Acute study of dose-dependent effects of (-)-epicatechin on vascular function in healthy male volunteers: A randomized controlled trial.

BACKGROUND & AIMS: There is convincing clinical evidence to suggest that flavanol-containing foods/beverages are capable of inducing improvements in human vascular function. However, whilst (-)-epicatechin has been tested for efficacy, a full dose-dependency has yet to be established, particularly at doses below 1 mg/kg BW. The current study examined the dose-dependent effects of (-)-epicatechin on human vascular function with concurrent measurement of plasma (-)-epicatechin metabolites and levels of circulating nitrite and nitrate species, NOx. METHODS: An acute, double-blind, placebo-controlled, crossover intervention trial was conducted in 20 healthy males with 4 treatment arms: water-based (-)-epicatechin (0.1, 0.5 and 1.0 mg/kg BW) and a water only as control. Vascular function was assessed by flow-mediated dilatation (FMD) measured at the brachial artery, laser Doppler imaging with iontophoresis (LDI) at the subcutaneous capillaries of the forearm (response to Ach and SNP) and peripheral blood pressure (BP) at baseline, 1, 2, 4 and 6 h post-intervention. Plasma analysis of epicatechin metabolites was conducted by LC-MS and circulating plasma of nitrite and nitrate species were performed using an HPLC-based system (ENO-30). RESULTS: Significant increases in % FMD were found to occur at 1 and 2 h following intake of 1 mg/kg BW, and at 2 h for the 0.5 mg/kg BW intake. There were no significant changes in LDI or BP at any time-points or intake levels. Increases in FMD over the 6 h timeframe were closely paralleled by the appearance of total plasma (-)-epicatechin metabolites. Non-significant changes in circulating NOx was observed. CONCLUSIONS: Our data add further evidence that (-)-epicatechin is a causal vasoactive molecule within flavanol-containing foods/beverages. In addition, we show for the first time that intake levels as low as 0.5 mg/kg BW are capable of inducing acute improvements in vascular function (FMD) in healthy volunteers.

Neuroprotective Effects of Selected Microbial-Derived Phenolic Metabolites and Aroma Compounds from Wine in Human SH-SY5Y Neuroblastoma Cells and Their Putative Mechanisms of Action.

Moderate wine consumption has shown the potential to delay the onset of neurodegenerative diseases. This study investigates the molecular mechanisms underlying the protective effects of wine-derived phenolic and aroma compounds in a neuroinflammation model based on SIN-1 stress-induced injury in SH-SY5Y neuroblastoma cells. Cell pretreatment with microbial metabolites found in blood after wine consumption, 3,4-dihydroxyphenylacetic (3,4-DHPA), 3-hydroxyphenylacetic acids and salicylic ß-d-O-glucuronide, at physiologically concentrations (0.1-10 µM) resulted in increased cell viability versus SIN-1 control group (p < 0.05). Results also showed significant decreases in mitogen-activated protein kinase (MAPK) p38 and ERK1/2 activation as well as in downstream pro-apoptotic caspase-3 activity by some of the studied compounds. Moreover, pretreatment with p38, MEK, and ERK1/2-specific inhibitors, which have a phenolic-like structure, also resulted in an increase on cell survival and a reduction on caspase-3 activity levels. Overall, these results contribute with new evidences related to the neuroprotective actions of wine, pointing out that wine-derived human metabolites and aroma compounds may be effective at protecting neuroblastoma cells from nitrosative stress injury by inhibiting neuronal MAPK p38 and ERK1/2, as well as downstream caspase 3 activity.

Assessment of flavanol stereoisomers and caffeine and theobromine content in commercial chocolates.

Assessment of the flavanol composition of 41 commercial chocolates was by HPLC-DAD. Among individual flavonols ranged from 0.095 to 3.264mgg(-1), epicatechin was the predominant flavanol accounting for 32.9%. Contrary to catechin, epicatechin was a reliable predictive value of the polyphenol content. Conversely the percentage of theobromine used as a proxy measure for nonfat cocoa solids (NFCS) was not a good predictor of epicatechin or flavanol content. In a further chiral analysis, the naturally occurring forms of cocoa flavanols, (-)-epicatechin and (+)-catechin, was determined joint the occurrence of (+)-epicatechin and (-)-catechin due to the epimerization reactions produced in chocolate manufacture. (-)-Epicatechin, the most bioactive compound and predominant form accounted of 93%. However, no positive correlation was found with% cocoa solids, the most significant quality parameter.

