Catecholaminergic and cholinergic systems of mouse brain are modulated by LMN diet, rich in theobromine, polyphenols and polyunsaturated fatty acids.

The possible modulatory effect of the functional LMN diet, rich in theobromine, polyphenols and polyunsaturated fatty acids, on the catecholaminergic and cholinergic neurotransmission, affecting cognition decline during aging has been studied. 129S1/SvlmJ mice were fed for 10, 20, 30 and 40 days with either LMN or control diets. The enzymes involved in catecholaminergic and cholinergic metabolism were determined by both immunohistological and western blot analyses. Noradrenalin, dopamine and other metabolites were quantified by HPLC analysis. Theobromine, present in cocoa, the main LMN diet component, was analysed in parallel using SH-SY5Y and PC12 cell lines. An enhanced modulatory effect on both cholinergic and catecholaminergic transmissions was observed on 20 day fed mice. Similar effect was observed with theobromine, besides its antioxidant capacity inducing SOD-1 and GPx expression. The enhancing effect of the LMN diet and theobromine on the levels of acetylcholine-related enzymes, dopamine and specially noradrenalin confirms the beneficial role of this diet on the "cognitive reserve" and hence a possible reducing effect on cognitive decline underlying aging and Alzheimer's disease.

Human bioavailability of flavanols and phenolic acids from cocoa-nut creams enriched with free or microencapsulated cocoa polyphenols.

Human bioavailability of cocoa flavanols and phenolic acids from a cocoa-nut cream (CC) and from CC enriched with a 1·5 % (w/w) cocoa polyphenol extract in free form (FPC) or encapsulated with a gastric-resistant high-amylose maize starch (EPC), was studied. In a randomised cross-over protocol, with 1-week wash-out in between, twelve healthy volunteers had three portions/d of each cream, providing approximately 190 µmol/d of total flavanols and 12 µmol/d of total phenolic acids with CC and 385 and 28 µmol/d with both FPC and EPC, respectively. Blood, urine and faecal samples were analysed by HPLC/MS/MS. Serum (epi)catechin was absent at baseline and after CC consumption, while 22·1 (SEM 2·62) and 1·59 (SEM 0·22) nmol (P <0·05) were found after FPC and EPC, respectively. The EPC increased faecal excretion of total flavanols compared to FPC (151·0 (SEM 54·6) v. 28·0 (SEM 14·0) nmol; P <0·05). Within 6 h after consumption, serum phenolic acid content was 50-fold higher than (epi)catechin; no difference between CC and FPC was observed, but a significant reduction after EPC (1954 (SEM 236·3) and 1459 (SEM 137·6) v. 726·8 (SEM 73·4) nmol, P <0·05) was recorded. Short-term phenolic acid urinary excretions were significantly higher after FPC than CC and EPC, the values being 11·4 (SEM 5·1) v. 3·1 (SEM 1·7) and 0·9 (SEM 0·5) µmol, respectively. Faecal phenolic acids were approximately 60-fold reduced after FPC (8·1 (SEM 0·13) nmol) and EPC (14·7 (SEM 2·7) nmol) consumption compared to CC (641·4 (SEM 99·1) nmol) consumption. The data demonstrated that: (i) (epi)catechin was absorbed from CC; (ii) cocoa polyphenols' consumption increased circulating phenolic acids; and (iii) encapsulated ingredient increased flavanol delivering into the gut. Further studies should evaluate whether encapsulated cocoa polyphenols may be a functional prebiotic ingredient.

Distribution of procyanidins and their metabolites in rat plasma and tissues in relation to ingestion of procyanidin-enriched or procyanidin-rich cocoa creams.

