Identification and characterization of bioactivity of simulated gastrointestinal digested indigenous southern African honey samples

Abstract

Development of disease in the gastrointestinal tract is predominantly due to oxidative damage, infection and inflammation which if left unresolved lead to conditions such as inflammatory bowel disease. Functional foods can prevent/reduce the effects of oxidative damage, infection and inflammation. Honey is a well described functional food due to its several bioactivities. However, a functional food needs to be bioavailable and some bioactive molecules found in honey such as methylglyoxal (MGO) and polyphenols have been reported to have reduced activity following digestion. Therefore, the aim of this study was to determine the effects of digestion on the antioxidant, antibacterial and anti-inflammatory activities of southern Africa honey samples.

Manuka (control) and three honey samples from the southern African region, Agricultural Eucalyptus (AE), south eastern Mozambique (SEMh), Western Cape, Fynbos (WC) were subjected to simulated gastroduodenal digestion and the bioactivities of the undigested (UD), gastric digested (GD) and gastroduodenal (GDD) fractions were determined.

Total polyphenol content, Trolox equivalent antioxidant capacity and oxygen radical absorbance capacity were determined. Cellular antioxidant activity (CAA) using the human colon adenocarcinoma (Caco-2) cells and the dichlorofluorescein diacetate assay was also determined. A polyphenol mixture (PP) and a synthetic honey (sugars and polyphenols) were also digested and the antioxidant properties determined. Antibacterial effects against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) and Gram-positive Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) bacteria were then evaluated using the microbroth dilution, minimum inhibitory concentration (MIC) assay. The contribution of sugars, MGO, H2O2, polyphenols and cationic peptides to antibacterial activity was also evaluated. Cytotoxicity against the mouse fibroblast (L929) cells, inhibition of nitric oxide (NO) formation, inhibition of E. coli induced NO levels in L929 cells and inhibition of LPS induced activation of human platelets was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay, sodium nitroprusside assay and scanning electron microscopy, respectively.

All honeys had antioxidant activity similar to Manuka honey. With GD the antioxidant properties of all honey fractions was unchanged. Following GDD, antioxidant activity was either unchanged or increased. In contrast, for CAA a strong pro-oxidant effect was observed. Using PP mixtures, findings were that this effect was not due to pH dependent polyphenol degradation associated with an increase in hydrogen peroxide (H2O2) levels, but possibly due to the presence of β-Carotene, which in the presence of O2 forms the carotenoid peroxyl radical.

All honeys had MIC’s between 25 – 30% (v/v) against all tested bacteria, with the exception of MANc 6.25% (v/v) against S. aureus and AE 40% (v/v) against B. subtilis. With GD, the antibacterial activity, was unchanged. With GDD the antibacterial activity of MANc was retained. The activity of AE, SEMh and WC was unchanged against P. aeruginosa and S. aureus but was reduced against E. coli and B. subtilis. The MIC of MGO was 0.8 - 1.2 mM against all bacteria while the MIC for H2O2 was 9 mM for all bacteria except B. subtilis (90 mM). Honey sugars did not achieve MIC. Therefore, the activity of AE, SEMh and WC was concluded not to be due to MGO or H2O2 as higher levels than what is present in honey were needed to achieve MIC, but due to sugars in combination with an unidentified component.

At honey concentrations that did not lead to cytotoxicity (<3% v/v), NO formation was reduced by 50%, 62%, 63% and 50% for MAN UMF10+, AE, SEMh and WC, respectively. E. coli induced NO formation in L929 cells and platelet activation was inhibited to varying degrees, with AE and SEMh honeys being the most effective.

In conclusion, at physiologically relevant levels, the beneficial effects of honey in a gastric environment was the reduction of oxidative damage and inflammation while in a gastroduodenal environment the predominant effect was anti-inflammatory.

Keywords: Antibacterial, anti-inflammatory, antioxidant, bioactive, digestion, functional food, gastroduodenal, Manuka honey, oxidative damage, polyphenol.