New insights into the combined toxicity of aflatoxin B1 and fumonisin B1 in HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are widely (co-)detected in food and known for their hepatotoxicity in humans. Still, their combined toxicity needs to be investigated, especially the impact on mitochondria. In our previous work, we examined the effect of short-term exposure to different doses of AFB1, FB1, and their binary mixture (MIX) on the bioenergetic status of HepG2 cells, a well-recognized in vitro model system for studying liver cell function. In the current work, we further investigated the (combined) effect of AFB1 and FB1 on the mitochondrial and glycolytic activity of HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing. The results showed that the co-exposure, especially at high doses, is more toxic due to a more inhibition of all parameters of mitochondrial respiration. However, FB1 contributes more to the MIX effects than AFB1. RNA transcriptome sequencing showed that the p53 signaling pathway, a major orchestrator of mitochondrial apoptosis, was differentially expressed. Moreover, the co-exposure significantly downregulated the genes encoding for Complexes I, II, III, and IV, representing the onset of the suppressed mitochondrial respiration in HepG2 cells.

Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model.

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease worldwide and is impacted by an unhealthy diet with excessive calories, although the role of sugars in NAFLD etiology remains largely unexplored. Rare sugars are natural sugars with alternative monomers and glycosidic bonds, which have attracted attention as sugar replacers due to developments in enzyme engineering and hence an increased availability. We studied the impact of (rare) sugars on energy production, liver cell physiology and gene expression in human intestinal colorectal adenocarcinoma (Caco-2) cells, hepatoma G2 (HepG2) liver cells and a coculture model with these cells. Fat accumulation was investigated in the presence of an oleic/palmitic acid mixture. Glucose, fructose and galactose, but not mannose, l-arabinose, xylose and ribose enhanced hepatic fat accumulation in a HepG2 monoculture. In the coculture model, there was a non-significant trend (p = 0.08) towards higher (20-55% increased) median fat accumulation with maltose, kojibiose and nigerose. In this coculture model, cellular energy production was increased by glucose, maltose, kojibiose and nigerose, but not by trehalose. Furthermore, glucose, fructose and l-arabinose affected gene expression in a sugar-specific way in coculture HepG2 cells. These findings indicate that sugars provide structure-specific effects on cellular energy production, hepatic fat accumulation and gene expression, suggesting a health potential for trehalose and l-arabinose, as well as a differential impact of sugars beyond the distinction of conventional and rare sugars.

Quercetin Mitigates Endothelial Activation in a Novel Intestinal-Endothelial-Monocyte/Macrophage Coculture Setup.

Atherosclerosis initiation is associated with a pro-inflammatory state of the endothelium. Quercetin is a flavonoid abundantly present in plant-based foods, with a possible impact on cardiovascular health. In this study, the effects of quercetin on lipopolysaccharide (LPS)-mediated endothelial inflammation and monocyte adhesion and migration, which are initial steps of the atherogenic process, are studied. Novel in vitro multicellular models simulating the intestinal-endothelial-monocytes/macrophages axis allowed to combine relevant intestinal flavonoid absorption, metabolism and efflux, and the consequent bioactivity towards peripheral endothelial cells. In this triple coculture, quercetin exposure decreased monocyte adhesion to and macrophage migration through an LPS-stressed endothelium, and this was associated with significantly lower levels of soluble vascular cell adhesion molecule-1 (sVCAM-1). Furthermore, quercetin decreased the pro-inflammatory cell environment upon LPS-induced endothelial activation, in terms of tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and sVCAM-1 expression. These findings highlight a mode-of-action by which quercetin may positively impact the initial states of atherosclerosis under more physiologically relevant conditions in terms of quercetin concentrations, metabolites, and intercellular crosstalk.

Bioavailability and Health Impact of Ingested Amyloid-like Protein Fibrils and their Link with Inflammatory Status: A Need for More Research?

