Chocolate: Modern Science Investigates an

Ancient Medicine

A Dose-Response Effect from Chocolate Consumption on Plasma

Epicatechin and Oxidative Damage1,2
Janice F. Wang,* Derek D. Schramm,* Roberta R. Holt,* Jodi L. Ensunsa,*
Cesar G. Fraga,** Harold H. Schmitz and Carl L. Keen*3
Departments of *Nutrition and Internal Medicine, University of California, Davis, California 95616-8669;
**Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires,
1113 Buenos Aires, Argentina and Analytical & Applied Sciences, Mars Incorporated, Hackettstown,
New Jersey 07840

ABSTRACT Evidence from epidemiological studies suggests that a diet high in plant foods and rich in polyphe-
nols is inversely associated with a risk for cardiovascular and other chronic diseases. Chocolate, like red wine and

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green tea, is a polyphenol-rich food, primarily containing procyanidin polyphenols. These polyphenols are hypoth-
esized to provide cardioprotective effects due to their ability to scavenge free radicals and inhibit lipid oxidation.
Herein, we demonstrate that 2 h after the ingestion of a procyanidin-rich chocolate containing 5.3 mg total
procyanidin/g, of which 1.3 mg/g was ()-epicatechin (epicatechin), plasma levels of epicatechin increased 133
27, 258 29 and 355 49 nmol/L in individuals who consumed 27, 53 and 80 g of chocolate, respectively. That
the rise in plasma epicatechin levels was functionally significant is suggested by observations of trends for
dose-response increases in the plasma antioxidant capacity and decreases in plasma lipid oxidation products. The
above data support the theories that in healthy adults, 1) a positive relationship exists between procyanidin
consumption and plasma procyanidin concentration and 2) the rise in plasma epicatechin contributes to the ability
of plasma to scavenge free radicals and to inhibit lipid peroxidation. J. Nutr. 130: 2115S2119S, 2000.

Numerous epidemiological studies have provided evidence
for the concept that dietary flavonoids can decrease the risk of
death from coronary heart disease (Hertog et al. 1993 and
1995, Renaud and de Lorgeril 1992), cancer (Steinmetz and
Potter 1991) and stroke (Sirving et al. 1996). In addition to
select fruits and vegetables, and beverages such as red wine and
green tea, chocolate has been identified as a food that is rich
in flavonoids, particularly the flavan-3-ol ()-epicatechin
(epicatechin) and its polymers (Adamson et al. 1999, Ham-
merstone et al. 1999). Recent studies have demonstrated that
in vitro, the flavonoids derived from chocolate can act as
potent antioxidants (Arteel and Sies, 1999, Bearden et al.
2000, Kondo et al. 1996). Consistent with this, it has been
reported that complex mixtures of these compounds can pro-
tect LDL from oxidation in vitro (Waterhouse et al. 1996) and
inhibit LDL oxidation ex vivo (Kondo et al. 1996). Through
this mechanism, flavonoids have been hypothesized to reduce
the rate of progression of numerous disease processes (Kinsella
et al. 1993).
1
Published as part of a supplement to The Journal of Nutrition. Guest editors
for the supplement publication were John W. Erdman, Jr., University of Illinois at
Urbana-Champaign; Jo Wills, Mars, United Kingdom and DAnn Finley, University
of California, Davis.
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 acid reactive substances.
Before accepting the hypothesis that the procyanidins in
procyanidin-rich cocoa and chocolate products are a compo-
nent that afford significant protection against cardiovascular
disease, it must be demonstrated that the bioavailability of the
procyanidins found in chocolate is sufficient to provide health-
ful physiological responses. The bioavailability of procyanidins
depends on their absorption and metabolism, which in turn
are determined by their chemical structure, the vehicle of
delivery, as well as the degree of their conjugation, glycosyla-
tion, sulfonation and acylation (Bravo 1998, Wiseman 1999.)
Flavonoids, as antioxidant molecules, can participate in
free radical scavenging, as well as in the chelation of redox
active metals. The degree to which dietary procyanidins con-
tribute to the overall oxidant defense system of the body in
vivo relative to other antioxidant molecules, such as tocoph-
erol, ascorbate, carotenoids and urate, and antioxidant en-
zymes such as superoxide dismutase, catalase and glutathione
peroxidase, also must be established. In theory, this should
result in a reduction in free radicalinduced molecular damage
(de Groot and Rauen 1998, Rice-Evans et al. 1995).
Recently, biomarkers associated with cardiovascular dis-
ease, including select oxidant defense molecules and enzymes,
LDL oxidation state and platelet function, have been assessed
after the acute consumption of chocolate or cocoa (Kondo et
al. 1996, Rein et al. 2000a and 2000b). In a recent study with
healthy adults, we demonstrated that after the acute consump-
tion of 80 g of procyanidin-rich chocolate, plasma epicatechin

0022-3166/00 $3.00 2000 American Society for Nutritional Sciences.

