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Given the central role of the transcription factor NF-kappaB in inflammation, molecules that can inhibit NF-kappaB are being actively investigated. The present work characterize potential interactions between dimeric procyanidins [B-type (B1 and B2) and A-type (A1 and A2)] and NF-kappaB proteins. B1 and B2, inhibited tumor necrosis factor alpha (TNFalpha)- and phorbol 12-myristate 13-acetate (PMA)-induced transactivation of NF-kappaB-driven genes and the increase of NF-kappaB-DNA nuclear binding in Jurkat T cells. B1 and B2, added in vitro to nuclear fractions, inhibited NF-kappaB binding to its DNA consensus sequence. B1 and B2 prevented the binding of RelA and p50 recombinant proteins to its DNA consensus sequence. All these effects were not observed with A1 and A2. Putative molecular models for possible interactions of B1, B2, A1 and A2, with NF-kappaB proteins were constructed, indicating that B-type dimeric procyanidins have higher possibilities of chemical interactions with NF-kappaB than A-type dimeric procyanidins. The results support the concept that B-type dimeric procyanidins can provide anti-inflammatory benefits due to their ability to reduce NF-kappaB binding to the DNA.

Hexameric procyanidins inhibit TNFalpha-induced NF-kappaB activation in Caco-2 cells. Most of the physiological actions of high molecular weight procyanidins could be limited to the gut lumen. Transcription factor NF-kappaB plays a central role in inflammation including human intestinal bowel disease. We investigated the capacity of a hexameric procyanidin fraction (Hex) to prevent tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation as related to oxidation and membrane interactions. In Caco-2 cells, Hex (2.5-20 microM) inhibited TNFalpha-induced NF-kappaB activation (IkappaB phosphorylation and degradation, p50 and RelA nuclear translocation, and NF-kappaB-DNA binding), inducible nitric oxide synthase expression, and cell oxidant increase. The effects on NF-kappaB activation persist beyond the period of direct exposition of cells to Hex. N-Acetylcysteine and alpha-lipoic acid inhibited TNFalpha-induced oxidant increase but did not affect NF-kappaB activation. In summary, Hex can inhibit NF-kappaB activation by interacting with the plasma membrane of intestinal cells, and through these interactions preferentially inhibits the binding of TNFalpha to its receptor and the subsequent NF-kappaB activation.

OBJECTIVES: Our goal was to test feasibility and efficacy of a dietary intervention based on daily intake of flavanol-containing cocoa for improving vascular function of medicated diabetic patients. BACKGROUND: Even in fully medicated diabetic patients, overall prognosis is unfavorable due to deteriorated cardiovascular function. Based on epidemiological data, diets rich in flavanols are associated with a reduced cardiovascular risk. METHODS: In a feasibility study with 10 diabetic patients, we assessed vascular function as flow-mediated dilation (FMD) of the brachial artery, plasma levels of flavanol metabolites, and tolerability after an acute, single-dose ingestion of cocoa, containing increasing concentrations of flavanols (75, 371, and 963 mg). In a subsequent efficacy study, changes in vascular function in 41 medicated diabetic patients were assessed after a 30-day, thrice-daily dietary intervention with either flavanol-rich cocoa (321 mg flavanols per dose) or a nutrient-matched control (25 mg flavanols per dose). Both studies were undertaken in a randomized, double-masked fashion. Primary and secondary outcome measures included changes in FMD and plasma flavanol metabolites, respectively. RESULTS: A single ingestion of flavanol-containing cocoa was dose-dependently associated with significant acute increases in circulating flavanols and FMD (at 2 h: from 3.7 +/- 0.2% to 5.5 +/- 0.4%, p < 0.001). A 30-day, thrice-daily consumption of flavanol-containing cocoa increased baseline FMD by 30% (p < 0.0001), while acute increases of FMD upon ingestion of flavanol-containing cocoa continued to be manifest throughout the study. Treatment was well tolerated without evidence of tachyphylaxia. Endothelium-independent responses, blood pressure, heart rate, and glycemic control were unaffected. CONCLUSIONS: Diets rich in flavanols reverse vascular dysfunction in diabetes, highlighting therapeutic potentials in cardiovascular disease.