The use of amyloid-like protein fibrils (ALFs) in food formulations looks very promising in terms of improving techno-functional properties, but raises some concerns in terms of food safety, because of their structural resemblance to disease-related endogenous amyloids. This review focuses on the biological fate and potential health implications of ingested ALF structures in both healthy and predisposed individuals. A comprehensive overview of ALF gastrointestinal digestion, intestinal absorption, and systemic dissemination is provided, in addition to a thorough assessment of potential ALF cross-seeding of endogenous precursor proteins linked to (non)neurodegenerative amyloidosis. In general, this study concludes that the health impact of ALF consumption remains widely understudied and merits additional research efforts to determine the exact extent to which ALF ingestion may influence the general health status.

Flavonoids and cellular stress: a complex interplay affecting human health.

Flavonoid consumption has beneficial effects on human health, however, clinical evidence remains often inconclusive due to high interindividual variability. Although this high interindividual variability has been consistently observed in flavonoid research, the potential underlying reasons are still poorly studied. Especially the knowledge on the impact of health status on flavonoid responsiveness is limited and merits more investigation. Here, we aim to highlight the bidirectional interplay between flavonoids and cellular stress. First, the state-of-the-art concerning inflammatory stress and mitochondrial dysfunction is reviewed and a comprehensive overview of recent in vitro studies investigating the impact of flavonoids on cellular stress, induced by tumor necrosis factor α, lipopolysaccharide and mitochondrial stressors, is given. Second, we critically discuss the influence of cellular stress on flavonoid uptake, accumulation, metabolism and cell responses, which has, to our knowledge, never been extensively reviewed before. Next, we advocate the innovative insight that stratification of the general population based on health status can reveal subpopulations that benefit more from flavonoid consumption. Finally, suggestions are given for the development of future cell models that simulate the physiological micro-environment, including interindividual variability, since more mechanistic research is needed to establish scientific-based personalized food recommendations for specific subpopulations.

A two-front nutritional environment fuels colorectal cancer: perspectives for dietary intervention.

Colorectal cancer (CRC) develops and progresses in a nutritional environment comprising a continuously changing luminal cocktail of external dietary and microbial factors on the apical side, and a dynamic host-related pool of systemic factors on the serosal side. In this review, we highlight how this two-front environment influences the bioenergetic status of colonocytes throughout CRC development from (cancer) stem cells to cancer cells in nutrient-rich and nutrient-poor conditions, and eventually to metastatic cells, which, upon entry to the circulation and during metastatic seeding, are forced to metabolically adapt. Furthermore, given the influence of diet on the two-front nutritional environment, we discuss dietary strategies that target the specific metabolic preferences of these cells, with a possible impact on colon cancer cell bioenergetics and CRC outcome.

Chemical composition, antioxidant, antimicrobial and antiproliferative activities of the extracts isolated from the pomace of rowanberry (Sorbus aucuparia L.).

In the present work, acetone, ethanol and water extracts of rowanberry (Sorbus aucuparia L.) pomace were evaluated for their antiproliferative, antimicrobial and antioxidative effects. Chemical composition of the extracts was determined by UPLC-ESI-MS/MS and spectrophotometric methods. Neochlorogenic and chlorogenic acids were the major phenolic compounds. The water extract contained the highest total proanthocyanidins content (301 ± 18.9 mg/g) and demonstrated the highest antioxidant activity in the all assays (DPPH, FRAP and ORAC). Extracts isolated from rowanberry pomace effectively inhibited the growth of undesirable microorganisms, especially Gram-positive bacteria. Acetone extract was the strongest antimicrobial agent followed by water and ethanol extracts. Acetone and water extracts demonstrated also higher cytotoxic potential in cell viability assays (SRB and MTT) using Caco-2 cells. In general, the results suggest that rowanberry pomace is a promising source of natural compounds with antioxidant and biological activities.

Cell line-dependent increase in cellular quercetin accumulation upon stress induced by valinomycin and lipopolysaccharide, but not by TNF-α.

