Food Chemistry 174 (2015) 214218

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Short communication

Effects of bioactive constituents in functional cocoa products

on cardiovascular health in humans
Beatriz Sarri a,, Sara Martnez-Lpez a, Jos Luis Sierra-Cinos b, Luis Garcia-Diz b, Luis Goya a,
Raquel Mateos a, Laura Bravo a
a
Department of Metabolism and Nutrition, Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Spanish National Research Council (CSIC), Jos Antonio Novais 10,
28040 Madrid, Spain
b
Department of Nutrition and Bromathology I, School of Pharmacy, Complutense University of Madrid (UCM), Ciudad Universitaria, s/n, 28040 Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Cocoa manufacturers are producing novel products increasing polyphenols, methylxanthines or dietary
Received 29 April 2014 bre to improve purported health benets. We attempt to explain the contribution of cocoa bioactive
Received in revised form 10 September compounds to cardiovascular effects observed in previous studies, placing particular emphasis on
2014
methylxanthines. We focused on a soluble cocoa product rich in dietary bre (DFCP) and a product rich
Accepted 1 November 2014
Available online 7 November 2014
in polyphenols (PPCP). Effects of regularly consuming DFCP (providing daily 10.17 g, 43.8 mg and
168.6 mg of total-dietary-bre, avanols and methylxanthines, respectively) as well as PPCP (providing
daily 3.74 g, 45.3 mg and 109.8 mg of total-dietary-bre, avanols and methylxanthines, respectively)
Chemical compounds studied in this article:
( ) Catechin (PubChem CID: 73160)
on cardiovascular health were assessed in two controlled, cross-over studies in free-living normocholes-
( ) Epicatechin (PubChem CID: 72276) terolemic and moderately hypercholesterolemic subjects. Both products increased HDL-cholesterol
Procyanidin B1 (PubChem CID: 11250133) concentrations, whereas only DFCP decreased glucose and IL-1b levels in all subjects. Flavanols appeared
Procyanidin B2 (PubChem CID: 122738) to be responsible for the increase in HDL-cholesterol, whereas insoluble-dietary-bre and theobromine in
Theobromine (3-7 Dimethylxanthine, DFCP were associated with the hypoglycemic and anti-inammatory effects observed.
PubChem CID: 5429) 2014 Elsevier Ltd. All rights reserved.
Caffeine (Methyltheobromine, PubChem
CID: 2519)
Theophylline (1,3-dimethylxanthine,
PubChem CID: 2153)

Keywords:
Soluble cocoa products
Dietary bre
Polyphenols
Methylxanthines
Cardiovascular health
Hypoglycemic
Inammation

1. Introduction
In recent years drastic changes have taken place in dietary hab-
its throughout Mediterranean populations, and a marked increase
in cardiovascular disease mortality is occurring (Gomez-Huelgas
et al., 2011). In Spain, the consumption of meat, sh, fruit and dairy
products has increased at the expense of consuming less vegeta-
bles, cereals and legumes (Carbajal, 2013). In contrast, cocoa
remains a popular foodstuff worldwide. Soluble cocoa products
Corresponding author. Tel.: +34 915492300; fax: +34 915493627.
E-mail address: beasarria@ictan.csic.es (B. Sarri).
have been popular in Spain, among other countries, being con-
sumed twice a day, at breakfast and as part of an evening break
known as merienda. Cocoa is an important source of avonoids,
mainly avanols, epicatechin and catechin and low molecular
weight procyanidins such as procyanidin B1 and B2, methylxan-
thines (mainly theobromine), and magnesium; all are biologically
active substances that may also affect human health positively
(Ellam & Williamson, 2013). Cocoa is also a relevant source of die-
tary bre (DF) in contrast to chocolate (Sarri et al., 2012).
The cardio-protective effects of cocoa are well established as
several comprehensive reviews recently published have shown
(Arranz et al., 2013; Ellam & Williamson, 2013). In an attempt to
improve potential health properties, cocoa manufacturers are

