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2
Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 75390-9073, and Departments of
*
Pathology and
Internal Medicine, UC Davis Medical Center, Sacramento, CA 95817
2To whom correspondence should be addressed. E-mail: ishwarlal.jialal{at}ucdmc.ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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, high sensitivity C-reactive protein and P-selectin. In conclusion, cocoa products supplementation in humans affects LDL oxidizability, but not urinary F2 isoprostanes or markers of inflammation.
KEY WORDS: • cocoa • chocolate • flavonoids • oxidation • inflammation • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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). Epidemiologic evidence suggests that dietary flavonoids are correlated with a reduced risk of death from cardiovascular disease, and a growing body of in vitro and human clinical evidence suggests that at least some dietary flavonoids may in fact enhance important and specific aspects of cardiovascular health (2
–4
). In this context, flavanols, (e.g., epicatechin, catechin) and their related oligomers, (e.g., procyanidins and other proanthocyanidins) have shown especially promising health potential. A number of different foods and beverages can contribute large amounts of flavanols and procyanidins to human diets, including chocolate, cocoa, tea, grapes, apples, nuts and red wine. Among these, chocolate and cocoa may be of particular interest because of their potential to contribute especially high levels of procyanidins to the diet (5
,6
).
Cocoa is derived from the beans of Theobroma cacao, a tree native to South America (7
). Cocoa represents a potentially rich dietary source of flavanoids. High concentrations of flavonoids are present in certain cocoas, predominantly as the flavonol monomers (-)-epicatechin (epicatechin) and (+)-catechin (catechin), and as oligomers of these monomeric base units, which are known as the procyanidins (5
,6
).
Numerous researchers are currently studying the biologic effects of cocoa and its flavanol and oligomer components. In vitro studies have shown that cocoa, and isolated cocoa flavonols and their oligomers, can increase the antioxidant capacity of solutions and slow the oxidation of LDL (8
–11
). A cup of hot cocoa (250 mL) contains 8.2 ± 2.9 µmol/g phenols, milk chocolate, 52.2 ± 20.4 µmol/g and dark chocolate, 126 ± 17.4 µmol/g phenols (12
). Kondo et al. (9
) showed that cocoa powder extract prolongs the lag time of LDL oxidation in a concentration-dependent manner. Waterhouse et al. (8
) found that at 5 µmol gallic acid equivalents/L, cocoa phenols inhibited LDL oxidation by 75%, whereas red wines inhibited LDL oxidation by 37–65%. Vinson et al. (13
) showed that chocolates had a higher flavonoid antioxidant quantity-quality index than did fruit, vegetables, red wine and black tea. Other in vitro studies have shown that cocoa flavonols may also induce endothelium-dependent vessel relaxation (14
), reduce the production of inflammatory cytokines, while increasing the production of anti-inflammatory cytokines (15
,16
), and increase the synthesis of prostacyclin, while reducing the production of the proinflammatory cysteinyl leukotrienes (17
). Cocoa polyphenol oligomers have been reported to protect against peroxynitrate-dependent oxidation and nitration reactions (18
), and decrease the expression of the activated conformation of glycoprotein IIb/IIIa and CD62P (P-selectin) on epinephrine-activated platelets (19
).