Substantial data suggest that flavonoid-rich food could help prevent cardiovascular disease and cancer. Cocoa is the richest source of flavonoids, but current processing reduces the content substantially. The Kuna living in the San Blas drink a flavanol-rich cocoa as their main beverage, contributing more than 900 mg/day and thus probably have the most flavonoid-rich diet of any population. We used diagnosis on death certificates to compare cause-specific death rates from year 2000 to 2004 in mainland and the San Blas islands where only Kuna live. Our hypothesis was that if the high flavanoid intake and consequent nitric oxide system activation were important the result would be a reduction in the frequency of ischemic heart disease, stroke, diabetes mellitus, and cancer--all nitric oxide sensitive processes. There were 77,375 deaths in mainland Panama and 558 deaths in the San Blas. In mainland Panama, as anticipated, cardiovascular disease was the leading cause of death (83.4 +/- 0.70 age adjusted deaths/100,000) and cancer was second (68.4 +/- 1.6). In contrast, the rate of CVD and cancer among island-dwelling Kuna was much lower (9.2 +/- 3.1) and (4.4 +/- 4.4) respectively. Similarly deaths due to diabetes mellitus were much more common in the mainland (24.1 +/- 0.74) than in the San Blas (6.6 +/- 1.94). This comparatively lower risk among Kuna in the San Blas from the most common causes of morbidity and mortality in much of the world, possibly reflects a very high flavanol intake and sustained nitric oxide synthesis activation. However, there are many risk factors and an observational study cannot provide definitive evidence.

Procyanidins can exert cytoprotective, anti-inflammatory, and anticarcinogenic actions in the gastrointestinal tract. Previous evidence has shown that procyanidins can interact with synthetic membranes and protect them from oxidation and disruption. Thus, in this study we investigated the capacity of a hexameric procyanidin fraction (Hex) isolated from cocoa to protect Caco-2 cells from deoxycholic (DOC)-induced cytotoxicity, cell oxidant increase, and loss of monolayer integrity. Hex interacted with the cell membranes without affecting their integrity, as evidenced by a Hex-mediated increase in the transepithelial electrical resistance, and inhibition of DOC-induced cytotoxicity. DOC induced an increase in cell oxidants, alterations in the paracellular transport, and redistribution of the protein ZO-1 from cell-cell contacts into the cytoplasm. Hex partially inhibited all these events at concentrations ranging from 2.5 to 20 microM. Similarly, Hex (5-10 microM) inhibited the increase in cell oxidants, and the loss of integrity of polarized Caco-2 cell monolayers induced by a lipophilic oxidant (2,2'-azobis (2,4-dimethylvaleronitrile). Results show that the assayed procyanidin fraction can interact with cell membranes and protect Caco-2 cells from DOC-induced cytotoxicity, oxidant generation, and loss of monolayer integrity. At the gastrointestinal tract, large procyanidins may exert beneficial effects in pathologies such us inflammatory diseases, alterations in intestinal barrier permeability, and cancer.

A proteomic analysis of procyanidin B(2) isolated from cocoa against oxidized low-density lipoprotein-induced lipid-laden macrophage formation was performed. Of approximately 400 detected proteins, 12 were differentially expressed as a result of B(2) treatment. They were subsequently identified by liquid chromatography-electrospray ionization-tandem mass spectrometry and the SWISS-PROT database. Further reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that B(2) strongly inhibited arachidonic acid inflammatory reactions, apoptosis, and their coupled mitogen-activated protein kinase and NF-kappaB pathways. To highlight proteins or genes with similar expressed patterns and similarly biological function induced by B(2) in lipid-laden macrophages, a cluster and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed. The data were mapped to multiple pathways. Further validation of the bioinformatic results revealed that activation of Wnt signaling may contribute to the cardioprotection of B(2). The differentially expressed genes and proteins mentioned above induced by B(2) are through regulating nuclear transcription factors, activating peroxisome proliferator-activated receptor-gamma and inhibiting AP-1 mRNA expressions. These in vitro data help to interpret the beneficial effects of B(2) in reducing the risk of atherosclerosis after consumption of flavonoid-rich foods. Many differentially expressed genes induced by B(2) help to uncover novel targets and may help to target disease interactions in atherosclerosis in the future.

