Dietary polyphenols protect against metabolic syndrome, despite limited absorption and digestion, raising questions about their mechanism of action. We hypothesized that one mechanism may involve the gut microbiota. To test this hypothesis, C57BL/6J mice were fed a high-fat diet (HFD) containing 1% Concord grape polyphenols (GP). Relative to vehicle controls, GP attenuated several effects of HFD feeding, including weight gain, adiposity, serum inflammatory markers (tumor necrosis factor [TNF]α, interleukin [IL]-6, and lipopolysaccharide), and glucose intolerance. GP lowered intestinal expression of inflammatory markers (TNFα, IL-6, inducible nitric oxide synthase) and a gene for glucose absorption (Glut2). GP increased intestinal expression of genes involved in barrier function (occludin) and limiting triglyceride storage (fasting-induced adipocyte factor). GP also increased intestinal gene expression of proglucagon, a precursor of proteins that promote insulin production and gut barrier integrity. 16S rRNA gene sequencing and quantitative PCR of cecal and fecal samples demonstrated that GP dramatically increased the growth of Akkermansia muciniphila and decreased the proportion of Firmicutes to Bacteroidetes, consistent with prior reports that similar changes in microbial community structure can protect from diet-induced obesity and metabolic disease. These data suggest that GP act in the intestine to modify gut microbial community structure, resulting in lower intestinal and systemic inflammation and improved metabolic outcomes. The gut microbiota may thus provide the missing link in the mechanism of action of poorly absorbed dietary polyphenols.

The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread. When expressed alone, E4orf4 kills transformed cells but not normal human cells. The only clear target of E4orf4 in mammalian cells is the Ba (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the eects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B' subunits, respectively. E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit its eect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55. These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A. However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function aecting cell growth. Oncogene (2001) 20, 5279 ± 5290.

The TNO intestinal model (TIM-1) of the human upper gastrointestinal tract was used to compare intestinal absorption/bioaccessibility of blueberry anthocyanins under different digestive conditions. Blueberry polyphenol-rich extract was delivered to TIM-1 in the absence or presence of a high-fat meal. HPLC analysis of seventeen anthocyanins showed that delphinidin-3-glucoside, delphinidin-3-galactoside, delphinidin-3-arabinoside and petunidin-3-arabinoside were twice as bioaccessible in fed state, whilst delphinidin-3-(6″-acetoyl)-glucoside and malvidin-3-arabinoside were twice as bioaccessible under fasted conditions, suggesting lipid-rich matrices selectively effect anthocyanin bioaccessibility. TIM-1 was fed blueberry juice (BBJ) or blueberry polyphenol-enriched defatted soybean flour (BB-DSF) containing equivalent amounts of free or DSF-sorbed anthocyanins, respectively. Anthocyanin bioaccessibility from BB-DSF (36.0 ± 10.4) was numerically, but not significantly, greater than that from BBJ (26.3 ± 10.3). Ileal efflux samples collected after digestion of BB-DSF contained 2.8-fold more anthocyanins than same from BBJ, suggesting that protein-rich DSF protects anthocyanins during transit through upper digestive tract for subsequent colonic delivery/metabolism.

The present study demonstrated that defatted soybean flour (DSF) can sorb polyphenols from blueberry and cranberry juices while separating them from sugars. Depending on DSF concentration and juice dilution, the concentration of blueberry anthocyanins and total polyphenols sorbed to DSF ranged from 2 – 22 mg/g and 10 – 95 mg/g, respectively while the concentration of anthocyanins and proanthocyanidins in cranberry polyphenol-enriched DSF ranged from 2.5 – 17 mg/g and 21 – 101 mg/g, respectively. Blueberry polyphenols present in one serving of fresh blueberries (73g) were delivered in just 1.4 g of blueberry polyphenol-enriched DSF. Similarly, one gram of cranberry polyphenol-enriched DSF delivered the amount of proanthocyanidins available in three 240 ml servings of cranberry juice cocktail. The concentration of blueberry anthocyanins and total polyphenols eluted from DSF remained constant after 22 weeks of incubation at 37°C, demonstrating the high stability of the polyphenol-DSF matrix. LC-MS analysis of eluates confirmed DSF retained major cranberry and blueberry polyphenols remained intact. Blueberry polyphenol-enriched DSF exhibited significant hypoglycemic activities in C57bl/6J mice, and cranberry polyphenol-enriched DSF showed anti-microbial and anti-UTI activities in vitro, confirming its efficacy. The described sorption process provides a means to create protein-rich food ingredients containing concentrated plant bioactives without excess sugars, fats and water that can be incorporated in a variety of scientifically validated functional foods and dietary supplements.

