Flavan-3-ol/Procyanidin Metabolomics in Rat Urine Using HPLC-Quadrupole TOF/MS.

SCOPE: Several studies have demonstrated that flavan-3-ol/procyanidins are associated with biological functions in the prevention of various chronic diseases, including obesity and diabetes. Knowledge of their mechanisms, including bioavailability, has significantly progressed in the last decade. However, the differences of the metabolic signatures among flavan-3-ol/procyanidins remain ambiguous. METHODS AND RESULTS: The metabolites in urine over time after acute administration of three typical flavan-3-ol/procyanidins ((epi)catechin [EPC], epigallocatechin gallate [EGCG], and procyanidin dimer [PC]) in view of the chemical structure were analyzed by HPLC-quadrupole TOF/MS. Several bile acid and amino acid derivatives including tryptophan and tyrosine, as well as flavan-3-ol/procyanidin conjugates and phenolic acid degradation products generated by the gut microbiota were observed in rat urine. CONCLUSION: Multivariate statistical analyses suggest that the exogenous and endogenous metabolites of flavan-3-ol/procyanidins greatly differ, although the chemical structures of three typical flavan-3-ol/procyanidins-EPC, EGCG, and PC-are similar. Thus, metabolomic differences likely affect their biological functions and health benefits.

A Simple, Enzymatic Biotransformation Method Using Fresh Green Tea Leaves Efficiently Generates Theaflavin-Containing Fermentation Water That Has Potent Physiological Functions in Mice and Humans.

The polyphenolic compound theaflavin, the main red pigment in black tea, possesses many beneficial properties, such as fat-reducing and glucose-lowering capabilities. To produce theaflavin-containing fermentation water on a large scale, we have developed a simple, inexpensive, and selective enzymatic biotransformation method to obtain sufficient levels from fresh green tea leaves. Subsequent administration of theaflavin-containing fermentation water to obese mice on a high-fat diet inhibited body weight gain, decreased casual blood glucose and fasting blood glucose levels, and lowered mesenteric and total fat composition. To note, there were no significant differences observed in food consumption between the experimental and control (water without theaflavin) mice groups. Next, we investigated the effect of this water on blood glucose levels in healthy humans and found that it significantly inhibited blood glucose levels. Thus, we showed that theaflavin-containing fermentation water can be efficiently generated from fresh green tea leaves and demonstrated its significantly potent effects in vivo.

Short-term biomarkers of apple consumption.

SCOPE: Urinary biomarkers are used to estimate the nutritional intake of humans. The aim of this study was to distinguish between low, medium, and high apple consumption by quantifying possible intake biomarkers in urine samples after apple consumption by HPLC-MS/MS. Apples were chosen as they are the most consumed fruits in Germany. METHODS AND RESULTS: Thirty subjects took part in 7-day study. They abstained from apples and apple products except for one weighed apple portion resembling one, two, or four apples. Before apple consumption and during the following days spot urine samples were collected. These urine samples were incubated with ß-glucuronidase, diluted, and directly measured by HPLC-MS/MS. Phloretin, epicatechin, procyanidin B2, and quercetin were detected in urine using Scheduled MRMTM mode. Phloretin was confirmed as a urinary biomarker of apple intake and had the ability to discriminate between low or medium (one or two apples) and high apple consumption (four apples). The groups also differ in the excretion of epicatechin and procyanidin B2. CONCLUSION: Apple consumption can be monitored by urinary biomarkers for a period of at least 12 h after consumption. Furthermore the amount of apples consumed can be estimated by the concentration of certain biomarkers.

Effect of consuming a grape seed supplement with abundant phenolic compounds on the oxidative status of healthy human volunteers.

