Lepteridine: properties, bioactivities and possible applications of a unique mānuka honey compound

Abstract

Mānuka honey is a premium food product produced exclusively in New Zealand. Traditionally, mānuka honey was authenticated using the MGO/DHA system. However, these compounds are unstable during prolonged storage, which has led to many concerns in the New Zealand honey industry. The New Zealand Ministry for Primary Industries (MPI) has recently established new criteria to authenticate mānuka honey using a combination of chemical markers and DNA pollen analysis. The identification of more reliable mānuka honey marker compounds would benefit the ongoing improvement of the current MPI criteria. Apart from the well-known antibacterial activity, mānuka honey also possesses potent anti-inflammatory activity. The anti-inflammatory bioactivity cannot be solely explained by the removal of pathogenic bacteria or the level of known phenolic compounds. The mechanisms underlying the anti-inflammatory properties of mānuka honey are also not fully understood. This thesis mainly focused on lepteridine, which was discovered as a unique compound in mānuka honey. The first part of this project aims to investigate the properties of lepteridine and its potential in mānuka honey authentication. The second part of this project aims to investigate whether lepteridine contributes to the anti-inflammatory bioactivity of mānuka honey through MMP-9 inhibition. The findings of this study demonstrated the potential of lepteridine as a reliable chemical and fluorescence marker compound of mānuka honey. Lepteridine is derived from the Leptospermum nectar and showed good stability over prolonged storage at 37 °C. Lepteridine was identified as the principal compound responsible for the MM2 signature fluorescence (ex330 nm–em470 nm) exhibited by mānuka honey, and the concentration was accurately determined using fluorescence spectroscopy. The concentration of lepteridine in honey was quantified using HPLC and LC-MS/MS. The minimum lepteridine threshold concentration for mānuka honey was determined using a mass spectrometry approach. These studies showed that the lepteridine-based analyses can effectively determine the authenticity of mānuka honey and compared favourably with the MPI criteria. An apparent inhibitory effect of MMP-9 was initially observed with mānuka honey phenolic extracts using a fluorometric assay. Lepteridine was identified as a contributor to MMP-9 inhibition with an IC50 of 11.5 μg/mL. However, the fluorometric spectroscopy technique does not directly measure the enzyme activity, and the output signal can be influenced by fluorescence quenching or the autofluorescence nature of mānuka honey. Further experiments were carried out using an absorbance-based chromogenic substrate and gelatin zymography. Lepteridine showed more potent inhibition on the gelatinase activity of MMP-9 than the peptide cleavage activity (chromogenic substrate). These findings suggest that lepteridine may interact with the exosite of MMP-9 by disrupting the binding of gelatin. The results from molecular docking analysis further supported the binding of lepteridine at the exosite of MMP-9 located within the fibronectin type II domain. These findings suggest that lepteridine might be of beneficial value for the treatment of conditions such as gastric ulcers where inhibition of aberrant MMP-9 activity is desirable.