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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.

The consumption of a diet rich in certain flavonoids, including the flavanol sub-class, has been associated with a reduced risk for vascular disease. We evaluated the effects of the regular consumption (14 d) of a flavanol-containing milk chocolate (FCMC) or cocoa butter chocolate (CBC) on variables related to vascular disease risk, oxidative stress and physical activity. Twenty-eight free-living, young (18–20 years old) male soccer players consumed daily 105 g of FCMC (168mg of flavanols) or CBC (,5mg of flavanols), as part of their normal diet. The consumption of FCMC was significantly associated with a decrease in diastolic blood pressure (25mmHg), mean blood pressure (25mmHg), plasma cholesterol (211%), LDLcholesterol (215%), malondialdehyde (212%), urate (211%) and lactate dehydrogenase (LDH) activity (211%), and an increase in vitamin E/cholesterol (þ12%). No relevant changes in these variables were associated with CBC consumption. No changes in the plasma levels of (2)-epicatechin were observed following analysis of fasting blood samples. In conclusion, FCMC consumption was associated with changes in several variables often associated with cardiovascular health and oxidant stress. The presence of significant quantities of flavanols inFCMC is likely to have been one of the contributing factors to these
results.

Fruits and vegetables have historically been considered rich sources of essential dietary micronutrients, soluble fiber, and antioxidants. More recently they are have been recognized as important sources for a wide array of phytochemicals that individually, or in combination, may benefit vascular health. Flavonoids are the largest, and most widely distributed class of phytochemicals, and can be further subdivided into several different sub-classes. Several epidemiology studies have observed an inverse association between flavonoid intake and risk of cardiovascular mortality. One sub-class of flavonoids, the flavanols, is found in foods such as grapes, red wine, tea, cocoa and chocolate; however, it is important to note that common food processing practices can significantly reduce the levels of these compounds found in finished food products. Recent studies have examined the potential of flavanol-rich cocoa and chocolates to influence vascular health. In this review, we discuss evidence for the hypothesis that the consumption of flavanol-rich cocoa can reduce the risk for cardiovascular disease through a multiplicity of mechanisms, including changes in oxidant defense mechanisms, vascular reactivity, cytokine production, and platelet function.

Procyanidins extracted from cocoa play a role in the defense against oxidative stress, as well as in vascular and immune functions. We previously reported that pentameric procyanidins isolated from cocoa inhibit the expression of the tyrosine kinase ErbB2 gene, thus slowing the growth of cultured human aortic endothelial cells. We herein investigate the further consequences of such inhibition by cocoa procyanidins, particularly regarding the protein level in phosphorylation patterns and the effects on the proliferation of human dermal microvascular endothelial cells (HDMECs) following angiogenic stimulation with low-level H2O2. We report herein that both the pentameric and octameric procyanidin fractions of cocoa inhibit the proliferation of HDMECs, whereas the pentameric fraction modulates the activity of several crucial proteins in angiogenic signaling by altering their tyrosine phosphorylation. Similar to aortic endothelial cells, the pentameric procyanidin fraction down-regulates the expression of ErbB2 tyrosine kinase in HDMECs. In conclusion, we report evidence suggesting that polyphenols may influence endothelial growth signaling, thus affecting angiogenesis in vitro. If these observations are applicable in vivo, they suggest a beneficial effect for cells overexpressing ErbB2, such as in specific neoplasias

There has been a long-standing interest in the relation between what we eat and cardiovascular risk. Over the years, attention has been given to calories, total fat, saturated fat, cholesterol, omega-3 polyunsaturated fatty acids, trans fatty acids, folic acid, antioxidants and, most recently, flavanols. Flavanol concentrations can be moderately high in a number of foods that have been associated with a reduction in cardiovascular risk including red wine, and black and green tea. Some cocoa and chocolate products are extraordinarily rich in flavanols but, as with other flavanol-containing foods, certain post-harvesting and processing procedures can have a striking influence on the flavanol content of chocolate and cocoa. Endothelial dysfunction with a consequent reduction in nitric oxide production has achieved a central conceptual role in the pathogenesis of atherosclerosis and coronary artery disease, diabetes mellitus and hypertension. Recent evidence that flavanol-rich cocoa activates vascular nitric oxide synthesis in the intact human raises an interesting possibility of a therapeutic potential.

