
cohort, in particular, habitual intake of cocoa has been found to be inversely associated with
approximately 50 % reduction of 15-year cardiovascular and all-cause mortality (Buijsse et al.,
2006). Individuals from the highest tertile of cocoa also had lower blood pressure compared to those
from lowest tertile (Buijsse et al., 2006). Similar observations have been made in the Potsdam
European Prospective Investigation into Cancer and Nutrition cohort (Buijsse et al., 2010).
Consumption of polyphenol-rich chocolate has been associated with lower risk of cardiac mortality

DOI:10.1080/10408398.2012.657921
6
after first myocardial infarction in the Stockholm Heart Epidemiology Program (Janszky et al.,
2009). Nonetheless, in a more recent cohort, only moderate habitual intake (1-2 servings per week
or 1-3 servings per month) was found to be protective against heart failure while no effect was
observed for cohorts members who consumed more than 3 servings of chocolate per week
(Mostofsky et al., 2010). In general, the value of epidemiological findings is limited by the possible
correlation between individual food or nutrient intake and other dietary and non-dietary factors that
may confound associations. This may explain some of the differences in findings. Likewise, data
from epidemiological studies could often under-estimate the true effect of cocoa and chocolate
consumption since most cocoa products available on the market are poor in polyphenols. As such
evidence from cohorts studies need to be reinforced by randomised controlled trials.
To date, findings from randomised controlled trials suggest a potential role for flavanol-rich
chocolate and cocoa products in non-pharmacological treatment of high blood pressure (Hernández
et al., 2011; Sanchez et al., 2010; Corti et al., 2009; Selmi et al., 2008). A recent literature
evaluation by Ried et al. (2010) has concluded that dark chocolate is superior to placebo in reducing
systolic hypertension or diastolic prehypertension. Taubert et al. (2007) were amongst the first
researchers to report the blood-pressure lowering effects of chocolate in a randomised controlled
trial. In their trial, 14-day consumption of polyphenol-rich chocolate induced a 5.1 (SD 2.4) and 1.8
(2.0) mm Hg reduction in systolic and diastolic blood pressure compared to white chocolate,
respectively. This study was followed by a series of evaluations confirming on the consumption of
100g dark chocolate containing 500mg polyphenols was found to reduce blood pressure in healthy
adults (Grassi et al., 2005a) and patients with essential hypertension (Grassi et al., 2005b, 2008).
Recently efforts have been to develop commercially available polyphenol-rich cocoa products. One
such product, CocoanOx, has been shown to produce similar reduction in blood pressure in rats to
Captopril (Cienfuegos-Jovellanos et al., 2009).

DOI:10.1080/10408398.2012.657921
7
Much speculation exists as to the cocoa constituent responsible for the blood-pressure
lowering effects of cocoa. Earlier, studies used white chocolate as placebo which apart from not
permitting effective blinding of human volunteers also differs in its nutrient composition since it
does not contain minerals like magnesium and methyxanithines. In a recent study by van den
Bogaard et al. (2010), theobromine-enriched cocoa was found to significantly increase 24-hour
ambulatory systolic blood pressure while lowering central systolic blood pressure. These findings
suggest that caution need to be exercised when interpreting the findings from earlier cross-sectional
studies. Moreover, the use of high quantities of chocolate, sometimes 100g per day, might not be
feasible because of the high-fat content and energy density of chocolate. The effective polyphenol
dose required to induce clinically relevant reduction in blood pressure also remains unclear.
Recently, Almoosawi et al. (2010) has also shown that in overweight subjects intake of 20g of
polyphenol-rich chocolate containing 500mg polyphenols produces similar reductions in blood
pressure as 20g of polyphenol-rich chocolate containing 1000mg polyphenols chocolate. Regardless
of these findings, evidence from long-term studies suggest that use of doses as low as 6.5g per day
induces clinically relevant reduction in blood pressure by increasing bioactive nitric oxide levels
(Taubert et al., 2007).
In relation to the mechanism by which polyphenol-rich chocolate reduces blood pressure it
appears to be related to increased nitric oxide bioavailability, or a maintenance of optimal nitric
oxide levels, that could be associated with lowering superoxide anion production in the vasculature
(Fraga et al., 2011); which then translated into improvement in arterial stiffness and endothelial
function, as outlined below.
Endothelium function
Cocoa and chocolate produce their cardio-protective effects via modulation of nitric oxide
bioavailability, hence endothelium function. In-vitro and in-vivo studies have shown that flavanol-

DOI:10.1080/10408398.2012.657921
8
rich cocoa reduces the activity of arginase, the main enzyme involved in the inactivation of nitric
oxide (Schnorr et al., 2008). This results in a rise in the plasma concentration of nitrose compounds,
which can then be expressed as improved brachial artery flow-mediated dilation (Yokoi et al., 2011;
Sies et al., 2005). Endothelial dysfunction is an early marker of atherosclerosis development. Thus,
the ability of polyphenol-rich cocoa and its products to reverse endothelial dysfunction suggests that
such products could be used at early stages of disease to prevent or delay progression to
cardiovascular events such as stroke. Evidence of the beneficial effects of cocoa and its related
products on endothelium function are consistent and range from effects on healthy volunteers
(Fisher et al., 2006a; Schroeter et al., 2006) to individuals with high cardiovascular risk factors such
as hypertensives (Grassi et al., 2005b), insulin-resistant (Grassi et al., 2008) and obese subjects
(Davison et al., 2008). Currently there is even data to suggest that polyphenol-rich chocolate
consumption improves endothelium-dependent responses in the coronary circulation, in addition to
decreasing shear stress-induced platelet adhesion in heart transplant recipients (Flammer et al.,
2007). Interestingly, flavanol-rich cocoa appears to improve blood pressure and endothelial
function to a greater extent in the elderly compared to younger healthier subjects (Fisher et al.,
2006a). This is unsurprising elderly (Fisher et al., 2006b). Currently debate still exists as to
whether polyphenol-rich products can be of any benefit to healthy young adults. What remains
evident is that the timing of dietary intervention is critical to ensuring maintenance of health and
disease prevention, since the use of diet, on its own, may be less effective at more advanced stages
of disease.
Lipid profile and peroxidation
In a meta-analysis of eight short-term trials, consumption of cocoa was found to elicit a 5.87
mg/dL (95% CI: −11.13, −0.61; P < 0.05) reduction in LDL cholesterol (Jia et al., 2011). This
effect was only observed in individuals who consumed low doses of cocoa and in those with high

DOI:10.1080/10408398.2012.657921
9
cardiovascular disease risks. By contrast, only a marginal reduction in total cholesterol (mean
reduction = -5.82 mg/dL; 95% CI: −12.39, 0.76; P = 0.08) was detected, and no significant change
in HDL cholesterol. Similar observation, have been made in a recent systematic review which
included 10 short-term trials (Tokede et al., 2011). However, in the latter meta-analysis, a
significant reduction in total cholesterol was seen (mean reduction = -6.23 mg/dl (−11.60, −0.85
mg/dl). Moreover, short-term trials showed greater improvements in lipid profile compared to long-
term clinical trials, possibly implying a potential physiological adaptation to high polyphenol intake
with long-term intake. The minor differences between the two meta-analyses could be related to
differences in the studies included in analysis. In the meta-analysis conducted by Tokede et al.
(2011), both short-term (2 week) and long-term trials (up to 12 weeks) as well as studies on
overweight individuals (Almoosawi et al., 2010, Davison et al., 2008) and elderly were included
(Crews et al., 2008). Alternatively, inter-individual variations in the absorption of cocoa
polyphenols could dictate the extent of the lipid-lowering effects of cocoa products, which may then
produce differences in results. In a randomised-controlled trial involving forty-two volunteers with
high cardiovascular risk, consumption of 40g of polyphenol-rich cocoa dissolved in 500ml of
skimmed milk reduced oxidised LDL levels and increased HDL cholesterol (Khan et al., 2011).
However, the increment in HDL cholesterol and the reduction in oxidised LDL were found to
strongly correlate with urinary cocoa polyphenol metabolites excretion. More recently, consumption
of polyphenol-rich cocoa was shown to even protect Type 2 diabetes patients against an atherogenic
lipid profile when cocoa is consumed as part of a balanced diet. Thus, consumption of 45g of
polyphenol-rich chocolate for 16 weeks was found to significantly reduce HDL cholesterol
(1.16 ± 0.08 vs 1.26 ± 0.08 mmol/l, P = 0.05) and cholesterol: HDL ratio (4.4 ± 0.4 vs
4.1 ± 0.4 mmol/l, P = 0.04) in patients with type 2 diabetes, without producing any changes in
weight or glycaemic control (Mellor et al., 2010).

