Kangaroo Island Propolis Kangaroo Island Propolis Improved Characterisation and Assessment of Chemistry and Botanical Origins through Metabolomics Douglas Iain King BPharm (Hons) MPS JP A thesis submitted in fulfilment of the requirements of the degree of Doctor of Philosophy Faculty of Pharmacy, The University of Sydney August, MMXVII Declaration The work described in this thesis was conducted by the author under the supervision of Dr Colin Duke and auxiliary supervision of Dr William (Bret) Church at the Faculty of Pharmacy, The University of Sydney. The author hereby declares that the research presented here is his own original work and has not been submitted to any other institution for the award of a degree. Douglas I. King 4th August 2017 ii Dedication I dedicate this thesis to my fiancée and future wife: My sweet girl, Anna Sanders Robinson Every day with you, my dearest, is a pleasure; you have always brought full support, comfort and love to me in all my endeavours. I trust I have provided you with the full support, comfort and love you so richly deserve in turn. I rejoice that I will soon be able to give you the time and attention I have had to divert to my research: these have been occasionally trying times for us both. I met you, asked you out, became your boyfriend, moved in and proposed to you all during the length of my candidature – it’s been an exciting and adventurous four years. Know that whatever adventures life brings us in the future, my sweet girl, I will always love you as you love me. iii Acknowledgements First and foremost, words cannot express my deep and lasting gratitude to my supervisor, A/Prof Colin Duke. I have had the extreme honour to have had Colin supervise both my Honours and Doctoral studies: a more helpful, available, interested and (above all) forgiving supervisor is not to be found. His natural products knowledge is immense; I have been lucky indeed to learn what little I could from him. As he approaches retirement from academia (but never research), I wish him continuing success with Australian propolis and look forward to a long collaboration. I am most grateful to Dr Rujee Duke and Dr Van Tran, whom I have also had the privilege to work closely with. Rujee’s knowledge of pharmacology and cell biology is encyclopaedic; I am extremely appreciative for her advice in these areas, where I have always struggled. Van, also a past supervisor of my Honours work, has always maintained an interest in my research and has made several helpful comments over the years. Dr Bret Church, roped in at last minute as my auxiliary supervisor, has kept a close eye on my progress and given much insight into the politics of academia – I thank him for both. I heartily thank those students I’ve shared the lab with, particularly my fellow researchers in the Duke Group: Dr Noushin Aminimoghadamfarouj and Dr Kaiser Hamid. Our collaboration has always been tight, and I’d like to think we’ve all provided useful tips and tricks to one another. Certainly, I’ve been the recipient of many. I’d also like to thank Noushin’s husband, Dr Reza Nematollahi, not strictly from our lab, but always present and a scholar and a gentleman. I had the honour of being the guest of Noushin and Reza for their graduation as Doctors (Sydney, 2016); I thank them for this mark of friendship and wish them both long and productive careers. For our shared work on genus Lepidosperma, I am grateful to our botanist and taxonomist collaborators Dr Russell Barrett (University of Western Australia), Karen Wilson (Royal Botanic Garden, Sydney), Prof Jeremy Bruhl and George Plunkett (University of New England). I particularly thank George; his doctoral research on chemotaxonomy of sedges has helped mine and my doctoral research, I trust, has helped his. I thank Dr Nick Proschogo of the School of Chemistry Mass Spectrometry Facility for performing mass spectrometry on DK-2, DK-3 and other samples at very short notice, and to Eurofins Panlabs for the performance of the cellular assays on DK-1. Turning from academe, I thank most deeply the love and support shown to me by my mother, Joanne King, and father, Mark King. Mum, Dad, a man could have no finer parents: know I love you dearly and know this thesis will make you proud – not that you weren’t already. I also thank my grandmother, Dawn King, for her love, support and ongoing interest in my studies. My siblings, Alison King and Dr Malcolm King (Melb, 2017), have always been supportive and I thank them deeply. I especially thank Mal, who submitted his thesis at The University of Melbourne last year (1), for being, in many ways, the ‘scouting party’ as to what I should iv expect over the course of my studies. I can only hope my thesis is half as robust and scholarly as his was. I also thank Mal and Ally for the gifts of a Mini Express One stovetop coffee maker (Bialetti Industrie Spa, Brescia, Lombardy, Italy) and a džezva for making Serbian coffee; both have both proved extremely useful in these last few thesis-writing months. I’d also like to treasure the memory of my good mate and loyal companion: my dog, Syd (2000-2016). He was a good boy to the very end and is sorely missed by the whole family. In the extracurricular world of Sydney University, I like to thank all my colleagues and friends at the Sydney University Postgraduate Representative Association, where I had the honour of being Treasurer for two years; and the Sydney University Musical and Madrigal Societies. It’s been a real pleasure and escape from the lab to sing with fellow students. I’d also like to thank the Faculty of Pharmacy postgraduate coordinator, Prof Dai Hibbs, who has always been good for a chat and a joke with this very Saesneg Australian. I thank all my fellow staff and the patients of Helensburgh Pharmacy, who are always curious as to my studies and take some pride in their ‘Dr’ Doug. It’s a wonderful town and place to work in. An especial thanks to the fellow members of the Society for Classic Pubs: Scott Ferguson, Tony Perusco, Alex Rae, and Dr Cass Stuart (Sydney, 2016) (2). This thesis hasn’t been quite an ‘open misère’, but regular meetings of the SCP have certainly eased it along. Good friends, good beer, good times. And, of course and always, all my gratitude and love to my dearest sweet girl, Anna. v Contents Declaration................................................................................................................................ ii Dedication ................................................................................................................................ iii Acknowledgements ................................................................................................................. iv Contents ................................................................................................................................... vi List of figures ........................................................................................................................... xi List of tables........................................................................................................................... xiv List of abbreviations ............................................................................................................ xvii Publications and conferences ................................................................................................ xx Abstract .................................................................................................................................. xxi Chapter One: A Review of Propolis Literature ............................................................................................. 1 1.1 General introduction and history ......................................................................................... 2 1.2 Physical nature of propolis................................................................................................... 7 1.3 Collection, manufacture and use of propolis by bees .......................................................... 8 1.4 Chemical variability of propolis ........................................................................................ 11 1.4.1 Early research...................................................................................................... 11 1.4.2 Major propolis types ........................................................................................... 12 1.4.2.1 Temperate propolis .............................................................................. 13 1.4.2.2 Mediterranean propolis ........................................................................ 15 1.4.2.3 Tropical propolis .................................................................................. 16 1.4.2.4 Pacific propolis..................................................................................... 17 1.4.3 Australian propolis .............................................................................................. 18 1.4.4 Kangaroo Island propolis .................................................................................... 19 1.5 Biological activity and medicinal use of propolis.............................................................. 23 1.5.1 Antimicrobial and antiparasitic activity .............................................................. 23 1.5.2 Antioxidant activity ............................................................................................ 26 1.5.3 Anti-inflammatory and immunomodulatory activity .........................................