BACKGROUND: Procyanidins are extensively metabolized via phase-II and microbial enzymes. However, their distribution in the body is not well characterized. AIM: This study investigates the distribution of procyanidins (monomers and dimers) and their phase-II metabolites in plasma and tissues (thymus, heart, liver, testicle, lung, kidney, spleen and brain). METHODS: Wistar rats were fed with 1 g of cocoa cream (CC), 50 mg of procyanidin hazelnut skin extract (PE) and 50 mg PE in 1 g CC (PECC). The rats were killed at 0, 1, 1.5, 2, 3, 4 and 18 h after gavage, and the plasma and tissues were analyzed by UPLC-MS/MS. RESULTS: Epicatechin-glucuronide was the main metabolite in the plasma after the CC intake, with C(max) at 423 nM and t(max) at 2 h, and methyl catechin-glucuronide (301 nM, 2 h) was the main metabolite in the plasma after the PE intake. As a result of the PECC enrichment, epicatechin-glucuronide (452 nM, 1.5 h) and catechin-glucuronide (297 nM, 2 h) were the main metabolites in the plasma. Methyl catechin-glucuronide was found in the liver after PE (8 nmol/g tissue, 4 h) and PECC (8 nmol/g, 1.5 h). The kidney was found to contain a high concentration of phase-II metabolites of procyanidins and is therefore thought to be the main site of metabolism of the compounds. Methyl catechin-sulfate (6.4 nmol/g, 4 h) was only quantified in the brain and after PE intake. Catechin metabolites were not found in the spleen or heart. Phenolic acids were detected in all tissues. CONCLUSIONS: The formulation of a product enriched or fortified with procyanidins is a way to increase their bioavailability, with clear effects on the plasmatic pharmacokinetics, and a greater accumulation of phenolic metabolites in such tissues as the liver, kidney, lung and brain.

Inhibition of the transcription factor c-Jun by the MAPK family, and not the NF-κB pathway, suggests that peanut extract has anti-inflammatory properties.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is involved in inflammatory responses in atherosclerosis. We propose an in vitro cellular assay to evaluate the anti-inflammatory mechanisms of potential modifiers such as food extracts. In the current model we assessed an anti-inflammatory effect of polyphenol-rich peanut extract in lipopolysaccharide (LPS)-induced THP-1 monocytes. METHODS: THP-1 monocytes were incubated with peanut extract (5, 25, 50 and 100 µg/mL) consisting of 39% flavonols, 37% flavanols and 24% phenolic acid (or BAY 11-7082 (5 µM) as experiment control) for 1 h and then stimulated with LPS (500 ng/mL) for 4 h. Cytotoxicity was measured as lactate dehydrogenase (LDH) activity release. NF-κB and MAPK family were determined by TransAm kit while TNF-α mRNA levels and its mRNA stability by RT-PCR. Intra- and extracellular TNF-α protein was measured by ELISA, and TNF-α converting enzyme (TACE) activity by a fluorimetric assay. RESULTS: Peanut extract inhibited the maximal LPS-induced extracellular TNF-α protein secretion by 18%, 29% and 47% at 25, 50 and 100 µg/mL, respectively (P<0.05). LPS stimulation revealed that 85% of TNF-α was released extracellularly while 15% remained intracellular. Peanut extract did not modify NF-κB but, instead, reduced c-Jun transcription factor activity (P<0.05), decreased TNF-α mRNA (albeit non-significantly) and had no effect on mRNA stability and TACE activity. CONCLUSION: Polyphenol-rich peanut extract reduces extracellular TNF-α protein by inhibiting c-Jun transcription factor from MAPK family, suggesting an anti-inflammatory effect. The proposed THP-1 monocyte model could be used to assess food extract impact (site and size effects) on the inflammation pathway.

Alpha-tocopherol and BAY 11-7082 reduce vascular cell adhesion molecule in human aortic endothelial cells.

BACKGROUND: In endothelial dysfunction, vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression (collectively termed cell adhesion molecules; CAMs) increase at sites of atherosclerosis and are stimulated by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α). METHODS: We evaluated the effect of alpha-tocopherol (AT; 10-150 µM) and BAY 11-7082 (BAY; 0.1 or 1 µM) on CAMs mRNA expression as well as their protein in soluble release form (sCAMs) in human aortic endothelial cells (HAECs) activated by TNF-α (1 or 10 ng/ml). Also, we determined the extent of lymphocyte adhesion to activated HAECs. RESULTS: BAY reduced VCAM-1, E-selectin and ICAM-1 mRNA expression by 30, 30 and 10%, respectively. Furthermore, protein reduction of sVCAM-1 by 70%, sE-selectin by 51% and sICAM-1 by 25% compared to HAECs stimulated by TNF-α was observed (p < 0.05). AT (50, 75 and 150 µM) decreased VCAM-1 mRNA expression by 30% and sVCAM-1 protein by 33% compared to HAECs stimulated by TNF-α (p < 0.05). TNF-α-activated HAEC adhesion to human Jurkat T lymphocytes was higher compared to nonactivated HAECs (p < 0.05). BAY (2 and 5 µM) reduced this lymphocyte adhesion (p < 0.05). CONCLUSION: BAY reduces all the CAMs studied as well as cell adhesion, while AT selectively inhibits VCAM-1; both induce endothelial dysfunction improvement.

