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Flavonoid-Rich Dark Chocolate Improves Endothelial Function and Increases Plasma Epicatechin Concentrations in Healthy Adults

Laboratory of Cardiovascular Physiology, Department of Physiological Nursing (M.B.E., M.M.E., A.B., E.Y.C., M.L.M.-S.), Boston, Massachusetts
Office of Research (S.M.P.), Boston, Massachusetts
School of Nursing, Cardiovascular Research Institute (M.J.M.), Boston, Massachusetts
School of Medicine, University of California, San Francisco, California, Antioxidant Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University (C.Y.C., H.-K.K., P.M., J.B.), Boston, Massachusetts

Address reprint requests to: Dr. Mary B. Engler, University of California, San Francisco, 2 Koret Way, Suite N631, San Francisco, California 94143-0610. E-mail: mary.engler{at}nursing.ucsf.edu

	ABSTRACT

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

 
Background: Dark chocolate derived from the plant (Theobroma cacao) is a rich source of flavonoids. Cardioprotective effects including antioxidant properties, inhibition of platelet activity, and activation of endothelial nitric oxide synthase have been ascribed to the cocoa flavonoids.

Objective: To investigate the effects of flavonoid-rich dark chocolate on endothelial function, measures of oxidative stress, blood lipids, and blood pressure in healthy adult subjects.

Design: The study was a randomized, double-blind, placebo-controlled design conducted over a 2 week period in 21 healthy adult subjects. Subjects were randomly assigned to daily intake of high-flavonoid (213 mg procyanidins, 46 mg epicatechin) or low-flavonoid dark chocolate bars (46 g, 1.6 oz).

Results: High-flavonoid chocolate consumption improved endothelium-dependent flow-mediated dilation (FMD) of the brachial artery (mean change = 1.3 ± 0.7%) as compared to low-flavonoid chocolate consumption (mean change = –0.96 ± 0.5%) (p = 0.024). No significant differences were noted in the resistance to LDL oxidation, total antioxidant capacity, 8-isoprostanes, blood pressure, lipid parameters, body weight or body mass index (BMI) between the two groups. Plasma epicatechin concentrations were markedly increased at 2 weeks in the high-flavonoid group (204.4 ± 18.5 nmol/L, p 0.001) but not in the low-flavonoid group (17.5 ± 9 nmol/L, p = 0.99).

Conclusion: Flavonoid-rich dark chocolate improves endothelial function and is associated with an increase in plasma epicatechin concentrations in healthy adults. No changes in oxidative stress measures, lipid profiles, blood pressure, body weight or BMI were seen.

Key words: flavonoid, endothelial, cholesterol, oxidized LDL, 8-isoprostanes, chocolate, epicatechin

	INTRODUCTION

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

 
Several studies provide evidence suggesting that dietary flavonoids may be protective against cardiovascular disease [1–5]. Other epidemiological studies demonstrate that flavonoid-rich diets high in fruits and/or vegetables also lower the risk of coronary heart disease [7–9]. This beneficial cardiovascular effect of flavonoids has been attributed to their natural antioxidant properties and their role in conserving tocopherols in biological membranes [10].

Recent advances in analytical methodology have shown that dark chocolate, a food of plant origin (Theobroma cacao), is a rich source of flavonoids [11–13]. Cocoa flavonoids are classified as flavanols and include the monomers, (–)-epicatechin, (+)-catechin, and procyanidins, the oligomers of these monomeric units. Specific cardioprotective effects recently ascribed to the cocoa flavonoids include: decreased susceptibility of low density lipoprotein (LDL) oxidation and sparing of alpha-tocopherol in vitro and ex vivo [14–17], and inhibition of platelet activation and aggregation [18, 19]. Furthermore, an increase in plasma antioxidant capacity and a decrease in plasma oxidation products are associated with elevated epicatechin concentrations [20, 21]. Schramm et al. [22] recently reported that the cocoa flavonoids decrease plasma leukotriene-prostacyclin ratios in human plasma and aortic endothelial cells. Other experimental investigations have shown that cocoa flavonoids activate endothelial nitric oxide synthase (eNOS) and enhance endothelium-dependent relaxation in vitro [23], inhibit human cytokine transcription and secretion [24], and inhibit mammalian 15-lipoxygenase activity [25].

These properties may impede atherogenesis since oxidative modification of LDL is believed to contribute to endothelial dysfunction and vascular disease [26, 27]. Macrophage scavenger receptors internalize oxidized LDL particles resulting in foam cell formation in the arterial wall. Oxidized LDL induces an inflammatory response with the production of endothelial leukocyte adhesion molecules and cytokines [28]. Nitric oxide (NO), an endogenous vasodilator, has many antiatherosclerotic properties including the inhibition of LDL oxidation, adhesion molecule expression, smooth muscle proliferation and contraction, platelet activation and aggregation, monocyte adhesion, and endothelin production [29]. Increased oxidative stress reduces the bioavailability of NO either by decreased NO production and/or increased NO degradation.

