Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte hemolysis

Excessive peroxidation of biomembranes is thought to contribute to the initiation and progression of numerous degenerative diseases. The present study examined the inhibitory effects of a cocoa extract, individual cocoa flavanols (-)-epicatechin and (+)-catechin, and procyanidin oligomers (dimer to decamer) isolated from cocoa on rat erythrocyte hemolysis. In vitro, the flavanols and the procyanidin oligomers exhibited dose-dependent protection against 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis between concentrations of 2.5 and 40 microM. Dimer, trimer, and tetramer showed the strongest inhibitory effects at 10 microM, 59.4%, 66.2%, 70.9%; 20 microM, 84.1%, 87.6%, 81.0%; and 40 microM, 90.2%, 88.9%, 78.6%, respectively. In a subsequent experiment, male Sprague-Dawley rats (approximately 200 g; n = 5-6) were given a 100-mg intragastric dose of a cocoa extract. Blood was collected over a 4-hr time period. Epicatechin and catechin, and the dimers (-)-epicatechin-(4beta>8)-epicatechin (Dimer B2) and (-)-epicatechin-(4beta>6)-epicatechin (Dimer B5) were detected in the plasma with concentrations of 6.4 microM, and 217.6, 248.2, and 55.4 nM, respectively. Plasma antioxidant capacity (as measured by the total antioxidant potential [TRAP] assay) was elevated (P < 0.05) between 30 and 240 min following the cocoa extract feeding. Erythrocytes obtained from the cocoa extract-fed animals showed an enhanced resistance to hemolysis (P < 0.05). This enhanced resistance was also observed when erythrocytes from animals fed the cocoa extract were mixed with plasma obtained from animals given water only. Conversely, plasma obtained from rats given the cocoa extract improved the resistance of erythrocytes obtained from rats given water only. These results show cocoa flavanols and procyanidins can provide membrane protective effects.     

Interactions between cocoa flavanols and inorganic nitrate: Additive effects on endothelial function at achievable dietary amounts

Dietary intervention studies have shown that flavanols and inorganic nitrate can improve vascular function, suggesting that these two bioactives may be responsible for beneficial health effects of diets rich in fruits and vegetables. We aimed to study interactions between cocoa flavanols (CF) and nitrate, focusing on absorption, bioavailability, excretion, and efficacy to increase endothelial function. In a double-blind randomized, dose–response crossover study, flow-mediated dilation (FMD) was measured in 15 healthy subjects before and at 1, 2, 3, and 4 h after consumption of CF (1.4–10.9 mg/kg bw) or nitrate (0.1–10 mg/kg bw). To study flavanol–nitrate interactions, an additional intervention trial was performed with nitrate and CF taken in sequence at low and high amounts. FMD was measured before (0 h) and at 1 h after ingestion of nitrate (3 or 8.5 mg/kg bw) or water. Then subjects received a CF drink (2.7 or 10.9 mg/kg bw) or a micro- and macronutrient-matched CF-free drink. FMD was measured at 1, 2, and 4 h thereafter. Blood and urine samples were collected and assessed for CF and nitric oxide (NO) metabolites with HPLC and gas-phase reductive chemiluminescence. Finally, intragastric formation of NO after CF and nitrate consumption was investigated. Both CF and nitrate induced similar intake-dependent increases in FMD. Maximal values were achieved at 1 h postingestion and gradually decreased to reach baseline values at 4 h. These effects were additive at low intake levels, whereas CF did not further increase FMD after high nitrate intake. Nitrate did not affect flavanol absorption, bioavailability, or excretion, but CF enhanced nitrate-related gastric NO formation and attenuated the increase in plasma nitrite after nitrate intake. Both flavanols and inorganic nitrate can improve endothelial function in healthy subjects at intake amounts that are achievable with a normal diet. Even low dietary intake of these bioactives may exert relevant effects on endothelial function when ingested together.     