http://dx.doi.org/10.1016/j.foodchem.2014.11.004
0308-8146/ 2014 Elsevier Ltd. All rights reserved.
 B. Sarri et al. / Food Chemistry 174 (2015) 214218 215

producing new functional cocoa products enriched with bioactive Table 1

components, with reduced energy (fat and sugar content). Flavanol, dietary bre and methylxanthine composition of the cocoa product rich in
dietary bre (DFCP) and the product rich in avanols (PPCP).
Recently, we have shown that two soluble cocoa products rich in
avanols and DF increased serum HDL-cholesterol concentrations DFCP PPCP
in healthy and moderately hypercholesterolemic (20002400 mg/ Total polyphenols (mg equiv gallic acid/g 15.75 0.67 34.04 2.28
L) subjects, the DF rich product also decreased glucose, IL-1b and product)
IL-10 levels (Martnez-Lpez et al., 2014; Sarri et al., 2014). Total avanols (mg/g dry matter) 1.46 0.32 3.02 0.35
IL-1b is a pro-inammatory cytokine that induces transient and Epicatechin (mg/g dry matter) 0.31 0.09 1.26 0.18
reversible endothelial dysfunction in humans as IL-1b can activate Catechin (mg/g dry matter) 0.60 0.12 0.47 0.03
Procyanidin B1 (mg/g dry matter) n.d. 0.20 0.04
NF-jB, which binds to specic sites on the promoter regions of tar- Procyanidin B2 (mg/g dry matter) 0.55 0.11 1.09 0.10
get genes, and this, in turn, modulates the endothelial synthesis of
Total dietary bre (%) 33.90 1.80 24.90 1. 64
other proinammatory cytokines (IL-1, IL-6 and TNF-a) and che- Soluble dietary bre (%) 1.68 0.13 3.13 0.59
mokines such as IL-8 and MCP-1 (Koer, Nickel, & Weis, 2005). This Neutral sugars (%) 0.69 0.04 2.46 0.43
work elaborates on the relative contribution of bioactive com- Uronic acid (%) 0.99 0.09 0.67 0.16
pounds, namely avonoids, DF and methylxanthines, from soluble Insoluble dietary bre (%) 32.22 1.67 21.77 1.05
Neutral sugars (%) 19.06 1.60 10.49 0.96
cocoa products, to the observed health benet.
Uronic acid (%) 1.26 0.07 1.47 0.09
Klason lignin (%) 11.90 0.28 9.81 0.21

2. Material and methods Total methylxanthines (mg/g dry matter) 5.62 0.19 7.32 0.93
Theobromine (mg/g dry matter) 5.11 0.14 6.43 0.84
Theophylline (mg/g dry matter) n.d. 0.01 0.01
2.1. Cocoa powder products Caffeine (mg/g dry matter) 0.51 0.05 0.88 0.08

The commercialised soluble cocoa products used in the studies Values represent mean SD, n = 6; n.d.: not detected.