DOI:10.1080/10408398.2012.657921
10
It is important to note that it is the polyphenol composition of cocoa and not the amount of
cocoa solids that determine the beneficial properties of cocoa and chocolate (Efraim et al, 2011;
Coper et al., 2008; Gu et al., 2006; Miller et al., 2006). In young (18-20 years old) male soccer
players, consumption of flavonol-rich milk chocolate, with a low cocoa solids content, has been
shown to be associated with improvements in several parameters associated with cardiovascular
health and oxidant stress in healthy young soccer players (Fraga et al., 2005). Thus consumption of
milk chocolate was found to decrease diastolic blood pressure (- 5 mm Hg), mean blood pressure (-
5 mm Hg), plasma cholesterol (-11%), LDL-cholesterol (-15%), malondialdehyde (- 12%), urate (-
11%) and lactate dehydrogenase (LDH) activity (- 11%), and to increase in vitamin E/cholesterol (+
12%) (Fraga et al., 2005). The fatty acid compsotion of cocoa butter may also explain the lipid-
lowering or neutral effects of chocolate. The main saturated fatty acid found in cacao butter is
stearic acid. Stearic acid has a neutral effect on blood lipids (Hannum and Erdman, 2000). Cocoa
also contains high amounts of monounsaturated fatty acids which are known to have a favorable
effect on lipid profile.
In addition to improving lipid profile, cocoa polyphenols can influence LDL oxidation.
Oxidative modification of LDL is a critical stage of atherogenesis, and one of the mediators is the
pro-inflammatory pro-atherogenic enzyme myeloperoxidase. Micromolar concentrations of (-)-
epicatechin or other flavonoids can suppress myeloperoxidase-induced lipid peroxidation of LDL
(Sies et al., 2005). Consumption of cocoa fiber has also been shown to reduce lipid peroxidation in
animal models (Lecumberri et al., 2007). Earlier, it was observed that soluble cocoa fiber product
in an animal model of dietary-induced hypercholesterolemia, diminished the negative impact of the
cholesterol-rich diet, buffering the decrease of high density lipoprotein-cholesterol, and the increase
of total and low density lipoprotein-cholesterol levels, and lipid peroxidation (malondialdehyde
levels) induced by the fatty diet. The soluble fiber product also decreased triglyceride levels to
values lower than those in the group fed the cholesterol-free diet (Ramos et al., 2008)

DOI:10.1080/10408398.2012.657921
11
Anti-platelet
Platelet activation and aggregation play a key role in inflammatory processes and form part
of the initial stages of arterial thrombosis development. Factors such as ADP, thrombin or collagen
activate platelets and stimulate their aggregation while prostaglandins inhibit these processes.
Cocoa, like aspirin, possesses potential anti-thrombotic properties. The ability of
polyphenol-rich cocoa and chocolate to modulate platelet function has been observed both ex-vivo
(Rein et al, 2000a-c) and in-vivo (Schramm et al., 2003; Holt et al., 2002; Pearson et al., 2005).
Some inconsistencies remain with regard to the magnitude of cocoa’s anti-platelet effects.
According to Pearson et al. (2005) cocoa has a less profound inhibitory effect on epinephrine-
stimulated platelet activation and function than aspirin. However, the combination of the two
produces an additive effect (Pearson et al., 2005). Heptinstall et al. (2006), on the other hand,
reported similar inhibitory effects of cocoa flavanols/ metabolites and aspirin on platelet
activation/aggregation, platelet-monocyte conjugate formation platelet-neutrophil conjugate
formation in-vitro, with no additive effect when combining cocoa flavanols and aspirin.
Differences in the results could be attributed to differences in the techniques used to assess platelet
function or variations in the products used. Regardless of these inconsistencies, several studies
have revealed potential mechanisms by which cocoa ad its related products can protect against
thromboemolic diseases. Cocoa procyanidins can inhibit platelet activation by altering eicosanoid
synthesis. Treatment of aortic endothelial cells with chocolate procyanidins doubles keto-
prostaglandin F
1α
production and reduces leukotriene production by 16% (Schramm et al., 2003).
When high-procyanidin chocolate (37g containing 4.0 mg/g procyanidins) is acutely consumed, this
produces a 37% increase and a 29% decrease in plasma prostacyclin and leukotrienes
concentrations, respectively, in healthy volunteers (Schramm et al., 2003). Cocoa can also diminish
platelet function by significantly reducing P selectin expression and by lowering ADP- and

DOI:10.1080/10408398.2012.657921
12
collagen-induced aggregation (Murphy et al., 2003, Innes et al., 2003). This effect correlates with a
rise in plasma ascorbic acid, epicatechin and catechin concentrations (Murphy et al., 2003). Pre-
treatment of human aortic endothelial cells with catechin and quercetin metabolites has also been
shown to influence monocyte adhesion (Koga and Meydani, 2001). According to Koga and
Meydani (2001), the flavonoid metabolites, as opposed to the intact flavonoids, are responsible for
the ability of flavonoids to modulate platelet function. In a double-blind, randomized study
involving 22 heart transplant recipients, consumption of 40 g of dark (70% cocoa) chocolate
reduced platelet adhesion decreased from 4.9+/-1.1% to 3.8+/-0.8% (P=0.04) (Flammer et al.,
2007).
In a large cross-sectional analysis assessing habitual chocolate consumption in 1535
subjects, chocolate consumers showed longer platelet function analyzer closure times (130 vs 123
seconds, P=.005) and decreased 11-dehydro thromboxane B2 (175 vs 290 ng/mol creatinine, P=.03)
(Bordeaux et al., 2007). Chocolate remained an independent predictor of both ex vivo and in vivo
platelet function testing after controlling for age, sex, education level, race, cigarette smoking, and
BMI and values of glucose, blood pressure, total cholesterol, fibrinogen, and Von Willbrand Factor.
Bordeaux et al. (2007) concluded that consumption of small quantities of chocolate (5.9g) can
improve platelet function.
Neurological/ Nervous System
Interest in the neurological and neuroprotective effects of cocoa and chocolate polyphenols
stemmed from findings that cocoa and chocolate can enhance vascular function by modulating
nitric oxide bioavailability. Nitric oxide is central to the regulation of peripheral vascular.
However, it also plays a key role in the cerebral circulation where it promotes brain perfusion.

DOI:10.1080/10408398.2012.657921
13
Studies suggest that daily intake of cocoa flavanols can enhance blood flow and perfusion of the
brain via its stimulatory effect on nitric oxide bioavailability (Pate et al., 2008). Indeed, in a study
conducted on thirty-four healthy elderly volunteers aged 72±6 years, Sorond (2008) used
transcranial Doppler ultrasound to measure blood flow velocity in the middle cerebral artery.
Sorond (2008) observed a consistent increase in blood flow velocity from 8% after 1 week of cocoa
consumption to 10% following 2-week consumption of flavanol-rich cocoa. These findings were
validated in a later study in which changes in middle cerebral artery flow, assessed by transcranial
Doppler ultrasound, strongly correlated with changes in perfusion measured by gadolinium-
enhanced MRI and arterial spin labelling (Sorond et al., 2010). Increased blood oxygenation and
blood flow to brain grey matter have also been reported (Field et al., 2011; Francis et al., 2006).
These findings suggest a potential role for cocoa flavanols in the treatment and prevention of
cerebrovascular diseases such as dementia and stroke.
Indeed, several studies indicate that polyphenols can reduce the risk of neurodegenerative
diseases particularly those stemming from oxidative stress, such as Alzheimer's and Parkinson's
disease. Incubation of neuronal cells with cocoa extract or (-)-epicatechin dose-dependently
reduces reactive oxygen species production and down regulates the stress-kinases: pJNK and p38,
which are involved in activation mitogen-activated protein kinase pathway (Ramiro-Puig and
Castell, 2009). In models of Parkinson’s disease, pretreatment of male adult Sprague-Dawley rats
with cocoa extract at a dose of 100 mg/kg/day has also been reported to diminish oxidative stress-
induced 6-hydroxydopamine-induced dopaminergic loss (Datla et al., 2007). Pre-treatment of
neurons with cocoa extract has also been reported to reduce expression and release of calcitonin
gene-related peptide expression, a factor that promotes neural inflammation (Abbey et al., 2008)
and migraine development (Li et al., 2008). Cocoa also exerts protective effects against amyloid β
protein-induced neurotoxicity (Yasuda et al., 2011; Heo and Lee, 2005), which is relevant to
Alzheimer’s disease, a neurological dementia caused by accumulation of amyloid plaques and