Cacao liquor polyphenol (CLP), 3 a major component of chocolate, has been found to inhibit both hydrogen peroxide and superoxide anion, typical reactive oxygen species (ROS) production by phorbol myristate acetate-activated granulocytes or menadione-activated peripheral blood lymphocytes, mitogen-induced proliferation of T cells, and polyclonal immunoglobulin production by B cells, interleukin (IL)-2 mRNA expression, and IL-2 secretion by T cells. In ethanol-induced peptic ulcer, cacao liquor water-soluble crude polyphenols (CWSP) inhibited xanthine oxidase but not myeloperoxidase, the results suggested that the CWSP was not only a radical scavenger, but that it also modulated leukocyte function. These in vitro findings suggest that cocoa has antioxidative and immunoregulatory effects (20
). The purpose of our study was to determine the effects of supplementation with cocoa products on markers of inflammation and oxidative stress in humans.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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651 mg procyanidins/d) for 6 wk and at the end of a 6-wk washout period. The composition of cocoa products is shown in Table 1
. The study protocol was approved by the institutional review board and all subjects gave informed consent. The subjects included men and premenopausal women between the ages of 20 and 60 y. Subjects were recruited if they fulfilled the following criteria: nonsmokers; nondiabetics; not on antioxidant or vitamin supplementation; no chronic disease or gastrointestinal problems; no bleeding diathesis; normal complete blood count; normal renal and liver function; alcohol intake <30 mL/d; not on hypolipidemic drugs, thyroid drugs, nonsteroidal anti-inflammatory drugs, oral contraceptives or anticoagulants.
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Fasting blood samples (60 mL) were obtained at each of the three time points. Plasma and serum samples were stored at -70°C and analyzed at the end of the study. A complete blood count, serum lipid profile, serum renal (creatinine) and liver function [aspartate aminotransferase (AST), alanine aminotransferase (ALT)] tests, serum glucose and serum thyroid-stimulating hormone were assayed at these time points as a safety measure by standard laboratory techniques in the Clinical Pathology Laboratory.
Sample preparation.
The chocolate was prepared for analysis as described by Adamson et al. (21
). In brief, the lipid was extracted from 15 g of sample through exhaustive extraction with hexane (4 x 45 mL). One gram of the dried, lipid-free solids was extracted with 5 mL of an extraction solvent composed of acetone/water/acetic acid in a ratio of 70:29.5:0.5 (v/v/v), respectively. The resulting slurry was pelleted by centrifugation at 1500 x g, and then the supernatant was filtered through a 0.45-µm nylon filter. For the determination of the percentage fat composition, AOAC Official Method 920.177 was used (22
). The chocolate sample was extracted and analyzed in duplicate.
Separation and quantification of procyanidins.
Sample extracts were analyzed by HPLC using the method described by Adamson et al. (21
) and Hammerstone et al. (23
). In brief, chromatographic analyses were performed using an HP 1100 Series HPLC (Hewlett-Packard, Palo Alto, CA) equipped with an autoinjector, quaternary HPLC pump, column heater, diode array detector, fluorescence detector and HP ChemStation for data collection and manipulation. Fluorescence detection was recorded at excitation wavelength of 276 nm, emission wavelength of 316 nm and UV detection at 280 nm. Normal phase separations of the procyanidin oligomers were performed using a Phenomenex (Torrance, CA) 5-µm Lichrosphere silica column (250 x 4.6 mm) at 37°C with a 5-µL injection volume. The ternary mobile phase consisted of A) dichloromethane, B) methanol and C) acetic acid and water (1:1 v/v). Separations were effected by a series of linear gradients of B into A with a constant 4% C at a flow rate of 1 mL/min as follows: elution starting with 14% B in A; 14–28.4% B in A, 0–30 min; 28.4–39.2% B in A, 30–45 min; and 39.2–86% B in A, 45–50 min.
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Measures of oxidative stress |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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Fasting blood (60 mL) anticoagulated with EDTA was obtained for studies of LDL oxidation. LDL (d = 1.019–1.063 kg/L) was isolated by rapid two-step method ultracentrifugation from plasma collected in EDTA (1 g/L) as described previously (24
). NaBr-EDTA salts were used to adjust density, and ultracentrifugation was performed at 543,000 x g using a TI-100 tabletop ultracentrifuge (Beckman Coulter, Fullerton, CA). The EDTA and salts were removed by passage of the LDL sample through a 10-mL Sephadex DG-10 column (Amersham Pharmacia Biotech, Piscataway, NJ). Samples were purged with nitrogen, refrigerated at 4°C until the protein concentration was measured and used for oxidation experiments within 24 h of isolation.