BACKGROUND: Diverse enzymatic and non-enzymatic antioxidants provide protection against reactive oxygen species in humans and other organisms. The nonenzymatic antioxidants include low molecular mass molecules such as plant-derived phenols. AIM OF STUDY: This study identified the major phenolic compounds of a grape seed extract by HPLC and analyzed the effect of consumption of biscuits enriched with this extract on the urinary oxidative status of healthy subjects by measurement of urine redox potential. METHODS: The major phenolic compounds were characterized in a red grape seed extract separated by HPLC with detection by a photodiode array (PDA), fluorescence (FL) and quadrupole mass spectrometer (MS). A nutritional study in a healthy volunteers group was done. Each volunteer ate eight traditional biscuits with no red grape seed extract supplementation. The second day each volunteer ate eight traditional biscuits supplemented with 0.6% (wt/wt) of grape seed extract. An overnight urine sample was obtained for each treatment. The redox potential was measured at 25 °C using a potentiometer in each urine sample. RESULTS: Epicatechin, catechin, procyanidin dimers B1 to B4, and the procyanidin trimer C2 were the major phenolic components in the extract. Epicatechin gallate and procyanidin dimers B1-3-G and B2-3'-G were the major galloylated flavan-3-ols. The forty-six healthy volunteers each shown a reduction of the urine redox potential after the treatment by traditional biscuits supplemented with the grape seed extract. CONCLUSIONS: This simple dietary intervention significantly reduced (33%) the urine redox potential, reflecting an overall increase in antioxidant status. Incorporation of plant-derived phenols in the diet may increase anti-oxidative status.

Effect of tea theaflavins and catechins on microvascular function.

Beneficial effects of flavonoid-rich black and green tea on macrocirculation have been well established. Theaflavins are unique to black tea as they are formed from catechins during the enzymatic oxidation of tea leaves. The study was performed to gain more insight into the effects of theaflavins on microcirculation and to compare effects with another important flavonoid class, the green tea derived catechins, which have been reported to improve vascular function. Twenty-four healthy subjects were included in a double-blind, placebo-controlled, randomised, cross-over study. On six different days, subjects received capsules with a single dose of catechins (500 mg), four varying doses of theaflavins (100 to 500 mg) or placebo. Microcirculation was assessed after each treatment by Pulse Amplitude Tonometry (EndoPAT) at baseline and 2, 4 and 6 h after test product intake. The EndoPAT reactive hyperemia response was improved by 500 mg catechins (reactive hyperemia index (RHI): 0.2; p = 0.04) and by 500 mg theaflavins (RHI: 0.19; p = 0.06) compared to placebo. Also, 300 mg theaflavins increased the RHI (0.28; p = 0.02), but no effects were observed at lower doses. The study suggests moderate effects of single doses of catechins and theaflavins on peripheral microcirculation.

Systemic absorption and metabolism of dietary procyanidin B4 in pigs.

SCOPE: Procyanidins (PCs) are among the most abundant polyphenols in the human diet and they are reported to exhibit several beneficial health effects. However, the knowledge about their metabolic fate is rather limited. To investigate the systemic absorption and metabolism of dietary PC B4, a kinetic study using pigs as model system has been performed. METHODS AND RESULTS: After oral application of a single dose of 10 mg/kg body weight PC B4, urine and plasma were collected over a period of 48 h. PC B4 and its possible metabolites were analyzed in physiological samples using HPLC-MS/MS and GC-MS. PC B4 was detected as intact molecule in urine as well as in plasma. Maximum reached plasma concentration of PC B4 (cmax ) was 2.13 ng/mL (3.68 nM) and mean total urinary excretion related to the administered dose was 0.008 ± 0.003%. In addition to that the monomeric structural units catechin and epicatechin were determined as degradation products. Furthermore, methylated and conjugated monomeric metabolites were identified. Monomeric metabolites were identified to be the major fraction occurring in the systemic circulation. The analysis of phenolic acids did not show an increase of these possible further metabolites. CONCLUSION: After oral administration, PC B4 is absorbed as intact molecule and it is excreted in urine. In addition, it is degraded to the monomeric subunits that are then further metabolized to methylated and glucuronidated conjugates in pigs.

A significant inhibitory effect on advanced glycation end product formation by catechin as the major metabolite of lotus seedpod oligomeric procyanidins.

Several lines of evidence suggested that B-type procyanidin oligomers from lotus seedpod (LSOPC) may effectively modulate the formation of advanced glycation end products (AGEs). In vivo, LSOPC is metabolized by intestinal flora to become various kinds of phenolic compounds that possess potent antioxidant activities. However, few reports of the absorption and metabolism of LSOPC have been revealed. In the present study, rats were orally administered with LSOPC at a dose of 300 mg/kg body weight. The metabolites of LSOPC in urine were elucidated by HPLC-MS/MS analysis 24 h post-administration. Eight major metabolites were significantly increased by the administration of 300 mg/kg of LSOPC (p < 0.01). The anti-glycative activity of LSOPC and its metabolites were investigated. The results showed that LSOPC and catechin had greater anti-glycative activities than other metabolites, which were positively correlated to their carbonyl scavenging activities and antioxidant capacities.