DOI:10.1080/10408398.2012.657921
14
neurofibrillary tangles in the brain and which is characterised by memory loss and progressive
decline in cognitive function. Indeed, cocoa can protect against cognitive decline associated with
normal aging. In old Wister-Unilever rats, 1-year oral administration of a cocoa polyphenolic
extract, at a dose of 24 mg/kg per day, delays the onset of age-related cognitive deficits and
maintains high urinary free dopamine levels (Bisson et al., 2008b). In a large trial involving 2031
human subjects, habitual chocolate consumption predicted improved cognitive performance (Nurk
et al., 2009). In fact, the association between chocolate consumption and cognitive performance was
dose-dependent, with maximum beneficial effect achieved at intakes of ∼10 g of chocolate per day
(Nurk et al., 2009).
It is important to state that chocolate is also rich in fat and contains amines such as tyramine,
histamine and phenylethylamine that can be linked to headaches. In a double blind study of
headache was performed using chocolate as the active agent and carob as the placebo. Sixty-three
women with chronic headache (50% migraine, 37.5% tension-type, 12.5% combined migraine and
tension-type) participated in the study. Diaries were maintained by the subjects throughout the
study, monitoring diet and headache. But contrary to the commonly held belief of patients and
physicians, chocolate didn’t play a significant role in trigger headaches in typical migraine, tension-
type, or combined headache sufferers (Marcus, 1997). However, in patients with migraine who
believed that chocolate could provoke their attacks, chocolate ingestion was followed by a typical
migraine episode in 5 out of 12 patients, while none of the 8 patients challenged with placebo had
an attack (p = 0.051) (Gibb, 1991). And in other study with four hundred twenty-nine patients that
had migraine, 16.5% reported that headaches could be precipitated by cheese or chocolate, and
nearly always by both. There was a statistical association between sensitivity to cheese/chocolate
and to red wine in patients with migraine, and related more to migraine than to more chronic
tension-type headache (Peatweld, 1995). Therefore, evidence of the association between chocolate
consumption and headaches remains conflicting.

DOI:10.1080/10408398.2012.657921
15
In addition to age-associated neurodegenerative diseases, cocoa and chocolate consumption
can play a role in the prevention of neurological disorders such as depression. Depression is a
common condition that affects a large proportion of the population nowadays. A recent study found
that the addition of high-flavanol cocoa extract given to rats in a forced swimming test reduces
depression (Messaoudi et al., 2008). This effect has been attributed to the conversion of tryptophan
from cocoa into serotonin and the presence of some compounds in cocoa that alleviate mood,
thereby protecting against depression. Raikkonen et al., (2004) evaluated prenatal frequency of
chocolate consumption and its relation to intensity of psychological stress in 305 mothers. The
temperament of infants at 6 months postpartum was also evaluated. Chocolate was found to
produce subjective feelings of psychological well being, to reduce maternal stress and improve
infant temperant. Similar observations have been made in elderly men where consumption of
chocolate was associated with improved overall health and better psychological well-being
(Strandberg et al., 2008). Together this evidence favors a potential role for cocoa and chocolate
polyphenols in maintaining cognitive function through the life course and preventing age-related
development of cerebrovascular disease.
Endocrine
Growing evidence suggests that polyphenols in cocoa can modulate the endocrine system.
In studies on diabetic-obese mice, administration of cocoa liquor procyanidins for 3 weeks have
been shown to dose-dependently reduce fasting glucose and fructosamine levels (Tomaru et al.,
2007). Likewise, acute reductions in postprandial glucose have been reported by Jalil et al. (2008),
with this effect coinciding with an improvement in antioxidant defense mechanism as demonstrated
by enhanced superoxide dismutase activity and reduction in 8-isoprostanes. Cocoa supplementation

DOI:10.1080/10408398.2012.657921
16
can also reduce free fatty acids, which is important since fatty acids can impair glucose metabolism.
Despite this evidence, no changes in fasting glucose have yet been reported with long-term
administration of cocoa extract (4 weeks) in neither mice nor humans. The reason behind these
differences in effect remains to be clarified, but could be related to the short half-lives of cocoa
polyphenols which leads to rapid metabolism and excretion. As a result, the benefit of cocoa
consumption may lie in its transient effects on postprandial glucose levels.
Despite this evidence, recent trials in humans show that consumption of polyphenol-rich
dark chocolate can improve insulin sensitivity in healthy subjects (Grassi et al., 2005a),
hypertensives (Grassi et al., 2005b), insulin-resistant individuals (Grassi et al., 2008) and
overweight subjects (Almoosawi et al., 2010). These effects could be attributed to the ability of
polyphenol-rich dark chocolate to improve endothelium function and antioxidant status. Only two
studies have so far investigated the effect of cocoa consumption on glucose and insulin regulation in
diabetics. None of these studies observed any significant improvement in glucose levels or insulin
resistance. Several explanations could be provided to explain such inconsistencies. First, most
previous trials conducted on non-diabetic patients compared the effect of polyphenol-rich dark
chocolate to polyphenol-deficient white chocolate. This implies that volunteers were not blinded to
treatment allocation which may have biased the results. Additionally, white chocolate differs in its
macro- and micro-nutrient composition to dark chocolate since it does not contain methylxanithines
or minerals such as magnesium. This implies that differences in the effect of dark chocolate and
white chocolate may be related to components other than polyphenols. Most of the above trials
were also of short duration (2 weeks) and used large amounts of chocolate (100g). Thus, the benefit
of cocoa or chocolate consumption may lie in its ability to reduce diabetes-related metabolic
complication as opposed to improving glycaemic control. This notion could be supported by
several studies. For instance, administration of proanthocyanidins derived from cacao has been
shown to inhibit diabetes-induced cataract formation, possibly by improving antioxidant activity

DOI:10.1080/10408398.2012.657921
17
(Osakabe et al., 2004). Similarly, supplementing the diet with 1-2% cocoa extract of diabetic rats
has been shown to increase HDL-cholesterol and the reduction in LDL-cholesterol (Ruzuadi et al.,
2005). In humans, Balzer et al. (2008) demonstrated that thrice-daily consumption of polyphenol-
rich cocoa for 30 days reversed vascular dysfunction in medicated diabetic patients. Similarly, in a
study on type 2 diabetics, daily intake of cocoa for 16 weeks raised HDL-cholesterol and reduced
total cholesterol-to-HDL ratio (Mellor et al., 2010). All of these findings suggest that cocoa
consumption may benefit diabetics by reducing cardiovascular risk factors.
Cocoa consumption can also be of benefit to obese individuals because of its low calorie
density in comparison to chocolate. Consistent with this, cocoa polyphenols have been shown to
prevent diet-induced obesity by modulating lipid metabolism, especially by decreasing fatty acid
synthesis and transport systems, and enhancing thermogenesis in hepatic and white adipose cells
(Matsui et al., 2005). In humans, long-term ingestion of a polyphenol-rich cocoa drink has been
reported to improve endothelial function (Davison et al., 2008). Certain constituents in cocoa also
have the potential to modulate glucocorticoid metabolism which could be of relevance to obesity-
related complications (Almoosawi, 2011).
Oral health
Addai et al. (2002) screened a large selection of foods for their acidogenic effects on teeth.
To their surprise, they found that the Golden Tree brand of milk chocolate produced in Ghana,
which contained 30% cocoa solids, was not acidogenic. This finding prompted researchers to
conduct further trials in an attempt to understand the mechanisms underlying their observations.
Evidence of the protective effect of cocoa against dental caries has been, in fact,
documented as early as 1985, when Paolino and Kashket observed that cocoa inhibited plaque

DOI:10.1080/10408398.2012.657921
18
accumulation and caries formation by reducing polysaccharide production. Since then, several
studies have shown the ability of cocoa to reduce the risk of dental caries and prevent periodontal
disease. Rats infected with Streptococcus sobrinus showed a significant reduction in caries scores
after administration of a water-soluble cacao extract compared to infected rats given a control diet
(Ito et al., 2003). This protective effect was attributed to the inhibitory activity of cocoa extract on
glucan synthesis by Streptococcus. Polymeric fractions from cocoa, in particular, appear to have
immunomudulatory effects on the production of cytokines IL-1β (IL-1β), IL-2 and IL-4 (Hirao et
al., 2010; Mao et al., 2002). Larger oligomeric fractions (hexamer through decamer) have also been
reported to inhibit IL-5 release, which may promote immunoglobulin A (Mao et al., 2002). More
recently, Percival et al. (2006) demonstrated that cocoa polyphenols can delay acid production by
Streptococcus mutans. The growth of streptococcus sanguins and formation of biofilm have also
been shown to be inhibited by cocoa dimer, tetramer and pentamer polymers (Percival et al., 2006).
Cocoa also contains tannins which possess anti-bacterial and anti-enzymatic properties (Beckett,
2008). Recently, Ferrazzano et al. (2009) reviewed the mechanisms of the anti-cariogenic effects of
cocoa polyphenols and concluded that products like cocoa may have potential applications in the
prevention of dental caries. Despite this evidence, it is important to highlight that findings from the
above experiments need to be interpreted with caution since the majority of cocoa and chocolate
products currently available on the market are low in polyphenols and high in simple sugars which
can be detrimental to oral health. Thus, the use of cocoa polyphenols as means of improving
periodontal health will only be possible once appropriate formulations and products are developed.
Lymphatic and Immunological

