Effects of (poly)phenol-rich diets on cardiometabolic disease prevention have been largely investigated. However, the lack of adequate analytical approaches and understanding of potential individual-dependent differences in the metabolism of these plant bioactives have been obstacles in exploiting their beneficial properties on human health.

An acute intervention study which involved 298 subjects (57% women, 40.7 y (SD ± 16.3)) was designed and conducted in an Italian cohort of healthy volunteers. Biological samples (i.e., urine, feces, and plasma), as well as clinical, dietary, and lifestyle data were collected. Subjects underwent an oral (poly)phenol challenge test (OPCT) consisting in the qualitative and quantitative standardised supplementation of fifteen classes of (poly)phenols. Urine samples were collected before and over the 24-h following the OPCT. Samples were analysed using an untargeted metabolomics approach via ultra-performance liquid chromatography coupled with ion mobility separation-high resolution mass spectrometry (UHPLC-IMS-HRMS). Identification of features (i.e., mass_retention time) resulting as discriminant between sub-groups of samples will be attempted using in-house MS-library combined with data available in public databases. Metabolic phenotypes in the metabolism of (poly)phenols are also considered.

Results obtained in this study aim at contributing to the elucidation of the inter-individual differences in the metabolism of dietary (poly)phenols and consequent effect on the possibility to define personalised nutrition initiatives aimed at preventing cardiometabolic diseases.

Funding Sources: This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (PREDICT-CARE study, Grant Agreement N. 950050).

Consumption of high-fat diets (HFD) is one of the main factors associated with the development of obesity-associated onset and progression of many pathologies. HFD-associated comorbidities are attributed to the metabolic endotoxemia resulting from the transport of intestinal lumen lipopolysaccharides (LPS) across the mucosa via their incorporation into nascent chylomicrons. In rodents, dietary (-)-epicatechin (EC) supplementation decreases HFD-induced hyperlipemia and endotoxemia (increased blood LPS). This work investigated whether EC, (+)-catechin (CT), procyanidins B2 dimer (B2), and hexamer (Hex) can decrease: i) triglyceride (TG) transport/CM formation and ii) the transcellular transport of LPS, induced by oleic acid (OA) in vitro (Caco-2 cells) and by olive oil in C57BL/6 mice.  For the in vitro experiments, differentiated Caco-2 cells monolayers were incubated for 3-12 hours with 0.1-1 µM EC, CT, B2 or Hex and OA, added to the apical side. For in vivo experiments, C57BL6/J male mice were i.p. injected with tyloxapol, to inhibit plasma lipolysis, followed by gavage with EC (5-40 mg/kg body weight) and olive oil. Blood was collected at baseline and 1, 2, and 3 h post gavage. In Caco-2 cells we observed that, while not affecting intestinal monolayer integrity, EC, CT, B2, and Hex decreased OA-stimulated basolateral ApoB48 secretion (chylomicron marker) in a dose-response manner. In addition, EC, CT, B2, and Hex inhibited select steps in chylomicrons formation. All compounds prevented OA-mediated and chylomicron-dependent transcellular transport of LPS. In vivo, upon olive oil gavage, plasma TG increased over-time (1-3 h). Three hours post-gavage, EC decreased olive oil-induced postprandial hypertriglyceridemia and endotoxemia in a dose-dependent manner. In summary, findings suggest that the capacity of foods rich in EC, CT, and PCAs to ameliorate HFD-associated pathologies can be in part related to their ability to mitigate HFD-induced chylomicron formation, hyperlipemia, and endotoxemia.

Objective: To investigate the effect of walnut consumption on cardiometabolic biomarkers, gut microbiota and inflammation in overweight/obese individuals. Methods: Adults (n=30) 45-75 yo, BMI >25.0 kg/m2 and LDL- C 130-189 mg/dL were included in this 10-week RCT. Intervention: 2 oz of walnuts vs. a no-walnut diet for 3 weeks, with 1-week run-in and 3-week washout. Blood, urine and fecal samples were collected at the beginning and end of each intervention. Primary outcome: blood lipids (LDL-C, HDL-C, TC, etc.). Secondary outcomes: blood inflammatory markers (PAI-1, ICAM-1, VCAM-1, IL-6), fecal pH, moisture, consistency, microbial metabolites (urolithins, short-chain fatty acids (SCFAs), bile acids (BAs)), fecal calprotectin (CPN) & lipocalin-2 (NGAL), and 16S microbiota analysis. Results: Walnuts did not affect blood inflammatory markers but there was a decrease in blood LDL-C and TC (both p-values < 0.01), fecal NGAL and CPN (both p < 0.05) after walnuts vs. controls. A decrease in fecal lithocholic BA (p = 0.01) and several SCFAs was also observed for the walnut group. There was an increase in percent relative abundance of Roseburia and a decrease in Bifidobacteria and Alistipes genera after walnuts only (all p  < 0 .05). Participants exhibited differential ability to produce various microbial-derived Uro metabolites (e.g., Uro-A, Uro-B, IsoUro-A, etc.), which allowed for clustering into Uro metabotypes (UM-A, UM-B and UM-0). Correlation analyses revealed several associations between specific plasma/urine Uro metabolites and blood lipids, as well as fecal BAs. Conclusion: Daily walnuts for 3 weeks decreased blood TC and LDL-C as well as fecal NGAL, CPN and lithocholic BA, while also resulting in shifts in several gut microbial genera. Three Uro metabotypes were detected and levels of various plasma/urine Uros showed low/moderate correlations with blood lipids and fecal BAs. This supports the hypothesis that health responses to walnuts may be mediated by gut microbiome and Uro metabotype.

Background: Cranberries contain high levels of polyphenols, including flavanols, flavan-3-ols, and tannins, known for their anti-inflammatory properties and diverse health benefits. However, the advantages of consistent consumption of cranberry polyphenols at low concentrations for individuals living with overweight and obesity remain unclear. This study aimed to investigate the effects of daily cranberry juice cocktail consumption over six weeks on inflammation and intestinal health in 45 adults with overweight and obesity, using a double-blind, randomized, placebo-controlled design.

Methods: The study involved adults aged 18-65 years with a BMI of 28-35 kg/m² and a body fat percentage over 18% for males and 25% for females. Participants were randomly assigned to receive either 16 oz of a placebo or cranberry juice cocktail daily for six weeks. Data were collected through blood and stool samples, as well as intestinal health questionnaires, at both baseline and the end of the study period.

Results: There were no significant changes in serum inflammatory cytokine levels in either the cranberry or placebo groups over the six weeks. However, AGACHAN scores, which assess constipation severity, significantly improved in the cranberry group (p = 0.0068), whereas no changes were observed in the placebo group. Furthermore, the placebo group showed an increase in evacuation categorization scores on the Bristol Stool scale, indicating worsening stool consistency and frequency from week three to six (p = 0.0236), while the cranberry group did not exhibit such increases.

Conclusions: The daily intake of cranberry juice cocktail for six weeks was well tolerated and effectively reduced symptoms of constipation in adults affected by overweight and obesity. These results support the potential role of cranberry polyphenols in enhancing intestinal health, without impacting systemic inflammation.

Tyrosine phenol–lyase (TPL) expressed in intestinal bacteria catalyzes the β-elimination of L-Tyr to produce phenol. Phenol and phenyl sulfate (PS), sulfate conjugate of phenol, are known as protein-bound uremic toxins. The elevation of the PS levels in blood is implicated in the etiology of uremia. In this study, we explored the TPL inhibitors in dietary (poly)phenols. Here, we present that epicatechin gallate, epigallocatechin gallate, and gallic acid strongly exhibit an inhibition of TPL activity. As a result of the structure-activity relationship between gallic acid and TPL, the carboxy group and meta-hydroxy group in gallic acid play an essential role in TPL inhibition. We have consequently identified 3,5-dihydroxybenzoic acid (3,5DHBA) as the strongest inhibitor among the hydroxybenzoic acids. TPL enzyme kinetics and X-ray crystal structure analysis demonstrated that 3,5DHBA competitively inhibited TPL activity (Ki = 13 µM) and the carboxy group and hydroxy group of 3,5DHBA forming ionic and hydrogen bonds with Y71, R100, R381, and R404 in the active site. Additionally, TPL-expressing bacteria (Citrobacter koseri and Morganella morganii) were incubated in M9 salt medium containing 4 mM L-Tyr and 100 or 500 µM 3,5DHBA for 30 min, 1 h, and 3 h, and the phenol concentration in the medium was determined. 3,5DHBA significantly inhibited phenol production in time- and dose-dependent manner. Furthermore, we have observed that the increased levels of fecal phenol in C57BL/6J mice induced by the administration of Tyr–rich diet (5%) for 14 days were significantly reduced (56.8±4.4%) by the addition of 0.2% 3,5DHBA for 2 days. Our findings suggested that 3,5DHBA could be the most promising phenolic metabolite to inhibit phenol production. Since phenol and PS have low reduction rates by hemodialysis, the oral administration of the strong TPL inhibitors or their parental (poly)phenols could be an effective strategy for phenol and PS–induced uremia.
 

