Identifying iron-binding antioxidants in New Zealand honeydew honey
Iron is an essential element for human physiological function. However, excess free iron could participate in the formation of reactive oxygen species via the Fenton reaction and is thought to be the primary mechanism for the pathogenesis of diseases related to iron excess. Prevention of the oxidative damage caused by reactive oxygen species can be accomplished by using selective iron-binding compounds. Beech honeydew honey has been found to have an exceptionally high level of iron-binding antioxidant activity. Due to that, the focus of the research in this thesis has been to isolate the compound(s) responsible for the activity and identify their molecular structure. The stability of the antioxidants on exposure to heat and high and low pH was investigated to get a better understanding of the conditions which could be used when developing suitable techniques for the isolation of the iron-binding antioxidants. The isolation of the antioxidants was first attempted with XAD-2 resin. The results of solid phase extraction using XAD-2 resin showed that the activity is mainly found in the honey effluent, the fraction not retarded by the resin, indicating that hydrophilic components contributed to the iron-binding antioxidant activity. The phenolic compounds isolated from this resin did not exhibit iron-binding antioxidant activity. When the antioxidants could not be isolated from the XAD-2 resin, size exclusion chromatography was used to separate the honey components based on their molecular sizes. The technique was successsful in separating antioxidants from free sugars in honey. However, size exclusion chromatography did not give any significant information regarding the identity of the antioxidants. Thus, two types of chromatography (reversed phase and anion exchange) were used in sequence to aid the separation of components of the active fraction from size exclusion chromatography. This procedure however was not compatible with subsequent mass spectrometry since a high level of sodium chloride existed in the final fraction, even after desalting using reversed phase column chromatography. Due to that, other means of chromatography were trialled in order to obtain a salt-free fraction prior to mass spectrometry. The first procedure was modified by switching the sequence of the columns used, and different mobile phases without salt were used. This second procedure was successful in isolating the antioxidants and the compounds have been characterised by mass spectrometry. Mass spectrometry revealed that the antioxidants isolated were compounds of low molecular weight (i.e. 255, 283 and 227) which exhibited peaks with high intensity. Nuclear magnetic resonance (NMR) spectroscopy of the active fraction showed that the antioxidant may possess a 1,4-disubstituted aromatic ring. However, full elucidation of the structure of the compound was not possible because of the very low mass of the material that had been isolated. Other than the information gained from mass spectrometry and NMR, the antioxidant also could be characterised by forming the negative ion at pH 9.5–10. The UV absorbance at 220 and 280 nm, but not at 360 nm is indicative that the antioxidant possess a phenolic ring but not a flavonoid in the molecular structure. Complete structural elucidation of the iron-binding antioxidants in honeydew honey could initiate a better understanding of the mechanism of action of the antioxidants. This could increase the use of honeydew honey in pharmaceutical and nutraceutical products as an iron-binding antioxidant. There is also the possibility that the identified compound(s) could be synthesised for pharmaceutical use, or serve as lead compounds for the development of other pharmaceutical iron-binding antioxidants.
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