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,3
*
Department of Nutrition, University of California, Davis, California 95616;
Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina and
**
Analytical and Applied Sciences, Mars Incorporated, Hackettstown, New Jersey 07840
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • antioxidant • flavonoids • bioavailability • cardiovascular disease • procyanidins • chocolate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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,
Renaud and de Lorgeril 1992
), stroke (Keli et al. 1996
) and cancer (Steinmetz and Potter 1991
).
Certain cocoas and chocolates contain substantial amounts of
flavonoids, especially the flavan-3-ol (-)-epicatechin (epicatechin)
and procyanidin oligomers (Adamson et al. 1999
). It is
postulated that dietary flavonoids, along with other dietary substances
such as tocopherols, ascorbate and carotenoids, can decrease
cardiovascular risk via a number of mechanisms, including the
protection of target molecules (lipids, proteins and nucleic acids)
from oxidative damage, suppressing the inflammatory response and
modulating vascular homeostasis (Demrow et al. 1995
,
Diaz et al. 1997
, Rein et al. 2000
,
Rice-Evans et al. 1995
).
The endogenous oxidant defense system is thought to be composed of
numerous enzymes, small molecule antioxidants and select vitamins that
act in concert (Haramaki et al. 1998
). Within this
defense system, additional antioxidant protection may be provided by
dietary flavonoids. However, there is a paucity of data regarding the
accurate estimation of flavonoid intake and the retention of the active
flavonoid metabolites in plasma (Bravo 1998
). If
flavonoids are absorbed from foods in sufficient quantity, their
physiological antioxidant activity could at least in part explain the
epidemiological observation of an inverse association between plant
food consumption and the incidence of several chronic diseases.
Indeed, the antioxidant properties of selected flavonoids have been
demonstrated in studies with synthetic liposomes (Salah et al. 1995
), ex vivo human plasma (Cherubini et al. 1999
, Lotito and Fraga 1998
), cell culture
models (Duthie and Dobson 1999
) and animal models
(Da Silva et al. 1998
, Fraga et al. 1987
).
Oligomeric procyanidins isolated from cocoa have been shown to possess
biological activities potentially relevant to oxidant defenses and
immune function. Bearden et al. (2000
) demonstrated that
oligomeric procyanidin fractions have potent activity with respect to
inhibition of LDL oxidation in vitro, and Kondo et al. (1996
) showed that the ingestion of cocoa mass can protect
against LDL oxidation ex vivo. In addition, oligomeric procyanidins
isolated from cocoa have demonstrated protection against in vitro
peroxynitrite-mediated protein damage (Arteel and Sies 1999
), as well as decreased rate of oxidation in synthetic
liposomes (Lotito et al., unpublished results). The potential to
modulate the immune response has been suggested by in vitro studies
using purified cocoa procyanidins (Mao et al. 1999
) and
a crude cocoa extract (Sanbongi et al. 1997
) by virtue
of altered cytokine transcription.
In the present study, we used a selective and sensitive method to measure the rise in plasma epicatechin, which occurred after acute chocolate consumption. In addition, we related the above changes in plasma epicatechin to changes in plasma antioxidant capacity and the presence of plasma lipid oxidation products.