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 < or = 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.

Flavonoids isolated from cocoa have biological activities relevant to oxidant defenses, vascular health, tumor suppression, and immune function. The intake of certain dietary flavonoids, along with other dietary substances such as tocopherols, ascorbate, and carotenoids, is epidemiologically associated with a reduced risk of cardiovascular disease. Flavonoids have also been shown to modulate tumor pathology in vitro and in animal models. We took advantage of the conserved sequences found in tyrosine kinases to study the influence of cocoa fractions and controls on gene expression. We report that the pentameric procyanidin (molecular weight of 1442 daltons) fraction isolated from cocoa was a potent inhibitor of tyrosine kinase ErbB2 expression, a receptor important in angiogenesis regulation. Consistent with this primary observation, the cocoa flavonoid fraction also suppressed human aortic endothelial cell (HAEC) growth and decreased expression of two tyrosine kinases responsive to ErbB2 modulation, namely VEGFR-2/KDR and MapK 11/p38beta2. These inhibitory effects were observed when HAECs were treated with the flavonol fraction (molecular weight 280 daltons) isolated from cocoa, which comprise the structural subunits from which the procyanidin flavonoid subclass is biosynthetically constructed. Down-regulation of ErbB2 and inhibition of HAEC growth by cocoa procyanidins may have several downstream implications, including reduced vascular endothelial growth factor (VEGF) activity and angiogenic activity associated with tumor pathology. These results suggest specific dietary flavonoids are capable of selectively inhibiting ErbB2 and therefore may offer important insight into the design of therapeutic agents that target tumors overexpressing ErbB2.

Proanthocyanidins (PAs) have been shown to have potential health benefits. However, no data exist concerning their dietary intake. Therefore, PAs in common and infant foods from the U.S. were analyzed. On the bases of our data and those from the USDA's Continuing Survey of Food Intakes by Individuals (CSFII) of 1994-1996, the mean daily intake of PAs in the U.S. population (>2 y old) was estimated to be 57.7 mg/person. Monomers, dimers, trimers, and those above trimers contribute 7.1, 11.2, 7.8, and 73.9% of total PAs, respectively. The major sources of PAs in the American diet are apples (32.0%), followed by chocolate (17.9%) and grapes (17.8%). The 2- to 5-y-old age group (68.2 mg/person) and men >60 y old (70.8 mg/person) consume more PAs daily than other groups because they consume more fruit. The daily intake of PAs for 4- to 6-mo-old and 6- to 10-mo-old infants was estimated to be 1.3 mg and 26.9 mg, respectively, based on the recommendations of the American Academy of Pediatrics. This study supports the concept that PAs account for a major fraction of the total flavonoids ingested in Western diets.

The capacity of the flavan-3-ols [(-)-epicatechin (EC) and (+)-catechin (CT)] and a B dimeric procyanidin (DP-B) to modulate phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in Jurkat T cells was investigated. The classic PMA-triggered increase in cell oxidants was prevented when cells were preincubated for 24 h with EC, CT, or DP-B (1.7-17.2 microM). PMA induced the phosphorylation of IKKbeta and the subsequent degradation of IkappaBalpha. These events were inhibited in cells pretreated with the flavonoids. PMA induced a 4.6-fold increase in NF-kappaB nuclear binding activity in control cells. Pretreatment with EC, CT, or DP-B decreased PMA-induced NF-kappaB binding activity and the transactivation of the NF-kappaB-driven gene IL-2. EC, CT, and DP-B inhibited, in vitro, NF-kappaB binding to its DNA consensus sequence, but they had no effect on the binding activity of CREB or OCT-1. Thus, EC, CT, or DP-B can influence the immune response by modulating NF-kappaB activation. This modulation can occur at early (regulation of oxidant levels, IKK activation) as well as late (binding of NF-kappaB to DNA) stages of the NF-kappaB activation cascade. A model is presented for possible interactions between DP-B and NF-kappaB proteins, which could lead to the inhibition of NF-kappaB binding to kappaB sites.