Background: Loss of functional capacity is one of the hallmarks in cardiovascular aging. Cocoa flavanols (CF) exert favorable effects on endothelial function, blood pressure, and inflammation. These cardiovascular health markers worsen with increasing age and limit functional exercise capacity. Aim: To investigate the effect of CF on cardiorespiratory-fitness in healthy elderly people. Methods: In a randomized, double-masked, placebo-controlled, parallel-group dietary intervention trial, 68 healthy elderly people (55-79 years, 28 female) received either 500 mg of CF or a nutrient-matched control capsule twice a day for 30 days. Primary endpoint was defined as peak oxygen consumption (VO2) in a cardiopulmonary exercise test (CPET). Secondary endpoints were oxygen pulse (VO2 per heart rate (HR)), resting blood pressure (BP), and resting vascular function. Results: After 30 days of CF intake peakVO2 increased by 190 ml min-1 (95% CI 1-371 ml min-1) and peakVO2 per kg by 2.5 ml (min kg)-1 (95% CI 0.30-4.2 ml (min kg)-1). O2-pulse increased by 1.7 ml (95% CI 0.29-3.2 ml) and max exercise capacity by 9.6 W (95% CI 2.1-17.7 W). CF decreased resting systolic and diastolic BP by 5.4 mmHg (95% CI -10.7 to -0.1 mmHg) and 2.9 mmHg (95% CI -5.5 to -0.4 mmHg), respectively. Flow-mediated vasodilation (FMD) increased by an absolute 1.3% (95% CI 0.76-1.79%) in the CF group. Indexes of pulmonary function were not affected. No changes for primary and secondary endpoints were detected in control. Conclusion: CF substantially improve markers of cardiorespiratory fitness in healthy elderly humans highlighting their potential to preserve cardiovascular health with increasing age.

A substantial body of evidence highlights the beneficial effects of (poly)phenols on non-communicable diseases. However, apart from recent recommendations on flavan-3-ol intake for optimal cardiometabolic health, no dietary guidelines exist for these compounds. The UK Nutrition Society Special Interest Group on Phytochemicals and Health has designed a framework to assess evidence for dietary recommendations on phytochemicals, including (poly)phenols. 

The framework comprises three phases. Phase 1, the Scoping Phase, involves a non-systematic, comprehensive review of existing literature to frame the compounds of interest and inform systematic evaluations. This phase includes defining the compounds, compiling dietary sources, and estimating intake and bioavailability using databases and literature searches. It assesses the validity of intake data through dietary assessment tools and biomarkers, and summarizes Absorption, Distribution, Metabolism, and Excretion (ADME) data, considering inter-individual variations and interactions with other dietary components. Preliminary safety and efficacy evaluations include reviews of international and national safety reports and non-systematic literature searches to assess the volume and scope of available research, including adverse event reports and nutrient and drug interactions. 

Phase 2 involves a thorough review of existing evidence, conducting systematic reviews as defined in Phase 1 and assessing the quality using tools like AMSTAR and the Cochrane risk of bias tool. The certainty of the evidence is evaluated using a combination of the UK Scientific Advisory Committee on Nutrition Framework and the GRADE framework, considering study limitations, inconsistency, indirectness, imprecision, and publication bias. 

Phase 3 aims to develop clear, evidence-based dietary recommendations based on the data reviewed in Phases 1 and 2. This includes summarizing evidence, highlighting the relationship between polyphenol intake and health outcomes, and developing actionable recommendations. This framework offers a structured approach to developing dietary recommendations for phytochemicals, ensuring scientific rigor and practical applicability. 

Macro and micronutrients, like polyphenols have been extensively studied for their effects on vascular and consequently on their role in developing or preventing onsets of cardiometabolic and neurological diseases. Recent data indicate that in biological systems, these nutrients act primarily through genomic and epigenomic mechanisms. The molecular mechanisms underlying their health effects are still not well identified, mainly due to the use of physiologically non-relevant conditions, but also due to the use of targeted genomic approaches aiming to evaluate the effect only on few specific genes, thus preventing to decipher detailed molecular mechanisms involved. The use of state-of-the-art untargeted analytical methods represents a significant breakthrough in nutrigenomics, as these methods enable detailed insights into the effects at each specific omics level. Moreover, the implementation of multi-omics approaches allows integration of different levels of regulation of cellular functions, to obtain a comprehensive picture of the molecular mechanisms of action. In combination with bioinformatics and the methods of machine learning, multi-omics has potential to make a huge contribution to the nutrition science. The aim of this presentation is to provide an overview of the use of the omics, multi-omics, and integrative approaches in studying the vascular effects of macro and micronutrients as well as address the potentials for use of the machine learning in nutrigenomics.

Preclinical research in inbred rodents consistently demonstrates strong anti-obesity activities of (−)-epigallocatechin gallate (EGCG), but results from clinical trials are variable. Questions remain regarding the genes critical for efficacy of EGCG in humans. Lack of genetic diversity in preclinical models may contribute to poor translational success. The effect of EGCG is a phenotype that depends on the genetics of the consuming organism. We studied the impact of high-fat feeding alone and with added EGCG on obesity using a longitudinal design in 50 Diversity Outbred mice. We hypothesized that the onset of obesity, and the effect of EGCG, would be highly variable due to genetic variability in the mice. When EGCG was introduced, mouse weight gain exhibited extreme variation. Additionally, 22% of mice lost body fat when EGCG was introduced. We propose that EGCG primarily explains fat loss and the slowing of weight gain, and that genetics explain the variability in the effect of both high fat diet and EGCG. Given the disconnect between inbred rodents and human genetic variability coupled with the poor track record of phytochemical translation to human clinical efficacy, use of genetically diverse rodents for preclinical discovery warrants consideration. Advances in rodent populations and the genetic tools for genotyping and QTL mapping make controlled variability an advantage: differentiation of phenotype (or response to stimuli) between hyper- and hypo-responders allows identification of the genes responsible if N is large enough. Such studies are needed in the future. Authors:  Michael Sweet, North Carolina State University; Lisard Iglesias-Carres, Fytexia; Jared Carter, Brigham Young University; Peter Ellsworth, Brigham Young University; Dahlia Nielsen, North Carolina State University; David Aylor, North Carolina State University; Jeffert Tessem, Brigham Young University; Andrew Neilson, North Carolina State University

Polyphenols are known for improving metabolic health and modulating gut microbiota. However, the combined effects of polyphenol-rich cranberry extract and dietary fibers like agavins on health are not fully understood. This research primarily aims to explore how polyphenol-rich cranberry extract (CP) and agavins (AG) affect metabolic health and gut microbiota in adults at cardiometabolic risk, with a special emphasis on assessing their impact on endotoxemia, as a primary outcome. Secondary objectives include examining changes in microbiota composition, low-grade inflammation, and overall metabolic parameters. To explore this, a randomized, double-blind, placebo-controlled trial was conducted. 112 participants were assigned to one of four groups: CP (240mg), AG (7g), CP+AG combination, or a maltodextrin placebo, for 10 weeks. Measurements taken before and after intervention included anthropometry, fecal samples, blood samples, and oral glucose tolerance tests (OGTT). Analyses covered plasma metabolic markers, inflammation, metabolic endotoxemia, fecal volatile fatty acids (VFA), and gut microbiota composition using 16S rRNA gene sequencing. The results indicate that the dietary interventions did not alter plasma glucose, low-grade inflammatory and endotoxemia markers as well as VFA levels in the overall group. Nonetheless, participants showed two distinct responses (M1 and M2) based on VFA profiles and gut microbiota composition. M1 sub-group exhibited a significant increase in SCFA and a significant decrease in BCFA levels after the intervention, as compared to M2 (p < 0 .01). M1 sub-group also had significantly higher pre-intervention levels of Bacteroides (p < 0 .02) and Ruminococcus (p < 0 .01) than M2 sub-group. M2 sub-group showed post-intervention increases in certain gut bacteria, such as Bacteroides uniformis (p=0.004), L. NK4A136 (p=0.006), Oscillibacter (p=0.0003) and P. merdae (P=0.009). These findings suggest that individual variability outweighed the influence of CP and AG on endotoxemia, overall metabolic parameters, low-grade inflammation, and microbiota composition. Interestingly, our results led us to classify participants into two profiles based on VFA and microbiota.

