The aim of the present study was to examine the antioxidant and antibacterial activity of 21 types of honey derived from Mount Olympus (Mt. Olympus), a region with great plant biodiversity. The antibacterial activity was examined against the growth of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) by the agar well diffusion assay and the determination of the minimum inhibitory concentration (MIC). The antioxidant activity was assessed by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS•+) free radical scavenging assays. These activities were compared to Manuka honey which is used as an alternative medicine. The results revealed that all tested honey types exhibited antibacterial activity against S. aureus and P. aeruginosa. The MIC of the tested honey types against S. aureus ranged from 3.125 to 12.5% (v/v), while MIC of Manuka honey was determined to be 6.25% (v/v). The MIC values of the tested honey types against P. aeruginosa ranged from 6.25 to 12.5% (v/v) and the MIC of Manuka honey was determined at 12.5% (v/v). Moreover, the results suggested that the presence of hydrogen peroxide and proteinaceous compounds in the honey types accounted, at least in part, for the antibacterial activity. In addition, the total polyphenolic content (TPC) of the honey types seemed to contribute to the antibacterial activity against P. aeruginosa. Furthermore, some of the tested honey types exhibited potent free radical scavenging activity against DPPH and ABTS•+ radicals, which was greater than that of Manuka honey. The results indicated that not only the quantity, but also the quality of the polyphenols were responsible for the antioxidant activity. Moreover, four honey types exhibiting great antioxidant activity were converted to powder using a freeze drying method. The results indicated that following conversion to powder all honey types, apart from one, retained their antioxidant activity, although their TPC was reduced. On the whole, and at least to the best of our knowledge, the present study is the first that extensively examined the bioactivities of different types of honey derived from Mt. Olympus.

Pine honey is a unique type of honeydew honey produced exclusively in Eastern Mediterranean countries like Greece and Turkey. Although the antioxidant and anti-inflammatory properties of pine honey are well documented, few studies have investigated so far its antibacterial activity. This study investigates the antibacterial effects of pine honey against P. aeruginosa PA14 at the molecular level using a global transcriptome approach via RNA-sequencing. Pine honey treatment was applied at sub-inhibitory concentration and short exposure time (0.5× of minimum inhibitory concentration –MIC- for 45 min). Pine honey induced the differential expression (>two-fold change and p ≤ 0.05) of 463 genes, with 274 of them being down-regulated and 189 being up-regulated. Gene ontology (GO) analysis revealed that pine honey affected a wide range of biological processes (BP). The most affected down-regulated BP GO terms were oxidation-reduction process, transmembrane transport, proteolysis, signal transduction, biosynthetic process, phenazine biosynthetic process, bacterial chemotaxis, and antibiotic biosynthetic process. The up-regulated BP terms, affected by pine honey treatment, were those related to the regulation of DNA-templated transcription, siderophore transport, and phosphorylation. Pathway analysis revealed that pine honey treatment significantly affected two-component regulatory systems, ABC transporter systems, quorum sensing, bacterial chemotaxis, biofilm formation and SOS response. These data collectively indicate that multiple mechanisms of action are implicated in antibacterial activity exerted by pine honey against P. aeruginosa.

Pine honey is a honeydew honey produced in the East Mediterranean region (Greece and Turkey) from the secretions of the plant sucking insect Marchalina hellenica (Gennadius) (Coccoidea: Marchalini-dae) feeding on living parts of Pinus species. Nowadays, honeydew honey has attracted great attention due to its biological activities. The aim of this study was to study unifloral pine honey samples produced in Greece regarding their physicochemical parameters and antioxidant and antibacterial activity against five nosocomial and foodborne pathogens. These honeys showed physicochemical and microscopic characteristics within the legal limits, except for diastase activity, a parameter known to be highly variable, depending on various factors. Substantially higher levels of H2O2 were estimated compared to other types of honeydew honey, whereas protein content was similar. The total phenolic content was 451.38 ± 120.38 mg GAE/kg and antiradical activity ranged from 42.43 to 79.33%, while FRAP values (1.87 to 9.43 mmol Fe+2/kg) were in general higher than those reported in the literature. Various correlations could be identified among these parameters. This is the first attempt to investigate in depth the antibacterial activity of pine honey from Greece and correlate it with honey quality parameters. All tested honeys exerted variable but significant antibacterial activity, expressed as MIC and MBC values, comparable or even superior to manuka honey for some tested samples. Although honey antibacterial activity is mainly attributed to hydrogen peroxide and proteins in some cases (demonstrated by elevated MICs after catalase and Proteinase K treatment, respectively), no strong correlation between the antibacterial activity and hydrogen peroxide concentration or total protein content was demonstrated in this study. However, there was a statistically significant correlation of moisture, antioxidant and antibacterial activity against Klebsiella pneuomoniae, as well as antioxidant and antibacterial activity against Salmonella ser. Typhimurium. Interestingly, a statistically significant negative correlation has been observed between diastase activity and Staphylococcus aureus antibacterial activity. Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey.

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