A colorimetric sensor array for detection and discrimination of antioxidants based on Ag nanoshell deposition on gold nanoparticle surfaces.

There is growing interest in developing a high-performance sensor array for detection and discrimination of antioxidants owing to their widespread use and essential role in the human body. The present work unveils a novel colorimetric sensor array for colorimetric discrimination of antioxidants based on the red, green, and blue alteration (ΔRGB) pattern recognition. In this sensor array, three concentrations of AgNO3 were used as sensing elements, and gold nanoparticles (AuNPs) were employed as a colorimetric probe. In the presence of antioxidants, the sensor array produces unique colorimetric response patterns for the discrimination of these antioxidants due to different reactivities between three different concentrations of AgNO3 and each antioxidant, leading to deposition of different quantities of Ag nanoshells on the surface of AuNPs, enabling an excellent discrimination of six antioxidants (catechin, epigallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, and gallocatechin) at a 20 nM level, when linear discriminant analysis (LDA), hierarchical cluster analysis (HCA), centroid diagram, spidergram, and color contour profiles were smartly combined. Furthermore, different concentrations of antioxidants and binary antioxidant mixtures, even ternary mixtures, could also be discriminated with this sensor array. Finally, the sensor array was successfully used for the discrimination of antioxidants in serum samples, demonstrating its potential applications in the diagnosis of antioxidant-related diseases.

A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols and theaflavins in rat plasma: Application to a pharmacokinetic study of Da Hong Pao tea.

A method based on ultra-performance liquid chromatography-tandem mass spectrometry has been developed for the rapid and simultaneous determination of five catechins and four theaflavins in rat plasma using ethyl gallate as internal standard. The pharmacokinetic profiles of these compounds were compared after oral administration of five kinds of Da Hong Pao tea to rats. Biosamples processed with a mixture of ß-glucuronidase and sulfatase were extracted with ethyl acetate-isopropanol. Chromatographic separation was achieved by gradient elution using 10 mm HCOONH4 solution and methanol as the mobile phase. Analytes were detected using negative ion electrospray ionization in multiple reaction monitoring mode. The lower limits of quantification were 1.0, 0.74 and 0.5 ng/mL for theaflavins, two catechins and three catechins, respectively. The validation parameters were well within acceptable limits. The average half-lives (t1/2 ) in blood of the reference solution group was much shorter than those of tea samples. The values of AUC0-t and Cmax of the polyphenols and theaflavins exhibited linear pharmacokinetic characteristics which were related to the dose concentration.

Global testing of shifts in metabolic phenotype.

INTRODUCTION: Current metabolomics approaches to unravel impact of diet- or lifestyle induced phenotype variation and shifts predominantly deploy univariate or multivariate approaches, with a posteriori interpretation at pathway level. This however often provides only a fragmented view on the involved metabolic pathways. OBJECTIVES: To demonstrate the feasibility of using Goeman's global test (GGT) for assessment of variation and shifts in metabolic phenotype at the level of a priori defined pathways. METHODS: Two intervention studies with identified phenotype variations and shifts were examined. In a weight loss (WL) intervention study obese subjects received a mixed meal challenge before and after WL. In a polyphenol (PP) intervention study obese subjects received a high fat mixed meal challenge (61E% fat) before and after a PP intervention. Plasma samples were obtained at fasting and during the postprandial response. Besides WL- and PP-induced phenotype shifts, also correlation of plasma metabolome with phenotype descriptors was assessed at pathway level. The plasma metabolome covered organic acids, amino acids, biogenic amines, acylcarnitines and oxylipins. RESULTS: For the population of the WL study, GGT revealed that HOMA correlated with the fasting levels of the TCA cycle, BCAA catabolism, the lactate, arginine-proline and phenylalanine-tyrosine pathways. For the population of the PP study, HOMA correlated with fasting metabolite levels of TCA cycle, fatty acid oxidation and phenylalanine-tyrosine pathways. These correlations were more pronounced for metabolic pathways in the fasting state, than during the postprandial response. The effect of the WL and PP intervention on a priori defined metabolic pathways, and correlation of pathways with insulin sensitivity as described by HOMA was in line with previous studies. CONCLUSION: GGT confirmed earlier biological findings in a hypothesis led approach. A main advantage of GGT is that it provides a direct view on involvement of a priori defined pathways in phenotype shifts.

