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.

Tea Is a Significant Dietary Source of Ellagitannins and Ellagic Acid.

The ellagitannin composition and the total content of ellagitannins in different types of tea were studied by high-performance liquid chromatography/ion-trap mass spectrometry. Strictinin and seven other isomers, tellimagrandin I, and ellagic acid were identified from tea infusions. The ellagitannin content in tea infusions was determined after acid hydrolysis and ranged from 0.15 to 4.46 mg of ellagic acid equivalent/g of tea in the infusions. The intake of ellagic acid after drinking a cup of tea brewed with 4 g of tea could range between 0.59 and 17.89 mg. These results indicate that tea can be a significant contributor to the dietary intake of ellagitannins. Urolithins, the gut microbiota metabolites produced in vivo from ellagic acid and ellagitannins, were detected in human urine after dietary tea beverage intake. Urolithin metabotypes A, B, and 0 were identified in volunteers after tea intake. These results suggest that the daily intake of ellagitannins from tea can have a role in tea health effects.

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.

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.

Effects of ellagitannin-rich berries on blood lipids, gut microbiota, and urolithin production in human subjects with symptoms of metabolic syndrome.

Ellagitannins are polyphenols abundant in strawberries, raspberries, and cloudberries. The effects of a mixture of these berries were studied in a randomized controlled trial with subjects having symptoms of metabolic syndrome. The study focused on serum lipid profiles, gut microbiota, and ellagitannin metabolites. The results indicate that bioavailability of ellagitannins appears to be dependent on the composition of gut microbiota.

Method development for ß-glucogallin and gallic acid analysis: application to urinary pharmacokinetic studies.

A reversed phase high-pressure liquid chromatographic electrospray tandem mass spectrometry method was developed for the simultaneous detection and analysis of ß-glucogallin and gallic acid. This method used a C(18) column with ultraviolet detection at 285 nm, ionization in the negative ion mode for ß-glucogallin and gallic acid, and in the positive ion mode for the internal standard 3-morpholin-4-ylpropane-1-sulfonic acid. Mobile phase consisted of a mixture of water, methanol, and formic acid at a flow rate of 0.7 mL/min. This method was validated over a concentration range of 1-100 µg/mL in rat urine.

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.

Identification of urinary and intestinal bacterial metabolites of ellagitannin geraniin in rats.

Hydrolyzable tannins, including ellagitannins, occur in foods such as berries and nuts. Various biological activities, including antioxidant, antiviral, and antitumor activities, have been noted and reported for ellagitannins, but the absorption and metabolism of purified ellagitannins are poorly understood. We describe herein the characterization of urinary and intestinal microbial metabolites in rats after the ingestion of ellagitannins. Urine samples were collected after oral administration of ellagitannins such as geraniin ( 1), corilagin ( 2), and their related polyphenols. The suspension of rat intestinal microflora was anaerobically incubated with ellagitannins. Each sample was separated by column chromatography and/or preparative HPLC to give seven metabolites, M1- M7. The structures of these metabolites were determined on the basis of spectroscopic data and chemical evidence. These compounds, except for M1, were characterized as ellagitannin metabolites for the first time. Furthermore, among four major metabolites ( M1- M4) in urine, M2 showed an antioxidant activity comparable to intact geraniin and related polyphenols.

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.

Metabolism of antioxidant and chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in humans: identification of biomarkers and individual variability.

Ellagitannins (ETs) are dietary polyphenols, containing ellagic acid (EA) subunits, with antioxidant and cancer chemopreventive activities that might contribute to health benefits in humans. However, little is known about their metabolic fate. We investigate here the metabolism of different dietary ETs and EA derivatives in humans. Forty healthy volunteers were distributed in four groups. Each group consumed, in a single dose, a different ET-containing foodstuff, i.e., strawberries (250 g), red raspberries (225 g), walnuts (35 g), and oak-aged red wine (300 mL). After the intake, five urine fractions (F) were collected at 8 (F1), 16 (F2), 32 (F3), 40 (F4), and 56 (F5) h. Neither ETs nor EA were detected in urine after LC-MS/MS analysis. However, the microbial metabolite 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one (urolithin B) conjugated with glucuronic acid was detected along the fractions F3-F5 in all of the subjects, independently of the consumed foodstuff. The mean percentage of metabolite excretion ranged from 2.8 (strawberries) to 16.6% (walnuts) regarding the ingested ETs. Considerable interindividual differences were noted, identifying "high and low metabolite excreters" in each group, which supported the involvement of the colonic microflora in ET metabolism. These results indicate that urolithin B (a previously described antiangiogenic and hyaluronidase inhibitor compound) is a biomarker of human exposure to dietary ETs and may be useful in intervention studies with ET-containing products. The antioxidant and anticarcinogenic effects of dietary ETs and EA should be considered in the gastrointestinal tract whereas the study of potential systemic activities should be focused on the bioavailable urolithin B derivatives.

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.