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.

Resveratrol in primary and secondary prevention of cardiovascular disease: a dietary and clinical perspective.

Primary prevention of cardiovascular disease (CVD) aims to avoid a first event in subjects that are at risk but have not yet been diagnosed with heart disease. Secondary prevention of CVD aims to avoid new events in patients with established heart disease. Both approaches involve clinical intervention and implementation of healthy lifestyles. The grape and wine polyphenol resveratrol (3,5,4'-trihydroxy-trans-stilbene) has shown cardioprotective benefits in humans. Most of these approaches deal with rather high doses and short follow-ups, and do not address the issue of long-term resveratrol consumption safety, especially in medicated individuals. Here, we review the trials conducted with resveratrol in patients at risk for or with established CVD, focusing on the two longest human clinical trials reported so far (1-year follow-up). We also discuss the expectations for resveratrol from a dietary and clinical perspective in relation to CVD. However, statistically significant changes in CVD-risk markers do not necessarily equal clinical significance in the daily care of patients.

Grape resveratrol increases serum adiponectin and downregulates inflammatory genes in peripheral blood mononuclear cells: a triple-blind, placebo-controlled, one-year clinical trial in patients with stable coronary artery disease.

PURPOSE: The grape and wine polyphenol resveratrol exerts cardiovascular benefits but evidence from randomized human clinical trials is very limited. We investigated dose-depending effects of a resveratrol-containing grape supplement on stable patients with coronary artery disease (CAD) treated according to currently accepted guidelines for secondary prevention of cardiovascular disease. METHODS: In a triple-blind, randomized, placebo-controlled, one-year follow-up, 3-arm pilot clinical trial, 75 stable-CAD patients received 350 mg/day of placebo, resveratrol-containing grape extract (grape phenolics plus 8 mg resveratrol) or conventional grape extract lacking resveratrol during 6 months, and a double dose for the following 6 months. Changes in circulating inflammatory and fibrinolytic biomarkers were analyzed. Moreover, the transcriptional profiling of inflammatory genes in peripheral blood mononuclear cells (PBMCs) was explored using microarrays and functional gene expression analysis. RESULTS: After 1 year, in contrast to the placebo and conventional grape extract groups, the resveratrol-containing grape extract group showed an increase of the anti-inflammatory serum adiponectin (9.6 %, p = 0.01) and a decrease of the thrombogenic plasminogen activator inhibitor type 1 (PAI-1) (-18.6 %, p = 0.05). In addition, 6 key inflammation-related transcription factors were predicted to be significantly activated or inhibited, with 27 extracellular-space acting genes involved in inflammation, cell migration and T-cell interaction signals presenting downregulation (p < 0.05) in PBMCs. No adverse effects were detected in relation to the study products. CONCLUSIONS: Chronic daily consumption of a resveratrol-containing grape nutraceutical could exert cardiovascular benefits in stable-CAD patients treated according to current evidence-based standards, by increasing serum adiponectin, preventing PAI-1 increase and inhibiting atherothrombotic signals in PBMCs.

Alternative method for gas chromatography-mass spectrometry analysis of short-chain fatty acids in faecal samples.

Short-chain fatty acids are the major end products of bacterial metabolism in the large bowel. They derive mostly from the bacterial breakdown of carbohydrates and are known to have positive health benefits. Due to the biological relevance of these compounds it is important to develop efficient, cheap, fast, and sensitive analytical methods that enable the identification and quantification of the short-chain fatty acids in a large number of biological samples. In this study, a gas chromatography-mass spectrometry method was developed and validated for the analysis of short-chain fatty acids in faecal samples. These volatile compounds were extracted with ethyl acetate and 4-methyl valeric acid was used as an internal standard. No further cleanup, concentration, and derivatization steps were needed and the extract was directly injected onto the column. Recoveries ranged between 65 and 105%, and no matrix effects were observed. The proposed method has wide linear ranges, good inter- and intraday variability values (below 2.6 and 5.6%, respectively) and limits of detection between 0.49 µM (0.29 µg/g) and 4.31 µM (3.8 µg/g). The applicability of this analytical method was successfully tested in faecal samples from rats and humans.

Consumption of a grape extract supplement containing resveratrol decreases oxidized LDL and ApoB in patients undergoing primary prevention of cardiovascular disease: a triple-blind, 6-month follow-up, placebo-controlled, randomized trial.

