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Flavanols, a class of polyphenols present in certain plant-based foods, have received increasing attention for their putative anticancer activity. In vitro and in vivo studies, which have compared the effectiveness of various monomer flavanols, indicate that the presence of a galloyl residue on the 3 position on the C-ring enhances the cytotoxicity of these compounds. Procyanidins, oligomerized flavanols, have been reported to be more cytotoxic than monomer flavanols in a variety of human cancer cell lines. Given the above, we evaluated the potential anticancer properties of dimer procyanidins that contain galloyl groups. Specifically, the cytotoxicity of synthetic digalloyl dimer B1 and B2 esters {[3-O-galloyl]-(−)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (DGB1) and [3-O-galloyl]-(−)-epicatechin-(4β,8)-(+)-epicatechin-3-O-gallate (DGB2), respectively} were tested in a number of in vitro models. DGB1 produced significant cytotoxicity in a number of human cancer cell lines evaluated by three independent methods: ATP content, MTT and MTS assays. For the three most sensitive cell lines, exposure to DGB1 and DGB2 for 24, 48 or 72 h was associated with a reduction in cell number and an inhibition of cell proliferation. Digalloyl dimers exerted significantly higher cytotoxic effects than the structurally related flavanols, (−)-epicatechin, (+)-catechin, (−)-epicatechin gallate, (−)-epigallocatechin gallate, (−)-catechin gallate and dimer B1 and B2. These results support the concept that the incorporation of galloyl groups and the oligomerization of flavanols enhances the cytotoxic effects of typical monomer flavanols. The therapeutic value of these compounds and their derivative forms as anticancer agents merits further investigation in whole animal models.

Due to long-term toxicity of current Hodgkin's lymphoma (HL) treatment, the present challenge is to find new therapies that specifically target deregulated signaling cascades, including NF-kappaB, which are involved in Hodgkin (H) and Reed-Sternberg (RS) cell proliferation and resistance to apoptosis. We previously presented evidence that dimeric procyanidin B2 (B2) can interact with NF-kappaB proteins inhibiting the binding of NF-kappaB to DNA. Herein, we investigated if B2, acting at a late event in NF-kappaB signaling cascade, could be effective in inhibiting NF-kappaB in H-RS cells with different mechanisms of constitutive NF-kappaB activation. B2 caused a concentration-dependent inhibition of NF-kappaB-DNA binding to a similar extent (41-48% inhibition at 25 microM B2) in all the tested H-RS cell lines (L-428, KM-H2, L-540, L-1236 and HDML-2). This was associated with the inhibition of NF-kappaB-driven gene expression, including cytokines (IL-6, TNFalpha and RANTES) and anti-apoptotic proteins (Bcl-xL, Bcl-2, XIAP and cFLIP). The finding of similar amounts of RelA and p50 proteins in the nucleus, but decreased NF-kappaB-DNA binding, even in those H-RS cells characterized by mutations in the inhibitory IkappaB proteins, supports that B2 acts by preventing the binding of NF-kappaB to DNA. B2 did not inhibit AP-1 and STAT3 constitutive activation in H-RS cells, indicating that the moderate effects of B2 on cell viability are due to the complex signaling aberrations in HL. Thus, several signaling pathways should be targeted when designing therapeutics for HL. In this regard, the capacity of B2 to inhibit NF-kappaB could be valuable in a multi-drug approach.

Flavanols and their related oligomeric compounds, the procyanidins, have received increased attention during the past decade due to their reported health benefits. On the basis of compelling data published during the past decade demonstrating that the consumption of certain flavanol-rich foods can improve markers of cardiovascular health, additional clinical, and epidemiological research is clearly warranted to establish appropriate public health recommendations. However, recommendations on the consumption of these foods appropriate for use by health professionals can only be made on the basis of clinical investigations that accurately identify and quantify--through proper analytical measurement systems--the flavanols in the foods used in these investigations. This manuscript provides an overview of the strengths, weaknesses, and limitations of commonly used analytical methods to characterize the content of flavanols in foods. Two nonspecific measurements widely used by investigators, the Folin-Ciocalteu assay and the Oxygen Radical Absorbance Capacity (ORAC) measurement, are discussed in this context, as is the use of various high-performance liquid chromatography methods that provide more specific data related to the content of flavanols in foods. A comparison of the data obtained from these analytical methods to those of the more rigorous high-performance liquid chromatography analyses demonstrates that these nonspecific methods are ill-suited for providing unequivocal data necessary to evaluate the importance of dietary flavanols in the context of improving cardiovascular health. Meaningful dietary recommendations for the consumption of flavanol-rich foods will only be made possible by additional well-designed clinical and epidemiological studies enabled by detailed compositional data obtained through use of appropriate analytical methods.

