The effect of polyphenols on DNA methylation-assessed biological age attenuation: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Epigenetic age is an estimator of biological age based on DNA methylation; its discrepancy from chronologic age warrants further investigation. We recently reported that greater polyphenol intake benefitted ectopic fats, brain function, and gut microbiota profile, corresponding with elevated urine polyphenols. The effect of polyphenol-rich dietary interventions on biological aging is yet to be determined. METHODS: We calculated different biological aging epigenetic clocks of different generations (Horvath2013, Hannum2013, Li2018, Horvath skin and blood2018, PhenoAge2018, PCGrimAge2022), their corresponding age and intrinsic age accelerations, and DunedinPACE, all based on DNA methylation (Illumina EPIC array; pre-specified secondary outcome) for 256 participants with abdominal obesity or dyslipidemia, before and after the 18-month DIRECT PLUS randomized controlled trial. Three interventions were assigned: healthy dietary guidelines, a Mediterranean (MED) diet, and a polyphenol-rich, low-red/processed meat Green-MED diet. Both MED groups consumed 28 g walnuts/day (+ 440 mg/day polyphenols). The Green-MED group consumed green tea (3-4 cups/day) and Mankai (Wolffia globosa strain) 500-ml green shake (+ 800 mg/day polyphenols). Adherence to the Green-MED diet was assessed by questionnaire and urine polyphenols metabolomics (high-performance liquid chromatography quadrupole time of flight). RESULTS: Baseline chronological age (51.3 ± 10.6 years) was significantly correlated with all methylation age (mAge) clocks with correlations ranging from 0.83 to 0.95; p < 2.2e - 16 for all. While all interventions did not differ in terms of changes between mAge clocks, greater Green-Med diet adherence was associated with a lower 18-month relative change (i.e., greater mAge attenuation) in Li and Hannum mAge (beta = - 0.41, p = 0.004 and beta = - 0.38, p = 0.03, respectively; multivariate models). Greater Li mAge attenuation (multivariate models adjusted for age, sex, baseline mAge, and weight loss) was mostly affected by higher intake of Mankai (beta = - 1.8; p = 0.061) and green tea (beta = - 1.57; p = 0.0016) and corresponded with elevated urine polyphenols: hydroxytyrosol, tyrosol, and urolithin C (p < 0.05 for all) and urolithin A (p = 0.08), highly common in green plants. Overall, participants undergoing either MED-style diet had ~ 8.9 months favorable difference between the observed and expected Li mAge at the end of the intervention (p = 0.02). CONCLUSIONS: This study showed that MED and green-MED diets with increased polyphenols intake, such as green tea and Mankai, are inversely associated with biological aging. To the best of our knowledge, this is the first clinical trial to indicate a potential link between polyphenol intake, urine polyphenols, and biological aging. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03020186.

The hope and hype of ellagic acid and urolithins as ligands of SARS-CoV-2 Nsp5 and inhibitors of viral replication.

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A-D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.

The Role of Punicalagin and Its Metabolites in Atherosclerosis and Risk Factors Associated with the Disease.

Atherosclerotic cardiovascular disease (ACVD) is the leading cause of death worldwide. Although current therapies, such as statins, have led to a marked reduction in morbidity and mortality from ACVD, they are associated with considerable residual risk for the disease together with various adverse side effects. Natural compounds are generally well-tolerated; a major recent goal has been to harness their full potential in the prevention and treatment of ACVD, either alone or together with existing pharmacotherapies. Punicalagin (PC) is the main polyphenol present in pomegranates and pomegranate juice and demonstrates many beneficial actions, including anti-inflammatory, antioxidant, and anti-atherogenic properties. The objective of this review is to inform on our current understanding of the pathogenesis of ACVD and the potential mechanisms underlying the beneficial actions of PC and its metabolites in the disease, including the attenuation of dyslipidemia, oxidative stress, endothelial cell dysfunction, foam cell formation, and inflammation mediated by cytokines and immune cells together with the regulation of proliferation and migration of vascular smooth muscle cells. Some of the anti-inflammatory and antioxidant properties of PC and its metabolites are due to their strong radical-scavenging activities. PC and its metabolites also inhibit the risk factors of atherosclerosis, including hyperlipidemia, diabetes mellitus, inflammation, hypertension, obesity, and non-alcoholic fatty liver disease. Despite the promising findings that have emerged from numerous in vitro, in vivo, and clinical studies, deeper mechanistic insights and large clinical trials are required to harness the full potential of PC and its metabolites in the prevention and treatment of ACVD.

