Cocoa and Health: A decade of Research

Abstract

It has been over 10 years since the first mention in a medical journal about cocoa and chocolate as potential sources of antioxidants for health. During this time, cocoa has been found to improve antioxidant status, reduce inflammation and correlate with reduced heart disease risk; with these results, and its popularity, it has received wide coverage in the press. However, after 10 years of research, what is known about the potential health benefits of cocoa and what are the important next steps in understanding this decadent source of antioxidants?

Full text

Review Article
Cocoa and health: a decade of research
Karen A. Cooper
1
, Jennifer L. Donovan
2
, Andrew L. Waterhouse
3
and Gary Williamson
1
*
1
Nestle
´Research Center, Vers-Chez-les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland
2
Department of Psychiatry and Behavioural Sciences, Medical University of South Carolina, Charleston, SC 29425, USA
3
Department of Viticulture & Enology, University of California, Davis, CA 95616, USA
(Received 5 December 2006 – Revised 29 May 2007 – Accepted 31 May 2007)
It has been over 10 years since the first mention in a medical journal about cocoa and chocolate as potential sources of antioxidants for health.
During this time, cocoa has been found to improve antioxidant status, reduce inflammation and correlate with reduced heart disease risk; with these
results, and its popularity, it has received wide coverage in the press. However, after 10 years of research, what is known about the potential health
benefits of cocoa and what are the important next steps in understanding this decadent source of antioxidants?
Cocoa: Chocolate: Health: Polyphenols: Antioxident
Introduction: why cocoa and why the focus on CVD?
It is an appealing idea that a food commonly consumed for
pure pleasure could also bring tangible benefits for health.
Olive oil, green tea and red wine have been commonly
researched in the past
1–3
and now there is growing interest
in cocoa. Cocoa is rich in polyphenols, similar to those
found in green tea, and as polyphenols have been shown to
have beneficial effects on CVD, it has resulted in heart
health being the most common target for research on cocoa.
There are several excellent recent reviews on cocoa and so
this review is intended to be focused on lessons learned and
on improving future research
4,5
.
For cocoa, the terms that are used to describe the particular
compounds of interest are flavanols (also known as flavan-3-
ols or catechins). Flavanols are a subclass of flavonoids
which are, in turn, a subclass of polyphenols. Flavanols can
be monomeric and those found in cocoa beans are (2)-epica-
techin and (þ)-catechin (their isomers may also be present in
small quantities), dimeric (the most common in cocoa are B2
and B5, both made of two units of epicatechin with differing
linkages) or they can be polymeric combinations of these
monomers, and chains of up to and over 10 units have been
found in cocoa
6
. These polymers are known as procyanidins.
For ease of writing, the term of cocoa polyphenols here
encompasses the monomers and the procyanidins. All poly-
phenols exert an antioxidant action in vitro
7,8
, however, this
does not mean that all polyphenols have an antioxidant
effect in vivo. The use of the term antioxidant in the present
report reflects this and is not intended to imply that all
cocoa polyphenols have a proven antioxidant benefit in vivo.
Chocolate and cocoa are two different terms and are not
interchangeable. Cocoa is the non-fat component of cocoa
liquor (finely ground cocoa beans) which is used in chocolate
making or as cocoa powder (commonly 12 % fat) for cooking
and drinks. Cocoa liquor contains approximately 55 % cocoa
butter and together this comprises cocoa solids, often referred
to on chocolate packaging. Chocolate refers to the combi-
nation of cocoa, cocoa butter, sugar, etc. into a solid food
product.
A recent survey found that in Europe, 58 % of people ate
milk chocolate, closely followed by dark chocolate (43 %)
9
.
For the UK, these figures were 61 and 35 %, respectively. In
the USA, milk chocolate is also considered the most popular,
but the majority of their confectionery consumption (,87 %)
is not as pure chocolate but rather enrobed with nuts, wafer,
fruit, etc.
10
. Cocoa taken as a beverage is also popular in
some countries like Spain and so should also be taken into
account when surveying intake of chocolate and cocoa
products.
What has been done in the last 10 years?
Waterhouse and colleagues wrote a letter, which was pub-
lished in 1996, that described an in vitro experiment that
was to open up a whole new area of nutrition and health
11
.
Polyphenols were extracted from commercial cocoa and cho-
colate, and the polyphenol content and antioxidant activity
*Corresponding author: Dr Gary Williamson, fax þ41 21 785 8544, email gary.williamson@rdls.nestle.com
Abbreviations: FMD, flow-mediated dilation; NO, nitirc oxide.
British Journal of Nutrition (2008), 99, 1–11 doi: 10.1017/S0007114507795296
qThe Authors 2008
British Journal of Nutrition

against LDL oxidation was measured. They found a potent
inhibition by the cocoa polyphenols. At 5 mmol/l total poly-
phenols (expressed as gallic acid equivalents), LDL oxidation
was inhibited by 75 %, compared to red wine at 37 –65 %.
This was the first publication to state that the action and con-
tent of polyphenols from cocoa meant that it could be con-
sidered as a dietary source of antioxidants.
