Urolithins: Diet-Derived Bioavailable Metabolites to Tackle Diabetes.

Diabetes remains one of the leading causes of deaths and co-morbidities in the world, with tremendous human, social and economic costs. Therefore, despite therapeutics and technological advancements, improved strategies to tackle diabetes management are still needed. One of the suggested strategies is the consumption of (poly)phenols. Positive outcomes of dietary (poly)phenols have been pointed out towards different features in diabetes. This is the case of ellagitannins, which are present in numerous foodstuffs such as pomegranate, berries, and nuts. Ellagitannins have been reported to have a multitude of effects on metabolic diseases. However, these compounds have high molecular weight and do not reach circulation at effective concentrations, being metabolized in smaller compounds. After being metabolized into ellagic acid in the small intestine, the colonic microbiota hydrolyzes and metabolizes ellagic acid into dibenzopyran-6-one derivatives, known as urolithins. These low molecular weight compounds reach circulation in considerable concentrations ranging until micromolar levels, capable of reaching target tissues. Different urolithins are formed throughout the metabolization process, but urolithin A, isourolithin A, and urolithin B, and their phase-II metabolites are the most frequent ones. In recent years, urolithins have been the focus of attention in regard to their effects on a multiplicity of chronic diseases, including cancer and diabetes. In this review, we will discuss the latest advances about the protective effects of urolithins on diabetes.

Quantification of thonningianin a in rat plasma by liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study.

CONTEXT: Thonningianin A is an ellagitannin substance and displays multiple pharmacological activities. OBJECTIVE: This study investigated the pharmacokinetic characteristics of thonningianin A after oral administration in rats using a fully validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. MATERIALS AND METHODS: A sensitive and selective LC-MS/MS assay was developed for quantifying thonningianin A. Eighteen Wistar rats were randomly divided into three groups (n = 6), which were given at a single dose of 10, 20, or 40 mg/kg thonningianin A by gavage. Blood samples (200 µL) were collected from the orbit vein at designated time points and analyzed using the LC-MS/MS method to measure the levels of thonningianin A. RESULTS: Thonningianin A and internal standard (IS) were eluted at 1.5 and ∼3.0 min, respectively. The selected reaction mode transitions monitored were m/z 873.2 > 300.3 and 819.3 > 610.6 for thonningianin A and the IS, respectively. The calibration range was 10-1200 ng/mL. The intra- and the inter-day accuracy and precision met the acceptance criteria. No carryover and matrix effect were observed. The plasma concentrations of thonningianin A increased rapidly after oral administration of three dosages and reached the mean peak concentrations (Cmax) within 0.61-0.83 h. Meanwhile, AUC0-t/AUC0-∞ of the three dosage groups was more than 89.0% (10 mg/kg), 95.7% (20 mg/kg), and 97.0% (40 mg/kg). DISCUSSION AND CONCLUSIONS: The present method is the first report in terms of the simple precipitation procedure, high sensitivity, and high-throughput efficiency. This validated assay was successfully applied to determine the pharmacokinetic behaviours of thonningianin A in rats. This study should be helpful for providing references for understanding the action mechanism and further application of Penthorum chinense.

Effect of Hydrolyzable Tannins on Glucose-Transporter Expression and Their Bioavailability in Pig Small-Intestinal 3D Cell Model.

Intestinal transepithelial transport of glucose is mediated by glucose transporters, and affects postprandial blood-glucose levels. This study investigates the effect of wood extracts rich in hydrolyzable tannins (HTs) that originated from sweet chestnut (Castanea sativa Mill.) and oak (Quercus petraea) on the expression of glucose transporter genes and the uptake of glucose and HT constituents in a 3D porcine-small-intestine epithelial-cell model. The viability of epithelial cells CLAB and PSI exposed to different HTs was determined using alamarBlue®. qPCR was used to analyze the gene expression of SGLT1, GLUT2, GLUT4, and POLR2A. Glucose uptake was confirmed by assay, and LC-MS/ MS was used for the analysis of HT bioavailability. HTs at 37 µg/mL were found to adversely affect cell viability and downregulate POLR2A expression. HT from wood extract Tanex at concentrations of 4 µg/mL upregulated the expression of GLUT2, as well as glucose uptake at 1 µg/mL. The time-dependent passage of gallic acid through enterocytes was influenced by all wood extracts compared to gallic acid itself as a control. These results suggest that HTs could modulate glucose uptake and gallic acid passage in the 3D cell model.