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.

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.

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.

Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-alpha (TNF-alpha) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-alpha released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3-4 fold increase in TNF-alpha relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-alpha secretion in the range of 48-128% relative to the PHA control. The monomers and dimers were slightly inhibitory (-1.5 and -15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-alpha levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.

The polymeric procyanidins were fractionated from lowbush blueberry on a Sephadex LH-20 column. The degree of polymerization (DP) for the polymers was determined by thiolysis to be in a range of 19.9 to 114.1. Normal-phase HPLC analysis indicated that the polymeric procyanidins did not contain oligomeric procyanidins with DP < 10. The polymers eluted as a single peak at the end of the chromatogram. The normal-phase HPLC gradient was modified to improve the separation of procyanidin monomers through decamers and to elute all the polymers beyond those as a distinct peak. Monomers through decamers were quantified individually. All the polymers (DP > 10) were quantified using a mixture of purified polymers as an external standard. Polymers were found to be the dominant procyanidins in brown sorghum bran, cranberry, and blueberry. Thiolysis of the polymer peaks indicated that epicatechin was present as extension units in these foods, however, the composition of terminal units varied considerably between catechin and epicatechin, or an A-type dimer linkage in the case of cranberry.

Cocoa flavanols and procyanidins possess wide-ranging biological activities. The present study investigated the stability of the cocoa monomers, (-)-epicatechin and (+)-catechin, and the dimers, epicatechin-(4beta-8)-epicatechin (Dimer B2) and epicatechin-(4beta- 6)-epicatechin (Dimer B5), in simulated gastric and intestinal juice and at different pH values. The dimers were less stable than the monomers at both acidic and alkaline pH. Incubation of Dimer B2 and Dimer B5 in simulated gastric juice (pH 1.8) or acidic pH resulted in degradation to epicatechin and isomerization to Dimer B5 and Dimer B2, respectively. When incubated in simulated intestinal juice or at alkaline pH, all four compounds degraded almost completely within several hours. These results suggest that the amount, and type, of flavanols and procyanidins in the gastrointestinal tract following the consumption of cocoa can be influenced by the stability of these compounds in both acidic and alkaline environments.

The Kuna Amerinds reside chiefly in the San Blas islands (Kuna Yala) on the Caribbean coast of Panama. The diet of this population has not previously been described in detail and composition data for certain foods consumed by this population does not exist or is lacking for certain important nutrients. The protein, fat, moisture, fiber, sugar, mineral and procyanidin content was determined in foods selected because of the frequency with which they are consumed by this population. For that reason, emphasis was given to Tule Masi, a typical Kuna stew-like dish, and cocoa-containing beverages. The foods analyzed were generally low in fat and protein. Potassium and magnesium are present in Tule Masi, common beverages and certain fruits and vegetables at significant levels when considering the preliminary reports on the daily intake of these foods. In addition, preliminary reports indicate that salt use is common, an observation that is supported by the sodium content of the Tule Masi. The cocoa and cocoa beans used in the preparation of beverages are rich in several minerals and procyanidins, as expected. This analysis will allow for the estimation of nutrient intake and subsequent investigations into the relationship between diet and health in this population.

Blueberries and cranberries were analyzed for procyanidins using normal-phase HPLC/MS. Monomers, identified as (+)-catechin and (-)-epicatechin, and a series of oligomers were detected in blueberries, and MS data confirmed that the oligomers consisted of (epi)catechin units that were exclusively singly linked (B-type). The procyanidin "fingerprints" were similar for Tifblue and Rubel but higher than that for lowbush blueberries. In whole cranberries, (-)-epicatechin was present, along with a complex series of oligomers. Both A-type (contained only one double linkage per oligomer) and B-type oligomers were present. Two commercial cranberry juices exhibited similar procyanidin profiles, except that one contained increased quantities. There were processing effects on the procyanidin content of cranberry extract and juices when compared to those of the unprocessed fruits. Monomer, dimers, and A-type trimers were the primary procyanidins, with only trace levels of the B-type trimers and A-type tetramers and with an absence of the higher oligomers in cranberry extract and juices.

