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The factors responsible for the varying levels of UMF® in mānuka (Leptospermum scoparium) honey A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biological Sciences at the University of Waikato by Jonathan McD C Stephens University of Waikato Hamilton, New Zealand February 2006 © 2006 i Abstract The variability in the level of the non-peroxide antibacterial component (UMF®) of mānuka honey produced in New Zealand was studied. A field analysis confirmed considerable variability existed in the honeys, and a number of hypotheses to explain this variability were proposed and examined. Nectar derived from Leptospermum scoparium (mānuka), was confirmed to be the source of UMF®. The dilution of mānuka honey with nectar derived from other floral sources was found to proportionally reduce the UMF® in monofloral mānuka honey. The utilisation of the thixotropic properties of mānuka honey allowed the degree of dilution in the field samples to be established, and an adjustment of the field results to account for the dilution of UMF® by other honey types revealed all monofloral mānuka honey contains UMF®. However, in the monofloral mānuka honey, significantly different levels of UMF® activity were found to come from reasonably well-defined geographic regions. The cause of the variable levels of UMF® activity in mānuka honey would appear to be the different varieties of L. scoparium being harvested by the honeybees, and the environmental parameters influencing nectar production or another species interacting with L. scoparium do not appear to influence UMF® activity. ii Three methods were used to establish genetic variability within regions of the North Island of New Zealand that gave rise to the various levels of UMF® activity. Analyses of morphological characteristics, chemotaxonomic essential oil profiles, and population genetics of L. scoparium populations were conducted, and the conclusions that were drawn from each of these were very similar. Two major divisions were identified, each divided into two varieties. The northern division, which contained the core populations from Northland and Waikato, represented the previously described L. scoparium var. incanum and L. scoparium var. linifolium. This division yielded mānuka honey with high UMF® activity. The southern division, which contained the core populations from the Central North Island and East Coast, represented the previously described L. scoparium var. myrtifolium and an unnamed variety. The latter, growing principally on the East Coast, uniquely contains triketones essential oils. The southern division yielded mānuka honey with low UMF® activity. Hybridisation between these varieties will occur, leading to a continuum of UMF® activity in mānuka honey. The data indicated multiple dispersions of L. scoparium to New Zealand from the evolutionary centre of the persistent-capsule Leptospermum group in south-east Australia, and later regional dispersal in New Zealand. From this study two hypotheses were accepted: the variability in the UMF® activity of mānuka honey is due to both the dilution of mānuka honey by other honey types and the variety of L. scoparium harvested. iii Acknowledgements Thanks must firstly go to my principal university supervisor, Professor Peter Molan. Professor Molan established the need for this study, and made the initial arrangements. Subsequently Professor Molan has provided assistance and advice in the duration of the study. In particular Professor Molan initially suggested the use of the thixotropic characteristics of mānuka honey as a potential method for determining the purity of a mānuka honey derived from unknown floral sources. A number of the academic staff at the university also provided assistance; chiefly Dr Janis Swann for assistance with the viscosity method, Dr Chrissen Gemmill with the population genetics work, Drs Brendan Hicks and Ray Littler with statistical analyses, and Associate Professor Bruce Clarkson for guidance with Leptospermum scoparium habitats and distribution. The chemotaxonomic work was completed in association with the university Department of Chemistry; Professor Alistair Wilkins provided much guidance, and his doctoral student Mahima Senanayake completed the practical analyses. Also at the university, the Honey Research Unit technician Kerry Allen and the Population Genetics