The anti-diabetic effect of eight Lagerstroemia speciosa leaf extracts based on the contents of ellagitannins and ellagic acid derivatives.

Previously, we have reported the opposite effects of compounds isolated from Lagerstroemia speciosa leaves on a glucose transport (GLUT4) assay. Ellagitannins from L. speciosa activated GLUT4, while ellagic acid derivatives showed an inhibitory effect. As part of our continuing research on anti-diabetic nutritional supplements, we herein compared the anti-diabetic effects of several extracts (LE1-8) from leaves of L. speciosa using different manufacturing processes based on the contents of ellagitannins and ellagic acid derivatives. Their anti-diabetic effects were evaluated through glucose uptake and adipocyte differentiation in 3T3-L1 cells in vitro as well as alloxan induced diabetic mice in vivo. These extracts were given to mice by gavage at doses of 0.25, 1.0, and 4.0 g per kg body weight once a day for 21 consecutive days. Results showed that LE1 (1.0 g kg-1), LE3 (1.0 or 4.0 g kg-1), LE4 (1.0 or 4.0 g kg-1), LE5 (0.25 or 1.0 or 4.0 g kg-1) and LE7 (1.0 or 4.0 g kg-1) showed significant anti-diabetic effects in alloxan-induced diabetic mice as indicated by the decreased levels of fasting blood glucose, body weight, serum biomarkers, tissue weight and body fat, and increased final insulin levels. LE8 (1.0 g kg-1) showed a moderate anti-diabetic effect as illustrated by the reduced fasting blood glucose level while LE2 and LE6 showed slight effects in alloxan-induced diabetic mice. The potential correlation of the content of ellagitannins, ellagic acid derivatives, and corosolic acid with the anti-diabetic activity was discussed.

A liquid chromatography-tandem mass spectrometry method for preclinical pharmacokinetics and tissue distribution of hydrolyzable tannins chebulinic acid and chebulagic acid in rats.

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of chebulinic acid and chebulagic acid in rat plasma and tissues and well used in the pharmacokinetic and tissue distribution studies after intraperitoneal injection administration. Samples were processed with methanol by protein precipitation, and chromatographic separation was performed on an Agilent Zorbax SB-C18 column (50 × 2.1 mm, 1.8 µm) with a mobile phase consisting of methanol and water containing 0.1% formic acid (60:40, v/v). Quantification was performed by selected reaction monitoring with m/z 977.1 → 806.8 for chebulagic acid, m/z 979.0 → 808.7 for chebulinic acid and m/z 851.2 → 704.9 for the internal standard. Good linearity was observed over their respective concentration range. The pharmacokinetic study showed that both compounds reached their peak concentration values (605.8 ± 35.6 ng/mL for chebulinic acid and 1327.1 ± 118.6 ng/mL for chebulagic acid) at the same time of 0.9 h following intraperitoneal injection administration. The two compounds could be detected in blood-abundant tissues. The kidney had the highest concentrations (462.6 ± 138.5 ng/g for chebulinic acid and 1651.7 ± 167.7 ng/g for chebulagic acid) at 1 h post-dose, followed by the heart, liver, spleen and lung.

Punicalagin Exerts Beneficial Functions in 6-Hydroxydopamine-Treated SH-SY5Y Cells by Attenuating Mitochondrial Dysfunction and Inflammatory Responses.