2115S
2116S SUPPLEMENT
levels increased from baseline values of 22 4 nmol/L to
values of 257 66 nmol/L after 2 h. Importantly, this increase
in epicatechin was associated with an increase in the antiox-
idant capacity of the plasma and a decrease in plasma 2-thio-
barbituric acid reactive substances (TBARS) (Rein et al.
2000a). In the current work, we extend these observations by
examining dose-response changes in plasma epicatechin,
plasma antioxidant capacity, plasma TBARS and 8-isopros-
tane after the acute consumption of different amounts of a
procyanidin-rich chocolate.
METHODS
Subjects. Twenty volunteers (14 women and 6 men, age range
20 56 years, weight 67 2.7 kg, body mass index 23.8 0.79 kg/m2)
composed the subject pool from which subjects were randomly drawn
to participate in each of four chocolate treatment groups. Ten to 12
subjects were studied on four separate occasions, separated by 1-week
intervals. Subjects were nonsmokers and were free from any apparent
diseases. Volunteers signed informed consent before beginning the
study, and all procedures were conducted in accordance with the
guidelines of the Human Subjects Review Committee of the Univer-
sity of California, Davis.
Chemicals. Chemicals were purchased from Sigma Chemical
Co. (St. Louis, MO) unless otherwise stated.
Study design. Participants were asked to refrain from taking
vitamin supplements, nonsteroidal anti-inflammatory medications
and foods rich in polyphenols for 24 h and to fast overnight for 12 h
before each feeding trial. Blood was collected at baseline (0 h) and 2
and 6 h after the first blood draw. Subjects were instructed to
consume the test foods (chocolate and bread, or bread only) within
15 min of the first blood draw. All subjects were asked to consume an
additional 95 g of bread during the next 2 h.
Test foods provided 0, 27, 53 or 80 g of procyanidin-rich chocolate
in the form of M&Ms Semi-Sweet Chocolate Mini Baking Bits made
with Cocoapro cocoa (Mars Incorporated, Hackettstown, NJ) and
included a serving of 47 g of bread. The 27-g chocolate portion was
in the form of candy-coated M&Ms baking bits, which provided
0.732 MJ, 0.021 g caffeine and 0.18 g theobromine. Each 27-g
chocolate portion contained 46 mg epicatechin and a total of 186 mg
of procyanidins. The bread (bagel) provided 0.544 MJ, 0.75 g total
fat, 25 g carbohydrate and 4.5 g protein in a 47-g serving.
Collection of plasma samples. Eighteen milliliters of venous
blood was drawn into Vacutainer tubes containing EDTA or sodium
heparin (Becton Dickinson, Franklin Lakes, NJ). Plasma was sepa-
rated by low-speed centrifugation (1500 g at 4C for 10 min) and
stored at 80C until analysis. For lipid oxidation determinations,
aliquots of plasma samples were added with 4% w/v butylated hy-
droxytoluene/ethanol (for a final ratio of 4:1) before freezing.
Assessment of epicatechin in plasma. Plasma concentration of
epicatechin was determined as described by Richelle et al. (1999),
with some modifications. In brief, 200 l of heparinized human
plasma was vortexed with a solution of 0.2 g/ml ascorbic acid, 1
mg/ml EDTA and 20 mol -glucuronidase suspension (2000 U of
glucuronidase activity and 80 U of sulfatase activity). After a 45-min
incubation at 37C, 0.5 ml of acetonitrile was added, and the samples
were vortexed and centrifuged at 10,000 g for 5 min. The super-
natant fraction was combined with 50 mg of alumina and 1 ml of 50
mmol/L Tris-HCl (pH 7.6), vortexed and centrifuged. After discard-
ing the supernatant, the alumina was washed with 1 ml of 50 mmol/L
Tris-HCl (pH 7.6), followed by a final wash with 1 ml of methanol,
and centrifuged as indicated. After discarding the methanol, residual
methanol was evaporated under nitrogen, and epicatechin was ex-
tracted from the alumina with 250 l of 0.25 mol/L perchloric acid.
The sample was centrifuged (10,000 g, 1 min, 4C), and the
supernatant fraction was filtered through a 0.2-m polyethersulfone
membrane (Nalgene).
