Validation of a high-throughput method for the quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma by UPLC-MS.

Nutritional biomarkers are critical tools to objectively assess intake of nutrients and other compounds from the diet. In this context, it is essential that suitable analytical methods are available for the accurate quantification of biomarkers in large scale studies. Recently, structurally-related (-)-epicatechin metabolites (SREMs) and 5-(3',4'-dihydroxyphenyl)-γ-valerolactone metabolites (gVLMs) were identified as biomarkers of intake of flavanols and procyanidins, a group of polyphenol bioactives. This study aimed at validating a high throughput method for the quantification of SREMs and gVLMs in plasma along with methylxanthines (MXs), dietary compounds known to interact with flavanol and procyanidin effects. To accomplish this, a full set of authentic analytical standards were used to optimize a micro solid phase extraction method for sample preparation coupled to HPLC-MS detection. Isotopically-labelled standards for all analytes were included to correct potential matrix effects on quantification. Average accuracies of 101%, 93% and 103% were obtained, respectively, for SREMs, gVLMs and MXs. Intra- and inter-day repeatability values were <15%. The method showed linear responses for all analytes (>0.993). Most SREMs and gVLMs had limits of quantifications <5 nM while limits of quantification of MXs were 0.2 µM. All analytes were stable under different tested processing conditions. Finally, the method proved to be suitable to assess SREMs, gVLMs and MXs in plasma collected after single acute and daily intake of cocoa-derived test materials. Overall, this method proved to be a valid analytical tool for high throughput quantification of flavanol and procyanidin biomarkers and methylxanthines in plasma.

Quantitative combination of natural anti-oxidants prevents metabolic syndrome by reducing oxidative stress.

Insulin resistance and abdominal obesity are present in the majority of people with the metabolic syndrome. Antioxidant therapy might be a useful strategy for type 2 diabetes and other insulin-resistant states. The combination of vitamin C (Vc) and vitamin E has synthetic scavenging effect on free radicals and inhibition effect on lipid peroxidation. However, there are few studies about how to define the best combination of more than three anti-oxidants as it is difficult or impossible to test the anti-oxidant effect of the combination of every concentration of each ingredient experimentally. Here we present a math model, which is based on the classical Hill equation to determine the best combination, called Fixed Dose Combination (FDC), of several natural anti-oxidants, including Vc, green tea polyphenols (GTP) and grape seed extract proanthocyanidin (GSEP). Then we investigated the effects of FDC on oxidative stress, blood glucose and serum lipid levels in cultured 3T3-L1 adipocytes, high fat diet (HFD)-fed rats which serve as obesity model, and KK-ay mice as diabetic model. The level of serum malondialdehyde (MDA) in the treated rats was studied and Hematoxylin-Eosin (HE) staining or Oil red slices of liver and adipose tissue in the rats were examined as well. FDC shows excellent antioxidant and anti-glycation activity by attenuating lipid peroxidation. FDC determined in this investigation can become a potential solution to reduce obesity, to improve insulin sensitivity and be beneficial for the treatment of fat and diabetic patients. It is the first time to use the math model to determine the best ratio of three anti-oxidants, which can save much more time and chemical materials than traditional experimental method. This quantitative method represents a potentially new and useful strategy to screen all possible combinations of many natural anti-oxidants, therefore may help develop novel therapeutics with the potential to ameliorate the worldwide metabolic abnormalities.

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.

Free radical-scavenging activities of oligomeric proanthocyanidin from Rhodiola rosea L. and its antioxidant effects in vivo.

This study aimed to determine the antioxidant activity of oligomeric proanthocyanidin from Rhodiola rosea L. (OPCRR). The free radical-scavenging activities exhibited by OPCRR, as determined by using 1,1-diphenyl-2-picrylhydrazyl, hydroxyl radical and superoxide anion (√[Formula: see text]) scavenging assays, were greater than that of vitamin C. The effects of OPCRR on the antioxidant enzymes activity and lipid peroxide content in vivo were evaluated through three observation biomarkers, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in serum, heart, liver and brain tissues in mice. The OPCRR significantly enhanced the SOD and GSH-Px activities, and reduced the MDA content in mice. These results indicated that the OPCRR has a great potential to be a natural antioxidant due to its considerable antioxidant activities in vitro and in vivo.

