NMR Spectroscopic Identification of Urolithin G, a Novel Trihydroxy Urolithin Produced by Human Intestinal Enterocloster Species.

Urolithins are gut microbiota metabolites of ellagic acid. Here, we have identified and chemically characterized a novel urolithin produced from urolithin D (3,4,8,9-tetrahydroxy urolithin) by in vitro incubation with different human gut Enterocloster species under anaerobic conditions. Urolithin G (3,4,8-trihydroxy urolithin) was identified by 1H NMR, 13C NMR, UV, HRMS, and 2D NMR. For the identification, NMR spectra of other known urolithins were also recorded and compared. Urolithin G was present in the feces of 12% of volunteers in an overweight-obese group after consuming an ellagitannin-rich pomegranate extract. The production of urolithin G required a bacterial 9-dehydroxylase activity and was not specific to the known human urolithin metabotypes A and B. The ability to produce urolithin G could be considered an additional metabolic feature for volunteer stratification and bioactivity studies. This is the first urolithin with a catechol group in ring A while having only one hydroxyl in ring B, a unique feature not found in human and animal samples so far.

Gut Bacteria Involved in Ellagic Acid Metabolism To Yield Human Urolithin Metabotypes Revealed.

We aimed to elucidate the gut bacteria that characterize the human urolithin metabotypes A and B (UM-A and UM-B). We report here a new bacterium isolated from the feces of a healthy woman, capable of producing the final metabolites urolithins A and B and different intermediates. Besides, we describe two gut bacterial co-cultures that reproduced the urolithin formation pathways upon in vitro fermentation of both UM-A and UM-B. This is the first time that the capacity of pure strains to metabolize ellagic acid cooperatively to yield urolithin profiles associated with UM-A and UM-B has been demonstrated. The urolithin-producing bacteria described herein could have potential as novel probiotics and in the industrial manufacture of bioactive urolithins to develop new ingredients, beverages, nutraceuticals, pharmaceuticals, and (or) functional foods. This is especially relevant in UM-0 individuals since they cannot produce bioactive urolithins.

Separation of Isomeric Forms of Urolithin Glucuronides Using Supercritical Fluid Chromatography.

Urolithins are gut microbiota metabolites produced in humans after consuming foods containing ellagitannins and ellagic acid. Three urolithin metabotypes have been reported for different individuals depending on the final urolithins produced. After absorption, they are conjugated with glucuronic acid (phase II metabolism), and these are the main circulating metabolites in plasma and reach different tissues. Different regioisomeric isomers of urolithin glucuronides have been described. Still, their identification and quantification in humans have not been properly reported due to resolution limitations in their analysis by reversed-phase high-performance liquid chromatography. In the present study, we report a novel method for separating these isomers using supercritical fluid chromatography. With this method, urolithin A 3- and 8-glucuronide, isourolithin A 3- and 9- glucuronide, and urolithin B 3-glucuronide (8-hydroxy urolithin 3-glucuronide; 3-hydroxy urolithin 8-glucuronide; 3-hydroxyurolithin 9-glucuronide; 9-hydroxyurolithin 3-glucuronide; and urolithin 3-glucuronide) were separated in less than 15 min. The proposed method was applied to successfully analyze these metabolites in urine samples from different volunteers belonging to different metabotypes.

Effect of Storage Conditions on the Stability of Polyphenols of Apple and Strawberry Purees Produced at Industrial Scale by Different Processing Techniques.

During a food product's life, storage conditions affect its composition of nutrients, bioactive compounds, and sensory attributes. In this research, strawberry and apple purees were selected as a model to examine how the storage of various purees industrially produced with different technologies affect the bioactive phenolic compounds, color, and sensory attributes. Specifically, fruit products processed on an industrial scale by different technologies including freezing, thermal treatment (mild and standard), and high-pressure processing were studied, as well as storage for up to 12 months at -20, 4, and 24 °C. In strawberry puree, storage conditions had a stronger impact on phenolic compound levels, particularly on anthocyanins, whereas in apple puree, the initial processing techniques exerted a greater influence than storage conditions, mainly caused by the hot or cold crushing processes. In general, proanthocyanidins were the major phenolic group and the most stable during storage, while anthocyanins were the group most affected by both processing and storage. Apple flavonols and dihydrochalcones were quite stable, while strawberry ellagitannins suffered higher degradations during storage. Through our analysis, it is found that during storage, the stability of polyphenols in each fruit is different, and processing and storage can be either detrimental or even beneficial. The selection of the ideal storage conditions (time and temperature) is a key factor to maintaining the polyphenol content in sensitive fruits such as strawberries. However, storage conditions are in some cases more important to minimizing the polyphenol losses than how the product is processed.

