Formulating poorly soluble drugs with polymers in the form of solid dispersions has been widely used for improving drug dissolution. Endogenous surface-active species present in the gut, such as bile salts, lecithin and other phospholipids, have been shown to play a key role in facilitating lipids and poorly soluble drugs solubilisation in the gut. In this study, we examined the possible occurrence of interactions between a model bile salt, sodium taurocholate (NaTC), and model spray dried solid dispersions comprising piroxicam and Hydroxypropyl Methylcellulose (HPMC), a commonly used hydrophilic polymer for solid dispersion preparation. Solubility measurements revealed the good solubilisation effect of NaTC on the crystalline drug, which was enhanced by the addition of HPMC, and further boosted by the drug formulation into solid dispersion. The colloidal behaviour of the solid dispersions upon dissolution in biorelevant media, with and without NaTC, revealed the formation of NaTC-HPMC complexes and other mixed colloidal species. Cellular level drug absorption studies obtained using Caco-2 monolayers confirmed that the combination of drug being delivered by solid dispersion and the presence of bile salt and lecithin significantly contributed to the improved drug absorption. Together with the role of NaTC-HPMC complexes in assisting the drug solubilisation, our results also highlight the complex interplay between bile salts, excipients and drug absorption.

The increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all samples contained detectable amounts of bacterial DNA with a concentration that varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly associated with the serum levels of zonulin, a marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected from the same subjects. 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of the genus Pseudomonas . Several control samples were also analyzed to assess the influence of contaminant bacterial DNA potentially originating from reagents and materials. The data reported here suggest that para-cellular permeability of epithelial (and, potentially, endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

Background During aging, alterations of the intestinal microbial ecosystem can occur contributing to immunosenescence, inflamm-aging and impairment of intestinal barrier function (increased intestinal permeability; IP). In the context of a diet-microbiota-IP axis in older subjects, food bioactives such as polyphenols may play a beneficial modulatory role. Methods MaPLE is a project centered on a randomized, controlled cross-over dietary intervention trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] targeted to older people (≥ 60 y) living in a well-controlled setting (i.e. nursing home). The 8-week interventions are separated by an 8-week wash-out period. Three small portions per day of selected polyphenol-rich foods are consumed during intervention in substitution of other comparable products within the C-diet. Biological samples are collected before and after each treatment period to evaluate markers related to IP, inflammation, vascular function, oxidative stress, gut and blood microbiomics, metabolomics. A sample size of 50 subjects was defined based on IP as primary outcome. Discussion Evidence that increasing the consumption of polyphenol-rich food products can positively affect intestinal microbial ecosystem resulting in reduced IP and decreased translocation of inflammogenic bacterial factors into the bloodstream will be provided. The integration of data from gut and blood microbiomics, metabolomics and other IP-related markers will improve the understanding of the beneficial effect of the intervention in the context of polyphenols−microbiota−IP interactions. Finally, findings obtained will provide a proof of concept of the reliability of the dietary intervention, also contributing to future implementations of dietary guidelines directed to IP management in the older and other at risk subjects. Trial registration The trial is registered at ( ISRCTN10214981 ); April 28, 2017.

