Roberta R. Holt

Epicatechin in Human Plasma: In Vivo Determination and Effect of Chocolate Consumption on Plasma Oxidation Status

Diets that are rich in plant foods have been associated with a decreased risk for specific disease processes and certain chronic diseases. In addition to essential macronutrients and micronutrients, the flavonoids in a variety of plant foods may have health-enhancing properties. Chocolate is a food that is known to be rich in the flavan-3-ol epicatechin and procyanidin oligomers. However, the bioavailability and the biological effects of the chocolate flavonoids are poorly understood. To begin to address these issues, we developed a method based on HPLC coupled with electrochemical (coulometric) detection to determine the physiological levels of epicatechin, catechin and epicatechin dimers. This method allows for the determination of 20 pg (69 fmol) of epicatechin, which translates to plasma concentrations as low as 1 nmol/L. We next evaluated the absorption of epicatechin, from an 80-g semisweet chocolate (procyanidin-rich chocolate) bolus. By 2 h after ingestion, there was a 12-fold increase in plasma epicatechin, from 22 to 257 nmol/L (P < 0.01). Consistent with the antioxidant properties of epicatechin, within the same 2-h period, there was a significant increase of 31% in plasma total antioxidant capacity (P < 0.04) and a decrease of 40% in plasma 2-thiobarbituric acid reactive substances (P < 0.01). Plasma epicatechin and plasma antioxidant capacity approached baseline values by 6 h after ingestion. These results show that it is possible to determine basal levels of epicatechin in plasma. The data support the concept that the consumption of chocolate can result in significant increases in plasma epicatechin concentrations and decreases in plasma baseline oxidation products.

A Dose-Response Effect from Chocolate Consumption on Plasma Epicatechin and Oxidative Damage

Evidence from epidemiological studies suggests that a diet high in plant foods and rich in polyphenols is inversely associated with a risk for cardiovascular and other chronic diseases. Chocolate, like red wine and green tea, is a polyphenol-rich food, primarily containing procyanidin polyphenols. These polyphenols are hypothesized 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.

Cocoa antioxidants and cardiovascular health

An increasing body of epidemiologic evidence supports the concept that diets rich in fruits and vegetables can promote health and attenuate, or delay, the onset of various diseases. Epidemiologic data support the idea that these health benefits are causally linked to the consumption of certain flavonoids present in fruit and vegetables. In the context of cardiovascular health, a particular group of flavonoids, namely, the flavan-3-ols (flavanols), has received attention. Flavanol-rich, plant-derived foods and beverages include wine, tea, and various fruits and berries, as well as cocoa and cocoa products. Numerous dietary intervention studies in humans and animals indicate that flavanol-rich foods and beverages might exert cardioprotective effects with respect to vascular function and platelet reactivity. This review discusses the bioactivity of flavanols in the context of cardiovascular health, with respect to their bioavailability, their antioxidant properties, and their vascular effects.

The effects of flavanol-rich cocoa and aspirin on ex vivo platelet function

BACKGROUND Flavanols modulate platelet function in vitro, but less is known of their in vivo effects and how they compare to pharmacological platelet inhibitors. We investigated the effect of a flavanol-rich cocoa beverage (897 mg/ml) in combination with and in comparison to aspirin on platelet function and activation in healthy subjects. METHODS AND RESULTS On separate test days in a crossover design, 16 healthy adults consumed aspirin (81 mg), cocoa (as a beverage), or aspirin plus cocoa. Platelet activation was measured by surface expression of P-selectin and PAC-1 binding to the activated conformation of the GPIIb/IIIa receptor (GPIIb/IIIa-act). Platelet function was measured on an analyzer (the PFA-100) that measures shear stress-induced platelet plug formation in response to collagen-epinephrine or collagen-ADP. Plasma epicatechin concentrations peaked approximately 2 h after subjects were given either the cocoa or aspirin plus cocoa. After 6 h, cocoa inhibited epinephrine-induced platelet function. Epinephrine-induced platelet function was inhibited 2 and 6 h after aspirin, and after aspirin plus cocoa. Epinephrine-stimulated P-selectin expression was inhibited by aspirin at 6 h, and after 2 and 6 h by aspirin plus cocoa. ADP-stimulated P-selectin expression was not affected by the treatments. Cocoa and aspirin, given separately, reduced epinephrine-stimulated GPIIb/IIIa-act expression at 2 and 6 h, respectively, and at 2 and 6 h when given together, suggesting an additive effective. ASA plus cocoa inhibited ADP-stimulated GPIIb/IIIa-act expression at 6 h. CONCLUSIONS Flavanol-rich cocoa inhibited epinephrine-stimulated platelet activation and function. These effects were qualitatively similar to aspirin, but less profound. These results emphasize the need to further examine the effects of food flavonoids for platelet modulating effects.

