Michèle M. Iskandar

High hydrostatic pressure pre-treatment of whey proteins enhances whey protein hydrolysate inhibition of oxidative stress and IL-8 secretion in intestinal epithelial cells

Background High hyperbaric pressure treatment of whey protein isolate (WPI) causes changes in the protein structure that enhances the anti-oxidant and anti-inflammatory effects of WPI. Objective The aim of this study was to compare the anti-oxidant and anti-inflammatory effects of pressurized whey protein isolate (pWPI) vs. native WPI (nWPI) hydrolysates in Caco-2 cells exposed to hydrogen peroxide (H2O2). Design Cells were cultured with different concentrations of pWPI or nWPI hydrolysates either 1 h before or 1 h after H2O2. Cell viability, IL-8 secretion, intracellular reactive oxygen species (ROS), and the medium anti-oxidant capacity (FRAP assay) were measured. Results Prior to and after H2O2 exposure, pWPI and nWPI hydrolysates inhibited IL-8 secretion and ROS generation, and increased FRAP activity in a dose-dependent manner. The maximal inhibition of H2O2-induced IL-8 secretion was greater with 2000 µg mL−1 of pWPI (50%) vs. nWPI (30%) hydrolysates. At the latter concentration, inhibition of H2O2-induced ROS formation reached 76% for pWPI, which was greater than for nWPI hydrolysates (32.5%). Conclusions These results suggest that WPI hydrolysates can alleviate inflammation and oxidative stress in intestinal cells exposed to oxidative injury, which is further enhanced by hyperbaric pressure pre-treatment of WPI.

Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4

UNLABELLED Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity. IN CONCLUSION (1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.

Down-regulation of cytokine-induced interleukin-8 requires inhibition of p38 mitogen-activated protein kinase (MAPK) via MAPK phosphatase 1 dependent and independent mechanisms

Down-regulation of overabundant interleukin (IL)-8 present in cystic fibrosis (CF) airways could ease excessive neutrophil burden and its deleterious consequences for the lung. IL-8 production in airway epithelial cells, stimulated with e.g. inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, is regulated by several signaling pathways including nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). We previously demonstrated that the anti-inflammatory drugs dexamethasone and ibuprofen suppress NF-κB; however, only dexamethasone down-regulates cytokine-induced IL-8, highlighting the importance of non-NF-κB mechanisms. Here, we tested the hypothesis that down-regulation of cytokine-induced IL-8 requires modulation of the MAPK phosphatase (MKP)-1/p38 MAPK/mRNA stability pathway. The effects of dexamethasone (5 nm) and ibuprofen (480 μm) on this pathway and IL-8 were studied in CF (CFTE29o−, CFBE41o−) and non-CF (1HAEo−) airway epithelial cells. We observed that dexamethasone, but not ibuprofen, destabilizes IL-8 mRNA and up-regulates MKP-1 mRNA. Further, siRNA silencing of MKP-1, via p38 MAPK, leads to IL-8 overproduction and diminishes the anti-IL-8 potential of dexamethasone. However, MKP-1 overexpression does not significantly alter IL-8 production. By contrast, direct inhibition of p38 MAPK (inhibitor SB203580) efficiently suppresses IL-8 with potency comparable with dexamethasone. Similar to dexamethasone, SB203580 decreases IL-8 mRNA stability. Dexamethasone does not affect p38 MAPK activation, which excludes its effects upstream of p38 MAPK. In conclusion, normal levels of MKP-1 are necessary for a full anti-IL-8 potential of pharmacological agents; however, efficient pharmacological down-regulation of cytokine-induced IL-8 also requires direct effects on p38 MAPK and mRNA stability independently of MKP-1.

Biotransformation of anthocyanins from two purple-fleshed sweet potato accessions in a dynamic gastrointestinal system

Cooked, milled purple-fleshed sweet potato (PFSP) accessions, PM09.812 and PM09.960, underwent digestion in a dynamic human gastrointestinal (GI) model that simulates gut digestive conditions to study the bioaccessibility and biotransformation of anthocyanins. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry showed accession-dependent variations in anthocyanin release and degradation. After 24h, more anthocyanin species were detected in the small intestinal vessel relative to other vessels for accession PM09.960 whereas more species appeared in the ascending colonic vessel for accession PM09.812. The ferric reducing antioxidant power was increased in the small intestinal vessel for PM09.960 and in the ascending colonic vessel for accession PM09.812, corresponding to the appearance of a majority of anthocyanins for each accession. These results show that intestinal and colonic microbial digestion of PFSP leads to an accession-dependent pattern for anthocyanin bioaccessibility and degradation.

