The antioxidant activity of catechin monomers and procyanidin (dimers to hexamers) fractions purified from cocoa was studied in two in vitro systems: liposomes and human LDL. Liposome oxidation (evaluated as formation of 2-thiobarbituric acid reactive substances) was initiated with 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN), iron/ascorbate, or UV-C; LDL oxidation (evaluated as formation of conjugated dienes) was initiated with Cu(2+) or AAPH. Catechin monomers and procyanidin fractions inhibited both liposome and LDL oxidation. Monomers, dimers, and trimers fractions were the most effective antioxidants when liposome oxidation was initiated in the aqueous phase. When oxidation was initiated in the lipid domains, higher molecular weight procyanidins were the most effective. All fractions significantly inhibited Cu-mediated LDL oxidation; no significant effect of procyanidin molecular weight was observed. The hexamer fraction was the least effective with respect to preventing AAPH initiated LDL oxidation. Results reported herein give further evidence on the influence of the oligomer chain length on the antioxidant protection by procyanidins.
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Peroxynitrite is a mediator molecule in inflammation, and its biological properties are being studied extensively. Flavonoids, which are natural plant constituents, protect against peroxynitrite and thereby could play an anti-inflammatory role. Procyanidin oligomers of different sizes (monomer through nonamer), isolated from the seeds of Theobroma cacao, were recently examined for their ability to protect against peroxynitrite-dependent oxidation of dihydrorhodamine 123 and nitration of tyrosine and were found to be effective in attenuating these reactions. The tetramer was particularly efficient at protecting against oxidation and nitration reactions. Epicatechin oligomers found in cocoa powder and chocolate may be a potent dietary source for defense against peroxynitrite.
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.
There is speculation that dietary polyphenols can provide cardioprotective effects due to direct antioxidant or antithrombotic mechanisms. We report in vitro and postingestion ex vivo effects of cocoa procyanidins, a procyanidin-rich cocoa beverage and dealcoholized red wine (DRW) on human platelet activation. In a series of in vitro studies, cocoa procyanidin trimers, pentamers or DRW (3 and 10 micromol/L) were incubated with citrated peripheral whole blood in the presence and absence of platelet agonists. Platelet activation was detected using fluorescent-labeled monoclonal antibodies recognizing the fibrinogen binding conformation of GPIIb-IIIa (referred to herein as PAC-1 binding) and the activation-dependent platelet epitope CD62P (P-selectin). The percentage of CD42a-positive platelets coexpressing PAC-1 binding and/or CD62P was determined by multiparameter flow cytometry. Procyanidin trimers, pentamers and DRW added to whole blood in vitro increased PAC-1 binding and P-selectin expression. In contrast, procyanidin trimers, pentamers and DRW inhibited the platelet activation in response to epinephrine. The effects on platelet activation of cocoa beverage and DRW consumption were also studied in healthy subjects. Citrated blood was obtained before and 2 and 6 h after the ingestion of a cocoa beverage, a caffeine-containing beverage, DRW or water. Platelet activation was measured by flow cytometry. The consumption of DRW did not affect the expression of activation-dependent platelet antigens, either unstimulated or after ex vivo activation with epinephrine. However, the consumption of DRW increased PAC-1 binding in response to 100 micromol/L ADP ex vivo. Cocoa consumption reduced platelet response to agonists ex vivo. The ingestion of water had no effect on platelet activation, whereas a caffeine-containing beverage augmented the response of platelets to epinephrine. In summary, select cocoa procyanidins and DRW added to whole blood in vitro increased expression of platelet activation markers in unstimulated platelets but suppressed the platelet activation response to epinephrine. In contrast, cocoa consumption suppressed unstimulated and stimulated platelet activation in whole blood. This suppressive effect observed on platelet reactivity may explain in part the reported cardioprotective effects of dietary polyphenols.
The aim of this study was to examine the effects of procyanidins derived from cocoa on vascular smooth muscle. Two hypotheses were tested: 1) extracts of cocoa, which are rich in procyanidins, cause endothelium-dependent relaxation (EDR), and 2) extracts of cocoa activate endothelial nitric oxide synthase (NOS). The experiments were carried out on aortic rings obtained from New Zealand White rabbits. The polymeric procyanidins (tetramer through decamer of catechin) caused an EDR. In addition, the Ca(2+)-dependent NOS activity, measured by the L-arginine to L-citrulline conversion assay, was significantly increased in aortic endothelial cells exposed to polymeric procyanidins, whereas monomeric compounds had no such effect. These findings demonstrate that polymeric procyanidins cause an EDR that is mediated by activation of NOS.
