Jonathan M. Stephens

Arabinogalactan proteins contribute to the immunostimulatory properties of New Zealand honeys.

Context: Factors in honey that improve wound healing are poorly understood, but are thought to include lipopolysaccharide (LPS), apalbumin-1 and -2, and a 5.8 kDa component that stimulate cytokine release from macrophages. Objective: To characterize the ability of New Zealand honeys to elicit the release of tumor necrosis factor-α (TNF-α) from monocytic cell lines as a model for early events within a wound site. Materials and methods: The ability of kanuka (Kunzea ericoides), manuka (Leptospermum scoparium), and clover (Trifolium spp.) honeys to stimulate the release of TNF-α from monocytic cell lines THP-1 and U937 was assayed by ELISA. Results: All three honeys stimulated TNF-α release from THP-1 cells, with kanuka honey being the most active. The activity of kanuka honey was associated with a high molecular weight (>30 kDa) component that was partially heat labile and inhibitable with polymyxin B. LPS concentrations in the honeys were too low to adequately explain the level of immunostimulation. The contribution of type II arabinogalactan proteins (AGPs) we recently identified in kanuka honey was tested, as AGPs are known immunostimulators. AGPs purified from kanuka honey stimulated the release of TNF-α from THP-1 and U937 cells. Discussion: Here we demonstrated that AGPs we recently identified in kanuka honey have immunostimulatory activity. We propose that the immunostimulatory properties of individual honeys relate to their particular content of LPS, apalbumins, the 5.8 kDa component and AGPs. Conclusion: The immunostimulatory activity of kanuka honey may be particularly dependent on AGPs derived from the nectar of kanuka flowers.

Fluorescence markers in some New Zealand honeys.

The fluorescence characteristics of various New Zealand honeys were investigated to establish if this technique might detect signatures unique to manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) honeys. We found unique fluorescence profiles for these honeys which distinguished them from other New Zealand honey floral types. Two excitation-emission (ex-em) marker wavelengths each for manuka and kanuka honeys were identified; manuka honey at 270-365 (MM1) and 330-470 (MM2) nm and kanuka honey at 275-305 (KM1) and 445-525 (KM2) nm. Dilution of manuka and kanuka honeys with other honey types that did not possess these fluorescence profiles resulted in a proportional reduction in fluorescence signal of the honeys at the marker wavelengths. By comparison, rewarewa (Knightia excelsa), kamahi (Weinmannia racemosa), and clover (Trifolium spp.) honeys did not exhibit unique fluorescence patterns. These findings suggests that a fluorescence-based screening approach has potential utility for determining the monoflorality status of manuka and kanuka honeys.

Isolation, Structural Elucidation, and Synthesis of Lepteridine From Ma̅nuka (Leptospermum scoparium) Honey

Ma̅nuka honey, made from the nectar of Leptospermum scoparium, has garnered scientific and economical interest due to its nonperoxide antibacterial activity. Biomarkers for genuine ma̅nuka honey are increasingly in demand due to the presence of counterfeit ma̅nuka honey. This work reports the identification of a compound previously unreported in ma̅nuka honey by HPLC, and determination of the structure of the as 3,6,7-trimethyllumazine using NMR, MS, IR, and UV/vis spectroscopy. This assignment was confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in ma̅nuka honeys and could potentially serve as a biomarker for genuine ma̅nuka honey.

Leptosperin is a distinct and detectable fluorophore in Leptospermum honeys.

New Zealand manuka (Leptospermum scoparium) honey exhibits two unique fluorescence signatures that distinguish it from other honey types. One of these is the MM1 fluorescence marker (270-365nm excitation-emission) which we show is due to a Leptospermum nectar-derived compound, leptosperin. Synthetic or honey-purified leptosperin not only displayed an identical fluorescence spectrum, but supplementation of leptosperin into clover or artificial honeys generated the MM1 fluorescence signature. There was a quenching effect of the honey matrix on leptosperin fluorescence but otherwise leptosperin was chemically stable over prolonged storage at 37°C. Leptosperin was also present in the woody-fruited Australian Leptospermum species at elevated concentrations but virtually absent in Leptospermum subtenue suggesting its elevated expression developed following the mid-Miocene separation of the genus. These findings suggest that fluorescence spectroscopy could offer a rapid and high-throughput screening method for identification of Leptospermum honeys using the MM1 fluorescence marker.