Extract of Irish potatoes (Solanum tuberosum L.) decreases body weight gain and adiposity and improves glucose control in the mouse model of diet-induced obesity.

Both sexes of mice were fed a high fat diet (HFD) for 10 weeks without and with polyphenolic-rich potato extracts (PRPE) of cultivars Onaway and Russet Burbank. PRPE attenuated weight gain in male and female mice by as much as 63.2%, which was associated mostly with a reduction in adiposity. Mice receiving PRPE showed enhanced capacity for blood glucose clearance. Sex differences regarding the impact of HFD and PRPE on plasma levels of insulin, ghrelin, leptin, gastric inhibitory peptide, and resistin were noted. PRPE may serve as part of a preventative dietary strategy against the development of obesity and type 2 diabetes.

High Hydrostatic Pressure Pretreatment of Whey Protein Isolates Improves Their Digestibility and Antioxidant Capacity

Whey proteins have well-established antioxidant and anti-inflammatory bioactivities. High hydrostatic pressure processing of whey protein isolates increases their in vitro digestibility resulting in enhanced antioxidant and anti-inflammatory effects. This study compared the effects of different digestion protocols on the digestibility of pressurized (pWPI) and native (nWPI) whey protein isolates and the antioxidant and anti-inflammatory properties of the hydrolysates. The pepsin-pancreatin digestion protocol was modified to better simulate human digestion by adjusting temperature and pH conditions, incubation times, enzymes utilized, enzyme-to-substrate ratio and ultrafiltration membrane molecular weight cut-off. pWPI showed a significantly greater proteolysis rate and rate of peptide appearance regardless of digestion protocol. Both digestion methods generated a greater relative abundance of eluting peptides and the appearance of new peptide peaks in association with pWPI digestion in comparison to nWPI hydrolysates. Hydrolysates of pWPI from both digestion conditions showed enhanced ferric-reducing antioxidant power relative to nWPI hydrolysates. Likewise, pWPI hydrolysates from both digestion protocols showed similar enhanced antioxidant and anti-inflammatory effects in a respiratory epithelial cell line as compared to nWPI hydrolysates. These findings indicate that regardless of considerable variations of in vitro digestion protocols, pressurization of WPI leads to more efficient digestion that improves its antioxidant and anti-inflammatory properties.

Effects of Simulated Human Gastrointestinal Digestion of Two Purple-Fleshed Potato Cultivars on Anthocyanin Composition and Cytotoxicity in Colonic Cancer and Non-Tumorigenic Cells

A dynamic human gastrointestinal (GI) model was used to digest cooked tubers from purple-fleshed Amachi and Leona potato cultivars to study anthocyanin biotransformation in the stomach, small intestine and colonic vessels. Colonic Caco-2 cancer cells and non-tumorigenic colonic CCD-112CoN cells were tested for cytotoxicity and cell viability after 24 h exposure to colonic fecal water (FW) digests (0%, 10%, 25%, 75% and 100% FW in culture media). After 24 h digestion, liquid chromatography-mass spectrometry identified 36 and 15 anthocyanin species throughout the GI vessels for Amachi and Leona, respectively. The total anthocyanin concentration was over thirty-fold higher in Amachi compared to Leona digests but seven-fold higher anthocyanin concentrations were noted for Leona versus Amachi in descending colon digests. Leona FW showed greater potency to induce cytotoxicity and decrease viability of Caco-2 cells than observed with FW from Amachi. Amachi FW at 100% caused cytotoxicity in non-tumorigenic cells while FW from Leona showed no effect. The present findings indicate major variations in the pattern of anthocyanin breakdown and release during digestion of purple-fleshed cultivars. The differing microbial anthocyanin metabolite profiles in colonic vessels between cultivars could play a significant role in the impact of FW toxicity on tumor and non-tumorigenic cells.