Endothelial dysfunction is characterized by dysregulation of NO metabolism and other important vasoactive molecules with resultant impairment of endothelium-dependent vascular relaxation. It can be demonstrated by diminished brachial artery flow-mediated dilation (FMD) which correlates with abnormal coronary artery endothelial responsiveness [30]. Endothelial dysfunction is associated with atherosclerosis and many cardiovascular risk factors including hypertension, hypercholesterolemia, smoking, increased age, and diabetes. It is not known whether cocoa flavonoids can improve endothelial function and measures of oxidative stress in human subjects. The present study was initiated to investigate these questions. A secondary aim of the study was to evaluate the effects of flavonoid-rich dark chocolate on plasma epicatechin concentrations, lipoprotein profiles and blood pressure.

	SUBJECTS AND METHODS

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

 
Subjects
Twenty-two healthy volunteer subjects (n = 11F and 11M) ages 21–55 years were enrolled in the study. Subjects were in good general health and English-speaking. Exclusion criteria included known cardiovascular disease, diabetes, hyperlipidemia, thyroid disorders, smoking, ± 20% ideal body weight, pregnancy, vegetarianism or extreme physical activity. Twenty-one subjects were included based on the above criteria. One subject was excluded due to hypercholesterolemia. The protocol was approved by the Institutional Review Board of the University of California, San Francisco and written informed consent was obtained.

Study Design
The study was a randomized, double-blind, placebo-controlled design conducted over a 2 week period. Subjects were assigned to daily intake of high-flavonoid (DOVE® Dark Chocolate, Mars, Incorporated, Hackettstown, NJ) or to low-flavonoid dark chocolate bars (46 g, 1.6 oz.) provided by the American Cocoa Research Institute (Vienna, VA). The nutrient composition of the chocolate bars is shown in Table 1.

A validated food frequency questionnaire (Block 98.2 BDDS, Block Dietary Data System, Berkeley, CA) [32] was completed at baseline to assess habitual dietary patterns. Three-day food records were also completed each week for the 2 week study and were analyzed using nutrition software, ESHA Food Processor for Windows, Version 7.11 (ESHA Research, Salem, OR). At each early morning visit, brachial artery endothelial function and blood pressure were measured followed by height and weight measurements. Venous blood was collected into vacutainer tubes containing EDTA or sodium heparin. Samples were analyzed for total cholesterol, triacylglycerol, LDL, high density lipoprotein (HDL), LDL oxidation, epicatechin, total antioxidant capacity, and 8-isoprostanes. Plasma was immediately processed and stored at –80°C until analysis. Plasma samples for 8-isoprostanes were immediately transferred to eppendorf tubes containing butylated hydroxytoluene (BHT) at a final concentration of 20 µmol/L, quickly frozen and stored at –80°C.

Endothelial Function
Endothelium-dependent FMD of the brachial artery was assessed at each early morning visit after 5–10 minutes of rest supine in a darkened room. All studies were performed by a single investigator blinded to treatment assignment using a 15 MHz linear array vascular transducer and a Sequoia C256 ultrasound system (Acuson, Mountain View, CA) as previously described [33]. Scans were recorded both digitally and on a Super VHS videotape for off-line imaging analyses using the Brachial Ultrasound Workstation (Medical Imaging Applications, Iowa City, IA). FMD was expressed as the peak change in arterial diameter from baseline within 2 minutes of hyperemia. Blood flow was determined from Doppler flow velocity and the cross sectional area of the artery.

Blood Pressure
Blood pressure was determined using the Dinamap (Johnson & Johnson Medical Inc., Tampa, FL.) automated blood pressure device with the subject supine after a 5-min. period of rest. Cuff pressure on the left arm was inflated 2 minutes after the initial reading. Both systolic and diastolic pressures were calculated as the mean value of 2 determinations, 2 minutes apart.

Lipid Profile
Blood was drawn into EDTA tubes and immediately stored on ice until centrifugation. Cholesterol was determined in plasma and lipoprotein fractions by an enzymatic technique and triacylglycerol was determined by a glycerokinase reaction [34]. Plasma levels of HDL cholesterol were determined after precipitation of LDL and VLDL with magnesium chloride and dextran sulfate. LDL cholesterol levels were calculated using the Friedwald equation [35] where LDL cholesterol = total cholesterol-(HDL cholesterol + triacylglycerol/5) with lipid concentrations measured as mmol/L.