Behavioral dynamics and neural grounding of a dynamic field theory of multi-object tracking.

The ability to dynamically track moving objects in the environment is crucial for efficient interaction with the local surrounds. Here, we examined this ability in the context of the multi-object tracking (MOT) task. Several theories have been proposed to explain how people track moving objects; however, only one of these previous theories is implemented in a real-time process model, and there has been no direct contact between theories of object tracking and the growing neural literature using ERPs and fMRI. Here, we present a neural process model of object tracking that builds from a Dynamic Field Theory of spatial cognition. Simulations reveal that our dynamic field model captures recent behavioral data examining the impact of speed and tracking duration on MOT performance. Moreover, we show that the same model with the same trajectories and parameters can shed light on recent ERP results probing how people distribute attentional resources to targets vs. distractors. We conclude by comparing this new theory of object tracking to other recent accounts, and discuss how the neural grounding of the theory might be effectively explored in future work.

Investigation into the efficacy of the acetylcholinesterase inhibitor, donepezil, and novel procognitive agents to induce gamma oscillations in rat hippocampal slices.

One of the major neuropathological hallmarks in Alzheimer's disease (AD) is the loss of cholinergic neurones of the nucleus basalis of Meynert (NbM). This consistent finding gave rise to the 'cholinergic' hypothesis of AD and lead to the subsequent development of acetylcholinesterase (AChE) inhibitors; the first class of drug to be approved for the treatment of AD. However, several studies have questioned the efficacy of using AChE inhibitors in AD. In this study we have investigated the ability of two AChE inhibitors, donepezil (Aricept) and physostigmine, to induce gamma oscillatory activity in rat hippocampal slices; network activity believed to play a role in higher cognitive function. We report here that donepezil is capable of inducing gamma oscillations in region CA3 of rat hippocampal slices, which may contribute to its procognitive action. However, donepezil-induced gamma oscillations are weak in comparison to physostigmine. We also explore the activity of novel agents with known procognitive activity, and show that one such agent, the M(1) muscarinic acetylcholine receptor agonist, 77-LH-28-1, can significantly enhance donepezil-induced gamma oscillations. These data support the notion that it should be possible to find a more efficacious AChE inhibitor or an adjunctive approach, to provide a better therapeutic intervention in AD.

Extra virgin olive oil phenolics: absorption, metabolism, and biological activities in the GI tract.

Olive oil, a typical ingredient of the Mediterranean diet, possesses many beneficial health effects. The biological activities ascribed to olive oil consumption are associated in part to its phenolics constituents, and mainly linked to the direct or indirect antioxidant activity of olive oil phenolics and their metabolites, which are exerted more efficiently in the gastrointestinal (GI) tract, where dietary phenolics are more concentrated when compared to other organs. In this regard, we present a brief overview of the metabolism, biological activities, and anticancer properties of olive oil phenolics in the GI tract.

Neuroinflammation and its modulation by flavonoids.

There is increasing evidence to suggest that neuroinflammatory processes contribute to the cascade of events that lead to the progressive neuronal damage observed in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Therefore, treatment regimes aimed at modulating neuroinflammatory processes may act to slow the progression of these debilitating brain disorders. Recently, a group of dietary polyphenols known as flavonoids have been shown to exert neuroprotective effects in vivo and in neuronal cell models. In this review we discuss the evidence relating to the modulation of neuroinflammation by flavonoids. We highlight the evidence which suggests their mechanism of action involves: 1) attenuation of the release of cytokines, such as interleukin-1beta (IL-1beta and tumor necrosis factor-alpha (TNF-alpha); 2) an inhibitory action against inducible nitric oxide synthase (iNOS) induction and subsequent nitric oxide (NO(*)) production; 3) inhibition of the activation of NADPH oxidase and subsequent reactive oxygen species generation; 4) a capacity to down-regulate the activity of pro-inflammatory transcription factors such as nuclear factor-kappaB (NF-kappaB); and 5) the potential to modulate signalling pathways such as mitogen-activated protein kinase (MAPK) cascade. We also consider the potential of these dietary compounds to represent novel therapeutic agents by considering their metabolism in the body and their ability to access the brain via the blood brain barrier. Finally, we discuss future areas of study which are necessary before dietary flavonoids can be established as therapeutic agents against neuroinflammation.

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress.

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.

Peroxynitrite-modified collagen-II induces p38/ERK and NF-kappaB-dependent synthesis of prostaglandin E2 and nitric oxide in chondrogenically differentiated mesenchymal progenitor cells.