This study investigated the role of the gut microbiome in determining exposure to blueberry polyphenol metabolites using germ-free mice and those colonized with fecal microbiomes collected from humans that participated in a 12-week blueberry feeding intervention. Fecal samples were collected from postmenopausal women with above-normal blood pressure before (Pre-BB) and after 12 weeks of blueberry consumption (Post-BB), or 12 weeks post-placebo consumption (Post-PL). Pooled samples were used to colonize 6-week-old germ-free (GF) male C57BL/6 mice, while a control group remained germ-free (total n = 24, n = 6/group). Six weeks after gut microbiota colonization, mice were gavaged with 116.1 mg of freeze-dried blueberry powder, and blood was collected after 1 and 6 hours. Plasma polyphenol metabolites were analyzed using microelution solid-phase extraction coupled with liquid chromatography-mass spectrometry.&nbsp;At 1 hour, gut microbial metabolite 4-methylcatechol-1-sulfate was higher in colonized mice compared to GF mice (p < 0.05). At 6 hours, several gut microbial metabolites (e.g., 4-methylcatechol-1-sulfate, hippuric acid, 2,5-dihydroxybenzoic acid, hydroxyphenyllactic acid, isovanillic acid 3-O-sulfate, dihydro caffeic acid 3-O-sulfate, and isoferulic acid 3-O-sulfate) were higher (p < 0.05) in colonized mice than GF mice. However, at 6 hours, several parent anthocyanins (i.e., malvidin 3-arabinoside, -galactoside, and -glucoside, peonidin 3-galactoside) were higher in GF mice than Post-BB and Post-PL groups (p < 0.05), as well as other compounds present in blueberries (e.g., chlorogenic acid, 3-feruloylquinic acid), which were higher (p < 0.05) in GF mice than in all colonized mice.&nbsp; These data identify which polyphenol metabolites result from gut microbiome metabolism and are critical for identifying how these microbes influence blueberry polyphenol metabolite exposure. Next steps include evaluation of chronic blueberry consumption on gut microbial polyphenol metabolizing capacity and understanding the role of these metabolites in improving endothelial function and predicting individual responses to blueberry intake.

Flavonoids exert vasculoprotective effects in humans, but interindividual variability in their action has also been reported. This study aims to identify genes that are associated with vascular health effects of flavonoids and whose polymorphisms could explain interindividual variability in response to their intake. Applying predetermined literature search criteria, we identified five human intervention studies reporting positive effects of flavonoids on vascular function together with global genomic changes analyzed using microarray methods. Genes involved in vascular dysfunction were identified from genome-wide association studies (GWAS). By extracting data from the eligible human intervention studies, we obtained 5807 differentially expressed genes (DEGs). The number of identified upstream regulators (URs) varied across the studies, from 227 to 1407. The search of GWAS Catalog revealed 493 genes associated with vascular dysfunction. An integrative analysis of transcriptomic data with GWAS genes identified 106 candidate DEGs and 42 candidate URs, while subsequent functional analyses and literature search identified 20 top priority candidate genes: ALDH2, APOE, CAPZA1, CYP11B2, GNA13, IL6, IRF5, LDLR, LPL, LSP1, MKNK1, MMP3, MTHFR, MYO6, NCR3, PPARG, SARM1, TCF20, TCF7L2, and TNF. In conclusion, this integrated analysis identifies important genes to design future nutrigenetic studies for development of precision nutrition for polyphenols.

Flavan-3-ols are the most consumed flavonoids in Western diets. Evidence suggests that circulating flavan-3-ol metabolites may act as mediators of their beneficial effects on health. The chemical structure of parent flavan-3-ols may affect their metabolism, with a putative impact on biological response. This study aims to evaluate the influence of flavan-3-ol structure on the quali-quantitative urinary excretion of phenolic metabolites. A double-blind, randomized, cross-over, acute intervention trial was performed in 11 healthy subjects consuming 1 mol of total flavan-3-ols from three different sources: green tea extract (GT), grape seed extract with high monomer content (GSM), and grape seed extract with high procyanidin content (GSP). Baseline spot urine and urine produced during 24 hours (48 hours for 6 subjects) after extract administration were collected. A total of 75 flavan-3-ol metabolites were quantified in urine samples, with phenyl-&gamma;-valerolactones being the main class. Flavan-3-ols from GT showed the highest bioavailability, while flavan-3-ols from GSP displayed the lowest value. GT consumption resulted in the production of (epi)gallocatechin derivatives, 5-(3ʹ,4ʹ,5ʹ-trihydroxyphenyl)- and 5-(3ʹ,5ʹ-dihydroxyphenyl)-&gamma;-valerolactones, trihydroxybenzoic acids, trihydroxybenzenes, and 4ʹ-hydroxycinnamic acids. GSM consumption was characterized by (epi)catechins, monohydroxy- and 5-(3ʹ,4ʹ-dihydroxyphenyl)-&gamma;-valerolactones, phenylvaleric acids, phenylpropanoic acids, and dihydroxybenzenes. GSP administration was characterized by cinnamic acid, phenylacetic acid, benzoic acid, and benzaldehyde production. Moreover, the endogenous production of phenolic metabolites was evaluated, suggesting that small phenolic acids and benzene derivatives may determine biases in the bioavailability estimation. This work suggests that flavan-3-ol structure influences the bioavailability and metabolism of this (poly)phenol class, and it represents a key aspect to be considered in the investigation of the health effects derived from the intake of (poly)phenol-rich foods.

(Poly)Phenols pharmacological activity is difficult to demonstrate due to their mild physiological effects, and to the large inter-individual variability observed. Many (poly)phenols have little bioavailability and reach the colon almost unaltered. There they encounter the gut microbes resulting in a two-way interaction in which (poly)phenols modulate the gut microbiota composition, and the intestinal microbes catabolize phenolics to release metabolites that are often more active and better absorbed than the native (poly)phenols. The nature and amount of the metabolites produced in humans depend on the gut microbiota composition and function, and different metabotypes have been identified. Nevertheless, not all the metabolites have the same biological activity, and thus the observed effects of dietary (poly)phenols hang on the gut microbiota. Some examples have shown that the health effects of (poly)phenols differ with the gut microbiota composition and the gut microbiota metabotypes. This has been shown for tea flavan 3-ols, ellagitannins and ellagic acid from different sources, lignans, and stilbenoids. Stratification in clinical trials according to  individuals’ metabotypes is necessary to fully understand the health effects of phenolic compounds. Here we present and discuss the most significant and updated knowledge regarding the reciprocal interrelation of the gut microbiota with dietary (poly)phenols as a key factor that modulates their health effects.

Heterogeneous results are obtained from clinical trials investigating the health effects of flavan-3-ols. Since these molecules are poorly absorbed in the small intestine, this heterogeneity has been mainly attributed to the interindividual variability associated with the capacity of the gut microbiota to convert flavan-3-ols into smaller bioavailable and bioactive metabolites. While many studies looked at the microbial metabolism of monomeric flavan-3-ols, there is limited evidence regarding the capacity of the gut microbiota to metabolize larger flavan-3-ols and their modulating effects on the gut microbiota. The aim of our study is to evaluate the ability of the gut microbiota to convert oligomeric A-type flavan-3-ols from cranberry and polymeric B-type flavan-3-ols from aronia into metabolites and assess the impact of these molecules on the composition and function of the gut microbiota.

We conducted a clinical trial, in which 39 subjects consumed 480 mg of cranberry extract (containing 82.3 mg of flavan-3-ols) daily for 4 days. Fecal, plasma and 24h urine samples were collected at the beginning and end of the supplementation. With the fecal samples collected at the beginning, 24h fecal batch fermentations were performed with oligomeric A-type flavan-3-ols from cranberry and polymeric B-type flavan-3-ols from aronia.

Fecal batch fermentations showed that  < 1 % of both flavan-3-ols-rich fractions were converted into bioavailable microbial metabolites. Moreover, no significant difference was observed between the concentration of urinary flavan-3-ols microbial metabolites measured at the beginning and end of the supplementation. However, a strong bifidogenic effect was observed, along with the bloom of several butyrate-producing bacteria. Fecal and plasmatic ratio of acetate was decreased, while butyrate was increased.

In conclusion, the health effects of oligomeric/polymeric flavan-3-ols much likely stems from their capacity to positively modulate both the composition and function of the gut microbiota, rather than by their microbial metabolism and the subsequent absorption of metabolites.

Background and Objectives: Cocoa is a rich source of flavan-3-ols, whose beneficial effects on gut health are likely due to their metabolites including phenyl-&gamma;-valerolactones. Ileostomy studies provide unique insights into in vivo food digestion, allowing identification & bioactivity testing of physiologically relevant dietary phytochemicals and their metabolites. This study investigated the effects of in vivo digested cocoa (poly)phenols and their gut-derived metabolites on gene expression related to intestinal barrier function.

Methods: Ileal samples were obtained from a randomised, double-blinded, two-way crossover acute feeding study (NCT03765606). Ileal fluids collected from 5 volunteers before and after (0-8 hr) intake of a flavanol-containing cocoa-based drink (~600mg), underwent an anaerobic temperature & pH controlled faecal batch fermentation (24 hr). Samples collected after 0, 5, 10 and 24 hr were analysed for microbiome and metabolome composition and subsequently tested for bioactivity. A murine (black male C57BL/6 mouse) intestinal organoid model was used to assess (multiplex PCR) the effect of cocoa ileal fluid fermentates (CIFFs) on 56 genes associated with barrier function.

Results:  A significant proportion of the ingested cocoa flavan-3-ols reached the colon as methoxy- and/or sulphated-metabolites. Fermentation of cocoa enriched ileal fluid using an in vitro gut model resulted in increases in flavan-3-ol metabolites including 3’,4’-dihydroxyphenyl-&gamma;-valerolactones and were significantly correlated with gut microbiota genera including Faecalibacterium. CIFFs were non-cytotoxic and increased gene expression of MUC2 (mucosal defence), TJP1 (tight junctions) and other target genes in the intestinal organoid model.   

