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Department of Internal Medicine, University of California Davis, California 95616-8636
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • cocoa • flavonoids • procyanidins • endothelium-dependent relaxation • nitric oxide synthase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The broad spectrum of the different effects
produced by these compounds has engaged the attention of researchers
for several decades. One recurring theme in these properties is their
antioxidant and free radical–scavenging ability (Kandaswami and Middleton 1996
). However, they also have the capacity to
complex metals and to modulate the action of enzymes (Chen et al. 1990
, Robak and Gryglewski 1988
). When
viewed against this background, it would not be surprising if
flavonoids influenced the contractile state of vascular smooth muscle.
A recent study reported from this laboratory examined the effect of red
wine on aortic smooth muscle. It was shown that red wine induced
endothelium-dependent relaxation
(EDR)3
in rings of rabbit aorta in vitro (Cishek et al. 1997
). Alcohol alone, in comparable concentrations, had
no effect on these rings. Because red wine also contains a variety of
flavonoids, a parallel series of experiments was carried out to
determine whether some of the flavonoids extracted from grape seeds
could mimic the effects of red wine. The flavonoids, which were a
mixture of polymeric procyanidins, also caused EDR. This response was
shown to be mediated by nitric oxide (Cishek et al. 1997
).
Cocoa is a plant product that is particularly rich in procyanidins such as epicatechin and its polymers. It is not known whether the extracts of cocoa have the same effects on vascular smooth muscle as the extracts from grape seed. The current study was undertaken in rings of the rabbit aorta to test two hypotheses: 1) extracts of cocoa, which are rich in procyanidins, cause EDR and 2) extracts of cocoa activate endothelial nitric oxide (NO) synthase (NOS). This study was approved by the Animal Use and Care Committee of the University of California, Davis.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Rabbit aortic rings were obtained as previously described
(Kappagoda et al. 1991
). Fresh aortic rings in Krebs’
buffer were mounted within 20 min; one can expect the rings to be
viable in oxygenated Krebs’ buffer for several hours. The rings were
tested initially to establish that the endothelium was viable by
exposing them to acetylcholine after precontraction with norepinephrine
(10-5 mol/L). The rings can be exposed to repeated doses
of norepinephrine and acetylcholine and repeated washings and still be
viable. Three aortic rings with functional endothelium were
precontracted with norepinephrine (10-5 mol/L). When the
contraction reached a steady state, cumulative doses of cocoa
flavonoids (10-7 to 10-5 mol/L) were added. A
fourth precontracted ring was tested with acetylcholine
(10-7 to 10-5 mol/L) and served as a time
control. The usual variation from one ring to another of the same size
is normally <10%. All four rings were taken from the same rabbit. The
order of addition of cocoa flavonoids was done in increasing dose to
obtain a dose-response curve that is the standard method to
determine vasodilatory capacity of pharmacological compounds. The
curves are obtained as conventional cumulative
dose-response curves, so doses cannot be added in random order (it
is not possible to wash the tissues between doses). These experiments
evaluated the effects of acute exposure to flavonoids.
In selected rings, the effect of
N
-nitro-L-arginine methyl
ester (L-NAME), an inhibitor of NOS, on the responses to procyanidins
was tested. In three rings, the effect of atropine (10-5
mol/L) was examined.
Effect of prolonged exposure to procyanidins.
To define the effect of prolonged exposure to all three compounds, the rings were each incubated with procyanidin (10-5 mol/L) for 30 min, and then the EDR to cumulative doses of acetylcholine was determined. Also, the effect of prior incubation on dose-dependent EDR to procyanidins was examined.
Effect of cocoa procyanidins extract on NOS activity.
To determine the effects of cocoa procyanidins extract on NOS activity,
aortic thoracic segments of a similar size were suspended in
Krebs-Henseleit buffer in an organ bath maintained at 37°C and
under control conditions or exposed to the pentamer and catechin
monomer (10-5 mol/L). After 2 min, the tissues were
removed from the organ bath, and each segment was pinned to a cork
board and cut longitudinally. Endothelial cells were obtained by a
single scrape of the luminal surface of the aorta with a blade. The
cells were placed in Eppendorf tubes containing 70 µl of 50 mmol/L
Tris buffer (pH 7.4) and immediately frozen in liquid nitrogen. Before
freezing, 5-µl aliquots of each sample were mixed with 5 µl of
methylene blue. The stained cells in the suspension were counted in a
hemocytometer using a light microscope. Determination of the
endothelial NO production was carried out by a modification of a
previously described method (Sobey et al. 1995
). The
samples frozen in liquid nitrogen were alternatively thawed and
refrozen five times in a water bath (37°C) to break up intracellular
membranes. Duplicate determinations of the conversion of
L-arginine to L-citrulline were made on each
sample. The cell homogenate (30 µl) was added to the reaction buffer
consisting of 4 µCi of [3H]-L-arginine
(Amersham, Arlington Heights, IL), 2 mmol/L NADPH, 6 µmol/L FAD, 6
µmol/L FMN, 15 µmol/L tetrahydrobiopterin, 1 µmol/L calmodulin
and 1.25 mmol/L CaCl2. To determine whether citrulline
activity was due to NOS activity, a parallel aortic sample boiled at
100°C was analyzed. Samples were incubated for 30 min at 37°C, and
the reaction was terminated by adding 1 ml of ice-cold 100 mmol/L
HEPES buffer (pH 5.5) containing 10 mmol/L EGTA. The total volume was
applied to a 5-ml AG 50W-X8 column (NA+ form) that had been
equilibrated with HEPES buffer without EGTA. Citrulline was eluted
twice with 1.0 ml of HEPES buffer. The radioactivity of the eluate was
counted with a liquid scintillation counter. NOS activity was
quantified in terms of the amount of
[3H]-L-citrulline produced/min and the mean
number of endothelial cells in the sample. The data were expressed in
pmol citrulline
generated · min-1 · 10-6 cells.