DOI:10.1080/10408398.2012.657921
19
Chronic and acute inflammation underlies the molecular basis of many cardiovascular and
cerebrovascular diseases. The role of cocoa in modulating inflammatory markers and immune
responses has been reviewed by several researchers (Selmi et al., 2006; Selmi et al., 2008; Ramiro-
Puig and Castell, 2009). This role has been established in both in-vitro and in-vivo studies and
could be summarised to include effects on the innate and the acquired immune system.
Accordingly, isolated flavanols and procyanidins fractions have been shown to attenuate the
release of inflammatory cytokines: interleukin-1b and interleukin-2 from peripheral blood
mononuclear cells (Mao et al., 2002), and to promote the production of anti-inflammatory cytokines
such as interleukin-4 and interleukin-5 in an oligomer length-dependent manner (Mao et al., 2002).
It was also observed that the effect of cocoa flavanols and procyanidins on peripheral blood
mononuclear cells depends on the immunological status of an individual. Cocoa flavonols and
procyanidins stimulated peripheral blood mononuclear cells individuals with low cytokine
transforming growth factor
-1 but attenuated peripheral mononuclear cell isolated from individuals
with high baseline transforming growth factor
-1.
In addition to influencing peripheral mononuclear cell, cocoa flavonoids have been shown to
affect the release of inflammatory cytokines and chemokines from macrophages (Ramiro-Puig et
al., 2005). This inhibitory effect is achieved at the transcriptional level as evident by the down-
regulation of TNFα, interleukin (IL) 1α, and IL-6 mRNA expression.
Besides the innate immune response, cocoa can modulate the acquired immune response. In
a study conducted by Ramiro-Puig et al. (2005), incubation of cocoa extract with stimulated murine
EL4.BU.OU6 cells dose-dependently inhibited IL-2Rα (CD25) expression, an early marker of
lymphocyte T activation. This effect was likely to be modulated through epicatechin as both cocoa
extract and epicatechin produced similar levels of inhibition. Cocoa flavonoids also induced a 3-
fold rise in IL-4 release. These findings have been recently replicated in-vivo. Perez-Berezo et al.
(2009) demonstrated that supplementing the diet of adult Wistar rats with a cocoa-enriched diet for

DOI:10.1080/10408398.2012.657921
20
9 weeks attenuates the Th2 immune response leading to reduced antibody synthesis. In another
study, weaned rats were given cocoa (4% or 10% food intake), containing 32 mg flavonoids/g, for
3 weeks (Ramiro-Puig et al., 2007b). The diet with the highest cocoa content was found to diminish
TNF-α secretion by peritoneal macrophages. The high-cocoa diet also promoted lymphocyte
proliferation rate but attenuated T helper 2-related cytokines and Ig secretion and decreased the
number of Th cells. Interestingly, one study has shown that the highest uptake and accumulation of
cocoa metabolites occurs in lymphoid tissues, particularly the thymus, which reinforces a role for
cocoa polyphenols in modulating lymphocyte composition in this tissue (Urpi-Sarda et al., 2010).
The ability of cocoa polyphenols to affect both the innate immune system and the adaptive
immunity has also been recently reported by Kenny et al. (2007). Kenny et al. (2007) demonstrated
that the chain length of flavanols determines the extent of cytokine release from both unstimulated
and LPS-stimulated peripheral blood mononuclear cells. Long-chain flavanols, in particular, were
found to increase LPS-induced synthesis of IL-1β, IL-6, IL-10,
and TNF- . By contrast, both long-
chain and short-chain flavanols increased expression of the B cell markers CD69 and
CD83.
Cocoa polyphenols have been speculated to act as general leukotriene inhibitors. Schewe et
al. (2001) demonstrated that (-)-epicatechin and its low-molecular procyanidins, i.e. dimers and to a
lesser extent trimers through pentamers, inhibit both dioxygenase and LTA
4
synthase activities of
human 5-lipoxygenase. In earlier studies, Schewe et al. (2001) reported inhibition of 15-
lipoxygenase-1, an important catalyst of lipid peroxidation, by (–)-epicatechin and cocoa
procyanidins (Schewe et al., 2001). Here, higher oligomers exhibited greater inhibitory activity
than monomers and medium-sized oligomers. Cocoa flavonoids were also found to inhibit
mammalian 12-lipoxygenases (Schewe et al., 2001).
It is important to observe that the value of some of the above findings is limited by the in-
vitro design of these studies and the use of pharmacological doses of cocoa polyphenols.
Nevertheless, because macrophages play an important role in the innate immune response and

DOI:10.1080/10408398.2012.657921
21
inflammation and are implicated in the development of atherosclerotic lesions and cancer cell
proliferation, a potential role for cocoa polyphenols in preventing atherosclerosis and cancer
development could be speculated. Likewise, as suggested by Ramiro-Puig et al. (2007a), cocoa’s
capacity to modulate macrophage cytokine secretion and lymphocyte function could be relevant to
hypersensitivity and autoimmunity. Consequently, further research is required to replicate these
findings in humans and to evaluate the clinical implications of these findings. Finally, it could be
stated that some authors have also speculated a role for cocoa in the prevention of malaria by virtue
of cocoa’s ability and the ability of cocoa constituents (polyphenols, magnesium and zinc) to
improve nitric oxide bioavailability, increase antioxidant status and to boost the immune system
(Addai, 2010).
Immunostimulation have been also observed as a health-promoting effect of the microflora.
Among effective prebiotics, cacao-derived flavonols was identified affecting the growth of select
gut microflora in humans to a healthy composition (Tzounis, 2011).
Dermatological
In the past, chocolate has often been viewed as a food that promotes acne production.
However, this common belief has not been supported by scientific evidence. Among the first to
question the link between chocolate and acne were researchers from the University of Missouri in
the 1960s (Grant and Anderson, 1965). Grant and Anderson failed in their attempt to induce an acne
flare-up in eight individuals with mild to moderate acne by feeding them a large amount of
chocolate. The authors discredited the assertion that chocolate causes acne. In a study examining
the effect of chocolate intake on acne, 65 participants consumed 112g of dairy-free cocoa-enriched
bars of chocolate each day for 4 months. Researchers found no significant change in acne

DOI:10.1080/10408398.2012.657921
22
production (Ferdowsian, 2010; Fulton et al., 1969). These findings were consistent with other
studies (Anderson, 1971; Grant and Anderson, 1965). In an extensive review of research on
chocolate and acne (Fries, 1978), it was concluded that the general trend of published reports
suggested that chocolate ingestion was unrelated to the cause of acne. In last years it was observed
the effect of other foods in acne occurrence. A positive association with acne for intake of total milk
and skim milk was identified in a study involving 47,355 women (Adebamowo, 2005), later
reinforced in an evaluation with 6,094 girls, aged 9-15 (Adebamowo, 2006) and with 4273 boys,
where a positive association between intake of skim milk and acne was found (Adebamowo, 2008).
This finding suggested that skim milk contains hormonal constituents, or factors that influence
endogenous hormones, in sufficient quantities to have biological effects in consumers (Danby,
2005; Melnik, 2009). Therefore, other compounds from chocolate, but not cocoa, can promote acne
occurrence giving the mistaken conclusion of chocolate as an acne promoter.
More attention has been drawn to the dermatological properties of cocoa polyphenols
recently. Dietary flavanols from cocoa have been shown to contribute to endogenous
photoprotection, to improve dermal blood circulation, and to affect cosmetically relevant skin
surface and skin hydration (Heinrich et al., 2006). Accordingly, consumption of high-flavanol cocoa
for 12 weeks has been shown to decrease skin roughness and scaling compared to low-flavanol
cocoa (Heinrich et al., 2006). This effect is potentially achieved through an increase in blood flow
in cutaneous and subcutaneous tissues which leads to higher skin density and hydration (Heinrich et
al., 2006). In another study the acute effects of a single dose of flavanol-rich cocoa on dermal
microcirculation was investigated (Neukam et al., 2007). Flavanol-rich cocoa consumption acutely
increased dermal blood flow and oxygen saturation. Pre-treatment with cocoa polyphenolic extract
have also been demonstrated to confer a significant protection against oxidation of cultured human
HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH) (Martin et
al., 2008). There is also evidence that flavonoid-rich products contribute to the protection of skin