Ellagitannins, urolithins, and neuroprotection: Human evidence and the possible link to the gut microbiota.

Ellagitannins (ETs) and ellagic acid (EA) are dietary polyphenols poorly absorbed but extensively metabolized by the human gut microbiota to produce different urolithins (Uros). Depending on the individuals' microbial signatures, ETs metabolism can yield the Uro metabotypes A, B, or 0, potentially impacting human health after consuming ETs. Human evidence points to improved brain health after consuming ET-rich foods, mainly pomegranate juices and extracts containing punicalagin, punicalin, and different EA-derivatives. Although ETs and (or) EA are necessary to exert the effects, the precise mechanism, actual metabolites, or final drivers responsible for the observed effects have not been unraveled. The cause-and-effect evidence on Uro-A administration and the improvement of animal brain health is consistent but not addressed in humans. The Uro-A's in vivo anti-inflammatory, mitophagy, autophagy, and mitochondrial biogenesis activities suggest it as a possible final driver in neuroprotection. However, the precise Uro metabolic forms reaching the brain are unknown. In addition to the possible participation of direct effectors in brain tissues, the current evidence points out that improving blood flow, gut microbiota ecology, and gut barrier by ET-rich foods and (or) Uro-A could contribute to the neuroprotective effects. We show here the current human evidence on ETs and brain health, the possible link between the gut microbiota metabolism of ETs and their effects, including the preservation of the gut barrier integrity, and the possible role of Uros. Finally, we propose a roadmap to address what is missing on ETs, Uros, and neuroprotection.

Stability of phenolic compounds in apple and strawberry: Effect of different processing techniques in industrial set up.

Different technologies commonly employed by the fruit processing industry affect the phenolic content. This study aimed to establish the extent to which different processing techniques and conditions, tested at an industrial scale, affect phenolics, color, and sensory attributes of fresh strawberries and apples. The effects of freezing, thermal treatments, and high-pressure processing were investigated. In strawberries, mild and standard thermal treatments showed similar patterns for most phenolic groups; an increase in proanthocyanidins, no change in ellagic acid conjugates, and a major decrease in flavonols and anthocyanins. In apples, mild treatments and high-pressure processing had similar effects in all phenolic groups, with increases in dihydrochalcones, hydroxycinnamics, and proanthocyanidins and decreases in flavonols. However, the standard thermal treatment increased flavonols and dihydrochalcones concentrations. This study shows that each fruit behaves differently, and both technology and processing conditions should be customized accordingly to preserve or even increase the phenolic content.

Untargeted Metabolomics Reveals New Markers of Food Processing for Strawberry and Apple Purees.

In general, food processing and its conditions affect nutrients, bioactive compounds, and sensory characteristics of food products. This research aims to use a non-targeted metabolomics approach based on UPLC-ESI-QTOF-MS to determine how fruit processing can affect the metabolic profile of fruits and, through a comprehensive metabolic analysis, identify possible markers to assess their degree of processing. The present study uses a real case from the food industry to evaluate markers of the processing of strawberry and apple purees industrially elaborated with different processing techniques and conditions. The results from the multivariate analysis revealed that samples were grouped according to the type of processing, evidencing changes in their metabolic profiles and an apparent temperature-dependent effect. These metabolic profiles showed changes according to the relevance of thermal conditions but also according to the exclusively cold treatment, in the case of strawberry puree, and the pressure treatment, in the case of apple puree. After data analysis, seven metabolites were identified and proposed as processing markers: pyroglutamic acid, pteroyl-D-glutamic acid, 2-hydroxy-5-methoxy benzoic acid, and 2-hydroxybenzoic acid ß-d-glucoside in strawberry and di-hydroxycinnamic acid glucuronide, caffeic acid and lysoPE(18:3(9Z,12Z,15Z)/0:0) in apple purees. The use of these markers may potentially help to objectively measure the degree of food processing and help to clarify the controversial narrative on ultra-processed foods.

A novel combined analytical UV and MS approach for the quantification of oleuropein metabolites in human biological samples when authentic standards are not available.