As the interface between the luminal and internal environment, the intestinal epithelium is strongly exposed to food-related, host-related and microbial stress. Furthermore, the endothelial stress response plays an important role in vascular disease development, which may be improved upon consumption of dietary bioactives such as polyphenols. The impact of the latter, however, is largely individual-dependent and effects are, in most cases, only observed under mild diseased conditions. Here, it is hypothesized that the individual's stressor levels may contribute to this variable response. To this end, the impact of the stressors (i) valinomycin (as model for cereulide, food-related microbial metabolite), (ii) TNF-α (host-related) and (iii) lipopolysaccharide (gram-negative bacterial cell related) on flavonoid accumulation was investigated in several intestinal and endothelial cell lines. Flow cytometry, confocal microscopy and an in-house developed, robust and high-throughput spectrofluorometric method, showed that quercetin accumulated in all tested cell lines in a dose-dependent manner. Upon stress induced by valinomycin and to a lesser extent by lipopolysaccharide, but not by TNF-α, an increased quercetin accumulation was observed in proliferating intestinal and endothelial cells and not in differentiated intestinal or quiescent endothelial cells. Therefore, flavonoid accumulation may be a potential cellular stress response mechanism which strongly depends on the applied stressor, flavonoid, cell line and even growth conditions. This opens perspectives for further understanding the mechanisms by which cellular stress may shape the individual's response to bioactive compounds.

Synthesis of 1,4-Thiazepane-Based Curcuminoids with Promising Anticancer Activity.

Curcumin, the main component of turmeric (Curcuma longa) is known to display an interesting bioactivity profile, including pronounced anticancer properties. However, its low bioavailability, metabolic instability and nonspecific activity are concerns that have to be addressed before curcuminoids can be considered for therapeutic applications. Within that framework, intensive research has been carried out in the last decades to develop new curcumin derivatives, generally centered on standard modifications of the sp2 curcumin framework, with the aim to augment its bioavailability while maintaining or improving its anticancer properties. To find potential hit molecules by moving away from the classical flat curcumin framework, we investigated an unexplored modification to produce novel, out-of-plane 1,4-thiazepane-based curcuminoids and assessed the impact of this modification on the biological activity. In this way, 21 new, structurally diverse thiazepane scaffolds (4-aryl-1-(7-aryl-1,4-thiazepan-5-ylidene)but-3-en-2-ones) were synthesized, as well as some biologically interesting unexpected reaction products (such as 5-aryl-6-arylmethylene-3-ethoxycyclohex-2-en-1-ones and 4-acetyl-5-aryl-2-(3-arylacryloyl)-3-methylcyclohex-2-en-1-ones). All these analogues were subsequently tested on their antioxidant capacity, their cytotoxicity properties and their ROS (reactive oxygen species) production. Many compounds demonstrated interesting activities, with ten curcuminoids, whereof eight 1,4-thiazepane-based, showing better antiproliferative properties compared to their mother compounds, as well as an increased ROS production. This unprecedented 3D curcumin modification has thus delivered promising new hit compounds with good activity profiles eligible for further exploration.

The response of five intestinal cell lines to anoxic conditions in vitro.

BACKGROUND INFORMATION: In vivo oxygen levels in tissues range from 1% to 15%, while mechanistic cell culture studies employ an atmospheric oxygen level of 21% to grow cells. These oxygen concentrations are therefore not representative for conditions where the cell response is dependent on oxygen partial pressure. In pathological situation, such as (colon) cancer or chronic inflammation, tissue oxygenation is severely affected, and even under physiological conditions a steep oxygen gradient is present in the large intestine, where epithelial cells co-exist with microbial species, resulting in almost anoxia at the midpoint of the lumen. In these situations, a better characterisation of the essential cellular behaviour under hypoxia or anoxia is required. RESULTS: We have characterised the cellular response of commonly used cell cultures for the study of intestinal epithelial processes and colon cancer development (Caco-2, HT-29, SW480, HCT 116 and LoVo) under conventional normoxic conditions (21% O2 ) and in an anoxic (<0.1% O2 ) environment generated in an anaerobic chamber. In general, anoxic conditions led to lower levels of oxidative stress, a reduction in reduced glutathione/oxidised glutathione (GSH/GSSG) ratio, the shift of the redox status to oxidised glutathione levels, reduced cell proliferation, decreased barrier function and higher glycolysis rates at the expense of oxidative respiration. CONCLUSIONS: Continuous exposure to anoxic conditions, such as occurring at the host-microbe interface in the intestine, may create an adaptive metabolic cellular response of the cells. SIGNIFICANCE: Considering adequate oxygen levels is essential for creating more physiologically relevant models for the study of host-microbe interactions and colon cancer development.