Absorption and urinary excretion of A-type procyanidin oligomers from Litchi chinensis pericarp in rats by selected ion monitoring liquid chromatography-mass spectrometry.

Intervention studies with A-type oligomeric procyanidins from litchi (Litchi chinensis) pericarp (LPOPC) suggested its protective effect against cardiovascular diseases. However, there is no consensus on the absorption and metabolism of LPOPC. It was demonstrated that the main components in LPOPC were (-)-epicatechin, A-type procyanidin dimers, trimers and tetramers. Rats were orally administered different levels of LPOPC (150 and 300 mg/kgbw), the procyanidins and their microbial metabolites in urine were identified by HPLC-MS/MS analysis 18 h post-administration. Data indicated that seven aromatic acid metabolites excreted were significantly increased by 300 mg/kgbw of LPOPC (P<0.01). However, only (-)-epicatechin and its methylated derivatives were detected in rat plasma 1h after 300 mg/kgbw of LPOPC administration. The total EC content absorbed in plasma was only 2.54 ± 0.53 µmol/L, indicating that the biological properties of LPOPC should be probably explained by its microbial degraded phenolic acids.

Intake of dietary procyanidins does not contribute to the pool of circulating flavanols in humans.

BACKGROUND: Accumulating data show a causal role for flavanols in the mediation of cardiovascular benefits associated with the consumption of flavanol- and procyanidin-containing foods. Evidence for a direct causal role for procyanidins in this context is far less profound due to the poor absorption of procyanidins. However, it has been proposed that procyanidins may break down in the gastrointestinal tract, resulting in monomeric flavanols, which contribute to the systemic flavanol pool. Verification or rejection of this supposition could significantly affect the interpretation of epidemiologic and dietary intervention data and the design of food-content databases. OBJECTIVE: We assessed the respective contribution of flavanols and procyanidins to the systemic pool of flavanols and 5-(3,4-dihydroxyphenyl)-γ-valerolactone (γ-VL) in humans. DESIGN: Test drinks that contained only flavanols (D1), procyanidins with a degree of polymerization that ranged from 2 to 10 (D2-10), or flavanols and procyanidins with a degree of polymerization that ranged from 2 to 10 (D1-10) were consumed by subjects (n = 12) according to a randomized, double-masked, crossover design. Plasma and urine samples were collected postprandially and analyzed. RESULTS: The ingestion of D1-10 resulted in the systemic presence of flavanols (plasma concentration: 863 ± 77 nmol/L), γ-VLs (24-h urine: 93 ± 18 µmol), and minute concentrations of procyanidin B2. With correction for small residual amounts of flavanols present in D2-10, only negligible concentrations of circulating flavanols were detected after ingestion of the drink, whereas the intake of D1 resulted in circulating flavanol concentrations similar to those detected after D1-10 consumption. CONCLUSIONS: These outcomes show that dietary procyanidins do not contribute to the systemic pool of flavanols in humans. Thus, these data reject the notion that procyanidins, through their breakdown into flavanols and subsequent absorption, causally modulate vascular function.

Structural identification of mouse fecal metabolites of theaflavin 3,3'-digallate using liquid chromatography tandem mass spectrometry.

Black tea consumption has been associated with many health benefits including the prevention of cancer and heart disease. Theaflavins are the major bioactive polyphenols present in black tea. Unfortunately, limited information is available on their biotransformation. In the present study, we investigated the metabolic fate of theaflavin 3,3'-digallate (TFDG), one of the most abundant and bioactive theaflavins, in mouse fecal samples using liquid chromatography/electrospray ionization tandem mass spectrometry by analyzing the MS(n) (n=1-3) spectra. Four metabolites theaflavin, theaflavin 3-gallate, theaflavin 3'-gallate, and gallic acid were identified as the major mouse fecal metabolites of TFDG. Glucuronidated and sulfated, instead of methylated metabolites of theaflavin 3-gallate, theaflavin 3'-gallate, and TFDG were detected and identified as the minor mouse fecal metabolites of TFDG. Our results indicate that TFDG can be degraded in mice. Further studies on the formation of those metabolites in TFDG-treated mice in germ-free conditions are warranted. To our knowledge, this is the first report on the biotransformation of TFDG in mice.