The beneficial health effects of phytochemicals depend on their bioavailability and the form under which they reach systemic circulation, usually as phase II metabolites. The lack of authentic standards for these metabolites makes their quantification in biological samples challenging. A new analytical approach to get a more accurate quantification of oleuropein metabolites in biological samples after ingestion of olive leaf extract was proposed. This approach was based on the calculation of a response factor in QTOF MS for each metabolite, comparing their quantification in UV and MS using urine samples concentrated in the metabolites of interest. Glucuronide and sulfate conjugates of hydroxytyrosol and homovanillyl alcohol were more accurately quantified in plasma and urine and for the first time, oleuropein aglycone conjugates and their hydroxylated and hydrogenated derivatives were quantified after consumption of olive products. This approach could be extensible to the analysis of other phenolic metabolites when authentic standards are not available, opening a valuable method for bioavailability studies.

Novel Regioselective Synthesis of Urolithin Glucuronides─Human Gut Microbiota Cometabolites of Ellagitannins and Ellagic Acid.

Urolithins (dibenzo-pyran-[b,d]-6 one derivatives) are human gut microbiota metabolites produced from the natural food antioxidant ellagic acid. Urolithins are better absorbed than ellagic acid and demonstrate biological activities that suggest that they are responsible for the health effects observed after consuming ellagitannin- and ellagic acid-containing foods. Urolithins occur in the systemic circulation as glucuronide conjugates following phase II metabolism. These phase II conjugates are essential for testing the urolithin mechanisms of action in human cell line bioassays. Urolithin glucuronides are not commercially available, and their biosynthesis leads to mixtures of regional isomers. This study describes a novel and regioselective synthesis of urolithin A (3,8-dihydroxy urolithin) 3- and 8-glucuronides and isourolithin A (3,9-dihydroxy urolithin) 3- and 9-glucuronides. The metabolites were characterized using 1H and 13C NMR spectroscopy and UV spectrophotometry. The presence of these metabolites in human subjects belonging to different urolithin metabotypes was also investigated.

Technological and Biotechnological Processes To Enhance the Bioavailability of Dietary (Poly)phenols in Humans.

The health effects of (poly)phenols (PPs) depend upon their bioavailability that, in general, is very low and shows a high interindividual variability. The low bioavailability of PPs is mainly attributed to their low absorption in the upper gastrointestinal tract as a result of their low water solubility, their presence in foods as polymers or in glycosylated forms, and their tight bond to food matrices. Although many studies have investigated how technological and biotechnological processes affect the phenolic composition of fruits and vegetables, limited information exists regarding their effects on PP bioavailability in humans. In the present review, the effect of food processing (mechanical, thermal, and non-thermal treatments), oral-delivery nanoformulations, enzymatic hydrolysis, fermentation, co-administration with probiotics, and generation of postbiotics in PP bioavailability have been overviewed, focusing in the evidence provided in humans.

Urolithins: a Comprehensive Update on their Metabolism, Bioactivity, and Associated Gut Microbiota.

Urolithins, metabolites produced by the gut microbiota from the polyphenols ellagitannins and ellagic acid, are discovered by the research group in humans almost 20 years ago. Pioneering research suggests urolithins as pleiotropic bioactive contributors to explain the health benefits after consuming ellagitannin-rich sources (pomegranates, walnuts, strawberries, etc.). Here, this study comprehensively updates the knowledge on urolithins, emphasizing the review of the literature published during the last 5 years. To date, 13 urolithins and their corresponding conjugated metabolites (glucuronides, sulfates, etc.) have been described and, depending on the urolithin, detected in different human fluids and tissues (urine, blood, feces, breastmilk, prostate, colon, and breast tissues). There has been a substantial advance in the research on microorganisms involved in urolithin production, along with the compositional and functional characterization of the gut microbiota associated with urolithins metabolism that gives rise to the so-called urolithin metabotypes (UM-A, UM-B, and UM-0), relevant in human health. The design of in vitro studies using physiologically relevant assay conditions (molecular forms and concentrations) is still a pending subject, making some reported urolithin activities questionable. In contrast, remarkable progress has been made in the research on the safety, bioactivity, and associated mechanisms of urolithin A, including the first human interventions.

Synthesis and Characterization of a Novel Resveratrol Xylobioside Obtained Using a Mutagenic Variant of a GH10 Endoxylanase.

