Nitric Oxide 29 (2013) 13

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Nitric Oxide
journal homepage: www.elsevier.com/locate/yniox

Nitric oxide mediates the antihypertensive and vascular relaxing

effects of a soluble cocoa ber product in spontaneously hypertensive rats
S. Fernndez Vallinas a, N. Lpez Carreras a, M. Miguel b,, A. Aleixandre a
a
b

Dpto. Farmacologa, Facultad de Medicina, Universidad Complutense, Avda. Complutense s/n, 28040 Madrid, Spain
Instituto de Investigacin en Ciencias de Alimentacin (CSIC-UAM, CEI-UAM+CSIC), C/Nicols Cabrera 9, 28049 Madrid, Spain

a r t i c l e

i n f o

Article history:
Received 11 July 2012
Revised 9 October 2012
Available online 9 November 2012
Keywords:
Cocoa
Endothelium
Fiber
Hypertension
Nitric oxide
Spontaneously hypertensive rats

a b s t r a c t
The involvement of endothelial-relaxing factors on the vascular and antihypertensive effects of a cocoa
ber product (CFP) obtained from cocoa husks was studied. We carried out in vitro experiments with
aorta rings from untreated spontaneously hypertensive rats (SHR) and in vivo experiments with SHR.
CFP did not relax the endothelium denuded aorta rings and NW-nitro-L-arginine methyl ester (L-NAME)
partially blocked the vascular relaxing and antihypertensive effects of CFP. Nevertheless, indomethacin
did not modify these effects. Nitric oxide mediates therefore the antihypertensive and aorta relaxing
effects of CFP in SHR.
2012 Elsevier Inc. All rights reserved.

1. Introduction
Arterial hypertension is a chronic medical condition in which
the blood pressure in the arteries is elevated. This pathology is a
major risk factor for many other cardiovascular alterations and
one of the more important causes of clinical death in the world.
Even moderate elevation of arterial blood pressure is associated
with a shortened life experience. Drug treatment is often necessary
in hypertensive people, but dietary and lifestyle changes can also
improve blood pressure and decrease the risk of hypertension
associated health complications. Moreover, drugs are often ineffective or insufcient to control this variable and frequently have
undesirable side effects. The research of dietary strategies to control arterial blood pressure are therefore always desirable. It is well
known from many years ago that ber prevents cardiovascular disease [1,2] and ber could be particularly useful in patients with
metabolic syndrome [3]. Nevertheless, the effect of ber on arterial
blood pressure has been studied to a much lesser extent than other
healthy effects of ber. We have characterized the short- [4] and
long-term [5] antihypertensive effect of a cocoa ber product

Abbreviations: CFP, cocoa ber product; eNOS, endothelial NO synthase;

NW-nitro-L-arginine methyl ester; MDA, malonmalondialdehyde; NO,
nitric oxide; NOS, NO synthase; SBP, systolic blood pressure; SHR, spontaneously
hypertensive rats.
Corresponding author. Address: Instituto de Investigacin en Ciencias de
Alimentacin (CIAL, CSIC-UAM), C/Nicols Cabrera 9, 28049 Madrid, Spain.
E-mail address: marta.miguel@csic.es (M. Miguel).

L-NAME,

1089-8603/$ - see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.niox.2012.10.006

(CFP) obtained from cocoa husks via a patented enzymatic process

[6] in spontaneously hypertensive rats (SHR). The aim of this research was to elucidate the involvement of endothelial relaxing
factors in the antihypertensive and vascular relaxing effects of
CFP in these animals.
2. Material and methods
2.1. Animals
Male SHR obtained from Charles River Laboratories Spain were
used. The rats were maintained at a temperature of 23 C with 12 h
light/dark cycles, and consumed tap water and a standard diet
(A04 Panlab, Barcelona, Spain) ad libitum during the experiments.
They were used for in vitro and in vivo experiments.
2.2. In vitro experiments
For the in vitro experiments, we used aorta rings from untreated
2428 week-old SHR, that were mounted in traditional organ
baths. After the equilibration period, the rings were rst contracted
by 80 mmol/L KCl to assess their functionality and when the contraction had reached the steady state (about 15 min after the
administration), the preparations were washed until the basal tension was recovered. Then the rings were exposed to 10 5 M
methoxamine, and doseresponse curves to CFP (10 610 3 mg/
ml) were performed in the methoxamine-precontracted rings.