After this, research papers linking chocolate and health
began to appear, and patents on polyphenol content in cocoa
and potential benefit areas were released. In 1999, another
letter was published, this time explaining the contribution of
chocolate compared to tea in the Dutch population as sources
of catechins, finding that although tea was still the major
source (55 %), chocolate contributed significantly (20 %)
12
.
The first human bioavailability trial of polyphenols from cho-
colate found that with 40 g of black chocolate, epicatechin was
indeed absorbed into the blood
13
. Epicatechin was present in
plasma as metabolites conjugated with glucuronide and sul-
phate groups. These compounds exhibited a T
max
of 2 h in
the plasma and C
max
of over 100 ng/ml. The compounds
were still measurable after 8 h.
Table 1 shows human trials with interventions using cocoa
in different forms from 2000 to 2007. For each study, the
intervention and its polyphenol content (if available), the con-
trols, subject type and the main outcomes are described. Each
of these trials investigated at least one health-related end-
point. The end-points selected at the beginning of this
period were suppression of platelet activation
14
and improve-
ment of plasma antioxidant activity and lipid oxidation
15
.
These end-points were logical as they had been shown pre-
viously to be affected positively by other sources of poly-
phenols such as red wine
16 – 20
. The rate of publication has
generally been increasing since 2000. There were two
human trials published in 2001, three in 2002, five in 2003,
three in 2004, six in 2005 and five in 2006 (one so far in
2007). More often than not, the studies yield at least one posi-
tive and significant result although, as more than one end-
point is measured in most of the trials, secondary outcomes
are often unchanged. These trials do not include those inves-
tigating the metabolism and pharmacokinetics of chocolate
components. End-points included blood pressure, insulin sen-
sitivity and resistance, endothelial function and flow-mediated
dilation (FMD), platelet function, plasma antioxidant status
and oxidative stress, plasma lipids (levels and oxidation),
nitric oxide (NO) and haemolysis. There is one epidemiologi-
cal study that correlates long-term cocoa intake with lower
overall and cardiovascular mortality in elderly men
21
and a
prospective study in post-menopausal women which found a
borderline inverse association of chocolate intake and CVD
mortality
22
. The research is predominantly focused on effects
on the vascular system, however, there are other areas of
research on man in vivo which are not so extensively investi-
gated, such as those concerned with cognition
23
, cancer
24
and
diabetes
25
.
Bioavailability
Richelle et al.
13
first demonstrated the appearance of epicate-
chin in blood after consumption of black (dark) chocolate; and
3 years later a study demonstrated the presence of a dimer in
the plasma within 30 min post-consumption of flavanol-rich
cocoa
26
. Cocoa polyphenols are therefore absorbed but factors
such as low C
max
in the plasma, a short half-life and rapid
excretion all add to a relatively low bioavailability
27
. In gen-
eral, the smaller the polyphenol, the higher the concentration
in the blood, and the higher chance that it will reach its
target organ in the body. Intestinal perfusion studies have
shown that B2 and B5 can cross the enterocytes but to a
very limited extent
28
. Larger units than the dimer are unlikely
to be able to cross the gut barrier, although they could have an
action within the gut lumen or be cleaved by colonic bacteria
before absorption of the resulting metabolites. Monomers such
as epicatechin are metabolised to O-methylated forms or con-
jugated as glucuronides and sulphates, with 30-O-methylepica-
techin being investigated for its potential protective effects
29
.
This area of research is still relatively new and the breakdown
products of procyanidins have not been fully identified nor
characterised for possible effects. For the moment, taking
only current knowledge into account, it would be logical
that for chocolate or cocoa to confer health benefits, it
should have a high percentage of the smaller (monomeric)
polyphenols. In accordance with this, a recent study concluded
that the epicatechin content was likely to be the main expla-
nation for cocoa’s association with health
30
.
Bioavailability can also be affected by the matrix in which
the cocoa polyphenols are delivered. In the previous human
trials such matrices have been semi-sweet chocolate baking
bits, cocoa powder, dark chocolate, tablets, drinks, milk choc-
olate and even in a muffin. It is possible that these different
matrices affect the release of the polyphenols from the food,
making them more or less available for absorption.
Of the twenty-eight trials listed in Table 1, fifteen trials used
a one-off dose of polyphenols and thirteen trials used a
chronic intervention style, with periods of supplementation
lasting from 4 d to 6 weeks. There are benefits and shortcom-
ings of both types of trials, depending on what exactly is being
investigated. However as the effect of polyphenols is often
short lived, once an effect has been seen in the short term, it
would be logical to see if this effect can be maintained over
a longer period. There may even be adaptation to a regular
supply of a certain polyphenol, resulting in a more efficient
uptake and therefore a greater possibility of an effect.
One aspect of intervention studies is that inter-subject varia-
bility of bioavailability may obscure the true meaning of
results. Most (non-bioavailability) studies assume a postpran-
dial T
max
of 120 min. However, studies can show such vari-
ation in C
max
at this time
15
, that either the C
max
occurs
earlier or later and so is missed, or there truly is large interper-
sonal variation in absorption of a compound such as epicate-
chin. It may also happen that a person with a high plasma
value at 2 h may not have a correspondingly high response
in whichever health-related biomarker is measured, obscuring
any potential correlation between apparent bioavailability
and bioefficacy. The consequence of this is that a single
measurement of plasma levels at 2 h cannot be considered a
measurement of bioavailability, but rather only a check for
compliance, limiting the usefulness of this measure.