Bioavailability of polyphenols from peanut skin extract associated with plasma lipid lowering function.

Peanut skin is a rich source of polyphenols including procyanidins and is shown to have hypolipidemic properties. This study investigated the bioavailability of peanut skin polyphenols using a rat model. First, the bioavailability of peanut skin polyphenols in rat plasma was evaluated. Our results showed procyanidin A2 levels in plasma peaked within 30 min of ingestion. The results of a second study show that peanut skin extract supplemented in addition to oil gavage resulted in significant decrease in plasma triglyceride and VLDL within 5h. In the third study, rats were given a Western type diet for 5 weeks with peanut skin extract at a dose of 150 and 300 mg/kg body weight. The main effects observed were lowering of total blood lipid and reduction of the plasma fatty acids profile. Our results suggest that procyanidin A may impart a key role of hypolipidemic effect seen in peanut skin polyphenols.

Serum metabolites of proanthocyanidin-administered rats decrease lipid synthesis in HepG2 cells.

The regular consumption of flavonoids has been associated with reduced mortality and a decreased risk of cardiovascular diseases. The proanthocyanidins found in plasma are very different from the original flavonoids in food sources. The use of physiologically appropriate conjugates of proanthocyanidins is essential for the in vitro analysis of flavonoid bioactivity. In this study, the effect of different proanthocyanidin-rich extracts, which were obtained from cocoa (CCX), French maritime pine bark (Pycnogenol extract, PYC) and grape seed (GSPE), on lipid homeostasis was evaluated. Hepatic human cells (HepG2 cells) were treated with 25 mg/L of CCX, PYC or GSPE. We also performed in vitro experiments to assess the effect on lipid synthesis that is induced by the bioactive GSPE proanthocyanidins using the physiological metabolites that are present in the serum of GSPE-administered rats. For this, Wistar rats were administered 1 g/kg of GSPE, and serum was collected after 2 h. The semipurified serum of GSPE-administered rats was fully characterized by liquid chromatography tandem triple quadrupole mass spectrometry (LC-QqQ/MS(2)). The lipids studied in the analyses were free cholesterol (FC), cholesterol ester (CE) and triglycerides (TG). All three proanthocyanidin-rich extracts induced a remarkable decrease in the de novo lipid synthesis in HepG2 cells. Moreover, GSPE rat serum metabolites reduced the total percentage of CE, FC and particularly TG; this reduction was significantly higher than that observed in the cells directly treated with GSPE. In conclusion, the bioactivity of the physiological metabolites that are present in the serum of rats after their ingestion of a proanthocyanidin-rich extract was demonstrated in Hep G2 cells.

Flavanol metabolites distribute in visceral adipose depots after a long-term intake of grape seed proanthocyanidin extract in rats.

A considerable number of epidemiological investigations and intervention studies have supported an association between the intake of flavanol- and proanthocyanidin-containing foods and a decreased risk of metabolic diseases. Nonetheless, less is know about the capacity of tissues to accumulate flavanols and/or their metabolites. The main objective of the present study was to determine (n 20) plasma bioavailability and disposition in the liver, muscle, brown adipose tissue (BAT) and white adipose tissues (mesenteric and perirenal) in rats after a long-term consumption of three doses of grape seed phenolic extract (5, 25 and 50 mg/kg body weight) for 21 d in order to determine whether there is a dose-response relationship. Glucuronidated conjugates (total glucuronidated conjugates: C(5 mg/kg) 1·9; C(25 mg/kg) 6·4; C(50 mg/kg) 27·7 µmol/l plasma) followed by methyl glucuronidated conjugates (total methyl glucuronidated conjugates: C(5 mg/kg) 1·98; C(25 mg/kg) 4·48; C(50 mg/kg) 12·5 µmol/l plasma) were the main flavanol metabolites quantified in plasma, also detecting a dimer in its free form (C(25 mg/kg) 0·74; C(50 mg/kg) 0·79 µmol/l plasma). Each of the studied organs has a particular behaviour of accumulation and response to the assayed grape seed extract doses, with an exponential bioavailability-dose relationship in BAT, in which flavanols could play an important role in the reduction or prevention of obesity, modulating the functionality of that tissue.