LMN diet, rich in polyphenols and polyunsaturated fatty acids, improves mouse cognitive decline associated with aging and Alzheimer's disease.

We examined whether LMN diet, reported to induce neurogenesis in adult mice, was able to antagonize the age-related behavioural impairment and neuropathology in wild type (WT) mice and Tg2576 mice, a mouse model of Alzheimer's disease (AD). Thirteen-month-old mice (once the amyloid (Aß) plaques were formed) were fed with the LMN diet for 5 months, and in the last 2 months of the regimen they received a battery of behavioural tests. In general, both aging and (to a higher extent) Tg2576 genotype deteriorated sensorimotor reflexes, exploratory behaviour in the hole board, activity (but not anxiety) in the elevated plus-maze, ambulation in the home cage during the dark phase, and spatial learning in the Morris water maze. LMN diet did not affect the detrimental effects observed in sensorimotor reflexes, but clearly reversed the effects of both aging and Tg2576 genotype. This behavioural amelioration was correlated with a 70% increase in cellular proliferation in subventricular zone (SVZ) of the brain, but did not correlate with a decrease of amyloid plaques. In contrast, administration of LMN diet to 10 months old mice (before the plaques are formed) strongly suggested a putative delay in the formation of plaques, as indicated by a decreasing tendency of soluble and fibrillar Aß levels in hippocampus which correlated with a decrease in Aß (1-40, 1-42) plasma content. Herein we describe for the first time that LMN diet rich in polyphenols, dry fruits and cocoa, was able to decrease behavioural deterioration caused by aging and Tg2576 genotype and to delay the Aß plaque formation. These results corroborate the increasing importance of polyphenols as human dietary supplements in amelioration of the cognitive impairment during aging and neurological disorders such as AD.

Distribution of procyanidins and their metabolites in rat plasma and tissues after an acute intake of hazelnut extract.

Procyanidins are present in a wide range of dietary foods and their metabolism is well known. Nevertheless, the biological target and their distribution are topics lacking information. The purpose of the present work was to study the metabolism and distribution of procyanidins and their metabolites in rat plasma and different tissues, such as liver, brain, lung, kidney, intestine, testicle, spleen, heart and thymus, after 2 h of an acute intake of hazelnut extract rich in procyanidins (5 g kg(-1) of rat body weight). The interest of an acute intake of procyanidins instead of repeated low doses from daily ingestion of is to achieve a concentration of metabolites in the tissues that allows their detection and quantification. The results showed that catechin and epicatechin-glucuronide, methyl catechin and epicatechin-glucuronide and methyl catechin and epicatechin-sulphate were detected in plasma samples at the µmol level. On the other hand, catechin-glucuronide, methyl catechin-glucuronide and methyl catechin-sulphate were identified in some tissues, such as thymus, intestine, lung, kidney, spleen and testicle at the nmol level. Procyanidins with a low grade of polymerization (dimers and trimers) were detected in plasma samples and the intestine. Additionally, a wide range of simple aromatic acids from fermentation by the colonic microflora was detected in all tissues studied.

A diet enriched in polyphenols and polyunsaturated fatty acids, LMN diet, induces neurogenesis in the subventricular zone and hippocampus of adult mouse brain.