Another fairly new area for cocoa and bioavailability is that
of the chiral nature of polyphenols and the effect of chirality
on bioavailability. For instance, the (þ) form of catechin
tends to dominate in cocoa beans, and the (2) form in
chocolate
31
. One paper found that chocolate tended to contain
K. A. Cooper et al.2
British Journal of Nutrition

Table 1. Human intervention trials with cocoa
Intervention Polyphenol content Control Subjects Main outcomes
Industry-
funded Reference
1 Semi-sweet chocolate baking bits
(onedoseofa,27g;b,53g;c,
80 g)
Total procyanidins
(epicatechin)
(a) 186 mg (46 mg),
(b) 365 mg (90 mg),
(c) 551 mg (136 mg)
No chocolate 20 healthy adults (20–56
years)
Dose-dependent increase in plasma
epicatechin. Non-significant trend for
an increase in plasma antioxidant
activity and a decrease in TBARS
Partially Wang et al.
56
2 18·75 g procyanidin-rich cocoa
powder in 330 ml water (one
dose)
897 mg epicatechin and total
procyanidins
Caffeine and
sucrose hot
drink or
water
30 healthy adults (24–50
years), 10 per group
Suppression of platelet activation.
Aspirin-like effect on primary
hemostasis 6 h after consumption
Authors from
industry,
not stated
outright
Rein et al.
14
3 105 g (of which 80 g chocolate)
semi-sweet baking bits (one
dose)
557 mg total procyanidins (of
which 137 mg epicatechin)
Vanilla milk
chips
(isoenergetic)
10 healthy adults (26–49
years) þ3 healthy
adults (28–36 years)
consuming control
12-fold increase in plasma epicatechin
2 h later, increase in plasma total
antioxidant activity and decrease in
TBARS
Partially Rein et al.
15
4 12 g cocoa powder £3/d for 2
weeks
2610 mg total polyphenols/d (of
which 244 mg epicatechin)
Sugar 15 healthy men, 9 in
active group
(32.5 ^6.4 years)
Increase in LDL oxidation lag time,
no change in plasma lipids or
antioxidants. Higher excretion of
epicatechin/metabolites in urine
Authors from
industry,
not stated
outright
Osakabe
et al.
40
5 22 g cocoa powder and 16 g dark
chocolate/d for 4 weeks
466 mg procyanidins/d (of
which 111 mg monomers)
Average
American
diet
23 healthy adults (21–62
years)
Increase in LDL oxidation lag time,
increase in serum antioxidant
capacity, increase in HDL
cholesterol
No but indus-
trial authors
Wan et al.
46
6 18·75 g cocoa powder in 300 ml
water with sugar, with and
without aspirin (one dose)
897 mg epicatechin and
procyanidins
81 mg aspirin 16 healthy adults (22 –49
years)
After 6 h, cocoa inhibited epinephrine-
stimulated platelet activation and
function
Partially Pearson
et al.
62
7 36·9 g dark chocolate and 30·95 g
cocoa powder in a drink/d for 6
weeks
651 mg total procyanidins/d
(chocolate ¼168 mg/d,
cocoa ¼483 mg/d)
None 25 healthy adults (20–60
years)
LDL oxidisability was lower, but no
effect on inflammation markers, or
plasma antioxidant capacity
Partially Mathur
et al.
63
8 25 g semi-sweet chocolate chips
(one dose)
220 mg flavanols and
procyanidins
None 18 healthy adults Increase in plasma epicatechin after
2 h with concurrent increase in
prostacyclin–leukotriene ratio.
Reduction in platelet-related
haemostasis
Partially Holt et al.
34
9 100 g dark chocolate/d for 14 d 500 mg/d total polyphenols 90 g white
chocolate
13 elderly adults (55–64
years with mild
hypertension)
Lower systolic and diastolic blood
pressure
No Taubert
et al.
64
10 Cocoa flavanol/procyanidin
tablets for 28 d
234 mg flavanols and
procyanidins/d (6 £39 mg
tablets/d)
Placebo tablets 13 healthy adults (active
40 y ^9), 15 healthy
adults (control 47.4
years ^4)
Lower platelet aggregation and P-
selectin expression, higher plasma
ascorbic acid, no change in
oxidation/antioxidant status markers.
Increase in plasma epicatechin and
catechin
Partially Murphy
et al.
65
11 High polyphenol cocoa drink
4£230 ml/d for 4 d
821 mg/d total flavanols
(epicatechin, catechin and
related oligomers)
Low flavanol
cocoa drink
27 healthy adults (18–72
years)
Improved peripheral vasodilation after
4 d, large acute response after
90 min
Partially Fisher et al.
66
12 100 ml high cocoa polyphenol
drink (one dose)
176 mg total (70 mg monomers,
106 mg procyanidins)
Low flavanol
cocoa drink
20 adults (all with 1 CHD
risk factor) (41 years ^
14) (77 % were
smokers)
NO bioactivity and arterial FMD
increased
Partially Heiss et al.