S. Fernndez Vallinas et al. / Nitric Oxide 29 (2013) 13

Relaxant responses to CFP were expressed as a percentage of the

precontraction induced by methoxamine. The previously described
procedure was applied to intact and endothelium-denuded tissue.
It was also applied to another two groups of intact preparations;
one with the addition of NW-nitro-L-arginine methyl ester (LNAME) (10 4 M), and the other one with the addition of indomethacin (10 5 M). Both drugs were added to the organ bath 30 min before methoxamine administration. The denuded endothelium
preparations were prepared by gently rubbing the tissue before it
was cut into rings. The efcacy of the procedure used to remove
the endothelial cells was judged by the loss of acetylcholine-induced relaxation when the aorta preparations were precontracted
with methoxamine. According to the study published by Furchgott
in 1980 [7,8], the endothelial cells are necessary for this response.

0
10

RELAXATION (%)

20
30
40
50
a
a

60
70
6

CFP Log (ng/ml)

For the in vivo experiments, a total of thirty 1720 week-old

male SHR were used. They were divided into two groups of fteen
animals, that were respectively administered by gastric intubation
1 ml distilled water or 300 mg/kg CFP dissolved in the same
volume of distilled water. Four hours after these administrations,
ve of the animals in each group were intraperitoneally administered 1 ml saline. The remaining rats in both groups were divided
into another two groups of ve animals that were respectively
administered, by the same procedure, 30 mg/kg L-NAME or 5 mg/
kg indomethacin, both dissolved in 1 ml of saline. Systolic blood
pressure (SBP) was recorded in the different rats by the tail cuff
method [9] before the initial oral administration and also 6 h after.
All the above-mentioned experiments were performed as
authorized for scientic research (European Directive 86/609/CEE
and Royal Decree 223/1988 of the Spanish Ministry of Agriculture,
Fisheries and Food).

Fig. 1. Soluble cocoa ber product (CFP) relaxation in different aorta ring
preparations from spontaneously hypertensive rats: intact (j), endothelium
denuded (h), intact incubated with L-NAME (10 4 M) (d), intact incubated with
indomethacin (10 5 M) (N). The aorta rings were precontracted with 10 6 M
methoxamine. Data are mean values SEM. for at least 6 preparations and 6
animals. Similar letters represent no statistical differences (P > 0.05). P was
estimated by two-way ANOVA (Bonferroni Test).

15
a,c

SBP (mm Hg)

2.3. In vivo experiments

b,c
a,b

-5

-15

-25

2.4. Statistical analysis

-35

The results are expressed as mean values standard error of the

mean (SEM) and were analyzed by a one-way or two-way analysis
of variance (ANOVA), using the GraphPad Prism software. Differences between the groups were assessed by the Bonferroni test.
Differences between the means was considered to be signicant
when P < 0.05.

Fig. 2. Changes in systolic blood pressure (SBP) caused in spontaneously hypertensive rats after different treatments: water + saline (j), 300 mg/kg cocoa ber
product (CFP) + saline ( ), water + 30 mg/kg L-NAME (h), 300 mg/kg CFP + 30 mg/
kg L-NAME ( ). Data are expressed as mean SEM. The experimental groups
always have 5 animals. Similar letters represent no statistical differences (P > 0.05).
P was estimated by one-way ANOVA (Bonferroni Test).

3. Results and discussion

SBP (mm Hg)

CFP relaxed the intact aorta ring preparations in a dose dependent manner. This effect was almost abolished in the endotheliumdenuded preparations and partially blocked in the intact rings treated with L-NAME, but it was not modied by indomethacin (see
Fig. 1). As expected, in the in vivo experiments, the SHR that only
received water or saline did not modify their SBP. On the contrary,
CFP caused a signicant decrease in SBP. L-NAME caused a slight
increase in SBP in the rats, and the effect of CFP was not observed
in the SHR that were treated with L-NAME (see Fig. 2). Nevertheless, indomethacin treatment did not modify SBP in the SHR and
this drug failed also to modify the antihypertensive effect of CFP
in these animals (see Fig. 3).
For this study, we have used SHR, a well known experimental
model for essential hypertension in humans [10]. SHR are usually
used nowadays to evaluate ingredients for functional foods [11
13] and, according to our previous research carried out in these
animals, a clear decrease in arterial blood pressure was observed
6 h post-administration of 300 mg/kg CFP [4]. The present study
corroborates the short-term antihypertensive effect of CFP in
SHR. The benecial effect of cocoa and cocoa derivatives on cardio-

