Dose-Dependent Increases in Ellagitannin Metabolites as Biomarkers of Intake in Humans Consuming Standardized Black Raspberry Food Products Designed for Clinical Trials.

SCOPE: Black raspberry (BRB) phytochemicals demonstrate anti-carcinogenic properties in experimental models, including prostate cancer. Two BRB foods, a confection and nectar, providing a consistent and reproducible product for human clinical studies are designed and characterized. METHODS AND RESULTS: Men with clinically localized prostate cancer are sequentially enrolled to a control group or one of four intervention groups (confection or nectar, 10 or 20 g dose; n = 8 per group) for 4 weeks prior to prostatectomy. Primary outcomes include: safety, adherence, and ellagitannin metabolism. Adherence to the intervention is >96%. No significant (≥grade II) toxicities are detected. Urinary urolithins (A, B, C, and D) and dimethyl ellagic acid (DMEA) quantified by Ultra high performance liquid chromatography tandem mass spectroscopy (UPLC/MS/MS) indicate a dose-dependent excretion yet heterogeneous patterns among men. Men in the BRB confection groups have greater urinary excretion of the microbial urinary metabolites urolithin A and DMEA, suggesting that this food matrix provides greater colonic microflora exposure. CONCLUSION: Fully characterized BRB confections and nectar are ideal for food-based large phase III human clinical studies. BRB products provide a bioavailable source of BRB phytochemicals, however large inter individual variation in polyphenol metabolism suggests that host genetics, microflora, and other factors are critical to understanding bioactivity and metabolism.

Identification and characterization of in vitro inhibitors against UDP-glucuronosyltransferase 1A1 in uva-ursi extracts and evaluation of in vivo uva-ursi-drug interactions.

Uva-ursi leaf is widely used to treat symptoms of lower urinary tract infections. Here, we evaluated the in vitro inhibitory effects of uva-ursi extracts on 10 major human UDP-glucuronosyltransferases (UGT) isoforms. Of the 10 tested UGT isoforms, uva-ursi extracts exerted the strongest inhibitory effect on UGT1A1-mediated ß-estradiol 3-glucuronidation with the lowest IC50 value of 8.45 ±â€¯1.56 µg/mL. To identify the components of uva-ursi extracts showing strong inhibitory effects against UGT1A1, the inhibitory effects of nine major constituents of the extracts were assessed. Among the tested compounds, gallotannin exerted the most potent inhibition on UGT1A1, followed by 1,2,3,6-tetragalloylglucose; both demonstrated competitive inhibition, with Ki values of 1.68 ±â€¯0.150 µM and 3.55 ±â€¯0.418 µM. We found that gallotannin and 1,2,3,6-tetragalloylglucose also inhibited another UGT1A1-specific biotransformation, SN-38-glucuronidation, showing the same order of inhibition. Thus, in vitro UGT1A1 inhibitory potentials of uva-ursi extracts might primarily result from the inhibitory activities of gallotannin and 1,2,3,6-tetragalloylglucose present in the extracts. However, in rats, co-administration with uva-ursi extracts did not alter the in vivo marker for UGT1A1 activity, expressed as the molar ratio of AUCSN-38 glucuronide/AUCSN-38, because plasma concentrations of gallotannin and 1,2,3,6-tetragalloylglucose may be too low to inhibit the UGT1A1-mediated metabolism of SN-38 in vivo. The poor oral absorption of gallotannin and 1,2,3,6-tetragalloylglucose in uva-ursi extracts might cause the poor in vitro-in vivo correlation. These findings will be helpful for the safe and effective use of uva-ursi extracts in clinical practice.

Development and validation of a liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of urolithin C in rat plasma and its application to a pharmacokinetic study.

Urolithins are microflora human metabolites of dietary ellagic acid derivatives. There is now a growing interest in the biological activities of these compounds. Several studies suggest that urolithins have potential antioxidant, anti-inflammatory, anticancer and anti-glycative activities. Recently, our group investigated the role of urolithins as potential anti-diabetic treatments; among the four urolithins, urolithin C was the most promising compound. The purpose of this paper was to develop a rapid, sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urolithin C in rat plasma. To date, no method is reported for the quantification of urolithin C in any of the matrices. Plasma samples were extracted with ethyl acetate. Urolithin D was selected as the internal standard. The separation was carried out on a C18 Kinetex EVO column (2.1mm×150mm, 2.6µm) using a mobile phase of acetonitrile-1% aqueous formic acid solution (30:70, v/v). A triple quadrupole mass spectrometer in the negative ion mode was used for the determination of the target analyte. The monitored ion transitions were m/z 243→187 for urolithin C and m/z 259→213 for the internal standard. The calibration curve range was 4.95-1085µg/L (r2>0.994). The intra- and inter-day precisions were less than 10%; accuracies ranged from 96.6 to 109%. The mean extraction recovery of urolithins C and D was greater than 91%. No significant matrix effects and no carryover effects were observed. Small changes in LC-ESI-MS/MS conditions did not have significant effect on the determination of urolithin C. Stability tests under various conditions were also investigated. This highly specific and sensitive method was used to analyze samples collected during preclinical pharmacokinetic studies in rats. Glucuronyl and sulfate conjugates of urolithin C were the main metabolites detected in plasma.