Laboratory technician Tracey Jones provided practical and timely advice. iv This study was completed with the support of the Bright Futures Scholarships Enterprise Awards scheme. Commercial sponsorship was originally made by Bee & Herbal (NZ) Ltd. However during the term of the study this company was acquired by Comvita (NZ) Ltd. Comvita (NZ) Ltd continued the support, and an emphasised thanks go to the CEO, Mr G. Boyd, and the chairman of the board, Mr B. Bracks, both now retired. More recently Mr G. Young, CFO, and the recently appointed CEO of Comvita New Zealand (2003) Ltd have continued to support the study. These members of the New Zealand honey industry have understood the value of this study, and have unstintingly provided the support needed for work of this nature, above all the funding for the environmental correlations with Landcare, the genetics analysis consumables, and lastly though not least, the majority of a scholarship. The ability to complete these expensive parts of this research in a timely manner is acknowledged. Thanks also go to Dr Jake Overton, who supervised the environmental correlation work at Landcare using the programs and data created principally under the supervision of Dr J. Leathwick. Many beekeepers throughout New Zealand provided samples for the study. For the most part the samples were appropriate and utilised in this study, and the apiarist’s ongoing support and encouragement has been appreciated. Without the initial sampling across New Zealand this study would not have been possible, and the supply of commercially sensitive site data was appreciated. It is hoped the collection of samples was not an onerous burden. The illustrations of the Leptospermum scoparium varieties highlighting the morphological differences were drawn by my mother, Janet Stephens. A time consuming task kindly completed, that undoubtedly improves this thesis. And finally to my wife, Lisa, who has assisted in many ways, particularly during proof-reading and writing, but also for being a reliable sounding board throughout the duration of this study. v Table of Contents page Abstract ii Acknowledgements iv Table of Contents vi List of Figures xii List of Tables xiv Chapter 1 Leptospermum scoparium (mānuka) honey 1 1.1 Pharmacological use of honey 2 1.2 Antibacterial activity in mānuka honey 3 1.3 Pharmacological development of mānuka honey 6 1.4 New Zealand honey 6 1.5 Mānuka honey resource 7 1.6 Intention of thesis 8 1.7 Hypotheses 9 Chapter 2 Leptospermum scoparium in New Zealand 11 2.1 Leptospermum scoparium 12 2.1.1 Taxonomic status 12 2.1.2 Leptospermum scoparium in New Zealand 14 2.1.3 Intraspecific variation in New Zealand 17 2.1.4 Genotypic and phenotypic variability 18 2.1.5 Chemotaxonomic analyses of Leptospermum scoparium 19 2.2 New Zealand distribution and habitats of Leptospermum scoparium 21 2.2.1 Permanent dominance of Leptospermum scoparium 22 2.2.2 Seral role of Leptospermum scoparium 24 vi Table of contents continued page Chapter 3 Field study of mānuka honey 27 3.1 Sampling strategy 28 3.2 Study period 28 3.3 Sample collection 28 3.3.1 Collection of mānuka honey 29 3.3.2 Site data 30 3.4 Sample receipt and storage 30 3.5 Testing method 31 3.5.1 UMF® assay 31 3.5.2 Sample preparation 31 3.5.3 Phenol and internal standards for assay 32 3.5.4 Assay plate preparation 32 3.5.5 Calculation of antibacterial activity 34 3.6 Re-testing of samples with non-detectable activity 34 3.7 Non-detectable samples 36 3.8 Field study results 37 3.8.1 New Zealand results 38 3.8.2 Northland region 39 3.8.3 Waikato region 40 3.8.4 Coromandel region 42 3.8.5 Taranaki region 44 3.8.6 East Coast region 45 3.8.7 Gisborne region 47 3.8.8 Hawkes Bay region 48 3.8.9 Wairarapa region 49 3.8.10 Northern South Island region 50 3.8.11 Eastern South Island region 52 3.8.12 West Coast region 53 3.9 Conclusion 55 Chapter 4 Nectar or by-product from another species is source of UMF® 56 4.1 Hypothesis 57 4.2 Nectar from another plant species 57 4.2.1 Indigenous New Zealand species 57 4.2.2 Surplus nectar collected by honeybees 58 4.2.3 Plant communities in regions producing honey with UMF® 60 4.2.4 Other plant species conclusion 64 4.3 By-product of another species 64 4.3.1 Scale insect distribution 65 4.3.2 Duration of production of honeydew 66 4.3.3 The composition of honeydew honeys 67 4.3.4 Other species conclusion 67 Chapter 5 Purity of mānuka honey