Dietary antioxidant capacity is associated with improved serum antioxidant status and decreased serum C-reactive protein and plasma homocysteine concentrations.

PURPOSE: To investigate the associations of dietary TAC from diet and supplements with serum antioxidant concentrations and serum C-reactive protein (CRP) and plasma total homocysteine (tHcy) in US adults. METHODS: This was a cross-sectional study. Food consumption data, serum antioxidant levels, and serum CRP and Plasma tHcy concentrations of 4,391 US adults aged ≥19 years in the National Health and Nutrition Examination Survey 2001-2002 were analyzed. The USDA flavonoid and proanthocyanidin databases and dietary supplement data as well as antioxidant capacities of 43 antioxidants were also utilized. RESULT: Serum CRP and plasma tHcy concentrations were higher in older adults, smokers, and those with lower non-leisure time physical activity levels (P < 0.05). Energy-adjusted daily total antioxidant capacity (TAC) from diet and supplements was positively associated with serum vitamin E and carotenoid concentrations (P < 0.05). Adjusted odds ratio (OR) for plasma tHcy >13 µmol/L significantly decreased across quartiles of TAC from diet and supplements (Q1 = 2.18 (1.56-2.77); Q2 = 1.30 (1.00-2.07); Q3 = 1.34 (0.84-2.28); Q4 = 1.00; P for linear trend <0.001). A negative trend across quartiles of TAC from diet and supplements was also observed in OR for serum CRP ≥3 mg/L (Q1 = 1.26 (0.97-1.70); Q2 = 1.21 (0.91-1.66); Q3 = 0.97 (0.80-1.24); Q4 = 1.00; P for linear trend <0.05). CONCLUSIONS: These findings indicated that dietary TAC provided an integrated conceptual tool in assessing serum antioxidants and investigating the associations between antioxidant intake and CVD risk. The implicated applicability of dietary TAC needs further validation in prospective cohort studies.

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.

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.

Glucuronidation and methylation of procyanidin dimers b2 and 3,3″-di-o-galloyl-b2 and corresponding monomers epicatechin and 3-o-galloyl-epicatechin in mouse liver.

PURPOSE: The 3,3″-di-O-galloyl ester of procyanidin B2 (B2G2) is a component of grape seed extract that inhibits growth of human prostate carcinoma cell lines. In preparation for studies in mice, its hepatic metabolism was examined in vitro and compared to B2 and the corresponding monomers, epicatechin (EC) and 3-O-galloyl-epicatechin (ECG). METHODS: Compounds were incubated with liver microsomes or cytosol containing cofactors for glucuronidation, sulfation or methylation, and products analyzed by liquid chromatography-mass spectrometry (LC-MS). B2G2 was administered orally to mice and plasma analyzed by LC-MS for unmodified procyanidin and metabolites. RESULTS: Glucuronides and methyl ethers of B2 and B2G2 were formed in small amounts. In contrast, EC and ECG were largely or completely converted to glucuronides, sulfates and methyl ethers under the same incubation conditions. B2G2 given orally to mice was partially absorbed intact; no significant metabolites were detected in plasma. CONCLUSIONS: Glucuronidation and methylation of procyanidins B2 and B2G2 occurred but were minor processes in vitro. B2G2 was partially absorbed intact in mice after oral dosing and did not undergo significant metabolism. Unlike the flavanol monomers EC and ECG, therefore, B2G2 bioavailability should not be limited by metabolism. These results paved the way for ongoing pharmacokinetic and efficacy studies.

Enzymatic synthesis of substituted epicatechins for bioactivity studies in neurological disorders.