Honey Production and Compositional Parameters

Abstract Manuka honey is produced from the nectar of Leptospermum scoparium and is of high therapeutic and commercial value due to its non-peroxide antibacterial activity, known as unique Manuka factor (UMF). These properties are attributable to the presence of methylglyoxal (MGO), which is derived from the precursor molecule dihydroxyacetone (DHA) present in the plant nectar, and potentially other components such as phenolic compounds. Despite the therapeutic importance of Manuka honey, relatively little is known about how its molecular composition and the MGO concentration are affected by aging and storage conditions. Here we describe findings using mass spectrometry showing that honey contains a diverse array of small-molecule compounds and that these vary depending on floral origin and storage conditions. We also describe how MGO levels change as Manuka honey matures as well as other components that might be useful as floral markers of this honey.

Lepteridine as a unique fluorescent marker for the authentication of manuka honey

The recent discovery of two unique manuka marker fluorescence wavelengths (MM1 and MM2) potentially offers a rapid and cost-effective approach for manuka honey authentication using spectroscopy. The fluorophore responsible for the MM1 marker has been identified as leptosperin. We investigated whether lepteridine may be responsible for the MM2 fluorescence. We quantified the lepteridine in manuka honey and manuka nectar, which ranged between 5-52mg/kg and 80-205mg/kg, respectively. Notably, the fluorescent spectrum of synthetic lepteridine matched the MM2 fluorescence signature. Fluorescence quenching was observed in the honey matrix but otherwise, lepteridine was stable over prolonged storage at 37°C. Lepteridine was also found in Australian Leptospermum honeys and nectars. Lepteridine concentration was positively correlated with concentrations of the MM1 fluorescence marker leptosperin in honeys. These findings identify lepteridine as the principle compound responsible for MM2 fluorescence, and support the utility as a marker compound for manuka honey authentication.

Soil influences on plant growth, floral density and nectar yield in three cultivars of mānuka (Leptospermum scoparium)

ABSTRACT Honey derived from the nectar of Leptospermum scoparium J.R. et G. Forst. Myrtaceae (mānuka) is a high-value product and there is considerable potential for economic growth in honey-growing regions of New Zealand through increased nectar yield from mānuka plantations. Leptospermum scoparium exhibits a significant amount of phenotypic plasticity throughout regions in New Zealand where it has established, although the influences on this plasticity are unknown. When assessing L. scoparium as a nectar source for honey in marginal land areas, the possible effect of soil on nectar chemistry and yield should be considered. We investigated whether phenological patterns of flowering, plant growth, nectar composition and nectar yield were influenced by soil composition. Three different cultivars of L. scoparium were grown on 10 different soils in glasshouse conditions. The soils chosen were representative of the range of New Zealand soils where mānuka is being considered as a commercial crop for the honey industry. ANOVA and general linear models revealed no significant effect of soils on nectar composition or production; however, significant but complex interactions between cultivars and soils influenced plant growth and flowering (P ≤ 0.05). Accordingly, the overall nectar yield was influenced by cultivar and soil interaction. Measured attributes of the soil such as cation exchange capacity, sulphate, iron, manganese, calcium and chloride were shown to influence the plant parameters assessed. Results allowed modelling of nectar potential against each soil type and established a mānuka soil index to determine the most appropriate soil for each cultivar. The results indicated that potential nectar yield increases will be dependent on cultivars being deployed according to the nature of the soil present. Furthermore, the mānuka cultivars displayed significantly greater growth in response to increased nutrients and some cultivars increased floral density, suggesting potential to improve nectar yield by greater plant growth using targeted fertilisation.

Fluorescence: A Novel Method for Determining Manuka Honey Floral Purity

Manuka honey, harvested from Leptospermum scoparium, is New Zealands most recog‐ nised honey type and commands a premium due to health‐related benefits. However, the plants distribution, relative to other species flowering simultaneously, allows hon‐ eybees to incorporate alternative nectars into the honey. Melissopalynological analysis in New Zealand is often unrepresentative due to the presence of many pollen‐bearing sources; consequently, alternative means of categorising manuka honey were examined. RP‐HPLC revealed that manuka honey contains distinct compounds, of which were rela‐ tively enriched and not present in the other New Zealand monofloral honeys. These main candidate compounds were isolated and have been described by mass spectrometry and nuclear magnetic resonance, synthesised to confirm structure, and as standards. These compounds, Leptosperin and Lepteridine, are a methyl syringate glycoside and pteridine derivative, respectively. Examination of these compounds revealed unique fluorescence signatures, this fluorescence could be detected in manuka honey samples the signal used to confirm that a honey was solely or predominantly consisted of L. scoparium nectar. Commercial manuka honeys were assessed by traditional analytical techniques, and comparisons were made with fluorescence signature; the fluorescence technique deter‐ mined the authenticity of the honeys accurately.