Health functions and related molecular mechanisms of ellagitannin-derived urolithins.

Ellagitannins are vital bioactive polyphenols that are widely distributed in a variety of plant-based foods. The main metabolites of ellagitannins are urolithins, and current research suggests that urolithins provide a variety of health benefits. This review focused on the role of the gut bacteria in the conversion of ellagitannins to urolithins. Based on the results of in vitro and in vivo studies, the health benefits of urolithins, including antioxidant, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, anti-aging, cardiovascular protective, neuroprotective, kidney protective, and muscle mass protective effects, were thoroughly outlined, with a focus on their associated molecular mechanisms. Finally, we briefly commented on urolithins' safety. Overall, urolithins' diverse health benefits indicate the potential utilization of ellagitannins and urolithins in the creation of functional foods and nutraceuticals to treat and prevent some chronic diseases.

Antioxidant and Antiglycation Effects of Cistus × incanus Water Infusion, Its Phenolic Components, and Respective Metabolites.

Reactive oxygen and carbonyl species promote oxidative and carbonyl stress, and the development of diabetes, metabolic syndrome, cardiovascular diseases, and others. The traditional herb Cistus × incanus is known for its antioxidant properties; therefore, the current study aimed to assess how the chemical composition of a C. incanus water infusion corresponds with its antioxidative and antiglycative effects in vitro. The composition of infusions prepared from commercial products was analyzed with UHPLC-ESI-qTOF-MS. Total phenolics, flavonoids, and non-flavonoid polyphenols were determined. Antioxidant activity of infusions and selected polyphenols was investigated using DPPH, ABTS, and FRAP. Fluorometric measurements and methylglyoxal capture were performed to investigate the antiglycation activity. PCA and PLS-DA models were applied to explore the correlation between chemical and antioxidant results. The principal flavonoids in C. incanus were flavonols. In vitro tests revealed that a stronger antioxidant effect was demonstrated by plant material from Turkey rich in flavonoids, followed by Albania and Greece. Flavonols and ellagic acid displayed stronger antiradical and reducing power than EA-derived urolithins. Hyperoside was the most potent inhibitor of glycation. The results indicate that flavonoids are primarily responsible for rock rose antioxidant and antiglycation properties. PLS-DA modeling can be used to identify the origin of plant material with sensitivity and specificity exceeding 86%.

Do Pomegranate Hydrolyzable Tannins and Their Derived Metabolites Provide Relief in Osteoarthritis? Findings from a Scoping Review.

Osteoarthritis (OA) is the most common form of arthritis affecting both the elderly and the middle-aged population. Although various therapeutics have been developed to arrest the structural deterioration of cartilage, the current treatments are limited to delay the progress of OA clinically. Therefore, it is pivotal to study new therapeutic agents for chondroprotection and the prevention of cartilage degeneration. Hydrolyzable tannin (HT)-containing foods aroused considerable interest in recent years for their relevant anti-inflammatory effects. The focus of this scoping review is to provide an overview of the evidence of the therapeutic potential of HTs and their metabolites in preventing or alleviating the course of OA. A broad search of PubMed and Scopus databases on this topic resulted in 156 articles. After the exclusion of reviews and not relevant records, 31 articles were retrieved. Although only some papers did not consider the biotransformation of HTs, most recent studies also have investigated the effect of HT metabolites. Further larger clinical trials, with an in-deep analysis of HT metabolization, are still needed to unravel the potential benefits of these compounds in OA, paving the way towards the development of a dietary strategy for the improvement of pro-inflammatory cytokine-induced chondrocyte dysfunctions and injuries.

The ellagic acid metabolites urolithin A and B differentially affect growth, adhesion, motility, and invasion of endometriotic cells in vitro.