Myeloperoxidase-induced lipid peroxidation of LDL in the presence of nitrite. Protection by cocoa flavanols

Lipid peroxidation (LPO) of low-density lipoprotein (LDL) is believed to be a pivotal process rendering this plasma lipoprotein atherogenic. Several endogenous factors have been proposed to mediate LPO of LDL, among them myeloperoxidase (MPO), which is active in atherosclerotic lesions, and the plasma level of which has been proposed to be a prognostic parameter for cardiac events. Nitrite, a major oxidation product of nitric oxide, is substrate of MPO and a cofactor of MPO-mediated LPO under physiological conditions. Dietary flavonoids including (-)-epicatechin, a major flavan-3-ol in cocoa products, grapes and wine, are substrates of MPO as well as potent inhibitors of LPO in LDL at micromolar concentrations. Moreover, they strongly suppress protein tyrosine nitration of LDL by MPO/nitrite or peroxynitrite. By blunting undesirable MPO-mediated actions of nitrite, presumably via scavenging of the strong prooxidant and nitrating *NO2 radical, dietary flavonoids modulate NO metabolism in a favorable direction and thus counteract endothelial dysfunction. This article gives a survey on recent progress in this field with special reference to own recently published work.     

The effects of cocoa swollen-shoot virus on the growth and yield of Amelonado and Amazon cocoa (Theobroma cacao) in Ghana

The virulent strain A of cocoa swollen-shoot virus (CSSV) severely decreased the growth and yield of Amelonado cocoa (Theobroma cacao) trees kept free of capsids (Distantiella theobroma and Sahlbergella singularis) and the dieback fungus (Calonectria rigidiuscula) in Ghana. Fifteen per cent of graft-inoculated Amelonado trees showed symptoms within 4 months, and 48, 80 and 100% within 6, 12 and 20 months, respectively. Infected trees, whether shaded or unshaded, began to decline 6 months after infection, and deteriorated rapidly during the next 27 months by which time 16 % had died and most others were moribund; fertilizer applications had no significant effect on the rate at which infected trees deteriorated. Yields of pods and dry cocoa were greatly reduced 2 yr after infection and were very low after 3 yr; yields were significantly reduced by virus infection but there were no significant further effects of applying fertilizer. These results confirm that CSSV strain A alone is very damaging and often eventually lethal to Amelonado trees in Ghana, and indicate that the conflicting results obtained previously in Ghana and Nigeria were probably due to differences in the virulence of the CSSV strains tested. In contrast, the virus had much less effect on cocoa trees of the Amazon type; only 3% of graft-inoculated Amazon trees showed symptoms within 4 months, and 43, 84 and 97% after 1, 2 and 3 yr, respectively. Slight deterioration of tree canopies was first detected c. 15 months after infection and, although it continued slowly during the next 21 months, the decline was much less severe than that of Amelonado trees. Yields of both unshaded and shaded trees were apparently reduced by virus infection, but yield losses were much smaller than those of Amelonado trees. These results support the present objectives of controlling the spread of CSSV in Ghana by roguing infected trees, and selecting cultivars with greater tolerance to infection for future use.     

A field experiment in Ghana on the tolerance of cocoa seedlings to cocoa swollen-shoot and cocoa mottle-leaf viruses

Seedings of Amelonado Cocoa and of two progenies obtained by crossing Iquitos (Upper Amazon) parents were infected with three strains of cocoa swollen-shoot virus (CSSV) and with cocoa mottle-leaf virus (CMLV). CSSV strain I A had the most severe effects on the growth and canopy condition of all varieties. All four virus isolates had more effect on the growth of Amelonado than on the Iquitos progenies, except that CSSV strain I W affected the growth of Amelonado and one of the Iquitos progenies equally, as did CMLV which caused the greatest decrease in the first crop of all varieties. The relative virulence of different CSSV strains in any one variety can be assessed from single criteria such as length of latent period or effect on growth; CMLV had a shorter latent period than CSSV Strain I A but less effect on growth.     

Field experiments on the resistance of cocoa to cocoa swollen-shoot virus

Progenies from crosses between different Upper Amazon cocoa types and between Upper Amazon and Amelonado were compared for their field resistance to infection with cocoa swollen-shoot virus (CSSV) virulent strain A. Among the intra-Amazon progenies, those from crosses of Scavina, Iquitos and Nanay groups showed most resistance. Progenies from crosses within these groups may have sufficient resistance to be of immediate practical value in reducing crop losses in areas where CSSV is widespread. Some Nanay progenies were more resistant than others and this provides scope for improvement by breeding. Progeny of crosses between Upper Amazon and Amelonado parents were less resistant than those from intra-Amazon crosses. The present results confirm those previously obtained in gauze-house tests on young plants.     

