Identification of Novel Urolithin Metabolites in Human Feces and Urine after the Intake of a Pomegranate Extract.

Urolithins are bioactive gut microbiota metabolites of ellagic acid. Here, we have identified four unknown urolithins in human feces after the intake of a pomegranate extract. The new metabolites occurred only in 19% of the subjects. 4,8,9,10-Tetrahydroxy urolithin, (urolithin M6R), was unambiguously identified by 1H NMR, UV, and HRMS. Three metabolites were tentatively identified by the UV, HRMS, and chromatographic behavior, as 4,8,10-trihydroxy (urolithin M7R), 4,8,9-trihydroxy (urolithin CR), and 4,8-dihydroxy (urolithin AR) urolithins. Phase II conjugates of the novel urolithins were detected in urine and confirmed their absorption, circulation, and urinary excretion. The production of the new urolithins was not specific of any of the known urolithin metabotypes A and B. The new metabolites needed a bacterial 3-dehydroxylase activity for their production, and this is a novel feature as all the previously known urolithins maintained the hydroxyl at 3 position. The ability of production of these "R" urolithins can be considered an additional metabolic feature for volunteer stratification.

An altered tissue distribution of flaxseed lignans and their metabolites in Abcg2 knockout mice.

Lignans are dietary polyphenols, which are metabolized by gut microbiota into the phytoestrogenic metabolites enterolignans, mainly enterolactone and enterodiol. Breast Cancer Resistance Protein (BCRP/ABCG2) is an efflux transporter that affects the plasma and milk secretion of several drugs and natural compounds. We hypothesized here that Abcg2 could influence the levels of lignans and their derived metabolites in target tissues. Consequently, we aimed to evaluate the role of Abcg2 in the tissue distribution of these compounds. We used Abcg2-/- knockout and wild-type male mice fed with a lignan-enriched diet for one week and analysed their plasma, small intestine, colon, liver, kidneys and testicles. High levels of lignans as well as enterolignans and their glucuronide and sulfate conjugates in the small intestine and colon were detected, with higher concentrations of the conjugates in the wild-type compared with Abcg2-/- mice. Particularly relevant was the detection of 24-fold and 8-fold higher concentrations of enterolactone-sulfate and enterolactone-glucuronide, respectively, in the kidney of Abcg2-/- compared with wild-type mice. In conclusion, our study showed that lignans and their derived metabolites were in vivo substrates of Abcg2, which affected their plasma and tissue levels. These results highlight the role of Abcg2 in influencing the health-beneficial properties of dietary lignans.

The gut microbiota metabolism of pomegranate or walnut ellagitannins yields two urolithin-metabotypes that correlate with cardiometabolic risk biomarkers: Comparison between normoweight, overweight-obesity and metabolic syndrome.

BACKGROUND & AIMS: Urolithins are microbial metabolites produced after consumption of ellagitannin-containing foods such as pomegranates and walnuts. Parallel to isoflavone-metabolizing phenotypes, ellagitannin-metabolizing phenotypes (urolithin metabotypes A, B and 0; UM-A, UM-B and UM-0, respectively) can vary among individuals depending on their body mass index (BMI), but correlations between urolithin metabotypes (UMs) and cardiometabolic risk (CMR) factors are unexplored. We investigated the association between UMs and CMR factors in individuals with different BMI and health status. METHODS: UM was identified using UPLC-ESI-qToF-MS in individuals consuming pomegranate or nuts. The associations between basal CMR factors and the urine urolithin metabolomic signature were explored in 20 healthy normoweight individuals consuming walnuts (30 g/d), 49 healthy overweight-obese individuals ingesting pomegranate extract (450 mg/d) and 25 metabolic syndrome (MetS) patients consuming nuts (15 g-walnuts, 7.5 g-hazelnuts and 7.5 g-almonds/d). RESULTS: Correlations between CMR factors and urolithins were found in overweight-obese individuals. Urolithin-A (mostly present in UM-A) was positively correlated with apolipoprotein A-I (P ≤ 0.05) and intermediate-HDL-cholesterol (P ≤ 0.05) while urolithin-B and isourolithin-A (characteristic from UM-B) were positively correlated with total-cholesterol, LDL-cholesterol (P ≤ 0.001), apolipoprotein B (P ≤ 0.01), VLDL-cholesterol, IDL-cholesterol, oxidized-LDL and apolipoprotein B:apolipoprotein A-I ratio (P ≤ 0.05). In MetS patients, urolithin-A only correlated inversely with glucose (P ≤ 0.05). Statin-treated MetS patients with UM-A showed a lipid profile similar to that of healthy normoweight individuals while a poor response to lipid-lowering therapy was observed in MB patients. CONCLUSIONS: UMs are potential CMR biomarkers. Overweight-obese individuals with UM-B are at increased risk of cardiometabolic disease, whereas urolithin-A production could protect against CMR factors. Further research is warranted to explore these associations in larger cohorts and whether the effect of lipid-lowering drugs or ellagitannin-consumption on CMR biomarkers depends on individuals' UM. CLINICAL TRIAL REGISTRY NUMBERS AND WEBSITES: NCT01916239 (https://clinicaltrials.gov/ct2/show/NCT01916239) and ISRCTN36468613 (http://www.isrctn.com/ISRCTN36468613).