DOI:10.1080/10408398.2012.657921
23
against UV-induced damage at the molecular and cellular level, thereby improving overall skin
conditions (Stahl, 2011). This finding has also been observed by Jorge et al. (2011) who showed
that long term cocoa ingestion leads to an increased resistance against UV-induced erythema and a
lowered transepidermal water loss (Jorge et al., 2011). A cosmetic formulation elaborated with 10%
ozonized theobroma oil was also observed as protector against oxidant radiation in exerting
beneficial effects in the restoring of the antioxidant activity on the skin of rats previously irradiated
with ultraviolet light (Sanches et al., 2011).
Respiratory
This area of research has not been widely investigated. One study has reported that dietary
supplementation of cacao liquor proanthocyanidins prevents lung injury induced by diesel exhaust
particles in mice (Yasuda et al., 2008). This effect was achieved through down-regulation of
vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and correlated with a
decline in oxidative stress and lipid peroxidation (Yasuda et al., 2008). The authors concluded that
these findings have potential relevance to asthma, tuberculosis and others respiratory conditions.
Other evaluation, showed that flavanol-rich cocoa has been acted to reverse endothelial dysfunction,
measured as flow-mediated dilation (FMD) of the brachial artery, in smokers (Heiss et al., 2005).
Reproductive
Most of the beneficial effects of cocoa on the reproductive system have been observed in
animal models. The highest accumulation of epicaetechin metabolites occurs in the testes which

DOI:10.1080/10408398.2012.657921
24
has important implication for cancer prevention (Urpi-Sarda et al., 2010). Administration of a diet
containing 0.5 to 2% cocoa-rich flavanols to male rats for 2 weeks have been shown to dose-
dependently reduce oxidative DNA damage in rat testes (Urpi-Sarda et al., 2010). An in-vitro study
evaluated the inhibitory effect of different cocoa polyphenols extracts, alone or combined with beta-
sitosterol (a common phytosterol which plays a protective role in cancer development) on human
prostate cancer. The results showed that cocoa polyphenols extracts have antiproliferative effects on
prostate cancer cell growth (Jourdain et al., 2006). The effect of a commercially-available
polyphenol-rich cocoa on prostate carcinogenesis was evaluated in sixty Wistar-Unilever rats. Rats
were treated orally with cocoa powder 24mg/kg or 48mg/kg or vehicle, daily for nine months.
Cocoa markedly reduced prostate cancer incidence and increased the life span of the rats (Melnyk et
al., 2011; Bisson et al., 2008a; Bisson et al., 2006). The authors concluded that polyphenol-rich
cocoa can prevent prostate hyperplasia induced by testosterone propionate and therefore may be
beneficial in the treatment of benign prostatic hyperplasia.
Final Considerations
Chocolate induces, in general, a feeling of pleasure as postulate by scientists (Fung, 2011).
Because its composition cocoa mass remains solid at room temperature, but when consumed, its fat
content absorbs heat from the mouth and melts at body temperature, producing the effect of 'melt in
your mouth'.
Eating chocolate, as part of a healthful balanced diet, could potentially provide a beneficial
enjoyable way to improving wellbeing. Chocolate can be a very nutritional component in food and
the knowledge of its various medicinal properties represents a stimulus to those involved with its
production, processing and consumption.

DOI:10.1080/10408398.2012.657921
25
Recently science has advanced significantly in improving our understanding of the various
features of chocolate that contribute to its popularity, and its after-effects of consumption on human
health have also been extensively studied. Although its marketing as a health product is not a
priority, eating in moderation, mainly the darker forms could potentially have many beneficial
results. But one thing is certain, both from a scientific viewpoint as sensory, chocolate is enjoyed as
a favorite food for most of the people.
Literature Cited
Abbey, M. J., Patil, V. V., Vause, C. V., and Durham, P. L. (2008). Repression of calcitonin gene-
related peptide expression in trigeminal neurons by a Theobroma cacao extract. Journal of
Ethnopharmacology. 115: 238–248.
Addai, F. K, Nuamah, I. K., and Addo, J. (2002). Responses of saliva pH to ingestion of Golden
Tree chocolate and the effect of stick chewing. J Gh Sci Assoc. 4(1): 8–13.
Addai, F. K. (2010). Natural cocoa as diet-mediated antimalarial prophylaxis. Medical Hypotheses.
74: 825–830.
Adebamowo, C. A., Spiegelman, D., Berkey, C. S., et al. (2006) “Milk consumption and acne in
adolescent girls” Dermatol Online J. 12(4): 1.
Adebamowo, C. A., Spiegelman, D., Berkey, C. S., Danby, F. W., Rockett, H. H., Colditz, G. A.,
Willett, W. C. and Holmes, M. D. (2008) Milk consumption and acne in teenaged boys. J Am
Acad Dermatol. 58:787-93
Adebamowo, C. A., Spiegelman, D., Danby, F. W., Frazier, A. L., Willett, W. C., Holmes, M. D.
(2005) High school dietary dairy intake and teenage acne. J Am Acad Dermatol. 52(2):207-14.

DOI:10.1080/10408398.2012.657921
26
Almoosawi, S., Fyfe, L., Ho, C., and Al-Dujaili, E. (2010). The effect of polyphenol-rich dark
chocolate on fasting capillary whole blood glucose, total cholesterol, blood pressure and
glucocorticoids in healthy overweight and obese subjects. British Journal of Nutrition. 103:
842–850.
Almoosawi, S. (2011). Polyphenols in Green Coffee Bean and Chocolate: Effect on glucoregulatory
biomarkers blood pressure and lipid profile in obesity. VDM Verlag Dr. Müller, Saarbrücken,
Saarland, Germany.
Anderson, P. C. (1971). Foods as the cause of acne. Am Fam Physician. 3(3): 102-3.
Balzer, J., Rassaf, T., Heiss, C., et al. (2008). Sustained Benefits in Vascular Function Through
Flavanol-Containing Cocoa in Medicated Diabetic Patients. Journal of the American College of
Cardiology. 51(22): 2141–9.
Bayard, V., Chamorro, F., Motta, J., and Hollenberg, N. K. (2007). Does flavanols intake influence
mortality from nitric oxide-dependent processes? Ischemic heart disease, stroke, diabetes
mellitus, and cancer in Panama. Int J Med Sci. 4: 53– 8.
Beckett, S. T. (2008). Nutrition and Health. In: The Science of Chocolate. 2
nd
Edition. pp. 196-208.
Beckett, S. T., Ed., The Royal Society of Chemistry, Cambridge, UK.
Bisson, J. F., Guardia-Llorens M. A., Hidalgo S., et al. (2008a). Protective effect of Acticoa
powder, a cocoa polyphenolic extract, on prostate carcinogenesis in Wistar-Unilever rats. Eur J
Cancer Prev. 17(1): 54-61.
Bisson, J. F., Nejdi, A., Rozan, P., et al. (2008b). Effects of long-term administration of a cocoa
polyphenolic extract (Acticoa powder) on cognitive performances in aged rats. British Journal
of Nutrition 100: 94–101.
Bisson, J. F., Hidalgo, S., and Rozan, P. (2006). Messaoudi, M. Preventive Effects of ACTICOA
Powder, a Cocoa Polyphenolic Extract, on Experimentally Induced Prostate Hyperplasia in
Wistar-Unilever Rats. Journal of Medicinal Food. 10(4): 622-627.