The stereochemical configuration of flavanols influences the level and metabolism of flavanols in humans and their biological activity in vivo

Extensive epidemiological and clinical evidence associates diets high in flavanol-containing foods with cardiovascular health benefits in humans. Catechin and epicatechin, the most common flavanols in foods, are present in the diet in different enantiomeric forms. This study investigated the influence of the stereochemical configuration of flavanols on their absorption, metabolism, and biological activity. Healthy adult males were asked to consume equal amounts of the stereochemically pure flavanols (-)-epicatechin, (-)-catechin, (+)-catechin, and (+)-epicatechin (1.5mg/kg bw) in a well-defined cocoa-based, dairy-containing drink matrix, and flavanol levels were subsequently determined in plasma and 24-h urine. The results obtained show that the stereochemical configuration of flavanols has a profound influence on their uptake and metabolism in humans. In addition, we assessed the vasodilatory activity of each flavanol stereoisomer in vivo and found (-)-epicatechin to be the single stereoisomer capable of mediating a significant arterial dilation response. Importantly, this effect was independent of the classic antioxidant properties of flavanols. Overall, these results indicate that the proposed beneficial health effects associated with the consumption of flavanol-containing foods will significantly depend on the stereochemical configuration of the flavanols ingested.     

Immune effects of cocoa procyanidin oligomers on peripheral blood mononuclear cells

There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1beta, IL-6, IL-10, and TNF-alpha in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.     

Chromatographic separation and automated analysis of flavanols

Flavanols from barley or hops were separated chromatographically and assayed automatically by reaction with the chromogen, 4-dimethylaminocinnamaldehyde. For separating the flavanols on Sephadex G-25, gradient elution with water-methanol mixtures was necessary. The chromogen was specific for flavanols and well suited to AutoAnalyzer methods. The method appears generally applicable in flavanol analysis of plant materials.     

Membrane effects of cocoa procyanidins in liposomes and Jurkat T cells

We investigated the effects of the interaction between flavanols and related procyanidins (dimer to hexamer) with both cell and synthetic membranes, on bilayer fluidity and susceptibility to oxidation. Cocoa derived dimers (0.05 to 1 microg/ml) protected Jurkat T cells from AMVN-mediated oxidation and increased plasma membrane fluidity. These effects occurred in a concentration- and chain length-dependent manner. In liposomes, procyanidins prevented the Fe2+ -induced permeabilization of the membrane. Together, these results support the hypothesis that procyanidins could interact with the polar headgroup of lipids, increasing membrane fluidity and also, preventing the access of molecules that could affect membrane integrity.     

Food intake,absorption and conversion in the fishOphiocephalus striatus

Summary 1. In the fishOphiocephalus striatus Bloch feeding rate increased 2 to 3 times, when feeding was resumed after less than 20 days fasting; fasting periods of more than 20 days result in resumption-feeding rates that were only slightly higher (0.5 to 0.8 times) than the controls.2. Although feeding rates were very high after resumption of feeding following different periods of starvation (10 to 40 days) absorption efficiency remained practically identical to that of controls.3. Following a period of less than 20 days fasting resumption of feeding led conversion efficiencies almost twice as high as the normal values, showing over-compensation. Fish fed after a period of more than 20 days fasting showed a decrease in conversion efficiency resulting in partial compensatory growth.4. Responses to resumption of feeding following different periods of starvation may be of two types: (a) Rates of metabolism and feeding are restored to pre-starvation rates and conversion efficiency increases due to corresponding increase in feeding rates, e. g. O.striatus, and (b) rates of metabolism and feeding continue to remain low and the increase in conversion efficiency is due to a corresponding decrease in energy expended on metabolism e. g.,E. lucius.