Development and validation of an UPLC-PDA method for the determination of corilagin in rat plasma and its application to pharmacokinetic study.

Corilagin, which was isolated from several medical herbs, has been reported to exert many pharmacological activities. A simple and rapid liquid ultra-performance liquid chromatography (UPLC) coupled to photodiode array (PDA) method has been developed to quantify corilagin in rat plasma. In this study, plasma samples were prepared by ethyl acetate extraction. Separation was performed on a HSS T3 (100mm×2.1mm, 1.8µm) column by using a mobile phase of acetonitrile and water with 0.1% trifluoroacetic acid (v/v). Corilagin and internal standard epicatechin were detected at a wavelength of 266nm. The calibration curve was linear (r>0.998) over a concentration range of 0.2µg/mL to 20µg/mL with a lower quantification limit of 0.2µg/mL. Both intra and inter-day precision values were within 5.7% and extraction recovery were greater than 81.0%. Stability tests showed that corilagin and IS remained stable during the analytical procedure. The validated UPLC-PDA method was then used to analyze the pharmacokinetics of corilagin administered to rats intravenously (10mg/kg) or orally (50mg/kg). Oral bioavailability of corilagin was calculated to be 10.7%, indicating that this component is not suitable for oral administration. The results provide basis for further preclinical studies on corilagin.

Serum pharmacochemistry for tracking bioactive components by UPLC-Q-TOF-MS/MS combined chromatographic fingerprint for quality assessment of Sanziguben Granule.

To more reasonably and effectively control the quality of Sanziguben Granule, chromatographic fingerprinting and serum pharmacochemistry of this traditional Chinese medicine compound were performed. A comprehensive comparison and evaluation of 15 batches of Sanziguben Granule was successfully conducted by using high performance liquid chromatography (HPLC) fingerprint analysis. After administering a set amount of Sanziguben Granule orally to rats, blood samples were collected and tested 4 times at intervals of 30min, 1h, 2h, and 4h using UPLC-Q-TOF-MS/MS. The blood showed presence of gallic acid and corilagin indicating the pharmacological significance of these two chemical compounds. According to the result, above mentional chemical compounds were designated biomarkers for quality control of Sanziguben Granule. Therefore, a purposeful and efficient method for quality control of Sanziguben Granule was established in the present study.

Urinary metabolites from mango (Mangifera indica L. cv. Keitt) galloyl derivatives and in vitro hydrolysis of gallotannins in physiological conditions.

SCOPE: The absorption, metabolism, and excretion of mango galloyl derivatives (GD) has not yet been investigated in humans, and studies investigating repeated dosages of polyphenols are limited. METHODS AND RESULTS: In this human pilot trial, healthy volunteers (age = 21-38 y, n = 11) consumed 400 g/day of mango-pulp (cv. Keitt) for 10 days, and seven metabolites of gallic acid (GA) were characterized and quantified in urine excreted over a 12 h period. Pyrogallol-O-sulfate and deoxypyrogallol-O-sulfate were found to be significantly more excreted between days 1 and 10 (p < 0.05) from 28.5 to 55.4 mg and 23.6 to 47.7 mg, respectively. Additionally, the in vitro hydrolysis of gallotannins (GTs) was monitored at physiological pH and temperature conditions, and after 4 h a significant (p < 0.05) shift in composition from relativity high to low molecular weight GTs was observed. CONCLUSION: Seven metabolites of GA were identified in the urine of healthy volunteers, and two microbial metabolites were found to be significantly more excreted following 10 days of mango consumption. Mango GTs were also found to release free GA in conditions similar to the intestines. GTs may serve as a pool of pro-GA compounds that can be absorbed or undergo microbial metabolism.