Synthesis of Non-Symmetrical Nitrogen-Containing Curcuminoids in the Pursuit of New Anticancer Candidates.

Curcumin is known to display pronounced anticancer effects and a variety of other biological activities. However, the low bioavailability and fast metabolism of this molecule present an issue of concern with respect to its medicinal applications. To address this issue, structural modifications of the curcumin scaffold can be envisioned as a strategy to improve both the solubility and stability of this chemical entity, without compromising its biological activities. Previous work in our group targeted the synthesis of symmetrical azaheteroaromatic curcuminoids, which showed better solubility and cytotoxicity profiles compared to curcumin. In continuation of that work, we now focused on the synthesis of non-symmetrical nitrogen-containing curcuminoids bearing both a phenolic and an azaheteroaromatic moiety. In that way, we aimed to combine good solubility, antioxidant potential and cytotoxic properties into one molecule. Some derivatives were selected for further chemical modification of their rather labile ß-diketone scaffold to the corresponding pyrazole moiety. In this way, thirteen new non-symmetrical aza-aromatic curcuminoids and four pyrazole-based analogues were successfully synthesized in a yield of 11-69 %. All newly synthesized analogues were evaluated for their antioxidant properties, reactive oxygen species (ROS) production, water solubility and anticancer activities. Several novel derivatives displayed good cytotoxicity profiles compared to curcumin, in combination with an improved water solubility and stability, and were thus identified as potential hit scaffolds for further optimization studies.

Aronia (Aronia melanocarpa) Polyphenols Modulate the Microbial Community in a Simulator of the Human Intestinal Microbial Ecosystem (SHIME) and Decrease Secretion of Proinflammatory Markers in a Caco-2/endothelial Cell Coculture Model.

SCOPE: To explore the mechanisms behind the health effects of Aronia (Aronia melanocarpa), the microbial community modulating and anti-inflammatory effects of Aronia polyphenols are investigated by combining the similutor of the human intestinal microbial ecosystem (SHIME) with a coculture of intestinal and endothelial cells. RESULTS: Administration of Aronia juice (6.5g L-1 ) to the SHIME for 2 weeks increases the abundance of firmicutes to 92% in the ascending colon (AC), 85% in the transverse colon (TC), and 82% in the descending colon (DC; p < 0.001), proteobacteria (6.7% in AC, p < 0.001), and Akkermansia (14% in TC and 18% in DC, p < 0.001) and decreases the abundance of Bifidobacterium species, associated with a decrease of acetate and increase of propionate and butyrate, whereas no significant difference is observed upon placebo juice treatment. After addition of the digests to TNF-α challenged Caco-2/endothelial cocultures, intercellular adhesion molecule (ICAM)-1, IL-8, and monocyte chemoattractant protein-1 levels are significantly downregulated. Interestingly, Aronia juice treats digests from each colon compartment resulting in a stronger decrease of the ICAM-1 secretion (up to 73%, p < 0.001) compared to their corresponding placebo treated digests, thereby pointing to a polyphenol-dependent effect. CONCLUSIONS: Aronia polyphenols modulate intestinal microbial composition, induce beneficial short chain fatty acid production, and prevent inflammatory stress in endothelial cells. This opens perspectives for the use of Aronia polyphenols as prebiotics in the context of intestinal and cardiovascular health.

Synthesis of Novel Aza-aromatic Curcuminoids with Improved Biological Activities towards Various Cancer Cell Lines.