The proanthocyanidins in three foods (pinto beans, plums and cinnamon) were studied with electrospray ionization (ESI) mass spectrometry (MS) in the negative mode following separation by normal-phase high-performance liquid chromatography. The MS/MS analysis demonstrated that the major ions derived from heterocyclic ring fission and retro-Diels-Alder reaction of flavan-3-ol provided information about the hydroxylation pattern and type of interflavan bond. The connection sequence of the oligomers was identified through diagnostic ions derived from quinone methide (QM) cleavage of the interflavan bond. Novel heterogeneous B-type proanthocyanidins containing (epi)afzelechin as subunits were identified in pinto beans. Proanthocyanidins with interestingly different A-type linkages were identified in plums and cinnamon. In efforts aimed at extending the identification capacity of ESI-MS to polymers, we found that the polymeric procyanidins fragmented readily instead of forming multiply charged ions in the negative ESI mode. Fragmentation patterns were proposed based on our data obtained by ESI-MS/MS and ESI time-of-flight MS.

A normal-phase HPLC-MS/MS method was applied to screen for proanthocyanidins in 88 different kinds of foods. Thirty-nine foods were found to contain proanthocyanidins. These foods include 19 kinds of fruits, eight cereals/beans, seven nuts, two beverages, two spices, and one vegetable. Twenty-five kinds of foods were found to contain both oligomeric (DP </= 10) and polymeric proanthocyanidins (DP > 10), and the other 14 foods contained only oligomers. Procyanidins with B-type linkages were detected as the only components in 21 foods and also as principal components in the others. Propelargonidins were identified in pinto bean, raspberry, strawberry, and almond, etc. Plum, avocado, peanut, curry, and cinnamon were identified as potential sources of A-type proanthocyanidins in addition to cranberry. Thiolytic degradation and MS/MS analyses indicated that the A-type linkages are present as a terminal unit in plum or between the extension units in curry, cinnamon, and avocado, whereas A-type linkages exist at both positions in cranberry and peanut.

BACKGROUND:Consumption of flavonoid-rich beverages, including tea and red wine, has been associated with a reduction in coronary events, but the physiological mechanism remains obscure. Cocoa can contain extraordinary concentrations of flavanols, a flavonoid subclass shown to activate nitric oxide synthase in vitro. OBJECTIVE: To test the hypothesis that flavanol-rich cocoa induces nitric-oxide-dependent vasodilation in humans. DESIGN: The study prospectively assessed the effects of Flavanol-rich cocoa, using both time and beverage controls. Participants were blinded to intervention; the endpoint was objective and blinded. METHODS: Pulse wave amplitude was measured on the finger in 27 healthy people with a volume-sensitive validated calibrated plethysmograph, before and after 5 days of consumption of Flavanol-rich cocoa [821 mg of flavanols/day, quantitated as (-)-epicatechin, (+)-catechin, and related procyanidin oligomers]. The specific nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) was infused intravenously on day 1, before cocoa, and on day 5, after an acute ingestion of cocoa. RESULTS: Four days of flavanol-rich cocoa induced consistent and striking peripheral vasodilation (P = 0.009). On day 5, pulse wave amplitude exhibited a large additional acute response to cocoa (P = 0.01). L-NAME completely reversed this vasodilation (P = 0.004). In addition, intake of flavanol-rich cocoa augmented the vasodilator response to ischemia. Flavanol-poor cocoa induced much smaller responses (P = 0.005), and none was induced in the time-control study. Flavanol-rich cocoa also amplified the systemic pressor effects of L-NAME (P = 0.005). CONCLUSION: In healthy humans, flavanol-rich cocoa induced vasodilation via activation of the nitric oxide system, providing a plausible mechanism for the protection that flavanol-rich foods induce against coronary events.