Observational data suggests a J-shaped curve in relation to coffee consumption and health protection with the most benefit being between ~3 and 5 cups of coffee per day.

It is likely that these benefits are driven by bioactives such as chlorogenic acids. However, not all coffee is made equal; the levels of bioactives are likely to vary depending on how it is prepared. To try and better define a ‘cup of coffee’, we have performed the first comprehensive analysis of coffee brews prepared by different methods as commonly consumed.

Coffee roasted to a low, medium and high extent and ground based on the brewing method (fine, medium or coarse) was used. Coffee was prepared in triplicate using bottled mineral water, by the following preparation methods; French press, stove espresso, cold brew, espresso, boiled (Skandinavian), Aeropress, coffee bag and paper filter. A UPLC-MSn method was developed using authentic standards and is being validated to quantify 3-caffeoylquinic acid, 4-caffeoylquinic acid, 5-caffeoylquinic acid, trigonelline, caffeine, N-methylpyridinium, niacin and hydroxyhydroquinone and an HPLC method was developed to quantify diterpenes.

The full data set will be presented.

Understanding how coffee preparation impacts the bioactive profile will provide insight as to the most favourable coffee brew for health benefits.

Metabolic homeostasis is linked to circadian rhythms. Disrupting these rhythms due to mistimed eating can increase the risk of metabolic diseases. Evening carbohydrate consumption increases postprandial glycemia, a major risk factor for type 2 diabetes, more so than in the morning. Evidence suggests green tea, high in (-)-epigallocatechin-3-gallate (EGCG), reduces postprandial glycemia when consumed with starchy foods. Combining (poly)phenols with evening meals is a valuable approach to consider. However, limited human research exists on the impact of (poly)phenols on circadian-metabolic responses. Our scoping review identified limited cell-based studies showing that (poly)phenols can restore glucose metabolism via clock components, although most studies used EGCG at supraphysiological concentrations (˃ 10 μM). BMAL1, a key circadian clock protein, was shown to improve glucose metabolism in mouse hepatocytes treated with 10 μM EGCG. We tested the hypothesis that combining green tea extract with a starchy meal could lower postprandial glucose and insulin levels in the morning and evening by conducting a randomized, double-blind, controlled, crossover study. Fasted healthy volunteers (n= 14) attended four visits (two at 8:00 AM and two at 6:00 PM) and consumed either a control (rice) or test meal (rice with incorporated green tea). Postprandial blood glucose and insulin levels were measured over three hours and incremental areas under the curve were calculated. Although green tea extract did not reduce postprandial glycemia, it significantly decreased insulin concentrations and peak insulin levels 30 minutes after the morning meal but not in the evening. These results suggest that green tea polyphenols may influence insulin secretion through circadian mechanisms, independent of carbohydrate digestion and absorption. Further studies are needed to understand how (poly)phenols in meals at different times of the day can improve metabolic health.

Coffee is reported to have some beneficial effects on cognitive function and glycaemic regulation including reduced risk of cognitive decline, improved performance on cognitive tests and reduced risk of type 2 diabetes, which are also a population that is at a higher risk of cognitive decline (1). These effects have been observed for both caffeinated and decaffeinated coffee, and have been associated with the polyphenol chlorogenic acid (CGA) (2). This polyphenol has been found to be more concentrated and bioavailable in certain coffees, known as “specialty coffee,” a term defined by the Specialty Coffee Association of America indicating a coffee that has met rigorous quality standards from growing to roasting (3). This current project has built on previous findings to explore the effects of specialty coffee on cognitive function in people with type 2 diabetes through a randomised, controlled, acute, crossover trial with two conditions taking place over 24 hours on two occasions separated by one week: a high CGA specialty coffee group and a conventional coffee control group. Participants (n=38) were included if they were 30 years or older, consumed 1-4 cups of coffee daily, and had a Type 2 Diabetes diagnosis for at least 1 year. Participants consumed each beverage 1 week apart in a random order and the test battery was completed before consumption, 5 hours after consumption, and 24 hours after consumption based on findings that CGA metabolism takes up to 36 hours. The test battery included questionnaires relating to health and mood measures, and cognitive tasks assessing memory and processing speed thought to be sensitive to the consumption of CGA-rich coffee (4, 5). Data collection is ongoing with 31 participants enrolled and an expected completion date of July 2024. Testing is 50% complete and analyses are projected to begin by late spring.

Objective: The microbial-mammalian metabolite trimethylamine N-oxide (TMAO) is a potential risk factor for atherosclerosis, thrombosis, and cardiovascular disease. Reduction of circulating TMAO levels may represent a mechanism by which cocoa protects cardiovascular health. In rats, short-term consumption of cocoa flavanols may alter choline metabolism and reduce urinary TMAO levels. However, evidence is limited whether cocoa extract (CE) changes TMAO and choline levels over time. We conducted a pilot study to evaluate the effect of CE on 2-year changes in TMAO and choline.

Methods: Participants were randomized into the COcoa Supplement and Multivitamin Outcomes Study (COSMOS), a randomized, placebo-controlled, 2x2 factorial trial testing a CE supplement (containing 500 mg/d flavanols) and a daily multivitamin (MVM) for chronic disease prevention in 21,442 older adults. We randomly selected 40 COSMOS participants with blood samples at baseline and year 2. TMAO and Choline were measured in blood samples employing an Opentrons OT-2 liquid handling robot for sample prep coupled with a Waters Acquity UPLC-TQD instrument for LC-MS/MS analysis. We excluded 3 participants with extreme TMAO values ≥50. In intention-to-treat analyses, linear mixed effect models assessed the effect of CE, adjusting for age, sex, and the MVM intervention.

Results: Overall, we included 20 women and 20 men with mean age of 78 years at baseline. Randomization successfully distributed baseline demographic, clinical, behavioral, and dietary characteristics by treatment group.Among 37 COSMOS participants, those assigned a CE supplement versus placebo had borderline significant 2-year increases in serum TMAO (+0.82 ± 0.42 vs +0.09 ± 0.48; p=0.04) and choline (+1.23 ± 0.30 vs +0.62 ± 0.26; p=0.03) levels versus placebo.  

Conclusions: Daily CE supplementation was associated with statistically significant 2-year increases in serum TMAO and choline levels in a pilot study in COSMOS. Additional analyses are needed to understand the interrelationships among CE, TMAO, and choline to improve our understanding of mechanisms that explain how CE supplementation may modify risk of CVD.  

Background: Proanthocyanidins (PAC) and oligosaccharides from cranberry exhibit multiple bioactive health properties and persist intact in the colon post-ingestion. Their interaction with the microbiome, that varies over time and across gut regions, remains inadequately understood. This study aimed to investigate the impact of cranberry extract on gut microbiota ecology and function, and the resulting effects on the gut epithelium.

Methods: The effect of cranberry extract was studied on six healthy participants over two weeks using the ex vivo artificial fermentation system TWIN-M-SHIME to replicate luminal and mucosal niches of the ascending and transverse colon. Fermentation effluents from four donors were incubated on murine intestinal organoids to investigate the effects on genes involved in epithelial cell differentiation (ATOH1, HES1, KLF4) and intestinal barrier integrity and defensive response (MUC2, TJP1, DEFA20, GPR55).

Results: Cranberry extract supplementation significantly influenced gut microbiota ecology under ex vivo conditions, altering bacterial metabolism. Bifidobacterium adolescentis flourished in the mucus of the ascending colon, accompanied by reduced Proteobacteria adhesion. Bacterial metabolism shifted from acetate to propionate and butyrate production. Although microbiota modulation varied among the six donors, the butyrogenic effect was consistent. This metabolic shift was associated with increased relative abundance of several short-chain fatty acid producing bacterial genera and the formation of a consortium of key butyrogenic bacteria in the transverse colon mucus. Incubations of fermentation effluents and organoids showed increased expression of genes involved in goblet cells differentiation, mucus production and intestinal barrier integrity, consistent with the effects of pure PAC. This response remained consistent across all donors, indicating functional stability despite variations in microbiota composition. However, it diminished after two weeks, suggesting that both the microbiota and the epithelium to the treatment.

Conclusions: These observations suggest that cranberry extract supplementation can modulate gut microbiota and host response to improve gut barrier function and potentially promote overall gut health.