Samples (50 l) were chromatographed using a Hewlett Packard
(Wilmington, DE) 1100 series system equipped with an ESA
(Chelmsford, MA) Coulochem II coulometric detector with a 5011
analytical cell set as follows: guard cell 550 mV, conditioning cell
100 mV and analytical cell 400 mV. Chromatography was carried
out using an Alltima C-18 column (5 m, 150 4.6 mm; Deerfield,
IL) with a 0.5-m Frit precolumn filter (Upchurch, WA). The
mobile phase was composed of two solvent solutions: solvent A, 40%
methanol/60% 50 mmol/L sodium acetate, pH 5.8; and solvent B, 7%
methanol/93% 100 mmol/L sodium acetate, pH 5.2.
Epicatechin separation was achieved with a flow rate of 1 ml/min
and a gradient elution. For gradient elution, 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.
Assessment of plasma TBARS. Plasma TBARS determination
was conducted as previously described (Oteiza et al. 1997), with
modifications for using a plate reader. Briefly, 100 l of plasma, 200
l of 3% sodium dodecyl sulfate, 800 l of 0.1 mol/L HCl, 100 l
10% (wt/v) phosphotungstic acid and 400 l of 0.7% (wt/v) 2-thio-
barbituric acid were combined and placed at 95C for 30 min.
Samples were cooled on ice and mixed with 1 ml of 1-butanol. After
centrifugation (1800 g, 10 min, 4C), a 200-l aliquot of the
butanol phase was separated and analyzed spectrofluorometrically
(excitation 515 nm and emission 555 nm) using a plate reader
attachment (PerkinElmer Cetus, Norwalk, CT). TBARS are ex-
pressed as malondialdehyde equivalents.
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Assessment of plasma antioxidant capacity. Plasma samples
(510 l) were assayed for their ability to inhibit the chemilumines-
cence produced by a mixture of 3 ml of 5.4 mg/ml 2,2-azo-bis(amidi-
nopropane) in 0.1 mmol/L phosphate-buffered saline, pH 7.4
(GIBCO BRL, Life Technologies, Grand Island, NY) and 10 l 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 (Lissi et
al. 1995). This lag time is proportional to the cumulative amount of
antioxidants present in the samples. A reference lag time was ob-
tained by using a known amount of 6-hydroxy-2,5,7,8-tetramethoxy-
chroman-2-carboxylic acid (Trolox; Aldrich Chemical Co, Milwau-
kee WI).
Assessment of 8-isoprostane. 8-Isoprostane (8-epi-prostaglandin
F2) was determined in plasma using 25 l of plasma added to 40 l
of the supplied enzyme immunoassay buffer from the 8-Isoprostane
Enzyme Immunoassay Kit (Cayman Chemical, Ann Arbor, MI.).
Plates were read using a Shimadzu (Columbia, MD) spectrophotom-
eter at 450 nm.
Statistical analysis. Results in the text and tables are expressed
as means SEM. Changes between the baseline (0 h) and the 2- and
6-h time points among treatment groups were examined using re-
peated-measures ANOVA, with control for multiple measurements
on the same subjects. Where appropriate, multiple comparisons were
made using Tukey-Kramer corrections.
Statistical significance was assessed at the 5% level. All statistical
analyses were performed using SAS for Windows Release 7 (SAS
Institute, Cary, NC).
RESULTS
Before the consumption of the test foods, 82% of the
subjects had baseline values of plasma epicatechin that were
nondetectable (1 nmol/L). The subjects with detectable
levels of epicatechin in plasma showed values ranging from 5.5
to 28.3 nmol/L.
After the consumption of the test foods, plasma epicatechin
levels were higher than baseline values for the three test
groups that consumed the procyanidin-rich chocolate. At the
2-h point, the plasma epicatechin concentrations averaged
133, 258 and 355 nmol/L for the subjects who consumed 27,
53 and 80 g of the procyanidin-rich chocolate, respectively
(Table 1). There were no significant changes in plasma epi-
catechin levels in the subjects who consumed bread only. In
the procyanidin-rich chocolate groups, the average decrease in
epicatechin concentration between the 2- and 6-h point was
 CHOCOLATE, EPICATECHIN DOSE-RESPONSE AND ANTIOXIDANTS 2117S