Gastrointestinal Simulation Model TWIN-SHIME Shows Differences between Human Urolithin-Metabotypes in Gut Microbiota Composition, Pomegranate Polyphenol Metabolism, and Transport along the Intestinal Tract.

A TWIN-SHIME system was used to compare the metabolism of pomegranate polyphenols by the gut microbiota from two individuals with different urolithin metabotypes. Gut microbiota, ellagitannin metabolism, short-chain fatty acids (SCFA), transport of metabolites, and phase II metabolism using Caco-2 cells were explored. The simulation reproduced the in vivo metabolic profiles for each metabotype. The study shows for the first time that microbial composition, metabolism of ellagitannins, and SCFA differ between metabotypes and along the large intestine. The assay also showed that pomegranate phenolics preserved intestinal cell integrity. Pomegranate polyphenols enhanced urolithin and propionate production, as well as Akkermansia and Gordonibacter prevalence with the highest effect in the descending colon. The system provides an insight into the mechanisms of pomegranate polyphenol gut microbiota metabolism and absorption through intestinal cells. The results obtained by the combined SHIME/Caco-2 cell system are consistent with previous human and animal studies and show that although urolithin metabolites are present along the gastrointestinal tract due to enterohepatic circulation, they are predominantly produced in the distal colon region.

A rosemary extract enriched in carnosic acid improves circulating adipocytokines and modulates key metabolic sensors in lean Zucker rats: Critical and contrasting differences in the obese genotype.

SCOPE: Carnosic acid (CA) and rosemary extracts (REs) have antiobesity effects but the mechanisms are not understood. We investigated some of the potential mechanisms contributing to the metabolic effects of an RE enriched in CA. METHODS AND RESULTS: An RE (∼40% CA) was administered to lean (Le, fa/+) and obese (Ob, fa/fa) female Zucker rats for 64 days. Several adipocytokines, brain-derived neurotrophic factor, phosphorylated AMP-activated protein kinase, and hepatic gene expression changes were investigated. The RE significantly decreased circulating tumor necrosis factor alpha (RE/CT = 0.36, p < 0.0003), IL-1ß (0.48, p < 0.032), and leptin (0.48, p < 0.002), and upregulated adiponectin (1.47, p < 0.045) in the Le rats. The RE also induced phase I and phase II gene expression and the peroxisome proliferator-activated receptor gamma coactivator 1-alpha. Notably, the RE decreased adipose phosphorylated AMP-activated protein kinase and did not affect hepatic peroxisome proliferator-activated receptor gamma coactivator 1-alpha in the Ob rats. CONCLUSION: Our results show that an RE rich in CA exerts anti-inflammatory effects and affects hepatic metabolism in normal Le rats. We report significant differences in the expression and regulation of key metabolic sensors between Le and Ob rats that may contribute to explain the different ability of the two genotypes to respond to the RE.

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.

Bioavailability of the major bioactive diterpenoids in a rosemary extract: metabolic profile in the intestine, liver, plasma, and brain of Zucker rats.