67
Cocoa and health 3
British Journal of Nutrition

Table 1. Continued
Intervention Polyphenol content Control Subjects Main outcomes
Industry-
funded Reference
13 100 g dark chocolate (with and
without 200 ml milk) (one dose)
Polyphenols not stated but
FRAP values were
147·4 mmol FE/100 g)
200 g milk
chocolate
(FRAP
78·3 mmol
FE/100 g)
12 healthy adults (25–35
years)
Dark chocolate increased plasma anti-
oxidant capacity and epicatechin.
Consuming milk with it reduced
these effects. Milk chocolate had
less effect than both these treat-
ments
No Serafini
et al.
51
14 75 g dark chocolate or high
phenolic dark chocolate for 3
weeks
Dark ¼274 mg/d (114 mg/d
epicatechin). High ¼418
mg/d (170 mg/d epicatechin)
75 g white
chocolate
45 healthy adults (19–49
years)
Both dark chocolates increased HDL
cholesterol and lipid peroxidation
decreased (but also with white
chocolate control). No change in
plasma antioxidant capacity
Partially Mursu et al.
58
15 46 g/d high phenolic dark
chocolate for 14 d
213 mg/d total procyanidins (of
which 46 mg/d epicatechin)
Low phenolic
dark
chocolate
21 healthy adults (21–55
years)
Improved endothelium-dependent
FMD, no change in blood pressure,
oxidative markers or blood lipids.
Higher plasma epicatechin
No Engler et al.
57
16 High polyphenol cocoa drink,
100 ml (one dose)
187 mg total monomers and
oligomeric procyanidins
Low phenolic
cocoa drink
20 healthy males (20–40
years)
F2 isoprostanes improved 2 and 4 h
after exercise
No but indus-
trial involve-
ment
Wiswedel
et al.
54
17 Dark chocolate, 100 g (one dose) 500 mg total polyphenols 90 g white
chocolate
15 healthy adults
(34 ^7.6 years)
Insulin sensitivity higher and insulin
resistance lower. Systolic blood
pressure lower
No Grassi et al.
25
18 Flavonoid-rich drink at 0·25,
0·375, 0·5 g/kg body weight)
(one dose)
12·2 mg/g monomers, 9·7 mg/g
dimers, 28·2 mg/g
procyanidins
Bread and
water
8 healthy males (26 ^2
years)
Reduction in the rate of free radical-
induced haemolysis
Partially Zhu et al.
68
19 105 g/d milk chocolate for 14 d 168 mg/d flavanols (of which
39 mg monomers and
126 mg polymers)
Cocoa butter
chocolate
28 healthy males (18–20
years) under exercise
stress
Decrease in diastolic and mean blood
pressure, plasma cholesterol, LDL,
malondialdehyde, urate and lactate
dehydrogenase activity, increase in
vitamin E–cholesterol ratio. No
change in plasma epicatechin but
samples were fasting
No but
industrial
involvement
(via author-
ship)
Fraga et al.
69
20 100 g dark chocolate (one dose) 2·62 g (of which 0·54 g
monomers and dimers,
0·76 g trimer-heptamers)
Sham chewing
and water
17 healthy adults (24–32
years)
Increase in resting and hyperaemic
brachial artery diameter. Increase in
FMD at 60 min. Aortic augmentation
index decreased. No significant
change in malondialdehyde, and
total antioxidant capacity and pulse
wave velocity
No Vlachopoulos
et al.
37
21 100 g/d dark chocolate for 15 d 88 mg/d flavanols (22 mg
catechin, 66 mg epicatechin)
90 g white
chocolate
20 never-treated adults
with essential
hypertension (44 ^8
years)
Insulin sensitivity improved, lower
systolic and diastolic blood pressure
and LDL, and improved FMD
No Grassi et al.
70
22 High polyphenol cocoa drink,
100 ml (one dose)
176 –185 mg flavanols (70–
74 mg monomers, 20 – 22 mg
epicatechin, 106– 111 mg
procyanidins)
Low phenolic
cocoa drink
11 adult smokers
(average 31 years)
Increased circulating NO, FMD, both
correlated to increases in flavanol
metabolites. Effects were reversed
with NG-monomethyl-l-arginine to
prove link to NO
Yes Heiss et al.
59
K. A. Cooper et al.4
British Journal of Nutrition

Table 1. Continued
Intervention Polyphenol content Control Subjects Main outcomes
Industry-
funded Reference
23 300 ml high polyphenol cocoa
drink (one dose)
917 mg flavanols (19 %
epicatechin)
300 ml low
polyphenol
cocoa drink
16 healthy males (25–32
years)
Acute elevations in levels of circulating
NO species, an enhanced FMD
response of conduit arteries, and an
augmented microcirculation
Partially Schroeter
et al.
30
24 40 g dark chocolate (one dose) Not stated but same brand as
used for Vlachopoulos et al.
37
White
chocolate
20 male smokers (age
not given)
Improved FMD after 2 h lasting for 8 h.
Reduction in platelet function.
Increased plasma total antioxidant
status
No Hermann
et al.
