Intestinal Cell Monolayers Research Articles

Short communication: Inhibition of angiotensin 1-converting enzyme by peptides derived from variants of bovine β-casein upon apical exposure to a Caco-2 cell monolayer

This study investigated the consequence of genetically contingent amino acid substitutions in bovine β-casein (CN) genetic variants A1, A2, B, and I on the structure and bioactive potential of peptides following in vitro digestion. The β-CN variants were digested in vitro using pepsin and pancreatin, and a peptide profile was obtained by liquid chromatography tandem mass spectrometry, revealing among others, the β-casomorphin precursor peptides VYPFPGPIHN and VYPFPGPIPN, derived from variant A1/B and from A2/I, respectively. These 2 peptides were synthesized and assessed for angiotensin 1-converting enzyme (ACE) inhibitory capacity before and after incubation with a monolayer of Caco-2 intestinal cells. The VYPFPGPIHN was a stronger ACE inhibitor than VYPFPGPIPN, with the concentration needed to reach half-maximal inhibition (IC50) of 123 ± 14.2 μM versus 656 ± 7.6 μM. Exposure to a Caco-2 intestinal cell monolayer did not affect ACE inhibition by VYPFPGPIHN, but resulted in an almost 2-fold increase in inhibition by VYPFPGPIPN after incubation. Subsequent tandem mass spectrometric analysis identified the truncated peptide VYPFPGPIP, suggesting hydrolysis by a cell membrane associated peptidase. Thus, genetic variation in bovine β-CN results in the generation of peptides that differ in bioactivity, and are differently affected by intestinal brush border peptidases.

Orally Available Collagen Tripeptide: Enzymatic Stability, Intestinal Permeability, and Absorption of Gly-Pro-Hyp and Pro-Hyp.

Collagen-derived small peptides, such as Gly-Pro-Hyp (GPH) and Pro-Hyp (PH), play a role in various physiological functions. Although collagen degrades in the gastrointestinal tract randomly and easily, it is not readily cleaved into bioactive peptides. To increase the bioavailability of bioactive peptides, a collagen tripeptide (CTP) was prepared from fish scales by the digestion method using collagenase from nonpathogenic Bacillus bacteria. It was demonstrated that Hyp-containing peptides-GPH and PH-were better absorbed and reached higher plasma levels after the oral administration of CTPs in rats compared to high molecular weight collagen peptide (H-CP). GPH and PH were stable in gastrointestinal fluid and rat plasma for 2 h, and GPH was able to be transported across the intestinal cell monolayer. These results suggest that the ingestion of CTP is an efficient method for taking bioactive peptides orally due to the enzymatic stability and intestinal permeability of GPH and PH.

The 5A apolipoprotein A-I (apoA-I) mimetic peptide ameliorates experimental colitis by regulating monocyte infiltration.

New therapies for inflammatory bowel disease (IBD) are highly desirable. As apolipoprotein (apo)A-I mimetic peptides are beneficial in several animal models of inflammation, we hypothesized that they might be effective at inhibiting murine colitis. Daily injections of 5A peptide, a synthetic bihelical apoA-I mimetic dissolved in PBS, or PBS alone were administered to C57BL/6 mice fed 3% (w v(-1) ) dextran sodium sulfate (DSS) in drinking water or healthy controls. Daily treatment with 5A peptide potently restricted DSS-induced inflammation, as indicated by improved disease activity indices and colon histology, as well as decreased intestinal tissue myeloperoxidase levels and plasma TNFα and IL-6 concentrations. Additionally, plasma levels of monocyte chemoattractant protein-1 and the monocyte expression of adhesion-mediating molecule CD11b were down-regulated, pro-inflammatory CD11b(+) /Ly6c(high) monocytes were decreased, and the number of intestinal monocytes was reduced in 5A peptide-treated animals as determined by intravital macrophage-related peptide-8/14-directed fluorescence-mediated tomography and post-mortem immunhistochemical F4/80 staining. Intravital fluorescence microscopy of colonic microvasculature demonstrated inhibitory effects of 5A peptide on leukocyte adhesion accompanied by reduced plasma levels of the soluble adhesion molecule sICAM-1. In vitro 5A peptide reduced monocyte adhesion and transmigration in TNFα-stimulated monolayers of human intestinal microvascular endothelial cells. Increased susceptibility to DSS-induced inflammation was noted in apoA-I(-/-) mice. The 5A peptide is effective at ameliorating murine colitis by preventing intestinal monocyte infiltration and activation. These findings point to apoA-I mimetics as a potential treatment approach for IBD.