Extraction of flavonoids from cocoa.
The extracts used in the present study were provided by Mars
Incorporated (Hackettstown, NJ). Approximately 230 g of cocoa
beans was frozen in liquid nitrogen and ground to a fine powder in a
laboratory mill. The powder was extracted three times with 750 ml of
hexane to remove lipids. The procedure yielded ~100 g of defatted
cocoa powder. The defatted cocoa powder was extracted with 1:l of a
mixture of acetone and water (70:30 v/v). The aqueous extract was
reextracted with hexane to remove residual lipids. The hexane layer was
discarded, and the acetone layer was evaporated at 45°C under partial
vacuum to a final volume of 200 ml. The resulting solution was
freeze-dried to yield ~20 g of acetone extract. Further
purification was undertaken with HPLC using methods previously
described by Adamson et al. (1999
) and Lazarus et al. (1999
). The following extracts were used in this study:
1) procyanidin mixture containing monomers, dimers,
trimers and tetramers (combination 1), 2) procyanidin
mixture containing pentamers through decamers (combination 2) and
3) pure procyanidin (monomer through decamer). Each of
the latter compounds had a purity of >90%.
Statistical analysis.
Relaxations are expressed as a percent of the contraction in response to norepinephrine. All drug concentrations are expressed as the final concentration in the tissue bath fluid. Group data were expressed as means ± SEM of n experiments. Analysis of covariance was used to compare dose-response curves. A P-value of <0.05 was considered statistically significant. The Student’s t test was carried out on NOS data to establish significance, and the P-value was corrected with a Bonferroni adjustment.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Acetylcholine invariably caused relaxation of aortic rings, and this
relaxation was abolished by the removal of the endothelium. Certain
cocoa procyanidins also evoked a relaxation of precontracted aortic
rings that was abolished by removal of endothelium. Combination 1 had
no effect on the aortic rings, whereas procyanidin combination 2 caused
a dose-dependent EDR of the rings similar to acetylcholine
(n = 5) (Fig. 1
). The responses to procyanidin combination 2 were abolished by
L-NAME (n = 3). Atropine, which abolished the
effect of acetylcholine, had no effect on the responses to procyanidin
combination 2 (n = 3).
|
). The tetramers through decamers elicited EDR. The failure of
combination 1, which contained a small quantity of the tetramer, to
elicit an EDR was probably due to a low concentration of the compound
in the mixture.
|
Preincubation of the tissues for 30 min with the pentamer and
combination 2 (10-5 mol/L) attenuated the
relaxations evoked by acetylcholine and the extract (Fig. 3
). The maximum relaxation to acetylcholine before incubation was 49
± 5.1%, and after incubation with combination 2, it was 0.83
± 0.8% (n = 5). The maximum relaxation to the
pentamer was 46.5 ± 4.5%, and after incubation with the
pentamer, the response to acetylcholine was 2.1 ± 1.4%
(n = 5). To determine whether L-arginine
could reverse this inhibition, the tissues were exposed to
L-arginine (10-4 mmol/L) for an
additional 30 min after the initial incubation with the flavonoids. The
tissues were then retested with acetylcholine (Fig. 4
) and the extracts. The maximum relaxations observed after arginine were
18.3 ± 5.7% for the pentamer and 21.5 ± 5.7% for
acetylcholine. Similar results were obtained with the responses to
combination 2. These observations were similar to those observed
previously with an extract of grape seed (Karim et al. 1998
) (data not shown).
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Endothelial NOS activity was measured in tissues in the basal state and
after exposure to the pentamer and monomer (Fig. 5
). It was found that endothelial NOS activity was increased
significantly by exposure to the pentamer (181 ± 21 pmol of
citrulline · 1 million endothelial cells-1 ·
min-1, n = 6) compared with
tissues that were maintained in the basal state (110 ± 10 pmol of
citrulline · 1 million endothelial cells-1 ·
min-1, n = 9; P
< 0.05). In contrast, cells obtained from tissue exposed to the
monomer showed an activity that was significantly lower than control
(36 ± 6 pmol of citrulline · 1 million endothelial
cells-1 · min-1,
n = 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These effects
cover a variety of processes, including inflammation (Kandaswami and Middleton 1984), platelet function (Pellegrini et al. 1996
), angiogenesis (Fotsis et al. 1998
) and
the regulation of vascular tone (Duarte et al. 1993
,
Fitzpatrick et al. 1993
). The investigation reported in
this paper has focused on the effects of these compounds on EDR.
