The Antioxidant and Anticancer Potentials of New Zealand Manuka Honey and Thyme Honey Ji He A thesis submitted to the Faculty of Health and Environment Sciences Auckland University of Technology in fulfillment of the requirements for the Degree of Master of Science August 2016 School of Applied Sciences Auckland, New Zealand. The Antioxidant and Anticancer Potentials of New Zealand Manuka Honey and Thyme Honey Approved by: Supervisors: Dr. Yan Li (Primary Supervisor) Dr. Jun Lu (Secondary Supervisor) Table of Content List of Figures .................................................................................................................. iv List of Tables .................................................................................................................... vi Attestation of Authorship ................................................................................................ vii Acknowledgements ........................................................................................................ viii Abbreviations ................................................................................................................... ix Abstract ............................................................................................................................ xi Chapter 1: General Introduction and Literature Review ................................................... 1 1.1 Introduction to Honey .......................................................................................... 1 1.1.1 Composition of Honey ............................................................................... 3 1.1.2 Manuka Honey ........................................................................................... 6 1.1.3 Thyme Honey ............................................................................................. 8 1.2 Oxidants ............................................................................................................... 9 1.2.1 Free Radicals ............................................................................................ 10 1.2.2 Reactive Oxygen Species (ROS) ............................................................. 11 1.2.3 The Two Sides of ROS ............................................................................. 13 1.2.4 Oxidative Stress and Disease ................................................................... 15 1.3 Antioxidants in General...................................................................................... 15 1.4 Antioxidants in Honey ....................................................................................... 19 1.5 Mechanism of Antioxidant Activity ................................................................... 21 1.5.1 Primary Antioxidants ............................................................................... 21 1.5.2 Secondary Antioxidants ........................................................................... 22 1.6 Cancer and Its Common Treatments .................................................................. 23 1.7 Colorectal Cancer (CRC) ................................................................................... 25 1.8 Anticancer Activity of Honey and Its Mechanisms............................................ 26 1.9 Other Therapeutic Effects of Honey .................................................................. 34 1.9.1 Antimicrobial Activity of Honey .............................................................. 34 1.9.2 Wound Healing and Anti-inflammatory Effects of Honey ....................... 37 1.9.3 Nutrition and Health Effects of Honey .................................................... 40 1.9.3.1 Oral Health ..................................................................................... 40 1.9.3.2 Gastroenterology ............................................................................ 40 1.9.3.3 Cardiovascular Health .................................................................... 42 1.9.3.4 Physiological Effects ...................................................................... 42 1.9.3.5 Different Health Enhancing Effects ............................................... 43 1.9.3.6 Allergy ............................................................................................ 43 1.9.3.7 Infants ............................................................................................. 44 1.9.3.8 Athletic Performance ...................................................................... 45 1.10 Antioxidant Capacity Assays ........................................................................... 45 1.10.1 Hydrogen Atom Transfer Assays ........................................................... 45 1.10.2 Electron Transfer Assays ........................................................................ 46 1.10.2.1 CUPRAC Assay and Its Limitations ............................................ 47 1.10.2.2 DPPH Assay and Its Limitations .................................................. 50 1.11 Caco-2 Cell Line as a Model of Colon Cancer ................................................ 53 i 1.12 MTT Cell Proliferation Assay and Its Limitations ........................................... 54 1.13 Objectives ......................................................................................................... 55 Chapter 2: Materials and Methods .................................................................................. 56 2.1 Materials ............................................................................................................. 56 2.2 DPPH Assay ....................................................................................................... 56 2.2.1 Scavenging Capacity versus Reaction Time ............................................ 56 2.2.2 Scavenging Capacity versus Concentrations of Honey ........................... 58 2.3 Methods for CUPRAC Assay ............................................................................ 59 2.4 Methods for Cell Viability MTT Assay .............................................................. 60 2.4.1 Cell Culture .............................................................................................. 