Evaluation of the digestion and absorption of certain phenolic compounds by Caco-2 cells (Utilization two novel methods)

De nombreux effets santé sont attribués aux polyphénols, phytomicronutriments importants de l'alimentation. Leur biodisponibilité, assez peu connue, fait d'objet de beaucoup de recherches. Notre étude porte sur trois composés phénoliques : la quercétine, l'acide sinapique et l'hespéridine. Ils ont été choisis pour leurs structures chimiques (aglycone ou glycosylée). Nous avons pu visualiser grâce à de nouvelles approches (fluorescence et absorbance) la migration et/ou la transformation de ces composés dans les cellules Caco-2 et appréhender leurs interaction avec une matrice alimentaire. Les techniques de microscopie à épifluorescence et confocale nous ont permis de mettre en évidence l'autofluorescence de ces composés au sein d'une monocouche cellulaire. Leur visualisation par le microscope confocal a demandé l'optimisation de plusieurs paramètres. Nous avons montré que le passage de la quercétine pourrait être transcellulaire et plus rapide que celui de l'acide sinapique tandis que l'hespéridine migre par un mécanisme transcellulaire au départ et paracellulaire ensuite. La cinétique de ces composés au contact d'une monocouche cellulaire intestinale a été examinée par la spectroscopie UV/visible. La quercétine disparait plus rapidement que l'hespéridine à concentration identique (250 uM) mais toutes deux montrent une évolution exponentielle. L'intervention de certains mécanismes a été également étudiée. L'impact d'une matrice alimentaire complexe et/ou de jus de fruit sur l'évolution de l'hespéridine, montre l'existence d'un certain nombre d'interactions par les modifications de l'absorbance du produit. Une partie de ces résultats, contrôlés par dosage HPLC, ont fait apparaitre une bonne corrélation avec ces deux nouvelles approches. Ces approches alternatives doivent permettre d'appréhender plus rapidement la biodisponibilité des composés phénoliques et doivent être approfondiesPhenolic compounds are the most abundant health benefits in human diet. However, the bioavailability of these compounds still remains ambiguous. In the present study, three phenolic compounds (quercetin, hesperidin and sinapic acid) chosen according to their different chemical structures (aglycone or glycoside), were used to evaluate the localization from their natural fluorescence properties in human intestinal Caco-2 cells and to characterize possible interactions between food matrix and these phenolic compounds during their permeation across intestinal cells via the observation by new ways: fluorescence and absorbance. Through the fluorescence and confocal microscopic techniques, such compounds could exhibit a specific fluorescence. Their visualization by the confocal microscope was required for the optimization of several parameters. The microscopic results showed that the passage of quercetin might occur transcellularly and across the apical membrane rapider than the sinapic acid while the hesperidin also migrated by transcellular mechanism at first and then by paracellular mechanism. In addition, some mechanisms and their kinetics were investigated by UV/visible spectroscopy. Quercetin is remarkably reduced faster than the sinapic and hesperidin before all of them will approach to constant rate at the final period. However, quercetin, sinapic acid and hesperidin clearly display in the exponential manner for their uptake by enterocytes. Moreover, the impacts of food matrix or Clementine juice on the hesperidin evolution shows the existence of certain interaction by modification of the product absorbance. A part of these results were disputed by HPLC quantitative method. In addition, the correlation with cellular uptake between phenolic compound visualization by confocal microscopy and the absorbance was observed. These results provide the alternative ways to understand rapidly the bioavailability of phenolic compoundsMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Flavonoïdes de pépins de raisin (comportement dans un modèle de digestion "in vitro" associé aux cellules caco-2 et effet sur la différenciation cellulaire)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Impact de traitements à haute pression isostatique ou dynamique sur des systèmes protéiques modèles (recherche d'indicateurs biologiques de traitement)