Atherosclerosis is the major cause for chronic vascular diseases. The key event in the pathogenesis of atherosclerosis is believed to be dysfunction of the endothelium and disruption of endothelial homeostasis, leading to vasoconstriction, inflammation, leukocyte adhesion, thrombosis, and proliferation of vascular smooth muscle cells. Endothelium-derived nitric oxide (NO) plays a major role in vascular homeostasis and a decrease in NO-bioavailability accelerates the development of atherosclerosis. Given that endothelial dysfunction is at least in part reversible, the characterization of endothelial function and therapeutical approaches have gained much attention over the past years. Recent studies demonstrated that especially the consumption of plant-derived foods rich in certain flavonoids can improve endothelial function in both compromised and healthy humans. Furthermore, various physiologic and biochemical measures have been used previously as biomarkers for the assessment of the proposed beneficial effects of flavonoids in this context. More recently, the analysis of plasma nitros(yl)ated species (RXNOs), referred to as the circulating NO pool, has gained recognition, especially as a marker for endothelial function. This review is aimed at evaluating the suitability of quantifying this NO pool as a biomarker for cardiovascular function in humans, in particular during dietary interventions with flavonoid-rich foods.

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.

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.

We report an improved synthesis of bis(5,7,3',4'-tetra-O-benzyl)epicatechin 4beta,8-dimer (3) from 5,7,3',4'-tetra-O-benzylepicatechin (1) and 5,7,3',4'-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin (2) by replacing the previously employed Lewis acid, titanium tetrachloride, with the clay mineral Bentonite K-10. Under the same conditions, the benzyl-protected all-4beta,8-trimer, -tetramer, and -pentamer were obtained regioselectively from their lower homologues, albeit in rapidly decreasing yields. Reaction of 2 with an organoaluminum thiolate generated from 2-mercaptobenzothiazole and trimethylaluminum followed by acetylation produced 3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3',4'-tetra-O-benzylepicatechin (12). Medium-sized protected oligomers with 4beta,8-interflavan linkages are obtained in improved yields by using this compound as the electrophile and silver tetrafluoroborate as activator and are isolated by reversed-phase HPLC. Their deprotection by ester saponification followed by hydrogenolysis yielded the free procyanidins, which were characterized as their peracetates. The synthetic procyanidins are identical by normal-phase HPLC with fractions isolated from cocoa. The principle of chain extension by two members was demonstrated using a dimeric electrophile obtained by self-condensation of compound 12. Both the synthetic and natural pentamer 32 inhibit the growth of several breast cancer cell lines. Using the MDA MB 231 line, it was established that this outcome is based on the induction of cell cycle arrest in the G0/G1 phase. Subsequent cell death is more likely necrotic rather than apoptotic. Control experiments demonstrate that the polyphenol itself, rather than hydrogen peroxide potentially formed by its autoxidation, is the causative agent.

BACKGROUND: Epidemiologic studies have linked flavonoid-rich foods with a reduced risk of cardiovascular mortality. Some cocoas are flavonoid-rich and contain the monomeric flavanols (-)-epicatechin and (+)-catechin and oligomeric procyanidins formed from these monomeric units. Both the monomers and the oligomers have shown potential in favorably influencing cardiovascular health in in vitro and preliminary clinical studies. Although previous investigations have shown increasing concentrations of (-)-epicatechin in human plasma after cocoa consumption, no information is available in the published literature regarding the presence of procyanidins in human plasma. OBJECTIVE: This study sought to determine whether procyanidins can be detected and quantified in human plasma after acute consumption of a flavanol-rich cocoa. DESIGN: Peripheral blood was obtained from 5 healthy adult subjects before (baseline, 0 h) and 0.5, 2, and 6 h after consumption of 0.375 g cocoa/kg body wt as a beverage. Plasma samples were analyzed for monomers and procyanidins with the use of reversed-phase HPLC with coulometric electrochemical array detection and liquid chromatography-tandem mass spectrometry. RESULTS: Procyanidin dimer, (-)-epicatechin, and (+)-catechin were detected in the plasma of human subjects as early as 0.5 h (16 +/- 5 nmol/L, 2.61 +/- 0.46 micro mol/L, and 0.13 +/- 0.03 micro mol/L, respectively) after acute cocoa consumption and reached maximal concentrations by 2 h (41 +/- 4 nmol/L, 5.92 +/- 0.60 micro mol/L, and 0.16 +/- 0.03 micro mol/L, respectively). CONCLUSION: Dimeric procyanidins can be detected in human plasma as early as 30 min after the consumption of a flavanol-rich food such as cocoa.