|
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless
otherwise stated. Plasma samples were treated as described by
Richelle et al. (1999
), with the following
modifications. In brief, 200 µl heparinized plasma was mixed with 20
µl of a solution containing 0.2 g/ml ascorbic acid, 1 mg/ml EDTA and
20 µl ß-glucuronidase suspension (~2000 U glucuronidase activity
and 80 U sulfatase activity) and vortexed for 30 s. After a 45-min
incubation at 37°C, 0.5 ml acetonitrile was added, and the resulting
mixture was vortexed briefly and then centrifuged at 10,000 x g for 5 min at 4°C. The upper fraction (water phase)
was combined with 1 ml of a suspension of 50 mg alumina in 50 mmol/L
Tris-HCl, pH 7.0 (the alumina was previously activated by exposure
to the referred buffer for 30 min). The resulting mixture was incubated
for 30 min at room temperature, with intermittent vortexing. The
resulting suspension was then centrifuged at 10,000 x g for 5 min at 4°C. After discarding the supernatant
fraction, the alumina was washed with 1 ml of 50 mmol/L Tris-HCl,
pH 7.0, vortexed and centrifuged as indicated, and the supernatant was
discarded. Then, the alumina was washed with 1 ml of methanol, vortexed
and centrifuged as indicated. After discarding the methanol, the
alumina was dried under nitrogen to eliminate residual methanol, after
which 250 µl of 0.25 mol/L perchloric acid were added, and the
mixture was vortexed. The aqueous phase was separated and centrifuged
at 10,000 x g for 1 min at 4°C, and the
supernatant was filtered before HPLC analysis using a PVDF syringe
filter (Whatman, Clifton, NJ). The resulting solution was analyzed for
epicatechin by reversed phase HPLC with electrochemical (coulometric)
detection. Chromatography was carried out using an HP 1100 HPLC system,
equipped with a quaternary pump, temperature-controlled
autosampler, column oven and diode array detector (Hewlett Packard,
Wilmington, DE), in series with an ESA (Chelmsford, MA) Coulochem II
coulometric detector with a 5011 analytical cell used as single
electrode or with an ESA CoulArray 5600 multielectrode array detector.
Data were collected using Chemstation data acquisition software
(Hewlett Packard) and CoulArray for Windows data acquisition software
(ESA) when using the multielectrode array detector. A reversed phase
Alltima C18 (5 µm, 150 mm x 4.6 mm column with a C18 5-µm
guard column; Alltech Associates, Deerfield, IL) was used to effect the
separation. The mobile phase was comprised of two solvent solutions
that were mixed according to the detection method used: solvent A, 40%
methanol, 60% 50 mmol/L sodium acetate, pH 5.8; and solvent B, 7%
methanol, 93% methanol/100 mmol/L sodium acetate, pH 5.2. For
isocratic elution (1 ml/min) with single electrode detection, the
mobile phase was 60% A and 40% B. For gradient elution with single
electrode detection, the composition of the mobile phase was first set
at 80% B, which was linearly decreased to 60% B by 1 min. This was
followed by another linear decrease to 20% B by 3.5 min. The mobile
phase composition was maintained at 20% B until 20 min, when B was
linearly increased to 80% by 30 min. For gradient elution with
multielectrode detection, the composition of the mobile phase was first
set at 80% B, which was linearly decreased to 60% B by 1 min and held
until 3 min. This was followed by another linear decrease to 20% B by
7 min, followed by another linear decrease to 0% B by 9 min. The
mobile phase composition was maintained at 100% A until 15 min when B
was linearly increased to 20% by 18 min. B was then increased linearly
to 40% by 20 min, to 60% by 23 min and finally to 80% by 25 min.
Coulometric detection with the single electrode detector was carried
out with a guard cell setting of +800 mV, cell 1 set to clean at +100
mV and cell 2 set at +400 mV for analysis. For electrochemical
detection, using the multielectrode array detector, the following
potentials were used: -50, +150, +185, +200, +250, +300, +700 and +
800 mV.
Subjects and clinical study design.
Thirteen healthy, nonsmoking adults with no history of heart disease or hemostatic disorders participated in the study. Their current health status was evaluated via a questionnaire. All participants gave written informed consent before their participation in the study, which was approved by the Human Subjects Review Committee (University of California, Davis, California).