BACKGROUND & AIM:Increased intestinal permeability (IP) can occur in older people and contribute to the activation of the immune system and inflammation. Dietary interventions may represent a potential strategy to reduce IP. In this regard, specific food bioactives such as polyphenols have been proposed as potential IP modulator due to their ability to affect several critical targets and pathways that control IP. The trial aimed to test the hypothesis that a polyphenol-rich dietary pattern can decrease serum zonulin levels, an IP surrogate marker involved in tight junction modulation, and can beneficially alter the intestinal microbiota, and IP-associated biochemical and clinical markers in older subjects.METHODS:A randomised, controlled, cross-over intervention trial was performed. Sixty-six subjects (aged ≥ 60 y) with increased IP based on serum zonulin levels, were randomly allocated to one of the two arms of the intervention consisting of a control diet (C-diet) vs. a polyphenol-rich diet (PR-diet). Each intervention was 8-week long and separated by an 8-week wash out period. At the beginning and at the end of each intervention period, serum samples were collected for the quantification of zonulin and other biological markers. Faecal samples were also collected to investigate the intestinal microbial ecosystem. In addition, anthropometrical/physical/biochemical parameters and food intake were evaluated.RESULTS:Fifty-one subjects successfully completed the intervention and a high compliance to the dietary protocols was demonstrated. Overall, polyphenol intake significantly increased from a mean of 812 mg/day in the C diet to 1391 mg/day in the PR-diet. Two-way analysis of variance showed a significant effect of treatment (p = 0.008) and treatment × time interaction (p = 0.025) on serum zonulin levels, which decreased after the 8-week PR-diet. In addition, a treatment × time interaction was observed showing a reduction of diastolic blood pressure (p = 0.028) following the PR-diet, which was strongest in those not using antihypertensive drugs. A decrease in both diastolic (p = 0.043) and systolic blood pressure (p = 0.042) was observed in women. Interestingly, a significant increase in fibre-fermenting and butyrate-producing bacteria such as the family Ruminococcaceae and members of the genus Faecalibacterium was observed following the PR intervention. The efficacy of this dietary intervention was greater in subjects with higher serum zonulin at baseline, who showed more pronounced alterations in the markers under study. Furthermore, zonulin reduction was also stronger among subjects with higher body mass index and with insulin resistance at baseline, thus demonstrating the close interplay between IP and metabolic features.CONCLUSIONS:These data show, for the first time, that a PR-diet can reduce serum zonulin levels, an indirect marker of IP. In addition, PR-diet reduced blood pressure and increased fibre-fermenting and butyrate-producing bacteria. These findings may represent an initial breakthrough for further intervention studies evaluating possible dietary treatments for the management of IP, inflammation and gut function in different target populations. THIS STUDY WAS REGISTERED AT WWW.ISRCTN.ORG AS:ISRCTN10214981.

Background: The reported effects of flavanol-rich foods such as cocoa, dark chocolate, and apples on blood pressure and endothelial function may be due to the monomeric flavanols [mainly (-)-epicatechin (EC)], the oligomeric flavanols [procyanidins (PCs)], or other components. Reports of well-controlled intervention studies that test the effects of isolated oligomeric flavanols on biomarkers of cardiovascular health are lacking. Objective: We studied the acute and chronic effects of an EC-rich apple flavanol extract and isolated apple PCs on systolic blood pressure (BP) and other cardiometabolic biomarkers. Design: Forty-two healthy men and women with moderately elevated BP completed this randomized, double-blind, placebo-controlled, 4-arm crossover trial. Participants ingested a single dose of an apple flavanol extract (70 mg monomeric flavanols, 65 mg PCs), a double dose of this extract (140 mg monomeric flavanols, 130 mg PCs), an apple PC extract (130 mg PCs, 6.5 mg monomeric flavanols), or placebo capsules once daily for 4 wk, in random order. Biomarkers of cardiovascular disease risk and vascular function were measured before and 2 h after ingestion of the first dose and after the 4-wk intervention. Results: Compared with placebo, none of the isolated flavanol treatments significantly (P < 0.05) changed systolic or diastolic BP (peripheral and aortic), plasma nitric oxide (NO) reaction products, or measures of arterial stiffness (carotid femoral pulse wave velocity, brachial-ankle pulse-wave velocity, or Augmentation Index) after 2 h or 4 wk of the intervention. There were no changes in plasma endogenous metabolite profiles or circulating NO; endothelin 1; total, HDL, or LDL cholesterol; triglycerides; fasting glucose; fructosamine; or insulin after 4 wk of the intervention. Conclusions: Our data suggest that, in isolation, neither monomeric flavanols nor PCs affect BP, blood lipid profiles, endothelial function, or glucose control in individuals with moderately elevated BP. The reported benefits of consuming flavanol-rich cocoa, chocolate, and apple products appear to be dependent on other components, which may work in combination with monomeric flavanols and PCs.

Elevated circulating cholesterol levels are a risk factor for CVD which is also associated with sub-optimal vascular function. There is emerging evidence that anthocyanins can cause beneficial cardio-protective effects by favourably modulating lipoprotein profiles. We compared the effects of blood orange juice which is rich in anthocyanins and blonde orange juice without anthocyanins on LDL-cholesterol and other biomarkers of CVD risk, vascular function and glycaemia. In all, forty-one participants (aged 25-84 years) with a waist circumference >94 cm (men) and >80 cm (women) completed a randomised, open label, two-arm cross-over trial. For 28 d participants ingested (i) 500 ml blood orange juice providing 50 mg anthocyanins/d and (ii) 500 ml blonde orange juice without anthocyanins. There was a minimum 3-week washout period between treatments. LDL-cholesterol and other biomarkers associated with CVD risk and glycaemia were assessed at the start and end of each treatment period. No significant differences were observed in total, HDL- and LDL-cholesterol, TAG, glucose, fructosamine, nitric oxide, C-reactive protein, aortic systolic blood pressure and diastolic blood pressure or carotid-femoral and brachial-ankle pulse wave velocity after 28 d ingestion of blood orange juice compared with standard orange juice. The lack of effect on LDL-cholesterol may be due to the modest concentration of anthocyanins in the blood orange juice.