BACKGROUND: Epidemiologic studies have shown inverse associations between dietary polyphenols and mortality from coronary heart disease. However, the basis for this protective association is uncertain. Food polyphenols reportedly have antioxidant properties and decrease platelet function in vitro. OBJECTIVE: This study sought to evaluate whether consumption of a polyphenol-rich cocoa beverage modulates human platelet activation and primary hemostasis. DESIGN: Peripheral blood was obtained from 30 healthy subjects before and 2 and 6 h after ingestion of a cocoa beverage (n = 10), a caffeine-containing control beverage (n = 10), or water (n = 10). Platelet activation was measured in terms of expression of activation-dependent platelet antigens and platelet microparticle formation by using fluorescent-labeled monoclonal antibodies and flow cytometry. Primary platelet-related hemostasis was measured with a platelet function analyzer. RESULTS: Ex vivo epinephrine- or ADP-stimulated expression of the fibrinogen-binding conformation of glycoprotein IIb-IIIa was lower 2 and 6 h after consumption of cocoa than before consumption. Cocoa consumption also decreased ADP-stimulated P-selectin expression. In contrast, epinephrine-induced platelet glycoprotein IIb-IIIa expression increased after consumption of the caffeine-containing beverage but not after water consumption. Platelet microparticle formation decreased 2 and 6 h after cocoa consumption but increased after caffeine and water consumption. Primary hemostasis in response to epinephrine in vitro was inhibited 6 h after cocoa consumption. The caffeine-containing beverage inhibited ADP-induced primary hemostasis 2 and 6 h after consumption. CONCLUSIONS: Cocoa consumption suppressed ADP- or epinephrine-stimulated platelet activation and platelet microparticle formation. Cocoa consumption had an aspirin-like effect on primary hemostasis.
Chocolate and cocoa are extensively used in many cultures. Although their composition has been studied, the functional significance of the components has not been as well defined. There are indications that cocoa constituents exert beneficial effects on human health, and therefore cocoa and chocolate may be considered functional foods. The use of functional foods to modulate human health has gained greater significance in recent years, and chocolate is widely consumed throughout society. We performed an extensive review of literature in both animal and human systems with respect to composition, bioavailability, comparative analysis with other food products and, especially, implications for cardiovascular disease and the human immune system. Although chocolate contains a high amount of saturated fats, the two major fatty acids are palmitic and stearic acid, which appear to have fewer implications for progression of coronary artery disease than other saturated fatty acids. In addition, the implications of flavonoids and other polyphenols in chocolate as antioxidants are significant, and their ability to modulate the immune system may also be applicable to infection and neoplasia. In this review, we attempt to place these issues in perspective and to provide the reader with an extensive summary of the literature on chocolate and cocoa and their potential mechanisms of action with respect to human health.
Nutrients exert measurable effects on biological processes and are among many factors that optimize health by helping to prevent, cure, treat or slow the progression of chronic diseases. Certain plant components (i.e., phytochemicals) may not be considered essential by traditional measures, but are increasingly recognized for their beneficial health effects. In particular, dietary flavonoids may make an important contribution to cardiovascular health. Epidemiological studies have shown that intake of flavonoids may be inversely associated with long-term mortality from coronary heart disease in epidemiological studies. Research with flavonoid-rich foods such as red wine, tea, blueberries and chocolate has demonstrated their antioxidant capacity. However, different flavonoids appear to have varying degrees of effect (e.g., inhibiting the oxidation of low-density lipoprotein cholesterol) and most of the flavonoid research has been limited to a few simple flavonoids, rather than a comprehensive investigation of all flavonoids present in the diet or a particular foodstuff. Well-controlled clinical studies are needed to determine whether flavonoids offer true benefits to cardiovascular health and to understand other potential mechanisms, in addition to antioxidant activity, which may be responsible for their protective action.
Given the widespread ingetion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the antiinflammatory cytokine, interleukin (IL-4). Both resting and phytohemagluttinin (PHA)- stimulated peripheral mononuclear blood cells (PMBC) were investigated at the protein secretion level. The smaller-sized cocoa fractions (tetramer or less) were unable to induce an IL-4 response (i.e. values fell below the detection limit of 0.25 pg/ml). The larger oligomeric procyanidines (penatmer or greater) stimulated secretion of IL-4 in resting PBMC by as much as 1.42 pg/ml, as shown by the nonamer. However only the hexameric, heptameric and decameric fractions proved to be statistically significant. Cells co-incubated with PBA showed an immense increase in IL-4 (21.1 ± 1.1 pg/ml). Only the monomeric fraction was able to induce the PHA-induced secretion of 48%. The other procyanidin oligomers supressed IL-4 production; in particular the hexameric, heptameric and octameric fractions significantly inhibited mitogen-stimulated secretion of IL-4 by 55%, 61% and 71%, respectively. This study offers additional data for the consideration of the health benefits of polyphenols from a wide variety of foods, including those benefits associated specifically with cooca and chocolate consumption.
Recent epidemiological research indicates that diets rich in flavonoid-containing foods may be associated with a reduced risk for cardiovascular disease. This protective effect is attributed, in part, to the ability of flavonoids to act as antioxidants. Certain chocolates and cocoas contain substantial amounts of procyanidins, and thus belong in the category of flavonoid-rich foods. Recent advancements in the identification and isolation of procyanidins, especially oligomeric procyanidins, from chocolate and cocoa have facilitated the investigation of individual procyanidin fractions with regard to their potential cardiovascular health benefits. In the following paper, we report on the antioxidant capacity of a cocoa as determined by the Oxygen Radical Absorbance Capacity (ORAC) assay, and the ability of individual procyanidin fractions from this same cocoa to inhibit low-density lipoprotein (LDL) oxidation in vitro. In addition, mechanisms are discussed by which flavonoids in chocolate and cocoa may enhance cardiovascular health.