Determination of corilagin in rat plasma using a liquid chromatography-electrospray ionization tandem mass spectrometric method.

A sensitive and simple liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method for the determination of corilagin in rat plasma has been developed. Samples were prepared with protein precipitation method and analyzed with a triple quadrupole tandem mass spectrometer. We employed negative electrospray ionization as the ionization source and the analytes were detected in multiple reaction monitoring mode. Separation was achieved on a C8 column eluted with mobile phase consisting of methanol-0.1% formic acid in a gradient mode at the flow rate of 0.3 mL/min. The total run time was 7.0 min.This method was proved to have good linearity in the concentration range of 2.5-1000.0 ng/mL. The lower limit of quantification of corilagin was 2.5 ng/mL. The intra- and inter-day relative standard deviationa across three validation runs for four concentration levels were both <9.8%. The relative error was within ±6.0%. This assay offers advantages in terms of expediency and suitability for the analysis of corilagin in rat plasma. The practical utility of this new HPLC-MS/MS method was confirmed in pilot plasma concentration studies in rats following oral administration.

A validated liquid chromatography-tandem mass spectrometry method for the determination of methyl gallate and pentagalloyl glucopyranose: application to pharmacokinetic studies.

Methyl gallate (MG) and pentagalloyl glucopyranose (PGG) are bioactive phenolic compounds that are widely distributed in herbs and plant foods. Their potential activities include anti-oxidant, anti-inflammatory, anti-cancer, anti-bacterial and anti-viral activities. However, knowledge concerning the pharmacokinetic characteristics of MG and PGG is limited. The purpose of this study was to develop a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to simultaneously quantify MG and PGG in rat blood samples. The linear response ranges for MG and PGG were 0.0195-20 and 0.0390-20 µM, respectively. The lower limit of quantification was 0.0195 µM for MG and 0.0390 µM for PGG. The intra- and inter-day variances were less than 15%, and accuracy was within 80-120%. This assay was successfully applied to pharmacokinetic studies in Sprague-Dawley rats after intraperitoneal administration of MG and PGG (20 mg/kg). The values of areas under the blood concentration time curves (AUC0₋24 h) for MG and PGG were 109.9 ± 73.40 and 38.78 ± 24.53 h*µM, respectively. The maximum blood concentrations (Cmax) of MG and PGG were 34.72 ± 17.32 and 6.39 ± 4.25 µM, respectively. The time required to reach the maximum concentration (Tmax) was 0.85 ± 0.70 h for both MG and PGG. The values of the elimination rate constant (Ke), elimination half-life (t1/2), volume of distribution (Vd), clearance (Cl) and mean resident time (MRTlast) were 0.056 ± 0.032 h(-1), 17.50 ± 12.25 h, 530.95 ± 247.54 L/kg, 159.91±76.05L/h/kg, 8.71 ± 2.53 h for MG and 0.023 ± 0.012 h(-1), 38.66 ± 22.89 h, 7838.89 ± 3474.72 L/kg, 30.98 ± 21.73 L/h/kg, 12.47 ± 2.77 h for PGG, respectively. In conclusion, a UPLC-MS/MS method was fully validated over a wide linear range and used to quantify the levels of MG and PGG in pharmacokinetic studies of MG and PGG in rats. The main advantages of this method are the use of small blood volumes (10 µL), rapid analysis (5 min) and excellent recoveries.

Pilot walnut intervention study of urolithin bioavailability in human volunteers.

A pilot intervention study was conducted in human volunteers (n = 4) to establish the bioavailability of urolithins, which are the terminal end-products of ellagitannin metabolism by the gastrointestinal microflora. Biospecimens (blood, feces, and urine) along with urolithins purified therefrom were analyzed for their antioxidant capacity in a range of in vitro assays. Urolithin metabolites were identified and quantitated in the biospecimens by negative ion mode HPLC-ESI-MS analysis. The data in this pilot study show that the metabolism of ellagitannins in the four volunteers gave rise to a diverse profile and a highly variable concentration of urolithins in urine. The concentration of glucuronidated urolithins in blood and urine did not correlate with antioxidant capacity. However, the antioxidant capacity of urine, but not plasma biospecimens, was highly correlated with uric acid concentration. The antioxidant capacity of fecal extracts correlated positively with the concentration of urolithin D in both the DPPH and FRAP assays, but not in the ORAC assay, which was entirely consistent with the in vitro assays for pure urolithin D.

Targeted metabolic profiling of pomegranate polyphenols and urolithins in plasma, urine and colon tissues from colorectal cancer patients.