OBJECTIVE: Peroxynitrite (ONOO(-)) is formed in the inflamed and degenerating human joint. Peroxynitrite-modified collagen-II (PMC-II) was recently discovered in the serum of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Therefore we investigated the cellular effects of PMC-II on human mesenchymal progenitor cells (MPCs) as a model of cartilage and cartilage repair cells in the inflamed and degenerating joint. DESIGN: MPCs were isolated from the trabecular bone of patients undergoing reconstructive surgery and were differentiated into a chondrogenic lineage. Cells were exposed to PMC-II and levels of the proinflammatory mediators nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) measured. Levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated mitogen activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappaB) activation were measured by enzyme linked immunosorbent assay (ELISA) together with specific MAPK and NF-kappaB inhibitors. RESULTS: PMC-II induced ()NO and PGE(2) synthesis through upregulation of iNOS and COX-2 proteins. PMC-II also lead to the phosphorylation of MAPKs, extracellularly regulated kinase 1/2 (ERK1/2) and p38 [but not c-Jun NH(2)-terminal kinase (JNK1/2)] and the activation of proinflammatory transcription factor NF-kappaB. Inhibitors of p38, ERK1/2 and NF-kappaB prevented PMC-II induced ()NO and PGE(2) synthesis, iNOS and COX-2 protein expression and NF-kappaB activation. CONCLUSION: iNOS, COX-2, NF-kappaB and MAPK are known to be activated in the joints of patients with OA and RA. PMC-II induced iNOS and COX-2 synthesis through p38, ERK1/2 and NF-kappaB dependent pathways suggesting a previously unidentified pathway for the synthesis of the proinflammatory mediators, ()NO and PGE(2), further suggesting that inhibitors of these pathways may be therapeutic in the inflamed and degenerating human joint.

Transgenic mice over-expressing human beta-amyloid have functional nicotinic alpha 7 receptors.

A potentially major factor in the development of Alzheimer's disease is the enhanced production of soluble beta-amyloid peptide fragments amyloid beta peptide(1-40) and amyloid beta peptide(1-42). These amyloid peptides are generated by cleavage of the amyloid-precursor protein and aggregate spontaneously to form amyloid plaques, which are a classical pathological hallmark in Alzheimer's disease. Although the precise mechanisms are unknown, it is widely believed that amyloid peptides initiate the degenerative process, resulting in subsequent cognitive decline. One interaction of amyloid beta peptide that may contribute to an impairment of cognition is its high affinity binding to the alpha 7 nicotinic receptor; a receptor shown to be important for cognition in a number of studies. There is some controversy, however, whether amyloid beta peptide inhibits or activates this receptor. We have cloned and stably expressed the human alpha 7 receptor and investigated its interaction with amyloid beta peptide using patch clamp electrophysiology. Human alpha 7 was activated in a concentration-dependent fashion by nicotine, acetylcholine and choline and potently inhibited by methyllycaconitine citrate. The responses were inwardly rectifying and exhibited rapid activation, desensitization and deactivation. Amyloid beta peptide(1-42) antagonized human alpha7 responses in a partially reversible fashion; no agonist effects of amyloid beta peptide(1-42) were detected. A similar inhibition of mouse alpha 7 was also observed. In addition, we have assessed the function of native alpha 7 receptors in hippocampal slices prepared from transgenic mice that over-express human amyloid. Despite this clear inhibition of recombinant receptors, hippocampal GABAergic interneurones in slices from beta-amyloid over-expressing mice still possess alpha 7 receptor-mediated currents.

Modulation of hippocampal excitability by 5-HT4 receptor agonists persists in a transgenic model of Alzheimer's disease.