Chronic consumption of flavanol-rich cocoa improves endothelial function and decreases vascular cell adhesion molecule in hypercholesterolemic postmenopausal women

Endothelial dysfunction characterizes many disease states including subclinical atherosclerosis. The consumption of flavanol-rich cocoa and cocoa-based products has been shown to improve endothelial function in both compromised and otherwise normal, healthy individuals when administered either acutely or over a period of several days, or weeks. Women experience increased risk for cardiovascular disease after menopause, which can be associated with endothelial dysfunction. Whether a flavanol-rich cocoa-based product can improve endothelial function in hypercholesterolemic postmenopausal women is not known. The purpose of the present study was to determine whether chronic dietary administration of flavanol-rich cocoa improves endothelial function and markers of cardiovascular health in hypercholesterolemic postmenopausal women. Thirty-two postmenopausal hypercholesterolemic women were randomly assigned to consume a high-flavanol cocoa beverage (high cocoa flavanols (CF)—446 mg of total flavanols), or a low-flavanol cocoa beverage (low CF—43 mg of total flavanols) for 6 weeks in a double-blind study (n=16 per group). Endothelial function was determined by brachial artery-reactive hyperemia. Plasma was analyzed for lipids (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol), hormones (follicle-stimulating hormone), total nitrate/nitrite, activation of cellular adhesion markers (vascular cell adhesion molecule 1, intercellular adhesion molecule 1, E-Selectin, P-Selectin), and platelet function and reactivity. Changes in these plasma markers were then correlated to brachial reactivity. Brachial artery hyperemic blood flow increased significantly by 76% (P<0.05 vs. baseline) after the 6-week cocoa intervention in the high CF group, compared with 32% in the low CF cocoa group (P=ns vs. baseline). The 2.4-fold increase in hyperemic blood flow with high CF cocoa closely correlated (r2=0.8) with a significant decrease (11%) in plasma levels of soluble vascular cell adhesion molecule-1. Similar responses were not observed after chronic use of low CF. There were no significant differences between high and low CF in other biochemical markers and parameters measured. This study is the first to identify beneficial vascular effects of flavanol-rich cocoa consumption in hypercholesterolemic postmenopausal women. In addition, our results suggest that reductions in plasma soluble vascular cell adhesion molecule-1 after chronic consumption of a flavanol-rich cocoa may be mechanistically linked to improved vascular reactivity.

Food effects on the absorption and pharmacokinetics of cocoa flavanols.

Macronutrients in food and gastric acid are known to have a pronounced effect on the metabolism of many xenobiotics, an effect that impacts their efficacy as bioactive agents. In this investigation we assessed the impact of select food treatments and the histamine H(2)-receptor antagonist Famotidine (Pepcid-AC) on flavanol absorption and metabolism. Four crossover intervention studies were conducted with 6 subjects each. Volunteers consumed sugar-free, flavanol-rich cocoa (0.125 g/kg body wt) alone, with macronutrient-rich foods (8.75 or 17.5 kJ/kg subject body wt) or Famotidine (Pepcid-AC). Blood samples were drawn at 5 time points including baseline. Plasma samples were analyzed for epicatechin and catechin flavanols by HPLC. Pharmacokinetic parameters were assessed using non-compartmental methodology. When provided at 17.5 kJ/kg subject body weight (approximately 4 kcal/kg), sugar and bread test meals increased flavanol area under the curve (AUC) values to 140% of control values (P < 0.05). A corresponding tendency for plasma antioxidant capacity to increase was observed for the cocoa treatment at 1.5 and 2.5 h (P < 0.17, P < 0.06, respectively). The ability of treatment meals to affect AUC values was positively correlated with treatment carbohydrate content (r = 0.83; P< 0.02). In contrast to carbohydrate rich meals, lipid and protein rich meals and Famotidine treatment had minimal effects on flavanol absorption. Based on C(max) and AUC values, this data suggests that the uptake of flavanols can be increased significantly by concurrent carbohydrate consumption.