Brazil’s rich biodiversity, encompassing the Atlantic Forest and Cerrado, is home to numerous native fruits that are excellent sources of polyphenols, known for their protective roles against non-communicable diseases. Passiflora tenuifila, a native species of passion fruit, is particularly rich in proanthocyanidins and fibers. This study investigated its effects on hyperlipidemic, overweight individuals through a crossover-controlled clinical trial involving 15 men and women. Participants consumed either P. tenuifila or a fiber control for two weeks, with a one-month washout period between interventions. Blood samples were collected on days 1 and 15 of each arm, and both biochemical parameters and transcriptomic changes in PBMC were analyzed. Biochemical analyses revealed that P. tenuifila consumption significantly reduced biomarkers associated with insulin resistance, including aspartate aminotransferase, insulin, and IL-6 levels. At the transcriptomic level, gender-specific differences in gene expression were observed following P. tenuifila consumption. In women, the expression of 5895 genes was significantly modulated, with enriched pathways involving peroxisome function, ABC transporters, TNF signaling, and cell signaling pathways such as PI3K-Akt and MAPK. These pathways are crucial in lipid metabolism, inflammation, and cellular signaling processes. In men, 3017 genes were modulated, with pathways linked to carbohydrate, sugar, cholesterol metabolism, HDL function, and vascular endothelial function, notably through VEGFA-VEGFR2 and STAT3 signaling. The findings suggest that P. tenuifila exerts beneficial effects on both insulin resistance biomarkers and gene expression profiles in a gender-specific manner, highlighting the importance of personalized nutrition. The modulation of key metabolic and inflammatory pathways underscores the potential of P. tenuifila as a functional food in the management of metabolic disorders, with distinct molecular responses in men and women. This study was funded by FAPESP (São Paulo Research Foundation) Project number 2022/05463-8.

Evidence has been reported on the crucial role of (poly)phenols in preventing chronic diseases. However, their bioavailability greatly differs across individuals, causing uneven physiological responses.

Here, we present the first genome-wide association studies (GWAS) on the main dietary (poly)phenols-related traits, aiming to gain genetic insights into inter-individual differences and unravel their potential pathophysiological impact.  Three hundred healthy individuals were recruited, and biological samples, anthropometric measurements, health status and lifestyle information were collected.  Urinary phenolic metabolites were used to identify clusters of individuals sharing similar metabolic phenotypes, as computed by principal components (PC) and sparse k-means algorithm (SKM). Moreover, individuals were divided by enterolactone metabolic capacity (EL) as an example for a given family of (poly)phenols.  After genotyping and quality controls, 287 individuals and 511908 markers were retained (median age: 38 years old; 57% female).

To empower genetic data, samples underwent genotype imputation on the Michigan Imputation Server, obtaining 6022639 variants.  The resulting clusters served as phenotypes (PC, SKM and EL) to perform the GWAS using PLINK2.  The GWAS pointed out about 1500 significant variants for each trait. In silico functional analyses were performed using FUMAGWAS: variants were annotated by SNP2GENE, and the role of the associated genes was assessed by GENE2FUNC.

Genes were first correlated with expression data from the GTEx catalog: differential expression was observed in the brain, the blood, and the colon. Moreover, pathways were investigated: PC showed associations with glucuronidation and different metabolic pathways; SKM with the neuronal system; EL with the ephrin signaling. Associations with miRNAs were also identified in all the traits. Finally, correlations with the GWAS catalog were investigated: significant hits were observed in the gut microbiome for all the traits; other hits were observed for cardiovascular diseases (PC), metabolites (SKM), and obesity and insulin level (EL).

Background: Preeclampsia, affecting 2-8% of pregnancies, is a major contributor to maternal and perinatal health risks. Maternal diet plays a crucial role in both maternal and offspring health and a dietary intervention based on a high polyphenol Mediterranean diet may mitigate the risk of preeclampsia.

Objective: To investigate whether a structured life-style intervention based on a Mediterranean diet high in polyphenols or Stress Reduction in high-risk pregnancies for small-for-gestational age (SGA) newborns can reduce the incidence of preeclampsia.

Methods: In a randomized clinical trial with parallel-group conducted at a University Hospital in Barcelona, Spain (2017-2020), 1221 pregnant women at high-risk for SGA newborns were randomly allocated at 19-23 weeks’ gestation into three groups: a Mediterranean diet intervention, a Stress reduction program or usual care (UC). Participants in the Mediterranean diet group (n=407) received monthly individual and group educational sessions, and free supply of extra-virgin olive oil and walnuts. Women in the Stress reduction group (n=407) underwent an 8-week mindfulness program adapted for pregnancy. While the main outcome was the prevalence of SGA, early (delivery  < 3 4 weeks) and late (delivery ³34 weeks) preeclampsia were analyzed as secondary outcomes.

Results: Among 1182 participants included for the intention-to-treat-analysis (n=392 Mediterranean diet; n=390 Stress reduction; n=400 UC), preeclampsia occurred in 83 women (7%), and in the majority (n=77, 6.5%) occurred late. Late preeclampsia was significantly reduced in the Mediterranean diet group compared to UC: n=20 (5.1%) vs. 36 (9%); OR 0.54, 95% CI 0.31-0.96; p=0.035. In the Stress reduction group (vs. UC, there was a non-significant trend for a lower risk of late preeclampsia (n=22 (5.5%), p=0.054).

Conclusion: A structured intervention during pregnancy based on a high polyphenol Mediterranean diet supplemented with extra-virgin olive oil and nuts reduced remarkably the incidence of late preeclampsia.

Silybum marianum (milk thistle) fruit extract silymarin contains the flavonolignans silybin, isosilybin, silychristin, silydianin, and their 2,3-dehydroderivatives, together with other minor flavonoids and a “polymeric” fraction and is a popular component of food supplements. Biotransformation of individual silymarin components by human microbiota was studied ex vivo, using batch incubations inoculated by fecal slurry and UHPL/MS. The initial experiment at 200 mg/L showed that flavonolignans are resistant to the metabolic action of intestinal microbiota. At 10 mg/L, biotransformation of flavonolignans was much slower than that of taxifolin. While silybin, isosilybin, and 2,3-dehydrosilybin underwent mostly demethylation, silychristin was predominantly reduced. Silydianin, 2,3-dehydrosilychristin and 2,3-dehydrosilydianin were reduced, as well, and decarbonylation and cysteine conjugation proceeded. No low-molecular-weight phenolic metabolites were detected. The interaction of the whole silymarin complex with microbiota was investigated with a focus on aging populations. Using advanced techniques such as NGS, NMR and LC-MS, we analyzed the dual impact of the microbiome on silymarin metabolism and the effect of silymarin on the microbiome’s structure and function. Silymarin significantly altered the metabolism of the gut microbiota, decreasing short-chain fatty acid production and glucose utilization. Healthy elders (70–80 years) showed a significant increase in a specific catabolite associated with Oscillibacter. Conversely, healthy young donors (12–45 years) exhibited faster breakdown of silymarin components, particularly isosilybin B, which negatively correlated with higher abundance of Faecalibacterium and Erysipelotrochaceae UCG-003. Finally, in 33 healthy adult male volunteers, who received 200 mg of silymarin orally twice daily for three months, bacterial composition of feces was investigated by NGS. A correlation was found between the number of metabolites and the composition of the intestinal microbiota. In all studies, strong inter-individual differences were observed among the individual donors/volunteers.

Supported by GAČR 23-04654S and 2304655S and MEYS CZ.02.01.01/00/22_008/0004597.

Introduction: Higher habitual dietary flavonoid intakes have been associated with lower mortality and chronic disease risk, but the role of flavonoid diversity has never been investigated.

Objective: To examine associations of quantity and diversity of dietary flavonoid intake with risk of all-cause mortality and major chronic disease.

Methods: 124,806 UK Biobank participants [mean (SD) age: 60.2 (7.8) years; ~55% female] were followed for up to 11.8 years. Flavonoid intake was estimated from repeated 24-hour dietary assessments and diversity was calculated using an algorithm which factored for both variety and distribution of consumption. Associations with all-cause mortality and incidence of cardiovascular disease (CVD), type 2 diabetes (T2DM), total cancer, respiratory disease, and neurodegenerative disease were assessed using Cox models.

Results: Both a higher quantity and a higher diversity of dietary flavonoid intake associated (following mutual adjustment and after accounting for socio-demographic, lifestyle and dietary confounders) with a lower risk of all-cause mortality and several chronic diseases: comparing higher (Q5) to lower intakes (Q1), the HRs (95% CIs) for quantity and diversity respectively were 0.83 (0.76–0.91) and 0.85 (0.77–0.94) for all-cause mortality, 0.87 (0.80–0.95) and 0.88 (0.80–0.96) for CVD, 0.76 (0.67–0.86) and 0.81 (0.70–0.92) for T2DM, 0.92 (0.85–0.99) and 0.91 (0.85–0.99) for total cancer, and 0.90 (0.84–0.96) and 0.91 (0.85–0.97) for respiratory disease; only intake quantity associated with neurodegenerative disease risk [Q5 vs Q1: 0.79 (0.67–0.93)]. Examining individual subclasses, for several, only intake quantity predicted risk, though for some, both quantity and diversity inversely associated including flavan-3-ols with all-cause mortality and T2DM, and flavones with T2DM. Diversity of flavonoid-rich food intake also inversely associated with all-cause mortality and several types of chronic disease.

Conclusion: These findings suggest that increasing both quantity and diversity of dietary flavonoid intake is better for long-term health than increasing either aspect alone. 

