BackgroundTraditional Asian and African medicine use immature okra fruits (Abelmoschus esculentus) as mucilaginous food to combat gastritis. Its effectiveness is due to polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach tissue. The present study investigates the antiadhesive effect in mechanistic detail.MethodologyA standardized aqueous fresh extract (Okra FE) from immature okra fruits was used for a quantitative in vitro adhesion assay with FITC-labled H. pylori J99, 2 clinical isolates, AGS cells, and fluorescence-activated cell sorting. Bacterial adhesins affected by FE were pinpointed using a dot-blot overlay assay with immobilized Lewisb, sialyl-Lewisa, H-1, laminin, and fibronectin. 125I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron nanoparticles with different coatings were used to investigate the influence of the charge-dependence of an interaction on the H. pylori surface.Principal findingsOkra FE dose-dependently (0.2 to 2 mg/mL) inhibited H. pylori binding to AGS cells. FE inhibited the adhesive binding of membrane proteins BabA, SabA, and HpA to its specific ligands. Radiolabeled compounds from FE bound non-specifically to different strains of H. pylori, as well as to BabA/SabA deficient mutants, indicating an interaction with a still-unknown membrane structure in the vicinity of the adhesins. The binding depended on the charge of the inhibitors. Okra FE did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors.ConclusionNon-specific interactions between high molecular compounds from okra fruits and the H. pylori surface lead to strong antiadhesive effects.

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Polysaccharide containing extracts from immature fruits of okra (Abelmoschus esculentus) are known to exhibit antiadhesive effects against bacterial adhesion of Helicobacter pylori (H. pylori) to stomach tissue. The present study investigates structural and functional features of polymers responsible for this inhibition of bacterial attachment to host cells. Ammonium sulfate precipitation of an aqueous extract yielded two fractions at 60% and 90% saturation with significant antiadhesive effects against H. pylori, strain J99, (FE60% 68% ± 15%; FE90% 75% ± 11% inhibition rates) after preincubation of the bacteria at 1 mg/mL. Sequential extraction of okra fruits yielded hot buffer soluble solids (HBSS) with dose dependent antiadhesive effects against strain J99 and three clinical isolates. Preincubation of H. pylori with HBSS (1 mg/mL) led to reduced binding to 3ʹ-sialyl lactose, sialylated Le a and Le x . A reduction of bacterial binding to ligands complementary to BabA and SabA was observed when bacteria were pretreated with FE90%. Structural analysis of the antiadhesive polysaccharides (molecular weight, monomer composition, linkage analysis, stereochemistry, and acetylation) indicated the presence of acetylated rhamnogalacturonan-I polymers, decorated with short galactose side chains. Deacetylation of HBSS and FE90% resulted in loss of the antiadhesive activity, indicating esterification being a prerequisite for antiadhesive activity.

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The Gram-negative bacterium Helicobacter pylori is the most common bacterial pathogen in humans, infecting 24-79% of the population at any time. Standard eradication protocols involve multi-target therapy including combinations of antibiotics, which has promoted the emergence of resistant strains. To address this challenge, we prepared antibiotic-free colloidal nanoparticles designed to interfere with the adhesion mechanisms of H. pylori and thus prevent both the onset and recurrence of infection. Our colloidal particles comprised a nanocapsule (NC) formulation based on an oil-core nanoemulsion co-stabilized with lysozyme and lecithin, coated with negatively-charged lowmolecular-weight (DexS40-NC) or high-molecular-weight (DexS500-NC) dextran sulfate, or positively-charged chitosan (CSHMC+30-NC). The oil core of all NC formulations was also loaded with curcumin, a model lipophilic drug with well-documented documented anti-inflammatory and antitumor activities. Our proof-of-principle experiments showed that the DexS40-NC formulation inhibited the adhesion of H. pylori to AGS stomach cells in a dose-dependent manner. DexS40-NC achieved more potent inhibition than DexS500-NC or uncoated control nanoemulsions, whereas the effect of CSHMC+30-NC was not clearcut given the ability of this formulation to aggregate bacteria. DexS40-NC, unlike DexS500-NC, showed no cytotoxic effects against AGS, Caco-2, or MDCK cell lines. DexS40-NC is, therefore, a promising candidate for further development as an alternative or complementary therapy against H. pylori infections.

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