5-HT(4) receptors are widely distributed in both peripheral and central nervous systems where they couple, via a G-protein, to the activation of adenylate cyclase. In the brain, the highest 5-HT(4) receptor densities are found in the limbic system, including the hippocampus and frontal cortex. It has been suggested that activation of these receptors may be of therapeutic benefit in diseases that produce cognitive deficits such as Alzheimer's disease (AD). Previous electrophysiological studies have shown that the 5-HT(4) agonist, Zacopride, can increase population spike amplitude recorded in region CA1 of rat hippocampal slices in a cyclic AMP (cAMP)/cAMP-dependent protein kinase A-dependent manner. We report here that the 5-HT(4) agonist, Prucalopride, and the 5-HT(4) partial agonist, SL65.0155, produce a similar effect in rat hippocampal slices and that the specific 5-HT(4) antagonist, GR113808, blocks these effects. To investigate the potential use of 5-HT(4) agonists in the treatment of AD, Prucalopride was applied to hippocampal slices from a transgenic mouse line that overexpresses the Abeta peptide. Despite the deficit in synaptic transmission present in these mice, the percentage increase of the CA1 population spike induced by Prucalopride was the same as that observed in wild-type mice. These data support 5-HT(4) receptors as a target for cognitive enhancement and suggest that a partial agonist would be sufficient to produce benefits, while reducing potential peripheral side effects. In addition, we show that 5-HT(4) receptors remain functional in the presence of excess Abeta peptide and may therefore be a useful target in AD.

Tests of a dynamic systems account of the A-not-B error: the influence of prior experience on the spatial memory abilities of two-year-olds.

Recently, Smith, Thelen, and colleagues proposed a dynamic systems account of the Piagetian "A-not-B" error in which infants' errors result from general processes that make goal-directed actions to remembered locations. Based on this account, the A-not-B error should be a general phenomenon, observable in different tasks and at different points in development. Smith, Thelen, et al.'s proposal was tested using an A-not-B version of a sandbox task. During three training trials and three "A" trials, 2-year-olds watched as a toy was buried in a sandbox at Location A. Following a 10-s delay, children searched for the object. Across five experiments, children's (total N = 92) performance on the A trials was accurate. After the A trials, children watched as a toy was hidden at Location B, 8 to 10 inches from Location A. In all experiments, children's searches after a 10-s delay were significantly biased in the direction of Location A. Furthermore, this bias toward Location A decreased with repeated trials to Location B, as well as when children completed fewer trials to Location A. Together, these data suggest that A-not-B-type errors are pervasive across tasks and development.

Contrasting influences of glucuronidation and O-methylation of epicatechin on hydrogen peroxide-induced cell death in neurons and fibroblasts.

The purpose of this study was to examine the comparative mechanisms by which the dietary form of the flavonoid epicatechin and its predominant in vivo metabolite, epicatechin glucuronide, influence oxidative stress-induced cell death in fibroblasts and neurons. The results demonstrate the contrasting influences of in vivo glucuronidation and methylation on the bioactivity of epicatechin.

Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3.

Oxidative stress has been associated with neuronal loss in neurodegenerative diseases and during age-associated cognitive decline. Flavonoids have been proposed to play a useful role in protecting the central nervous system against oxidative and excitotoxic stress, although the mechanism of action is unknown. Using oxidized low-density lipoprotein (oxLDL) as the oxidative insult we investigated the mechanism of neurotoxicity and attempted to identify possible sites of action of two of the most potent protective flavonoids, epicatechin and kaempferol, in cultured primary neurons. Using cultured striatal neurons and selective phosphospecific antibodies we addressed the potential role of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). OxLDL stimulated a Ca(2+)-dependent activation of both ERK1/2 and JNK that was strongly inhibited by pre-treatment with low micromolar concentrations of epicatechin. Neurotoxicity induced by oxLDL, however, was neither reduced nor enhanced by inhibiting ERK1/2 activation with mitogen-activated protein kinase kinase (MEK) inhibitors, suggesting that this cascade is unlikely to be involved in either oxLDL toxicity or the protective effects of flavonoids. oxLDL caused a sustained activation of JNK that resulted in the phosphorylation of the transcription factor c-Jun, which was abolished in neurons pre-treated with flavonoids. Furthermore, oxLDL induced the cleavage of procaspase-3 and increased caspase-3-like protease activity in neurons, an effect which was strongly inhibited by pre-exposure to either epicatechin or kaempferol. In addition, a caspase-3 inhibitor reduced oxLDL-induced neuronal death, implicating an apoptotic mechanism. A major in vivo metabolite of epicatechin, 3'-O-methyl-epicatechin was as effective as epicatechin in protecting neurons. Thus dietary flavonoids might have potential as protective agents against neuronal apoptosis through selective actions within stress-activated cellular responses, including protein kinase signalling cascades.

Epicatechin is the primary bioavailable form of the procyanidin dimers B2 and B5 after transfer across the small intestine.