The aim was to incorporate vegetables containing the phytochemicals quercetin, apigenin, glucoraphanin and carotenoids into a processed potato-based snack and assess their bioaccessibility and bioavailability. Three different processing routes were tested for incorporation and retention of phytochemicals in snacks using individually quick frozen or freeze-dried vegetables. No significant differences in the uptake or transport of quercetin or apigenin between a vegetable mix or snacks were observed using the CaCo-2 transwell model. Simulated in vitro digestions predicted a substantial release of quercetin and apigenin, some release of glucoraphanin but none for carotenes from either the snack or equivalent steamed vegetables. In humans, there were no significant differences in the bioavailability of quercetin, apigenin or glucoraphanin from the snack or equivalent steamed vegetables. We have shown that significant quantities of freeze-dried vegetables can be incorporated into snacks with good retention of phytochemicals and with similar bioavailability to equivalent steamed vegetables.

The liver plays a critical role in food and drug metabolism and detoxification and accordingly influences systemic body homeostasis in health and disease. While the C57BL/6 and ApoE − / − mouse models are widely used to study gene expression changes in liver disease and metabolism, currently there are no validated stably expressed endogenous genes in these models, neither is it known how gene expression varies within and across liver lobes. Here we show regional variations in the expression of Ywhaz , Gak , Gapdh , Hmbs and Act-β endogenous genes across a liver lobe; Using homogeneous samples from the four liver lobes of 6 C57BL/6 mice we tested the stability of 12 endogenous genes and show that Act-β and Eif2-α are the most stably expressed endogenous genes in all four lobes and demonstrate lobular differences in the expression of Abca1 cholesterol efflux gene. These results suggest that sampling from a specified homogeneous powdered liver lobe is paramount in enhancing data reliability and reproducibility. The stability of the 12 endogenous genes was further tested using homogeneous samples of left liver lobes from 20 ApoE − / − mice on standard or high polyphenol diets. Act-β and Ywhaz are suitable endogenous genes for gene expression normalisation in this mouse model.

We have studied the uptake of quercetin aglycone into CaCo-2/TC7 cells in the presence and absence of mixed micelles that are present in the human small intestine. The micelles inhibited the transport of quercetin into the cells. To gain an understanding of why this is the case we examined the solubilisation of quercetin in micelles of differing composition and into pure lipid phases. We did this by using the environmental sensitivity of quercetin's UV-visible absorption spectra and measurement of free quercetin by filtration of the micellar solutions. The nature of the micelles was also studied by pyrene fluorescence. We found that the partitioning of quercetin into simple bile salt micelles was low and for mixed micelles was inhibited by increasing the bile salt concentration. The affinity of quercetin decreased in the order egg phosphatidylcholine (PC) = lysoPC > mixed micelles > bile salts. These results, together with the innate properties of quercetin, contribute to an understanding of the low bioavailability of quercetin.

Scope: We recently reported potent inhibition of VEGF signalling by two flavanols at sub-micromolar concentrations, mediated by direct binding of the flavanols to VEGF. The aim of this study was to quantify the inhibitory potency and binding affinity of a wide range of dietary polyphenols and determine the structural requirements for VEGF inhibition.Methods and results: The concentration of polyphenol required to cause 50% inhibition (IC50) of VEGF-dependent VEGFR-2 activation in HUVECS was determined after pretreating VEGF with polyphenols at various concentations. Binding affinities and binding sites on VEGF were predicted using in-silico modelling. Ellagic acid and 15 flavonoids had IC50 values <= 10 mu M while 28 other polyhenols were weak/non-inhibitors. Structural features associated with potent inhibition included 3-galloylation, C-ring C2=C3, total OH, B-ring catechol, C-ring 3-OH of flavonoids. Potency was not associated with polyphenol hydrophobicity. There was a strong correlation between potency of inhibition and binding affinities, and all polyphenols were predicted to bind to a region on VEGF involved in VEGFR-2 binding.Conclusion: Specific polyphenols bind directly to a discrete region of VEGF and inhibit VEGF signalling, and this potentially explains the associations between consumption of these polyphenols and CVD risk.