STUDY QUESTION: What are the effects of plant-derived antioxidant compounds urolithin A (UA) and B (UB) on the growth and pathogenetic properties of an in vitro endometriosis model? SUMMARY ANSWER: Both urolithins showed inhibitory effects on cell behavior related to the development of endometriosis by differentially affecting growth, adhesion, motility, and invasion of endometriotic cells in vitro. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common benign gynecological diseases in women of reproductive age and is defined by the presence of endometrial tissue outside the uterine cavity. As current pharmacological therapies are associated with side effects interfering with fertility, we aimed at finding alternative therapeutics using natural compounds that can be administered for prolonged periods with a favorable side effects profile. STUDY DESIGN, SIZE, DURATION: In vitro cultures of primary endometriotic stromal cells from 6 patients subjected to laparoscopy for benign pathologies with histologically confirmed endometriosis; and immortalized endometrial stromal (St-T1b) and endometriotic epithelial cells (12Z) were utilized to assess the effects of UA and UB on endometriotic cell properties. Results were validated in three-dimensional (3D) in vitro co-culture spheroids of 12Z and primary endometriotic stroma cells of one patient, and organoids from 3 independent donors with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects on cell growth were measured by non-radioactive colorimetric assay to measure cellular metabolic activity as an indicator of cell viability (MTT assay) and flow cytometric cell cycle assay on primary cultures, St-T1b, and 12Z. Apoptosis analyses, the impact on in vitro adhesion, migration, and invasion were evaluated in the cell lines. Moreover, Real-Time Quantitative Reverse Transcription polymerase chain reaction (RT-qPCR) assays were performed on primary cultures, St- T1b and 12Z to evaluate a plausible mechanistic contribution by factors related to proteolysis (matrix metalloproteinase 2, 3 and 9 -MMP2, MMP3, MMP9-, and tissue inhibitor of metalloproteinases -TIMP-1-), cytoskeletal regulators (Ras-related C3 botulinum toxin substrate 1 -RAC1-, Rho-associated coiled-coil containing protein kinase 2 -ROCK2-), and cell adhesion molecules (Syndecan 1 -SDC1-, Integrin alpha V-ITGAV-). Finally, the urolithins effects were evaluated on spheroids and organoids by formation, viability, and drug screen assays. MAIN RESULTS AND THE ROLE OF CHANCE: 40 µM UA and 20 µM UB produced a significant decrease in cell proliferation in the primary endometriotic cell cultures (P < 0.001 and P < 0.01, respectively) and in the St-T1b cell line (P < 0.001 and P < 0.05, respectively). In St-T1b, UA exhibited a mean half-maximum inhibitory concentration (IC50) of 39.88 µM, while UB exhibited a mean IC50 of 79.92 µM. Both 40 µM UA and 20 µM UB produced an increase in cells in the S phase of the cell cycle (P < 0.01 and P < 0.05, respectively). The same concentration of UA also increased the percentage of apoptotic ST-t1b cells (P < 0.05), while both urolithins decreased cell migration after 24 h (P < 0.001 both). Only the addition of 5 µM UB decreased the number of St-T1b adherent cells. TIMP-1 expression was upregulated in response to treating the cells with 40 µM UA (P < 0.05). Regarding the 12Z endometriotic cell line, only 40 µM UA decreased proliferation (P < 0.01); while both 40 µM UA and 20 µM UB produced an increase in cells in the G2/M phase (P < 0.05 and P < 0.01, respectively). In this cell line, UA exhibited a mean IC50 of 40.46 µM, while UB exhibited a mean IC50 of 54.79 µM. UB decreased cell migration (P < 0.05), and decreased the number of adherent cells (P < 0.05). Both 40 µM UA and 20 µM UB significantly decreased the cellular invasion of these cells; and several genes were altered when treating the cells with 40 µM UA and 10 µM UB. The expression of MMP2 was downregulated by UA (P < 0.001), and expression of MMP3 (UA P < 0.001 and UB P < 0.05) and MMP9 (P < 0.05, both) were downregulated by both urolithins. Moreover, UA significantly downregulated ROCK2 (P < 0.05), whereas UB treatment was associated with RAC1 downregulation (P < 0.05). Finally, the matrix adhesion receptors and signaling (co)receptors SDC1 and ITGAV were downregulated upon treatment with either UA or UB (P < 0.01 and P < 0.05, respectively in both cases). Regarding the effects of urolithins on 3D models, we have seen that they significantly decrease the viability of endometriosis spheroids (80 µM UA and UB: P < 0.05 both) as well as affecting their area (40 µM UA: P < 0.05, and 80 µM UA: P < 0.01) and integrity (40 µM UA and UB: P < 0.05, 80 µM UA and UB: P < 0.01). On the other hand, UA and UB significantly inhibited organoid development/outgrowth (40 and 80 µM UA: P < 0.0001 both; 40 µM UB: P < ns-0.05-0.001, and 80 µM UB: P < 0.01-0.001-0.001), and all organoid lines show urolithins sensitivity resulting in decreasing viability (UA exhibited a mean IC50 of 33.93 µM, while UB exhibited a mean IC50 of 52.60 µM). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed on in vitro endometriosis models. WIDER IMPLICATIONS OF THE FINDINGS: These in vitro results provide new insights into the pathogenetic pathways affected by these compounds and mark their use as a potential new therapeutic strategy for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded EU MSCA-RISE-2015 project MOMENDO (691058). The authors have no conflicts of interest to declare.