Phenolic metabolites in plasma and tissues of rats fed with a grape pomace extract as assessed by liquid chromatography-tandem mass spectrometry.

Grape pomace extract (GPE) is a rich and relatively low-cost source of phenolic compounds. However, little is known about the main GPE metabolites in mammals, which could help explain the observed health-promoting effects. This study investigated the presence of parent compounds from flavanol, flavonol and stilbene families and their metabolites in rat plasma and tissues after an acute intake of GPE in doses of 300 and 600 mg kg/body weight. The measurement of free compounds and their metabolites was performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results showed the presence of epicatechin, epicatechin methyl-glucuronide, epicatechin methyl-sulphate, catechin, catechin-glucuronide, quercetin methyl-glucuronide, resveratrol-3-glucuronide, resveratrol-4-glucuronide and resveratrol-3-sulphate in plasma, which was dose dependent. The most abundant measured compound in plasma was epicatechin-glucuronide. The presence of glucuronidated and methyl-glucuronidated forms of catechin were observed in the liver at both doses, while epicatechin-glucuronide and methyl-glucuronide were detected only upon intake of 600 mg GPE/kg body weight. At this dose epicatechin-glucuronide and methyl-glucuronide were also detected in muscle, and catechin methyl-glucuronide in adipose tissue. Results show the main GPE metabolites present in rat tissues after oral consumption, contributing to better understand the health benefits of GPE and its potential utilization as a functional ingredient.

Lack of pharmacokinetic interaction between fluvastatin and green tea in healthy volunteers.

PURPOSE: The objective of this study is to assess the effects of green tea and its major catechin component, (-)-epigallocatechin gallate (EGCG), on CYP2C9-mediated substrate metabolism in vitro, and the pharmacokinetics of fluvastatin in healthy volunteers. METHODS: The metabolism of diclofenac and fluvastatin in human recombinant CYP2C9 was investigated in the presence of EGCG. In a randomized three-phase crossover study, 11 healthy volunteers ingested a single 20-mg dose of fluvastatin with green tea extract (GTE), containing 150 mg of EGCG, along with water (300 mL), brewed green tea (300 mL), or water (300 mL) after overnight fasting. Plasma concentrations of fluvastatin and EGCG were measured by ultra-performance liquid chromatography with fluorescence detection and a single mass spectrometer. RESULTS: EGCG inhibited diclofenac 4'-hydroxylation and fluvastatin degradation with IC50 of 2.23 and 48.04 µM, respectively. Brewed green tea used in the clinical study also dose-dependently inhibited the metabolism of diclofenac and fluvastatin in vitro. However, no significant effects of GTE and brewed green tea were observed in plasma concentrations of fluvastatin. The geometric mean ratios with 90% CI for area under the plasma concentration-time curve (AUC0-∞) of fluvastatin were 0.993 (0.963-1.024, vs. brewed green tea) and 0.977 (0.935-1.020, vs. GTE). CONCLUSIONS: Although in vitro studies indicated that EGCG and brewed green tea produce significant inhibitory effects on CYP2C9 activity, the concomitant administration of green tea and fluvastatin in healthy volunteers did not influence the pharmacokinetics of fluvastatin.

Development and validation of a high-throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra-batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high-throughput sample analysis studies.

Quantitative Analysis of Four Catechins from Green Tea Extract in Human Plasma Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry for Pharmacokinetic Studies.