Epidemiologic studies have linked flavonoid-rich foods with a reduced risk of cardiovascular mortality. Some cocoas are flavonoid-rich and contain the monomeric flavanols (-)-epicatechin and (+)-catechin and oligomeric procyanidins formed from these monomeric units. Both the monomers and the oligomers have shown potential in favorably influencing cardiovascular health in in vitro and preliminary clinical studies. Although previous investigations have shown increasing concentrations of (-)-epicatechin in human plasma after cocoa consumption, no information is available in the published literature regarding the presence of procyanidins in human plasma. OBJECTIVE: This study sought to determine whether procyanidins can be detected and quantified in human plasma after acute consumption of a flavanol-rich cocoa. DESIGN: Peripheral blood was obtained from 5 healthy adult subjects before (baseline, 0 h) and 0.5, 2, and 6 h after consumption of 0.375 g cocoa/kg body wt as a beverage. Plasma samples were analyzed for monomers and procyanidins with the use of reversed-phase HPLC with coulometric electrochemical array detection and liquid chromatography-tandem mass spectrometry. RESULTS: Procyanidin dimer, (-)-epicatechin, and (+)-catechin were detected in the plasma of human subjects as early as 0.5 h (16 +/- 5 nmol/L, 2.61 +/- 0.46 micro mol/L, and 0.13 +/- 0.03 micro mol/L, respectively) after acute cocoa consumption and reached maximal concentrations by 2 h (41 +/- 4 nmol/L, 5.92 +/- 0.60 micro mol/L, and 0.16 +/- 0.03 micro mol/L, respectively). CONCLUSION: Dimeric procyanidins can be detected in human plasma as early as 30 min after the consumption of a flavanol-rich food such as cocoa.

Epidemiologic studies suggest an inverse association of tea consumption with cardiovascular disease. The antioxidant effects of flavonoids in tea (including preventing oxidative damage to LDL) are among the potential mechanisms that could underlie the protective effects. Other possible mechanisms include attenuating the inflammatory process in atherosclerosis, reducing thrombosis, promoting normal endothelial function, and blocking expression of cellular adhesion molecules. Cocoa and chocolate can also be rich sources of flavonoids. Flavanols and procyanidins isolated from cocoa exhibit strong antioxidant properties in-vitro. In acute feeding studies, flavanol-rich cocoa and chocolate increased plasma antioxidant capacity and reduced platelet reactivity. Based on limited data, approximately 150 mg of flavonoids is needed to trigger a rapid antioxidant effect and changes in prostacyclin. Some dose-response evidence demonstrates an antioxidant effect with approximately 500 mg flavonoids. Brewed tea typically contains approximately 172 mg total flavonoids per 235 ml (brewed for 2 min); hence, consumption of 1 and 3.5 cups of tea would be expected to elicit acute and chronic physiologic effects, respectively. Chocolate is more variable with some products containing essentially no flavonoids (0.09 mg procyanidin/g), whereas others are high in flavonoids (4 mg procyanidin/g). Thus, approximate estimates of flavonoid rich chocolate needed to exert acute and chronic effects are 38 and 125 g, respectively. Collectively, the antioxidant effects of flavonoid-rich foods may reduce cardiovascular disease risk.

