Gallic acid mitigates intestinal inflammation and loss of tight junction protein expression using a 2D-Caco-2 and RAW 264.7 co-culture model.

A 2D-intestinal epithelial Caco-2/RAW 264.7 macrophage co-culture model was developed to demonstrate the relative efficacy of different phenolic acids to mitigate changes in Caco-2 epithelial cell redox state initiated both directly by autoxidation products, H2O2, and indirectly through cell communication events originating from cytokine stimulated macrophage. An inducer cocktail (lipopolysaccharide + interferon gamma) was used to activate RAW 264.7 cells in the 2D- Caco-2/RAW co-culture and intracellular changes in Caco-2 cell redox signaling occurred in response to positive changes (p < 0.05) in inflammatory biomarkers derived in macrophage that included IL-6, TNF-α, nitric oxide and peroxynitrite, respectively. Phenolic acids varied in relative capacity to reduce NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in cocktail inflamed induced macrophage. This response in addition to the relative predisposition of gallic acid (GA) to undergo autoxidation to generate H2O2 activity (p < 0.05), culminated in downstream cell signaling in Caco-2 nuclear factor erythroid 2-related factor (Nrf2) activity (increase 26.9 %), altered monolayer integrity (increase 33.7 %), and release of interleukin 8 (IL-8) (decrease 80.5 %) (p < 0.05). It can be concluded that the co-culture model described herein was useful to assess the importance of communication between cytokine stimulated macrophage and intestinal cells. Moreover, the relative unique efficacy of GA, compared to other phenolic acids tested to protect against activated macrophage induced changes related to intestinal dysfunction were particularly relevant to epithelial redox signaling, intestinal permeability and regulation of tight junction proteins. This study concludes that phenolic acids are not equal in the capacity to protect against intestinal cell dysfunction despite some indication of biological activity.

Application of a HyPer-3 sensor to monitor intracellular H2O2 generation induced by phenolic acids in differentiated Caco-2 cells.

Intestinal epithelial cells (IECs) are an important point of contact between dietary food components consumed and subsequent whole-body utilization for body maintenance and growth. Selective bioactive phenolic acids, widely present in fruits, vegetables and beverages can generate hydrogen peroxide (H2O2) and contribute to the cellular redox balance, hence influencing well-known cellular antioxidant and pro-oxidant mechanisms. Our findings have showed that increasing extracellular H2O2 resulted in associated changes in intracellular H2O2 levels in Caco-2 cells (p < 0.05) which was facilitated by activity of a family of water channel membrane proteins, termed aquaporins (AQPs). To demonstrate this, a HyPer-3 genetically encoded fluorescent H2O2 sensitive indicator was used to enable fluorescent real-time imaging of intracellular H2O2 levels as a measure of changes occurring in extracellular H2O2 in differentiated Caco-2 cells exposed to different phenolic acids. The use of confocal microscopy and flow cytometry, respectively, captured visualization and quantification of H2O2 uptake in differentiated Caco-2 cells. DFP00173, an aquaporin 3 (AQP3) inhibitor was effective at inhibiting the intracellular uptake of H2O2 and was sensitive to varied levels of H2O2 generated when different phenolic acids were added to the culture media. In summary, HyPer-3 was shown to be an effective technique to demonstrate relative capabilities of structurally different dietary phenolic acids that have potential to alter intestinal redox balance by changing intracellular H2O2, and either antioxidant or pro-oxidant activity, respectively.

Bacteriophage-Insensitive Mutants of Antimicrobial-Resistant Salmonella Enterica are Altered in their Tetracycline Resistance and Virulence in Caco-2 Intestinal Cells.

Bacteriophages have shown promise as therapeutic alternatives to antibiotics for the control of infectious bacteria, including the human pathogen Salmonella. However, the development of effective phage-based applications requires the elucidation of key interactions between phages and target hosts, particularly since host resistance to phage is inevitable. Little is known about the alteration of host phenotypes following the development of resistance to phage. The aim of this study is to evaluate the antibiotic susceptibility and virulence of a Salmonella isolate following the development of resistance to bacteriophage SI1. We observed enhanced susceptibility to tetracycline and decreased invasion capacity in a differentiated Caco-2 intestinal cell line. Whole genome sequence analysis revealed an array of mutations, most notably, truncations in vgrG1_2, a core gene involved in Type VI secretion and mutations in the lipopolysaccharide, thereby indicating the plausible attachment site of phage SI1. These findings shed light on understanding the underlying mechanism for phage immunity within the host. Importantly, we reveal an associated genetic cost to the bacterial host with developing resistance to phages. Taken together, these results will aid in advancing strategies to delay or eliminate the development of host resistance when designing informed phage-based antimicrobials.