Curcumin, a natural compound extracted from the rhizomes of Curcuma longa, displays pronounced anticancer properties but lacks good bioavailability and stability. In a previous study, we initiated structure modification of the curcumin scaffold by imination of the labile ß-diketone moiety to produce novel ß-enaminone derivatives. These compounds showed promising properties for elaborate follow-up studies. In this work, we focused on another class of nitrogen-containing curcuminoids with a similar objective: to address the bioavailability and stability issues and to improve the biological activity of curcumin. This paper thus reports on the synthesis of new pyridine-, indole-, and pyrrole-based curcumin analogues (aza-aromatic curcuminoids) and discusses their water solubility, antioxidant activity, and antiproliferative properties. In addition, multivariate statistics, including hierarchical clustering analysis and principal component analysis, were performed on a broad set of nitrogen-containing curcuminoids. Compared to their respective mother structures, that is, curcumin and bisdemethoxycurcumin, all compounds, and especially the pyridin-3-yl ß-enaminone analogues, showed better water solubility profiles. Interestingly, the pyridine-, indole-, and pyrrole-based curcumin derivatives demonstrated improved biological effects in terms of mitochondrial activity impairment and protein content, in addition to comparable or decreased antioxidant properties. Overall, the biologically active N-alkyl ß-enaminone aza-aromatic curcuminoids were shown to offer a desirable balance between good solubility and significant bioactivity.

A Critical Evaluation of In Vitro Hesperidin 2S Bioavailability in a Model Combining Luminal (Microbial) Digestion and Caco-2 Cell Absorption in Comparison to a Randomized Controlled Human Trial.

SCOPE: Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. METHOD AND RESULTS: We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. CONCLUSIONS: In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability.

Cu isotope fractionation response to oxidative stress in a hepatic cell line studied using multi-collector ICP-mass spectrometry.

Reactive oxygen species (ROS) are generated in biological processes involving electron transfer reactions and can act in a beneficial or deleterious way. When intracellular ROS levels exceed the cell's anti-oxidant capacity, oxidative stress occurs. In this work, Cu isotope fractionation was evaluated in HepG2 cells under oxidative stress conditions attained in various ways. HepG2 is a well-characterised human hepatoblastoma cell line adapted to grow under high oxidative stress conditions. During a pre-incubation stage, cells were exposed to a non-toxic concentration of Cu for 24 h. Subsequently, the medium was replaced and cells were exposed to one of three different external stressors: H2O2, tumour necrosis factor α (TNFα) or UV radiation. The isotopic composition of the intracellular Cu was determined by multi-collector ICP-mass spectrometry to evaluate the isotope fractionation accompanying Cu fluxes between cells and culture medium. For half of these setups, the pre-incubation solution also contained N-acetyl-cysteine (NAC) as an anti-oxidant to evaluate its protective effect against oxidative stress via its influence on the extent of Cu isotope fractionation. Oxidative stress caused the intracellular Cu isotopic composition to be heavier compared to that in untreated control cells. The H2O2 and TNFα exposures rendered similar results, comparable to those obtained after mild UV exposure. The heaviest Cu isotopic composition was observed under the strongest oxidative conditions tested, i.e., when the cell surfaces were directly exposed to UV radiation without apical medium and in absence of NAC. NAC mitigated the extent of isotope fractionation in all cases.

Protein hydrolysate from canned sardine and brewing by-products improves TNF-α-induced inflammation in an intestinal-endothelial co-culture cell model.

PURPOSE: The anti-inflammatory activity of sardine protein hydrolysates (SPH) obtained by hydrolysis with proteases from brewing yeast surplus was ascertained. METHODS: For this purpose, a digested and desalted SPH fraction with molecular weight lower than 10 kDa was investigated using an endothelial cell line (EA.hy926) as such and in a co-culture model with an intestinal cell line (Caco-2). Effects of SPH <10 kDa on nitric oxide (NO) production, reactive oxygen species (ROS) inhibition and secretion of monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), chemokine IL-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) were evaluated in TNF-α-treated and untreated cells. RESULTS: Upon TNF-α treatment, levels of NO, MCP-1, VEGF, IL-8, ICAM-1 and endothelial ROS were significantly increased in both mono- and co-culture models. Treatment with SPH <10 kDa (2.0 mg peptides/mL) significantly decreased all the inflammation markers when compared to TNF-α-treated control. This protective effect was more pronounced in the co-culture model, suggesting that SPH <10 kDa Caco-2 cells metabolites produced in the course of intestinal absorption may provide a more relevant protective effect against endothelial dysfunction. Additionally, indirect cross-talk between two cell types was established, suggesting that SPH <10 kDa may also bind to receptors on the Caco-2 cells, thereby triggering a pathway to secrete the pro-inflammatory compounds. CONCLUSION: Overall, these in vitro screening results, in which intestinal digestion, absorption and endothelial bioactivity are simulated, show the potential of SPH to be used as a functional food with anti-inflammatory properties.

