Effects of DAPP on oxidative stress or LPS-induced inflammation on inflammatory markers in Caco-2/15 cells.

<p>Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) for 6 h at 37°C as described in Materials and Methods. Protein expression of the inflammatory markers TNF-α (A to C) and IL-6 (D to F) was determined by Western blot, respectively. Results represent the means ± SEM of n = 3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Ctrl; ^###<i>P</i><0.001 vs. Fe/Asc;^†<i>P</i><0.05, ^†††<i>P</i><0.001 vs. LPS; ^$$$<i>P</i><0.001 vs. Fe/Asc+LPS.</p

Identification of procyanidins in DAPP.

<p>Representative chromatograms of DAPP (25 mg/mL) from AB powder (A) or JC-047 (B) were obtained by normal phase analytical HPLC using an Agilent 1260/1290 Infinity system coupled to a fluorescence detector.</p

Separation and identification of polyphenolic compounds in DAPP crude AB powder.

<p>Extracted ion chromatograms of some identified polyphenolic compounds in AB powder using accurate mass measurement; (A). Extracted ion chromatograms of quercetin glycosides and dihydrochalcone (B) and their related mass spectra (C): quercetin 3-O-glucoside, m/z 463.0905 (1A); quercetin 3-O-galactoside, m/z 463.09018 (1B); quercetin 3-O-arabinoside, m/z 433.07914 (1C); quercetin 3-O-xyloside, m/z 447.07977 (1D); quercetin 3-O- rhamnoside, m/z 447.09506 (1E); and phloridzin, m/z 435.13171 (1F) obtained with negative ion electrospray ionisation. These polyphenols are provided from a mixture of 250 µg of crude extract DAPP in 1 mL Optima grade water.</p

Identification of procyanidins in DAPP.

<p>Experimental mass measurement and empirical formula calculation for quercetin glycosides and dihydrochalcone. A good agreement between the theoretical and the experimental m/z values was obtained for all compounds examined (<5 ppm). Separations were performed on an 1100 LC system coupled to an ESI-MSD-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA).</p

Separation and identification of polyphenolic compounds in DAPP purified JC-047 fraction.

<p>Extracted ion chromatograms of some identified polyphenolic compounds in JC-047 using accurate mass measurement (A). Extracted ion chromatograms of catechin, epicatechin and trimeric flavan-3-ol oligomers C1 to C4 (B) and their related mass spectra (C): (+)-catechin, m/z 289.07541 (1A) and (-)-epicatechin, m/z 289.07659 (1B) and that trimeric flavan-3-ol oligomers C1 to C4 (2A to 2D) obtained with negative ion electrospray ionisation. These polyphenols provided of a mixture of 250 µg of purified fraction DAPP in 1 mL Optima grade water.</p

Effects of DAPP on cell integrity in Caco-2/15 cells.

<p>Integrity of the monolayer was determined by cell viability, morphology (data not shown), differentiation and tight junction assays using fully differentiated Caco-2/15 cells. Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) for 6 h at 37°C as described in Materials and Methods. MTT (A), villin protein mass (B), transepithelial resistance (C) and occludin protein expression (D) were assessed. Results represent the means ± SEM of n = 3 independent experiments. *<i>P</i><0.05 vs. Ctrl; ^##<i>P</i><0.01 vs. Fe/Asc.</p

Heterogeneity of fractionated procyanidin oligomers and polymers of DAPP on normal-Phase HPLC.

<p>The procyanidin composition of DAPP from 25 mg/mL crude extract (AB powder) and purified fraction (JC-047) was analyzed by normal phase analytical HPLC using an Agilent 1260/1290 Infinity system coupled to a fluorescence detector. Individual procyanidins with degrees of polymerization (DP) from DP1 to DP>10 were quantified using external calibration curve of (−)-epicatechin, taking into account their relative response factors in fluorescence. The results were expressed as mg/100 g of extract weight ± SEM. *<i>P</i><0.05, ***<i>P</i><0.001 vs. AB powder.</p

Effects of DAPP on fatty acid composition in Caco-2/15 cells.

<p>After 10 days differentiation, Caco-2/15 cells were incubated for 6 h at 37°C in the absence or presence of Fe/Asc (200 µM/2 mM) with DAPP from 250 µg/mL AB powder or JC-047 and collected for fatty acid (FA) composition. Data represent the means ± SEM of two experiments, each done in duplicate (n = 4). Student’s <i>t</i> test (two-tailed) was used to compare differences between means (X±SEM). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Ctrl; ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. Fe/Asc.</p><p>AA: arachidonic acid, ALA: alpha-linolenic acid, DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid, LA: linoleic acid, PUFA: polyunsaturated fatty acids.</p

Effects of DAPP on oxidative stress or LPS-induced inflammation on NF-κB in Caco-2/15 cells.

<p>Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) in the presence or absence of 50 µM caffeic acid (CAPE, a specific NF-κB inhibitor) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) (A) and LPS (200 µg/mL) (B) for 6 h at 37°C, and LPS (200 µg/mL) (C) for 24 h at 37°C to mimic a chronic inflammation as described in Materials and Methods. Results represent the means ± SEM of n = 3 independent experiments. **<i>P</i><0.01, ***<i>P</i><0.001 vs. Ctrl;^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. Fe/Asc; ^†††<i>P</i><0.001 vs. LPS.</p

Effects of DAPP on oxidative stress and LPS-induced inflammation on prostaglandin E2 in Caco-2/15 cells.

<p>Crude AB powder (250 µg/mL) and purified JC-047 fraction (250 µg/mL) were added to the apical compartment of differentiated Caco-2/15 cells for 24 h before incubation with Fe/Asc (200 µM/2 mM) and LPS (200 µg/mL) (A), as well as indomethacin heptyl ester (0.4 µM) (B), as a selective cyclooxygenase (COX)-2 inhibitor, for 6 h at 37°C as described in Materials and Methods. PGE2 was determined by enzymatic immunoassay. Results represent the means ± SEM of N = 3 independent experiments. ***<i>P</i><0.001 vs. Ctrl; ^†††<i>P</i><0.001 vs. LPS; ^$$$<i>P</i><0.001 vs Fe/Asc+LPS.</p