Lecithin-Based Nano-emulsification Improves the Bioavailability of Conjugated Linoleic Acid.

In this study, we investigated the effects of lecithin-based nano-emulsification on the heat stability and bioavailability of conjugated linoleic acid (CLA) in different free fatty acid (FFA) and triglyceride (TG) forms. CLA nano-emulsion in TG form exhibited a small droplet size (70-120 nm) compared to CLA nano-emulsion in FFA form (230-260 nm). Nano-emulsification protected CLA isomers in TG form, but not in free form, against thermal decomposition during the heat treatment. The in vitro bioavailability test using monolayers of Caco-2 human intestinal cells showed that nano-emulsification increased the cellular uptake of CLA in both FFA and TG forms. More importantly, a rat feeding study showed that CLA content in small intestinal tissues or plasma was higher when CLA was emulsified, indicating an enhanced oral bioavailability of CLA by nano-emulsification. These results provide important information for development of nano-emulsion-based delivery systems that improve thermal stability and bioavailability of CLA.

Carboxymethyl starch mucoadhesive microspheres as gastroretentive dosage form

Carboxymethyl starch microspheres (CMS-MS) were produced from carboxymethyl starch powder (CMS-P) with a degree of substitution (DS) from 0.1 to 1.5 in order to investigate the influence of DS on physicochemical, drug release and mucoadhesion properties as well as interactions with gastrointestinal tract (GIT) epithelial barrier models. Placebo and furosemide loaded CMS-MS were obtained by emulsion-crosslinking with sodium trimetaphosphate (STMP). DS had an impact on increasing equilibrium water uptake and modulating drug release properties of the CMS-MS according to the surrounding pH. The transepithelial electrical resistance (TEER) of NCI-N87 gastric cell monolayers was not influenced in presence of CMS-MS, whereas that of Caco-2 intestinal cell monolayers decreased with increasing DS but recovered initial values at about 15h post-treatment. CMS-MS with increasing DS also enhanced furosemide permeability across both NCI-N87 and Caco-2 monolayers at pH gradients from 3.0 to 7.4. Mucoadhesion of CMS-MS on gastric mucosa (acidic condition) increased with the DS up to 55% for a DS of 1.0 but decreased on neutral intestinal mucosa to less than 10% with DS of 0.1. The drug release, permeability enhancement and mucoadhesive properties of the CMS-MS suggest CMS-MS with DS between 0.6 and 1.0 as suitable excipient for gastroretentive oral delivery dosage forms.

Enhancing Nutraceutical Performance Using Excipient Foods: Designing Food Structures and Compositions to Increase Bioavailability

AbstractThe oral bioavailability of many bioactives (pharmaceuticals, dietary supplements, nutrients, and nutraceuticals) is limited because of physicochemical and physiological events that occur within the gastrointestinal tract (GIT) after their ingestion. These events include: (i) restricted liberation from drugs, supplements, or foods; (ii) extensive metabolism or chemical transformation during passage through the GIT; (iii) low solubility in intestinal fluids; (iv) low permeation through the intestinal cell monolayer; and (v) efflux from epithelium cells. Bioactive bioavailability can often be improved by designing the composition and structure of food matrices to control their liberation, transformation, solubilization, transport, absorption, and efflux in the GIT. This article reviews the potential impact of food composition and structure on the oral bioavailability of bioactives, and then shows how this knowledge can be used to design excipient foods that can improve the bioavailability profile of bioactives. The bioactive may be incorporated within an excipient food or co‐ingested with an excipient food. The suitability of oil‐in‐water emulsions as excipient foods is highlighted. The utilization of excipient foods may provide a new strategy for improving the efficacy of nutraceuticals, supplements, and pharmaceuticals.

Microfluidic Assembly of a Multifunctional Tailorable Composite System Designed for Site Specific Combined Oral Delivery of Peptide Drugs.