DOI:10.1080/10408398.2012.657921
27
Bordeaux, B., Yanek, L. R., Moy, T. F., et al. (2007). Casual chocolate consumption and inhibition
of platelet function. Prev Cardiol. 10(4): 175-80.
Buijsse, B., Feskens, E. J., Kok, F. J., Kromhout, D. (2006). Cocoa intake, blood pressure, and
cardiovascular mortality: the Zutphen Elderly Study. Arch Intern Med. 166(4): 411-7.
Buijsse, B., Weikert, C., Drogan, D., et al. (2010). Chocolate consumption in relation to blood
pressure and risk of cardiovascular disease in German adults. European Heart Journal. 31 (13):
1616-1623.
Cienfuegos-Jovellanos, E., Quinones, M. D. M., Muguerza, B., et al. (2009). Antihypertensive
effect of a polyphenol-rich cocoa powder industrially processed to preserve the original
flavonoids of the cocoa beans. J. Agric. Food Chem. 57: 6156–6162.
Cooper, K.A., Campos-Giménez, E., Alvarez, D.J., Rytz, A., Nagy, K., Williamson, G. (2008).
Predictive relationship between polyphenols and nonfat cocoa solids content of chocolate.
Journal of Agricultural and Food Chemistry. 56(1): 260-265.
Cooper, K. A., Donovan, J. L., Waterhouse, A. L., Williamson, G. (2008). Cocoa and Health: A
Decade of Research. British Journal of Nutrition. 99:1-11.
Corti, R., Flammer, A. J., Hollenberg, N. K., and Lusche, T.F. (2009). Cocoa and cardiovascular
health. Circulation. 119: 1433-1441.
Crews, W. D Jr., Harrison, D. W., and Wright, J. W. (2008). A double-blind, placebo-controlled,
randomized trial of the effects of dark chocolate and cocoa on variables associated with
neuropsychological functioning and cardiovascular health: clinical findings from a sample of
healthy, cognitively intact older adults. Am J Clin Nutr. 87: 872-80.
Crozier, S. J., Preston, A. G., Hurst, J. W., Payne, M. J., Mann, J., Hainly, L., Miller, D. L. (2011)
Cacao seeds are a “Super Fruit": A comparative analysis of various fruit powders and products.
Chemistry Central Journal 5:5

DOI:10.1080/10408398.2012.657921
28
Danby, F. W. (2005) Acne and milk, the diet myth, and beyond. J Am Acad Dermatol. 52: 360-2
Datla, K. P., Zbarsky, V., Rai, D., et al. (2007). Short-term supplementation with plant extracts rich
in flavonoids protect nigrostriatal dopaminergic neurons in a rat model of Parkinson's disease. J
Am Coll Nutr. 26(4): 341-9.
Davison, K., Coates, A. D., Buckley, J. D., and Howe, P. R. C. (2008). Effect of cocoa flavanols
and exercise on cardiometabolic risk factors in overweight and obese subjects. International
Journal of Obesity. 32: 1289–1296.
Efraim, P., Alves, A. B., Jardim, D. C. P. (2011). Polifenóis em cacau e derivados: teores, fatores de
variação e efeitos na saúde. Braz. J. Food Technol., 14(3): 181-201.
Ferdowsian, H. R., and Levin, S. (2010). Does Diet Really Affect Acne? Skin Therapy Letter.
15(3): 1-2.
Ferrazzano, G. F., Amato, I., Ingenito, A., and Pollio, A. N. A. (2009). Anti-cariogenic effects of
polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia. 80: 255-262.
Field, D. T., Williams, C. M., and Butler, L. T. (2011). Consumption of cocoa flavanols results in
an acute improvement in visual and cognitive functions. Physiology and Behavior. 103(4): 255-
260.
Fisher, N. D. L., and Hollenberg, N. K. (2006a). Aging and vascular responses to flavanolrich
cocoa. J Hypertens. 24: 1575–80.
Fisher, N. D. L., Sorond, F. A., and Hollenberg, N. K. (2006b). Cocoa flavanols and brain
perfusion. J Cardiovasc Pharmacol. 47(2): 210-214.
Flammer, A., Hermann, F., Sudano, I., et al. (2007). Dark chocolate improves coronary vasomotion
and reduces platelet reactivity. Circulation. 116: 2376–82.
Fraga, C. G., Actis-Goretta, L., Ottaviani, J. I., et al. (2005). Regular consumption of a flavanol-rich
chocolate can improve oxidant stress in young soccer players. Clin Dev Immunol. 12(1): 11-7.

DOI:10.1080/10408398.2012.657921
29
Fraga, C. G., Litterio, M. C., Prince, P. D., Calabro, V., Piotrkowski, B. and Galleano, M. (2011)
Cocoa flavanols: effects on vascular nitric oxide and blood pressure, J. Clin. Biochem. Nutr. 48:
63–7.
Francis, S. T., Head, K., Morris, P. G., and Macdonald, I. A. (2006). The effect of flavanol-rich
cocoa on the fMRI response to a cognitive task in healthy young people. J Cardiovasc
Pharmacol. 47(2): 215-220.
Fries, J. H. (1978) Chocolate: A review of published reports of allergic and other deleterious effects
real or presumed. Annals of Allergy. 41:195-207.
Fulton Jr., J. E., Plewig, G., and Kligman, A.M. (1969). Effect of chocolate on acne vulgaris. Jama.
210(11): 2071-4.
Fung, T. (2011). Healthy Eating: A guide to the new nutrition. Harvard School of Public Health -
Nutrition Department. 48 pages.
Gibb, C., Davies, P., Glover, V., et al. (1991) Chocolate is a migraine-provoking agent. Cephalalgia
11:93-5.
Grant, J. D., and Anderson, P. C. (1965). Chocolate as a Cause of Acne: a Dissenting View. Mo
Med. 62: 459-60.
Grassi, D., Lippi, C., Necozione, S., et al. (2005a). Short-term administration of dark chocolate is
followed by a significant increase in insulin sensitivity and a decrease in blood pressure in
healthy persons. Am J Clin Nutr. 81(3): 611-4.
Grassi, D., Necozione, S., Lippi, C., et al. (2005b). Cocoa reduces blood pressure and insulin
resistance and improves endothelium-dependent vasodilation in hypertensives. Hypertension.
46: 398–405.
Grassi D., Desideri G., Necozione S., Lippi C., Casale R., Properzi G., Blumberg J. B., and Ferri C.
(2008). Blood pressure is reduced and insulin sensitivity increased in glucose-intolerant,

DOI:10.1080/10408398.2012.657921
30
hypertensive subjects after 15 days of consuming high-polyphenol dark chocolate. J Nutr. 138:
1671–1676.
Gu, L., House, S. E., Wu, S., Ou, B., Prior, R. L. (2006). Procyanidin and Catechin Contents and
Antioxidant Capacity of Cocoa and Chocolate Products. Journal of Agricultural and Food
Chemistry. 53(11): 4057-4061.
Hannum, S. M. and Erdman, J. W. (2000). Emerging Health benefits from cacao and chocolate.
Journal of Medicinal Food. 3(2): 73-75.
Heinrich, U., Neukam, K., Tronnier, H., et al. (2006). Long-term ingestion of high flavanol cocoa
provides photoprotection against UV-induced erythema and improves skin condition in women.
J Nutr. 136(6): 1565-9.
Heiss, C., Kleinbongard, P., Dejam, A., et al. (2005). Acute consumption of flavanol-rich cocoa
and the reversal of endothelial dysfunction in smokers. J Am Coll Cardiol. 46: 1276–83.
Heo, H. J., and Lee, C. Y. (2005). Epicatechin and catechin in cocoa inhibit amyloid beta protein
induced apoptosis. J Agric Food Chem. 53(5): 1445-8.
Heptinstall, S., May, J., Fox, S., et al. (2006). Cocoa flavanols and platelet and leukocyte function:
recent in vitro and ex vivo studies in healthy adults. Journal of Cardiovascular Pharmacology.
47(2): 197-205.
Hernández, P. M., Gutierrez, J. J. O.; Martínez, A. C., et al. (2011). Efecto de una bebida lactea a
base de cocoa sobre la presión arterial y la función endotelial en pacientes con insuficiencia
cardiaca: ensayo clínico aleatorizado, a doble ciego, controlado con placebo. Revista Cubana
de Cardiología y Cirugía Cardiovascular. 17: 52-61.
Hirao, C., Nishimura, E., Kamei, M., et al. (2010). Antibacterial effects of cocoa on periodontal
pathogenic bacteria. Journal of Oral Biosciences. 52(3): 283-291.