were provided by Nutrexpa S.L. As described in Bravo and Saura-

Calixto (1998), cocoa extracts were obtained by washing 1 g of
defatted cocoa with 40 mL of 50% aqueous methanol (HPLC grade;
Panreac, Castellar del Valls, Spain) containing 0.8% of 2 mol/L
hydrochloric acid (Panreac) for 1 h at room temperature with con-
stant shaking. Afterwards, samples were centrifuged (10 min,
3000g) and supernatants were collected. Residues obtained were
successively extracted with 40 mL of 70% acetone (v/v; Panreac)
in water (1 h, constant shaking). Then samples were centrifuged
(10 min, 3000g) and supernatants were collected. Finally, superna-
tants obtained after each extraction step were combined and made
up to 100 mL. Polyphenolic and methylxanthine composition was
characterised by high-performance liquid chromatography (HPLC)
with diode-array detection (DAD) using an Agilent 1200 series
equipment (Martin et al., 2008). Quantication was achieved in
comparison with known standards [catechin, epicatechin, procy-
anidin B1 and theobromine were purchased from SigmaAldrich
(St. Louis, USA), procyanidin B2 from Extrasynthese (Genay,
France), and caffeine from Fluka Chemika (Buchs, Switzerland)].
Total polyphenols were measured spectrophotometrically using
the FolinCiocalteu reagent and gallic acid (SigmaAldrich), as
standard. Total DF analysis was carried out following the same
procedure described in Sarri et al. (2014). The total polyphenol,
avanol, DF and methylxanthine composition of the soluble cocoa
product rich in DF (DFCP) and the soluble cocoa product rich in
polyphenols (PPCP) is shown in Table 1.
in polyphenol intake and to be able to attribute the results
observed to the cocoa products. The daily consumption of ava-
nols, DF and methylxanthines provided by DFCP and PPCP is shown
in Table 2.
2.3. Subjects
The studies were conducted according to the guidelines laid
down in the Declaration of Helsinki and all procedures were
approved by the Clinical Research Ethics Committee of Hospital
Universitario Puerta de Hierro Majadahonda in Madrid (Spain).
Volunteer recruitment was carried out through placing advertise-
ments in the Universidad Complutense campus as well as through
giving short talks between lectures. The inclusion criteria were:
total cholesterol of <2000 and >20002400 mg/L (<5.172 and
>5.1726.206 mmol/L) for the normocholesterolemic and hyper-
cholesterolemic groups, respectively, being non-vegetarian,
non-smoker, men and women (not including pregnant women),
between 18 and 55 y old, not suffering from any chronic pathology
or gastrointestinal disorder. None had taken dietary supplements,
laxatives, or antibiotics six months before the start of the study.
Their body mass index was under 30 kg/m2.
Fifty subjects were initially accepted to participate in the studies
and gave informed written consent; however, only 44 completed
both interventions (baseline characteristics are given in Table 3).

Table 2
2.2. Study design Daily intake of the cocoa product rich in dietary bre (DFCP) and the product rich in
polyphenols (PPCP) and the corresponding daily intake of bioactive compounds.
Consecutively, two controlled studies were carried out in DFCP PPCP
free-living healthy and moderately hypercholesterolemic (2000
Cocoa dose (g) 30.0 15.0
2400 mg/L; 5.1726.206 mmol/L) subjects to assess the effects of
consuming DFCP (Sarri et al., 2014) and PPCP (Martnez-Lpez Total avanols (mg) 43.80 45.30
Epicatechin (mg) 9.30 18.90
et al., 2014) on markers of cardiovascular health. Each study con- Catechin (mg) 18.00 7.05
sisted of a two-week run-in, four-week milk stage (control) and Procyanidin B1 (mg) 0.00 3.00
four-week cocoa intervention. The doses of cocoa consumed in Procyanidin B2 (mg) 16.50 16.35
both studies corresponded with consumption patterns as recom- Total dietary bre (g) 10.17 3.74
mended by the manufacturer: 15 g per serving of the DFCP twice Soluble dietary bre (g) 0.50 0.47
a day (30 g/day), and half that amount of PPCP, 7.5 per serving of Insoluble dietary bre (g) 9.67 3.27
PPCP twice per day (15 g/day) since this product had a strong cocoa Total methylxanthines (mg) 168.60 109.80
avour. For the duration of the studies, other cocoa products, cer- Theobromine (mg) 153.30 96.45
Theophylline (mg) 0.00 0.15
tain fruits and vegetables rich in polyphenols, and their derived
Caffeine (mg) 15.30 13.20
beverages, were restricted to reduce inter-individual differences
216 B. Sarri et al. / Food Chemistry 174 (2015) 214218

Table 3
Baseline characteristics of the participants in the study.

Normocholesterolemic (n = 24) Hypercholesterolemic (n = 20)

Men (n = 11) Women (n = 13) Men (n = 9) Women (n = 11)
Age (y) 28.1 7.9 26.1 6.1 35.7 11.2 25.4 6.8
Weight (kg) 75.2 10.6 60.3 7.7 81.0 14.9 58.8 5.5
BMI (kg/m2) 24.1 3.6 22.0 2.6 26.2 4.2 22.4 2.3
Systolic BP (mmHg) 117.4 10.2 115.1 9.1 122.9 15.3 118.9 10.4
Diastolic BP (mmHg) 71.8 8.0 72.8 8.4 79.8 10.3 73.9 8.4
Heart rate (bpm) 65.1 10.1 69.1 9.8 63.8 9.5 82.2 8.2

SD = standard deviation. BMI = body mass index. BP = blood pressure. bpm = beats per minute.