Plasma Epicatechin
The bioavailability of dark chocolate polyphenols was assessed by identifying (–)-epicatechin in the plasma by HPLC with electrochemical detection (ECD) according to slight modification of the method described by Milbury [36]. Briefly, 20 µL vitamin C-EDTA (200 mg vitamin C and 1 mg EDTA in 1 mL 0.4 mol/L NaH₂PO₄) and 20 µL glucuronidase/sulfatase type II (Sigma, St. Louis, MO) was added to 200 µL plasma and incubated at 37°C for 45 minutes. Flavonoids were extracted by addition of 500 µL acetonitrile with immediate vortexing. The mixture was centrifuged at 10,000 x g for 5 minutes at room temperature and 500 µL supernatant removed, dried under purified nitrogen air, and reconstituted in 100 µL of the aqueous HPLC mobile phase. After centrifugation at 14,000 x g for 5 minutes at room temperature, 50 µL supernatant was injected into the HPLC column for separation, detection, and analysis. Plasma epicatechin concentration was calculated based on a standard curve created by authentic epicatechin (Sigma, St. Louis, MO) through the same procedures as the plasma samples.

Ex Vivo LDL Oxidation
The effect of chocolate polyphenols on the resistance of LDL against Cu²⁺-induced oxidation was measured according to a slight modification of the method described by Esterbauer et al. [37]. To preserve the oxidation properties of LDL, sucrose was added to EDTA-containing plasma samples at a final concentration of 0.6% and stored at –80°C for a maximum of 8 weeks [38]. Briefly, LDL (density = 1.019–1.063 g/mL) was separated from plasma according to Havel et al. [39]. One mL plasma was mixed with 0.52 g KBr and centrifuged at 330,000 x g for 90 minutes at 4°C using a Beckman NVT-90 rotor in a Beckman L8-M centrifuge (Palo Alto, CA). The LDL band was aspirated from the centrifuge tube and EDTA was removed using a PD-10 column according to the manufacturer’s instructions (Amershan Pharmacia Biotech, Sweden). LDL protein was determined with a BCA protein assay kit (Pierce; Rockford, IL). The oxidation reaction was initiated by 10 µmol/L CuSO₄ at a final concentration of 100 µg/mL LDL protein in a volume of 1 mL. Formation of conjugated dienes was monitored by absorbance at 234 nm at 37°C over 3 hours using a Shimadzu UV1601 spectrophotometer (Japan) equipped with a six-position automated sample changer. Results from the LDL oxidation and the conjugated diene formation are expressed in terms of lag time (defined as the intercept at the abscissa in the diene-time plot) [40].

Oxygen Radical Absorbance Capacity (ORAC)
The total antioxidant capacity of plasma was assessed by ORAC assay using a Fluo Star Optima Microplate reader (BMG Labtechnologies, Inc. Durham, NC) and a modification of the method described by Huang et al. [41]. Heparinized plasma samples were used for ORACtotal and ORACpca. Plasma ORACpca samples were prepared with 0.5 M perchloric acid (1:1, v/v) and centrifuged at 10,000 x g for 10 minutes to obtain the protein free supernatant. The ORAC value (µmol Trolox Equivalent) represents the area under the quenching curve of fluorescein initiated by 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) in the presence of plasma antioxidants relative to that of Trolox, a water-soluble vitamin E analog (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid). One ORAC unit equals the net protection produced by 1 µmol/L Trolox.

Plasma 8-Isoprostanes
8-isoprostanes, a biomarker of in vivo peroxidation of arachidonic acid independent of cyclooxygenase activity, was measured by an enzyme immunoassay (EIA) microtitre plate assay kit (Cayman, Ann Arbor, MI) based on the method developed by Maclouf et al. [42] and using a Fluo Star Optima Microplate reader (BMG Labtechnologies, Inc. Durham, NC). Following Folch extraction and KOH digestion, total 8-isoprostanes from 0.5 mL plasma were extracted by use of a solid phase extraction (SPE) 8-isoprostanes affinity column (Cayman Chemical Company). Recoveries averaged >90% with a variance of <20%. Subsequently, total 8-isoprostanes were measured by EIA. This assay is based on the competition between 8-isoprostane and an 8-isoprostane-acetylcholinesterase conjugate (8-isoprostane tracer) for a limited number of 8-isoprostane-specific rabbit antiserum binding sites. The assay employs an Ellman’s reaction producing an absorbance at 412 nm proportional to the amount of 8-isoprostane tracer bound to the well and inversely proportional to free 8-isoprostane.