Ten subjects (four men and six premenopausal women, age range 26–49
years, body mass index 23.2 ± 1.2 kg/m2) consumed
80 g of procyanidin-rich chocolate in the form of 105 g
of M&M’s Semi-Sweet Chocolate Mini Baking Bits made with Cocoapro
cocoa (Mars Incorporated, Hackettstown, NJ), and three subjects (one
man and two women, age range 28–36 years, body mass index 21.1 ± 0.3 kg/m2) consumed isocaloric amounts of vanilla milk
chips (low-procyanidin food) (Guittard Chocolate Company; Burlingame,
CA). The procyanidin-rich chocolate provided 557 mg total
procyanidins, of which 137 mg (470 µmol) was epicatechin, as
determined by Adamson et al. (1999
), whereas the vanilla
milk chips did not contain detectable levels of procyanidins or
epicatechin. The procyanidin-rich chocolate provided 27 g of
fat. Participants were instructed to abstain from vitamin supplements,
alcoholic beverages and caffeine- or theobromine-containing foods
for at least 24 h before and during the test day. Subjects fasted
at least 8 h before test food consumption. Venous blood (10 ml)
was obtained from all subjects between 0800 and 0900 h in two 5-ml
Vacutainer tubes containing EDTA or sodium heparin as anticoagulant
(Becton Dickinson, Franklin Lakes, NJ). All of the determinations
reported here were performed using heparinized blood. Plasma was
separated by low-speed centrifugation (1500 x g at 4°C for 10 min) and stored at -80°C until
analysis. Immediately after the blood draw, the subjects consumed the
test foods (procyanidin-rich chocolate or low-procyanidin vanilla
milk chips). Additional blood samples were taken 2 and 6 h later
and processed as indicated. All subjects were given a light meal of
bread and cream cheese between 1200 and 1300 h. Systolic and
diastolic blood pressures were determined before each blood draw.
Clinical and antioxidants determinations.
Plasma triglycerides, total- and HDL-cholesterol, vitamin E and vitamin C were determined by the Clinical Nutrition Research Unit at the University of California, Davis. Uric acid was determined spectrophotometrically using a commercial kit (Sigma Chem., St. Louis, MO).
2-Thiobarbituric acid reactive substance determination.
Thiobarbituric acid reactive substances
(TBARS)4
were assayed using frozen plasma as previously described
(Oteiza et al. 1997
). A 50-µl aliquot of 4% (wt/v)
butylhydroxytoluene/ethanol was added to each plasma sample (200 µl)
to avoid artifactual oxidation during the procedure. TBARS were
determined spectrofluorometrically, and sample values are expressed as
equivalents of malondialdehyde per plasma triglycerides.
Plasma antioxidant capacity determination.
Plasma antioxidant capacity was essentially determined as described by
Lissi et al. (1995
). Plasma samples (5–10 µl) were
assayed for their ability to inhibit the chemiluminescence produced by
a mixture of 3 ml of 5.4 mg/ml 2,2'-azo-bis(amidinopropane) in 0.1
mmol/L phosphate-buffered saline, pH 7.4 (GIBCO BRL, Life
Technologies, Grand Island, NY) and 10 µl of 1 mg/ml luminol. The
chemiluminescence was measured in a liquid scintillation counter
(Wallac 1410; Wallac Oy, Turku, Finland). The plasma antioxidant
capacity value was calculated as the lag time before an increase in the
chemiluminescence was observed. This lag time is proportional to the
cumulative amount of antioxidants present in the samples (Lissi et al. 1995
). A reference lag time was obtained by using a
known amount of 6-hydroxy-2,5,7,8-tetramethoxychroman-2-carboxylic acid
(Trolox; Aldrich Chemical Co., Milwaukee, WI).
Statistical analyses.
Results in the text and tables are expressed as means ± SE. Changes between the baseline (0 h) and the 2- and 6-h time points within a diet group were examined by paired t test. Regression analysis was used to compare correlations between the variables. Statistical significance was assessed at the 5% level. Analysis was performed using routines available in StatView for Windows Version 5.0.1. (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We developed an HPLC method that was based on procedures described by
Lee et al. (1995
) and modified by Ho et al. (1995
) and Richelle et al. (1999
). Both HPLC and
the detection conditions were modified, with the result that
qualitative and quantitative advantages were obtained.
The use of a stepwise gradient allowed an improvement in the baseline
slope compared with the isocratic run (Fig. 1A, B
). By using the gradient conditions and a multielectrode array detector,
it was possible to simultaneously determine epicatechin and catechin
(Fig. 1C
). The multielectrode array detector allowed for the
determination of compounds with different oxidation potentials, with a
high selectivity afforded by comparing the coulometric response given
by standards and samples. The response of the coulometric detection was
linear for pure epicatechin and catechin, between 20 and 120, and 40
and 120 pg, respectively (Fig. 2
). When plasma with negligible amounts of epicatechin or catechin was
spiked with pure compounds, the typical recovery of epicatechin and
catechin from the sample was between 70 and 90%. The obtained peak
areas showed a linear relationship with the amount spiked (Fig. 2)
.