Multifunctional tailorable composite systems, specifically designed for oral dual-delivery of a peptide (glucagon-like peptide-1) and an enzymatic inhibitor (dipeptidyl peptidase 4 (DPP4)), were assembled through the microfluidics technique. Both drugs were coloaded into these systems for a synergistic therapeutic effect. The systems were composed of chitosan and cell-penetrating peptide modified poly(lactide-co-glycolide) and porous silicon nanoparticles as nanomatrices, further encapsulated in an enteric hydroxypropylmethylcellulose acetylsuccinate polymer. The developed multifunctional systems were pH-sensitive, inherited by the enteric polymer, enabling the release of the nanoparticles only in the simulated intestinal conditions. Moreover, the encapsulation into this polymer prevented the degradation of the nanoparticles' modifications. These nanoparticles showed strong and higher interactions with the intestinal cells in comparison with the nonmodified ones. The presence of DPP4 inhibitor enhanced the peptide permeability across intestinal cell monolayers. Overall, this is a promising platform for simultaneously delivering two drugs from a single formulation. Through this approach peptides are expected to increase their bioavailability and efficiency in vivo both by their specific release at the intestinal level and also by the reduced enzymatic activity. The use of this platform, specifically in combination of the two antidiabetic drugs, has clinical potential for the therapy of type 2 diabetes mellitus.

Interaction exists between matriptase inhibitors and intestinal epithelial cells

The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.

Controlled Dissolution of Griseofulvin Solid Dispersions from Electrosprayed Enteric Polymer Micromatrix Particles: Physicochemical Characterization and in Vitro Evaluation.

The oral bioavailability of a poorly water-soluble drug is often inadequate for the desired therapeutic effect. The bioavailability can be improved by enhancing the physicochemical properties of the drug (e.g., dissolution rate, permeation across the gastrointestinal tract). Other approach include shielding the drug from the gastric metabolism and targeted drug release to obtain optimal drug absorption. In this study, a poorly water-soluble model drug, griseofulvin, was encapsulated as disordered solid dispersions into Eudragit L 100-55 enteric polymer micromatrix particles, which were produced by electrospraying. Similar micromatrix particles were also produced with griseofulvin-loaded thermally oxidized mesoporous silicon (TOPSi) nanoparticles dispersed to the polymer micromatrices. The in vitro drug dissolution at pH 1.2 and 6.8, and permeation at pH 7.4 across Caco-2/HT29 cell monolayers from the micromatrix particles, were investigated. The micromatrix particles were found to be gastro-resistant, while at pH 6.8 the griseofulvin was released very rapidly in a fast-dissolving form. Compared to free griseofulvin, the permeability of encapsulated griseofulvin across the intestinal cell monolayers was greatly improved, particularly for the TOPSi-doped micromatrix particles. The griseofulvin solid dispersions were stable during storage for 6 months at accelerated conditions. Overall, the method developed here could prove to be a useful oral drug delivery solution for improving the bioavailability of poorly water-soluble or otherwise problematic drugs.

Transepithelial transport of milk derived bioactive peptide VLPVPQK

The transepithelial transport of an antioxidative and ACE inhibitory peptide, VLPVPQK (named peptide C) derived from casein hydrolysates was investigated along with extensively studied opioid peptide β-casomorphin using a human intestinal cell (Caco-2) monolayer. The susceptibility to the brush-border peptidases and route of transepithelial transport were observed to be the primary factors influencing the transport of these peptides. The apical to basal transport mechanism was studied using bradykinin as control as it shows resistance to cellular peptidases and its route of transepithelial transport had been established. VLPVPQK and BCM 5 were hydrolyzed by cellular peptidases while bradykinin was found intact. The transport of VLPVPQK (1.0%) was found to be relatively much higher than BCM 5 (0.03%) and bradykinin (0.1%). Interestingly the effect of some inhibitors on the transport of VLPVPQK suggested involvement of PepT1 like transporters/SOPT2 while BCM 5, its hydrolytic product and bradykinin were suggested to be transported mainly via the intracellular transcytosis pathway.

Indomethacin co-crystals and their parent mixtures: does the intestinal barrier recognize them differently?