8
25 High polyphenol cocoa drink 4 £
230 ml/d for 4 – 6 d
Per 100 ml, 9·2 mg epicatechin,
10·7 mg catechin and
69·3 mg flavanol oligomers
(821 mg/d)
None 15 young (,50 years)
and 19 older (.50
years)
NO synthesis after cocoa was
suppressed in older volunteers.
FMD was enhanced in both groups
but more in older group. Pulse wave
amplitude enhanced in both groups,
with acute rises with cocoa
ingestion, more robustly in older
subjects. No change in BP
Partially Fisher & Hol-
lenberg
71
26 22 g cocoa powder and 16 g dark
chocolate (in a muffin)
111 mg monomers and 466
procyanidins
Cocoa butter
equivalent in
muffin
4 (30–49 years)
normolipidaemic
subjects (pilot trial)
Dark chocolate increased resistance of
LDL and VLDL to oxidation whilst
cocoa butter alone decreased
resistance. Noted after examination
of dietary data that chocolate is third
highest contributor of antioxidants to
the American diet
No Vinson
et al.
10
27 41 g/d of high polyphenol dark
chocolate either with or without
almonds 60 g/d for 6 weeks
plus dietary advice
Not stated No intervention
except same
dietary
advice
49 women with choles-
terol 4·1– 7·8 mmol/l
(22–65 years)
Dark chocolate decreased TAG by
21 %, 19 % when eaten with
almonds, 13 % with almonds alone
and 11 % with no intervention.
Circulating intercellular adhesion
molecule with dark chocolate alone
No. Industry
supplied
chocolate
only
Kurlandsky &
Stote
72
28 High flavanol cocoa drink 100 ml
£3/d for 1 week
Per 100 ml, 59 mg epicatechin,
15 mg catechin and 232 mg
flavanol oligomers (918 mg/d
procyanidins)
Low phenolic
cocoa drink
6 male smokers with
smoking-related
endothelial dysfunction
(11 total) (22–32
years)
Daily continual FMD increases at
baseline (fasted) and a sustained
FMD augmentation at 2 h post-
ingestion. A dose-dependent effect
also seen with FMD and nitrate.
Biomarkers for oxidative stress
unaffected.
Yes Heiss et al.
39
FE, Ferric equivalents; FMD, flow-mediated dilation; FRAP, ferric-reducing ability of plasma; TRAP, ferric reducing ability of plasma (or antioxidant potential ) TBARS, thiobarbituric acid reactive substances.
Cocoa and health 5
British Journal of Nutrition

predominantly (2)-epicatechin and (2)-catechin, with only
small amounts of (þ)-catechin and negligible (þ)-epicate-
chin
32
. The same paper indicated that the (þ) form of catechin
was almost 10 times more absorbed than the (2) form using a
rat perfusion model, which may explain why catechin from
cocoa is not as well absorbed as from other foods
33,34
.
Mechanism of action
Multiple approaches have been used to investigate the mech-
anism of action of cocoa polyphenols including clinical, pre-
clinical and in vitro studies. Cocoa polyphenols have been
investigated predominantly for their effect on the vascular
system, with NO concentrations being a central target
(Fig. 1). One of these effects is on endothelial function,
which is an extremely promising biomarker to calculate
heart attack risk
35,36
. Several clinical studies have shown
improved endothelial function after cocoa consumption
37 – 39
,
but it is not known if these improvements are due to a
subtle combination of mild effects rather than a single targeted
effect. Other effects related to reduced CVD risk include
decreased susceptibility of LDL to oxidation
40
, and inhibition
of platelet activation and aggregation
14
. As shown in Table 1,
although many different biomarkers have been measured, the
results consistently show changes in biomarkers related to oxi-
dative status and/or vascular function (thiobarbituric acid reac-
tive substances (TBARS), LDL oxidation, F2-isoprostanes,
platelet aggregation and FMD).
How much chocolate is enough?
Chocolate is predominantly a food for pleasure, and many
people incorporate it into part of a healthy, varied and
balanced diet. However, there is controversy over whether it
should be recommended for its health benefits. Furthermore,
it is difficult establish how much chocolate and what type to
recommend for health benefits. High cocoa content dark cho-
colate tends to be richest in polyphenols, although each choco-
late is different in polyphenol content
41
. Polyphenols are
known to be destroyed by harsh processing of the cocoa
bean and so percentage cocoa content should be considered
a guideline only to polyphenol content. There are no long-
term intervention studies addressing the health benefits of cho-
colate consumption. Most previous short-term studies have
given a single ‘dose’ of chocolate, which is probably more
than one person would normally consume, and demonstrated
an effect: decreased plasma leukotriene –prostacyclin ratios
in human plasma and aortic endothelial cells
42
, activation of
endothelial nitric oxide synthase and enhanced endothelium
relaxation in vitro
43
, inhibition of human cytokine transcrip-
tion and secretion
44
, and inhibition of mammalian 15-lipoxy-
genase activity
45
. These multiple effects lend credence to
the opinion that a ‘simple’ antioxidant mechanism in vivo is
not likely, but more probably via inhibition of inflammatory
pathways, leading to reduced risk of a chronic disease state.