Perfusion of isolated small intestine with the procyanidin dimers B2 and B5 extracted from cocoa indicated that both forms of dimer are transferred to the serosal side of enterocytes but only to a very small extent (<1% of the total transferred flavanol-like compounds). However, perfusion of dimer mainly resulted in large amounts of unmetabolised/unconjugated epicatechin monomer being detected on the serosal side (95.8%). The cleavage of dimer during transfer seemed to be energy-dependent, requiring an intact cell system, as incubation with jejunal homogenates failed to yield epicatechin. Low levels methylated dimer were also detected (3.2%), but no conjugates and metabolites of epicatechin indicating that metabolism of monomer and dimer is limited during dimer cleavage/translocation. The methylation of dimer may be by catechol-O-methyltransferase, however, at high concentrations of dimer COMT activity is reduced leading to an inhibition of both monomer and dimer O-methylation.

Medications in the breast-feeding mother.

Prescribing medications for a breast-feeding mother requires weighing the benefits of medication use for the mother against the risk of not breast-feeding the infant or the potential risk of exposing the infant to medications. A drug that is safe for use during pregnancy may not be safe for the nursing infant. The transfer of medications into breast milk depends on a concentration gradient that allows passive diffusion of nonionized, non-protein-bound drugs. The infant's medication exposure can be limited by prescribing medications to the breast-feeding mother that are poorly absorbed orally, by avoiding breast-feeding during times of peak maternal serum drug concentration and by prescribing topical therapy when possible. Mothers of premature or otherwise compromised infants may require altered dosing to avoid drug accumulation and toxicity in these infants. The most accurate and up-to-date sources of information, including Internet resources and telephone consultations, should be used.

Novel biomarkers of the metabolism of caffeic acid derivatives in vivo.

The purpose of this study was to investigate biomarkers of the bioavailability and metabolism of hydroxycinnamate derivatives through the determination of the pharmacokinetics of their urinary elimination and identification of the metabolites excreted. Coffee was used as a rich source of caffeic acid derivatives and human supplementation was undertaken. The results show a highly significant increase in the excretion of ferulic, isoferulic, dihydroferulic acid (3-(4-hydroxy-3-methoxyphenyl)-propionic acid), and vanillic acid postsupplementation relative to the levels presupplementation. Thus, ferulic, isoferulic, and dihydroferulic acids are specific biomarkers for the bioavailability and metabolism of dietary caffeic acid esters. Isoferulic acid is a unique biomarker as it is not a dietary component, however, dihydroferulic acid may well derive from other flavonoids with a structurally related B-ring. 3-Hydroxyhippuric acid has also been identified as an indicator for bioavailability and metabolism of phenolic compounds, and shows a highly significant excretion increase postsupplementation. The results reveal isoferulic acid (and possibly dihydroferulic acid) as novel markers of caffeoyl quinic acid metabolism.

An internal ribosome entry segment in the 5' untranslated region of the mnt gene.

Mnt is a transcriptional repressor related to the Myc/Mad family of transcription factors. It is expressed in proliferating, resting and differentiating cells and is believed to antagonize the function of Myc. Here we have characterized the major transcription initiation site of the mnt gene. In doing so we noted a remarkable level of sequence conservation between the murine and human 5' untranslated regions. Our experiments revealed that this sequence contains an internal ribosome entry segment (IRES). In addition, we show that sequences at both the 5' and 3' end of the IRES are essential for its function. These findings indicate that mnt can be translated by internal initiation. Such a mechanism may allow efficient Mnt synthesis when cap-dependent translation initiation is reduced.

Epicatechin and its in vivo metabolite, 3'-O-methyl epicatechin, protect human fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation.

There is considerable current interest in the cytoprotective effects of natural antioxidants against oxidative stress. In particular, epicatechin, a major member of the flavanol family of polyphenols with powerful antioxidant properties in vitro, has been investigated to determine its ability to attenuate oxidative-stress-induced cell damage and to understand the mechanism of its protective action. We have induced oxidative stress in cultured human fibroblasts using hydrogen peroxide and examined the cellular responses in the form of mitochondrial function, cell-membrane damage, annexin-V binding and caspase-3 activation. Since one of the major metabolites of epicatechin in vivo is 3'-O-methyl epicatechin, we have compared its protective effects with that of epicatechin. The results provide the first evidence that 3'-O-methyl epicatechin inhibits cell death induced by hydrogen peroxide and that the mechanism involves suppression of caspase-3 activity as a marker for apoptosis. Furthermore, the protection elicited by 3'-O-methyl epicatechin is not significantly different from that of epicatechin, suggesting that hydrogen-donating antioxidant activity is not the primary mechanism of protection.