Resveratrol is a natural polyphenol with antioxidant activity and numerous health benefits. However, in vivo application of this compound is still a challenge due to its poor aqueous solubility and rapid metabolism, which leads to an extremely low bioavailability in the target tissues. In this work, rXynSOS-E236G glycosynthase, designed from a GH10 endoxylanase of the fungus Talaromyces amestolkiae, was used to glycosylate resveratrol by using xylobiosyl-fluoride as a sugar donor. The major product from this reaction was identified by NMR as 3-O-ꞵ-d-xylobiosyl resveratrol, together with other glycosides produced in a lower amount as 4'-O-ꞵ-d-xylobiosyl resveratrol and 3-O-ꞵ-d-xylotetraosyl resveratrol. The application of response surface methodology made it possible to optimize the reaction, producing 35% of 3-O-ꞵ-d-xylobiosyl resveratrol. Since other minor glycosides are obtained in addition to this compound, the transformation of the phenolic substrate amounted to 70%. Xylobiosylation decreased the antioxidant capacity of resveratrol by 2.21-fold, but, in return, produced a staggering 4,866-fold improvement in solubility, facilitating the delivery of large amounts of the molecule and its transit to the colon. A preliminary study has also shown that the colonic microbiota is capable of releasing resveratrol from 3-O-ꞵ-d-xylobiosyl resveratrol. These results support the potential of mutagenic variants of glycosyl hydrolases to synthesize highly soluble resveratrol glycosides, which could, in turn, improve the bioavailability and bioactive properties of this polyphenol.

High-Pressure Processing vs. Thermal Treatment: Effect on the Stability of Polyphenols in Strawberry and Apple Products.

Polyphenols are important bioactive compounds that are affected by processing. The consumer's demand for minimally processed products contributes to the increase in non-thermal technologies such as high-pressure processing (HPP) in the food industry. This review is aimed at critically discussing the positive and negative effects of thermal treatment (TT) and HPP on the stability of different polyphenol families in agro-food products obtained from strawberry and apple, two of the most used fruits in food processing. Our findings show that the phenolic content was affected by processing, fruit type, polyphenol family, and storage conditions (time and temperature) of the final product. To increase shelf life, manufacturers aiming to preserve the natural content of polyphenols need to find the sweet spot between polyphenol stability and product shelf-life since the residual enzyme activity from HPP can affect polyphenols negatively.

Endocrine disruption in Crohn's disease: Bisphenol A enhances systemic inflammatory response in patients with gut barrier translocation of dysbiotic microbiota products.

The relevance of environmental triggers in Crohn's disease remains poorly explored, despite the well-known association between industrialization and disease onset/progression. We have aimed at evaluating the influence of endocrine disrupting chemicals in CD patients. We performed a prospective observational study on consecutive patients diagnosed of CD. Serum levels of endocrine disruptors, short-chain fatty acids, tryptophan and cytokines were measured. Bacterial-DNA and serum endotoxin levels were also evaluated. Gene expression of ER-α, ER-ß and GPER was measured in PBMCs. All patients were genotyped for NOD2 and ATG16L1 polymorphisms. A series of 200 CD patients (140 in remission, 60 with active disease) was included in the study. Bisphenol A was significantly higher in patients with active disease versus remission and in colonic versus ileal disease. GPER was significantly increased in active patients and correlated with BPA levels. BPA was significantly increased in patients with bacterial-DNA and correlated with serum endotoxin levels, (r = 0.417; P = .003). Serum butyrate and tryptophan levels were significantly lower in patients with bacterial-DNA and an inverse relationship was present between them and BPA levels (r = -0.491; P = .001) (r = -0.611; P = .001). Serum BPA levels correlated with IL-23 (r = 0.807; P = .001) and IL-17A (r = 0.743; P = .001). The multivariate analysis revealed an independent significant contribution of BPA and bacterial-DNA to serum levels of IL-23 and IL-17A. In conclusion, bisphenol A significantly affects systemic inflammatory response in CD patients with gut barrier disruption and dysbiotic microbiota secretory products in blood. These results provide evidence of an endocrine disruptor playing an actual pathogenic role on CD.

Data sharing in PredRet for accurate prediction of retention time: Application to plant food bioactive compounds.

Prediction of retention times (RTs) is increasingly considered in untargeted metabolomics to complement MS/MS matching for annotation of unidentified peaks. We tested the performance of PredRet (http://predret.org/) to predict RTs for plant food bioactive metabolites in a data sharing initiative containing entry sets of 29-103 compounds (totalling 467 compounds, >30 families) across 24 chromatographic systems (CSs). Between 27 and 667 predictions were obtained with a median prediction error of 0.03-0.76 min and interval width of 0.33-8.78 min. An external validation test of eight CSs showed high prediction accuracy. RT prediction was dependent on shape and type of LC gradient, and number of commonly measured compounds. Our study highlights PredRet's accuracy and ability to transpose RT data acquired from one CS to another CS. We recommend extensive RT data sharing in PredRet by the community interested in plant food bioactive metabolites to achieve a powerful community-driven open-access tool for metabolomics annotation.