15

-5

-15

-25
b

-35
Fig. 3. Changes in systolic blood pressure (SBP) caused in spontaneously hypertensive rats after different treatments: water + saline (j), 300 mg/kg cocoa ber
product (CFP) + saline ( ), water + 5 mg/kg Indomethacin (h), 300 mg/kg
CFP + 5 mg/kg Indomethacin ( ). Data are expressed as mean SEM. The experimental groups always have 5 animals. Similar letters represent no statistical
differences (P > 0.05). P was estimated by one-way ANOVA (Bonferroni Test).

vascular disease, and in particular in the hypertensive pathology,

has been extensively studied. Cocoa and cocoa derivatives are

S. Fernndez Vallinas et al. / Nitric Oxide 29 (2013) 13

known as a signicant source of antihypertensive avonoids, particularly avan-3-ols and procyanidins, and some studies have
demonstrated the endothelium dependent effect of these compounds [14,15]. CFP, a cocoa husk derived product, was characterized as a soluble ber with 22.4 1.4 mg/g dry matter polyphenol
content [5] and we have previously proposed that the polyphenols
of this ber product are responsible, at least in part for its antihypertensive properties [4,5]. Therefore, we hypothesized that CFP
could also have endothelium dependent effects. Nitric oxide (NO)
was identied in 1987 as an important endothelial relaxing factor
[16], and the role of NO in arterial tone was clearly established
soon later [17]. This study actually demonstrates the involvement
of the endothelial relaxing factor NO in the blood pressure lowering effect and aorta relaxing effects of CFP in SHR. The results obtained in the endothelium denuded aorta rings obtained from
these animals clearly indicate that this tissue is necessary for the
vascular relaxing effect of the ber product. It was accepted that
the source of endothelial NO was a guanidinium nitrogen of L-arginine and that the enzyme responsible for its formation was an oxygenase called endothelial NO synthase (eNOS) [18]. L-NAME is an
in vivo and in vitro inhibitor of NO synthase (NOS) [19,20] and in
the present study, we have also observed a clear decrease in the
aorta relaxing effects of CFP when the rings were incubated with
L-NAME. A clear increase in SBP was observed in the L-NAME treated SHR. The inhibition of basal NO synthesis by L-NAME in these
animals could justify these results, but what was more important
in the in vivo experiments in order to corroborate our hypothesis
for the present study was the observed impairment of the CFP antihypertensive effect in the L-NAME treated SHR. Except for some
apparent inconsistencies, it become generally accepted that NO
was the main endothelium derived relaxing factor [21]. Nevertheless, the endothelium secretes other vasodilator agents different
from NO, such as prostacyclin. The rst endothelium-derived
relaxing substance described was in fact prostacyclin, which is produced by the action of the cyclo-oxygenase enzyme [22]. In this
study, we have also evaluated the effect of CFP in indomethacin
incubated aorta rings and in SHR intraperitoneally injected this
inhibitor of the cyclo-oxygenase enzyme and the endothelial prostanoid biosynthesis. However, indomethacin treatment did not
modify SBP in the SHR, and this compound clearly failed to modify
CFP antihypertensive effect. In addition, the effect of CFP in the aorta preparations was neither modied by indomethacin. We could
therefore discard the endothelial prostacyclin release as a mechanism implicated in the vascular relaxing and antihypertensive effects of this ber product in SHR. NO is actually an important
mediator of blood pressure homeostasis, and the increase in arterial tone that characterizes the hypertensive state implies frequently an excess of free radicals that destroy this mediator.
Enhanced endothelial superoxide anion production has been
described in hypertension and these effects are related to
impairment of endothelium-dependent relaxation [23,24]. CFP
has demonstrated a high antioxidant capacity in the SHR [25].
CFP also decreased malondialdehyde, a metabolite that could be
considered a biomarker of free radical production, in the plasma
of SHR [5]. We could therefore propose that this ber may decrease
arterial blood pressure in SHR by an increase in the synthesis and/
or the bioavailability of NO. Further investigation should be carried
out nevertheless to clarify the molecular pathways involved in the
benecial effects of CFP to control arterial blood pressure and it
seems clear that future studies should provide more information
in order to use this ber as an antihypertensive food ingredient.
It is also true that further investigation should be carried out to
demonstrate its antihypertensive efciency in humans, but the

present study represents a valuable contribution to clarify the

mechanisms implicated in the antihypertensive effect of CFP.
Acknowledgments
This study was supported by Natraceutical Group (206/2006
UCM Project). We also thank Manuel Bas Caro, Technician in Pharmacology, for his excellent care of the rats. M Miguel holds a Ramon and Cajal work contract.
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