Glucuronidated and/or methylated metabolites of the proanthocyanidin (PA) monomer (-)-epicatechin are detected in both blood and brain following feeding of rodents with a monomeric grape seed PA extract shown to reduce symptoms in a mouse model of Alzheimer's disease. To generate metabolites for future mechanistic studies, we investigated the ability of recombinant human glucuronosyl transferases of the UGT1A and UGT2B families to glucuronidate epicatechin or 3'-O-methyl epicatechin in vitro. Of twelve enzymes tested, UGT1A9 was the most efficient, producing epicatechin 3'-O-glucuronide as the major product. Incubation of UGT1A9 with 3'-O-methyl-epicatechin resulted in two major products, one of which was identified as 3'-O-methyl-epicatechin 5-O-glucuronide, a major metabolite found in blood plasma and brain tissue of the rodents following feeding with a grape seed extract. We also investigated in vitro methylation of epicatechin and epicatechin glucuronides by human catechol O-methyltransferase. Enzymatic production of 3'-O-methyl-epicatechin 5-O-glucuronide was optimized to 50% overall yield. These studies form a basis for generation of mg quantities of pure epicatechin (methyl) glucuronides of biological significance, and provide clarification of structure of previously identified epicatechin metabolites.

Influence of formulation and processing on absorption and metabolism of flavan-3-ols from tea and cocoa.

Flavan-3-ols are a major subclass of the class of plant phytochemicals known as flavonoids. Flavan-3-ols are commonly found in fruit, vegetable, and botanical products, including tea, cocoa, grapes, and apples. Both monomeric catechins and polymeric procyanidins are common in the diet, along with several derivatives produced by degradation of these species during processing. Both epidemiological and biological evidence suggests a health-protective role for dietary flavan-3-ols, leading to increased interest in the bioavailability of these compounds from foods. Flavan-3-ol bioavailability depends on numerous factors, including digestive release, absorption, metabolism, and elimination. In addition to these in vivo factors, the complexity of whole-food systems (physical form, flavan-3-ol form and dose, macronutrient and micronutrient profile, processing, etc.) influences the absorption efficiency and circulating profile of flavan-3-ols. An understanding of how food matrices may influence flavan-3-ol absorption will provide a framework to design and develop functional products that positively affect flavan-3-ol absorption and, by extension, potential bioactivity.

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.

Effect of whole-crop pea (Pisum sativum L.) silages differing in condensed tannin content as a substitute for grass silage and soybean meal on the performance, metabolism, and carcass characteristics of lambs.

Two experiments were conducted to investigate the effect of inclusion of whole-crop pea (WCP) silages, differing in condensed tannin content, as a substitute for grass silage (GS) and soybean meal on lamb metabolism, performance, plasma metabolites, digestibility, and carcass characteristics. In both experiments lambs were offered either solely GS or a 50:50 mix on a DM basis of GS with either low-tannin (LTPS) or high-tannin (HTPS) pea silage ad libitum. Each forage mix was fed with either 400 g/d of low-protein (LP) concentrate or 400 g/d of LP with an additional 200 g/d of pelletized soybean meal (HP), resulting in 6 dietary treatments. Experiment 1 examined the effects of the diets on metabolism, digestibility, and N balance using 6 lambs in 4 periods of 21 d in an incomplete crossover design. Experiment 2 used 48 lambs and examined the effects of the diets on ADG, plasma metabolites, and carcass characteristics over 56 d. Both experiments were analyzed using a 3 × 2 factorial arrangement of treatments. In Exp. 1, lambs offered the LTPS diets had a greater (P < 0.05) digestibility of DM and OM than those offered the GS diets. Lambs offered the WCP silages had an increased (P < 0.05) N intake, N output, and digestibility of GE compared with those offered GS. Mean N digestibility was greatest (P < 0.05) in lambs offered LTPS. Lambs offered HP diets had increased (P < 0.001) digestibility of DM, OM, GE and N, and N- intake, output, retention, and digestibility compared with those offered the LP diets. In Exp. 2, there was no effect (P > 0.05) of forage type on intake, slaughter BW, or feed conversion efficiency (FCE). However, lambs offered the LTPS had a greater (P < 0.05) ADG than those offered the GS diets. Feeding diets containing HP increased (P < 0.001) total DMI, slaughter BW, ADG, and FCE. Lambs offered the WCP had a greater (P < 0.05) plasma ß-hydroxybutyrate and urea concentration compared with those offered the GS diets. Feeding lambs HP diets increased (P < 0.05) plasma urea and total protein. Forage mix had no effect (P > 0.05) on carcass composition except for fat depth, which was greater (P < 0.05) in lambs offered WCP silage. Diets containing the HP increased (P < 0.05) carcass weight, hind leg circumference, chop dimensions, and kidney weight. It was concluded that lambs offered LTPS performed better than those offered GS and that LTPS has a concentrate sparing effect. Additionally, the increased tannin concentration in HTPS did not increase performance over lambs offered either GS or LTPS.

