The Flavonoid Profile of New Zealand Manuka Honey
Deadman, B. J. (2009). The Flavonoid Profile of New Zealand Manuka Honey (Thesis, Master of Science (MSc)). The University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/5443
Permanent Research Commons link: http://hdl.handle.net/10289/5443
Flavonoids are a class of natural products derived from plants which are incorporated into honeys through propolis, nectar and pollen. Research has shown that many honeys possess characteristic flavonoid profiles which can be used as markers for the geographical and floral origin of honey. The objective of this study has been to determine the flavonoid profile of New Zealand manuka (Leptospermum scoparium) honey, a honey which is internationally recognised for its unique medicinal properties. While all honeys are antibacterial due to the production of peroxides, manuka is unique because it exhibits an additional non-peroxide antibacterial activity due to high levels of methylglyoxal. The established extraction method, which utilises Amberlite XAD-2 resin to extract phenolics from honey, has been modified to permit extraction of phenolics from samples as small as five grams, with no measurable loss of extraction reproducibility. This development opens up a much larger collection of honey samples to flavonoid profiling. Measurement of the recovery rates for this extraction method has been further developed in this study in an attempt to account for the matrix effects of the high carbohydrate and phenolic acid content, relative to the flavonoids, of honey. This was achieved by extraction of flavonoid standards from an artificial honey matrix. The recovery rates of 10 6%, 16 7% and 19 9% for quercetin, chrysin and kaempferol respectively, were significantly lower than rates (28-60%) measured using the accepted procedure of extracting flavonoids from a simple solution. However, it should be noted that the recovery rates measured for extraction from solution were lower than those reported by other research groups. This has been partly attributed to an additional filtration step using a sacrificial HPLC column which was implemented to protect the analytical HPLC system from an unknown contaminant in the phenolic extracts.Having evaluated the reproducibility and reliability of the modified extraction method, it was then applied to the analysis of 31 manuka honeys and 8 other non-manuka honeys from New Zealand. The results have shown that the honeys studied have a common flavonoid profile consisting mainly of the flavanone pinocembrin and the dihydroflavonol pinobanksin. These flavonoids are derived from propolis and are a common feature of honeys from temperate regions of the Northern Hemisphere. The manuka honeys analysed had total flavonoid content ranging between 0.59-2.24mg/100g of honey, with an average level of 1.16 0.16mg per 100g of honey. The flavonoid profile of these samples consisted mainly of pinobanksin (0.27 0.04mg/100g honey), pinocembrin (0.17 0.02mg/100g honey), luteolin (0.14 0.02mg/100g honey) and chrysin (0.13 0.02mg/100g honey), together accounting for 61% of the total flavonoid content. Manuka honey was distinguishable from the other honeys studied by its high total flavonoid content, high luteolin content (greater than 0.05mg/100g honey) and high levels of an unidentified (unknown compound 01) which was found to elute with the flavonoids on HPLC but did not appear to be a flavonoid from its UV spectrum. Statistical analysis showed that a positive correlation existed between the levels of unknown compound 01 in manuka honeys and their non-peroxide antibacterial activity. A similar correlation was also observed for luteolin. The progress of this research has been hampered by the limited range of flavonoid standards available for comparison with HPLC chromatogram peaks. A total of eight flavonoids were found in manuka honey, and a further seven in the non-manuka honeys, which did not coincide with any of the flavonoid standards available. Two of unknown flavonoids were subsequently extracted from fifteen kilograms of manuka honey using Amberlite XAD-2 resin and liquid-liquid extraction, and isolated by a combination of Sephadex-LH20 column chromatography and HPLC. Characterisation of these flavonoids was achieved using a combination of UV absorption spectroscopy, 1H, 13C and HMBC NMR spectroscopy, and LDI-TOF mass spectrometry. The isolated flavonoids were identified as pinobanksin and 8-methoxykaempferol, both flavonoids which have been previously found in honeys.
The University of Waikato
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