Exploring In Vitro the Combination of Cistus × incanus L. and Castanea sativa Mill. Extracts as Food Supplement Ingredients against H. pylori Infection

Abstract

Cistus spp. have been traditionally used for inflammatory and infectious disorders, including gastrointestinal ailments, in the Mediterranean area. Among them, Cistus × incanus L. is one of the most frequently cited species in the literature for a variety of biological activities which include inflammatory diseases. Cistus spp. aerial parts are rich in polyphenols such as condensed and hydrolysable tannins, procyanidins, and flavonoids, which show gastroprotective activities. The purpose of the present study is to investigate the biological activities of a hydroalcoholic extract from Cistus × incanus L. aerial parts in gastric epithelial cells (GES-1) infected with H. pylori. The extracts inhibited IL-8 and NF-κB induced by H. pylori and showed antibacterial activity after simulated digestion. Since our previous paper reported interesting results on the ability of Castanea sativa Mill. leaf extract to decrease inflammatory conditions in H. pylori-infected gastric cells, the combination of Castanea sativa and Cistus × incanus extracts was also investigated, showing strong anti-inflammatory activity and inhibition of bacterial adhesion. This association of botanicals is proposed herein as a novel food supplement capable of counteracting gastric inflammatory conditions.

Keywords: Cistus; Cistus × incanus; Helicobacter pylori; gastritis; inflammation.

Figure A1

TLC analysis of polyphenols from Cistus × incanus L. (CI). 1. Chlorogenic acid (light blue) and rutin (orange) (10 μg); 2. Hyperoside (10 μg); 3. Epicatechin (10 μg); 4. CI (500 μg); 5. CI Dig. (500 μg).

Figure A2

Effect of Cistus × incanus L. extract on GES-1 viability (MTT test). GES-1 cells were treated with CI or CI Dig. for 6 h. Results (n = 3) were expressed as a percentage (%) of viability (absorbance at 595 nm) ± SEM, relative to untreated cells. The dashed line represents the cut-off for acceptable value of viability, arbitrarily considered above 80%.

Figure 1

Effect of Cistus × incanus L. extracts on IL-8 release induced by H. pylori in GES-1 cells. GES-1 cells were infected with H. pylori (50:1) and treated with CI (A) or CI Dig. (B) for 6 h. EGCG (50 μM) was used as a reference inhibitor. IL-8 secretion was measured through ELISA assay. Results (n = 3) were expressed as a percentage (%) of the mean of IL-8 release (pg/mL) ± SEM, relative to H. pylori infection (black bar). ** p < 0.01, *** p < 0.001 vs. H. pylori infection. ^a p < 0.0001 of CI dig with respect to CI.

Figure 2

Effect of Cistus × incanus L. extracts on the nuclear translocation of NF-κB (p65) induced by H. pylori in GES-1 cells. GES-1 cells were infected with H. pylori (50:1) and treated with CI or CI Dig. for 1 h. NF-κB (subunit p65, red) translocation into nuclei (blue) was measured by immunofluorescence (63× objective, 50 μm). Merged images include β-actin (green) and bacterial staining (CFSE, white).

Figure 3

Effect of Cistus × incanus L. extracts on the adhesion of H. pylori to GES-1 cells. GES-1 cells were infected with H. pylori–CSFE (50:1) and treated with CI (A,C) or CI Dig. (B,D) for 1 h, before (A,B) or after (C,D) infection. Procyanidin A2 (PA, 500 μM) was used as a reference inhibitor. Bacterial adhesion was measured by FACS analysis. Results (n = 3) were expressed as a percentage (%) of the mean bacterial adhesion (MFI) ± SEM, relative to untreated H. pylori (black bar). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. H. pylori infection. MFI, median fluorescence intensity.

Figure 4

Effect of Cistus × incanus L. extracts on H. pylori growth and biofilm formation. Bacteria were treated with CI (A) or CI Dig. (B,C) for 72 h. Tetracycline (MIC = 0.125 μg/mL) was used as a reference antibiotic in each experiment. MIC values (A,B) were obtained by microbroth dilution, while biofilm formation was revealed by crystal violet assay. Results (n = 3) were expressed as a percentage (%) of the mean bacterial growth (absorbance at 600 nm) ± SEM, relative to untreated H. pylori (black bar). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. H. pylori infection.

Figure 5

Effect of Cistus × incanus L. extract on H. pylori urease activity. Adherent H. pylori to GES-1 (A) or H. pylori suspension (B) were treated with CI Dig. for 1 h. NaF (1 mM) was used as a reference urease inhibitor. The enzymatic activity was assessed using a colorimetric assay based on pH indicator. Results (n = 3) were expressed as a percentage (%) of pH increase (absorbance at 570 nm) ± SEM, relative to untreated H. pylori (black bar). * p < 0.05, ** p < 0.01 vs. H. pylori infection.

Figure 6

Effect of combinations (mix) of Cistus × incanus L. (CI) and Castanea sativa Mill. (CS) extracts on IL-8 release induced by H. pylori in GES-1 cells. GES-1 cells were infected with H. pylori (50:1) and treated with mix (A) and mix Dig. (B) for 6 h. EGCG (50 μM) was used as a reference inhibitor. IL-8 release was measured by ELISA assay. Mix 1, combination of CI/CS 75:25; Mix 2, combination of CI/CS 50:50; Mix 3, combination of CI/CS 25:75. Results (n = 3) were expressed as a percentage (%) of the mean of IL-8 release (pg/mL) ± SEM, relative to H. pylori infection (black bar). ** p < 0.01, *** p < 0.001 vs. H. pylori infection.

Figure 7

Effect of a combinations (Mix 2 Dig., 50:50) of Cistus × incanus L. (CI Dig.) and Castanea sativa Mill. (CS Dig.) extracts on the nuclear translocation of NF-κB (p65) induced by H. pylori in GES-1 cells. GES-1 cells were infected with H. pylori (50:1) and treated with Mix 2 Dig for 1 h. NF-κB (subunit p65, red) translocation into nuclei (blue) was measured by immunofluorescence (63× objective, 50 μm). Merged images include β-actin (green) and bacterial staining (CFSE, white).

Figure 8

Effect of Cistus × incanus L. (CI Dig.) and Castanea sativa Mill. (CS Dig.) extracts on the adhesion of H. pylori to GES-1 cells. GES-1 cells were infected with H. pylori–CSFE (50:1) and treated with CI Dig. (vertical lines, 50 μg/mL), CS Dig. (horizontal lines, 50 μg/mL), or their combination (Mix, 100 μg/mL) for 1 h during infection. Procyanidin A2 (PA, 500 μM) was used as a reference inhibitor. Bacterial adhesion was measured by FACS analysis. Mix 1, combination of CI/CS Dig. 25:75; Mix 2, combination of CI/CS Dig. 50:50; Mix 3, combination of CI/CS Dig. 25:75. Results (n = 3) were expressed as a percentage (%) of the mean bacterial adhesion (MFI) ± SEM, relative to untreated H. pylori (black bar). * p < 0.05, *** p < 0.001 vs. H. pylori; ## p < 0.01 vs. CS Dig. MFI, median fluorescence intensity.