Previous studies reported in the literature relating to the effect of
these compounds on blood vessels have yielded conflicting results. For
instance, in the rat aorta, Fitzpatrick et al. (1993
)
observed an EDR while Duarte et al. (1993)
observed a
relaxation which was not dependent upon the presence of endothelium.
These studies were undertaken with both crude extracts and pure
monomeric flavonoids, such as quercetin and catechin. Subsequently,
Cishek et al. (1997
) demonstrated that extracts derived
from grape seed elicited EDR in aortic rings of New Zealand White
rabbits fed a nonpurified stock diet. The active agent in the extract
were the polymeric procyanidins. The monomers derived from grape seeds
were inactive, despite their widely recognized antioxidant activity
(Cishek et al. 1997
). The current study is the first investigation that has examined the effects of cocoa procyanidins on EDR in a systematic manner. The main conclusion from these findings is that tetramers and higher polymers of epicatechin induce EDR in rabbit aortic rings in vitro. The monomers, dimers and trimers were not capable of eliciting an EDR. This finding is of particular interest in the context of the antioxidant properties of these compounds.
The procyanidins scavenge free radicals, and those derived from cocoa
have been shown to protect the oxidation of LDL in vitro through such a
mechanism (Kondo et al. 1996
). These observations raise
the possibility that the EDR observed in the present study was the
result of an "NO-sparing" effect of these compounds (secondary to
their antioxidant activity). If such a mechanism were the basis for the
EDR observed in this study, a relaxant response would have been evident
with the monomeric procyanidins as well because their antioxidant
capacity is also well documented (Chen et al. 1990
).
Thus, it is necessary to seek an alternative explanation.
This investigation has clearly demonstrated that the polymeric
compounds have the capacity to activate endothelial NOS, whereas the
monomeric compounds do not. The phenomena associated with this
activation are of interest. There is a similarity between the responses
observed with grape seed extracts and cocoa extracts. Monomers from
both sources did not evoke an EDR, whereas the polymers did so.
Polymeric procyanidins from grape seeds increased cGMP activity in
aortic rings (Cishek et al. 1997
) and activated
endothelial NOS (Karim et al., 1999a
). L-NAME
blocked the responses evoked by both extracts. Thus, it would be
reasonable to conclude that the EDR evoked by the polymeric
procyanidins in vitro is due to activation of endothelial NOS.
The EDR evoked by both extracts was not blocked by atropine, indicating another mode of activation besides the muscarinic receptors in the endothelial cells. These observations raise the intriguing possibility that the effects of the polymeric procyanidins are mediated by a unique receptor mechanism. Alternatively, a non–receptor-mediated mechanism similar to the action of a calcium ionophore could be involved. An antioxidant effect per se appears unlikely to be the explanation because of the failure of monomeric extracts to elicit EDR.
Finally, the effect of prolonged exposure of aortic rings to
procyanidins merits comments. It was evident that the EDR in these
rings was greatly attenuated. This effect was in part due to loss of
the substrate arginine and in part due to inactivation of the NOS. It
raises the possibility that the EDR elicited by procyanidins in vitro
may not be the best indicator of their effects on the circulatory
system in vivo. Indeed, there is no evidence at this time that
oligomeric procyanidins are absorbed as such when given orally,
although plasma catechin concentrations show an increase after the
consumption of cocoa beverages containing polymeric procyanidins. In
the absence of direct measurements of polymeric procyanidins in blood,
it is not possible to extrapolate findings obtained in vitro to the
intact circulation. However, recent studies have shown that EDR is
preserved in rabbits fed a nonpurified stock diet enriched with grape
seed extracts containing polymeric procyanidins (Karim et al. 1999b
).
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FOOTNOTES |
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2 To whom reprint requests should be addressed.
3 Abbreviations used: EDR, endothelium-dependent relaxation; L-NAME, N-nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Fitzpatrick D. F., Hirschfield S. L., Coffey R. G. Endothelium-dependent vasorelaxing activity of wine and other grape products. Am. J. Physiol. 1993;265:H774-H778
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R. R Holt, S. A Lazarus, M C. Sullards, Q. Y. Zhu, D. D Schramm, J. F Hammerstone, C. G Fraga, H. H Schmitz, and C. L Keen Procyanidin dimer B2 [epicatechin-(4{beta}-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa Am. J. Clinical Nutrition, October 1, 2002; 76(4): 798 - 804. [Abstract] [Full Text] [PDF]
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T. Schewe, H. Kuhn, and H. Sies Flavonoids of Cocoa Inhibit Recombinant Human 5-Lipoxygenase J. Nutr., July 1, 2002; 132(7): 1825 - 1829. [Abstract] [Full Text] [PDF]
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