Comparative studies of urolithins and their phase II metabolites on macrophage and neutrophil functions.

PURPOSE: Ellagitannins are high molecular weight polyphenols present in high quantities in various food products. They are metabolized by human and animal gut microbiota to postbiotic metabolites-urolithins, bioavailable molecules of a low molecular weight. Following absorption in the gut, urolithins rapidly undergo phase II metabolism. Thus, to fully evaluate the mechanisms of their biological activity, the in vitro studies should be conducted for their phase II conjugates, mainly glucuronides. The aim of the study was to comparatively determine the influence of urolithin A, iso-urolithin A, and urolithin B together with their respective glucuronides on processes associated with the inflammatory response. METHODS: The urolithins obtained by chemical synthesis or isolation from microbiota cultures were tested with their respective glucuronides isolated from human urine towards modulation of inflammatory response in THP-1-derived macrophages, RAW 264.7 macrophages, PBMCs-derived macrophages, and primary neutrophils. RESULTS: Urolithin A was confirmed to be the most active metabolite in terms of LPS-induced inflammatory response inhibition (TNF-α attenuation, IL-10 induction). The observed strong induction of ERK1/2 phosphorylation has been postulated as the mechanism of its action. None of the tested glucuronide conjugates was active in terms of pro-inflammatory TNF-α inhibition and anti-inflammatory IL-10 and TGF-ß1 induction. CONCLUSION: Comparative studies of the most abundant urolithins and their phase II conjugates conducted on human and murine immune cells unambiguously confirmed urolithin A to be the most active metabolite in terms of inhibition of the inflammatory response. Phase II metabolism was shown to result in the loss of urolithins' pharmacological properties.

Design, synthesis, and biological evaluation of new urolithin amides as multitarget agents against Alzheimer's disease.

A series of urolithin amide (i.e., URO-4-URO-10 and THU-4-THU-10) derivatives was designed and synthesized, and their chemical structures were confirmed with spectroscopic techniques and elemental analysis. The title compounds and synthesis intermediates (THU-1-THU-10 and URO-1-URO-10) were evaluated for their potential to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and monoamine oxidase B (MAO-B). Compounds THU-4 and THU-8 were found to be the most potent inhibitors for the cholinesterases and MAO-B, respectively. The docking studies were also employed to evaluate the binding modes of the most active compounds with AChE, BuChE, and MAO-B. Furthermore, the moderate-to-strong activities of the compounds were also displayed in amyloid-beta inhibition and antioxidant assay systems. The results pointed out that the urolithin scaffold can be employed in drug design studies for the development of multitarget ligands acting on various cascades shown to be important within the pathophysiology of Alzheimer's disease.

Natural Dibenzo-α-Pyrones: Friends or Foes?