Statistical Analysis
Differences between the two groups in the baseline dietary characteristics shown in Table 3 were examined by independent sample t-tests, but if the assumption of equal variance was not met, the separate variances t-test (Welch test) was used. Statistical significance of the outcomes was determined by applying a repeated measures analysis of variance (RMANOVA) at alpha = 0.05 with one between subject’s factor (group) with two levels (experimental and control) and one within subject’s factor (time) with two levels (baseline and post intervention). This design allowed for three tests: the main effect of group, the main effect of time, and the interaction of group by time. If the test of the interaction was significant, tests of simple main effects were done to examine the difference between baseline and post intervention within each group. All results are shown as mean ± SEM. Eta-squared (eta²) represents the percent of explained variance.

	RESULTS

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

View larger version (31K): [in this window] [in a new window]   Fig. 1. Flow-mediated dilation (FMD %) of the brachial artery in the low-flavonoid and high-flavonoid dark chocolate groups.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Effect of low-flavonoid and high-flavonoid dark chocolate on plasma epicatechin. Mean ± SEM.

	DISCUSSION

1

In the current study, no changes in biomarkers of antioxidant and oxidative stress were demonstrated following the intervention. Our results are consistent with an acute chocolate feeding study by Wang et al., which showed that plasma antioxidant capacity, concentrations of 8-isoprostanes and 2-thiobarbituric acid reactive substances (TBARS) were unchanged at 2 and 6 hour time points after chocolate consumption [21]. Another study by Rein and colleagues [20] did report a significant increase in plasma total antioxidant capacity and a decrease in TBARS at 2 hours after ingestion of 80 g flavonoid-rich chocolate concomitant with a significant increase in plasma epicatechin (257 nmol/L) in thirteen healthy subjects. This concentration of epicatechin was approximately 50 nmol/L higher than our levels after 2 weeks intervention with 46 g. Osakabe et al. [15] also found a reduction in LDL susceptibility to oxidation in healthy subjects after consumption of cocoa powder daily for 2 weeks. In two recent long-term studies, dietary supplementation for 4–6 weeks with flavonoid-rich chocolate and cocoa powder [16, 17] increased LDL oxidation lag time, but did not affect plasma ORAC or urinary F₂ isoprostanes.

Although, previous studies have demonstrated an increase in plasma epicatechin concentrations following consumption of flavonoid-rich products, the findings relative to measurements of oxidative stress are conflicting with our results. We saw higher baseline epicatechin concentrations (17–25 nmol/L) in our study subjects as compared to Wang’s study (1–4 nmol/L). Some variances in the baseline measurements among the studies may be the result of differences in the baseline diets or in the sensitivity for detecting low concentrations of epicatechin using different HPLC coupled with electrochemical (coulometric detection) methodologies. In addition, the increment of epicatechin concentrations 2 hours after chocolate consumption compared to baseline was approximately eightfold greater in our study using 46 g chocolate, 65-fold in the study by Wang et al. (53 g chocolate), and twelvefold in the Rein et al. study (80 g chocolate). The variability in oxidative stress measures may be related to the duration of the intervention as well as the type and amount of the chocolate or cocoa powder and flavonoids consumed. A recent 6-week study on the effects of increased intake of fruits and vegetables, other plant sources of flavonoids, with varied intakes of unsaturated fatty acids (linoleic or oleic acid) also showed no measurable effects on lipid peroxidation (ex vivo LDL oxidation, TBARS, 8-isoprostanes) or lipoprotein metabolism [44]. It is acknowledged that adequate assessment of oxidative stress requires several independent measurements [45].

It is notable that the subjects’ lipid profiles did not change adversely following the flavonoid-rich or low-flavonoid chocolate intervention. In previous long-term studies of chocolate supplementation, neutral and/or beneficial cholesterol effects have been observed [46, 47]. The high content of stearic acid in cocoa butter which is readily converted to oleic acid [48] could help explain these effects. Dietary supplementation with isoflavonoids, a class of flavonoids derived from soybean-based foods, similarly has no effect on lipoprotein parameters in normocholesterolemic individuals [49, 50].

	CONCLUSION

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

 
In summary, flavonoid-rich dark chocolate improves endothelium-dependent vasodilation. This effect is associated with increased plasma epicatechin concentrations in healthy adults, though it is possible that flavonoids are a marker for some other bioactive constituent of chocolate. This could be evaluated by the administration of purified flavonoids. It is also acknowledged that the long-term clinical significance of the changes in endothelial function detected in the study are not known. Our findings suggest a possible cardioprotective effect by flavonoid-rich chocolate, independent of changes in measures of oxidative stress and lipid profiles. Further larger, long-term clinical trials with food sources rich in flavonoids, including chocolate, are certainly warranted.

	FOOTNOTES

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

 
This study was funded by the University of California, San Francisco, School of Nursing. The chocolate for the study was provided by the American Cocoa Research Institute, Vienna, VA.

Received June 11, 2003. Accepted October 29, 2003.

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TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION REFERENCES

 

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