Megan N.C. Grainger

Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey

Using HPLC a fraction of New Zealand manuka honey has been isolated, which gives rise to the non-peroxide antibacterial activity. This fraction proved to be methylglyoxal, a highly reactive precursor in the formation of advanced glycation endproducts (AGEs). Methylglyoxal concentrations in 49 manuka and 34 non-manuka honey samples were determined using a direct detection method and compared with values obtained using standard o-phenylenediamine derivatisation. Concentrations obtained using both the methods were similar and varied from 38 to 828 mg/kg.

Kinetics of the conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey: Part II – Model systems

The kinetics of conversion of dihydroxyacetone (DHA) to methylglyoxal (MGO) were investigated in mānuka honeys and DHA-doped clover honeys stored between 4 and 37°C. Both the disappearance of DHA and appearance of MGO were confirmed as overall, first order reactions, albeit probably composites of multiple reactions. Increasing the storage temperature accelerated the rate of DHA loss and the initial rate of formation of MGO, but better conversion efficiency was observed at lower temperature. At 37°C, more MGO was lost at later times in mānuka honey compared to DHA-doped-clover honey. Thirty-seven New Zealand mānuka honeys and four clover honeys were analysed for various chemical and physical properties; comparison of rate constants and these parameters identified some positive correlations.

Effect of high pressure processing on the conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka (Leptospermum scoparium) honey and models thereof.

The effect of high pressure processing (HPP) on the conversion of dihydroxyacetone (DHA) to methylglyoxal (MGO) was examined in New Zealand mānuka honey and models thereof. The objective was to confirm that previously reported increases of MGO with HPP treatment originated from conversion of DHA. RP-HPLC was used to quantify DHA, MGO and hydroxymethylfurfural (HMF) after derivatisation with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) or (in the case of MGO) separately with o-phenylenediamine (OPD). Fresh and stored mānuka honey, clover honey with DHA added and artificial 26 honey with DHA added were subjected to nine different pressures and holding times and compared to untreated samples. There was no consistent trend of decrease in DHA or increase in MGO for any of the samples with any treatment. Samples showed random change generally within 5-10% of an untreated sample for MGO, DHA and HMF. HPP does not accelerate the conversion of DHA to MGO in honey.

Isolation of maltol glucoside from the floral nectar of New Zealand mānuka (Leptospermum scoparium).

Maltol glucoside (3-(β-D-glucopyranosyloxy)-2-methyl-4H-pyran-4-one), 1, was isolated from a preparation of the floral nectar from the New Zealand mānuka tree (Leptospermum scoparium). 1 eluted just after dihydroxyacetone in HPLC of underivatized nectar and showed a UV absorbance maximum of 258 nm. The structure of 1 was confirmed by NMR and high resolution mass spectrometry.

Kinetics of conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey: Part III--A model to simulate the conversion.

A kinetic model for the conversion of dihydroxyacetone (DHA) to methylglyoxal (MGO) in honey is proposed; a building block approach was used to create the model. Artificial honeys doped with DHA and individual perturbants were fitted first, then multiple perturbants (alanine, proline and iron, and combinations of these) were fitted before comparing the simulation to real honey samples (doped clover and mānuka honey). The main responses in the prediction model were DHA, MGO, proline, primary amino acids, acidity, 3-phenyllactic acid and 4-methoxyphenyllactic acid. Three temperatures (20, 27 and 37°C) were studied and the conversion of DHA to MGO was monitored over at least 1year. Differences in the conversion between clover doped with DHA and mānuka honey were observed. The simulation fitted well for the honeys tested.

Kinetics of conversion of dihydroxyacetone to methylglyoxal in New Zealand mānuka honey: Part IV – Formation of HMF

During a study of the conversion of dihydroxyacetone (DHA) to methylglyoxal (MGO) in maturing New Zealand mānuka honey, the kinetics of formation of 5-(hydroxymethyl)furfural (HMF) was studied at temperatures from 4 to 37°C. Formation of HMF was first-order during an induction period and zero-order thereafter indicating that the mechanism includes the formation of certain critical intermediates and that these require time to build up; the duration of the induction period depended primarily upon temperature. The zero-order rate constant at 37°C was the same for mānuka honey and clover honey doped with 2000 or 10,000mg/kg DHA and for artificial honey with 2000mg/kg of DHA and either alanine or proline and alanine added. Zero-order rate constants for artificial honey with added amino acids were less than for a control without amino acids. A simulation was created to predict the formation of HMF over time at 37°C in mānuka honey.

Determination of Menaquinone-7 by a Simplified Reversed Phase- HPLC Method

BACKGROUND An efficient and accurate HPLC method was developed for the determination of menaquinone-7 (MK-7) in microbial fermentation using 2-propanol and n-hexane as extraction solvents as well as the eluent. METHODS Extraction was carried out with 2-propanol and n-hexane (2:1, v/v) after enzymatic hydrolysis with 1% (w/v) lipase and ethanol water treatment prior to quantification in order to remove interfering lipids and denatured proteins. Chromatographic separation of MK-7 was accomplished isocratically on a C 18 Gemini column using a mobile phase mixture of 2- propanol: n-hexane (2:1, v/v) with a flow rate of 0.5 mL/min. UV detection was carried out from 200-400 nm and the chromatogram was extracted at a wavelength of 248 nm. A linear response was shown by the method with a coefficient of determination (R2) value of 0.9982. RESULTS The recoveries of MK-7 were greater than 94% and the intra and inter day R.S.D values were less than 2%, demonstrating the accuracy of the method. The lower limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/mL and 0.29 µg/mL, respectively. CONCLUSION The general usefulness of the described method is demonstrated by the application of this method in the analysis of MK-7 from Bacillus species. Under these conditions, the analysis of MK-7 was achieved in less than 8 minutes with a retention time of 7.19 ± 0.1 minutes.