Co-crystals are crystalline complexes of two or more molecules bound together in crystal lattices through noncovalent interactions. The solubility and dissolution properties of co-crystals can allow to increase the bioavailability of poorly water-soluble active pharmaceutical ingredients (APIs). It is currently believed that the co-crystallization strategy should not induce changes on the pharmacological profile of the APIs, even if it is not yet clear whether a co-crystal would be defined as a physical mixture or as a new chemical entity. In order to clarify these aspects, we chose indomethacin as guest poorly aqueous soluble molecule and compared its properties with those of its co-crystals obtained with 2-hydroxy-4-methylpyridine (co-crystal 1), 2-methoxy-5-nitroaniline (co-crystal 2), and saccharine (co-crystal 3). In particular, we performed a systematic comparison among indomethacin, its co-crystals, and their parent physical mixtures by evaluating via HPLC analysis the API dissolution profile, its ability to permeate across intestinal cell monolayers (NCM460), and its oral bioavailability in rat. The indomethacin dissolution profile was not altered by the presence of co-crystallizing agents as physical mixtures, whereas significant changes were observed by the dissolution of the co-crystals. Furthermore, there was a qualitative concordance between the API dissolution patterns and the relative oral bioavailabilities in rats. Co-crystal 1 induced a drastic decrease of the transepithelial electrical resistance (TEER) value of NCM460 cell monolayers, whereas its parent mixture did not evidence any effect. The saccharin-indomethacin mixture induced a drastic decrease of the TEER value of monolayers, whereas its parent co-crystal 3 did not induce any effects on their integrity, being anyway able to increase the permeation of indomethacin. Taken together, these results demonstrate for the first time different effects induced by co-crystals and their parent physical mixtures on a biologic system, findings that could raise serious concerns about the use of co-crystal strategy to improve API bioavailability without performing appropriate investigations.

Secretion of angiopoietin-like 4 protein from intestinal cells

Background: Angiopoietin-like 4 (ANGPTL4) has been suggested to play a role in lipid metabolism as a regulatory protein of lipoprotein lipase activity. Intestinal secretion of ANGPTL4, which is regulated by fatty acids, may inhibit the activity of circulating lipoprotein lipase; but, recent studies suggest that it could also inhibit pancreatic lipase in the gut and thereby reduce intestinal uptake of lipids. Secretion of the ANGPTL4 protein to either the lumen or tissue/blood side of the intestinal epithelial layer would indicate possible modes of action.Methods: Caco-2 cells were grown on permeable membranes and cultured for 21 days to spontaneously differentiate into an intact monolayer of intestinal cells, mimicking the epithelial cell layer lining the intestinal wall. Cells were treated with 9 mM butyrate and the time dependent gene expression and protein secretion to the apical and basolateral side was analysed over a time-course of 24 hours. Possible feedback from ANGPTL4 protein was investigated by adding 0.25 ng/ml recombinant ANGPTL4 protein to culture media.Results: Butyrate-induced ANGPTL4 gene expression increased in Caco-2 cells after 2 hours, reaching a plateau of approximately 6 fold after 6-24 hours, while the ANGPTL4 protein secretion to both the apical and basolateral sides was increased 18-24 hours after stimulation. A negative feedback on apical and basolateral secretion was observed in the presence of recombinant ANGPTL4 on the apical and basolateral sides, respectively.Conclusion: The present study indicates that, upon exposure to butyrate, the monolayer of epithelial cells secretes the ANGPTL4 protein to both the tissue/blood (basolateral) side and the luminal (apical) side of the monolayer which, in an in vivo situation, may be interpreted as potential inhibition of both the circulating and pancreatic lipase.Keywords: Angiopoietin-like 4, Caco-2, Permeable membrane, Protein, Secretion, Intestine, Negative feedback

POF1B Localizes to Desmosomes and Regulates Cell Adhesion in Human Intestinal and Keratinocyte Cell Lines