SCOPE: Urolithins are bioactive metabolites produced by the gut microbiota from ellagitannins (ETs) and ellagic acid (EA). We investigated whether urolithins could be detected in colon tissues from colorectal cancer (CRC) patients after pomegranate extract (PE) intake. METHODS AND RESULTS: CRC patients (n = 52) were divided into controls and PEs consumers (900 mg/day for 15 days) before surgical resection. PEs with low (PE-1) and high (PE-2) punicalagin:EA ratio were administered. Twenty-three metabolites, but no ellagitannins, were detected in urine, plasma, normal (NT) or malignant (MT) colon tissues using UPLC-ESI-QTOF-MS/MS (UPLC, ultra performance liquid chromatography; QTOF, quadrupole TOF). Free EA, five EA conjugates, gallic acid and 12 urolithin derivatives were found in colon tissues. Individual and total metabolites levels were higher in NT than in MT, independently of the PE consumed. The maximal mean concentration (1671 ± 367 ng/g) was found in NT after consumption of PE-1 and the lowest concentration (42.4 ± 10.2 ng/g) in MT with PE-2. Urolithin A or isourolithin A were the main urolithins produced (54 and 46% patients with urolithin A or isourolithin A phenotype, respectively). High punicalagin content (PE-2) hampered urolithins formation. CONCLUSION: Significant levels of EA derivatives and urolithins are found in human colon tissues from CRC patients after consumption of pomegranate. Further studies are warranted to elucidate their biological activity.

A fast, sensitive, and high-throughput method for the simultaneous quantitation of three ellagitannins from Euphorbiae pekinensis Radix in rat plasma by ultra-HPLC-MS/MS.

A fast, sensitive, and high-throughput ultra-HPLC-MS/MS method has been developed and validated for the simultaneous determination of three main active constituents of Euphorbiae pekinensis Radix in rat plasma. After addition of the internal standard, plasma samples were extracted by liquid-liquid extraction with ethyl acetate/isopropanol (1:1, v/v) and separated on a CAPCELL PAK C18 column (100 × 2.0 mm, 2 µm, Shiseido, Japan), using a gradient mobile phase system of methanol/water. The detection of the analytes was performed on a 4000Q UHPLC-MS/MS system with turbo ion spray source in the negative ion and multiple reaction-monitoring mode. The linear range was 1.0-1000 ng/mL for 3,3'-di-O-methyl ellagic acid-4'-O-ß-D-glucopyranoside (i), 1.5-1500 ng/mL for 3,3'-di-O-methyl ellagic acid-4'-O-ß-D-xylopyranoside (ii), and 5.0-5000 ng/mL for 3,3'-di-O-methyl ellagic acid (iii). The intra- and interday precision and accuracy of all the analytes were within 15%. The extraction recoveries of the three analytes and internal standard from plasma were all more than 80%. The validated method was first successfully applied to the evaluation of pharmacokinetic parameters of compounds 1, 2, and 3 in rat plasma after intragastric administration of the Euphorbiae pekinensis Radix extract.

Preparation of penta-O-galloyl-ß-D-glucose from tannic acid and plasma pharmacokinetic analyses by liquid-liquid extraction and reverse-phase HPLC.

The gallotannin penta-O-galloyl-beta-D-glucose (PGG) has many biological activities including in vivo anti-cancer efficacy. We present in this paper a scaled-up protocol for its preparation in high purity from tannic acid by acidic methanolysis with typical yield of 15%. We also describe a method for the analysis of PGG in mouse plasma by HPLC and its application in preliminary pharmacokinetic studies. A liquid-liquid extraction (LLE) protocol was optimized for the extraction of PGG from mouse plasma. The extraction efficiency for PGG at 1 µg/mL in mouse plasma was 70.0±1.3% (n=5). The limit of detection (LOD) for PGG was approximately 0.2 µg/mL. Preliminary pharmacokinetic parameters of PGG following a single i.p. injection with 5% ethanol/saline vehicle in mice were established. The peak plasma PGG concentrations (C(max)) were approximately 3-4 µM at a dose of 0.5 mg per mouse (∼20 mg/kg) at 2 h post-injection (T(max)).

Bioavailability of anthocyanins and ellagitannins following consumption of raspberries by healthy humans and subjects with an ileostomy.