Diets rich in flavonoids have been associated with reduced risk for cardiovascular disease. This may be due, in part, to flavonoid-induced alterations in eicosanoid synthesis. Our objective was to identify plant-derived beverages that alter synthesis of prostacyclin in cultured human aortic endothelial cells (HAEC), and to determine if these beverages could alter in vivo 6-keto-prostaglandin F(1alpha) (a stable metabolite of prostacyclin) synthesis and platelet function. HAEC were treated with nine commonly consumed beverages to determine their effects on prostacyclin synthesis under acute and chronic treatment regimens. Orange, purple grape, and pomegranate juices and coffee (6-9 mL/kg) were then provided to 28 fasted, healthy adult subjects (eight men and 20 women) on five separate days. Plasma samples were collected immediately following juice consumption (baseline), and at 2 and 6 hours post-consumption. On an acute basis, administration of HAEC with pomegranate juice increased media prostacyclin. Chronic exposure to purple grape and pomegranate juice increased aortic endothelial cell prostacyclin synthesis (38% and 61%, respectively; P <.05). The consumption of purple grape, pomegranate, and orange juice prolonged epinephrine/collagen-induced clotting time (P <.05). Purple grape juice increased plasma 6-keto-prostaglandin F(1alpha) (20%; P <.05) at 2 hours; pomegranate and orange juice did not significantly influence plasma prostacyclin concentrations. Consistent with the in vitro data, coffee consumption did not influence clotting time or plasma prostacyclin concentrations. These results indicate that the HAEC model system can provide a qualitative means to screen food and food-derived products for biologic activity related to cardiovascular health.

In vitro studies have suggested that flavonoids may have specific vascular effects, but their mechanism of action has not been clarified. A subclass of flavonoids—flavan-3-ols and their oligomers (procyanidins)—are constituents of cocoa beans, which can be detected in human plasma after ingestion of cocoa. In turn, plant extracts rich in flavan-3-ols can increase the activity of nitric oxide synthase (NOS) in endothelial cells. Nitric oxide is an essential signaling molecule in vascular physiology. Nitric oxide bioactivity can be preserved in human plasma in a circulating pool via increases in a number of nitrosated compounds. Thus, it is possible that cocoa rich in flavan-3-ols may lead to improved endothelium-dependent dilation via an increase of nitric oxide bioactivity. However, commercially available cocoa drinks contain only small amounts of flavan-3-ols due to roasting and alkalization of cocoa beans, which are known to degrade flavan-3-ols. We tested the hypothesis that ingestion of flavan-3-ol rich cocoa can increase the circulating pool of nitric oxide in human plasma, thus increasing endothelium-dependent dilation. Participants were 26 outpatients with at least 1 cardiovascular risk factor, including history of coronary artery disease, hypertension, hyperlipidemia, diabetes, or current tobacco use. Individuals were excluded if they had C-reactive protein levels greater than 0.5 mg/dL, atrial fibrillation, acute coronary syndrome, or New York Heart Association class III or IV heart failure. Individuals were studied in the morning after a 12-hour fasting period. In an initial study involving the first 6 participants, we assessed the time course of flavan-3-ol effects on flow-mediated dilation (FMD). This was measured at 0, 2, 4, and 6 hours after ingestion of 100 mL of cocoa drink containing 176 mg of flavan-3-ols (70 mg of epicatechin plus catechin, 106 mg of procyanidins [The Positive Food Co, Wokingham, England]) (n = 6) or control (100 mL cocoa drink with <10 mg of flavan-3-ols [Dovedrink, Mars Inc, Hackettstown, NJ] or water) (n = 3). We then used these results to guide the timing of a double-blind crossover study. Twenty participants received 100 mL of cocoa drinks with high or low levels of flavan-3-ols, in random order, on 2 consecutive days. The sum of nitrosylated and nitrosated species (collectively referred to as RNO) was measured by reductive chemiluminescence assay 2 hours after ingestion on both days. Nitrate and nitrite levels were measured as previously described. Endothelium-dependent dilation was assessed by measuring FMD of the brachial artery. In addition, we measured a number of other vascular parameters that would not be expected to change as a result of flavan-3-ol, including blood pressure, heart rate, and plasma levels of nitrite and nitrate. Similarly, we measured endothelium-independent dilation of the brachial artery following sublingual application of 400 µg of glyceroltrinitrate, diameter of the brachial artery, and forearm blood-flow at rest and during reactive hyperemia, as assessed by venous occlusion plethysmography. (Technical details are available from the authors.) All variables except endothelium-independent dilation were measured both before and after ingestion of the cocoa. Endothelium-independent dilation was measured only after ingestion of each drink, as nitroglycerine could have interfered with measurement of the other variables. Differences were assessed by paired t tests, with P values for multiple comparisons adjusted by the Bonferroni criterion. Our study was approved by the ethics board of the Medical Faculty of the Heinrich Heine-University, and all participants gave written informed consent.  We found that a single dose of a cocoa drink rich in flavan-3-ols transiently increased nitric oxide bioactivity in human plasma and significantly reversed endothelial dysfunction. The correlation between FMD and levels of RNO suggests that flavan-3-ols induce arterial dilation via their effects on nitric oxide availability, a conclusion that is supported by the negative results for the other vascular variables. The long-term clinical effect of flavan-3-ols, however, remains to be established.