By means of morphological and biochemical criteria, we here provide evidence for the localization and function of premature ovarian failure, 1B (POF1B) in desmosomes. In monolayers of Caco-2 intestinal cells and in stratified HaCaT keratinocytes, endogenous POF1B colocalized with desmoplakin at desmosome plaques and in cytoplasmic particles aligned along intermediate filaments (IFs). POF1B predominantly co-fractionated with desmosomes and IF components and exhibited properties characteristic of desmosomes (i.e., detergent insolubility and calcium independence). The role of NH2 and COOH domains in the association of POF1B with desmosomes and IFs was revealed by transient expression of the truncated protein in Caco-2 cells and in cells lacking desmosomes. The function of POF1B in desmosomes was investigated in HaCaT keratinocytes stably downregulated for POF1B expression. Transmission electron microscopy analysis revealed a decrease in desmosome number and size, and desmosomes of the downregulated keratinocytes displayed weak electron-dense plaques. Desmosome alterations were associated with defects in cell adhesion, as revealed by the reduced resistance to mechanical stress in the dispase fragmentation assay. Moreover, desmosome localization of POF1B was restricted to granular layers in human healthy epidermis, whereas it largely increased in hyperproliferative human skin diseases, thus demonstrating the localization of POF1B also in desmosomes of multistratified epithelia.

Reinforcement of the intestinal mucus layer protects against Clostridium difficile intestinal injury in vitro.

Clostridium difficile infection is increasing in incidence and severity. Attributable factors include virulence factors, including C difficile toxins A and B, as well as host immunologic status. The mucus component of the intestinal barrier is impaired by malnutrition, shock insults, and alterations in the gut microbiome. Exogenous phosphatidylcholine (PC) administration results in reinforcement of the mucus layer and is of therapeutic benefit in chronic ulcerative colitis. We therefore studied the role of exogenous PC combined with secretory immunoglobulin A (IgA) in intestinal barrier function against C difficile infection invitro. Dimeric IgA was placed in the basal chambers of mucus-producing (HT29-methotrexate) and non-mucus-producing (HT29) strains of intestinal epithelial monolayers and allowed to undergo transcytosis and, in additional experiments, exogenous colostral IgA (30 ng/mL) was added to the apical media. After subsequent coculture with PC and C difficile toxin A in the apical chamber, tumor necrosis factor-α, interleukin-6, toxin A uptake, intestinal epithelial cell monolayer permeability, and necrosis were determined. A significant decrease of 4- to 5-fold in tumor necrosis factor-α and interleukin-6 levels and equally significant decreases in toxin A uptake and permeability changes in the intestinal cell monolayers with mucus or PC and transcytosed or colostral IgA vs control are shown. All groups analyzed also displayed a 2- to 3-fold reduction in necrosis. Mucus or "exogenous" mucus in the form of PC has a synergistic role with secretory IgA in barrier defense against C difficile toxin A. Exogenous PC administration can be a therapeutic adjunct in patients with severe or recalcitrant C difficile infection.

Chitosan-modified porous silicon microparticles for enhanced permeability of insulin across intestinal cell monolayers

Porous silicon (PSi) based particulate systems are emerging as an important drug delivery system due to its advantageous properties such as biocompatibility, biodegradability and ability to tailor the particles' physicochemical properties. Here, annealed thermally hydrocarbonized PSi (AnnTHCPSi) and undecylenic acid modified AnnTHCPSi (AnnUnTHCPSi) microparticles were developed as a PSi-based platform for oral delivery of insulin. Chitosan (CS) was used to modify the AnnUnTHCPSi microparticles to enhance the intestinal permeation of insulin. Surface modification with CS led to significant increase in the interaction of PSi microparticles with Caco-2/HT-29 cell co-culture monolayers. Compared to pure insulin, the CS-conjugated microparticles significantly improved the permeation of insulin across the Caco-2/HT-29 cell monolayers, with ca. 20-fold increase in the amount of insulin permeated and ca. 7-fold increase in the apparent permeability (Papp) value. Moreover, among all the investigated particles, the CS-conjugated microparticles also showed the highest amount of insulin associated with the mucus layer and the intestinal Caco-2 cells and mucus secreting HT-29 cells. Our results demonstrate that CS-conjugated AnnUnTHCPSi microparticles can efficiently enhance the insulin absorption across intestinal cells, and thus, they are promising microsystems for the oral delivery of proteins and peptides across the intestinal cell membrane.

Development of an integration mutagenesis system in Lactobacillus gasseri

Lactobacillus gasseri ATCC 33323 is a member of the acidophilus-complex group, microbes of human origin with significant potential for impacting human health based on niche-specific traits. In order to facilitate functional analysis of this important species, a upp-based counterselective chromosomal integration system was established and employed for targeting the lipoteichoic acid (LTA) synthesis gene, ltaS, in L. gasseri ATCC 33323. The ltaS gene encodes a phosphoglycerol transferase responsible for building the glycerol chain of LTA. No isogenic mutant bearing the deletion genotype was recovered, but an integration knockout mutant was generated with insertion inactivation at the ltaS locus. The ltaS deficient derivative exhibited an altered cellular morphology and significantly reduced ability to adhere to Caco-2 intestinal cell monolayers, relative to the wild-type parent strain.