From the Departments of Nutrition, Internal Medicine, and Food Science, University of California, Davis, and Analytic and Applied Sciences, MARS Inc, Hackettstown, NJ.

Background: Polyphenolic phytochemicals inhibit vascular and inflammatory processes that contribute to disease. These effects are hypothesized to result from polyphenol-mediated alterations in cellular eicosanoid synthesis.

Objective: The objective was to determine and compare the ability of cocoa procyanidins to alter eicosanoid synthesis in human subjects and cultured human aortic endothelial cells.

Design: After an overnight fast, 10 healthy subjects (4 men and 6 women) consumed 37 g low-procyanidin (0.09 mg/g) and high-procyanidin (4.0 mg/g) chocolate; the treatments were separated by 1 wk. The investigation had a randomized, blinded, crossover design. Plasma samples were collected before treatment and 2 and 6 h after treatment. Eicosanoids were quantitated by enzyme immunoassay. Endothelial cells were treated in vitro with procyanidins to determine whether the effects of procyanidin in vivo were associated with procyanidin-induced alterations in endothelial cell eicosanoid synthesis.

Results: Relative to the effects of the low-procyanidin chocolate, high-procyanidin chocolate induced increases in plasma prostacyclin (32%; P < 0.05) and decreases in plasma leukotrienes (29%; P < 0.04). After the in vitro procyanidin treatments, aortic endothelial cells synthesized twice as much 6-keto-prostaglandin F1 (P < 0.01) and 16% less leukotriene (P < 0.05) as did control cells. The in vitro and in vivo effects of procyanidins on plasma leukotriene-prostacyclin ratios in culture medium were also comparable: decreases of 58% and 52%, respectively.

Conclusion: Data from this short-term investigation support the concept that certain food-derived flavonoids can favorably alter eicosanoid synthesis in humans, providing a plausible hypothesis for a mechanism by which they can decrease platelet activation in humans.

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.

Procyanidins are a subclass of flavonoids found in commonly consumed foods that have attracted increasing attention due to their potential health benefits. However, little is known regarding their dietary intake levels because detailed quantitative information on the procyanidin profiles present in many food products is lacking. Therefore, the procyanidin content of red wine, chocolate, cranberry juice and four varieties of apples has been determined. On average, chocolate and apples contained the largest procyanidin content per serving (164.7 and 147.1 mg, respectively) compared with red wine and cranberry juice (22.0 and 31.9 mg, respectively). However, the procyanidin content varied greatly between apple samples (12.3-252.4 mg/serving) with the highest amounts on average observed for the Red Delicious (207.7 mg/serving) and Granny Smith (183.3 mg/serving) varieties and the lowest amounts in the Golden Delicious (92.5 mg/serving) and McIntosh (105.0 mg/serving) varieties. The compositional data reported herein are important for the initial understanding of which foods contribute most to the dietary intake of procyanidins and may be used to compile a database necessary to infer epidemiological relationships to health and disease.

Recent data has demonstrated that cacao liquor polyphenols (procyanidins) have antioxidant activity, inhibit mRNA expression of interleukin-2 and are potent inhibitors of acute inflammation. Given the widespread ingestion of cocoa in many cultures, we investigated whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate synthesis of the pro-inflammatory cytokine, interleukin-1 beta. Both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) were investigated at the levels of transcription and protein secretion. Individual cocoa fractions were shown to augment constitutive IL-1 beta gene expression, although values varied between subjects. Interestingly, the smaller fractions of cocoa (monomer-tetramer) consistently reduced IL-1 beta expression of PHA-stimulated cells by 1-15%, while the larger oligomers (pentamer-decamer) increased expression by 4-52%. These data, observed at the transcription level, were reflected in protein levels in PHA-induced PBMC. The presence or absence of PHA did not alter the effects of the cocoa procyanidins with the exception of the pentamer. This study offers additional data for the consideration of the health-benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa 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.

In the current study, we investigated the usefulness of reversed-phase and normal-phase chromatography for comparing the separation of low molecular weight flavonoids in green tea versus the oligomeric procyanidins in cocoa. The results of this study demonstrated that the reversed-phase technique was better suited for the separation of the flavan-3-ols and flavonols in green tea while the normal-phase method was superior for separation of flavan-3-ol oligomers in cocoa. Therefore, it was concluded that both techniques are required for a comprehensive survey of the flavonoid classes that are ubiquitous in nature.

Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.