Green tea is consumed as a beverage worldwide and has beneficial effects, such as a lower risk of cardiovascular disease and cancer. A quantitative analysis of the beneficial components in plasma is important for understanding the potential health benefits of green tea. Four catechins­epigallocatechin-3-gallate (EGCG), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epicatechin (EC)­which account for the majority of the components of green tea, were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, a validated method was optimized to obtain the blood concentrations after the one-time ingestion of 630 mg green tea extract with digoxin and then after the ingestion of 630 mg green tea repeatedly for 15 days. The calibration curve, including the LLOQ, was constructed over 1⁻500 ng/mL for EGCG, ECG, and EGC and 0.1⁻50 ng/mL for EC. The method for inter- and intra-validation was applied, acceptable for both accuracy and precision. We successfully developed an appropriate UPLC-MS/MS method for human plasma with good reproducibility and sensitivity. Thus, this method could be applied for future preclinical and clinical studies on EGCG, ECG, EGC, and EC.

Green Tea Polyphenols Inhibit Colorectal Tumorigenesis in Azoxymethane-Treated F344 Rats.

In studying the cancer-preventive activities of green tea polyphenols, we previously demonstrated that dietary administration of polyphenon E (PPE) inhibited the formation of aberrant crypt foci (ACF) in the colon of azoxymethane (AOM)-treated F344 rats. Herein, we reported cancer-preventive activity of PPE using colorectal cancer as an end point. F344 rats were given two weekly injections of AOM, and then maintained on a 20% high-fat diet with or without 0.24% PPE for 34 wk. In the control group, 83% of rats developed colorectal tumors. Dietary PPE treatment significantly increased the plasma and colonic levels of tea polyphenols, and decreased tumor multiplicity and tumor size. Histological analysis indicated that PPE significantly decreased the incidence of adenocarcinoma, and the multiplicity of adenocarcinoma as well as the multiplicity of adenoma. PPE treatment significantly decreased plasma levels of proinflammatory eicosanoids, prostaglandin E2, and leukotriene B4. It also decreased ß-catenin nuclear expression, induced apoptosis, and increased expression levels of RXRα, ß, and γ in adenocarcinomas. In conclusion, our results convincingly demonstrated the inhibitory effects of orally administered PPE on colon carcinogenesis in AOM-treated rats and suggested possible biomarkers for the biological effects of green tea polyphenols.

Effects of a diet containing dried grape pomace on blood metabolites and milk composition of dairy cows.

BACKGROUND: The effect of a diet containing 15% grape pomace (GP) on the general health status and milk quality of dairy cows was assessed by plasma biochemistry and total polyphenol (TP) content, milk polyphenols, milk composition and milk protein fractions. RESULTS: Among the polyphenols measured by liquid chromatography-mass spectroscopy in GP, in feed containing GP (GP+) or not containing GP (GP-), gallic acid and epicatechin were present in the highest concentrations (67.58 and 19.23 µg mL-1 , respectively). Higher amounts of TP were also detected in the blood plasma of GP+ cows (114.06 and 83.93 mg GAE L-1 , respectively) but not in their milk (233.17 and 245.75 mg GAE L-1 , respectively). Also a significant increase was found for lactose and ß-lactoglobulin, although there was no effect on α-lactalbumin, albumin, secretory components and caseins. CONCLUSION: Inclusion of 15% GP in the diets of dairy cows is beneficial for overall normal blood constituent metabolism and helps to maintain cow health. The milk of cows fed with a GP diet preserves the normal levels of fat, protein and caseins, and has increased levels of components that make this milk a versatile ingredient material for the food industry (e.g. model whey powders, stability of lactose-rich powders). © 2016 Society of Chemical Industry.

Dark chocolate attenuates intracellular pro-inflammatory reactivity to acute psychosocial stress in men: A randomized controlled trial.