Molecular Mechanisms That Define Redox Balance Function in Pathogen-Host Interactions-Is There a Role for Dietary Bioactive Polyphenols?

To ensure a functional immune system, the mammalian host must detect and respond to the presence of pathogenic bacteria during infection. This is accomplished in part by generating reactive oxygen species (ROS) that target invading bacteria; a process that is facilitated by NADPH oxidase upregulation. Thus, bacterial pathogens must overcome the oxidative burst produced by the host innate immune cells in order to survive and proliferate. In this way, pathogenic bacteria develop virulence, which is related to the affinity to secrete effector proteins against host ROS in order to facilitate microbial survival in the host cell. These effectors scavenge the host generated ROS directly, or alternatively, manipulate host cell signaling mechanisms designed to benefit pathogen survival. The redox-balance of the host is important for the regulation of cell signaling activities that include mitogen-activated protein kinase (MAPK), p21-activated kinase (PAK), phosphatidylinositol 3-kinase (PI3K)/Akt, and nuclear factor κB (NF-κB) pathways. An understanding of the function of pathogenic effectors to divert host cell signaling is important to ascertain the mechanisms underlying pathogen virulence and the eventual host-pathogen relationship. Herein, we examine the effectors produced by the microbial secretion system, placing emphasis on how they target molecular signaling mechanisms involved in a host immune response. Moreover, we discuss the potential impact of bioactive polyphenols in modulating these molecular interactions that will ultimately influence pathogen virulence.

The Role of Nitric Oxide in Regulating Intestinal Redox Status and Intestinal Epithelial Cell Functionality.

Important functions of intestinal epithelial cells (IECs) include enabling nutrient absorption to occur passively and acting as a defense barrier against potential xenobiotic components and pathogens. A compromise to IEC function can result in the translocation of bacteria, toxins, and allergens that lead to the onset of disease. Thus, the maintenance and optimal function of IECs are critically important to ensure health. Endogenous biosynthesis of nitric oxide (NO) regulates IEC functionality both directly, through free radical activity, and indirectly through cell signaling mechanisms that impact tight junction protein expression. In this paper, we review the current knowledge on factors that regulate inducible nitric oxide synthase (iNOS) and the subsequent roles that NO has on maintaining IECs' intestinal epithelial barrier structure, functions, and associated mechanisms of action. We also summarize important findings on the effects of bioactive dietary food components that interact with NO production and affect downstream intestinal epithelium integrity.

Changes in levels of enzyme inhibitors during soaking and cooking for pulses available in Canada.

The effects of processing (soaking and cooking) on enzyme inhibitors (α-amylase, trypsin and chymotrypsin inhibitors) in a range of pulses (4 peas, 9 lentils, 3 chickpeas, 2 faba beans and 4 beans) were investigated, using soybean as a control. Analysis of variance indicated that pulse type, treatment and their interaction had significant effects on levels of all enzyme inhibitors. Soybean contained the highest levels of trypsin inhibitory activity (TIA) and chymotrypsin inhibitory activity (CIA) among all seeds. α-Amylase inhibitory activity was absent from peas, lentils, chickpeas and faba beans, but was present in beans and soybean. TIA was found to be low in peas but high in beans. Beans contained relatively high CIA levels followed by chickpeas, lentils, peas and faba beans. Soaking markedly decreased the activity of enzyme inhibitors. Cooking of presoaked seeds was even more effective as greater reductions (78.7-100%) were observed for all pulses. The content of enzyme inhibitors in pulses varied widely, but levels of protease inhibitors were generally lower that those found in soybean. Processing, in particular heat treatments, drastically reduced these levels.

Hydrogen Peroxide Produced from Selective Phenolic Acids in Cell Culture Underlies Caco-2 Changes in Cell Proliferation Parameters.