The fate of anthocyanins, ellagic acid, and ellagitannins was studied following the consumption of 300 g of raspberries by healthy human volunteers and subjects with an ileostomy. Postingestion plasma and urine from the former and ileal fluid and urine from the latter group were collected and analyzed by HPLC-PDA-MS(2). Plasma from the healthy volunteers did not contain detectable quantities of either the native raspberry polyphenolics or their metabolites. The three main raspberry anthocyanins were excreted in urine in both healthy and ileostomy volunteers 0-7 h after ingestion, in quantities corresponding to <0.1% of intake. This indicates a low level of absorption in the small intestine. With ileostomy volunteers 40% of anthocyanins and 23% of the ellagitannin sanguiin H-6 were recovered in ileal fluid with the main excretion period being the first 4 h after raspberry consumption. The recovery of ellagic acid in ileal fluid was 241%, indicating hydrolysis of ellagitannins in the stomach and/or the small intestine. Urinary excretion of ellagic acid and an ellagic acid-O-glucuronide was <1% of intake. No intact or conjugated forms of ellagitannins were detected in urine from either healthy subjects or ileostomy volunteers. However, in healthy subjects, but not the ileostomists, ellagitannins were catabolized with the appearance of urolithin A-O-glucuronide, two of its isomers, and urolithin B-O-glucuronide in urine collected 7-48 h after raspberry consumption. There was marked variation in the urolithin profile of individual volunteers, indicating differences in the colonic microflora responsible for ellagitannin degradation.

Pomegranate juice and extracts provide similar levels of plasma and urinary ellagitannin metabolites in human subjects.

Pomegranate juice (PJ), a rich source of polyphenols including ellagitannins, has attracted much attention due to its reported health benefits. This has resulted in the consumption of liquid and powder pomegranate extracts as alternatives to PJ. Therefore establishing the bioavailability of polyphenols from these extract preparations is necessary. Sixteen healthy volunteers sequentially consumed, with a 1-week washout period between treatments, PJ (8 ounces, Wonderful fruit variety), a pomegranate polyphenol liquid extract (POMxl, 8 ounces), and a pomegranate polyphenol powder extract (POMxp, 1,000 mg). The three interventions provided 857, 776, and 755 mg of polyphenols as gallic acid equivalents, respectively. Plasma bioavailability, judged based on ellagic acid levels over a 6-hour period, did not show statistical differences in area under the curve for the three interventions: 0.14 +/- 0.05, 0.11 +/- 0.03, and 0.11 +/- 0.04 micromol . hour/L for PJ, POMxl, and POMxp, respectively. The time of maximum concentration was delayed for POMxp (2.58 +/- 0.42 hours) compared to PJ (0.65 +/- 0.23 hours) and POMxl (0.94 +/- 0.06 hours). Urolithin-A glucuronide, a urinary metabolite of ellagic acid, was not significantly different with the three interventions, reaching levels of approximately 1,000 ng/mL. This study demonstrates that ellagitannin metabolites, delivered from pomegranate fruits, as PJ, POMxl, and POMxp, reach equivalent levels with a delay in time of maximum concentration of POMxp compared to PJ and POMxl.

Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum l.) polyphenols after ingestion of a standardized extract in healthy human volunteers.

The intake of polyphenols has been demonstrated to have health-promoting and disease-preventive effects. The pomegranate (Punica granatum L.), which is rich in several polyphenols, has been used for centuries in ancient cultures for its medicinal purposes. The potential health benefits of pomegranate polyphenols have been demonstrated in numerous in vitro studies and in vivo experiments. This study investigated the absorption and antioxidant effects of a standardized extract from pomegranate in healthy human volunteers after the acute consumption of 800 mg of extract. Results indicate that ellagic acid (EA) from the extract is bioavailable, with an observed C(max) of 33 ng/mL at t(max) of 1 h. The plasma metabolites urolithin A, urolithin B, hydroxyl-urolithin A, urolithin A-glucuronide, and dimethyl ellagic acid-glucuronide were identified by HPLC-MS. The antioxidant capacity measured with the oxygen radical absorbance capacity (ORAC) assay was increased with a maximum effect of 32% after 0.5 h, whereas the generation of reactive oxygen species (ROS) was not affected. The inflammation marker interleukin-6 (IL-6) was not significantly affected after 4 h after the consumption of the extract. Overall, this study demonstrated the absorbability of EA from a pomegranate extract high in ellagitannin content and its ex vivo antioxidant effects.

Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours.