LDL oxidation indices.
Samples of freshly isolated LDL were diluted to 200 mg LDL protein/L with PBS (pH 7.4) and incubated at 37°C with 2.5 µmol/L copper at 37°C for 3 h. Formation of conjugated dienes was measured via continuous spectrophotometric monitoring at 234 nm (25
). Measurements were recorded every 10 min and used to assess LDL oxidation. Lag time, oxidation rate and maximum conjugated diene concentration produced during the 3-h time course were calculated and used to characterize the oxidation kinetics of each LDL sample.
In vivo lipid peroxidation.
In vivo lipid oxidation was determined by measuring urinary F2 isoprostanes by competitive ELISA (urinary F2 isoprostanes kit from Oxford Biomedical Research, Oxford, MI). An early morning urine sample was collected at the three visits and urinary F2 isoprostanes were determined as a direct measure of oxidative stress. The results obtained from ELISA were standardized to milligrams creatinine. Urinary F2 isoprostanes provide a sensitive, specific and noninvasive method for the assessment of in vivo lipid peroxidation in humans (26
).
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Antioxidant capacity |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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Total phenols.
Plasma total phenols were measured by Folin’s method described by Serafini et al. (27
). Briefly, 500 µL of plasma was acidified and after extraction of complexed phenols with alcoholic sodium hydroxide, proteins were precipitated using 0.75mol/L metaphosphoric acid, and reextracted with a mixture of acetone/water (1:1) before measurement of total phenol content using phenol as standard.
ORAC measurement.
The antioxidant potential of heparinized plasma was determined by the ORAC assay based on the procedure described by Cao and Prior (28
). The method utilizes ß-phycoerythrin (ß-PE) as an indicator protein and 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) as a peroxyl radical generator. Trolox was used as a control standard. Under appropriate conditions, the loss of PE fluorescence in the presence of reactive species is an index of oxidative damage of the protein. The inhibition by an antioxidant, which is reflected in the protection against the loss of PE fluorescence in the ORAC assay, is a measure of its antioxidant capacity. The ORAC assay has high specificity; it measures the capacity of an antioxidant to directly quench free radicals. The analyzer was programmed to record fluorescence of ß -PE every 5 min for 20 cycles after AAPH was added. All fluorescence measurements were expressed relative to the initial reading. The differences in areas under the ß -PE decay curves between the blank and the sample were used to calculate the final results and were expressed as µmol Trolox equivalents (TE)/L.
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Markers of inflammation |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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The cytokines IL-1ß, IL-6 and tumor necrosis factor (TNF)- were measured in the supernatant of lipopolysaccharide (1 mg/L) activated whole blood after a 24-h incubation at 37°C using a highly sensitive immunoassay (R&D Systems, Minneapolis, MN). Monoclonal antibody specific for IL-1ß, TNF- and IL-6 is coated on the plate and any IL-1ß, TNF-, and IL-6 present in the plasma is bound by the immobilized antibody. The intraassay CV was <4%.
High sensitivity C-reactive protein (hs-CRP).
As a sensitive marker of inflammation, serum CRP levels were determined quantitatively by means of particle-enhanced immunonephelometry using polystyrene particles coated with monoclonal antibodies to CRP (29
). This immunoassay is sensitive, precise and accurate in the range of 0.2–60 mg/L.
P-selectin.
P-selectin is derived exclusively from platelets and endothelium and was measured at all three time points by ELISA (30
). The CV was <5%.
Statistical methods.