Absorption and Metabolism of Phenolics from Digests of Polyphenol-Rich Potato Extracts Using the Caco-2/HepG2 Co-Culture System

The bioactivity of dietary polyphenols depends upon gastrointestinal and hepatic metabolism of secondary microbial phenolic metabolites generated via colonic microbiota-mediated biotransformation. A polyphenol-rich potato extract (PRPE) containing chlorogenic, caffeic, and ferulic acids and rutin was digested in a dynamic multi-reactor gastrointestinal simulator of the human intestinal microbial ecosystem (GI model). Simulated digestion showed extensive degradation of the parent compounds and the generation of microbial phenolic metabolites. To characterize the transport and metabolism of microbial phenolic metabolites following digestion, a co-culture of intestinal Caco-2 and hepatic HepG2 cells was exposed to the PRPE-derived digests obtained from the colonic vessels. Following a 2 h incubation of the digesta with the Caco-2/HepG2 co-cultures, approximately 10–15% of ferulic, dihydrocaffeic, and dihydroferulic acids and 3–5% of 3-hydroxybenzoic, 3-hydroxyphenylpropionic, and coumaric acids were observed in the basolateral side, whereas 3-hydroxyphenylacetic acid, phenylpropanoic acid, and cinnamic acid were not detected. Subsequent HepG2 cellular metabolism led to major increases in ferulic, dihydrocaffeic, 3-hydroxyphenylpropionic, and coumaric acids ranging from 160–370%. These findings highlight the importance of hepatic metabolism towards the generation of secondary metabolites of polyphenols despite low selective Caco-2 cellular uptake of microbial phenolic metabolites.

Inhibitory effects of apple peel polyphenol extract on the formation of heterocyclic amines in pan fried beef patties.

The efficacy of polyphenol-rich dried apple peel extract (DAPP) to inhibit the formation of heterocyclic aromatic amines (HCAs) during frying of beef patties was assessed after DAPP was applied at 0.1, 0.15 and 0.3% (w/w) either on the surface of the patties or mixed inside the patty prior to frying. 2-Amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx), 2-amino-1-ethyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-dimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) were quantified after frying. HCA concentrations decreased (p<0.05) upon both surface and mixed applications of DAPP at all of the tested doses. Surface application of 0.3% DAPP showed greater (p<0.05) inhibitory effects on HCA formation by 68% for MeIQx, 56% for 4,8-DiMeIQx and 83% for PhIP as opposed to 41%, 21% and 60% respectively, for the mixed DAPP application of 0.3%. The present study results indicate that surface application of DAPP in meat preparation prior to pan-frying can be a useful approach to minimize the formation of genotoxic HCAs in fried beef patties.

Microbial Biotransformation of a Polyphenol-Rich Potato Extract Affects Antioxidant Capacity in a Simulated Gastrointestinal Model

A multistage human gastrointestinal model was used to digest a polyphenol-rich potato extract containing chlorogenic acid, caffeic acid, ferulic acid, and rutin as the primary polyphenols, to assess for their microbial biotransformation and to measure changes in antioxidant capacity in up to 24 h of digestion. The biotransformation of polyphenols was assessed by liquid chromatography–mass spectrometry. Antioxidant capacity was measured by the ferric reducing antioxidant power (FRAP) assay. Among the colonic reactors, parent (poly)phenols were detected in the ascending (AC), but not the transverse (TC) or descending (DC) colons. The most abundant microbial phenolic metabolites in all colonic reactors included derivatives of propionic acid, acetic acid, and benzoic acid. As compared to the baseline, an earlier increase in antioxidant capacity (T = 8 h) was seen in the stomach and small intestine vessels as compared to the AC (T = 16 h) and TC and DC (T = 24 h). The increase in antioxidant capacity observed in the DC and TC can be linked to the accumulation of microbial smaller-molecular-weight phenolic catabolites, as the parent polyphenolics had completely degraded in those vessels. The colonic microbial digestion of potato-based polyphenols could lead to improved colonic health, as this generates phenolic metabolites with significant antioxidant potential.