By how much the change in biomarkers listed earlier influence
the actual risk of CVD is difficult to quantify at present. Endo-
thelial function is an excellent indicator of CVD risk
36
, but
effects of cocoa polyphenols are generally short lived, i.e.
for the duration of the presence of catechins in plasma.
Some studies have shown an effect on general antioxidant
markers in vivo, and antioxidant assays reflect specific bio-
chemical parameters in the plasma. For example, the Trolox
equivalent antioxidant capacity assay is dominated by the pre-
sence of albumin and uric acid, and the polyphenol itself at
low micromolar concentrations would not have a significant
effect by itself on the Trolox equivalent antioxidant capacity
value. However, on a regular daily basis, the overall effects
may potentially accumulate. Very recently it has been
shown that the effects of cocoa polyphenols on FMD (but
not markers of oxidative stress) can be cumulative if taken
in high doses on a daily basis for 1 week and with a return
to baseline after a week washout
39
. Seven of the studies
used approximately 100 g of chocolate and two used 920 ml
of a cocoa drink in 1 d, which would be difficult to justify
every day long term. There have been very few dose –response
studies and so it is difficult to judge exactly how much choco-
late is needed for an ‘antioxidant’ effect. However, for other
effects, very little might be needed. In smokers, 40 g of dark
chocolate improved FMD and platelet function (no polyphenol
content was stated)
38
. The most recent study on smokers
with endothelial dysfunction found that the dose needed for
Fig. 1. Diagram to show the how cocoa polyphenols might affect the vascular system, with nitric oxide (NO) as the target. eNOS, endothelial nitric oxide synthase.
K. A. Cooper et al.6
British Journal of Nutrition

a half-maximal FMD at 2 h post-consumption was 616 mg
total flavanols
39
. Another study found an increase in the pros-
tacyclin–leukotriene ratio and a reduction in platelet-related
haemostasis in healthy people with just 25 g of semi-sweet
chocolate bits containing 220 mg flavanols and procyanidins
34
.
The polyphenol content is of more importance and it is essen-
tial that, in future, all published trials give a full characteris-
ation of the chocolate or cocoa used and the calculated
dose. This characterisation should include a breakdown of
the types of polyphenols, especially monomer content. For
example, one study indicated that their dose was 500 mg
total polyphenols in 100 g dark chocolate/d
64
whereas another
gave 22 g cocoa powder and 16 g dark chocolate/d containing
466 mg procyanidins
46
. Although both these sets of infor-
mation are useful to some extent, they could be improved
by the use of a more comparable parameter such as epicate-
chin. It is also important to specify the methodology behind
the measurement, i.e. HPLC or colorimetry.
Fat and sugar are major components of chocolate, and pro-
vide significant energy that needs to be taken into account
when assessing possible risks and benefits of recommending
chocolate consumption for health purposes. Chocolate con-
tains fatty acids such as stearic, oleic and palmitic acids.
These particular fats appear to have a neutral effect on
blood lipid levels
47,48
, i.e. they do not raise blood cholesterol
levels. Chocolate, especially of the milk variety, contains high
amounts of sugar which obviously increases the energy value
and has possible implications for dental health and diabetes if
eaten in large amounts, although carbohydrates might play a
role in improving uptake of polyphenols
49
. Cocoa itself is
much easier to recommend on a health basis as it is not
high in sugar and fat. Populations that take cocoa compared
to genetically similar groups with less consumption, i.e.
island- v. mainland-dwelling Kuna Indians of Panama, have
been shown to excrete more NO metabolites, which is an indi-
cator of higher NO production, which is in turn associated
with lower incidence of CVD
30
. A more recent evaluation of
the causes of mortality between these two populations found
a substantially lower number of deaths between 2000 and
2004 from NO-dependent diseases such as CVD, cancer and
diabetes mellitus in the island v. mainland Kuna Indians
50
.
Dark or milk?
This is a question that is often asked when considering health
effects. Many countries around the world predominantly con-
sume cocoa as part of milk chocolate rather than dark. Also a
cocoa drink may be made with either water or milk. So we can
question whether these people are getting similar benefits as
those countries where dark chocolate or water-based cocoa
drinks are mainly consumed.
The polyphenols in chocolate come from the cocoa liquor.
Hence, as milk chocolate generally contains less cocoa
liquor than dark chocolate, it will contain less polyphenols.
White chocolate contains no cocoa liquor and hence no poly-
phenols at all. However, this is complicated by the fact that
polyphenols can be destroyed during the processing of the
raw cocoa depending on the manufacturing methods used.
So a chocolate may contain 70 % cocoa solids but due to pro-
cessing only contain the same content of polyphenols as a
normal milk chocolate. How would a consumer know that
the dark chocolate they are buying is a good source of poly-
phenols?