All statistical analyses were done by the biostatistician at the General Clinical Research Center at our institution using the SAS system (SAS Institute, Cary, NC). After ANOVA, pre- and postsupplementation variables were compared with paired t tests for parametric, and Wilcoxon-signed rank tests for nonparametric data, respectively. Results are expressed as mean ± SD and the level of significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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. The effect of cocoa product supplementation on the serum lipid profile and glucose levels of subjects is shown in Table 3
. There were no beneficial or deleterious effects of 6 wk of cocoa product supplementation on serum lipids and glucose concentrations compared with baseline and washout periods. Compliance was examined by two measures, i.e., measurement of total phenols and ORAC activity. There were no effects of cocoa intake on total phenols (baseline: 70.8 ± 11 µg/L phenol equivalents; 6 wk: 75.6 ± 15.6 µg/L phenol equivalents; 12 wk: 75.1 ± 18.5 µg/L phenol equivalents, P = 0.18) or ORAC (baseline: 3.5 ± 0.5 µmol/L TE; 6 wk: 3.3 ± 0.5 µmol/L TE; 12 wk: 3.4 ± 0.3 µmol/L TE, P = 0.26).
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. LDL oxidizability significantly decreased due to cocoa product supplementation as evidenced by a 9.8% longer lag time of conjugated diene formation compared with the baseline and washout phases. Serum total antioxidant capacity or total phenols and LDL oxidation lag time, rate of LDL oxidation or conjugated diene formation were not correlated.
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(P = 0.20) from lipopolysaccharide-activated mononuclear cells after supplementation did not differ from the baseline and washout periods (Table 4
). Plasma levels of hs-CRP and P-selectin were not affected by supplementation (Table 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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,9
,31
). However, little information is available about the absorption, metabolism and antioxidant activity of cocoa and chocolate flavonoids in humans after consumption.
The results of our study indicated favorable effects of cocoa powder and dark chocolate on LDL oxidation susceptibility. The lag phase of LDL oxidation corresponds to the time required for the endogenous antioxidant to be consumed. The length of lag time, thus, is an index of the level and quantity of antioxidants in LDL particles (32
). However, we were unable to detect epicatechin in plasma samples of these individuals (data not shown). It is likely that the timing of the blood draws explains the lack of epicatechin in the plasma because the blood samples in our study were taken after a 12-h fast. Several investigators have reported that the majority of absorbed epicatechin is cleared from the blood within 8 h (33
–35
). Our findings are similar to those of a study by Wan et al. (36
) who also observed a rapid clearance of epicatechin in subjects fed 22 g cocoa powder and 16 g dark chocolate. Those investigators also reported an 8% increase in LDL oxidation lag time. Osakabe et al. (37
) found a significant increase in lag time to LDL oxidation after 1 and 2 wk of cocoa consumption, independent of a concurrent presence of plasma epicatechin. This suggests that the protective effect on LDL oxidation may have been due to an effect of the cocoa flavanols on the amount of vitamin E, or other antioxidants, associated with LDL particles. Regardless of the mechanisms involved, these results provide support for the concept that the intake of dietary flavonoids can be associated with improvements in the oxidative defense system.
We also measured urinary F2 isoprostane levels, which is a more direct measure of in vivo lipid peroxidation. However, we did not find any effect on F2 isoprostanes. It could be because an early morning urine sample was used for analyses instead of a 24-h urine collection. Our findings are in line with the results of a study by Wang et al. (38
) who could not document a dose-response between chocolate consumption and plasma F2 isoprostane concentrations.
The finding that ORAC activity was not different at all three time points indicates that cocoa product supplementation did not affect total antioxidant capacity. Our results are not consistent with the findings of Wan et al. (36
) that an increase in plasma antioxidant capacity was associated with an increase in the consumption of procyanidin-rich chocolate and was positively correlated with LDL oxidation lag time. Their finding suggested that ORAC may be a poor marker of LDL oxidizability. In fact, Wang et al. (38
) clearly showed that the maximal increase in total antioxidant capacity occurs between 2 and 6 h of chocolate ingestion; thus, our sample obtained after an overnight fast may not have been appropriate to assess changes in ORAC activity.