The physicochemical property of phenolic acids to generate hydrogen peroxide (H2O2) in cell culture media has been underreported when describing multiple biological effects in vitro. Our aim was to focus on examining the relative capacity of four common phenolic acids widely consumed in the Western diet for autoxidation potential to generate H2O2 during in vitro culture. Furthermore, quantifying H2O2 derived from different phenolic acids cultured in Dulbecco's modified Eagle's medium (DMEM) was associated with changes in cell proliferation in non-differentiated human intestinal carcinoma cells. Results showed that the different percentage losses of phenolic acids, namely, caffeic (84.78 ± 1.51), chlorogenic (37.3 ± 0.38), ferulic (1.26 ± 0.78), and gallic (100%), paralleled a relative capacity to generate H2O2 when present in DMEM media for 24 h. The rate and total H2O2 generated was dependent on both phenolic acid type and concentration (p < 0.05). Gallic acid had the greatest capacity to generate H2O2 in culture without the presence of cells (p < 0.05). When cultured with non-differentiated Caco-2 cells, gallic acid evoked the greatest bioactivity that included cytotoxicity, anti-proliferation, apoptosis, and nuclear condensation, respectively (p < 0.05). Corresponding treatments with cells with phenolic acids in the presence of catalase confirmed that H2O2 generated from phenolic acid autoxidation was involved in cell proliferation and apoptosis.

Intestinal polyphenol antioxidant activity involves redox signaling mechanisms facilitated by aquaporin activity.

Ascertaining whether dietary polyphenols evoke an antioxidant or prooxidant activity, which translates to a functional role required to maintain intestinal cell homeostasis continues to be an active and controversial area of research for food chemists and biochemists alike. We have proposed that the paradoxical function of polyphenols to autoxidize to generate H2O2 is a required first step in the capacity of some plant phenolics to function as intracellular antioxidants. This is based on the fact that cell redox homeostasis is achieved by a balance between H2O2 formation and subsequent outcomes of antioxidant systems function. Maintaining optimal extracellular and intracellular H2O2 concentrations is required for cell survival, since low levels are important to upregulate endogenous antioxidant capacity; whereas, concentrations that go beyond homeostatic control typically result in an inflammatory response, growth arrest, or eventual cell death. Aquaporins (AQPs) are a family of water channel membrane proteins that facilitate cellular transportation of water and other small molecule-derived solutes, such as H2O2, in all organisms. In the intestine, AQPs act as gatekeepers to regulate intracellular uptake of H2O2, generated from extracellular polyphenol autoxidation, thus enabling an intracellular cell signaling responses to mitigate onset of oxidative stress and intestinal inflammation. In this review, we highlight the potential role of AQPs to control important underlying mechanisms that define downstream regulation of intestinal redox homeostasis, specifically. It has been established that polyphenols that undergo oxidation to the quinone form, resulting in subsequent adduction to a thiol group on Keap1-Nrf2 complex, trigger Nrf2 activation and a cascade of indirect intracellular antioxidant effects. Here, we propose a similar mechanism that involves H2O2 generated from specific dietary polyphenols with a predisposition to undergo autoxidation. The ultimate bioactivity is regulated and expressed by AQP membrane function and thus, by extension, represents expression of an intracellular antioxidant chemoprotection mechanism.

Prooxidant capacity of phenolic acids defines antioxidant potential.

Phenolic acids derived from vegetables, fruits and beverages are considered abundant sources of natural antioxidants consumed in the human diet. In addition to having well-known antioxidant activity, phenolic acids also exhibit pro-oxidant activity under selected conditions. We hypothesized that the availability of extracellular H2O2 derived from phenolic acid autoxidation will diffuse across cell membranes to participate as a messenger molecule to activate intracellular redox signaling in response to oxidative stress. We report on the relative activity of structurally different phenolic acids to generate specific changes in the extracellular - intracellular H2O2 flux that induces intracellular redox signaling corresponding to a function to reduce intracellular oxidative stress. HyPer-3 methodology was used to measure increases in intracellular H2O2 in differentiated Caco-2 intestinal cells in response to phenolic acid autoxidation and changes in extracellular H2O2 production. The potential for different phenolic acids to autoxidize and generate H2O2 was dependent on the structure and concentration of phenolic acid. Activation of nuclear factor erythroid 2-related factor (Nrf2) cell signaling was enhanced (p < 0.05) by phenolic acid induced H2O2 production, and mitigated when present along with catalase (p < 0.05), or, alternatively by blocking aquaporin 3 (AQP3) function (p < 0.05) using DFP00173 as the AQP3 inhibitor. The relative capacity of phenolic acids to generate H2O2 via autoxidation was structure specific and corresponded to the level of Nrf2 cell signaling in differentiated Caco-2 epithelial cells. The Nrf2-Keap1 response paralleled the extent of reduced oxidative stress observed in differentiated Caco-2 cells determined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Lysine-Derived Maillard Reaction Products Inhibit the Growth of Salmonella enterica Serotype Typhimurium.