SCOPE:Excessive concentrations of vascular endothelial growth factor (VEGF) drive angiogenesis and cause complications such as increased growth of tumours and atherosclerotic plaques. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signalling by polyphenols.METHODS AND RESULTS:We show that the polyphenols epigallocatechin gallate from green tea and procyanidin oligomers from apples potently inhibit VEGF-induced VEGF receptor-2 (VEGFR-2) signalling in human umbilical vein endothelial cells by directly interacting with VEGF. The polyphenol-induced inhibition of VEGF-induced VEGFR-2 activation occurred at nanomolar polyphenol concentrations and followed bi-phasic inhibition kinetics. VEGF activity could not be recovered by dialysing VEGF-polyphenol complexes. Exposure of VEGF to epigallocatechin gallate or procyanidin oligomers strongly inhibited subsequent binding of VEGF to human umbilical vein endothelial cells expressing VEGFR-2. Remarkably, even though VEGFR-2 signalling was completely inhibited at 1 μM concentrations of polyphenols, endothelial nitric oxide synthase was shown to still be activated via the PI3K/Akt signalling pathway which is downstream of VEGFR-2.CONCLUSION:These data demonstrate for the first time that VEGF is a key molecular target for specific polyphenols found in tea, apples and cocoa which potently inhibit VEGF signalling and angiogenesis at physiological concentrations. These data provide a plausible mechanism which links bioactive compounds in food with their beneficial effects.

Potent and specific inhibition of VEGF signalling mediated by slow tight‐binding of flavonoids to VEGF Paul A. Kroon1, Christina W.A. Moyle1, Ana B. Cerezo1, Mark S. Winterbone1, Paul W. Needs1, Wendy J. Hollands1, Rebecca L. Edwards1: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.Background: Previously, we reported that the predominant effect of a low dose of an apple procyanidin extract on gene expression in cultured human vascular endothelial cells was inhibition of angiogenic functions and associated signalling pathways. Vascular endothelial growth factor (VEGF) is the dominant driver of vascular angiogenesis, and excessive concentrations of VEGF drive angiogenesis and cause complications such as age‐related macular degeneration and increased growth of tumours and atherosclerotic plaques. Since VEGF signalling plays crucial roles in diseases for which flavonoids have been shown to be protective, we further investigated the interactions of flavonoids with VEGF signalling.Methods and Results: Polyphenols such as EGCg (green tea) and procyanidin oligomers (cocoa, apples) potently inhibited VEGFA‐mediated phosphorylation of VEGF receptor‐2 in HUVECs with IC50 values of 89 and 280 nM, respectively. Many other polyphenols including (‐)‐epicatechin were completely ineffective. We show that EGCg and a procyanidin tetramer inhibited VEGFR2 activation by directly binding to VEGFA and preventing ligand‐receptor binding. The polyphenol‐VEGFA complexes were formed as a result of slow, tight‐binding reactions from which VEGF activity could not be recovered by dialysis. Remarkably, even though VEGFR‐2 signalling was completely inhibited at 1 µM concentrations of EGCg and procyanidins, endothelial NO synthase (eNOS) was shown to still be activated via the PI3K/pAkt signalling pathway.Conclusion: These data show that polyphenols can potently inhibit VEGFR‐2 signalling while retaining the pro‐endothelial functions that occur downstream of PI3K/Akt.Grant Funding Source: Supported by the BBSRC (UK), EU FP7 (BACCHUS‐312090, VegFenol‐274885) and Coressence Ltd.

The major dietary flavonol quercetin, which has been shown to improve endothelial function and decrease blood pressure, is extensively metabolized during absorption. This study examined the relative abilities of quercetin and its human metabolites to modulate the expression of eNOS and ET-1, which are involved in regulating endothelial homeostasis. Quercetin aglycone significantly reduced both eNOS protein and gene expression in HUVEC, mirroring the effects of the pro-inflammatory cytokine TNFα. In the presence of TNFα the aglycone caused further reductions in eNOS, whereas the metabolites were without effect in either TNFα-stimulated or unstimulated cells. ET-1 expression was significantly reduced by quercetin in both TNFα-stimulated or unstimulated HUVECs. The metabolites had no effect on ET-1 expression with the exception of quercetin-3'-sulfate, which caused a moderate increase in TNFα-stimulated cells. These results suggest that metabolic transformation of quercetin prevents it from causing a potentially deleterious decrease in eNOS in endothelial cells.