Natural dibenzo-α-pyrones (DAPs) can be viewed from two opposite angles. From one angle, the gastrointestinal metabolites urolithins are regarded as beneficial, while from the other, the emerging mycotoxin alternariol and related fungal metabolites are evaluated critically with regards to potential hazardous effects. Thus, the important question is: can the structural characteristics of DAP subgroups be held responsible for distinct bioactivity patterns? If not, certain toxicological and/or pharmacological aspects of natural DAPs might yet await elucidation. Thus, this review focuses on comparing published data on the two groups of natural DAPs regarding both adverse and beneficial effects on human health. Literature on genotoxic, estrogenic, endocrine-disruptive effects, as well as on the induction of the cellular anti-oxidative defense system, anti-inflammatory properties, the inhibition of kinases, the activation of mitophagy and the induction of autophagy, is gathered and critically reviewed. Indeed, comparing published data suggests similar bioactivity profiles of alternariol and urolithin A. Thus, the current stratification into hazardous Alternaria toxins and healthy urolithins seems debatable. An extrapolation of bioactivities to the other DAP sub-class could serve as a promising base for further research. Conclusively, urolithins should be further evaluated toward high-dose toxicity, while alternariol derivatives could be promising chemicals for the development of therapeutics.

Bacterial metabolism as responsible of beneficial effects of phytoestrogens on human health.

Phytoestrogens (PE) are compounds found in plants such as soy (isoflavones), flax seeds and cereals (lignans) and pomegranates (ellagitannins). PE have shown estrogenic/antiestrogenic, antioxidant, anti-inflammatory, antineoplastic and apoptotic activities. The human studies are showing promising although inconsistent results about the beneficial effects of PE on ameliorating the menopausal symptoms or reducing the risk of certain cancers, cardiovascular disease or diabetes. The effects of PE on the organism are mediated by the intestinal microbiota, which transforms them into bioactive PE such as genistein, equol, enterolignans and certain urolithins. In this work, we review the most recent findings about the bacteria able to metabolize PE, together with the latest studies on the effects of PE on health. In addition, we describe the possible factors hindering the demonstration of the beneficial effect of PE on health, evincing the importance of measuring the actual circulating PE in order to encompass the variability of PE metabolism due to the intestinal microbiota. With this in mind, we also explore an approach to ensure the access to bioactive PE.

Synthesis, Characterization, Molecular Docking, and Biological Activities of Some Natural and Synthetic Urolithin Analogs.

Urolithins (that is, hydroxy substituted benzo[c]chromen-6-one derivatives) are formed within the gastrointestinal tract following to the exposure to various ellagitannin rich diet, particularly involving pomegranate, nuts, and berries. Regarding the bioavailability deficiency of ellagitannins, the biological activities obtained through the extracts of these dietaries are attributed to the urolithin compounds, since they are bioavailable. Particularly, there are studies indicating the importance of ellagitannin-rich food for protective and alternative treatment of Alzheimer's Disease (AD). From this perspective, within this study, the major urolithins (that is, urolithins A and B), their methyl ether metabolites, as well as some synthetic urolithin analogs have been synthesized and screened for their biological activities in various enzyme inhibition (acetylcholinesterase, butyrylcholinesterase, monoamine oxidase B, cyclooxygenase 1, and cyclooxygenase 2) and antioxidant (DPPH radical scavenging) assay systems. The results pointed out the potential of urolithins to act as inhibitors on these receptors. Docking studies were also performed to investigate the possible interactions.

Ellagic Acid and Urolithins A and B Differentially Regulate Fat Accumulation and Inflammation in 3T3-L1 Adipocytes While Not Affecting Adipogenesis and Insulin Sensitivity.