60 2.4.2 Cell Counting ........................................................................................... 60 2.4.3 Seeding Cells ............................................................................................ 61 2.4.4 Adding Honey as the Treatment ............................................................... 61 2.4.5 MTT Assay ............................................................................................... 62 2.4.6 IC Calculation Method .......................................................................... 62 50 2.5 Statistic Analysis ................................................................................................ 63 Chapter 3: Results ........................................................................................................... 63 3.1 The Stability of DPPH Solution Mixed with Distilled Water and Absolute Methanol .................................................................................................................. 63 3.2 Antioxidant Activity of Manuka Honey and Thyme Honey Tested by DPPH Assay ........................................................................................................................ 65 3.2.1 Time Course of Free Radical Scavenging Capacity ................................. 65 3.2.2 Concentration-Dependent Antioxidant Activity of Tested Honeys .......... 71 3.3 Antioxidant Activity of Manuka Honey and Thyme Honey Tested by CUPRAC Assay ........................................................................................................................ 73 3.4 Anticancer Activity of Manuka Honey and Thyme Honey ................................ 76 3.4.1 Concentration- and Time-Dependent Effects of Manuka Honey and Thyme Honey on Caco-2 Cell Viability ........................................................................ 76 3.4.2 Anticancer potency ................................................................................... 79 Chapter 4: Discussion ..................................................................................................... 82 4.1 Antioxidant Activity of Manuka Honey and Thyme Honey from New Zealand .................................................................................................................................. 82 4.1.1 Effects of Different Solvents on Antioxidant Ability of Manuka Honey and Thyme Honey .................................................................................................... 84 4.1.2 Time-dependent Antioxidant Activity of Manuka Honey and Thyme Honey ........................................................................................................................... 87 4.1.3 Concentration-dependent Antioxidant Activity of Manuka Honey and Thyme Honey .................................................................................................... 88 4.1.4 Comparison of Antioxidant Activity between Manuka Honey and Thyme Honey ................................................................................................................ 89 4.2 Anticancer Activity of Manuka Honey and Thyme Honey from New Zealand. 91 4.2.1 Anti-proliferative Effects of Manuka Honey ........................................... 91 4.2.2 Anti-proliferative Effects of Thyme Honey ............................................. 92 4.2.3 Comparison of Anti-Proliferative Effects between Manuka Honey and Thyme Honey .................................................................................................... 93 4.3 The Correlation between Antioxidant and Anticancer effects of honey ............ 95 ii Chapter 5: Conclusion ..................................................................................................... 97 5.1 Manuka Honey and Thyme Honey as Effective Antioxidants with Future Direction of Research ............................................................................................... 97 5.2 Manuka Honey and Thyme Honey as Effective Anticancer Agent with Future Direction of Research ............................................................................................... 98 References ....................................................................................................................... 99 iii List of Figures Figure 1. The basic structure of flavonoids. Redrawn from Bravo (1998) ............. 17 Figure 2. The two pathways of apoptosis. BID: BH3 interacting-domain death agonist; Cytochrome c: cytochrome complex; APAF-1: Apoptotic protease activating factor 1; IAP: inhibitor of apoptosis. Redrawn from S. Ahmed and Othman (2013) ................................................................................................. 29 Figure 3. The mechanism of honey leading to apoptosis in colon cancer cell lines. TNF: tumor necrosis factor; TRADD: TNF receptor associated death domain protein; Cyt. c: cytochrome complex; APAF-1: Apoptotic protease activating factor 1. Redrawn from Jaganathan and Mandal (2010) .................................. 30 Figure 4. Reactions in CUPRAC assay. Redrawn from Özyürek et al. (2011) ....... 48 Figure 5. Structures of DPPH. Redrawn from Kedare and Singh (2011) ............... 51 Figure 6. Seeding plan of Caco-2 cell line. ............................................................. 61 Figure 7. The absorbance of DPPH dissolved in distilled water and methanol. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same solvent at time zero. ................................................. 64 Figure 8. Time-dependent free radical scavenging capacity of Manuka honey dissolved in distilled water. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same concentration at time zero. .................................................................................................................. 65 Figure 9. Free radical scavenging capacity of Thyme honey dissolved in distilled water. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same concentration at time zero. ............................ 66 Figure 10. Free radical scavenging capacity of Manuka honey dissolved in methanol. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same concentration at time zero. ....................................... 