3. Results and discussion
The most signicant nding observed after consuming either
DFCP or PPCP was increased HDL-cholesterol (p < 0.001, Fig. 1).
This increment was greater in the normocholesterolemic subjects
in both interventions (77 and 91 mg/L with DFCP and PPCP, respec-
tively, compared to baseline) than in the dyslipidemic patients (51
and 60 mg/L with DFCP and PPCP, respectively).
To date, a body of scientic evidence points to polyphenols as
the compounds responsible for the benecial cardiovascular effects
associated with cocoa (Arranz et al., 2013; Baba et al., 2007; Ellam
& Williamson, 2013; Hooper et al., 2008; Khan et al., 2012; Shrime
et al., 2011). DFCP and PPCP provided similar daily amounts of
cocoa avanols (Table 2) since the avanol concentration in PPCP
was twice that of DFCP but the amount of cocoa daily consumed
was half that in the PPCP intervention; as a consequence, the daily
consumption of avanols was 45.3 mg (PPCP) and 43.8 mg (DFCP),
respectively and, thus, the intake of phenolic compounds was sim-
ilar. Interestingly, DFCP contained less epicatechin than PPCP (0.31
vs. 1.26 mg/g, respectively) whilst there was more catechin in
DFCP than in PPCP (0.60 vs. 0.47 mg/g, respectively). It is likely that
processing during manufacture is responsible for these differences,
as roasting is a crucial step in technical treatment of cocoa that
causes avanol losses and/or modication, especially the epimer-
ization of ( )-epicatechin to ( )-catechin (Kothe, Zimmermann,
& Galensa, 2013).
As for DF intake, during the DFCP intervention, subjects con-
sumed over 10 g cocoa DF/d in comparison with less than 4 g/
day in the PPCP (Table 2). This large difference in DF intake should
have resulted in distinctive changes in HDL-cholesterol values as
each intervention would be expected to have a direct effect on
HDL metabolism. These results contrast with those described by
(A) (B)
HDL-C (mg/L) HDL-C (mg/L)
800 800
b b
a 700 b
700
a b a
b b
600 a a 600
a
500 500
400 400
Normo-C Hyper-C Normo-C Hyper-C
Baseline Milk interv on (control) Cocoa interv
Fig. 1. Effects of consuming (A) the cocoa product rich in bre (DFCP) and (B) the
cocoa product rich in polyphenols (PPCP) on HDL-cholesterol (HDL-C) levels in
normocholesterolemic (Normo-C) and hypercholesterolemic (Hyper-C) subjects.
According to the general linear model of the variance for repeated measures
analysis, the effect of consuming DFCP (p < 0.001) and PPCP (p < 0.001) on HDL-C
was statistically signicant, not showing differences between the groups. Different
letters (a and b) denote signicant differences according to the Bonferroni test
within the normocholesterolemic or hypercholesterolemic group.
Jenkins et al. (2000) who reported a signicant increase in HDL-
cholesterol after consumption of a cocoa-bran (25 g DF/d) for two
weeks by healthy subjects, although they did not analyse the poly-
phenolic content. However, in a previous open intervention trial
(without control or randomization) among free-living, moderately