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Nahrungsaufnahme, Absorption und Konversion bei dem FischOphiocephalus striatus

Kurzfassung Individuen vonOphiocephalus striatus Bloch wurden nach verschieden langen Hungerzeiten gefüttert, um Aufschlüsse über Aufnahme, Absorption und Konversion der Nahrung zu erhalten. Nach einer Hungerperiode von weniger als 20 Tagen nahm die Nahrungsaufnahme um das Dreifache zu. Hungerten die Tiere länger als 20 Tage, so steigerte sich die Nahrungsaufnahme hingegen nur geringfügig. Trotz der erhöhten Nahrungsaufnahme war der Absorptions-Nutzeffekt dem der Kontrolltiere gleich. Der Konversions-Nutzeffekt verdoppelte sich bei Individuen, die weniger als 20 Tage gehungert hatten; daraus resultierte eine Überkompensation des Wachstums. Bei Tieren, die nach einer Hungerperiode von mehr als 20 Tagen wieder gefüttert wurden, konnte eine gewisse Verringerung des Konversions-Nutzeffektes sowie eine teilweise Kompensation festgestellt werden. Diese Ergebnisse werden im Hinblick auf ähnliche Versuche beiEsox lucius (Ivlev 1939) diskutiert. Es wird die Schlußfolgerung gezogen, daß sich bei Fischen, die nach verschieden langen Hungerzeiten erneut gefüttert werden, zwei Typen unterscheiden lassen: beim ersten Typ ist die Nahrungsaufnahme geringer als vor der Hungerzeit, der Konversions-Nutzeffekt jedoch durch Erniedrigung der für den Stoffwechsel aufgebrachten Energie erhöht; beim zweiten Typ erreichen Nahrungsaufnahme und Stoffwechselintensität sofort die Werte vor der Hungerzeit oder übertreffen diese sogar. Hierbei wird die Erhöhung der Nahrungsaufnahme von einer entsprechenden Vergrößerung des Konversions-Nutzeffektes begleitet.

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Part of a Ph. D. Thesis, University of Madras, India.     

Effect of cocoa pod age on egg distribution and egg parasitism of the cocoa pod borer in Malaysia

The effects of pod age on oviposition, and on egg and larval survival of the cocoa pod borer (CPB), Conopomorpha cramerella (Snellen), were investigated. CPB were allowed to oviposit on a batch of 50 pods for a period of 7 days over several exposure dates. Numbers of CPB eggs/pod and survival to larval emergence from the pods were recorded. Pods were harvested when ripe and analyzed for larval survival within the pod. The oviposition pattern varied with pod age. Older pods less than 7 weeks before ripening (WBR) were preferred, suggesting greater nutritional value or chemical attraction of the older pods. The proportion of eggs parasitized by Trichogrammtoidea bactrae fumata Nagaraja (TBF), the number of eggs lost and the proportion surviving to larval emergence from the pod were independent of pod age. Egg parasitism was low, implying that TBF was not an effective natural enemy. Mass-releases of TBF should be intensified during cropping periods with higher proportions of susceptible pods (<10 WBR). Survival of larvae in the presclerotic layer (preSCL) was high and independent of pod age. Larval mortality at the SCL (sclerotic layer) was dependent on pod age and was high in older pods. Larval density influenced the survival of larvae inside the pod and was dependent on pod age. Larval mortality associated with SCL hardness and thickness is one of the criteria for the selection of CPB resistant cocoa clones.     

Methods for assessing the tolerance and resistance of different types of cocoa to cocoa swollen-shoot virus

Different types of cocoa were compared for their resistance to infection and tolerance of infection with the virulent strain A of cocoa swollen-shoot virus, inoculated either by mealybugs or manually. The method of infection did not affect the ranking of the cocoa types for resistance, except at high percentages of infection, or for tolerance. In comparison with other methods, manual inoculation simplifies the screening of cocoa types for resistance and tolerance to virus infection, and provides a more sensitive test for resistance.     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Acid-base effects on intestinal Cl- absorption and vesicular trafficking