An emerging consumer trend to purchase minimally heated and ready-to-eat food products may result in processing methods that do not effectively reduce pathogenic populations. Crude Maillard reaction products (MRPs) are naturally generated compounds that have been shown to display antimicrobial effects against pathogens. Crude MRPs were generated from reducing sugars (fructose (Fru), glucose (Glc), ribose (Rib) or xylose (Xyl)) with lysine and the melanoidin equivalence was measured using an absorbance of 420 nm (Ab420). The relative antimicrobial activity of each MRP was measured by examining both the length of lag phase and maximum growth rate. MRPs were found to significantly shorten the lag phase and decrease the maximum growth rate of S. Typhimurium (p < 0.05). Glucose-lysine MRP (GL MRP) was determined to have the highest relative melanoidin (1.690 ± 0.048 at Ab420) and its efficacy against S. Typhimurium populations was measured at 37 °C and at pH 7.0 and estimated on xylose lysine deoxycholate (XLD) agar. GL MRP significantly reduced S. Typhimurium populations by >1 log CFU/mL at 8 and 24 h after inoculation (p < 0.05). GL MRPs also further decreased S. Typhimurium populations significantly under thermal stress condition (55 °C) compared to optimal (37 °C) by ~1 log CFU/mL (p < 0.05). Overall, GL MRP demonstrated effective antimicrobial activity against S. Typhimurium at 37 °C and 55 °C.

Molecular Basis for the Simultaneous Enhancement of the Aroma-Generating Capacity and Bioactivity of Maillard Reaction Precursors through Mechanochemistry.

Ball milling at ambient temperatures can accelerate the formation and accumulation of early-stage Maillard reaction intermediates considered important precursors of aromas and antioxidants. In this study, using chemical and biological assays, we explored the potential of sequential milling and heating to enhance the antioxidant and aroma-generating capacity of Maillard model systems. Milling (30 Hz/30 min) followed by dry heating (90 °C/30 min) of glycine or lysine with glucose significantly increased not only the intensity of their aroma-active compounds as analyzed by headspace-gas chromatography/mass spectrometry (HS-GC/MS) but also their free radical scavenging capacity as assessed by 2,2'-azino-bis-(3-ethylbenzothiazoneline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assays. This was attributed to the increased formation of redox-active endiol moieties and precursors of N,N-dialkyl-pyrazinium radical cation in the lysine system assessed by electrospray ionization-quadrupole time-of-flight/tandem mass spectrometry (ESI-QqTOF/MS/MS) analysis. The test samples also inhibited NO generation and cellular oxidative stress in RAW 264.7 murine macrophage cells, indicating size reduction induced by milling promoted paracellular absorption.

Characterization of phytochemical mixtures with inflammatory modulation potential from coffee leaves processed by green and black tea processing methods.

Our previous study reported that different tea processing methods along with the age of coffee leaves affected antioxidant and anti-inflammatory bioactivities; however, identification of phytochemical components or associated mixtures that contribute to the anti-/pro-inflammatory activities was not determined. Herein, we report results of additional experiments designed to characterize the phytochemical composition of fractionated coffee leaf extract, derived from Japanese-style-green-tea-process-young (JGTP-Y) and black-tea-process-mature (BTP-M) leaves and related these data to anti-/pro-inflammatory activities. The aqueous fraction of BTP-M coffee leaves induced nitric oxide (NO), iNOS, COX-2, IL-6 and IL-10 production in Raw 264.7 cells. A 40% methanol fraction possessed greatest anti-inflammatory activities in IFN-γ and LPS treated Raw 264.7 cells (P < 0.05). The anti-inflammatory activities of coffee leaf fractions could not only be attributed to chlorogenic acids, mangiferin, rutin, and caffeine content, but possibly subtle interactions of mixtures of bioactive molecules.