Cocoa procyanidin chain length does not determine ability to protect LDL from oxidation when monomer units are controlled.

Cocoa flavan-3-ols (catechin, epicatechin and oligomeric procyanidins) were tested for their ability to decrease LDL oxidative susceptibility and spare alpha-tocopherol (alpha-toc) in vitro. Physiologic concentration (0.10-0.50 &mgr;M) of flavanols were used. The flavanols increased LDL conjugated diene lag times dose-dependently from 23-207% and 15-143% in response to copper and AAPH oxidation, respectively, and delayed alpha-toc consumption. Sparing of LDL alpha-toc represents a possible mechanism for flavanols to enhance the resistance of plasma and LDL to oxidative stress. Procyanidins decreased LDL oxidative susceptibility with increasing chain length. However, when based on equivalent amounts of monomeric units, they inhibited LDL oxidation to a similar extent. This suggests that antioxidant activity of procyanidins with biologic substrates is not attributable to chain length or charge delocalization through polymeric linkages, but primarily to ring structures and catechol groups. Additionally, human plasma was analyzed for the presence of oligomeric procyanidins following consumption of a flavanol-rich cocoa product. Procyanidin dimers were detected in plasma concordant with the appearance of monomeric flavanols, with a peak of 0.08 +/- 0.01 &mgr;mol/L (n = 6) at two hours after consumption. Thus, this paper confirms the occurrence of procyanidins in human plasma, and extends previous structure-function observations regarding flavanoid protection of LDL.

Influence of cocoa flavanols and procyanidins on free radical-induced human erythrocyte hemolysis.

Cocoa can be a rich source of antioxidants including the flavan-3-ols, epicatechin and catechin, and their oligomers (procyanidins). While these flavonoids have been reported to reduce the rate of free radical-induced erythrocyte hemolysis in experimental animal models, little is known about their effect on human erythrocyte hemolysis. The major objective of this work was to study the effect of a flavonoid-rich cocoa beverage on the resistance of human erythrocytes to oxidative stress. A second objective was to assess the effects of select purified cocoa flavonoids, epicatechin, catechin, the procyanidin Dimer B2 and one of its major metabolites, 3ʹ-O-methyl epicatechin, on free radical-induced erythrocyte hemolysis in vitro. Peripheral blood was obtained from 8 healthy subjects before and 1, 2, 4 and 8 h after consuming a flavonoid-rich cocoa beverage that provided 0.25 g/kg body weight (BW), 0.375 or 0.50 g/kg BW of cocoa. Plasma flavanol and dimer concentrations were determined for each subject. Erythrocyte hemolysis was evaluated using a controlled peroxidation reaction. Epicatechin, catechin, 3ʹ-O-methyl epicatechin and (-)-epicatechin-(4β > 8)epicatechin (Dimer B2) were detected in the plasma within 1 h after the consumption of the beverage. The susceptibility of erythrocytes to hemolysis was reduced significantly following the consumption of the beverages. The duration of the lag time, which reflects the capacity of cells to buffer free radicals, was increased. Consistent with the above, the purified flavonoids, epicatechin, catechin, Dimer B2 and the metabolite 3ʹ-O-methyl epicatechin, exhibited dose-dependent protection against AAPH-induced erythrocyte hemolysis at concentrations ranging from 2.5 to 20 μM. Erythrocytes from subjects consuming flavonoid-rich cocoa show reduced susceptibility to free radical-induced hemolysis (p < 0.05).

Dietary flavanols and platelet reactivity.

Epidemiology studies suggest that the consumption of diets rich in flavonoids is associated with reduced risk of cardiovascular disease. Plant-derived foods and beverages, such as red wine, tea, grape and grape juice, cocoa and chocolate, can be rich in 1 particular class of flavonoid, the flavan-3-ols. There is now an increasing body of research that suggests that consuming flavanol-rich foods can positively affect hemostasis, through mechanisms that either directly affect platelet function or increase certain endothelium-derived factors that maintain platelet acquiescence or increase fibrinolysis. In this paper, we will review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent hemostasis. In addition, we will briefly review the body of literature with regard to other flavanol-rich foods and beverages, and possible mechanisms of action.

Flavanols and platelet reactivity.

Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP) IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg) aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.