In rat ileum and colon, apical membrane exchange and net Cl^- absorption are stimulated by increases in PCO₂ or . Because changes in PCO₂ stimulate colonic Na⁺ absorption, in part, by modulating vesicular trafficking of the Na⁺/H⁺ exchanger type 3 isoform to and from the apical membrane, we examined whether changes in PCO₂ affect net Cl^- absorption by modulating vesicular trafficking of the exchanger anion exchanger (AE)1. Cl^- transport across rat distal ileum and colon was measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at PCO₂ 70 mmHg than at PCO₂ 21 mmHg but was not affected by pH changes in the absence of CO₂. AE1 was internalized when PCO₂ was reduced and exocytosed when PCO₂ was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl^- absorption caused by an increase in PCO₂. Increases in PCO₂ stimulated ileal Cl^- absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by PCO₂. We conclude that CO₂ modulation of colonic, but not ileal, Cl^- absorption involves effects on vesicular trafficking of AE1. PCO₂; ileum; colon; anion exchanger 1; Na⁺/H⁺ exchanger type 3     

Age related differences in the plasma kinetics of flavanols in rats

Dietary flavanols produce beneficial health effects; once absorbed, they are recognized as xenobiotics and undergo Phase-II enzymatic detoxification. However, flavanols with a degree of polymerization greater than 2 reach the colon, where they are subjected to microbial metabolism and can be further absorbed and undergo Phase-II reactions. In this sense, flavanols' health-promoting properties are mainly attributed to their metabolic products. Several age-related physiological changes have been evidenced, and it is known that flavanols' bioavailability is affected by internal factors. Therefore, this study aimed to elucidate whether animals of different ages, specifically young and adult rats, exhibit differences in their flavanol metabolism and plasma bioavailability. To accomplish this, an acute dose of a grape seed polyphenol extract was administered to male rats; after 2, 4, 7, 24 and 48 h, flavanols and their Phase-II and microbial metabolites were quantified by HPLC-ESI-MS/MS in plasma. The results indicated important age-related quantitative differences in plasma flavanol metabolites. Interestingly, adult rats presented a remarkable reduction in flavanol absorption and Phase-II flavanol metabolism. Consequently, microbial-derived flavanol metabolism is triggered by higher flavanol affluence in the colonic tract. Furthermore, young rats presented a faster metabolic profile than adult rats. Hence, our results indicate that the physiological bioactivities of flavanols may depend on age.     

Coenzyme Q10: absorption, tissue uptake, metabolism and pharmacokinetics

Available data on the absorption, metabolism and pharmacokinetics of coenzyme Q10 (CoQ10) are reviewed in this paper. CoQ10 has a fundamental role in cellular bioenergetics. CoQ10 is also an important antioxidant. Because of its hydrophobicity and large molecular weight, absorption of dietary CoQ10 is slow and limited. In the case of dietary supplements, solubilized CoQ10 formulations show enhanced bioavailability. The T_max is around 6 h, with an elimination half-life of about 33 h. The reference intervals for plasma CoQ10 range from 0.40 to 1.91 μmol/l in healthy adults. With CoQ10 supplements there is reasonable correlation between increase in plasma CoQ10 and ingested dose up to a certain point. Animal data show that CoQ10 in large doses is taken up by all tissues including heart and brain mitochondria. This has implications for therapeutic applications in human diseases, and there is evidence for its beneficial effect in cardiovascular and neurodegenerative diseases. CoQ10 has an excellent safety record.     

The resistance and tolerance of different cocoa varieties to cocoa swollen-shoot virus in Ghana

The resistance and tolerance to infection with cocoa swollen-shoot virus (virulent strain A) of forty-five and thirty-eight cocoa progenies respectively was estimated using a manual inoculation method. Resistance was estimated from the proportion of inoculated seedlings which developed symptoms, and tolerance from the severity of these symptoms during a 4-month period. Resistance was found among Nanay, Iquitos and Scavina, all Upper Amazon types; and an inter-Nanay cross was outstandingly resistant and offers scope for improvement by breeding. Some Amazon-Amazon and Trinitario-Amazon progenies snowed tolerance, especially those with Trinitario T9/21 as a parent. Some progenies seem sufficiently promising for field testing to select varieties suitable for planting in those areas in Ghana where swollen-shoot disease is epidemic.