Flavanol-rich dark chocolate consumption relates to lower risk of cardiovascular mortality, but underlying mechanisms are elusive. We investigated the effect of acute dark chocolate consumption on inflammatory measures before and after stress. Healthy men, aged 20-50years, were randomly assigned to a single intake of either 50g of flavanol-rich dark chocolate (n=31) or 50g of optically identical flavanol-free placebo-chocolate (n=34). Two hours after chocolate intake, both groups underwent the 15-min Trier Social Stress Test. We measured DNA-binding-activity of the pro-inflammatory transcription factor NF-κB (NF-κB-BA) in peripheral blood mononuclear cells, as well as plasma and whole blood mRNA levels of the pro-inflammatory cytokines IL-1ß and IL-6, and the anti-inflammatory cytokine IL-10, prior to chocolate intake as well as before and several times after stress. We also repeatedly measured the flavanol epicatechin and the stress hormones epinephrine and cortisol in plasma and saliva, respectively. Compared to the placebo-chocolate-group, the dark-chocolate-group revealed a marginal increase in IL-10 mRNA prior to stress (p=0.065), and a significantly blunted stress reactivity of NF-κB-BA, IL-1ß mRNA, and IL-6 mRNA (p's⩽0.036) with higher epicatechin levels relating to lower pro-inflammatory stress reactivity (p's⩽0.033). Stress hormone changes to stress were controlled. None of the other measures showed a significant chocolate effect (p's⩾0.19). Our findings indicate that acute flavanol-rich dark chocolate exerts anti-inflammatory effects both by increasing mRNA expression of the anti-inflammatory cytokine IL-10 and by attenuating the intracellular pro-inflammatory stress response. This mechanism may add to beneficial effects of dark chocolate on cardiovascular health.

Effects of ω-3 Fatty Acids and Catechins on Fatty Acid Synthase in the Prostate: A Randomized Controlled Trial.

Animal and human studies suggest fish oil and green tea may have protective effect on prostate cancer. Fatty acid synthase (FAS) has been hypothesized to be linked to chemoprotective effects of both compounds. This study evaluated the independent and joint effects of fish oil (FO) and green tea supplement (epigallocatechin-3-gallate, EGCG) on FAS and Ki-67 levels in prostate tissue. Through a double-blinded, randomized controlled trial with 2 × 2 factorial design, 89 men scheduled for repeat prostate biopsy following an initial negative prostate biopsy were randomized into either FO alone (1.9 g DHA + EPA/day), EGCG alone (600 mg/day), a combination of FO and EGCG, or placebo. We used linear mixed-effects models to test the differences of prostate tissue FAS and Ki-67 by immunohistochemistry between pre- and post-intervention within each group, as well as between treatment groups. Results did not show significant difference among treatment groups in pre-to-post-intervention changes of FAS (P = 0.69) or Ki-67 (P = 0.26). Comparing placebo group with any of the treatment groups, we did not find significant difference in FAS or Ki-67 changes (all P > 0.05). Results indicate FO or EGCG supplementation for a short duration may not be sufficient to produce biologically meaningful changes in FAS or Ki-67 levels in prostate tissue.

Rapid and sustained systemic circulation of conjugated gut microbial catabolites after single-dose black tea extract consumption.

Gut microbial catabolites of black tea polyphenols (BTPs) have been proposed to exert beneficial cardiovascular bioactivity. This hypothesis is difficult to verify because the conjugation patterns and pharmacokinetics of these catabolites are largely unknown. The objective of our study was to identify, quantify, and assess the pharmacokinetics of conjugated BTP metabolites in plasma of healthy humans by means of an a priori untargeted LC-MS-based metabolomics approach. In a randomized, open, placebo-controlled, crossover study, 12 healthy men consumed a single bolus of black tea extract (BTE) or a placebo. The relative and, in several cases, absolute concentrations of a wide range of metabolites were determined using U(H)PLC-LTQ-Orbitrap-FTMS. Following BTE consumption, a kinetic response in plasma was observed for 59 BTP metabolites, 11 of these in a quantitative manner. Conjugated and unconjugated catechins appeared in plasma without delay, at 2-4 h, followed by a range of microbial catabolites. Interindividual variation in response was greater for gut microbial catabolites than for directly absorbed BTPs. The rapid and sustained circulation of conjugated catabolites suggests that these compounds may be particularly relevant to proposed health benefits of BTE. Their presence and effects may depend on individual variation in catabolic capacity of the gut microbiota.