DOI:10.1080/10408398.2012.657921
31
Hollenberg, N.K. (2006). Vascular action of cocoa flavanols in humans: the roots of the story. J
Cardiovasc Pharmacol. 47(2): 99-102.
Hollenberg, N. K., Martínez, G., Mccullogh, M., et al. (1997). Aging, acculturation, salt intake, and
hypertension in the Kuna of Panama. Hypertension. 29: 171–176.
Holt, R. R., Lazarus, S. A., Sullards, M. C., et al. (2002). Procyanidin dimer B2 [epicatechin-(4beta-
8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am J Clin
Nutr. 76: 798-804.
Innes, A. J., Kennedy G., McLaren M., et al. (2003). Dark chocolate inhibits platelet aggregation in
healthy volunteers. Platelets 14: 325-7.
Ito, K., Nakamura, Y., Tokunaga, T., et al. (2003). Anti-cariogenic properties of a water-soluble
extract from cacao. Biosci Biotechnol Biochem. 67(12): 2567-73.
Jalil, A. M. M., and Ismail, A. (2008). Polyphenols in Cocoa and Cocoa Products: Is There a Link
between Antioxidant Properties and Health? Molecules. 13: 2190-2219.
Jalil, A. M. M., Ismail, A., Pei Pei, C., et al. (2008). Effects of Cocoa Extract on Glucometabolism,
Oxidative Stress, and Antioxidant Enzymes in Obese-Diabetic (Ob-db) Rats. J. Agric. Food
Chem. 56: 7877–7884.
Janszky, I., Mukamal, K. J., Ljung, R., et al. (2009). Chocolate consumption and mortality
following a first acute myocardial infarction: the Stockholm Heart Epidemiology Program. J
Intern Med. 266: 248 –257.
Jia L., Liu X., Bai Y. Y., Li S. H., Sun K., He C., et al. (2010) Short-term effect of cocoa product
consumption on lipid profile: a meta-analysis of randomized controlled trials. Am J Clin Nutr.
92: 218-25.
Jorge, A. T. S., Arroteia, K. F., Lago, J. C., et al. (2011). A new potent natural antioxidant mixture
provides global protection against oxidative skin cell damage. International Journal of
Cosmetic Science. 33(2): 113-119.

DOI:10.1080/10408398.2012.657921
32
Jourdain, C., Tenca, G., Deguercy, A., et al. (2006). In-vitro effects of polyphenols from cocoa and
beta-sitosterol on the growth of human prostate cancer and normal cells. Eur J Cancer Prev.
15(4): 353-61.
Keeney, P. (1972). Various interactions in chocolate flavor. Journal of the American Oil Chemists'
Society. 49(10): 567-572.
Kenny, T. P., Keen, C. L., Schmitz, H. H., and Gershwin, M. E. (2007). Immune effects of cocoa
procyanidin oligomers on peripheral blood mononuclear cells. Exp Biol Med (Maywood)
232(2): 293-300.
Khan, N., Monagas, M., Andres-Lacueva, C. et al. (2011). Regular consumption of cocoa powder
with milk increases HDL cholesterol and reduces oxidized LDL levels in subjects at high-risk
of cardiovascular disease. Nutrition, Metabolism & Cardiovascular Diseases 00: 1-8.
Koga, T., and Meydani, M. (2001). Effect of plasma metabolites of (+)-catechin and quercetin on
monocyte adhesion to human aortic endothelial cells. Am J Clin Nutr. 73: 941– 8.
Lecumberri, E., Goya, L., Mateos, R., et al. (2007). A diet rich in dietary fiber from cocoa improves
lipid profile and reduces malondialdehyde in hypercholesterolemic rats. Nutrition. 23(4): 332-
41.
Li, J., Vause, C. V., and Durham, P. L. (2008). Calcitonin gene-related peptide stimulation of nitric
oxide synthesis and release from trigeminal ganglion glial cells. Brain Res. 1196:22-32.
Mao, T. K., Van de Water, J., Keen, C. L., et al. (2002). Effect of cocoa flavanols and their related
oligomers on the secretion of interleukin-5 in peripheral blood mononuclear cells. J Med Food.
5(1): 17-2.
Marcus, D. A., Scharff, L., Turk, D., Gourley, L. M. (1997). A double-blind provocative study of
chocolate as a trigger of headache. Cephalalgia 17: 855-862.
Martín, M. A., Ramos, S., Mateos, R., et al. (2008). Protection of Human HepG2 Cells against
Oxidative Stress by Cocoa Phenolic Extract. J. Agric. Food Chem. 56: 7765–7772. .

DOI:10.1080/10408398.2012.657921
33
Matsui, N., Ito, R., Nishimura, E., Yoshikawa, M., et al. (2005). Ingested cocoa can prevent high-fat
diet–induced obesity by regulating the expression of genes for fatty acid metabolism. Nutrition.
21: 594–601.
Mehrinfar, R., Frishman, W. (2008). Flavanol-Rich Cocoa: A Cardioprotective Nutriceutical.
Cardiology in Review. 16(3): 109-115.
Mellor, D. D., Sathyapalan, T., Kilpatrick, E. S., et al. (2010). Atkin, High-cocoa polyphenol-rich
chocolate improves HDL cholesterol in Type 2 diabetes patients. Diabet Med, 27: 1318–1321
Melnik, B. (2009) Milk consumption: aggravating factor of acne and promoter of chronic diseases
of Western societies. J Dtsch Dermatol Ges. 7(4): 364-370.
Melnyk, J. P., Marcone, M. F. (2011). Aphrodisiacs from plant and animal sources - A review of
current scientific literature. Food Research International. 44: 840-850.
Messaoudi, M., Lalonde, R., Schroeder, H., and Desor, D. (2008). Antidepressant-like Effects of a
Cocoa Polyphenolic Extract in Wistar-Unilever Rats. Nutritional Neuroscience. 11(6): 269-
276.
Miller, K. B., Stuart, D. A.; Smith, N. L.; Lee, C. Y.; McHale, N. L. Flanagan, J. A.; Ou, B., Hurst,
W. J. (2006) Antioxidant Activity and Polyphenol and Procyanidin Contents of Selected
Commercially Available Cocoa-Containing and Chocolate Products in the United States.
Journal of Agricultural and Food Chemistry. 54: 4062-4068.
Moffat, A. M., Swash, M., and Scott, D. F. (1974). Effect of Chocolate in Migraine: A Double
Blind Study. J Neurology, Neurosurgery and Psychiatry. 37(4): 445-448.
Mostofsky, E., Levitan, E.B., Wolk, A., et al. (2010). Chocolate intake and incidence of heart
failure: A population-based, prospective study of middle-aged and elderly women. Circ Heart
Fail. 3(5): 612-6.

DOI:10.1080/10408398.2012.657921
34
Murphy, K. J., Chronopoulos, A. K., Singh, I., et al. (2003). Dietary flavanols and procyanidin
oligomers from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr. 77(6):
1466-1473.
Neukam, K., Stahl, W., Tronnier, H., et al. (2007). Consumption of flavanol-rich cocoa acutely
increases microcirculation in human skin. Eur J Nutr. 46(1): 53–56.
Nurk, E., Refsum, H., Drevon, C. A., et al. (2009). Intake of Flavonoid-Rich Wine, Tea, and
Chocolate by Elderly Men and Women Is Associated with Better Cognitive Test Performance.
J. Nutr. 139: 120–127.
Osakabe, N., Yamagishi, M., Natsume, M., et al. (2004). Ingestion of proanthocyanidins derived
from cacao inhibits diabetes-induced cataract formation in rats. Exp. Biol. Med (Maywood)
229(1): 33-9.
Paolino, V. J., and Kashket, S. (1985). Inhibition by Cocoa Extracts of Biosynthesis of Extracellular
Polysaccharide by Human Oral Bacteria. Archs Oral Biol. 30(4): 359-363.
Pate, A.K., Rogers, J.T., and Huang, X. (2008). Cognitive Impairment, and Alzheimer’s Dementia.
Int J Clin Exp Med. 1: 181-191.
Pearson, D. A., Holt, R. R., Rein, D., et al. (2005). Flavanols and platelet reactivity, Clin. Dev.
Immunol. 12(1): 1–9.
Peatweld, R. C. (1995) Relationships between food, wine, and beer-precipitated migrainous
headaches. Headache 35: 355–357
Percival, R. S., Devine, D. A., and Duggal, M. S. (2006). The Effect of Cocoa Polyphenols on the
growth, metabolism, and biofilm formation by Streptococcus mutans and Streptococcus
sanguinis. Eur J Oral Sci. 114(4): 343-8.
Perez-Berezo, T., Ramiro-Puig, E., Perez-Cano F. J., et al. (2009). Influence of a cocoa-enriched
diet on specific immune response in ovalbumin-sensitized rats. Mol. Nutr. Food Res. 53: 389 –
397.