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Clincal study.
Having a sensitive and selective method to determine very low levels of epicatechin, we designed a clinical study that involved the consumption of a chocolate that was rich in epicatechin and procyanidin oligomers.
Mean values for six clinical variables that were evaluated at baseline
(zero time) and 2 and 6 h after the test food consumption are
shown in Table 1
. All of these variables were within the normal range. With respect to
the subjects fed the procyanidin-rich chocolate, no significant
changes were observed in mean arterial pressure over the study period.
Plasma total, LDL and HDL cholesterol concentrations were unchanged
over the 6-h period. At 2 h after the consumption of the test
food, there was an increase in plasma triglycerides with respect to
baseline values (44% in subjects fed the procyanidin-rich
chocolate and 32% in subjects fed the low-procyanidin food).
Plasma triglycerides returned to baseline values by 6 h in the
high-procyanidin group.
|
In subjects fed the procyanidin-rich chocolate, plasma epicatechin
concentrations increased by 12-fold (P < 0.007)
relative to baseline values (257 ± 66 vs 22 ± 4 nmol/L)
(Table 2
) within 2 h after consumption. At the 6-h time point, plasma
epicatechin levels in eight of the subjects were markedly lower than at
the 2-h time point (Fig. 3
). However, two of the subjects showed an increase in plasma epicatechin
concentration between the 2- and 6-h time points (Fig. 3)
. Subjects who
consumed the low-procyanidin vanilla chips had no significant
changes over the 6-h period.
|
|
. By the 6-h time
point, the mean plasma antioxidant capacity had returned to baseline
values. It should be noted that in 1 of the 10 subjects, the mean
plasma antioxidant capacity value at the 2-h time point was lower than
the value at baseline. Subjects who consumed the low-procyanidin
vanilla chips had no significant changes over the 6-h period.
At 2 h after the intake of procyanidin-rich chocolate, values
for plasma TBARS were 40% lower than at baseline (P = 0.003) (Table 2)
. By the 6-h time point, values for plasma TBARS were
still 30% lower than baseline values (P = 0.008). Data
were pooled across the three time points and adjusted for within
subject variation using general linear models. When the above was done,
there was a significant inverse association between plasma epicatechin
and TBARS for the subjects who consumed the procyanidin-rich
chocolate (r = -0.624; P = 0.002; n
= 30). Subjects that consumed the low-procyanidin vanilla
chips had no significant changes over the 6-h period.
Plasma concentrations of vitamin E, vitamin C and uric acid were not
influenced by the consumption of procyanidin-rich chocolate (Table 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The three different methods that were used for epicatechin
determination in this study did not show any significant differences
with respect to the obtained detection limits. Consistent with this,
the three methods yielded similar values for plasma epicatechin
concentration. However, the use of the gradient elution and the
multielectrode array detection method provides the additional
opportunity to simultaneously measure epicatechin and catechin. Thus,
we suggest that when possible, this latter method should be used. In
addition, the coulometric detection affords a detection limit that is
20 times that achieved using typical fluorescence (Ho et al. 1995
, Richelle et al. 1999
) or amperometric
(Lotito and Fraga 1999
) detectors.
By using the present methodology, glucuronide and sulfate conjugates of
epicatechin are degraded to free epicatechin by the
glucuronidase/sulfatase treatment. Thus, the measured levels of
epicatechin reflect the sum of free and conjugated epicatechin but not
the methylated metabolites. Orally administered epicatechin in mammals
is thought to largely undergo glucuronidation at the level of the
intestinal mucosa (Da Silva et al. 1998
). Thus, the rise
in epicatechin in plasma observed after the procyanidin-rich
chocolate intake may be due in part to epicatechin metabolites (in
particular, unmethylated glucuronide, glucuronide-sulfate and
unmethylated sulfate conjugates), as well as to free epicatechin.
Similar conclusions were drawn for catechin bioavailability in humans
consuming red wine (Donovan et al. 1999
).