Determination of exogenous epigallocatechin gallate peracetate in mouse plasma using liquid chromatography with quadrupole time-of-flight mass spectrometry.

A robust method for the quantitation of epigallocatechin gallate peracetate in plasma for pharmacokinetic studies is lacking. We have developed a validated method to quantify this compound using liquid chromatography with quadrupole time-of-flight mass spectrometry with isopropanol and tert-butyl methyl ether (3:10) extraction and thin-layer chromatography purification. The epigallocatechin gallate peracetate-1-(13) C8 isotope was used as an internal standard. The linear range (r(2) > 0.9950) was from 0.05 to 100.00 µg/mL. The lower limit of quantification of the method was 0.05 µg/mL. Reproducibility, coefficient of variation, was between 0.7 and 12.6% (n = 6), accuracy between 83.7 and 104.6% (n = 5), and recovery ranged from 82.4 to 109.0% (n = 4). Ion suppression was approximately 40%. No mass spectral peaks were found to interfere between the standard and internal standard or the blank plasma extracts. Epigallocatechin gallate peracetate in plasma was stably stored at -80°C over three months even after three freeze-thaw cycles. Extracts were stable in the sampler at 4°C for over 48 h. Plasma levels were maintained at 1.36 µg/mL for 360 min after intraorbital intravenous injection at 50 mg/kg in mice. This method can be used to reliably measure epigallocatechin gallate peracetate in plasma for pharmacokinetic studies.

Green tea extract suppresses N-methyl-N-nitrosourea-induced photoreceptor apoptosis in Sprague-Dawley rats.

BACKGROUND: Retinitis pigmentosa (RP) is a group of inherited neurodegenerative human diseases characterized by the loss of photoreceptor cells by apoptosis and eventual blindness. A single intraperitoneal (ip) injection of N-methyl-N-nitrosourea (MNU) causes photoreceptor cell apoptosis within 7 days in rats. Green tea extract (THEA-FLAN 90S; GTE) is a common herbal supplement with pluripotent properties including antioxidant activity. The purpose of the present study was to evaluate the efficacy of GTE against photoreceptor apoptosis in 7-week-old female Sprague-Dawley rats that received a single ip injection of 40 mg/kg MNU. METHODS: The oral administration of 250 mg/kg/day GTE was initiated 3 days prior to MNU injection and continued once daily throughout the experiment. Rats were sacrificed at 12, 24, and 72 h and 7 days after MNU injection, and the eyes were examined morphologically and morphometrically. The photoreceptor cell ratio, retinal damage ratio, and retinal preservation ratio were used to determine the structural and functional alterations. The number of apoptotic photoreceptor cells per mm(2) was determined in situ by TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL). Our results indicated that oral administration of GTE significantly suppressed the loss of photoreceptor cells morphometrically 7 days after MNU injection. The number of TUNEL-positive cells per mm(2) in MNU-exposed rat central retina with or without GTE administration was 981 vs. 2056 at 24 h after MNU injection. CONCLUSIONS: GTE structurally and functionally suppressed MNU-induced photoreceptor cell apoptosis. These findings indicate that GTE may help to ameliorate the onset and progression of human RP.

The profiling and identification of the metabolites of (+)-catechin and study on their distribution in rats by HPLC-DAD-ESI-IT-TOF-MS(n) technique.