Ellagic acid (EA) is a component of ellagitannins, present in crops such as pecans, walnuts, and many berries, which metabolized by the gut microbiota forms urolithins A, B, C, or D. In this study, ellagic acid, as well as urolithins A and B, were tested on 3T3-L1 preadipocytes for differentiation and lipid accumulation. In addition, inflammation was studied in mature adipocytes challenged with lipopolysaccharide (LPS). Results indicated that EA and urolithins A and B did not affect differentiation (adipogenesis) and only EA and urolithin A attenuated lipid accumulation (lipogenesis), which seemed to be through gene regulation of glucose transporter type 4 (GLUT4) and adiponectin. On the other hand, gene expression of cytokines and proteins associated with the inflammation process indicate that urolithins and EA differentially inhibit tumor necrosis factor alpha (TNFα), inducible nitric oxide synthase (iNOS), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Urolithins A and B were found to reduce nuclear levels of phosphorylated nuclear factor κB (p-NF-κB), whereas all treatments showed expression of nuclear phosphorylated protein kinase B (p-AKT) in challenged LPS cells when treated with insulin, indicating the fact that adipocytes remained insulin sensitive. In general, urolithin A is a compound able to reduce lipid accumulation, without affecting the protein expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer binding protein-α (c/EBPα), and PPARα, whereas EA and urolithin B were found to enhance PPARγ and c/EBPα protein expressions as well as fatty acid (FA) oxidation, and differentially affected lipid accumulation.

Recent Advances in the Production and Applications of Ellagic Acid and Its Derivatives. A Review.

Ellagitannins (ETs), characterized by their diversity and chemical complexity, belong to the class of hydrolysable tannins that, via hydrolysis under acidic or alkaline conditions, can yield ellagic acid (EA). They are mostly found as a part of extractives in angiosperms. As known antioxidants and chelators, EA and EA derivatives are drawing an increasing interest towards extensive technical and biomedical applications. The latter ones include possible antibacterial, antifungal, antiviral, anti-inflammatory, hepato- and cardioprotective, chemopreventive, neuroprotective, anti-diabetic, gastroprotective, antihyperlipidemic, and antidepressant-like activities, among others. EA's synthesis and production challenges prompt further research on new methods and alternative sources. Conventional and prospective methods and raw materials for the production of EA and its derivatives are reviewed. Among the potential sources of EA, the residues and industrial streams of the pulp industry have been highlighted and considered as an alluring alternative in terms of commercial exploitation.

Plasma and Urinary (Poly)phenolic Profiles after 4-Week Red Raspberry (Rubus idaeus L.) Intake with or without Fructo-Oligosaccharide Supplementation.

Red raspberries (RRB) are high in anthocyanin- and ellagitannin- type (poly)phenols. This study aimed to investigate the effect of 4-week RRB supplementation on (poly)phenolic metabolism in adults with prediabetes and insulin-resistance (PreDM-IR); and whether adding fructo-oligosaccharides (FOS), prebiotics, would augment the microbial metabolites of RRB (poly)phenols. In a randomized crossover clinical trial, subjects (n = 35: PreDM-IR, n = 25; healthy Reference group, n = 10) consumed 1 cup RRB (fresh weight equivalence) per day and RRB with 8 g FOS per day each for 4 weeks in random order separated by 4-week washout. Plasma and urinary (poly)phenolic metabolites were characterized after (0-24h) consuming a RRB-based test drink (2 cups RRB) at baseline/week 0 and again after 4-week supplementations. A total of 123 (poly)phenolic metabolites were quantified. After 4-week RRB supplementation, several metabolite groups were significantly increased (p < 0.05), including urolithins, phenyl-γ-valerolactones, and phenolic acids. Supplementing FOS with RRB for 4 weeks enhanced benzoic acid derivatives compared to the baseline (p < 0.05). Specific effects of supplementation by metabolic status indicated 4-week RRB supplementation significantly increased microbial metabolites that were lower in PreDM-IR group. Our results suggest alterations in the capacity of PreDM-IR group to metabolize and render bioavailable raspberry-derived (poly)phenols when consumed regularly.

Pomegranate ellagitannin-gut microbial-derived metabolites, urolithins, inhibit neuroinflammation in vitro.

