In Vivo Consumption of Cranberry Exerts ex Vivo Antiadhesive Activity against <i>FimH</i>-Dominated Uropathogenic Escherichia coli: A Combined in Vivo, ex Vivo, and in Vitro Study of an Extract from Vaccinium macrocarpon

For investigation of the molecular interaction of cranberry extract with adhesins of uropathogenic Escherichia coli (UPEC), urine from four volunteers consuming standardized cranberry extract (proanthocyanidin content = 1.24%) was analyzed within ex vivo experiments, indicating time-dependent significant inhibition of 40–50% of bacterial adhesion of UPEC strain NU14 to human T24 bladder cells. Under in vitro conditions a dose-dependent increase in bacterial adhesion was observed with proanthocyanidin-enriched cranberry Vaccinium macrocarpon extract (proanthocyanidin content = 21%). Confocal laser scanning microscopy and scanning electron microscopy proved that <i>V.m.</i> extract led to the formation of bacterial clusters on the outer plasma membrane of the host cells without subsequent internalization. This agglomerating activity was not observed when a PAC-depleted extract (<i>V.m.</i> extract^≠PAC) was used, which showed significant inhibition of bacterial adhesion in cases where type 1 fimbriae dominated and mannose-sensitive UPEC strain NU14 was used. <i>V.m.</i> extract^≠PAC had no inhibitory activity against P- and F1C-fimbriae dominated strain 2980. Quantitative gene expression analysis indicated that PAC-containing as well as PAC-depleted cranberry extracts increased the <i>fimH</i> expression in NU14 as part of a feedback mechanism after blocking <i>FimH</i>. For strain 2980 the PAC-containing extract led to up-regulation of P- and F1C-fimbriae, whereas the PAC-depleted extract had no influence on gene expression. <i>V.m.</i> and <i>V.m.</i> extract^≠PAC did not influence biofilm and curli formation in UPEC strains NU14 and 2980. These data lead to the conclusion that also proanthocyanidin-free cranberry extracts exert antiadhesive activity by interaction with mannose-sensitive type 1 fimbriae of UPEC

Xyloglucan from Tropaeolum majus Seeds Induces Cellular Differentiation of Human Keratinocytes by Inhibition of EGFR Phosphorylation and Decreased Activity of Transcription Factor CREB

Xyloglucan XG (molecular weight 462 kDa, 1,4-/1,4,6-(<i>p</i>Glc) linked backbone, side chains of 1-<i>p</i>Xyl, 1,2-<i>p</i>Xyl, 1-<i>p</i>-Gal) was isolated from the seeds of Tropaeolum majus. XG (100 μg/mL) induced terminal cellular differentiation of human keratinocytes, as demonstrated by immunofluorescence staining and Western blot using cytokeratin 10 and involucrin as marker proteins. Differentiation was also induced by XG-derived oligosaccharides (degree of polymerization 7–9). Quantitative real-time polymerase chain reaction (qPCR) revealed the induction of gene expression of typical differentiation markers (cytokeratin, filaggrin, involucrin, loricrin, transglutaminase) in a time-dependent manner. Whole human genome microarray indicated that most of upregulated genes were related to differentiation processes. Microarray findings on selected genes were subsequently confirmed by qPCR. For identification of the molecular target of xyloglucan PAGE of keratinocyte membrane preparations was performed, followed by blotting with fluorescein isothiocyanate-labeled XG. XG interacting proteins were characterized by MS. Peptide fragments of epidermal growth factor receptor (EGFR) and integrin β4 were identified. Subsequent phospho-kinase array indicated that phosphorylation of EGFR and transcription factor cAMP response element-binding protein (CREB) was decreased in the XG-treated cells. Thus, the XG-induced differentiation of keratinocytes is proposed to be mediated by the inhibition of the phosphorylation of EGFR, leading to a dimished CREB activation, which is essential for the activation of cellular differentiation

Influence of RA1 on <i>P</i>. <i>gingivalis</i>-mediated hemagglutination.

<p>Values from 3 independent experiments display the respective titer shifts related to the untreated control groups.</p><p>Influence of RA1 on <i>P</i>. <i>gingivalis</i>-mediated hemagglutination.</p

Influence of flavan-3-ols and proanthocyanidins from RA1 on the adhesion of <i>P</i>. <i>gingivalis</i> to KB cells in the coincubation adhesion assay.