BACKGROUND: Flavonoids may be partly responsible for some health benefits, including antiinflammatory action and a decreased tendency for the blood to clot. An acute dose of flavanols and oligomeric procyanidins from cocoa powder inhibits platelet activation and function over 6 h in humans. OBJECTIVE: This study sought to evaluate whether 28 d of supplementation with cocoa flavanols and related procyanidin oligomers would modulate human platelet reactivity and primary hemostasis and reduce oxidative markers in vivo. DESIGN: Thirty-two healthy subjects were assigned to consume active (234 mg cocoa flavanols and procyanidins/d) or placebo (< or = 6 mg cocoa flavanols and procyanidins/d) tablets in a blinded parallel-designed study. Platelet function was determined by measuring platelet aggregation, ATP release, and expression of activation-dependent platelet antigens by using flow cytometry. Plasma was analyzed for oxidation markers and antioxidant status. RESULTS: Plasma concentrations of epicatechin and catechin in the active group increased by 81% and 28%, respectively, during the intervention period. The active group had significantly lower P selectin expression and significantly lower ADP-induced aggregation and collagen-induced aggregation than did the placebo group. Plasma ascorbic acid concentrations were significantly higher in the active than in the placebo group (P < 0.05), whereas plasma oxidation markers and antioxidant status did not change in either group. CONCLUSIONS: Cocoa flavanol and procyanidin supplementation for 28 d significantly increased plasma epicatechin and catechin concentrations and significantly decreased platelet function. These data support the results of acute studies that used higher doses of cocoa flavanols and procyanidins.

BACKGROUND: Proanthocyanidins, the most abundant polyphenols in chocolate, are not depolymerized in the stomach and reach the small intestine intact, where they are hardly absorbed because of their high molecular weight. In vitro and in vivo studies using pure compounds as substrates suggest that proanthocyanidins and the related catechin monomers may be degraded into more bioavailable low-molecular-weight phenolic acids by the microflora in the colon. OBJECTIVE: The aim of the study was to estimate the amounts of phenolic acids formed by the microflora and excreted in the urine of human subjects after consumption of polyphenol-rich chocolate. DESIGN: After consumption of a polyphenol-free diet for 2 d and a subsequent overnight fast, 11 healthy subjects (7 men and 4 women) consumed 80 g chocolate containing 439 mg proanthocyanidins and 147 mg catechin monomers. All urine was collected during the 24 h before chocolate consumption and at 3, 6, 9, 24, and 48 h after chocolate consumption. Aromatic acids were identified in urine by gas chromatography-mass spectrometry and were quantified by HPLC-electrospray ionization tandem mass spectrometry. RESULTS: Chocolate intake increased the urinary excretion of the 6 following phenolic acids: m-hydroxyphenylpropionic acid, ferulic acid, 3,4-dihydroxyphenylacetic acid, m-hydroxyphenylacetic acid, vanillic acid, and m-hydroxybenzoic acid. CONCLUSION: The antioxidant and biological effects of chocolate may be explained not solely by the established absorption of catechin monomers but also by the absorption of microbial phenolic acid metabolites.