Des indicateurs biologiques de traitement ont été recherchés pour évaluer l'impact de traitements à haute pression sur des systèmes protéiques modèles tels que (i) l'entérotoxine A de Staphylococcus aureus (SEA) et (ii) un isolat de protéines lactosériques. Les effets de la pression isostatique appliquée à la SEA en solution dans un tampon Tris-HCl (20 mM, pH 7,4) en présence ou non d'agent chaotropique, dissociant ou chélateur (urée 8M, dodécyle sulfate de sodium 30 mM ou acide tétraacétique d'éthylène diamine 1 mM) ont été explorés. Parmi les méthodes étudiées (i.e. toxicité de la SEA sur des cellules Caco-2 ; superantigénicité de la SEA évaluée par la prolifération de lymphocytes T de rat ; immunoréactivité de la SEA), la superantigénicité de la toxine a montré les réponses les plus nettes après traitement de la SEA à 600 MPa pendant 15 min et 20C ou 45C. En parallèle, la fluorescence intrinsèque de la protéine a été étudiée par spectroscopie sous pression entre 10 et 600 MPa, à 20C ou 45C, ou bien en présence d'urée, à 20C. L'ensemble des résultats indique que la pressurisation jusqu'à 600 MPa à 20C entraîne des modifications de structure réversibles sans modification de la réponse superantigénique de la SEA, et donc une baro-résistance de la toxine. Par contre, le traitement à 600 MPa et 45C, ou bien la présence d'urée à 20C (0,1 ou 600 MPa) entraîne des modifications de structure partiellement réversibles accompagnées d'une augmentation de la réponse superantigénique de la toxine. La pression dynamique (aussi appelée homogénéisation à ultra-haute pression, UHPH) appliquée à des dispersions de protéines lactosériques à des niveaux de pression supérieurs à 200 MPa, entraîne la formation d'agrégats protéiques de taille submicronique et contrôlée. Le comportement de ces protéines sur des monocouches de cellules TC7 a été exploré à la fois par l'évaluation de la quantité de protéines transportées à travers les cellules et l'observation des coupes cellulaires par microscopie confocale à fluorescence. Les cinétiques de transport montrent la pénétration progressive de la beta-lactoglobuline (protéine majoritaire) et probablement des agrégats induits par le procédé, à travers la monocouche cellulaire en fonction de la durée d'incubation des cellules.Biological indicators for food processing were studied in view of characterising the effects of high pressure processing on model proteins such as (i) Staphylococcus aureus enterotoxin A (SEA) and (ii) whey protein isolate. The effects of isostatic high pressure applied to SEA solution in Tris-HCl buffer (20 mM, pH 7,4) in the presence or not of chatropic, dissociating or chelating agents (urea 8M, sodium dodecyl sulfate 30 mM or ethylene diamine tetraacetic acid 1 mM) have been investigated. Among the methods tested (i.e. SEA toxicity on Caco-2 cells; SEA superantigenicity as evaluated by the proliferation of rat T lymphocytes; SEA immunoreactivity), SEA superantigenicity displayed the finest changes in the biological responses that SEA could induce after processing at 600 MPa for 15 min and 20C or 45C. In parallel, the intrinsic protein fluorescence was studied by spectrofluorimetry under pressure between 10 and 600 MPa, at 20C or 45C, or in the presence of urea, at 20C. Pressurisation up to 600 MPa at 20C induced structural modifications that were reversible without changes in the SEA superantigenic response, and indicate some baro-resistance of the toxin. In contrast, processing at 600 MPa and 45C, or processing at 20C in the presence of urea (at 0.1 or 600 MPa) induced partially reversible structural changes accompanied by an increase in SEA superantigenicity. Dynamic high pressure (also called ultra-high pressure homogenization, UHPH) at pressure levels above 200 MPa applied to dispersions of whey protein isolate induced protein aggregates of submicron and controlled sizes. The protein behaviour on TC7 cell monolayers was investigated both by assessing the amount of transported proteins through the cells, and using confocal fluorescence spectroscopy. Transport kinetics indicated that beta-lactoglobulin (the major whey protein) and probably the protein aggregates induced by UHPH enter the cell layers and progress inside the cells as a function of exposure time.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Evaluation de la qualité nutritionnelle des jus d'agrumes (estimation in vitro de la biodisponibilité des caroténoïdes)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Iron and Zinc in Vitro Availability in Pearl Millet Flours (Pennisetum glaucum) with Varying Phytate, Tannin, and Fiber Contents

International audienceSimulations of gastro-intestinal digestion, used to estimate in vitro iron and zinc availability, were performed on two kinds of samples: (i) samples with decreased phytate contents from whole pearl millet flour and (ii) nondephytinized or dephytinized samples from two pearl millet grain fractions, a decorticated fraction with low fiber and tannin contents and a bran fraction with high fiber and tannin contents. Iron and zinc in vitro availabilities of whole pearl millet flour were significantly improved by phytate degradation, even if the IP6 were not all degraded. Total dephytinization of decorticated fraction led to a marked increase in iron and zinc in vitro availabilities, but that of bran fraction had no effect on either iron or zinc in vitro availability. Even if phytates are involved in reducing in vitro iron and zinc availability in pearl millet flour, fibers and tannins play an important role by chelating a high proportion of iron and zinc in grain hulls

Grapefruit Juices Impair the Bioaccessibility of β-Carotene from Orange-Fleshed Sweet Potato but Not Its Intestinal Uptake by Caco-2 Cells

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audienceAmong various factors influencing beta-carotene (Bc) bioavailability, information on interactions between carotenoids or other micronutrients such as flavonoids during a meal that contains different plant-derived foods is quite limited. Because orange-fleshed sweet potato (OFSP) is an important Bc-rich staple food, a source of vitamin A in developing countries, this study focused on the effect of citrus fruit juice carotenoids and flavonoids on Bc bioaccessibility from OFSP. In vitro digestion coupled with the Caco-2 cell culture model was used to evaluate the bioaccessibility and cellular uptake of Bc from OFSP in the presence of pink grapefruit (pGF) or white grapefruit (wGF) juices. The addition of grapefruit juices significantly decreased the bioaccessibility, by up to 30%, but not the cellular uptake of Bc from boiled OFSP. Lycopene, but more probably naringin, present in grapefruit juices was suspected to be responsible for the inhibitory effect of the citrus juices on Bc bioaccessibility. This inhibition was apparently due in part to competition for incorporation between Bc and naringin into mixed micelles during in vitro digestion. In contrast, Bc uptake from dietary micelles was not impaired by naringin

Effect of citrus flavanones on carotenoid uptake by intestinal caco-2 cells

Health benefits of fruits and vegetables could be attributed to the presence of a large diversity of phytochemicals, including carotenoids. The bioactivities of carotenoids greatly depend on their bioavailability that could be modulated by the presence of other dietary constituents. Because citrus juices contain diverse antioxidant phytochemicals, the effects of flavonoids and ascorbic acid on intestinal carotenoid uptake were investigated. Experiments were conducted by using a Caco-2 cell monolayer exposed to micelles enriched in beta-cryptoxanthin b-CX and beta-carotene b-C, in the presence of hesperetin HES, hesperidin HES-G, naringenin, acid ascorbic AA and iron. After 5 or 24 h incubation, both HES-G and HES significantly increased b-CX and b-C uptake by around 1.6-fold. Interestingly, AA was shown to eliminate the enhancing effect of HES-G by decreasing significantly the cellular uptake of b-CX and b-C by 10% after 5 h incubation. Iron decreased the carotenoid uptake, but HES-G was shown to counteract the iron effect, certainly due to its iron-chelating activity