In VitroCharacterization of the Intestinal Absorption of Methylmercury using a Caco-2 Cell Model

Methylmercury (CH3Hg) is one of the forms of mercury found in food, particularly in seafood. Exposure to CH3Hg is associated with neurotoxic effects during development. In addition, methylmercury has been classified by the International Agency for Research on Cancer as a possible human carcinogen. Although the diet is known to be the main source of exposure, few studies have characterized the mechanisms involved in the absorption of this contaminant. The present study examines the absorption process using the Caco-2 cell line as a model of the intestinal epithelium. The results indicate that transport across the intestinal cell monolayer in an absorptive direction occurs mainly through passive transcellular diffusion. This mechanism coexists with carrier-mediated transcellular transport, which has an active component. The participation of H(+)- and Na(+)-dependent transport was observed. Inhibition tests point to the possible participation of amino acid transporters (B(0,+) system, L system, and/or y(+)L system) and organic anion transporters (OATs). Our study suggests the participation in CH3Hg absorption of transporters that have already been identified as being responsible for the transport of this species in other systems, although further studies are needed to confirm their participation in intestinal absorption. It should be noted that CH3Hg experiences important cellular acumulation (48-78%). Considering the toxic nature of this contaminant, this fact could affect intestinal epithelium function.

Effect of wild-type Shigella species and attenuated Shigella vaccine candidates on small intestinal barrier function, antigen trafficking, and cytokine release.

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.

Stability and absorption of anthocyanins from blueberries subjected to a simulated digestion process

Numerous studies have shown that anthocyanins usually have better in vitro bioactivity than in vivo bioactivity. This may be due to physiochemical degradation during gastrointestinal digestion and their poor bioavailability in in vivo studies. Therefore, this study aims to investigate the effects of anthocyanin structure on their stability under simulated gastrointestinal digestion and to assess their absorption in the intestines using Caco-2 human intestinal cell monolayers. The results show that gastric digestion does not significant affect blueberry anthocyanins in terms of composition and antioxidative activity. However, approximately 42% of the total anthocyanin and 29% of the antioxidative activity were lost during intestinal digestion. Structural analysis indicated that fewer free hydroxyl groups and more methoxy groups in the B-ring improve anthocyanin stability. The absorption trials demonstrated that more hydrophobic anthocyanins have better absorption efficiency than more hydrophilic anthocyanins. Furthermore, the glycoside structure also determines the absorption efficiency of anthocyanins.

Effect of edible oils on quercetin, kaempferol and galangin transport and conjugation in the intestinal Caco-2/HT29-MTX co-culture model

Solubility and matrix play an important role in the gut lumen in delivering bioactive compounds to the absorptive surface of enterocytes. The purpose of this study was to determine the effect of certain commonly consumed lipids, soybean, olive and corn oil, on the transport and conjugation of flavonols (myricetin, quercetin, kaempferol and galangin) using the conjugation-competent co-cultured Caco-2/HT29-MTX intestinal cell monolayer model. To enable identification and quantification of conjugates, each flavonol was enzymatically glucuronidated or sulphated, then analysed by HPLC with triple quadrupole mass spectrometric detection. Quantification showed large differences in mass spectrometric peak area response factors between the aglycones and many of the conjugates, with galangin-sulphate for example ionising ∼15-fold better than galangin. Flavonol aglycones and conjugates were transported to the basolateral side of Caco-2/HT29-MTX co-cultures. The total amount of methyl, sulphate and glucuronide conjugates was in the order: galangin > quercetin > kaempferol > myricetin. All oils inhibited the transport and conjugation of galangin, the most hydrophobic flavonol, whereas they increased the sulphation, and to some extent glucuronidation, of quercetin and kaempferol. The results show that the lipid matrix has the potential to modify both transport and conjugation of dietary flavonols, but that the effect depends upon the structure and hydrophobicity.