hypercholesterolemic volunteers who consumed daily two serv-
ings of a bre-rich cocoa product, which provided a slightly more
total DF (12 g/day) than in our studies and 30% less soluble poly-
phenols (283 mg/day of polyphenols), HDL-cholesterol was not
signicantly increased 3% after eight weeks compared with base-
line values (Sarri et al., 2012).
Recently, Neungerl, Zebregs, Schuring, and Trautwein (2013)
found that theobromine independently increased HDL-cholesterol
in a two-centre, double-blind, randomized, placebo-controlled, full
factorial parallel design in healthy subjects, suggesting methylxan-
thine was responsible for increased HDL-cholesterol. In our studies,
DFCP increased daily theobromine intake by 60% compared with
PPCP (Table 2). However, HDL-cholesterol, compared with baseline,
was 13 and 9% higher in the normo- and hypercholesterolemic sub-
jects, respectively, after consuming the DFCP for four weeks, and
16% and 12% in healthy and dyslipidemic volunteers, respectively,
after the PPCP. Therefore, based on these results, theobromine did
not have a major contribution to the HDL-cholesterol increase,
since DFCP consumption did not increase HDL-cholesterol levels
higher than after consuming PPCP in spite of DFCP being a richer
source of methylxanthines.
From the cardiovascular perspective, our results point to cocoa
avonoids as responsible for the HDL-cholesterol enhancing effect,
with epicatechin and catechin having similar biological activity.
Flavanols may exert this effect through different mechanisms, such
as increased expression of scavenger receptor B type I (SR-BI), ste-
rol regulatory element binding proteins (SREBPs), ATP binding cas-
sette transporter A1 (ABCA1) or apolipoprotein A1, among others
(Martnez-Lpez et al., 2014).
The second most important effect was a decrease in fasting
plasma glucose (p = 0.029) after consuming DFCP, but not PPCP
(Fig. 2). This result is in agreement with the signicant decrease
in fasting plasma glucose (p = 0.019) observed in a previous study
with a DF bre-rich cocoa product (Sarri et al., 2012). Polyphenol-
rich cocoa products have been shown to reduce fasting and post-
prandial blood glucose as well as insulin resistance (Almoosawi,
Tsang, Ostertag, Fyfe, & Al-Dujaili, 2012; Grassi, Lippi, Necozione,
Desideri, & Ferri, 2005a, 2005b). However, the similar polyphenol
intake during both interventions does not support the different
responses for fasting glucose levels. Instead, differences in plasma
on glucose concentration points to a prominent effect of DF since it is
well known that DF rich foods delay glucose absorption from the
small intestine (Giacco et al., 2000) and improve insulin sensitivity
(Tokede, Gaziano, & Djouss, 2011).
Methylxanthines in DFCP, particularly theobromine, and to a
lesser extent caffeine, may have a role in lowering plasma glucose
as suggested by Kelly (2005), since methylxanthines inhibit phos-
phodiesterase and, thereby, increase intracellular concentration of
 B. Sarri et al. / Food Chemistry 174 (2015) 214218 217

