Production of pure mono-floral honey from Agastache rugosa (Fisch. et Mey.) O. Kuntze and its biological activities
Creators: name: ANAND, Sushil
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310602
subject: Bioprocessing, bioproduction and bioproducts

description: Growing resistance to antimicrobials has forced us to develop new antimicrobial products. Plant nectar-derived honey has various bioactive properties including antimicrobial activity, and its bioactivity is influenced not only by the source of the nectar but also by the conditions under which the honey is produced. Currently, marketed mono-floral honeys are usually produced in the wild or from single flowers but under uncontrolled conditions resulting in batch-to-batch variability in the concentration of the bioactive compounds. Consequently, it is difficult to monitor the source and the percentage of transfer of the bioactive compounds from flower nectar to honey. In addition, the potential compounds responsible for such activities must be identified before it can be approved for sale with regulatory authorities in order to allow standardisation. The research planned during the PhD project focused on producing monofloral pure honey from a medicinal plant, Agastache rugosa under highly controlled conditions and evaluating bioactivity of individual honeys including commercially-important Leptospermum honeys (Manuka, Jelly bush, Tea tree, and Super Manuka) and Jarrah honey together with identifying potential bioactive compounds responsible for their activity and source of the compounds.<br><br>
Mono-floral pure honey was produced in batches from 100 and 250 potted plants grown in the closed greenhouse, so that bees could only forage on desired plants. Both batches yielded 100 g and 500 g of honey. The quality of the harvested honey was characterized by its physicochemical parameters namely total phenolics, total flavonoids, moisture content, colour, pH, protein content and antioxidant capacity using DPPH, ABTS+, ORAC and FRAP methods. The results confirmed that the honey harvested was mature and of good quality containing 17% moisture content (below 20%). There was a positive correlation between colour, total phenolic content and DPPH scavenging activity for most of the honeys except Tea tree honey. The DPPH assay clearly indicated that the phenolic compounds contribute to the scavenging activity of the honeys. Nevertheless, all assays confirmed that Agastache honey had significant antioxidant capacity.<br><br>
The antimicrobial activity of honeys was evaluated against clinical and non-clinical strains of Staphylococcus aureus (methicillin-susceptible and methicillin-resistant strains), Pseudomonas aeruginosa, and Escherichia coli. The minimum inhibitory concentration (MIC) for Agastache honey was in the range of 6-25% (w/v) for all species examined. Staphylococci were more susceptible to all honeys than Pseudomonas aeruginosa and Escherichia coli. Similarly, the antifungal activity of honeys was tested against dermatophytes (T. mentagrophytes and T. rubrum) and C. albicans by agar well diffusion and micro-dilution (AWD and MD) methods. In AWD and MD assays, Agastache honey was effective at 40% concentration against dermatophytes (zone diameter, 19.5-20 mm) and C. albicans with the same MIC and MFC values indicating fungicidal activity. In addition, samples were treated with catalase to verify the antimicrobial role of hydrogen peroxide in individual honey, which revealed increased bacterial growth at MIC of Tea tree honey (35%), Super Manuka (15%), Jarrah honeys (12%), and Agastache honey (10%), indicating variable contributions of hydrogen peroxide to antimicrobial activity. This was further confirmed by measuring the accumulation of hydrogen peroxide at 40% of honey concentrations. The results were consistent with the finding that Tea tree produced highest amount of hydrogen peroxide (155 µM) followed by Jarrah (115 µM), Super Manuka (41 µM) and Agastache honey (5.13 µM). These findings clearly indicate that other factors than H2O2 production, for example, phenolic and volatile compounds, contribute to the antimicrobial and antifungal activities.<br><br>
In addition to transfer of the compounds from flower nectar to honey, the presence of phenolic compounds and volatiles in the samples and their relation to the bioactivity were investigated. To identify possible antimicrobial phenolic compounds, an LC-MS/MS method was developed, and HS-SMPE-GC/MS was used to identify possible volatile antifungal compounds in the honey samples. The results revealed that major phenolic compounds, namely chlorogenic acid, syringic acid, rosmarinic acid, ferulic acid, vanillic acid and proto-catechuic acid, while major volatile compounds, including estragole, Bicyclo undec-4-ene, 4,11,11-trimethyl-8-methylene-, and D-limonene were identified as being transferred from flower nectar to honey. The possible antimicrobial compounds found in Agastache honey were Phenyllactic acid, 4-Hydroxybenzoic acid, p-Coumaric acid and Gallic acid. Methyl syringate, a highly antioxidant compound was detected in high amounts. Several antifungal compounds were identified in Agastache honey, such as Estragole, Phenol-2, 4-bis (1, 1-dimethylethyl), Nonanoic acid, 2-propenoic acid, 3-phenyl, ethyl ester Ethyl cinnamate, 2-hydroxy-3-methoxybenzaldehyde, 1-Nonyl-1 H-benzo[d]imidazole. Overall, the present findings on the antioxidant and
 antimicrobial activities of Agastache honey were close to those of Leptospermum honeys (used for wound care) and greater antifungal activity, suggesting that Agastache honey products could be developed for the topical application against skin-infection causing bacteria, fungi and in wound care management.