BACKGROUND Parkinson's disease (PD) is a common age-related neurodegenerative disorder, but effective therapeutic agents for PD remain largely limited. MATERIAL AND METHODS In the present study, we evaluated the beneficial effects and underlying mechanisms of punicalagin (PN) in human neuroblastoma SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) to mimic PD in vitro. Cell viability was monitored by MTT assay and LDH release assay. Cell apoptosis was assayed by Annexin V-FITC/PI double-staining. Intracellular ROS production was assessed by DCFH-DA staining. The expression levels of protein and mRNA were determined by Western blotting and qRT-PCR analysis, respectively. RESULTS The results showed that pretreatment of SH-SY5Y cells with PN (50, 100, and 200 µM) prior to exposure to 200 µM 6-OHDA for 2 h resulted in increased cell viability and decreased cell apoptosis. PN also inhibited excessive oxidative stress in 6-OHDA-treated SH-SY5Y cells. Moreover, PN treatment effectively restored mitochondrial function and enhanced phosphorylation of AMPK. Furthermore, PN blocked 6-OHDA-induced NF-κB activation and IL-1ß expression. CONCLUSIONS Our study shows that PN exhibited neuroprotective effects on the 6-OHDA-treated SH-SY5Y cells, thus providing a potential theoretical insight for the clinical application of PN against PD.

Ellagitannins, Gallotannins and their Metabolites- The Contribution to the Anti-Inflammatory Effect of Food Products and Medicinal Plants.

The popularity of food products and medicinal plant materials containing hydrolysable tannins (HT) is nowadays rapidly increasing. Among various health effects attributable to the products of plant origin rich in gallotannins and/or ellagitannins the most often underlined is the beneficial influence on diseases possessing inflammatory background. Results of clinical, interventional and animal in vivo studies clearly indicate the antiinflammatory potential of HT-containing products, as well as pure ellagitannins and gallotannins. In recent years a great emphasis has been put on the consideration of metabolism and bioavailability of natural products during examination of their biological effects. Conducted in vivo and in vitro studies of polyphenols metabolism put a new light on this issue and indicate the gut microbiota to play a crucial role in the health effects following their oral administration. The aim of the review is to summarize the knowledge about HT-containing products' phytochemistry and their anti-inflammatory effects together with discussion of the data about observed biological activities with regards to the current concepts on the HTs' bioavailability and metabolism. Orally administered HT-containing products due to the limited bioavailability of ellagitannins and gallotannins can influence immune response at the level of gastrointestinal tract as well as express modulating effects on the gut microbiota composition. However, due to the chemical changes being a result of their transit through gastrointestinal tract, comprising of hydrolysis and gut microbiota metabolism, the activity of produced metabolites has to be taken into consideration. Studies regarding biological effects of the HTs' metabolites, in particular urolithins, indicate their strong and structure-dependent anti-inflammatory activities, being observed at the concentrations, which fit the range of their established bioavailability. The impact of HTs on inflammatory processes has been well established on various in vivo and in vitro models, while influence of microbiota metabolites on silencing the immune response gives a new perspective on understanding anti-inflammatory effects attributed to HT containing products, especially their postulated effectiveness in inflammatory bowel diseases (IBD) and cardiovascular diseases.

Pentagalloylglucose, a highly bioavailable polyphenolic compound present in Cortex moutan, efficiently blocks hepatitis C virus entry.