(A) (B) (A) (B)

Glucose (mg/L) Glucose (mg/L) IL-1 (pg/mL) IL-1 (pg/mL)

900 900 5 5
4 4
800 800
3 3
700 700 2 2
1 1
600 600
0 0
500 500 Normo-C Hyper-C Normo-C Hyper-C
Normo-C Hyper-C Normo-C Hyper-C
(C) (D)
Baseline Milk intervenon (control) Cocoa intervenon
IL-10 (pg/mL) IL-10 (pg/mL)
Fig. 2. Effects of consuming (A) the cocoa product rich in bre (DFCP) and (B) the 20 20
a
cocoa product rich in polyphenols (PPCP) on glucose levels in normocholestero- a
lemic (Normo-C) and hypercholesterolemic (Hyper-C) subjects. According to the 15 a 15
general linear model of the variance for repeated measures analysis, the effect of ab
b
consuming DFCP (p = 0.029) on glucose levels was statistically signicant, in 10 b 10
contrast to PPCP, not showing differences between the groups. According to the
Bonferroni test, there were no differences within the normocholesterolemic or 5 5
hypercholesterolemic group.
0 0
cyclic AMP (cAMP) in pancreatic cells, antagonizing P1 purine Normo-C Hyper-C Normo-C Hyper-C
receptors (Bruinsma & Taren, 1999). In contrast, methylxanthines
Baseline Milk intervenon (control) Cocoa intervenon
are negatively coupled to adenylate cyclase in hepatocytes and
induce a decrease in cAMP concentration (Doyle & Egan, 2003). Fig. 3. Effects of consuming (A) the cocoa product rich in bre (DFCP) and (B) the
Because both insulin secretion from pancreatic cells and liver glu- cocoa product rich in polyphenols (PPCP) on IL-1b and effects of consuming DFCP
cose output are directly dependent on the intracellular concentra- (C) and PPCP (D) on IL-10 levels in normocholesterolemic (Normo-C) and
tion of cAMP, it is reasonable to speculate that methylxanthines hypercholesterolemic (Hyper-C) subjects. According to the general linear model
of the variance for repeated measures analysis, the effects of consuming DFCP on IL-
may inuence glucose metabolism positively in humans (Grassi
1b was statistically signicant (p = 0.001), in contrast to PPCP, not showing
et al., 2005a). differences between the groups; whereas the effect of both DFCP and PPCP on IL-
The third most relevant outcome observed after regularly con- 10 (p = 0.001 and p = 0.022, respectively) were signicant. Different letters (a and b)
suming DFCP, in contrast to PPCP, was the decrease in IL-1b levels denote signicant differences according to the Bonferroni test within the normo-
(p = 0.001; Fig. 3). Again, the intake of cocoa avonoids in both cholesterolemic or hypercholesterolemic group. The absence of letters indicates
that there were no differences according to the paired test.
interventions would not support a direct effect of polyphenols.
There is evidence supporting an anti-inammatory effect of cocoa
polyphenols decreasing serum C-reactive protein (CRP) levels as although knowledge about the effects of methylxanthines on
well as different cytokines (IL-2, IL-5, TNF-a, TGF-b), although inammation is scarce, these compounds have shown to reduce
absence of any measurable impact has also been reported (revised allergic inammation (Tilley, 2011). Thus, perhaps, the higher
in Arranz et al., 2013; Ellam & Williamson, 2013). The use of cocoa theobromine content in DFCP may be responsible for the reduction
products with different phenolic composition in distinct sub-pop- in IL-1b. However, the effects of DFCP on inammation are not
ulation groups (healthy, obese, dyslipidemic, diabetic, etc.) could clear and should be investigated further as a decrease in anti-
account for these apparently contradictory ndings. inammatory cytokine IL-10 was also observed.
In the present work, the effect of DFCP on IL-1b levels is in
agreement with previous studies that show that a high-bre diet 4. Conclusions
is associated with lower plasma levels of other pro-inammatory
cytokines, IL-6, and TNF-R2, in the absence of an association Among the bioactive constituents in the two functional cocoa
between DF and CRP (Ma et al., 2008). In contrast, whole grain products, avanols seem to be responsible for increased HDL-
intake has been associated with lower concentrations of CRP in cholesterol, observed after consuming either product, whereas
epidemiological and intervention studies (Jonnalagadda et al., the insoluble DF and theobromine in the bre rich product were
2011). In fact, epidemiologic studies show that cereal bre, a mar- associated with the hypoglycemic and anti-inammatory effects
ker of whole-grain intake, and mainly insoluble DF, offer protection observed after consuming only the bre rich product, although
from CVD and diabetes (Du et al., 2010). To our knowledge, there is the effects on inammation need to be further claried.
no other study that associates the intake of insoluble DF with a
decrease in IL-1b. However, there are studies that show an associ- Acknowledgements
ation between insoluble DF intake and other pro-inammatory
molecules, such as Qi et al. (2006) who observed high whole grain This study was funded by Nutrexpa S.L. Project Consolider-
intake was associated with lower CRP and TNF-a levels among Ingenio (CSD2007-00063) from the Spanish Ministry of Science
women with type 2 diabetes. Other authors described an inverse and Innovation is also acknowledged. S.M.-L. thanks the Spanish
association between whole grain intake and CRP concentrations National Research Council for her predoctoral fellowship under
(Jensen et al., 2006; Lutsey et al., 2007). Accordingly, Katcher the JAE-Pre programme funded by the European Social Fund.
et al. (2008) observed a 38% reduction in CRP concentrations
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