<p>Data represent IC₅₀ resp. IC₇₅ for compound 5. IC₅₀: concentration of the test compound reducing the bacterial adhesion to 50%, related to the untreated control group; IC₇₅: concentration of the test compound 5 reducing the bacterial adhesion to 75%, related to the untreated control group.</p><p>Influence of flavan-3-ols and proanthocyanidins from RA1 on the adhesion of <i>P</i>. <i>gingivalis</i> to KB cells in the coincubation adhesion assay.</p

Docking scores for compounds 1 to 14 from RA1 for the proteolytic domain of Rgp and for the hemaglutinin (HA) domain and correlation of the respective docking scores for Rgp with Arg-gingipain activity as determined within the protease assay (Rgp activity at 50 μM in %, related to the untreated control groups).

<p>Docking scores for compounds 1 to 14 from RA1 for the proteolytic domain of Rgp and for the hemaglutinin (HA) domain and correlation of the respective docking scores for Rgp with Arg-gingipain activity as determined within the protease assay (Rgp activity at 50 μM in %, related to the untreated control groups).</p

Structural features of flavan-3-ols, proanthocyanidins and quercetin-3-O-glucuronid tested for antiadhesive and ant-gingipain activity against <i>P</i>. <i>gingivalis</i>; compounds not isolated from <i>Rumex acetosa</i> are marked by asterisk and have been used to obtain complete structure-activity relations; G: gallic acid.

<p>Structural features of flavan-3-ols, proanthocyanidins and quercetin-3-O-glucuronid tested for antiadhesive and ant-gingipain activity against <i>P</i>. <i>gingivalis</i>; compounds not isolated from <i>Rumex acetosa</i> are marked by asterisk and have been used to obtain complete structure-activity relations; G: gallic acid.</p

Representative fluorescence microscopy images of FITC labeled <i>P</i>. <i>gingivalis</i> adherent to murine oral mucosa sections from four independent experiments; data sets 4 and 5 originate as technical replicates from the same experiment to indicate intraassay reproducibility A 1–5: untreated control B 1–5: positive control, pretreated with 5 mM TLCK for 90 minutes C 1–5: RA1 100 μg/mL (preincubation of bacteria for 90 minutes).

<p>Magnification: 100 ×. Images are equalized in brightness, contrast and fluorescence intensity.</p

UHPLC of RA1 (1 mg/mL).

<p><b>7</b> epicatechin-(4β→8)-epicatechin, <b>14</b> epicatechin-(4β-→8)-epicatechin(4β→8,2β→O→7)-epicatechin, <b>5</b> epicatechin-3-O-gallate, <b>15</b> quercetin-3-O-glucuronide, <b>8</b> epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate, <b>IS</b> internal standard epigallocatechin-3-O-gallate.</p

Protein-ligand docking of epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate 10 into the binding cavity of Arg-gingipain.

<p>(<b>A</b>) 3D model; protein: green: hydrophobic, purple: polar, red: exposed; ligand: yellow: carbon, light grey: hydrogen, red: oxygen, blue: nitrogen; (<b>B</b>) 2D model.</p

Phytochemical Characterization of Low Molecular Weight Constituents from Marshmallow Roots (<i>Althaea officinalis</i>) and Inhibiting Effects of the Aqueous Extract on Human Hyaluronidase‑1

Extract <i>RE</i> was obtained from the roots of <i>Althaea officinalis</i> in a yield of 8.1%, related to the dried plant material, by extraction with MeOH–H₂O (1:1), followed by precipitation with EtOH to remove high molecular weight constituents. Phytochemical investigation of <i>RE</i> revealed the presence of <i>N</i>-phenylpropenoyl-l-amino acid amides <b>1</b>–<b>5</b>, 8% glycine betaine <b>6</b>, about 9% total amino acids with proline as the main compound, and about 61% mono- and oligomeric carbohydrates with sucrose as the main compound. Further fractionation revealed the presence of a hypolaetin diglycoside (<b>12</b>) and four hypolaetin glycosides (<b>7</b>–<b>9</b> and <b>11</b>) with <i>O</i>-sulfocarbohydrate moieties; additionally, 4′-<i>O</i>-methylisoscutellarein-8-<i>O</i>-β-d-(3″-<i>O</i>-sulfo)­glucuronopyranoside (<b>10</b>) and the diglycosylated coumarin haploperoside D (<b>13</b>) were identified. The hypolaetin-<i>O</i>-sulfoglycosides <b>7</b>–<b>10</b> are new natural products. <i>RE</i> inhibited the enzymatic activity of surface-displayed human hyaluronidase-1 on <i>Escherichia coli</i> F470 cells with an IC₅₀ of 7.7 mg/mL. <i>RE</i> downregulated mRNA expression of <i>hyal-1</i> in HaCaT keratinocytes at 125 and 250 μg/mL, respectively. These data contribute to a deeper phytochemical understanding of marshmallow root extracts and to the positive influence of extracts used for therapy of irritated and inflamed buccal tissue and cough