Background: (Poly)phenols are bioactive compounds naturally present in plant-based foods but they have been suggested to increase the prostate cancer (PCa) risk in retrospective case-control studies. Therefore, our aim is to prospectively evaluate these associations, using dietary questionnaires and biomarkers, including overall and clinically relevant subtypes of PCa.
Methods: EPIC, an on-going prospective cohort study, recruited 131,425 adult men, in the nineties, from 7 European countries. Almost 7,000 incident PCa cases were diagnosed during a mean follow-up of 14-y. In the entire cohort, dietary (poly)phenol intake was assessed by combining validated center/country-specific dietary questionnaires and the Phenol-Explorer database. Plasma (poly)phenol concentrations were analyzed in 851 matched PCa case&ndash;control pairs from the EPIC cohort through a targeted metabolomics approach, using HPLC-MS/MS. Multivariable-adjusted Cox proportional hazards models and multivariable conditional logistic regression were used to estimate hazard ratios (HR) and odds ratios and 95% confidence intervals (CI), respectively.
Results: No associations were observed between total dietary intake of (poly)phenols and risk of PCa, either overall (HRlog2 = 0.99, 95% CI 0.94&ndash;1.04) or by PCa subtype. Null associations were also found for all classes and subclasses of (poly)phenol intakes with the risk of PCa, overall and by PCa subtype. Plasma concentrations of gallic acid 4-sulfate (HRlog2 = 0.92, 95% CI 0.86&ndash;0.98), dihydroferulic acid 4-glucuronide (HRlog2 = 0.94, 95% CI 0.90&ndash;0.99), and dihydroferulic acid 4-sulfate (HRlog2 = 0.99, 95% CI 0.94&ndash;1.04) were inversely associated with overall PCa risk, while 3,4-dihydroxybenzoic acid (HRlog2 = 1.05, 95% CI 1.01&ndash;1.10) was positively associated with overall PCa risk.
Conclusions: Results of the current large prospective cohort study suggest that whereas dietary (poly)phenol intake may have null association with PCa risk; the presence of some microbial metabolites of (poly)phenols in plasma might indicate a protective role in PCa risk.

Flavanols have been the focus of broad-ranging health and nutrition research, including COSMOS. Based on the collective data, the Academy of Nutrition and Dietetics issued the first authoritative dietary guidance recommending dietary flavanols for cardiometabolic health. Consequently, quantifying the flavanol content of foods has become more important for agriculture, food manufacturers, as well as consumers. A complete analytical solution has been created specifically for cocoa flavanols (CF), including a CF reference material available from NIST and analytical methodologies fully accredited by AOAC as an Official Method of Analysis in 2020. These efforts can also serve as a starting point for methods aimed at quantifying flavanols in other foods and beverages. 

The AOAC 2020.05 method for CF analysis differs from the various historic methods, resulting in the same flavanol content being reported out differently. To ensure comparability of historic and contemporary analytical reports, and to support the continuity of CF content reporting and interpretations of clinical studies and food content databases, we developed an approach to address this challenge. We assessed method biases by analyzing samples reflecting a broad range of CF content and various matrices using key historic methods and AOAC 2020.05 side-by-side and constructed a comprehensive dataset. Three linear statistical models were built and cross-validated and the resulting models were applied to convert the CF content as reported based on historic methods to that of AOAC 2020.05, enabling a direct comparison between historic and contemporary data. 

The results of recent work highlight the ability to develop robust and transferable approaches for reconciling challenges related to differences in analytical methods for the quantifications of the selfsame bioactives. More importantly, it provides the basis for comparing historic and more recent studies of CF in the context of systematic reviews and meta-analyses by removing uncertainty and ambiguity introduced by analytical method evolution.

Background: A single exposure to mental stress can induce acute impairments in endothelial function, increasing the risk of cardiovascular events. During stressful periods, food choices often worsen, which can influence the consequences of stress on vascular health. For example, fat consumption can delay the recovery of endothelial function following stress and cocoa flavanols can improve endothelial function post-stress, although this has only been shown in a fasted state. Therefore, this study investigated whether cocoa flavanols consumed in combination with fat can mitigate the negative impact of fat on endothelial function following stress.

Methods: In a randomised, counterbalanced, cross-over, intervention study, 23 healthy males and females ingested a high-fat meal (56.5g fat) with high-flavanol cocoa (150mg (-)-epicatechin) or low-flavanol cocoa (< 6mg (-)-epicatechin) 1.5 hours before an 8-minute mental stress task. Vasodilatory responses (forearm blood flow, FBF), blood pressure (BP), and cardiovascular activity were assessed pre-meal at baseline, and post-meal at rest and during stress. Endothelial function (brachial artery flow-mediated dilatation, FMD) was assessed at pre-meal baseline and 30- and 90-minutes post-stress.

Results: FBF, BP and cardiovascular activity significantly increased during mental stress compared to baseline and rest (all p<.001) in both conditions. FMD was impaired at 30 (p=.003) and 90 (p=.017) minutes post-stress in the low-flavanol cocoa condition, showing a stress and fat-induced decline in endothelial function. High-flavanol cocoa attenuated the impairment in FMD at 30 minutes post-stress (no difference to baseline, p=.383) and improved FMD at 90 minutes post-stress (p=.004).

Conclusion: The mental stress task evoked cardiovascular and vasodilatory responses, which were consistent across conditions. Endothelial function was impaired following stress and fat consumption. However, high-flavanol cocoa attenuated the stress and fat-induced decline in endothelial function. These findings have implications for dietary interventions which could use flavanols to protect the vasculature from stress and stress-induced changes in food choices.

Measuring (poly)phenol intake accurately is challenging for several reasons including the lack of accurate data on food content, lack of validated assessment methods, and high variability in the (poly)phenol content of foods. Dietary assessment methods such as Food Frequency Questionnaires (FFQs) and food records have been widely used to estimate dietary intake. However, they have many limitations inherent to their self-reporting nature, while biomarkers provide a more objective approach. We have compared different methods for the estimation of (poly)phenol intake, including FFQ, 7-day food diaries (7DD), as well as plasma and urinary biomarkers. Our results showed that the agreement between FFQ and 7DD was moderate but the agreements with biomarkers measured in plasma and urine were poor. We have also developed a novel a priori (poly)phenol-rich dietary score (PPS) to measure adherence to (poly)phenol-rich dietary patterns. The PPS was positively associated with estimated intakes of most classes and subclasses of (poly)phenols but also with multiple urinary (poly)phenol metabolites, which suggests that PPS may be a good reflection of (poly)phenol intake and exposure levels, and it can be used to identify participants with higher compliance to a diet rich in (poly)phenols in a free-living population. We further developed and evaluated a metabolic signature based on the PPS using a targeted metabolomics method comprising multiple representative (poly)phenol metabolites. Our results showed that the metabolic signature comprising several metabolites from urine or plasma samples was significantly associated with the adherence to PPS, and (poly)phenol intake estimated from FFQs and 7DDs. Furthermore, a novel (Poly)phenol FFQ was also developed to capture the accurate habitual (poly)phenol intake in the UK population. The (Poly)phenol FFQ estimated (poly)phenol intake exhibited moderate agreements and associations with 7DDs and metabolites from plasma and urine samples. Strengths and limitations of using biomarkers and dietary assessment tools will be discussed.
 

Aortic blood vessels are lined with endothelial cells that regulate blood flow. Their dysfunction constitutes a crucial event in the physiopathology of atherosclerosis and cardiovascular disorders. Epicatechin, a polyphenol found in cocoa, apple or grapes, was observed to have capacity to prevent or delay the development of these diseases. However, molecular mechanisms of epicatechin on aortic endothelium are still unexplored. The objective of this study was to investigate the biologenomic effects of phase 2 and gut microbiome-derived metabolites of epicatechin in TNF-α-stimulated human aortic endothelial cells at low (nM) concentrations by evaluating their multi-omic modification (expression of mRNA, microRNA, and long non-coding RNAs). We observed that metabolites are biologically active and can modulate the expression of protein-coding and non-coding genes. In silicon 3D docking analyses revealed potential strong interaction between epicatechin metabolites and transcription factors. Integrative bioinformatics analysis of obtained data revealed complex networks of genomics modifications by acting at different levels of regulation. Metabolites modulate cellular pathways including cell adhesion, cytoskeleton organization, focal adhesion, signaling pathways, pathways regulating endothelial permeability, and interaction with immune cells. This study demonstrates multimodal mechanisms of action where epicatechin metabolites could preserve aortic endothelial cell integrity, presenting mechanisms of action underlying epicatechin’s cardioprotective properties.

Introduction: Polyphenolic compounds in cocoa extract (CE) may lower risk of chronic diseases such as cardiovascular disease (CVD) and incidence of certain cancers. However, the effects of CE supplementation among individuals with a history of cancer on CVD and cancer risks have not been fully evaluated.

Purpose: In post hoc analyses, we examined whether CE supplementation decreases cardiovascular events, incident cancer, and mortality among older adults with a history of cancer.

Methods: Participants were enrolled in the COcoa Supplement and Multivitamin Outcomes Study (COSMOS), a 2-by-2 factorial trial of CE supplementation and a multivitamin for preventing CVD and cancer among 21,442 older US adults. There were 3,443 participants with a self-reported baseline history of cancer diagnosed ≥2 years prior to randomization. In Cox proportional hazards regression models, we estimated hazard ratios (HR) and 95% confidence intervals (CI) for CE supplementation on major incident cardiovascular events (including nonfatal myocardial infarction, stroke, coronary revascularization, and CVD death), total CVD events (adding angina, peripheral artery disease, and carotid artery disease), all-cause mortality, and incident cancer in intention-to-treat analyses.