SCOPE: The cardioprotective role of resveratrol as part of the human diet is not yet clear. Our aim was to investigate the effect of a grape supplement containing 8 mg resveratrol in oxidized LDL (LDLox), apolipoprotein-B (ApoB), and serum lipids on statin-treated patients in primary cardiovascular disease prevention (PCP). METHODS AND RESULTS: A triple-blind, randomized, placebo-controlled trial was conducted. Seventy-five patients (three parallel arms) consumed one capsule (350 mg) daily for 6 months containing resveratrol-enriched grape extract (GE-RES, Stilvid®), grape extract (GE, similar polyphenolic content but no resveratrol), or placebo (maltodextrin). After 6 months, no changes were observed in the placebo group and only LDL cholesterol (LDLc) decreased by 2.9% (p = 0.013) in the GE group. In contrast, LDLc (-4.5%, p = 0.04), ApoB (-9.8%, p = 0.014), LDLox (-20%, p = 0.001), and LDLox/ApoB (-12.5%, p = 0.000) decreased in the Stilvid® group, whereas the ratio non-HDLc (total atherogenic cholesterol load)/ApoB increased (8.5%, p = 0.046). No changes were observed in hepatic, thyroid, and renal function. No adverse effects were observed in any of the patients. CONCLUSION: This GE-RES reduced atherogenic markers and might exert additional cardioprotection beyond the gold-standard medication in patients from PCP. The presence of resveratrol in the GE was necessary to achieve these effects.

One-year consumption of a grape nutraceutical containing resveratrol improves the inflammatory and fibrinolytic status of patients in primary prevention of cardiovascular disease.

The search for complementary treatments in primary prevention of cardiovascular disease (CVD) is a high-priority challenge. Grape and wine polyphenol resveratrol confers CV benefits, in part by exerting anti-inflammatory effects. However, the evidence in human long-term clinical trials has yet to be established. We aimed to investigate the effects of a dietary resveratrol-rich grape supplement on the inflammatory and fibrinolytic status of subjects at high risk of CVD and treated according to current guidelines for primary prevention of CVD. Seventy-five patients undergoing primary prevention of CVD participated in this triple-blinded, randomized, parallel, dose-response, placebo-controlled, 1-year follow-up trial. Patients, allocated in 3 groups, consumed placebo (maltodextrin), a resveratrol-rich grape supplement (resveratrol 8 mg), or a conventional grape supplement lacking resveratrol, for the first 6 months and a double dose for the next 6 months. In contrast to placebo and conventional grape supplement, the resveratrol-rich grape supplement significantly decreased high-sensitivity C-reactive protein (-26%, p = 0.03), tumor necrosis factor-α (-19.8%, p = 0.01), plasminogen activator inhibitor type 1 (-16.8%, p = 0.03), and interleukin-6/interleukin-10 ratio (-24%, p = 0.04) and increased anti-inflammatory interleukin-10 (19.8%, p = 0.00). Adiponectin (6.5%, p = 0.07) and soluble intercellular adhesion molecule-1 (-5.7%, p = 0.06) tended to increase and decrease, respectively. No adverse effects were observed in any patient. In conclusion, 1-year consumption of a resveratrol-rich grape supplement improved the inflammatory and fibrinolytic status in patients who were on statins for primary prevention of CVD and at high CVD risk (i.e., with diabetes or hypercholesterolemia plus ≥1 other CV risk factor). Our results show for the first time that a dietary intervention with grape resveratrol could complement the gold standard therapy in the primary prevention of CVD.

Metabolites and tissue distribution of resveratrol in the pig.

SCOPE: trans-Resveratrol (RES) and/(or) its metabolites exert many effects in vivo. Our aim was to study the metabolism and tissue distribution of RES using the pig, a mammal physiologically close to humans. METHODS AND RESULTS: Forty-seven tissues, organs and fluids were analyzed 6 h after intragastric RES administration (5.9 mg/kg body weight) using HPLC-MS/MS. Twelve RES and seven dihydroresveratrol (DH-RES) metabolites were detected. DH-RES was the main metabolite in cecum, colon and rectum, whereas RES-3-O-glucuronide was the most abundant one in fluids and organs. Approximately 74.5% of the total RES administered was recovered in the form of RES, DH-RES and derived metabolites (65.1% along the gastrointestinal tract, 7.7% in urine, 1.2% in bile and 0.5% in organs). We report here, for the first time, the occurrence of RES ribosyl-sulfate derivative, DH-RES diglucuronide, DH-RES sulfoglucuronide and DH-RES disulfate as well as the metabolic profile of RES and DH-RES in the aorta, lymph, lymph node, ovaries, uterus, cerebellum, pancreas, urinary bladder tissue, fat and muscle. CONCLUSION: This study contributes to the clarification of the metabolism and tissue distribution of RES and could help to further understand the mechanisms underlying its effects.

Ellagitannins, ellagic acid and vascular health.