In the presence of a H(2)O(2)-generating system, myeloperoxidase (MPO) caused conjugated diene formation in low-density lipoprotein (LDL), indicating lipid peroxidation which was dependent on nitrite but not on chloride. The oxidation of LDL was inhibited by micromolar concentrations of flavonoids such as (-)-epicatechin, quercetin, rutin, taxifolin and luteolin, presumably via scavenging of the MPO-derived NO(2) radical. The flavonoids served as substrates of MPO leading to products with distinct absorbance spectra. The MPO-catalyzed oxidation of flavonoids was accelerated in the presence of nitrite.

Evidence suggests that certain flavan-3-ols and procyanidins (FP) can have a positive influence on cardiovascular health. It has been previously reported that FP isolated from cocoa can potentially modulate the level and production of several signaling molecules associated with immune function and inflammation, including several cytokines and eicosanoids. In the present study, we examined whether FP fractions monomers through decamers modulate secretion of the cytokine transforming growth factor (TGF)-beta(1) from resting human peripheral blood mononuclear cells (PBMC). A total of 13 healthy subjects were studied and grouped according to their baseline production of TGF-beta(1). When cells from individuals with low baseline levels of TGF-beta(1) (n = 7) were stimulated by individual FP fractions (25 microg/ml), TGF-beta(1) release was enhanced in the range of 15%-66% over baseline (P < 0.05; monomer, dimer, and tetramer). The low-molecular-weight FP fractions (<or=pentamer) were more effective at augmenting TGF-beta(1) secretion than their larger counterparts (>or=hexamer), with the monomer and dimer inducing the greatest increases (66% and 68%, respectively). In contrast to the above, TGF-beta(1) secretion from high TGF-beta(1) baseline subjects (n = 6) was inhibited by individual FP fractions (P < 0.05; trimer through decamer). The inhibition was most pronounced with trimeric through decameric fractions (28%-42%), and monomers and dimers moderately inhibited TGF-beta(1) release (17% and 23%, respectively). Given the vascular actions associated with TGF-beta(1), we suggest that in healthy individuals, homeostatic modulation of its production by FP offers an additional mechanism by which FP-rich foods can potentially benefit cardiovascular health.

Cocoa flavanols and procyanidins have numerous biological activities. It is known that (-)-epicatechin, (+)-catechin, epicatechin-(4beta-8)-epicatechin (dimer B2), and epicatechin-(4beta-6)-epicatechin (dimer B5) are unstable at physiologic pH, degrading almost completely within several hours, whereas they are relatively stable at pH 5.0. The present study investigated the effects of ascorbic and citric acid on the stability of monomers and dimers in simulated intestinal juice (pH 8.5) and in sodium phosphate buffer (pH 7.4). The addition of ascorbic acid to the incubation mixture significantly increased the stability of the monomers and dimers, whereas the addition of citric acid provided no protective effects. LC-MS showed that with the degradation of dimer B2 and dimer B5, doubly linked A-type dimers were formed. The present results, although not directly transferable to in vivo conditions, suggest that ascorbic acid may stabilize cocoa flavanols and procyanidins in the intestine where the pH is neutral, or alkaline, before absorption.

BACKGROUND: Polyphenolic procyanidins are abundant flavonoid polymers in Western diets. In vitro biological activity has been reported for these compounds, but activity in vivo depends on the amount and chemical nature of the flavonoids reaching the gastrointestinal tract. Degradation of procyanidins under simulated gastric conditions at pH 2.0 has been reported in vitro. OBJECTIVE: The objective was to examine whether depolymerization of procyanidins occurs in the stomach of human subjects in vivo. DESIGN: After an overnight fast, 6 healthy subjects (3 men and 3 women) consumed 500 mL of a cocoa beverage containing 733 mg procyanidin polymers and 351 mg structurally related flavanol monomers. With the use of a nasogastric tube, stomach contents were collected every 10 min after beverage ingestion until the stomach was emptied. Flavanols and procyanidins (up to pentamers) were quantified by normal and reversed-phase HPLC. RESULTS: In all subjects, gastric transit lasted approximately 50-60 min. No change in the HPLC profile of procyanidins was observed during this period, showing that procyanidins were remarkably stable in the stomach environment. CONCLUSION: The results suggest that most ingested procyanidins reach the small intestine intact and are available for absorption or metabolism.