TABLE 1
Concentration of epicatechin in plasma of subjects fed different amounts of procyanidin-rich chocolate

Epicatechin

Procyanidin-rich chocolate food

consumed, g 0 27 53 80

Time, h nmol/L
0 1 1 (9)1 2 2 (13)1 4 2 (13)1 4 3 (10)1
2 19 14 (9)1 133 27 (13)3 258 29 (13)3 355 49 (10)3
6 1 1 (9)1 26 8 (13)2 66 8 (13)2 103 16 (10)2
Area under the curve,
mol h L1 0 (9) 0.5 (13) 1.0 (13) 1.5 (10)

Values are expressed as means SEM. The area under the curve for epicatechin versus time was calculated from the means of the different time
points. Values in parentheses indicate number of subjects. Means within a column not sharing a common superscript number are significantly different
at P 0.05.

81, 77 and 74% for the 27-, 53- and 80-g groups, respectively. With respect to lipid oxidation, there was an increase in
It can be noted that the 6-h epicatechin concentrations in the plasma TBARS that was dependent on the time after which
53- and 80-g procyanidin-rich chocolate groups were still subjects consumed bread only (0.63 0.12, 0.77 0.11 and

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markedly higher than the baseline concentrations (P
0.001), and they were higher than plasma epicatechin
concentrations in the subjects who consumed bread only (P
0.0001).
With respect to plasma antioxidant capacity, there was a
decrease in the time of inhibition produced by plasma in the
subjects who consumed the non chocolate-containing food
from 489 88 s (0 h) to 401 70 s (2 h) and 350 69 s (6
h). These decreases were ameliorated in the subjects who
consumed procyanidin-rich chocolate. Although there were
no trends toward increased plasma antioxidant capacity 2 h
after the consumption of chocolate, 6 h after the consumption
of chocolate, there was a trend for an increase in the time of
inhibition with an increase in the amount of procyanidin-rich
chocolate ingested (Fig. 1). Subjects who consumed 27 g had
an increase in total antioxidant capacity of 78 s at 2 h and a
decrease of 38 s at 6 h compared with baseline values. Subjects
who consumed 53 g had a decrease of 57 s at 2 h and an
increase of 138 s at 6 h compared with baseline values. Finally,
subjects who consumed 80 g of chocolate had increases of 36
and 253 s at 2 and 6 h compared with baseline values, respec-
tively.
0.83 0.11 mol/L at the 0-, 2- and 6-h time points, respec-
tively). There was a trend for the increase in plasma TBARS
to be reversed, in a dose-dependent manner, in the subjects
who consumed procyanidin-rich chocolate (Fig. 2). A second
marker of lipid oxidation, plasma 8-isoprostane, was also mea-
sured in the subjects. Plasma 8-isoprostane values for the
subjects who consumed the different amounts of the procya-
nidin-rich chocolate were not statistically different at the 2-
and 6-h time points (Table 2).
DISCUSSION
In this study, we confirm previous observations that in
healthy adult human subjects, epicatechin is rapidly absorbed
after the consumption of procyanidin-rich chocolate (Rein et
al. 1999 and 2000). Importantly, the data support the theory
that in healthy adults, a linear relationship exists between
consumption of procyanidin-rich chocolate and plasma pro-
cyanidin concentration. In addition, we have extended this
observation by showing dose-dependent increases in plasma
epicatechin that are associated with increases in plasma anti-
oxidant capacity and reductions in plasma lipid oxidation.
The highest plasma epicatechin concentration observed in