Results: Over a median of 3.75 years of follow-up, there were 155 deaths, 283 incident cancers, and 192 total cardiovascular and 170 major cardiovascular events. Participants randomized to CE did not differ from placebo for total or major cardiovascular events (Total: HR: 0.96, 95% CI: [0.73-1.28]; Major: 0.96, [0.71-1.30]). Additionally, those taking CE did not differ from placebo for all-cause mortality (1.08, [0.79-1.48]) or incident cancers (1.16, [0.92-1.47]).

Conclusions: CE supplementation did not significantly reduce risks of total or major cardiovascular events, incident cancer, or all-cause mortality among older adults with a history of cancer. Given the small number of events and length of follow-up, additional analyses will further clarify the health effects of CE among older adults with a history of cancer.
 

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. Despite the number of new NAFLD cases predicted to increase, there are no United States Food and Drug Administration-approved drugs for NAFLD. Bioactive dietary polyphenols such as proanthocyanidins (PAC) are potent in reducing the risk of NAFLD. The majority of dietary PAC are oligomeric PAC (OPAC) and polymeric PAC (PPAC), which are low in bioavailability. The biotransformation of OPAC and PPAC into bioavailable metabolites using probiotics can enhance their physiological benefits and can be considered the next generation of prebiotics. Initially, an ultrasonication-assisted aqueous-ethanol-based PAC extraction method was developed to obtain food-grade PAC suitable for use in the development of synbiotics. The ability of a mixture of Lactobacillus and Bifidobacterium probiotic bacteria (PB) together with novel PB Akkermansia muciniphila to biotransform PPAC was evaluated in the C57BL/6 mice. The biotransformed (BT)-OPAC and BT-PPAC was assessed against NAFLD using the palmitic acid (PA)-induced lipid accumulation in mouse hepatocytes (AML12). The regulation of lipogenesis and fatty acid β-oxidation were investigated. The optimum conditions to extract PAC from grape seeds were by using 47% aqueous-ethanol at 60 °C temperature, 10.14:1 solvent to solid ratio, and 53 min of sonication time. PB could biotransform PAC into bioavailable metabolites and improve the PPAC biotransformation in the mice. The biotransformed (BT)-OPAC and BT-PPAC significantly reduced the palmitic acid (PA)-induced lipid accumulation in mouse hepatocytes (AML12) by suppressing the de novo lipogenesis and promoting the fatty acid β-oxidation. Also, BT-PAC ameliorated PA-induced oxidative stress by activating the Nrf2 antioxidant pathway. BT-PAC significantly reduced PA and LPS-induced inflammation in AML12 cells by suppressing the activation of toll-like receptor 4-mediated inflammatory signaling pathways. This study presents a new ethanol-based PAC extraction process and a sustainable source for the extraction of food-grade PAC,
 

Old age is the major risk factor for sporadic Alzheimer’s disease (AD). We and others have described an association between the oxytosis/ferroptosis non-apoptotic regulated cell death pathway and aging. Based on this association, we incorporated protection against this pathway as part of a cell-based phenotypic screening approach to identify novel drug candidates for the treatment of AD. Using this approach, we identified the fisetin derivative CMS121 as a potent neuroprotective molecule that can maintain cognitive function in multiple pre-clinical models of AD. Furthermore, we identified a key target of CMS121 as fatty acid synthase, a protein which had not been previously considered in the context of AD. Importantly, not only does CMS121 maintain cognitive function in mouse models of familial AD and age-related dementia but it can also reduce or prevent a number of physiological changes that are associated with an increased risk of developing AD including type 2 diabetes, kidney dysfunction, liver inflammation and age-related hearing impairment. Together, our data suggest that CMS121 can act both in the CNS as well as in the periphery to reduce mulitple factors that contribute to the development and progression of AD and thus might serve as a new paradigm for AD treatment.

Several human behaviours/contexts within individuals’ daily lives can render the vasculature vulnerable. Experiencing an acute episode of psychological stress or prolonged sitting (1-8 h) can induce acute impairments in endothelial function up to 2%, as measured by Flow-mediated dilatation (FMD). Chronic exposure to mental stress and sedentary behaviours increase the risk of cardiovascular diseases. Stress and sitting are often associated with eating behaviours: food choices during these periods can either protect or worsen the impact of stress/sitting on vascular function. Flavonoid-rich foods, such as, cocoa and blueberries, have been widely shown to induce rapid, transient improvements in brachial FMD within 1 to 4 h of intake, making them interesting dietary strategies with which to target periods of vascular vulnerability. Conversely, healthier behaviours such as engaging in physical activity, may also be maximized by concomitant intake of flavonoid-rich foods.

We have conducted 5 randomised, counterbalanced, cross-over, double-blinded intervention studies in young and old healthy adults (N=20-60, gender balanced) to test whether flavonoid-rich interventions in the form of cocoa (695 mg flavanols) or blueberries (339 mg anthocyanins) can ameliorate endothelial function in the context of mental stress, prolonged sitting and low/moderate/high intensity exercise. We have also assessed if baseline levels of fitness (High/Low fit: VO2max > 50 or < 40 ml/kg/min, respectively) modify the vascular effects of flavonoid intake.

Our data suggest that flavonoids can prevent declines in brachial FMD after an episode of mental stress both in a fasted and postprandial state. Flavonoids also ameliorate (2 h) sitting induced impairments in both brachial and femoral FMD in both young and older adults. Importantly, they are equally effective in both high and low fit individuals. We are currently collecting data from the last RCT investigating impact of flavonoids on endothelial function post-exercise in high and low-fit individuals.

Both habitual consumption of high-fat diets (HFD) and a single high-fat meal (HFM) can cause endotoxemia and associated inflammation. With an adverse consequence being decreased expression of Brain Derived Neurotrophic Factor (BDNF), a protein not only critical for brain function, but its circulating concentrations have been inversely correlated with obesity, altered cognition, and cardiovascular disease. BDNF’s involvement in sustaining health underscores the relevance of finding strategies, e.g. diet, to mitigate decreases in circulating BDNF under proinflammatory conditions. We previously showed that consumption of anthocyanins (AC) mitigated HFD-induced intestinal permeability and associated inflammation. This work investigated if dietary supplementation with a cyanidin-and delphinidin-rich extract (CDRE) could counteract HFD/obesity-induced neuroinflammation and prevent BDNF downregulation. This was studied in the hippocampus of healthy male mice fed for 14 weeks either: i) a control diet containing 10% calories from fat; ii) a HFD containing 60% calories from fat (lard); and iii) the HFD supplemented with 2, 20 or 40 mg AC/kg body weight. HFD caused: i) endotoxemia (plasma LPS) and a 110% increase in the hippocampal expression of the LPS receptor (TLR4): ii) hippocampal microgliosis and an increased expression of genes involved in neuroinflammation, i.e. Iba-1, NOX4, TNFɑ, and IL-1β, and iii) decreased expression of hippocampal BDNF. CDRE-supplemented mice did not develop hippocampal inflammation and showed a dose dependent increase in BDNF mRNA levels. Moreover, a double-blinded placebo controlled cross-over study was conducted including healthy participants who consumed a HFM along with a CDRE supplement or placebo. CDRE supplementation mitigated the HFM-mediated postprandial endotoxemia, and significantly increased (64%) serum BDNF levels. BDNF and IL-1β mRNA levels in PBMCs were inversely correlated confirming a relationship between inflammation and decreased BDNF expression. In summary, AC supplementation could help mitigate negative effects associated with HFD in part by decreasing inflammation and preventing BDNF downregulation.

Many epidemiological studies and intervention trials have indicated that flavanols present in grapes and blueberries may reduce the risk of cardiovascular disease and dementia. However, due to the low bioavailability of these compounds, their mechanism of action remains unclear. The principal constituents of polyphenol-rich grape and blueberry extract (PEGB) are catechins and their dimers., In sensory tests performed by Quantitative Descriptive Analysis (QDA) using healthy panelists, PGEB exhibited marked astringency at concentrations where the taste of quercetin was barely detectable. The microcirculatory dynamics of rats following a single oral dose of PEGB were observed using laser Doppler techniques, demonstrating that PEGB significantly increased blood flow in testicular arterioles. In addition,  PEGB was administered orally to a rat model of Alzheimer's disease (AD) in which hippocampal neurodegeneration had been induced by administration of AlCl3 and D-galactose for 30 consecutive days. Behavioral tests were conducted the day before slaughter when brains were collected for analysis. PEGB significantly reduced behavioral changes and hippocampal neurodegeneration. Furthermore, PEGB reduced phosphorylated tau (p-Tau) levels, overexpression of amyloid precursor protein (APP), and β-amyloid (Aβ) accumulation. Furthermore, PEGB reduced the oxidant-promoting and pro-inflammatory hippocampal changes induced by Alzheimer's disease. The findings of these studies are pertinent to the prevention and treatment of Alzheimer's disease. PEGB effects on the neurodegeneration may be associated with altered circulatory dynamics, potentially due to increased sympathetic activity resulting from its astringency.