Approximately 142 million people worldwide are infected with hepatitis C virus (HCV). Although potent direct acting antivirals are available, high costs limit access to treatment. Chronic hepatitis C virus infection remains a major cause of orthotopic liver transplantation. Moreover, re-infection of the graft occurs regularly. Antivirals derived from natural sources might be an alternative and cost-effective option to complement therapy regimens for global control of hepatitis C virus infection. We tested the antiviral properties of a mixture of different Chinese herbs/roots named Zhi Bai Di Huang Wan (ZBDHW) and its individual components on HCV. One of the ZBDHW components, Penta-O-Galloyl-Glucose (PGG), was further analyzed for its mode of action in vitro, its antiviral activity in primary human hepatocytes as well as for its bioavailability and hepatotoxicity in mice. ZBDHW, its component Cortex Moutan and the compound PGG efficiently block entry of HCV of all major genotypes and also of the related flavivirus Zika virus. PGG does not disrupt HCV virion integrity and acts primarily during virus attachment. PGG shows an additive effect when combined with the well characterized HCV inhibitor Daclatasvir. Analysis of bioavailability in mice revealed plasma levels above tissue culture IC50 after a single intraperitoneal injection. In conclusion, PGG is a pangenotypic HCV entry inhibitor with high bioavailability. The low cost and wide availability of this compound make it a promising candidate for HCV combination therapies, and also emerging human pathogenic flaviviruses like ZIKV.

Proanthocyanidins and hydrolysable tannins: occurrence, dietary intake and pharmacological effects.

Tannins are a heterogeneous group of high MW, water-soluble, polyphenolic compounds, naturally present in cereals, leguminous seeds and, predominantly, in many fruits and vegetables, where they provide protection against a wide range of biotic and abiotic stressors. Tannins exert several pharmacological effects, including antioxidant and free radical scavenging activity as well as antimicrobial, anti-cancer, anti-nutritional and cardio-protective properties. They also seem to exert beneficial effects on metabolic disorders and prevent the onset of several oxidative stress-related diseases. Although the bioavailability and pharmacokinetic data for these phytochemicals are still sparse, gut absorption of these compounds seems to be inversely correlated with the degree of polymerization. Further studies are mandatory to better clarify how these molecules and their metabolites are able to cross the intestinal barrier in order to exert their biological properties. This review summarizes the current literature on tannins, focusing on the main, recently proposed mechanisms of action that underlie their pharmacological and disease-prevention properties, as well as their bioavailability, safety and toxicology. LINKED ARTICLES: This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.

Neuroprotective Effects of Ellagitannins: A Brief Review.

BACKGROUND: Neurodegenerative diseases are increasingly inevitable as the population gets older, with enormous socio-economic costs. The World Health Organization (WHO) has reported that by 2040, neurodegenerative diseases will overtake cancer to become the second leading cause of death, after cardiovascular disease. OBJECTIVE: Herein, this review outlines the neuroprotective effects and clinical implications of ellagitannins, a class of hydrolysable tannins, for the management and treatment of neurodegenerative disorders. RESULTS: Recent investigations suggest that the combination of genes and environmental toxins may contribute to the risk of developing neurodegenerative disorders. Thus, intervention strategies aimed at reducing exposure to risk factors such as life style, smoking, diet, vitamin deficiencies, chemical exposure and pollution are warranted. Modulation of dietary components including ellagitannins, as a therapeutic strategy to slow down or attenuate the progression of neuronal degeneration has become a focus of interest in the last few decades. CONCLUSION: Polyphenolic plant phytochemicals and ellagitannins in particular, contain an array of neuroprotective properties useful to improve human health and protect against neurodegeneration.

Application of a low polyphenol or low ellagitannin dietary intervention and its impact on ellagitannin metabolism in men.

SCOPE: Plant polyphenols are widespread in the American diet, yet estimated intake is uncertain. We examine the application of the Polyphenol Explorer® (PED) database to quantify polyphenol and ellagitannin (ET) intake of men with prostate cancer and tested the implementation of diets restricted in polyphenols or ETs. METHODS AND RESULTS: Twenty-four men enrolled in a 4-week trial were randomized to usual, low-polyphenol or low-ET diet. Estimated polyphenol and ET intakes were calculated from 3-day diet records utilizing the PED. Urine and plasma metabolites were quantified by UPLC-MS. Adherence to the restricted diets was 95% for the low polyphenol and 98% for low-ET diet. In the usual diet, estimated dietary polyphenol intake was 1568 ± 939 mg/day, with coffee/tea beverages (1112 ± 1028 mg/day) being the largest contributors and estimated dietary ET intake was 12 ± 13 mg/day. The low-polyphenol and low-ET groups resulted in a reduction of total polyphenols by 45% and 85%, respectively, and omission of dietary ETs. UPLC analysis of urinary host and microbial metabolites reflect ET intake. CONCLUSION: PED is a useful database for assessing exposure to polyphenols. Diets restricted in total polyphenol or ET intake are feasible and UPLC assessment of ET metabolites is reflective of dietary intake.

