Kangaroo Island Propolis: Improved Characterisation and Assessment of Chemistry and Botanical Origins through Metabolomics

Introduction: Propolis, a sticky substance produced by bees from plant resins, has a long history of safe use medicinally. Kangaroo Island, SA (KI) lacks many introduced European plants bees preferentially collect resin from; consequentially, propolis from KI is produced from resinous native plants. Several identifiably reproducible pure-source KI propolis types exist. Research into medical use of compounds from KI native plants is limited. Metabolomics is a growing field of interest in natural products chemistry, including beehive products. Metabolomic and similarity-scoring assessment of KI propolis, through statistical evaluation of 1D 1H-NMR fingerprints, provides an entry point for research into medical use of KI native plant compounds. Many avenues to product discovery in pharmaceutical chemistry are suffering diminishing returns: metabolomics-guided natural products assessment has the potential for further identification of novel therapeutic compounds from resinous plants. Aim: To assess and identify, via metabolomic investigation of NMR fingerprints, major propolis types on KI, and to produce, from this, similarity-scoring tools for assessment of propolis samples. Method: KI propolis samples, identified as pure-source by TLC, and resinous KI plants were analysed by 1H-NMR and HPLC. Data points of interest were normalised and binned to form individual sample ‘fingerprints’. Data from these fingerprints were analysed by hierarchical clustering and principal component analysis (PCA) to confirm provisionally-identified pure-source propolis types and identify subtypes within propolis and resinous plant species. From this, calculator tools were created to score similarity (out of 1000) of 1H-NMR fingerprints to the average spectrum of pure-source propolis types, as well as to calculated mixtures of these average spectra. Assessment of the chemistry of two major KI propolis types identified (CP- and F-type) was made by fractionation and NMR, with one compound, 6,8-diprenyleriodictyol, isolated from CP-type propolis in quantity, submitted for epigenetic and other biological assays. Results: Source resinous plants were demonstrated, through hierarchical clustering and PCA, to cluster with propolis types arising from these sources, with closely related plants and sub-chemotypes clustering separately, confirming specificity. A number of previously-identified pure-source propolis types and known botanical sources were shown to have very high similarity (> 800/1000) to the expected propolis type. Calculator tools were observed to accurately predict the content of mixed propolis samples to within ± 10%. A number of methylflavanones, and two novel terminally-hydroxylated prenyldihydrochalcones were isolated from F-type propolis. 6,8-diprenyleriodictyol demonstrated a range of promising activity in biological assays. Conclusion: Metabolomic evaluation of 1H-NMR fingerprints can reliably identify and assess pure-source KI propolis and identify botanical origin of source resins. Similarity scoring calculators can accurately identify mixed-source propolis samples. KI propolis types are a rich source of pharmaceutically-interesting flavanones and related compounds, many of which are prenylated. 6,8-diprenyleriodictyol displays strong anti-inflammatory and anticancer activity, especially against Burkitt’s lymphoma. A number of possible epigenetic pathways for this activity were observed