DOI:10.1080/10408398.2012.657921
35
Pucciarelli, D., and Grivetti, L. (2008). The Medicinal Use of Chocolate in Early North America.
Molecular Nutrition and Food Research. 52(10): 1215-1227.
Raikkonen, K., Pesonen, A. K., Jarvenpaa, A. L., and Strandberg, T. E. (2004). Sweet babies:
chocolate consumption during pregnancy and infant temperament at six months. Early Hum
Dev. 76(2): 139-45.
Ramiro-Puig, E., Casadesús, G., Lee, H., et al. (2009) Neuroprotective effect of cocoa flavonids on
in vitro oxidative stress. Eur J Nutr. 48(1): 54–61.
Ramiro-Puig, E., and Castell, M. (2009). Cocoa: antioxidant and immunomodulator. British Journal
of Nutrition. 101: 931–940.
Ramiro-Puig, E., Franch, A., Castellote, C., Rez-Cano, F. P. Permanyer, J., Izquierdo-Pulido, M.,
and Castell, M. (2005). Flavonoids from Theobroma cacao Down-Regulate Inflammatory
Mediators. J. Agric. Food Chem. 53: 8506-8511.
Ramiro-Puig, E., Perez-Cano, C., Ramírez-Santana, C., et al. (2007a). Spleen lymphocyte function
modulated by a cocoa-enriched diet. British Society for Immunology, Clinical and
Experimental Immunology. 149: 535–542.
Ramiro-Puig, E., Urpí-Sarda, M., Perez-Cano, F. J., et al. (2007b). Cocoa-Enriched Diet Enhances
Antioxidant Enzyme Activity and Modulates Lymphocyte Composition in Thymus from
Young Rats. J. Agric. Food Chem. 55(16): 6431-6438.
Ramos, S., Moulay, L., Granado-Serrano, A., Vilanova, O., Muguerza, B., Goya, L., Bravo, L.
(2008) Hypolipidemic effect in cholesterol-fed rats of a soluble fiber-rich product obtained
from cocoa husk. J Agric Food Chem 56(16): 6985–6993.
Rein, D., Lotito, S., Holt, R. R., et al. (2000a). Epicatechin in human plasma: in vivo determination
and effect of chocolate consumption on plasma oxidation status. J Nutr. 130: 2109S-14S.
Rein, D., Paglieroni, T. G., Pearson, D. A., et al. (2000b). Cocoa and wine polyphenols
modulate platelet activation and function. J. Nutr., 130: 2120S–2126S.

DOI:10.1080/10408398.2012.657921
36
Rein, D., Paglieroni, T. G., Wun, T., et al. (2000c). Cocoa inhibits platelet activation and
function. Am. J. Clin. Nutr. 72: 30-35.
Ried, K., Sullivan, T., Fakler, P., et al. (2010). Does chocolate reduce blood pressure? A meta-
analysis. BMC Medicine.8: 39.
Ruzaidi, A., Amin, I, Nawalyah A. G., et al. (2005). The effect of Malaysian cocoa extract on
glucose levels and lipid profiles in diabetic rats. J Ethnopharmacol. 98(1-2): 55-60.
Sanchez, M., Quiñones, L., Moulay, B., et al. (2010). Changes in Arterial Blood Pressure of a
Soluble Cocoa Fiber Product in Spontaneously Hypertensive Rats. J. Agric. Food Chem. 58(3):
1493–1501
Sánchez, Y., Díaz, M. F., Hernández, F., Gil, D., and García, G. (2011) Antioxidant effects of an
ozonized theobroma oil formulation on damaged-inflammatory rat skin. Grasas y aceites, 62
(1): 105-110
Sarmadi, B., Ismail, A., and Hamid, M. (2011). Antioxidant and angiotensin converting enzyme
(ACE) inhibitory activities of cocoa (Theobroma cacao L.) autolysates. Food Research
International. 44 (1): 290-296.
Sathyapalan, T., Beckett, S., Rigby, A.S., Mellor, D.D., Atkin, S.L. (2010) High cocoa polyphenol
rich chocolate may reduce the burden of the symptoms in chronic fatigue syndrome. Nutrition
Journal, 9: 55.
Schewe, T., Sadik, C., Klotz, L. O., et al. (2001). Polyphenols of cocoa: inhibition of mammalian
15-lipoxygenase. Biol. Chem. 382: 1687–1696.
Schnorr, O., Brossette, T., Momma, T. Y., et al. (2008). Cocoa flavanols lower vascular arginase
activity in human endothelial cells in vitro and in erythrocytes in vivo. Archives of
Biochemistry and Biophysics. 476: 211–215.
Schramm, D. D., Karim, M., Schrader, H. R., et al. (2003). Food effects on the absorption and
pharmacokinetics of cocoa flavanols. Life Sci. 73: 857– 69.

DOI:10.1080/10408398.2012.657921
37
Selmi, C., Cocchi, C. A., Lanfredini, M., et al. (2008). Chocolate at heart: the anti-inflammatory
impact of cocoa flavanols. Mol Nutr Food Res. 52: 1340-1348.
Selmi, C., Mao, T. K., Keen, C. L., et al. (2006). The anti-inflammatory properties of cocoa
flavanols. J Cardiovasc Pharmacol. 47(2): 163-71.
Sies H., Schewe T., Heiss C., and Kelm M. (2005). Cocoa polyphenols and inflammatory
mediators. Am J Clin Nutr 81:304–312.
Sorond, F. (2008). Cerebral Blood Flow Response to Flavanol-Rich Cocoa in Healthy Elderly
Humans. Neuropsychiatric Disease and Treatment. 4(2): 433-440.
Sorond, F., Hollenberg, N. K., Panych, L. P., and Fisher, N. D. L. (2010). Brain Blood Flow And
Velocity: Correlations Between Magnetic Resonance Imaging and Transcranial Doppler. J
Ultrasound Med. 29(7): 1017–1022.
Spencer, J. P., Schroeter, H., Rechner, A. R., and Rice-Evans, C. (2001). Bioavailability of flavan-
3-ols and procyanidins: gastrointestinal tract influences and their relevance to bioactive forms
in vivo. Antioxid Redox Signal. 3: 1023–39.
Stahl, W. (2011). Flavonoid-Rich Nutrients for the Skin. Nutriton for Healthy Skin. 2: 85-90.
Stoclet, J. C., and Schini-Kerth, V. (2011). Dietary flavonoids and human health. Annales
Pharmaceutiques Francaises. 69(2): 78-90.
Strandberg, T.E., Strandberg, A.Y., Pitkala, K., et al. (2008). Chocolate, well-being and health
among elderly men. Eur J Clin Nutr. 2(2): 247-53.
Taubert D., Roesen R., Lehmann C., et al. (2007). Effects of low habitual cocoa intake on blood
pressure and bioactive nitric oxide: a randomized controlled trial. JAMA. 298(1): 49-60.
Tokede, O. A., Gaziano, J. M., and Djousse, L. (2011). Effects of cocoa products / dark chocolate
on serum lipids: A meta-analysis. Eur J Clin Nutr. 65: 879 -886.

DOI:10.1080/10408398.2012.657921
38
Tomaru, M., Takano, H., Osakabe, N., et al. (2007). Dietary supplementation with cacao liquor
proanthocyanidins prevents elevation of blood glucose levels in diabetic obese mice. Nutrition.
23(4): 351–355.
Tzounis, X., Rodriguez-Mateos, A., Vulevic, J., et al. (2011). Prebiotic evaluation of cocoa-derived
flavanols in healthy humans by using a randomized, controlled, double-blind, crossover
intervention study. American Journal of Clinical Nutrition. 93(1): 62-72.
Urpi-Sarda, M., Ramiro-Puig, E., Khan, N., et al. (2010). Distribution of epicatechin metabolites in
lymphoid tissues and testes of young rats with a cocoa-enriched diet. British Journal of
Nutrition. 103: 1393-1397.
van den Bogaard B., Draijer R., Westerhof B. E., et al. (2010). Effects on peripheral and central
blood pressure of cocoa with natural or high-dose theobromine: a randomized, double-blind
crossover trial. Hypertension. 56(5): 839-846.
Wollgast, J., and Anklam, E. (2000). Polyphenols in chocolate: is there a contribution to human
health? Food Research International. 33: 449-459.
Yasuda, A., Takano, H., Osakabe, N., et al. (2008). Cacao liquor proanthocyanidins inhibit lung
injury induced by diesel exhaust particles. Int J Immunopathol Pharmacol. 21(2): 279-88.
Yasuda, A., Natsume, M., Osakabe, N., et al. (2011). Cacao polyphenols influence the regulation of
apolipoprotein in HepG2 and Caco2 cells. Journal of Agricultural and Food Chemistry. 59(4):
1470-1476.
Yokoi, K., Konomi, A., Otagi, M. (2011). Comparison of a Modified Hemoglobin Regeneration
Efficiency Method with a Slope-Ratio Assay in Measuring Relative Bioavailability of Cocoa
Powder IronUsing Rats. Biol Trace Elem Res. 127: 143–147.