Urolithins, the rescue of "old" metabolites to understand a "new" concept: Metabotypes as a nexus among phenolic metabolism, microbiota dysbiosis, and host health status.

Urolithins are dibenzo[b,d]pyran-6-one derivatives that are produced by the human gut microbiota from ellagitannins and ellagic acid (EA). These metabolites are much better absorbed than their precursors and have been suggested to be responsible for the health effects attributed to ellagitannins and EA that occur in food products as berries and nuts. In the present review, the role and potential of urolithins in human health are critically reviewed, and a perspective of the research approach needed to demonstrate these health effects is presented, based on the existing knowledge. The analytical methods available for urolithin analysis, their occurrence in different tissues and biological fluids, and their metabolism by human gut microbiota are considered. In addition, the interindividual variability observed for the production of urolithins (metabotypes) and its relationship with health status and dysbiosis are also reviewed. The potential mechanisms of action of urolithins are also critically discussed, paying attention to the concentration and the type of metabolites used in the in vitro and in vivo assays and the physiological significance of the results obtained. The gut microbiota metabolism of EA to urolithins and that of daidzein to equol, their individual variations, and the effects on health are also compared.

Anti-inflammatory activity of Punica granatum L. (Pomegranate) rind extracts applied topically to ex vivo skin.

Coadministered pomegranate rind extract (PRE) and zinc (II) produces a potent virucidal activity against Herpes simplex virus (HSV); however, HSV infections are also associated with localised inflammation and pain. Here, the objective was to determine the anti-inflammatory activity and relative depth penetration of PRE, total pomegranate tannins (TPT) and zinc (II) in skin, ex vivo. PRE, TPT and ZnSO4 were dosed onto freshly excised ex vivo porcine skin mounted in Franz diffusion cells and analysed for COX-2, as a marker for modulation of the arachidonic acid inflammation pathway, by Western blotting and immunohistochemistry. Tape stripping was carried out to construct relative depth profiles. Topical application of PRE to ex vivo skin downregulated expression of COX-2, which was significant after just 6h, and maintained for up to 24h. This was achieved with intact stratum corneum, proving that punicalagin penetrated skin, further supported by the depth profiling data. When PRE and ZnSO4 were applied together, statistically equal downregulation of COX-2 was observed when compared to the application of PRE alone; no effect followed the application of ZnSO4 alone. TPT downregulated COX-2 less than PRE, indicating that tannins alone may not be entirely responsible for the anti-inflammatory activity of PRE. Punicalagin was found throughout the skin, in particular the lower regions, indicating appendageal delivery as a significant route to the viable epidermis. Topical application of TPT and PRE had significant anti-inflammatory effects in ex vivo skin, confirming that PRE penetrates the skin and modulates COX-2 regulation in the viable epidermis. Pomegranates have potential as a novel approach in ameliorating the inflammation and pain associated with a range of skin conditions, including cold sores and herpetic stromal keratitis.

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.

In Vivo Metabolite Profiling of a Purified Ellagitannin Isolated from Polygonum capitatum in Rats.

Ellagitannin is a common compound in food and herbs, but there are few detailed studies on the metabolism of purified ellagitannins. FR429 is a purified ellagitannin with antitumor potential, which is from Polygonum capitatum Buch.-Ham.ex D. Don. The present study was designed to investigate the metabolic profiles of FR429 in rats in vivo. Using liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MS(n)-IT-TOF), total eight metabolites were found in rat bile and urine after intravenous administration of FR429, but could not be detected in plasma. These metabolites were ellagic acid, mono-methylated FR429, ellagic acid methyl ether glucuronide, ellagic acid methyl ether diglucuronide, ellagic acid dimethyl ether glucuronide, and ellagic acid dimethyl ether diglucuronide. It was concluded that methylation and subsequent glucuronidation were the major metabolic pathways of FR429 in rats in vivo. This is the first report on the in vivo metabolism of the purified ellagitannin in rats.