Butyrate-producing bacteria supplemented in vitro to Crohn's disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity.

The management of the dysbiosed gut microbiota in inflammatory bowel diseases (IBD) is gaining more attention as a novel target to control this disease. Probiotic treatment with butyrate-producing bacteria has therapeutic potential since these bacteria are depleted in IBD patients and butyrate has beneficial effects on epithelial barrier function and overall gut health. However, studies assessing the effect of probiotic supplementation on microbe-microbe and host-microbe interactions are rare. In this study, butyrate-producing bacteria (three mono-species and one multispecies mix) were supplemented to the fecal microbial communities of ten Crohn's disease (CD) patients in an in vitro system simulating the mucus- and lumen-associated microbiota. Effects of supplementation in short-chain fatty acid levels, bacterial colonization of mucus environment and intestinal epithelial barrier function were evaluated. Treatment with F. prausnitzii and the mix of six butyrate-producers significantly increased the butyrate production by 5-11 mol%, and colonization capacity in mucus- and lumen-associated CD microbiota. Treatments with B. pullicaecorum 25-3T and the mix of six butyrate-producers improved epithelial barrier integrity in vitro. This study provides proof-of-concept data for the therapeutic potential of butyrate-producing bacteria in CD and supports the future preclinical development of a probiotic product containing butyrate-producing species.

Egg-derived bioactive peptides with ACE-inhibitory properties: a literature update.

Egg proteins contain a wide set of peptide sequences which have an impact on cardiovascular health. Their modes-of-action involve, among others, the inhibition of angiotensin-converting enzyme (ACE) and antioxidant and anti-inflammatory properties. In this review, we focus particularly on ACE-inhibition and discuss recent findings in: (i) production methods for egg protein-derived ACE-inhibitory peptides, (ii) in vitro functionality of these peptides, (iii) their intestinal digestion and absorption in order to reach the target tissue, (iv) the impact of ACE-inhibitory egg-derived peptides in vivo and (v) future perspectives for the implementation of egg-derived ACE-inhibitory peptides as functional foods.

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

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

Ferulic acid-4-O-sulfate rather than ferulic acid relaxes arteries and lowers blood pressure in mice.

Consumption of foods rich in ferulic acid (FA) such as wholegrain cereals, or FA precursors such as chlorogenic acids in coffee, is inversely correlated with risk of cardiovascular disease and type 2 diabetes. As a result of digestion and phase II metabolism in the gut and liver, FA is converted predominantly into ferulic acid-4-O-sulfate (FA-sul), an abundant plasma metabolite. Although FA-sul is the main metabolite, very little has been reported regarding its bioactivities. We have compared the ex vivo vasorelaxing effect of FA and FA-sul (10-7-3.10-5M) on isolated mouse arteries mounted in tissue myographs. FA-sul, but not FA, elicited a concentration-dependent vasorelaxation of saphenous and femoral arteries and aortae. The FA-sul-mediated vasorelaxation was blunted by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a soluble guanylate cyclase (sGC) inhibitor. The role of sGC was confirmed in femoral arteries isolated from sGCα1(-/-) knockout mice. Furthermore, 4-aminopyridine, a specific inhibitor of voltage-dependent potassium channels, significantly decreased FA-sul-mediated effects. In anesthetized mice, intravenous injection of FA-sul decreased mean arterial pressure, whereas FA had no effect, confirming the results obtained ex vivo. FA-sul is probably one of the major metabolites accounting for the blood pressure-lowering effects associated with FA consumption.