OBJECTIVES: Urolithins, ellagitannin-gut microbial-derived metabolites, have been reported to mediate pomegranate's neuroprotective effects against Alzheimer's disease (AD), but there are limited data on their effects against neuroinflammation. Herein, we: (1) evaluated whether urolithins (urolithins A and B and their methylated derivatives) attenuate neuroinflammation in murine BV-2 microglia and human SH-SY5Y neurons, and (2) evaluated hippocampus of transgenic AD (R1.40) mice administered a pomegranate extract (PE; 100 or 200 mg/kg/day for 3 weeks) for inflammatory biomarkers. METHODS: Effects of urolithins (10 µM) on inflammatory biomarkers were evaluated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. In a non-contact co-culture cell model, SH-SY5Y cell viability was assessed after exposure to media collected from LPS-BV-2 cells treated with or without urolithins. Effects of urolithins on apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells were assessed. Hippocampal tissues of vehicle and PE-treated transgenic R1.40 mice were evaluated for gene expression of inflammatory biomarkers by qRT-PCR. RESULTS: Urolithins decreased media levels of nitric oxide, interleukin 6 (IL-6), prostaglandin E2, and tumor necrosis factor alpha from LPS-BV-2 microglia. In the co-culture cell model, media from LPS-BV-2 cells treated with urolithins preserved SH-SY5Y cell viability greater than media from cells treated without urolithins. Urolithins mitigated apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells. While not statistically significant, inflammatory biomarkers (TNF-α, COX-2, IL-1, and IL-6) appeared to follow a decreasing trend in the hippocampus of high-dose PE-treated animals compared to controls. DISCUSSION: The attenuation of neuroinflammation by urolithins may contribute, in part, toward pomegranate's neuroprotective effects against AD.

Plasma urolithin metabolites correlate with improvements in endothelial function after red raspberry consumption: A double-blind randomized controlled trial.

Raspberries are a rich source of ellagitannins and anthocyanins. The aim of this work was to investigate whether raspberry consumption can improve vascular function and to understand which phenolic metabolites may be responsible for the effects. A 3 arm double-blind randomized controlled crossover human intervention trial was conducted in 10 healthy males. Flow-mediated dilation (FMD) was measured at baseline, 2 h, and 24 h post-consumption of 200 g and 400 g of red raspberries containing 201 or 403 mg of total (poly)phenols, or a matched control drink. Raspberry (poly)phenol metabolites were analyzed in plasma and urine by UPLC-QTOF mass spectrometry using authentic standards. Significant improvements in FMD were observed at 2 h (1.6% (95%CI 1.2, 1.9) and 1.2% (95% CI 0.8, 1.5)) and 24 h (1.0% (95% CI 0.6, 1.2) and 0.7% (95%CI 0.2, 0.9)) post-consumption of the 200 and 400 g raspberry drinks as compared to control, respectively. Plasma ellagic acid, urolithin A-3-glucuronide and urolithin A-sulfate correlated with the improvements in FMD at 2 and 24 h post consumption, respectively. Consumption of dietary achievable amounts of red raspberries acutely improves endothelial function up to 24 h and ellagitannins may be responsible for the observed effect.

Trimethylamine-N-Oxide (TMAO)-Induced Impairment of Cardiomyocyte Function and the Protective Role of Urolithin B-Glucuronide.

One of the most recently proposed candidates as a potential trigger for cardiovascular diseases is trimethylamine-N-oxide (TMAO). Possible direct effects of TMAO on myocardial tissue, independent of vascular damage, have been only partially explored so far. In the present study, we assessed the detrimental direct effects of TMAO on cardiomyocyte contractility and intracellular calcium dynamics, and the ability of urolithin B-glucuronide (Uro B-gluc) in counteracting TMAO-induced cell damage. Cell mechanics and calcium transients were measured, and ultrastructural analysis was performed in ventricular cardiomyocytes isolated from the heart of normal adult rats. Cells were either untreated, exposed to TMAO, or to TMAO and Uro B-gluc. TMAO exposure worsened cardiomyocyte mechanics and intracellular calcium handling, as documented by the decrease in the fraction of shortening (FS) and the maximal rate of shortening and re-lengthening, associated with reduced efficiency in the intracellular calcium removal. Ultrastructurally, TMAO-treated cardiomyocytes also exhibited glycogen accumulation, a higher number of mitochondria and lipofuscin-like pigment deposition, suggesting an altered cellular energetic metabolism and a higher rate of protein oxidative damage, respectively. Uro B-gluc led to a complete recovery of cellular contractility and calcium dynamics, and morphologically to a reduced glycogen accumulation. We demonstrated for the first time a direct negative role of TMAO on cardiomyocyte functional properties and the ability of Uro B-gluc in counteracting these detrimental effects.