FIGURE 1 Antioxidant capacity of plasma at baseline and 2 and

6 h after the consumption of procyanidin-rich chocolate. Values are
expressed as changes in lag time(s) before chemiluminescence is ob-
served. A weak trend (P 0.5302) was observed for increasing anti-
oxidant capacity with increasing amounts of procyanidin-rich chocolate
consumed. Variability is represented as SEM. Please refer to Table 1 for
the number of subjects in each group.
FIGURE 2 Plasma TBARS at baseline and 2 and 6 h after the
consumption of procyanidin-rich chocolate. Values are expressed as
malondialdehyde equivalents (mol/L). A weak trend (P 0.3451) was
observed for decreasing plasma TBARS with increasing amounts of
procyanidin-rich chocolate consumed. Variability is represented as SEM.
Please refer to Table 1 for the number of subjects in each group.
2118S SUPPLEMENT
TABLE 2
8-Isoprostane in plasma of subjects fed different amounts
of procyanidin-rich chocolate
8-Isoprostane
Procyanidin-rich
chocolate food
consumed, g 0 27 53 80
Time, h mol/L
0 48 5 (7) 39 8 (8) 52 7 (10) 50 7 (7)
2 44 9 (7) 51 8 (8) 52 7 (10) 57 9 (7)
6 58 7 (7) 42 5 (8) 49 4 (10) 42 9 (7)
Values are expressed as means SEM. Values in parentheses
indicate number of subjects. There were no significant differences
across treatments or time.
this study was 562 nmol/L, which occurred 2 h after consump-
tion in a subject who consumed 80 g of the procyanidin-rich
chocolate. The average plasma epicatechin concentration at
the 2-h point in this group was 355 nmol/L. This concentra-
tion is similar to that reported by Richelle et al. (1999),
although it is higher than the average value of 285 nmol/L
achieved in a previous study by our group (Rein et al. 2000a).
In contrast to the present study, in the study by Rein et al.
(2000a), subjects did not consume any additional food (bread)
until 4 h after the chocolate test meal. Therefore, we suggest
that the difference in results between our two studies is due to
subtle changes in the experimental methods that were used.
This can be shown by examining the change in plasma epi-
catechin concentration between the 2- and 6-h time points
after chocolate consumption. In the present study, this differ-
ence was 263 nmol/L, whereas in the study of Rein et al.
(2000a), this difference was 104 nmol/L. The estimated area
under the curve of epicatechin concentration versus time is
similar between the two studies (1480 versus 1540 nmol h1
L1), suggesting that although maximum absorption time was
delayed with chocolate consumption alone, the total epicat-
echin absorbed after the consumption of 80 g of procyanidin-
rich chocolate was unchanged between the two studies.
The influence of the bread on the bioavailability of epicat-
echin from chocolate must be determined in future studies.
When green tea and black tea were consumed with semi-
skimmed milk, the addition of milk did not significantly affect
the blood catechin levels for black tea with milk (van het Hof
et al. 1998). In the current study, we can estimate the amount
of epicatechin present in the plasma, extrapolated to a 6-h
postconsumption time, to 1.6, 1.5 and 1.4% of the total
epicatechin consumed in 80, 53 and 27 g of procyanidin-rich
chocolate. These percentages can be compared with a plasma
catechin absorption of 330 mol h1 L1 and a recovery of
0.24% after subjects consumed 126 mol of ()-catechin in
reconstituted red wine (Bell et al. 2000). Collectively, the data
from the above studies suggest that catechins from chocolate
are more bioavailable than catechins from wine.
Similar to plasma epicatechin kinetics, there were also
differences in chocolate-induced changes in plasma antioxi-
dant capacity between our previous work (Rein et al. 2000a)
and the current study. In the former study, plasma antioxidant
capacity peaked at 2 h after chocolate consumption, whereas
in the present study, plasma antioxidant capacity showed a
further increase at the 6-h time point from that of the 2-h time
point. This response is consistent with the different pattern of
epicatechin absorption that was observed in the two studies.
It has been suggested that the measurement of plasma
concentrations of F2-isoprostanes can provide an additional
biomarker for oxidative stress in vivo (Pratico, 1999, Roberts
and Morrow, 2000). In addition, because of their function as
renal and pulmonary vasoconstrictors, the assessment of F2-
isoprostanes may provide insight into the effects of specific
food products on vascular health (Morrow and Roberts 1997,
Morrow et al. 1999, Practico, 1999). In the current study, we
could not document a dose-related change between chocolate
consumption and plasma F2-isoprostane concentrations.
Given the results of the current study, as well as the findings
of others, we suggest that the daily consumption of procyani-
din-rich foods, such as chocolate, can result in improvements
in the antioxidant potential of plasma. A potential mechanism
for such improvement would be a sparing capacity that in-
volved other antioxidant molecules such as ascorbate, -to-
copherols and carotenes (Lotito and Fraga 1998 and 1999 and
2000, Salah et al. 1995).
The physiological relevance of such an increase in plasma
epicatechin after the consumption of procyanidin-rich choc-
olate can be shown by the results presented in this study and
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in Rein et al. (2000a) on modifying select oxidative stress
variables. In addition, it can be speculated that changes in
oxidative stress may contribute to the suppressive effects of
procyanidins on platelet activation (Rein et al. 2000b). In
summary, the results obtained to date demonstrate an in vivo
physiological effect of chocolate consumption on several bio-
logical variables associated with vascular pathologies, includ-
ing cardiovascular disease.
ACKNOWLEDGMENT
We would like to thank Janet Peerson for her assistance in the
statistical analysis of these data.
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