Ellagitannins (ETs) from pomegranate juice (PJ) are reported to have numerous biological properties, but their absorption and metabolism in humans are poorly understood. To investigate the pharmacokinetics of pomegranate ETs, 18 healthy volunteers were given 180 mL of PJ concentrate, and blood samples were obtained for 6 h afterwards. Twenty-four-hour urine collections were obtained on the day before (-1), the day of (0), and the day after (+1) the study. Ellagic acid (EA) was detected in plasma of all subjects with a maximum concentration of 0.06 +/- 0.01 micromol/L, area under concentration time curve of 0.17 +/- 0.02 (micromol x h) x L(-1), time of maximum concentration of 0.98 +/- 0.06 h, and elimination half-life of 0.71 +/- 0.08 h. EA metabolites, including dimethylellagic acid glucuronide (DMEAG) and hydroxy-6H-benzopyran-6-one derivatives (urolithins), were also detected in plasma and urine in conjugated and free forms. DMEAG was found in the urine obtained from 15 of 18 subjects on d 0, but was not detected on d -1 or +1, demonstrating its potential as a biomarker of intake. Urolithin A-glucuronide was found in urine samples from 11 subjects on d 0 and in the urine from 16 subjects on d +1. Urolithin B-glucuronide was found in the urine of 3 subjects on d 0 and in the urine of 5 subjects on d +1. Urolithins, formed by intestinal bacteria, may contribute to the biological effects of PJ as they may persist in plasma and tissues and account for some of the health benefits noted after chronic PJ consumption. Whether genetic polymorphisms in EA-metabolizing enzymes (e.g., catechol-O-methyl transferase and glucuronosyl transferase) are related to variations in response to PJ remains to be established.

The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxy-6H-dibenzopyran-6-one derivatives by the colonic microflora of healthy humans.

BACKGROUND: The antiatherogenic activity of pomegranate juice has been attributed to its antioxidant polyphenols. The most potent in vitro antioxidant polyphenol from this juice is the ellagitannin punicalagin. However, the bioavailability of ellagitannins, including punicalagin, has not been previously described in humans. AIM OF THE STUDY: The present work aims to evaluate, in healthy humans, the bioavailability and metabolism of pomegranate juice ellagitannins, to assess their effect on several blood parameters (including cardiovascular risk disease markers) and to compare the antioxidant activity of punicalagin with that of the in vivo generated metabolites. DESIGN: Six healthy subjects (four men and two women) consumed 1 L of pomegranate juice daily (5.58 g/L polyphenols, including 4.37 g/L punicalagin isomers) for 5 days. The polyphenols and the in vivo generated metabolites were measured by HPLC-DAD-MS-MS. Fourteen haematological and twenty serobiochemical parameters including LDL, HDL and VLDL as well as cholesterol and triglycerides in each lipoprotein were evaluated. In vitro antioxidant activity of plasma (ABTS and FRAP assays) and urine (ABTS and DPPH) were determined. RESULTS: Neither punicalagin nor ellagic acid present in the juice were detected in both plasma and urine. Three microbial ellagitannin-derived metabolites were detected: 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide, an unidentified aglycone (tentatively, trihydroxy-6H-dibenzo[b,d]pyran-6-one) and hydroxy-6-H-dibenzo[b,d]pyran-6-one glucuronide. These metabolites could reach up to 18.6 microM in plasma, although a large inter-individual variability was observed. In urine, the same metabolites and their corresponding aglycones became evident after 1 day of juice consumption. Total urine excretion of metabolites ranged from 0.7 to 52.7% regarding the ingested punicalagin. No relevant effect was observed on any blood parameter. The metabolites did not show significant antioxidant activity compared to punicalagin from pomegranate juice. CONCLUSIONS: The potential systemic biological effects of pomegranate juice ingestion should be attributed to the colonic microflora metabolites rather than to the polyphenols present in the juice.

Determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography.

A method for the identification and determination of tannic acid and its phenolic metabolites in biological fluids by high-performance liquid chromatography was developed. Tannic acid and four phenolic compounds, namely gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid, were successfully extracted from the biological fluids by using ethyl acetate at acidic conditions. Gallic acid, pyrogallol and 4-O-methylgallic acid were found in the sheep urine, gallic acid, 4-O-methylgallic acid and ellagic acid in plasma, and gallic acid and ellagic acid in abomasal fluid after abomasal dosing of tannic acid. Tannic acid was found in the plasma apart from the abomasal fluid into which it was administered. The concentrations of tannic acid, gallic acid, pyrogallol, 4-O-methylgallic acid and ellagic acid in plasma, abomasal fluid and urine were measured. This method could be applied to measurement of other hydrolysable tannins and their phenolic metabolites in biological materials.