Urolithins in Human Breast Milk after Walnut Intake and Kinetics of Gordonibacter Colonization in Newly Born: The Role of Mothers' Urolithin Metabotypes.

The maternal-infant transmission of several urolithins through breast milk and the gut colonization of infants by the urolithin-producing bacterium Gordonibacter during their first year of life were explored. Two trials (proof-of-concept study: n = 11; validation study: n = 30) were conducted, where breastfeeding mothers consumed walnuts as a dietary source of urolithin precursors. An analytical method was developed and validated to characterize the urolithin profile in breast milk. Total urolithins ranged from 8.5 to 176.9 nM, while they were not detected in breast milk of three mothers. The mothers' urolithin metabotypes governed the urolithin profile in breast milk, which might have biological significance on infants. A specific quantitative polymerase chain reaction method allowed monitoring the gut colonization of infants by Gordonibacter during their first year of life, and neither breastfeeding nor vaginal delivery was essential for this. The pattern of Gordonibacter establishment in babies was conditioned by their mother's urolithin metabotype, probably because of mother-baby close contact.

Metabolism of different dietary phenolic compounds by the urolithin-producing human-gut bacteria Gordonibacter urolithinfaciens and Ellagibacter isourolithinifaciens.

Gordonibacter urolithinfaciens and Ellagibacter isourolithinifaciens are two human gut bacterial species that convert ellagic acid into urolithins. Urolithins are bioactive postbiotics produced by dehydroxylation reactions catalyzed by different catechol-dehydroxylases. The metabolic ability of these anaerobic bacteria on other dietary-phenolic compounds is unknown. In the present study, we evaluated the metabolism of flavonoids (quercetin, hesperetin, hesperidin, nobiletin, catechin, isoxanthohumol), isoflavonoids (daidzein), coumarins (esculetin, umbelliferone, scoparone), phenylpropanoids [caffeic acid; 3-(3',4'-dihydroxyphenyl)propanoic acid (dihydrocaffeic acid); rosmarinic acid, and chlorogenic acid], benzoic acid derivatives (gallic acid, ellagic acid), lignans (secoisolariciresinol diglucoside), stilbenes (resveratrol), and secoiridoids (oleuropein) by G. urolithinfaciens DSM 27213T and E. isourolithinifaciens DSM 104140T. Both strains metabolized ellagic acid leading to the characteristic urolithins. They also metabolized caffeic, dihydrocaffeic, rosmarinic, and chlorogenic acids. The rest of the phenolic compounds were not transformed. Catechol dehydroxylation and double bond reduction were prominent transformations observed during the incubations. The enzymatic activities seem to have a narrow substrate scope as many catechol- (quercetin, catechin, esculetin, gallic acid) and double bond-containing (resveratrol, esculetin, scoparone, umbelliferone) phenolics were not metabolized. The catechol-dehydroxylase activity was more efficient in E. isourolithinifaciens, while the reductase activity was more relevant in G. urolithinfaciens.

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.

Improving the reporting quality of intervention trials addressing the inter-individual variability in response to the consumption of plant bioactives: quality index and recommendations.

PURPOSE: The quality of the study design and data reporting in human trials dealing with the inter-individual variability in response to the consumption of plant bioactives is, in general, low. There is a lack of recommendations supporting the scientific community on this topic. This study aimed at developing a quality index to assist the assessment of the reporting quality of intervention trials addressing the inter-individual variability in response to plant bioactive consumption. Recommendations for better designing and reporting studies were discussed. METHODS: The selection of the parameters used for the development of the quality index was carried out in agreement with the scientific community through a survey. Parameters were defined, grouped into categories, and scored for different quality levels. The applicability of the scoring system was tested in terms of consistency and effort, and its validity was assessed by comparison with a simultaneous evaluation by experts' criteria. RESULTS: The "POSITIVe quality index" included 11 reporting criteria grouped into four categories (Statistics, Reporting, Data presentation, and Individual data availability). It was supported by detailed definitions and guidance for their scoring. The quality index score was tested, and the index demonstrated to be valid, reliable, and responsive. CONCLUSIONS: The evaluation of the reporting quality of studies addressing inter-individual variability in response to plant bioactives highlighted the aspects requiring major improvements. Specific tools and recommendations favoring a complete and transparent reporting on inter-individual variability have been provided to support the scientific community on this field.