(+)-Catechin, a potential beneficial compound to human health, is widely distributed in plants and foods. A high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry method was applied to profile and identify the metabolites of (+)-catechin in rats and to study the distribution of these metabolites in rat organs for the first time. In total, 51 phase II metabolites (44 new) and three phase I metabolites were tentatively identified, comprising 16 (+)-catechin conjugates, 14 diarylpropan-2-ol metabolites, 6 phenyl valerolactone metabolites and 18 aromatic acid metabolites. Further, 19 phase II metabolites were new compounds. The in vivo metabolic reactions of (+)-catechin in rats were found to be ring-cleavage, sulfation, glucuronidation, methylation, dehydroxylation and dehydrogenation. The numbers of detected metabolites in urine, plasma, small intestine, kidney, liver, lung, heart, brain and spleen were 53, 23, 27, 9, 7, 5, 3, 2 and 1, respectively. This indicated that small intestine, kidney and liver were the major organs for the distribution of (+)-catechin metabolites. In addition, eight metabolites were found to possess bioactivities according to literature. These results are very helpful for better comprehension of the in vivo metabolism of (+)-catechin and its pharmacological actions, and also can give strong indications on the effective forms of (+)-catechin in vivo.

Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

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.

Comparison of catechin profiles in human plasma and urine after single dosing and regular intake of green tea (Camellia sinensis).

Green tea (Camellia sinensis) catechin profiles in plasma and urine following single dosing and regular ingestion of green tea are not clear. We performed a placebo-controlled intervention study with sixteen healthy volunteers to determine changes in total and free catechins after a single dose and following 1 week of twice-daily green tea. Blood and urine samples were collected before (fasting) and after (60 and 120 min for blood; 90 and 180 min for urine) drinking 200 ml of 1.5% (w/v) green tea or water (n 8 each), and fasting samples were again collected after 7 d of 150 ml of 1% (w/v) supplemental green tea or water twice daily. After a 4-week washout, subjects were crossed onto the other treatment and procedures repeated. Plasma results at 1 h post-ingestion showed elevated (P < 0.05) mean epigallocatechin gallate (EGCG; 310 (SD 117) nmol/l; all in free form), epigallocatechin (EGC; 192 (SD 67) nmol/l; 30% free) and epicatechin gallate (ECG; 134 (SD 51) nmol/l; 75% free). Fasting plasma after 7 d of regular intake showed increased (P < 0.05) EGCG (80 v. 15 nmol/l at baseline) and ECG (120 v. 40 nmol/l), with > or =90% of both in their conjugated forms. Total EGC was < 10 nmol/l. Post-ingestion conjugation and renal loss of EGC and epicatechin were rapid and high, but were negligible for EGCG and ECG. In the green tea consumed, the content was EGCG > EGC > ECG, and the acute plasma response mirrored this. However, after chronic consumption there was almost no EGC found in fasting plasma, some EGCG was present, but a rather high level of ECG was maintained.

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.

Blood pressure and endothelial function in healthy, pregnant women after acute and daily consumption of flavanol-rich chocolate: a pilot, randomized controlled trial.

BACKGROUND: Several randomized clinical trials (RCTs) indicate that flavanol-rich chocolate has beneficial effects on flow-mediated dilation (FMD) and blood pressure (BP). However, no RCTs have evaluated these outcomes in pregnant women. The objective of this 2-group, parallel, double-blind RCT was to examine the effects of flavanol-rich chocolate on FMD and BP in pregnant women with normal BP. METHODS: Forty-four healthy, pregnant women were randomized to the high-flavanol (n = 23) or low-flavanol (n = 21) chocolate consumption for 12 weeks. At randomization (0, 60, 120 and 180 min after a single 40-g dose of chocolate), 6 and 12 weeks after daily 20-g chocolate intake, we evaluated plasma concentrations of flavanols and theobromine, as well as the FMD and BP. RESULTS: Plasma epicatechin was significantly increased (p < 0.001) 180 min after the consumption of 40-g high-flavanol chocolate compared to low-flavanol chocolate. Theobromine concentrations were significantly higher 180 min and 12 weeks after the intake of experimental chocolate or low-flavanol chocolate (p < 0.001). FMD was not different between the 2 groups at all pre-defined time periods. No other significant within-group or between-group changes were observed. CONCLUSION: These results confirm the feasibility of a large-scale RCT comparing daily consumption of flavanol-rich chocolate to an equivalent placebo during pregnancy and demonstrate higher plasma epicatechin and theobromine concentration in the intervention group after acute ingestion TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01659060.