SCOPE: Carnosic acid (CA) and derived diterpenes abundant in rosemary extracts (REs) exert anti-obesity effects. The aim of this study was to investigate the bioavailability of these compounds in a rat model of obesity. METHODS AND RESULTS: A total of 26 compounds were tentatively identified based on accurate mass information and the isotopic pattern provided by TOF-MS analyzer. The main metabolites detected in the gut content, liver, and plasma were the glucuronide conjugates of CA, carnosol, and rosmanol. Two other metabolites were also identified: CA 12-methyl ether and 5,6,7,10-tetrahydro-7-hydroxyrosmariquinone. All the metabolites were detected as early as 25 min following oral administration. Most of the compounds remained in the intestine, liver, and (or) plasma at substantial concentrations for several hours supporting their potential health benefits in these tissues. We also corroborated the presence of small quantities of CA and detected trace quantities of the main CA metabolites in the brain. Notably, we did not find significant differences in the metabolic profile between lean and obese rats. CONCLUSION: We report for the first time a comprehensive profile of metabolites in various organs following the oral consumption of an RE enriched in CA and contribute to establish the potential bioactive molecules.

Intestinal ellagitannin metabolites ameliorate cytokine-induced inflammation and associated molecular markers in human colon fibroblasts.

Pomegranate ellagitannins (ETs) are transformed in the gut to ellagic acid (EA) and its microbiota metabolites, urolithin A (Uro-A) and urolithin B (Uro-B). These compounds exert anti-inflammatory effects in vitro and in vivo. The aim of this study was to investigate the effects of Uro-A, Uro-B, and EA on colon fibroblasts, cells that play a key role in intestinal inflammation. CCD18-Co colon fibroblasts were exposed to a mixture of Uro-A, Uro-B, and EA, at concentrations comparable to those found in the colon (40 µM Uro-A, 5 µM Uro-B, 1 µM EA), both in the presence or in the absence of IL-1ß (1 ng/mL) or TNF-α (50 ng/mL), and the effects on fibroblast migration and monocyte adhesion were determined. The levels of several growth factors and adhesion cytokines were also measured. The mixture of metabolites significantly inhibited colon fibroblast migration (∼70%) and monocyte adhesion to fibroblasts (∼50%). These effects were concomitant with a significant down-regulation of the levels of PGE(2), PAI-1, and IL-8, as well as other key regulators of cell migration and adhesion. Of the three metabolites tested, Uro-A exhibited the most significant anti-inflammatory effects. The results show that a combination of the ET metabolites found in colon, urolithins and EA, at concentrations achievable in the intestine after the consumption of pomegranate, was able to moderately improve the inflammatory response of colon fibroblasts and suggest that consumption of ET-containing foods has potential beneficial effects on gut inflammatory diseases.

Induction of antioxidant flavonol biosynthesis in fresh-cut potatoes. Effect of domestic cooking.

The effect of fresh-cutting and subsequent cold storage on phenolic compounds from five long-term-stored potato cultivars (Agria, Cara, Liseta, Monalisa, and Spunta) was studied. Fresh-cutting induced the biosynthesis of three flavonols, which were identified by HPLC-DAD-ESIMS as quercetin 3-rutinoside, quercetin 3-diglucoside, and quercetin 3-glucosylrutinoside. The flavonols were detected after a lag period of 3 days of cold storage. The content ranged from 6 to 14 mg/100 g of fresh weight depending on the cultivar after 6 days of storage. Chlorogenic acid as the main caffeic acid derivative and the amino acids tyrosine and tryptophan were also quantified. The effect of cold storage under light or in dark was studied with new-season-harvested Monalisa potatoes. The flavonol induction was higher in fresh-cut potatoes stored under light than in the dark. However, caffeic acid derivatives were not affected. Domestic cooking such as boiling, microwaving, and frying provoked a partial loss of the flavonols, which were retained in the range of 4-16 mg per serving (213 g). Steam-cooking resulted in the highest retention of caffeic acid derivatives and aromatic amino acids compared with the other cooking methods studied. This means that due to the large amount of potatoes consumed in the Western diet, fresh-cut potatoes can be a significant source of health-promoting phenolics.