Of the twenty-eight clinical trials, only two used milk cho-
colate. One study published in Nature showed that 100 g plain
dark chocolate resulted in an increase in total antioxidant
capacity but was markedly reduced when consumed with
200 ml whole milk, or taken as milk chocolate (200 g)
51
.It
was also shown that absorption of epicatechin from chocolate
was significantly less when consumed with milk or as milk
chocolate. The hypothesis is that milk proteins bind to cocoa
polyphenols, which in turn prevents their absorption in the
gastrointestinal tract. However, this study generated much
controversy in the literature. Studies after this have not
found this reduction in epicatechin bioavailability when
cocoa was consumed with milk
52
, but also have not been
able to definitively explain why the original paper found
these results. Experimental differences, such as giving cocoa
powder in a drink comparing either water or milk as a
matrix, rather than as a solid chocolate may be one possible
reason. The fat differences between milk chocolate and a
cocoa and milk drink are considerable and may play a
role
53
. Matrix effects are becoming increasingly important
for food as new EU legislation Directive 2000/13/EC that
came into effect in January 2007 may make it essential that
any food labelling a high content of a beneficial compound
must be able to show evidence that it is bioavailable from
that food product, and is also effective in its implied benefit.
As many of the human studies used liquid-based cocoa for
their interventions and found positive effects, it indicates that
cocoa polyphenols taken as a liquid can be bioavailable,
though no direct comparison with solid cocoa or chocolate
has been made to date. As most studies have investigated
dark chocolate to avoid the possibility that milk might inter-
fere, it is hard to infer that milk chocolate will be just as effec-
tive as dark, even when strictly controlling for overall
polyphenol intake. One study did use milk chocolate and
found a positive effect on blood pressure, plasma cholesterol
and markers of oxidative stress on young exercising males
54
.
One other study has shown bioavailability from a milk
cocoa beverage common for children in Spain
55
. However,
we feel that this issue has not been resolved, as there has
been no definitive study confirming the bioavailability with
solid milk chocolate. It is important to resolve this issue as
milk chocolate is much more popular in many countries.
Designing smart clinical intervention trials
Of the twenty-eight human intervention trials, twenty-one were
with apparently healthy adults (one including exercise as a
stress parameter and one comparing older v. younger adults).
The other seven involved elderly hypertensives, healthy smo-
kers, smokers with endothelial dysfunction, subjects with one
CHD risk factor, women with high cholesterol and essential
hypertensives. Although most of the twenty-one trials had at
least one positive measurable change in a health biomarker,
one could question the usefulness of healthy and well-nour-
ished subjects for testing the efficacy of antioxidant supplemen-
tation. The age ranges used within the healthy adult trials were
also wide, often over 30 years. As polyphenolic antioxidants
may possibly be more useful in the ageing population, since
ageing can be considered to partly involve an ‘oxidative
Cocoa and health 7
British Journal of Nutrition

stress’, it is probable that effects in the older segment of sub-
jects may be missed by grouping all the data together.
When antioxidants such as polyphenols are given in an
optimal dose to a person with a sufficient dietary status, sup-
plementation is unlikely to make much of a measurable
difference to their health status (Fig. 2), for example, on
plasma antioxidant status or blood pressure
56 – 58
. However,
if the person is subject to a stress event, such as inflam-
mation, smoke inhalation or sunburn, or suffers from chronic
antioxidant deficiencies, then antioxidants may be able to
counteract the effect of the stress to return this individual
closer towards a healthy status. Hence using people who
are at risk of a disease (e.g. through elevated blood pressure,
ageing or poor diet) to look for an effect of polyphenolic
antioxidants is more likely to provide a measurable result
and therefore reveal the true potential of the compound
being studied.
Obviously, the use of a control group always strengthens
human intervention trials. However, if biological effects are
to be attributed to cocoa polyphenols rather than another com-
ponent of the cocoa, then the perfect control would be a dark
chocolate that contains everything other than polyphenols.
Most trials have been unable to do this, as it is not that
simple to make or find. Controls have ranged from white cho-
colate to bread and water. This may show the effect is due to
cocoa but not necessarily to cocoa polyphenols. Some trials
have been able to source a control which purports to be low
in polyphenols such as trials with cocoa drinks
30,39,54,59
and
one which uses a low polyphenol dark chocolate
57
. Perhaps
the best trial for this to date used epicatechin as a positive con-
trol and found the effects from both cocoa and epicatechin to
be of a similar magnitude, hence allowing the effects to be
more strongly attributed to epicatechin
30
. However, the
majority have not been able to control fully for this aspect,
and for these trials, the question does remain as to whether
the effects seen are from cocoa polyphenols, from some
other component such as caffeine or magnesium, or indeed
from a synergistic effect of several components from cocoa.
Potential for research bias?
As with any research there is the potential for bias. The field
of cocoa polyphenols has been dominated by industrially
funded research for the last 10 years. Of all the twenty-
eight listed publications, fifteen had partial or full industrial
funding and a further four had industrial involvement of
some type (supply of chocolate, etc.) not including those
that were helped by the American Cocoa Research Institute
which is a non-profit organisation dedicated to supporting
cocoa research and consisting of many industrial members.