1) Impellizzeri D, et al. Int J Mol Sci. 2023, 24, 6938.

2) Osakabe N et al. Molecules, in press

Type 2 receptors (T2Rs), which are bitter taste receptors, are expressed in taste buds and enteroendocrine cells. The activation of T2Rs has been shown to promote the secretion of gastrointestinal (GI) hormones, which suppress appetite and improve glucose tolerance. Many polyphenols taste bitter and potentially influence blood sugar levels1). However, the interaction between polyphenols and T2R, of which 25 types are known to exist in humans, remains unclear. The present study employed in silico simulation methods to investigate the interaction between T2R46, the first member of the T2Rs for which a detailed three-dimensional structure has been revealed2), and 489 polyphenols listed in Phenol-Explorer (http://phenol-explorer.eu/). As the molecular docking result using comprehensive chemical calculation software MOE, almost all the polyphenols showed negative binding energy scores, indicating that polyphenols can bind to T2R46. The interaction with amino acid residue W88 is important for the activation of T2R46. Many compounds classified as flavonoids interacted with W88. Next, we calculated the structure-activity relationship (QSAR) between the binding energy and the result of the molecular docking. The result of QSAR analysis follows the equation,

S =  -1.26777 -0.01161*vsurf_S +0.00007*weinerPath (1)

where S is the binding energy score, vsurf_S is the interaction field surface area, wienerPath is the Wiener path number (measure of molecular branching). This model showed the good fit (R-squared value=0.9359) to the test polyphenols, indicating that polyphenols with larger interaction surface areas and more molecular branching will likely bind to T2R46. To check the validity of the model, 68 known ligands listed in the Bitter DB (https://bitterdb.agri.huji.ac.il/dbbitter.php) were fitted. The resulting R-squared value was 0.9155. Combined with the molecular docking results, flavonoids with the characteristics above may activate T2R46, thereby regulating energy balance.


1.Osakabe N. et al. Biomolecules. 2024, 14, 234
2.Xu W. et al. Science. 2022, 377, 1298

A Mediterranean diet rich in (poly)phenols has been associated with positive effects on neurodegenerative disorders, such as Alzheimer’s and Parkinson’s. However, the (poly)phenols’ ability to directly modulate brain disorders likely depends on their accessibility to the brain. Thus, circulating phenolic metabolites are more likely to cross the blood-brain barrier (BBB) than food-occurring (poly)phenols. Herein, the distribution kinetics in the brain tissue of circulating phenolic metabolites in Sprague Dawley rats fed a polyphenol-rich extract from foods belonging to the Mediterranean diet (pomegranate, lemon, orange, grape seed, olive and resveratrol) at human equivalent doses (HED), and screening of their potential to attenuate microglia-mediated neuroinflammation were evaluated. Kinetic data revealed 41 phenolic metabolites in plasma, 22 in non-perfused and 19 in perfused brains. Among phenolic metabolites, the bioavailable ellagic acid-derived metabolite, urolithin A (Uro-A) and its derived conjugated forms can cross the BBB. Next, we investigated whether Uro-A and other main urolithins (Uro-B and IsoUro-A) and their phase-II conjugates exert anti-(neuro)inflammatory effects on human microglial cells (HMC3) stimulated by LPS or TNF-a. Cells were treated with 5 μM of each Uro in the presence of LPS (500 ng/mL) or TNF-α (50 ng/mL) for 24 hours, and the release of pro-inflammatory cytokines (IL-6 and IL-8) was evaluated. Furthermore, NF-kB nuclear translocation was evaluated using confocal microscopy. All Uros and their conjugates showed a significant reduction of IL-6 in LPS-activated HMC3 cells. However, none of the Uros or their conjugates exerted any effect in the presence of TNFα, suggesting that the anti-(neuro)inflammatory effects are stimulus-dependent. Overall, our results suggest that the consumption of sources containing ellagic acid, the polyphenol precursor of Uros (e.g. pomegranate, walnuts or berries), could be beneficial in attenuating neuroinflammation, potentially preventing or retarding the development of neurodegenerative diseases. Acknowledgements: This work was funded by the Fundación Séneca (grant 22030/PI/22) (Murcia, Spain).

(Poly)phenols, abundant in fruits and vegetables, have emerged as pleiotropic compounds against Parkinson's Disease (PD). We identified gut-derived polyphenol metabolites (PMs) as abundant in the bloodstream, capable of crossing the blood-brain barrier (BBB), being protective against neurodegeneration and neuroinflammation. However, PMs’ mechanisms in a comprehensive/translatable model of the full brain for Parkinson’s disease (PD) have never been explored. By taking advantage of novel microphysiological systems (MPS) we explored their effects on 3D neuronal system and currently we aim disclose PMs’ molecular potential against PD on a brain-on-chip. An overview of the evidence collected in PD models will be presented. In the end, we hope to provide physiologically relevant insights for the nutritional management of PD by unveiling PMs’ physiologic mode of action in an avant-garde cell model of the pathology.  

Evidence suggests that neuroinflammation is a key hallmark in the development of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In our group, we have focused on the potential of dietary and gut microbiota metabolites, the low molecular weight phenolic metabolites (LMWPs). These molecules reach human circulation and the brain, modulating microglia function, and attenuating neuroinflammation. In this presentation, we will show the screening of more than 70 LMWPs in microglia cells, demonstrating the capability of LMWPs to attenuate inflammatory markers, and identifying LMWPs with the highest potential. Proteomic studies have shown the modulation of key inflammatory proteins unveiling the mechanism of action of LMWPs. The most effective LMWP was validated using an animal model of neuroinflammation. Meanwhile, an animal dietary intervention showed the capability of a diet rich in (poly)phenols and LMWPs to reduce neuroinflammation and behavior hallmarks of neurodegenerative diseases. Overall, we will show the anti-inflammatory capability of LMWPs in microglia cells, attenuating neuroinflammation and ultimately hampering the impact of neuroinflammation in neurodegenerative diseases.      

The consumption of ellagitannins has been associated with several health benefits. After ingestion, these compounds are hydrolyzed to ellagic acid and are further metabolized to urolithins by the gut microbiota. Therefore, both the chemical form in which this ellagitannins are found in the food matrix, as well as the microbiota composition of the individual, may impact the associated health outcomes. Thus, we investigated the effect of acid hydrolysis of jabuticaba peel and seed powder (JPSP) on ellagitannins metabolism in normoweight volunteers. Twenty volunteers participated in a single-blinded randomized crossover clinical trial. Volunteers ingested 3 g of JPSP and hydrolyzed JPSP (HJPSP), with a 14-day washout period. Urine samples were collected at intervals of 0-4 h, 4-8 h, 8-12 h, 12-24 h, 24-36 h, 36-48 h and 48-72 h after ingestion. Volunteers followed a diet free of ellagitannins four days before and during the clinical trial. After JPSP and HJPSP consumption, five and six urolithins, respectively, were identified in urine, being urolithin A 3/8-glucuronide and isourolithin A 3/9-glucuronide the main metabolites (73% of total). HJPSP intake led to an earlier urinary excretion (0-4 h) of some urolithins compared to JPSP (8-12 h). Nevertheless, no significant differences were found between the maximum excretion time of urolithins after the consumption of both JPSP and HJPSP. Volunteers were classified as metabotype A (45%), B (40%), and 0 (15%). Urolithins excretion after HJPSP ingestion was 40%, 77%, and 26% higher than that observed after JPSP when intervals 0-36 h, 0-48 h, and 0-72 h were considered, respectively. Metabotype 0 volunteers had the lowest percent body fat (5.6%), followed by metabotype A (12.9%) and metabotype B volunteers (18.6%). Thus, the body composition of individuals should be considered, together with nutritional status, when investigating ellagitannins metabolism.

Backgrounds: Flavanols have been shown to have potential blood pressure (BP)-lowering effects in  shorter-term, smaller-scale trials, but its preventive effect on incident hypertension has not been examined in a large-scale randomized controlled trial (RCT). The COcoa Supplement and Multivitamin Outcomes Study (COSMOS) provided an opportunity to evaluate whether long-term cocoa extract supplementation had an effect on incident hypertension in older adults.
Methods: COSMOS is a 2X2 factorial, double-blind, placebo-controlled RCT testing cocoa extract (including 500 mg/day cocoa flavanols, with 80 mg/day (-)-epicatechin) and a multivitamin among 21,442 women aged ≥65y and men aged ≥60y. In 8,905 COSMOS participants free from self-reported hypertension at baseline, the effect of cocoa extract on incident hypertension was investigated with median follow-up of 3.4 years. Incident cases of hypertension were primarily identified as self-reported first-time physician diagnosis, initiation of anti-hypertensive medications, or elevated SBP ≥140 mmHg or DBP ≥ 90 mmHg.
Results: COSMOS participants free from hypertension at baseline were aged 71.1 (6.2) years and 58.8% women, with self-reported SBP  
Conclusion: In older adults, cocoa extract supplementation may play an important role in the prevention of hypertension among those with higher SBP levels.