Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey

Abstract

Honeydew honey is increasingly valued due to its pronounced antibacterial potential; however, the underlying mechanism and compounds responsible for the strong antibacterial activity of honeydew honey are still unknown. The aim of this study was to investigate the inhibition of bacterial growth of 23 honeydew honey samples. Activity of bee-derived glucose oxidase (GOX) enzyme, the content of defensin-1 (Def-1) and hydrogen peroxide (H₂O₂), and total polyphenol content were determined in the 23 honey samples. Our results demonstrated that antibacterial activity of honeydew honey was equivalent to medical-grade manuka and kanuka honey and was abolished by catalase. Although H₂O₂ is an important factor in the inhibition of bacterial growth, polyphenolic compounds and their interaction with H₂O₂ are the key factors responsible for high antibacterial activity of honeydew honey. In addition, our results indicated that the antibacterial activity of honeydew honey is not dependent on GOX-mediated production of H₂O₂ or the presence of Def-1.

Figure 1

Antibacterial activity of honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey against Staphylococcus aureus and Pseudomonas aeruginosa isolates. Activity was determined with a minimum inhibitory concentration (MIC) assay. The MIC was defined as the lowest concentration of honey solution (%) inhibiting bacterial growth. K, kanuka honey; M, manuka honey.

Figure 2

Glucose oxidase (GOX) activity and hydrogen peroxide (H₂O₂) production in honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey. (A) GOX activity was determined in 20% (w/v) honey solutions with a GOX assay kit. (B) H₂O₂ production was measured in 40% (w/v) honey samples with a modified GOX assay kit. The data are expressed as the mean values with standard error of the mean (SEM).

Figure 3

Content of defensin-1 (Def-1) and total polyphenol in honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey. (A) Def-1 content was quantified in honey samples using indirect competitive enzyme-linked immunosorbent assay and the results were expressed as a percentage of total protein. (B) Total polyphenol (TP) content was determined with a Folin Ciocalteu Phenolic Content Quantification Assay Kit in 20% (w/v) honeydew honey solutions. Gallic acid (GAE) was used as the reference standard compound and results were expressed as GAE equivalents (mg/ml). Data are expressed as the mean values with standard error of the mean (SEM).

Figure 4

Detection of glucose oxidase (GOX) and defensin-1 (Def-1) in honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following proteinase K treatment by immunoblotting. Aliquots (15 μl) of 50% (w/v) honey solution with or without proteinase K were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 16.5% Tricine-SDS-PAGE. After semi-dry blotting procedure, the blocked membrane was incubated overnight with a rabbit polyclonal antibody against honeybee GOX or Def-1 (1:2000). Shown are cropped blots. (The blots with indicated cropping lines are shown in Supplementary Fig. S2). Immunoreactive bands were detected in solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (GOX) or detected using an enhanced chemiluminescence Immobilon Western kit (Def-1).

Figure 5

Antibacterial activity of honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following catalase and proteinase K treatment against (A) Staphylococcus aureus and (B) Pseudomonas aeruginosa isolates. The 50% (w/v) honey solutions were treated with catalase (2000–5000 U/mg protein) at a final concentration ranging from 1000 to 2500 U/ml at room temperature for 2 h or proteinase K (30 U/mg) at a final concentration of 50 μg/ml at 37 °C for 30 min. The antibacterial activity was determined with a minimum inhibitory concentration (MIC) assay. The MIC was defined as the lowest concentration of honey solution (%) inhibiting bacterial growth. K, kanuka honey; M, manuka honey.

Figure 6

Graphical representation of the results of the general linear mixed model with response variable treatment type, bacterial species, and their two-way interaction. (Pseudomonas aeruginosa – red and Staphylococcus aureus – blue).