Rapid analysis of procyanidins and anthocyanins in plasma by microelution SPE and ultra-HPLC.

In the analysis of biological samples, such as plasma or serum, the quantity of sample available is a critical parameter in most cases. A good approach is the use of the microelution SPE (µSPE) plates as sample pre-treatment technique in which the loaded sample volume is low. An off-line µSPE and ultra-performance LC-ESI-MS/MS (UPLC-ESI-MS/MS) method was developed and validated to determine procyanidins and anthocyanins in spiked plasma samples. The sample pre-treatment µSPE allowed the simultaneous determination of procyanidins and anthocyanins from plasma by using a small sample volume (350 µL) and without an evaporation step previous to the chromatographic analysis. Moreover, the use of UPLC technique allowed to determine the studied compounds at low concentration levels in a short analysis time (12.5 min approximately). Then, the developed method was applied to determine the studied compounds, procyanidins and anthocyanins, and their metabolites in rat plasma samples. Previously, the rats had consumed 5000 mg/kg of a grape pomace extract and the plasma was extracted 4 h after administration. The procyanidins catechin and epicatechin glucuronide, methyl catechin and epicatechin glucuronide, and methyl catechin and epicatechin sulphate were detected at µM concentration level, and the parent anthocyanins at nM.

Proanthocyanidin metabolites associated with dietary fibre from in vitro colonic fermentation and proanthocyanidin metabolites in human plasma.

Proanthocyanidins (PAs) or condensed tannins, a major group of dietary polyphenols, are oligomers and polymers of flavan-3-ol and flavan-3, 4-diols widely distributed in plant foods. Most literature data on PAs' metabolic fate deal with PAs that can be extracted from the food matrix by aqueous-organic solvents ( extractable proanthocyanidins). However, there are no data on colonic fermentation of non-extractable proanthocyanidins (NEPAs), which arrive almost intact to the colon, mostly associated to dietary fibre (DF). The aim of the present work was to examine colonic fermentation of NEPAs associated with DF, using a model of in vitro small intestine digestion and colonic fermentation. Two NEPA-rich materials obtained from carob pod (Ceratonia siliqua L. proanthocyanidin) and red grapes (grape antioxidant dietary fibre) were used as test samples. The colonic fermentation of these two products released hydroxyphenylacetic acid, hydroxyphenylvaleric acid and two isomers of hydroxyphenylpropionic acid, detected by HPLC-ESI-MS/MS. Differences between the two products indicate that DF may enhance the yield of metabolites. In addition, the main NEPA metabolite in human plasma was 3,4-dihydroxyphenyl acetic acid. The presence in human plasma of the same metabolites as were detected after in vitro colonic fermentation of NEPAs suggests that dietary NEPAs would undergo colonic fermentation releasing absorbable metabolites with potential healthy effects.

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.

Determination of procyanidins and their metabolites in plasma samples by improved liquid chromatography-tandem mass spectrometry.

An off-line solid-phase extraction (SPE) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining procyanidins, catechin, epicatechin, dimer, and trimer in plasma samples. In the validation procedure of the analytical method, linearity, precision, accuracy, detection limits (LODs), quantification limits (LOQs), and the matrix effect were studied. Recoveries of the procyanidins were higher than 84%, except for the trimer, which was 65%. The LODs and LOQs were lower than 0.003 and 0.01 microM, respectively, for all the procyanidins studied, except for the trimers, which were 0.8 and 0.98 microM, respectively. This methodology was then applied for the analysis of rat plasma obtained 2h after ingestion of grape seed phenolic extract. Monomers (catechin and epicatechin), dimer and trimer in their native form were detected and quantified in plasma samples, and their concentration ranged from 0.85 to 8.55 microM. Moreover, several metabolites, such as catechin and epicatechin glucuronide, catechin and epicatechin methyl glucuronide, and catechin and epicatechin methyl-sulphate were identified. These conjugated forms were quantified, in reference to the respective unconjugated form, showing concentrations between 0.06 and 23.90 microM.

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.