We previously showed that C57BL/6J mice fed high-fat diet (HFD) supplemented with 1% grape polyphenols (GP) for 12 weeks developed a bloom of Akkermansia muciniphila with attenuated metabolic syndrome symptoms. Here we investigated early timing of GP-induced effects and the responsible class of grape polyphenols. Mice were fed HFD, low-fat diet (LFD) or formulations supplemented with GP (HFD-GP, LFD-GP) for 14 days. Mice fed HFD-GP, but not LFD-GP, showed improved oral glucose tolerance compared to controls. A. muciniphila bloom occurred earlier in mice fed LFD-GP than HFD-GP; however, timing was dependent on baseline A. muciniphila levels rather than dietary fat. Mice gavaged for 10 days with GP extract (GPE) or grape proanthocyanidins (PACs), each delivering 360 mg PACs/kg body weight, induced a bloom of fecal and cecal A. muciniphila, the rate of which depended on initial A. muciniphila abundance. Grape PACs were sufficient to induce a bloom of A. muciniphila independent of specific intestinal gene expression changes. Gut microbial community analysis and in vitro inhibition of A. muciniphila by GPE or PACs suggest that the A. muciniphila bloom in vivo occurs via indirect mechanisms.

Defatted soybean flour (DSF) can sorb and concentrate blueberry anthocyanins and other polyphenols, but not sugars. In this study blueberry polyphenol-enriched DSF (BB-DSF) or DSF were incorporated into very high fat diet (VHFD) formulations and provided ad libitum to obese and hyperglycemic C57BL/6 mice for 13 weeks to investigate anti-diabetic effects. Compared to the VHFD containing DSF, the diet supplemented with BB-DSF reduced weight gain by 5.6%, improved glucose tolerance, and lowered fasting blood glucose levels in mice within 7 weeks of intervention. Serum cholesterol of mice consuming the BB-DSF-supplemented diet was 13.2% lower than mice on the diet containing DSF. Compounds were eluted from DSF and BB-DSF for in vitro assays of glucose production and uptake. Compared to untreated control, doses of BB-DSF eluate containing 0.05 – 10 μg/μL of blueberry anthocyanins significantly reduced glucose production by 24% - 74% in H4IIE rat hepatocytes, but did not increase glucose uptake in L6 myotubes. The results indicate that delivery of blueberry polyphenols stabilized in a high-protein food matrix may be useful for the dietary management of pre-diabetes and/or diabetes.

Defatted soy flour (DSF), soy protein isolate (SPI), hemp protein isolate (HPI), medium roast peanut flour (MPF) and pea protein isolate (PPI) stably bind and concentrate cranberry (CB) polyphenols, creating protein/polyphenol-enriched matrices. Proanthocyanidins (PAC) in the enriched matrices ranged from 20.75 mg/g (CB-HPI) to 10.68 mg/g (CB-SPI). Anthocyanins (ANC) ranged from 3.19 mg/g (CB-DSF) to 1.68 mg/g (CB-SPI), while total phenolics (TP) ranged from 37.61 mg/g (CB-HPI) to 21.29 mg/g (CB-SPI). LC-MS indicated that the enriched matrices contained all identifiable ANC, PAC and flavonols present in CB juice. Complexation with SPI stabilized and preserved the integrity of the CB polyphenolic components for at least 15 weeks at 37 °C. PAC isolated from enriched matrices demonstrated comparable anti-adhesion bioactivity to PAC isolated directly from CB juice (MIC 0.4 to 0.16 mg/mL), indicating their potential utility for maintenance of urinary tract health. Approximately 1.0 g of polyphenol-enriched matrix delivered the same amount of PAC available in one cup (300 mL) of commercial CB juice cocktail; which has been shown clinically to be the prophylactic dose for reducing recurring urinary tract infections. CB-SPI inhibited gram- positive and gram-negative bacterial growth. Nutritional and sensory analyses indicated that the targeted CB-matrix combinations have high potential for incorporation in functional food formulations.

Cranberry pomace is a byproduct of cranberry processing and is comprised of seeds, skins and stems of the cranberry fruit. While cranberry pomace contains beneficial polyphenols, including proanthocyanidins and anthocyanins, it is not a palatable source of these compounds and is typically discarded. In this study, we have developed and optimized a method to extract polyphenols from cranberry pomace using aqueous ethanol, a food grade solvent. Biochemical characterization of the pomace extract showed the presence of a broad range of polyphenols also present in cranberry juice concentrate. By co-drying cranberry pomace extract with a protein-rich food matrix, such as soy protein isolate (SPI), we have developed a method to produce a cranberry polyphenol-SPI complex (CBP-SPI) containing 10% cranberry polyphenols. Unlike dried cranberry pomace extract alone, proanthocyanidins, anthocyanins and total polyphenols were found to be highly stable at 37 °C in the CBP-SPI powder. The extraction and stabilization of cranberry pomace polyphenols using SPI provides an innovative approach for utilizing pomace in the development of novel food ingredients.