The effects of cocoa powder and extracts with different amounts of flavanols and related procyanidin oligomers were investigated on the growth of Caco-2 cells. Treatment of the cells with 50 microg/ml of procyanidin-enriched (PE) extracts caused a 70% growth inhibition with a blockade of the cell cycle at the G2/M phase. PE extracts caused a significant decrease of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities, two key enzymes of polyamine biosynthesis. This led to a decrease in the intracellular pool of the polyamines. These observations indicate that polyamine metabolism might be an important target in the anti-proliferative effects of cocoa polyphenols.

Oligomeric procyanidins containing 4alpha-linked epicatechin units are rare in nature and have hitherto not been accessible through stereoselective synthesis. We report herein the preparation of the prototypical dimer, epicatechin-4alpha,8-epicatechin (6), by reaction of the protected 4-ketones 11a,b with aryllithium reagents derived by halogen/metal exchange from the aryl bromides 26a,b. Removal of the 4-hydroxyl group from the resulting tertiary benzylic alcohols 27a,b was effected by tri-n-butyltin hydride and trifluoroacetic acid in a completely stereoselective manner, resulting in hydride delivery exclusively from the beta face. If benzyl was chosen for protection of the 3-hydroxyls, all protective groups could subsequently be removed in a single step by hydrogenolysis. tert-Butyldimethylsilyl groups, on the other hand, permitted selective deprotection of the 3-hydroxyls in preparation for their subsequent acylation with tri-O-benzylgalloyl chloride. Only monogalloylation at the "bottom" 3-hydroxyl took place when 28c was acylated under the previously reported conditions, reflecting the increased steric hindrance of the "top" 3-hydroxyl group in 28c compared with its 4beta,8-isomer 3. The preparation of compounds 14 and 17 containing phloroglucinol trimethyl ether in the 4alpha and 4beta linkages to epicatechin is also described. The 8-position of the bromine atom in 19, previously conjectured in analogy to the structurally characterized tetramethyl ether 20, was confirmed by transformation of both compounds into the common derivative 25.

The assignment of interflavan bond regio- and stereochemistry in oligomeric proanthocyanidins has in the past relied on empirical spectroscopic techniques which are influenced by the conformation of the C rings. Only recently was the 4,8-regiochemistry of procyanidin B2 (3b) firmly established by 2-dimensional NMR methods. We describe herein the proof of 4beta-stereochemistry in 3b by oxidative degradation of the derivative 3d bearing differential (O-benzyl and O-methyl) protecting groups in its "top" and "bottom" epicatechin moieties, to (R)-(-)-2,4-diphenylbutyric acid. The key elements of the degradative process are (1) removal of the C-3 alcohol functions through a modified Barton deoxygenation employing hypophosphorous acid as the reducing agent; (2) deprotection of the "top" unit by hydrogenolysis, followed by exhaustive aryl triflate formation with N,N-bis(trifluoromethanesulfonyl)aniline and DBU in DMF; (3) hydrogenolytic deoxygenation of the "top" unit over Pearlman's catalyst with concomitant scission of the O-C2 bond; (4) selective oxidation of the "bottom" unit with NaIO4/RuCl3. The hitherto unreported absolute configuration of (-)-2,4-diphenylbutyric acid was established as R by X-ray crystal structure analysis of the (R)-(+)-alpha-methylbenzylamine salt. As a corollary, the selectivity of hydrogenolytic and solvolytic reactions of epicatechin-derived tetrasulfonates has been investigated.

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.

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.

Flavonoids, natural plant constituents, protect against peroxynitrite and can thereby play a role in defense against this mediator of inflammation. Procyanidin oligomers of different size (monomer through nonamer), isolated from the seeds of Theobroma cacao, were examined for their ability to protect against peroxynitrite-dependent oxidation of dihydrorhodamine 123 and nitration of tyrosine. By molarity, oligomers were more effective than the monomeric epicatechin; the tetramer was particularly efficient at protecting against oxidation and nitration reactions. These results suggest that epicatechin oligomers found in cocoa powder and chocolate may be a potent dietary source for defense against peroxynitrite.