At present it is widely accepted that there are at least two neurogenic sites in the adult mammalian brain: the subventricular zone (SVZ) of lateral ventricles and the subgranular zone (SGZ) of the hippocampus dentate gyrus. The adult proliferation rate declines with aging and is altered in several neurodegenerative pathologies including Alzheimer's disease. The aim of this work was to study whether a natural diet rich in polyphenols and polyunsaturated fatty acids (LMN diet) can modulate neurogenesis in adult mice and give insight into putative mechanisms. Results with BrdU and PCNA demonstrated that the LMN fed mice had more newly generated cells in the SVZ and SGZ, and those with DCX (undifferentiated neurons) and tyrosine hydroxylase, calretinin, and calbindin (differentiated neurons) immunostainings and western blots demonstrated a significant effect on neuronal populations, strongly supporting a positive role of the LMN diet on adult neurogenesis. In primary rat neuron cultures, the LMN cream dramatically protected against damage caused by both hydrogen peroxide and Abeta(1-42), demonstrating a potent antioxidant effect that could play a major role in the normal adult neurogenesis and, moreover, the LMN diet could have a significant effect combating the cognitive function decline during both aging and neurodegenerative diseases such as Alzheimer's disease.

Rapid determination of phenolic compounds and alkaloids of carob flour by improved liquid chromatography tandem mass spectrometry.

An improved chromatographic method was developed using ultra-performance liquid chromatography-tandem mass spectrometry to identify and quantify phenolic compounds and alkaloids, theobromine and caffeine, in carob flour samples. The developed method has been validated in terms of speed, sensitivity, selectivity, peak efficiency, linearity, reproducibility, limits of detection, and limits of quantification. The chromatographic method allows the identification and quantification of 20 phenolic compounds, that is, phenolic acids, flavonoids, and their aglycone and glucoside forms, together with the determination of the alkaloids, caffeine and theobromine, at low concentration levels all in a short analysis time of less than 20 min.

Obtention and characterization of phenolic extracts from different cocoa sources.

The aim of this study was to evaluate several cocoa sources to obtain a rich phenol extract for use as an ingredient in the food industry. Two types of phenolic extracts, complete and purified, from different cocoa sources (beans, nibs, liquor, and cocoa powder) were investigated. UPLC-MS/MS was used to identify and quantify the phenolic composition of the extracts, and the Folin-Ciocalteu and vanillin assays were used to determine the total phenolic and flavan-3-ol contents, respectively. The DPPH and ORAC assays were used to measure their antioxidant activity. The results of the analysis of the composition of the extracts revealed that the major fraction was procyanidins, followed by flavones and phenolic acids. From the obtained results, the nib could be considered the most interesting source for obtaining a rich phenolic cocoa extract because of its rich phenolic profile content and high antioxidant activity in comparison with the other cocoa sources.

Antioxidant activity of olive pulp and olive oil phenolic compounds of the arbequina cultivar.

The aim of this study was to characterize antioxidant activities of phenolic compounds that appear in olive pulp and olive oils using both radical scavenging and antioxidant activity tests. Antiradical and antioxidant activities of olive pulp and olive oil phenolic compounds were due mainly to the presence of a 3,4-dihydroxy moiety linked to an aromatic ring, and the effect depended on the polarity of the phenolic compound. Glucosides and more complex phenolics exhibited higher antioxidant activities toward oxidation of liposomes, whereas in bulk lipids aglycons were more potent antioxidants with the exception of oleuropein. Lignans acted as antioxidants only in liposomes, which could partly be due to their chelating activity, because liposome oxidation was initiated by cupric acetate. The antioxidant activity of virgin olive oil is principally due to the dialdehydic form of elenolic acid linked to hydroxytyrosol (3,4-DHPEA-EDA), a secoiridoid derivative (peak RT 36, structure unidentified), and luteolin.

Effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars.

The purpose of the work was to investigate the effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars. The level in the phenolic content of olive drupes declines rapidly during the black maturation phase. A general decreasing trend was observed too in the phenolic content of olive oils during the ripening process in the three varieties studied. Important differences in the high-performance liquid chromatography profile between varieties were observed. These included the presence of very low amounts of lignans in olive oils proceeding from the Morrut cultivar, and the presence of three peaks after elution of 3,4-DHPEA-EDA in the Farga and Morrut cultivars, which could be used as differentiating parameters. Sensory profile differences were observed between olive cultivars and due to the ripening process.