In vivo administration of urolithin A and B prevents the occurrence of cardiac dysfunction in streptozotocin-induced diabetic rats.

BACKGROUND: Emerging evidence suggests that specific (poly)phenols may constitute new preventative strategies to counteract cell oxidative stress and myocardial tissue inflammation, which have a key role in the patho-physiology of diabetic cardiomyopathy. In a rat model of early diabetes, we evaluated whether in vivo administration of urolithin A (UA) or urolithin B (UB), the main gut microbiota phenolic metabolites of ellagitannin-rich foods, can reduce diabetes-induced microenvironmental changes in myocardial tissue, preventing cardiac functional impairment. METHODS: Adult Wistar rats with streptozotocin-induced type-1 diabetes (n = 29) were studied in comparison with 10 control animals. Diabetic rats were either untreated (n  = â€Š9) or subjected to daily i.p. injection of UA (n = 10) or UB (n = 10). After 3 weeks of hyperglycaemia, hemodynamics, cardiomyocyte contractile properties and calcium transients were measured to assess cardiac performance. The myocardial expression of the pro-inflammatory cytokine fractalkine and proteins involved in calcium dynamics (sarcoplasmic reticulum calcium ATPase, phospholamban and phosphorylated phospholamban) were evaluated by immunoblotting. Plasma, urine and tissue distribution of UA, UB and their phase II metabolites were determined. RESULTS: In vivo urolithin treatment reduced by approximately 30% the myocardial expression of the pro-inflammatory cytokine fractalkine, preventing the early inflammatory response of cardiac cells to hyperglycaemia. The improvement in myocardial microenvironment had a functional counterpart, as documented by the increase in the maximal rate of ventricular pressure rise compared to diabetic group (+18% and +31% in UA and UB treated rats, respectively), and the parallel reduction in the isovolumic contraction time (-12%). In line with hemodynamic data, both urolithins induced a recovery of cardiomyocyte contractility and calcium dynamics, leading to a higher re-lengthening rate (+21%, on average), lower re-lengthening times (-56%), and a more efficient cytosolic calcium clearing (-32% in tau values). UB treatment also increased the velocity of shortening (+27%). Urolithin metabolites accumulated in the myocardium, with a higher concentration of UB and UB-sulphate, potentially explaining the slightly higher efficacy of UB administration. CONCLUSIONS: In vivo urolithin administration may be able to prevent the initial inflammatory response of myocardial tissue to hyperglycaemia and the negative impact of the altered diabetic milieu on cardiac performance.

Urolithins impair cell proliferation, arrest the cell cycle and induce apoptosis in UMUC3 bladder cancer cells.

Ellagitannins have been gaining attention as potential anticancer molecules. However, the low bioavailability of ellagitannins and their extensive metabolization in the gastrointestinal tract into ellagic acid and urolithins suggest that the health benefits of consuming ellagitannins rely on the direct effects of their metabolites. Recently, chemopreventive and chemotherapeutic activities were ascribed to urolithins. Nonetheless, there is still a need to screen and evaluate the selectivity of these molecules and to elucidate their cellular mechanisms of action. Therefore, this work focused on the antiproliferative effects of urolithins A, B and C and ellagic acid on different human tumor cell lines. The evaluation of cell viability and the determination of the half-maximal inhibitory concentrations indicated that the sensitivity to the studied urolithins varied markedly between the different cell lines, with the bladder cancer cells (UMUC3) being the most susceptible. In UMUC3 cells, urolithin A was the most active molecule, promoting cell cycle arrest at the G2/M checkpoint, increasing apoptotic cell death and inhibiting PI3K/Akt and MAPK signaling. Overall, the present study emphasizes the chemopreventive/chemotherapeutic potential of urolithins, highlighting the stronger effects of urolithin A and its potential to target transitional bladder cancer cells.