Hydrolysable tannins are phenolic phytochemicals that show high antioxidant and free-radical scavenging activities. For this reason their potential effects preventing oxidative related diseases, such as cardiovascular diseases, have been largely studied. In vitro studies show that ellagitannins, at concentrations in the range 10-100 µM, show some relevant anti-atherogenic, anti-thrombotic, anti-inflammatory and anti-angiogenic effects, supporting the molecular mechanisms for the vascular health benefits. While there is good evidence supporting the vascular effects in vitro, the evidence on animal models or humans is much scarcer. The in vitro results often do not match the findings in the in vivo studies. This could be explained by the low bioavailability of the antioxidant ellagitannins and ellagic acid. The main ellagitannin metabolites circulating in plasma are ellagic acid microbiota metabolites known as urolithins, and they have lost their free-radical scavenging activity. They are present in plasma as glucuronide or sulphate conjugates, at concentrations in the nM range. Future studies should focus in the bioavailable metabolites, urolithins, and in the form (conjugated with glucuronic acid or sulphate) and concentrations (nM range) in which they are found in plasma. In this review we critically discuss the role of ellagitannins and ellagic acid on vascular health.

Preventive oral treatment with resveratrol pro-prodrugs drastically reduce colon inflammation in rodents.

There is no pharmaceutical or definitive surgical cure for inflammatory bowel diseases (IBDs). The naturally occurring polyphenol resveratrol exerts anti-inflammatory properties. However, its rapid metabolism diminishes its effectiveness in the colon. The design of prodrugs to targeting active molecules to the colon provides an opportunity for therapy of IBDs. Herein we explore the efficacy of different resveratrol prodrugs and pro-prodrugs to ameliorate colon inflammation in the murine dextran sulfate sodium (DSS) model. Mice fed with a very low dose (equivalent to 10 mg for a 70 kg-person) of either resveratrol-3-O-(6'-O-butanoyl)-ß-D-glucopyranoside (6) or resveratrol-3-O-(6'-O-octanoyl)-ß-D-glucopyranoside (7) did not develop colitis symptoms and improved 6-fold the disease activity index (DAI) compared to resveratrol. Our results indicate that these pro-prodrugs exerted a dual effect: (1) they prevented the rapid metabolism of resveratrol and delivered higher quantities of resveratrol to the colon and (2) they reduced mucosal barrier imbalance and prevented diarrhea, which consequently facilitated the action of the delivered resveratrol in the colon mucosa.

Occurrence of urolithins, gut microbiota ellagic acid metabolites and proliferation markers expression response in the human prostate gland upon consumption of walnuts and pomegranate juice.

Epidemiology supports the important role of nutrition in prostate cancer (PCa) prevention. Pomegranate juice (PJ) exerts protective effects against PCa, mainly attributed to PJ ellagitannins (ETs). Our aim was to assess whether ETs or their metabolites ellagic acid and urolithins reach the human prostate upon consumption of ET-rich foods and to evaluate the effect on the expression of three proliferation biomarkers. Sixty-three patients with BPH or PCa were divided into controls and consumers of walnuts (35 g walnuts/day) or pomegranate (200 mL PJ/day) for 3 days before surgery. Independently of the ETs source, the main metabolite detected was urolithin A glucuronide, (3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide) (up to 2 ng/g) together with the traces of urolithin B glucuronide, (3-hydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide) and dimethyl ellagic acid. The small number of prostates containing metabolites was likely caused by clearance of the compounds during the fasting. This was corroborated in a parallel rat study and thus the presence of higher quantities of metabolites at earlier time points cannot be discarded. No apparent changes in the expression of CDKN1A, MKi-67 or c-Myc were found after consumption of the walnuts or PJ. Our results suggest that urolithin glucuronides and dimethyl ellagic acid may be the molecules responsible for the beneficial effects of PJ against PCa.

Up-regulation of tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 in human colon cancer Caco-2 cells following repetitive exposure to dietary levels of a polyphenol-rich chokeberry juice.

Consumption of berries and red fruits rich in polyphenols may contribute to the reduction of colon cancer through mechanisms not yet understood. In this study, we investigated the response of subconfluent Caco-2 cells (a human colon carcinoma model) to repetitive exposure (2 h a day for a 4-day period) of a subtoxic dose of a chokeberry (Aronia melanocarpa) juice containing mixed polyphenols. To mimic physiological conditions, we subjected the chokeberry juice to in vitro gastric and pancreatic digestion. The effects on viability, proliferation and cell cycle were determined, and changes in the expression of genes in response to the chokeberry treatment were screened using Affymetrix oligonucleotide microarrays. Exposure to the chokeberry juice inhibited Caco-2 cell proliferation by causing G(2)/M cell cycle arrest. We detected changes in the expression of a group of genes involved in cell growth and proliferation and cell cycle regulation, as well as those associated to colorectal cancer. A selection of these genes was further confirmed by quantitative RT-PCR. Among these, the tumor suppressor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), whose expression is known to be reduced in the majority of early adenomas and carcinomas, was up-regulated by the treatment both at the mRNA and protein levels (as shown by flow cytometry analysis). CEACAM1, with a significant regulatory role on cell proliferation of particular interest at early stages of cancer development, may be a potential target for chemoprevention by food components such as those present in polyphenol-rich fruits.