Because there are few data in this area at this dose, we examined the effect of cocoa products on biomarkers of inflammation. We did not detect anti-inflammatory effects of cocoa product supplementation because concentrations of IL-1 ß, IL-6, TNF-, hs-CRP and P-selectin did not differ at the three time points tested.
In summary, the present study showed favorable effects of cocoa powder and dark chocolate consumption on LDL oxidative susceptibility. The lack of effects on biomarkers of inflammation and F2 isoprostanes is ascribed to the short biological half-life of flavonoids from cocoa products because there were no effects on ORAC activity and total phenols. This contrasts with lipid-soluble antioxidants such as -tocopherol, which remains elevated in plasma and LDL during the supplementation phase after a 12-h fast (24
). Consequently, this result may indicate a decreased risk of cardiovascular disease when changes in susceptibility and extent of LDL oxidation are implicated as important causative factors. Data obtained to date on the biologic effects of flavonoid-rich cocoas and chocolate support the concept that the consumption of flavonoid-rich foods may be associated with positive cardiovascular effects. There is a need to devise strategies for increasing their intake within the context of a healthy diet that meets energy requirements.
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FOOTNOTES |
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3 Abbreviations used: AAPH, 2,2'-azobis (2-amidino-propane) dihydrochloride; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CLP, cocoa liquor polyphenols; CWSP, cacao liquor water-soluble crude polyphenols; hs-CRP, high sensitivity C-reactive protein; IL, interleukin; ORAC, oxygen radical absorbance capacity; PE, phycoerythrin; ROS, reactive oxygen species; TE, Trolox equivalents; TNF, tumor necrosis factor.
Manuscript received 10 July 2002. Initial review completed 30 July 2002. Revision accepted 6 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSMeasures of oxidative stressAntioxidant capacityMarkers of inflammationRESULTSDISCUSSIONLITERATURE CITED |
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O. K. Chun, S.-J. Chung, K. J. Claycombe, and W. O. Song Serum C-Reactive Protein Concentrations Are Inversely Associated with Dietary Flavonoid Intake in U.S. Adults J. Nutr., April 1, 2008; 138(4): 753 - 760. [Abstract] [Full Text] [PDF]
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 S. Devaraj, G. O'Keefe, and I. Jialal Defining the Proinflammatory Phenotype Using High Sensitive C-Reactive Protein Levels as the Biomarker J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4549 - 4554. [Abstract] [Full Text] [PDF]
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 D. Grassi, S. Necozione, C. Lippi, G. Croce, L. Valeri, P. Pasqualetti, G. Desideri, J. B. Blumberg, and C. Ferri Cocoa Reduces Blood Pressure and Insulin Resistance and Improves Endothelium-Dependent Vasodilation in Hypertensives Hypertension, August 1, 2005; 46(2): 398 - 405. [Abstract] [Full Text] [PDF]
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H. Sies, T. Schewe, C. Heiss, and M. Kelm Cocoa polyphenols and inflammatory mediators Am. J. Clinical Nutrition, January 1, 2005; 81(1): 304S - 312S. [Abstract] [Full Text] [PDF]
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 M. B. Engler, M. M. Engler, C. Y. Chen, M. J. Malloy, A. Browne, E. Y. Chiu, H.-K. Kwak, P. Milbury, S. M. Paul, J. Blumberg, et al. Flavonoid-Rich Dark Chocolate Improves Endothelial Function and Increases Plasma Epicatechin Concentrations in Healthy Adults J. Am. Coll. Nutr., June 1, 2004; 23(3): 197 - 204. [Abstract] [Full Text] [PDF]
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L. I. Mennen, D. Sapinho, A. de Bree, N. Arnault, S. Bertrais, P. Galan, and S. Hercberg Consumption of Foods Rich in Flavonoids Is Related to a Decreased Cardiovascular Risk in Apparently Healthy French Women J. Nutr., April 1, 2004; 134(4): 923 - 926. [Abstract] [Full Text] [PDF]
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