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.

Ellagitannins in Cancer Chemoprevention and Therapy.

It is universally accepted that diets rich in fruit and vegetables lead to reduction in the risk of common forms of cancer and are useful in cancer prevention. Indeed edible vegetables and fruits contain a wide variety of phytochemicals with proven antioxidant, anti-carcinogenic, and chemopreventive activity; moreover, some of these phytochemicals also display direct antiproliferative activity towards tumor cells, with the additional advantage of high tolerability and low toxicity. The most important dietary phytochemicals are isothiocyanates, ellagitannins (ET), polyphenols, indoles, flavonoids, retinoids, tocopherols. Among this very wide panel of compounds, ET represent an important class of phytochemicals which are being increasingly investigated for their chemopreventive and anticancer activities. This article reviews the chemistry, the dietary sources, the pharmacokinetics, the evidence on chemopreventive efficacy and the anticancer activity of ET with regard to the most sensitive tumors, as well as the mechanisms underlying their clinically-valuable properties.

Scope of Hydrolysable Tannins as Possible Antimicrobial Agent.

Hydrolysable tannins (HTs) are secondary metabolites from plants, which are roughly classified into gallotannins and ellagitannins having gallic acid and ellagic acid residues respectively attached to the hydroxyl group of glucose by ester linkage. The presence of hexahydroxydiphenoyl and nonahydroxyterphenoyl moieties is considered to render antimicrobial property to HTs. HTs also show considerable synergy with antibiotics. Nevertheless, they have low pharmacokinetic property. The present review presents the scope of HTs as future antimicrobial agent. Copyright © 2016 John Wiley & Sons, Ltd.

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.

In Vitro Bioaccessibility, Human Gut Microbiota Metabolites and Hepatoprotective Potential of Chebulic Ellagitannins: A Case of Padma Hepaten® Formulation.

Chebulic ellagitannins (ChET) are plant-derived polyphenols containing chebulic acid subunits, possessing a wide spectrum of biological activities that might contribute to health benefits in humans. The herbal formulation Padma Hepaten containing ChETs as the main phenolics, is used as a hepatoprotective remedy. In the present study, an in vitro dynamic model simulating gastrointestinal digestion, including dialysability, was applied to estimate the bioaccessibility of the main phenolics of Padma Hepaten. Results indicated that phenolic release was mainly achieved during the gastric phase (recovery 59.38%-97.04%), with a slight further release during intestinal digestion. Dialysis experiments showed that dialysable phenolics were 64.11% and 22.93%-26.05% of their native concentrations, respectively, for gallic acid/simple gallate esters and ellagitanins/ellagic acid, in contrast to 20.67% and 28.37%-55.35% for the same groups in the non-dialyzed part of the intestinal media. Investigation of human gut microbiota metabolites of Padma Hepaten and pure ChETs (chebulinic, chebulagic acids) established the formation of bioactive urolithins (A, B, C, D, M5). The fact of urolithin formation during microbial transformation from ChETs and ChET-containing plant material was revealed for the first time. Evaluation of the protective effect of ChETs colonic metabolites and urolithins on tert-butyl hydroperoxide (t-BHP)-induced oxidative injury in cultured rat primary hepatocytes demonstrated their significant reversion of the t-BHP-induced cell cytotoxicity, malonic dialdehyde production and lactate dehydrogenase leakage. The most potent compound was urolithin C with close values of hepatoprotection to gallic acid. The data obtained indicate that in the case of Padma Hepaten, we speculate that urolithins have the potential to play a role in the hepatic prevention against oxidative damage.