There are several reasons why this picture might appear
skewed and these are discussed in a recent commentary on
this subject
60
. In short, an industry-funded study is likely to
be conducted with a foodstuff already considered to be a
likely possibility for success as prior in vitro research and
product development would have narrowed down the poten-
tial candidates. If there has been a null or negative result in
a study, then it is likely the industry would possess the
resources to try again with a modified study. In addition,
journals tend to be less interested in publishing null results
and so this can distort the overall scientific area. However,
investigators already avoid such studies and rarely apply for
(and more rarely receive) grants that are designed to observe
little to no effect, even from public agencies. On the other
hand, industry support is certainly less likely to be requested
for studies into potentially negative effects unless there is a
health and safety issue. However, the other important issue
is that if industry had not been involved, would the area of
interest exist and would valuable faculty research capacity
be so directed? This may be especially true of cocoa polyphe-
nol research which has been obviously dominated by indus-
try-funded studies since its inception. Overall, the main
point to consider is that all the papers described here were
published in peer-reviewed journals and therefore must be
considered trustworthy and reliable; otherwise there is a
need to investigate the integrity of the review process. One
way for industry (and academia) to improve transparency of
ongoing human trials would be to formally register with
one of the public domain agencies, such as with the National
Institutes of Health (www.clinicaltrials.gov), at the beginning
of any study. The advantage is that a null or negative study
would still be public knowledge, and this could help bring
more balance to this area of research.
Future research directions
For the future, we recommend that since cocoa is accepted as
a dietary source of polyphenols, future studies should focus on
specific mechanisms of action, i.e. inflammatory pathways,
and not direct antioxidant effects, with more diversification
on non-vascular end-points. Human intervention trials should
be conducted that use a relevant amount (e.g. about 40 g/d,
10 % of a 8369 kJ/d (2000 kcal/d) diet) of chocolate, an
amount most people could readily incorporate into their diet.
In addition, the composition of the cocoa or chocolate must
be carefully defined with regard to the proportions of polyphe-
nols in the monomeric, oligomeric and polymeric forms, as
Fig. 2. A simplified representation of the hypothetical action of antioxidants
on the health status of an apparently healthy person or a person under
chronic stress. , The status of health, e.g. in relation to inflammation; ,
the status of health when the person is under chronic stress; , only a small
improvement in health status with an optimal dose of an antioxidant under
conditions of minimal stress; , the worsening health status after a stress
event such as UV exposure, smoke inhalation, inflammation or oxidative
stress; , the improvement in health status when an optimal dose of an anti-
oxidant is given whilst in a state of stress.
K. A. Cooper et al.8
British Journal of Nutrition

well as the concentrations of the fats, sugars and other com-
ponents such as proteins from milk solids. Further studies on
milk chocolate to settle the bioavailability debate are most
definitely required.
Separating the effects of specific compounds could also
prove fruitful as there is less information on the larger pro-
cyanidins and their health effects. There is also the question
of attributing beneficial effects to cocoa polyphenols v.
cocoa as a whole. Other compounds in cocoa are known to
be bioactive such as caffeine and theobromine
61
.
The bona fide health effect of cocoa polyphenols will not be
answered short of a large-scale epidemiological study or long-
term interventions. The only epidemiological papers to date
gave intriguing results
21,22
but without more corroboration
the question will remain unanswered. Whilst long-term inter-
ventions will be difficult or impossible to blind, it should pro-
ceed with the best controls possible, because without them,
conclusion of the benefits of chocolate on changing disease
risk will remain tenuous. To obtain results in a reasonable
time frame and with the most likelihood of a significant
result, we suggest targeting future trials to populations that
are under antioxidant stress or deficiency due to a poor diet,
chronic disease, ageing or have an elevated risk of CVD for
other reasons. Only with such results will it be possible to
assess definitively whether or not cocoa and chocolate,
which was originally only a decadent indulgence, can affect
public health.
We would like to suggest this checklist for future planning
of cocoa and chocolate trials, although it is not exhaustive and
designed only to help in future studies.
1. Where possible, conduct randomised, controlled, cross-
over, multi-dose trials.
2. Use well-defined cocoa or chocolate (if possible, for
industry to allow similar cocoa/chocolate to be available
for independent researchers for future studies/repeating
work).
3. Ensure bioavailability of the active component from its
matrix.
4. Use an appropriate control of no-polyphenol chocolate.
5. Recruit volunteers with at least one non-optimal bio-
marker or disease risk factor.
6. Use a dose of cocoa or chocolate that can readily be
incorporated into the daily diet, giving appropriate dietary
advice to volunteers on balancing energy.
7. Measure composition including the polyphenol profile of
the cocoa or chocolate before and after the trial (check for
stability on storage or batch variations).
8. Ensure the final publication contains the analytical results
along with the appropriate description of analytical
methodology.
9. Carefully assess the biological relevance of the chosen
biomarker, with special attention to antioxidant
biomarkers.
10. Strive for transparency by registering human trials before
they start with a recognised database, e.g. www.clinical-
trials.gov.
11. Attempt to publish null or negative results to enable bal-
ancing of the literature and preventing needless dupli-
cation of work. Challenge journals if papers are rejected
on this basis.
Acknowledgements
The idea for this article was generated by Gary Williamson.
All authors contributed equally for intellectual input and writ-
ing of the manuscript. G. Williamson and K. Cooper are
employed by the Nestle
´Research Center. J. Donovan receives
a research grant from the Nestle
´Research Center.
A. Waterhouse declares no conflict of interest. There was no
specific funding for this work.
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