There is accumulating evidence from epidemiological and human intervention studies that quercetin-rich diets can protect against cardiovascular diseases. Quercetin glycosides are modified during metabolism, and the forms reaching the systemic circulation are glucuronidated, sulfated, and methylated. The aim of this study was to analyse the potential beneficial effects of quercetin and its conjugated metabolites on vascular function on a co-culture model of human umbilical artery smooth muscle cells and human umbilical vein endothelial cells. We observed that physiologically relevant metabolites of quercetin were able to reduce ET-1 protein and gene expression and to increase accumulation of cGMP in TNF-α-induced HUASMCs co-cultured with HUVECs. This is the first study to demonstrate an ability of quercetin and its conjugated metabolites, at physiologically achievable concentrations, to modulate vascular function in a co-culture model comprising human vascular endothelial and smooth muscle cells.

Dietary flavonoids have been shown to have a number of anti-inflammatory properties, including decreasing the expression of adhesion molecules. Flavonoids however, are metabolised during absorption and the forms reaching the systemic circulation are glucuronidated, sulfated and methylated. Most previous studies of the effects of flavonoids have used the parent compounds rather than the metabolites found in blood plasma and we have recently shown that metabolites of quercetin can retain some of the anti-inflammatory properties of the parent aglycone when used to treat human umbilical endothelial cells (HUVEC).Using both physiologically achievable (2 mu M) and Supraphysiological (10 mu M) concentrations, we investigated the ability of quercetin and its predominant human metabolites to attenuate the expression of vascular cell adhesion molecule-1 (VCAM-I), intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in human umbilical artery smooth muscle cells (HUASMC) activated by tumor necrosis factor-alpha (TNF alpha). Quercetin was able to reduce TNF alpha-induced upregulation of VCAM-I, ICAM-1 and MCP-1 at both the protein and transcript (mRNA) level in HUASMC. However the quercetin metabolites, quercetin 3'-sulfate, quercetin 3-glucuronide and 3'-methylquercetin 3-glucuronide, had no effect on TNF alpha-induced tip regulation of adhesion molecule or chemokine expression, at either concentration tested. These data do not support the notion that the vascular anti-inflammatory effects of quercetin consumption are mediated through effects on smooth muscle cells. (C) 2008 Elsevier Ireland Ltd. All rights reserved,

As a part of a larger study investigating the effects of α-tocopherol on gene expression in type 2 diabetics we observed a pro-oxidant effect of α-tocopherol which we believe may be useful in interpreting outcomes of large intervention trials of α-tocopherol.

OBJECTIVE:Telomeres are DNA sequences necessary for DNA replication, which shorten at cell division at a rate related to levels of oxidative stress. Once shortened to a critical length, cells are triggered into replicative senescence. Type 2 diabetes is associated with oxidative DNA damage, and we hypothesized that telomere shortening would characterize type 2 diabetes.RESEARCH DESIGN AND METHODS:We studied 21 male type 2 diabetic subjects (mean age 61.2 years, mean HbA(1c) 7.9%) selected to limit confounding effects on telomere length and 29 matched control subjects. Telomere length was measured in peripheral venous monocyte and T-cells (naïve and memory) by fluorescent in situ hybridization and oxidative DNA damage by flow cytometry of oxidized DNA bases. Peripheral insulin resistance (homeostasis model assessment) and high-sensitivity C-reactive protein (hsCRP) were measured.RESULTS:Mean monocyte telomere length in the diabetic group was highly significantly lower than in control subjects (4.0 [1.1] vs. 5.5 [1.1]; P < 0.0001), without significant differences in lymphocyte telomere length. There was a trend toward increased oxidative DNA damage in all diabetes cell types examined and a significant inverse relationship between oxidative DNA damage and telomere length (r = -0.55; P = 0.018) in the diabetic group. Telomere length was unrelated to plasma CRP concentration or insulin resistance.CONCLUSIONS:Monocyte telomere shortening in type 2 diabetes could be due to increased oxidative DNA damage to monocyte precursors during cell division. This data suggests that monocytes adhering to vascular endothelium and entering the vessel wall in type 2 diabetes are from a population with shorter telomeres and at increased risk of replicative senescence within vascular plaque.