In vivo bioavailability, absorption, excretion, and pharmacokinetics of [14C]procyanidin B2 in male rats.

Procyanidins are important biologically active compounds, but the pathway and extent of absorption and metabolism are controversial. We conducted a mass balance study to evaluate the total radioactivity excreted in urine and feces after oral administration of [(14)C]procyanidin B2 to male rats (n = 5). Urine and feces were collected daily from 0 to 96 h. Absolute bioavailability of (14)C from [(14)C]procyanidin B2 was calculated as approximately 82% using the values for total urinary (14)C. A pharmacokinetic study measured total radioactivity in the blood (n = 9). Blood samples were collected at designated time intervals (0.5-24 h) after administration. Three treatments were used: 1) intravenous, 2) oral higher dose (21 mg/kg b.wt.), and 3) oral lower dose (10.5 mg/kg). Blood concentration of total (14)C reached a maximum at approximately 6 h after ingestion of [(14)C]procyanidin B2 (groups II and III), and area under the curve (AUC) was dependent on oral dose. After intravenous or oral administration the terminal half-lives were similar, whereas 8-fold larger values were obtained after oral dosing for total clearance and the apparent volumes of distribution. These pharmacokinetic differences explain the apparently lower (14)C bioavailability (8-11%) for [(14)C]procyanidin calculated from blood [AUC((0-24))] values. After oral administration of [(14)C]procyanidin B2, 63% was excreted via urine within 4 days. The data suggest that much of the parent compound administered orally is degraded by the gut microflora before absorption and that these microbial metabolites have a different distribution from the compounds circulating after the intravenous dose.

Disposition of 14C-labelled ENDOTELON in humans.

In order to study the disposition of ENDOTELON in humans, this compound was labelled with 14C by photosynthesis. ENDOTELON consists of a complex of procyanidolic oligomers extracted from the seeds of a variety of vine cultivated in the Bordeaux wine-growing region, and is prescribed for the treatment of chronic venous insufficiency and retinal lesions. Considering the difficulty in labelling the various constituents of the product, the labelling procedure was based on providing radioactive CO2 to the plant. After isolation and purification, 150 mg of active material (50 microCi) was administered orally to six healthy volunteers. Radioactivity was measured in the blood over time until 72 and 120 hours in the same subjects after drug administration. Urinary and faecal elimination was measured for a period of 167 hours. Urinary elimination of the radioactive compounds represented 12 to 27% of the administered dose and faecal elimination represented 47 to 75% depending on the subject. The radioactivity of the 14CO2 eliminated in the breath was also measured, and represented around 8% of the total radioactivity for the 72-hour period after administration. Although the disposition of ENDOTELON is based on the total radioactivity measured over time, this technique allows the evaluation of the elimination rate of the product and its metabolites from the human body.

Sorghum extrusion increases bioavailability of catechins in weanling pigs.

Catechins and procyanidins are beneficial for human health; however, their bioavailability is low. The effect of food processing on catechin bioavailability from sources containing predominantly procyanidins has not been studied. The sumac sorghum mixture (50% whole grain+50% bran) used in this study contained catechins, procyanidins dimers, and polymers at 0.08, 0.6, and 26.4 mg/g, respectively. Extrusion decreased the polymeric procyanidins by 48% to 22 mg/g while increasing catechins (50%) and dimers (64%) to 0.12 and 1.0 mg/g, respectively. Six weanling pigs (8.9+/-1.1 kg) received a single dose by gavage of the sorghum mixture (7 g/kg0.75), the sorghum mixture extrudate, or white sorghum (50% whole grain+50% bran) in a randomized crossover design. Treatments were separated by a 7-day washout period. Blood was drawn at 0, 1, 2, and 4 h. Plasma catechin, 3'-O-methylcatechin, 4'-O-methylcatechin, epicatechin, 3'-O-methylepicatechin, and 4'-O-methylepicatechin peaked at 1 h and were 18, 43, 1, 0.7, 0.7, and 0.3 nmol/L for pigs receiving sorghum, respectively. Plasma levels in pigs receiving extruded sorghum were 66, 110, 2, 16, 8, and 11 nmol/L, respectively. Plasma levels of catechin, 3'-O-methylcatechin, and the total catechins were higher in pigs fed extruded sorghum at 1, 2, and 4 h postdose (P