The present study shows that a plasma epicatechin level of 260 nmol/L
can be achieved within 2 h after the consumption of 557 mg of
procyanidins containing 137 mg of epicatechin from a
procyanidin-rich chocolate. The above plasma value of epicatechin
is similar to that measured after the consumption of comparable
flavonoid quantities from tea (Lee et al. 1995
), onions
(Hollman et al. 1996
) and black chocolate
(Richelle et al. 1999
). In the latter study, 2–3 h
after the ingestion of chocolate, epicatechin attained peak plasma
concentrations of 380 nmol/L after an intake of 0.9 g of
polyphenols (gallic acid equivalent) and 700 nmol/L after an intake of
1.7 g.
That an increase in plasma epicatechin of the magnitude reported in
this report is physiologically significant is suggested by the
concurrent increase that we observed in plasma antioxidant capacity and
the concomitant decrease in plasma TBARS. These changes in plasma
antioxidant/oxidation profiles were not related to diurnal variations,
as similar changes were not observed in the subjects fed the
low-procyanidin meal. Similar to the present findings, an increase
in the plasma antioxidant capacity was determined in humans after the
consumption of a green tea infusion containing 400 mg of catechins
(Pietta et al. 1998
). By contrast, no protection to in
vitro plasma or LDL oxidation was observed in people consuming black
tea (Cherubini et al. 1999
) or red wine
(Abu-Amsha Caccetta 2000
), respectively. Taken together,
these data could suggest a differential protection depending on the
ability of the food to deliver the flavonoid into the body. In
addition, it could reflect a differential protection of different
flavonoids/polyphenols.
The in vitro antioxidant activity of some flavonoids has been rated
considerably higher than that of ascorbic acid and 
-tocopherol
(Bagchi et al. 1999
, Rice-Evans et al. 1995
). However, the in vivo antioxidant activity of a compound
depends on its concentration in the plasma, or in the target tissue,
and its capacity to react with a radical that is dictated by its redox
potential. Considering physiological plasma concentrations,
epicatechin, including some of its metabolites, clearly can reach high
nanomolar concentrations (200–400 nmol/L). These values are ~1/200
of the hydrophilic ascorbate and ~1/150 of the lipid-associated
vitamin E. Concerning chemical reactivity, the standard redox potential
for epicatechin can be considered to be ~430 mV (Jovanovic et al. 1995
), implying that epicatechin semiquinone radical can be
reduced by ascorbate (Bors and Michel 1999
) and that
epicatechin can reduce vitamin E (redox potential = 500 mV)
(Buettner 1993
). Therefore, when considering both
reactivity and concentrations, epicatechin and related catechins seem
to be less important physiological antioxidants than vitamin E or
ascorbate in humans. However, we, along with others, have shown that
when plasma is subjected to an in vitro oxidation, epicatechin and
related catechins can prevent -tocopherol depletion, acting as an
antioxidant of intermediate reactivity between ascorbate and
-tocopherol (Lotito and Fraga 1998
, 1999
and 2000
,
Salah et al. 1995
). Thus, the insertion of epicatechin
into a physiological antioxidant network may explain the observed
increase in plasma antioxidant capacity and the reduction in plasma
TBARS that occurred after the consumption of epicatechin-rich
meals.
The clinical parameters that were followed in this study were not
markedly affected by either the procyanidin-rich meal or the
low-procyanidin meal over the 6-h period. In this study, mean
arterial pressure was unaffected by the chocolate meal, a finding that
is consistent with the recent report by Baron et al. (1999
), who found no effect of the consumption of ~100 g of
chocolate on blood pressure in young healthy individuals.
In summary, we have confirmed the previous observation by
Richelle et al. (1999
) regarding the bioavailability of
epicatechin from chocolate. Of greater importance, in association with
the increase in plasma epicatechin, we documented an increase in plasma
antioxidant capacity and a decrease in the concentration of plasma
oxidation products. Procyanidin-associated changes in the oxidative
defense system may contribute to the health benefits associated with
the consumption of these procyanidin-rich foods.
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FOOTNOTES |
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2 This work was supported in part by grants from NIH (DK-35747) and Mars Incorporated.
3 To whom reprint requests should be addressed.
4 Abbreviation used: TBARS, 2-thiobarbituric reactive substances.
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