67 Figure 11. Free radical scavenging capacity of Thyme honey dissolved in methanol. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same concentration at time zero. ....................................... 68 Figure 12. The difference of scavenging capacity between Manuka honey dissolved in distilled water and methanol. Data are expressed as means ± SD (n = 3). .. 69 Figure 13. The difference of scavenging capacity between Thyme honey dissolved in distilled water and methanol. Data are expressed as means ± SD (n = 3). .. 70 Figure 14. Comparison of scavenging capacity of Manuka honey, Thyme honey and Ascorbic acid dissolved in distilled water. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the concentration of 0 g/ml. ............................................................................. 71 Figure 15. Comparison of scavenging capacity of Manuka honey, Thyme honey and Ascorbic acid dissolved in methanol. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the concentration of 0 g/ml. ................................................................................... 72 Figure 16. Comparison of reducing ability of Manuka honey, Thyme honey, and ascorbic acid dissolved in distilled water at different concentrations. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the concentration of 0 g/ml. .................. 74 iv Figure 17. Comparison of reducing ability of Manuka honey, Thyme honey, and ascorbic acid dissolved in methanol at different concentrations. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the concentration of 0 g/ml. .................. 74 Figure 18. Comparison between Figure 16 and 17. Data are expressed as means ± SD (n = 3) ......................................................................................................... 75 Figure 19. Growth inhibitory effects of Manuka honey and Thyme honey on Caco- 2 cells treated for 48 hours. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the lowest concentration (0.003125 g/ml). ........................................................................ 76 Figure 20. Growth inhibitory effects of Manuka honey and Thyme honey on Caco- 2 cells treated for 72 hours. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the lowest concentration (0.003125 g/ml). ........................................................................ 77 Figure 21. Growth inhibitory effects of Thyme honey on Caco-2 cells treated for 48 and 72 hours. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the lowest concentration (0.003125 g/ml). ............................................................................................... 77 Figure 22. Growth inhibitory effects of Manuka honey on Caco-2 cells treated for 48 and 72 hours. Data are expressed as means ± SD (n = 3) * P < 0.05, compared with the values determined for the same treatment at the lowest concentration (0.003125 g/ml). ............................................................................................... 78 Figure 23. IC value of Manuka honey .................................................................. 79 50 Figure 24. IC value of Manuka honey .................................................................. 79 50 Figure 25. IC value of Thyme honey .................................................................... 80 50 Figure 26. IC value of Thyme honey .................................................................... 80 50 Figure 27. Comparison of IC values of Manuka honey and Thyme honey incubated 50 for 48 hours ...................................................................................................... 80 Figure 28. Comparison of IC values of Manuka honey and Thyme honey incubated 50 for 72 hours ...................................................................................................... 80 Figure 29. Comparison of IC values of Manuka honey and Thyme honey on Caco- 50 2 cell growth for 48 and 72 hours. Data are expressed as means ± SD (n = 3) 81 v List of Tables Table 1. The average composition of honey (%) ....................................................... 3 Table 2. Main constituents of honey .......................................................................... 4 Table 3. Flavonoid categories .................................................................................. 17 Table 4. Structure of phenolic acids ........................................................................ 18 Table 5. The discovered bacteria that sensitive to honey ........................................ 35 Table 6. DPPH assay results with solvent of distilled water (%). Data are expressed as means ± SD (n = 3). ..................................................................................... 72 Table 7. DPPH assay results with solvent of methanol (%). Data are expressed as means ± SD (n = 3). ......................................................................................... 72 Table 8. CUPRAC assay R2 values of Manuka honey, Thyme honey, and ascorbic acid ................................................................................................................... 75 Table 9. The differences of IC values of Manuka honey and Thyme honey treatment 50 after 48 and 72 hours, Unit: g/ml. Data are expressed as means ± SD (n = 3); a and b, P < 0.01, compared with Manuka honey at 48 and 72 hours respectively. .......................................................................................................................... 81 vi Attestation of Authorship I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person (except where explicitly defined in the acknowledgements), nor material which to a substantial extent has been submitted for the award of any other degree or diploma of a university or other institution of higher learning. Signed Name Ji He Date 12th August/ 2016. vii Acknowledgements At the outset, I am extremely indebted to my primary supervisor Dr. Yan Li who provides me with the opportunity to devote myself to this incredibly fantastic project, and helps me with thoughtful ideas and suggestions when difficulties were encountered. His marvelous patience, language, guidance and encouragement always impress me from start to finish. Secondly, I am truly grateful to my secondary supervisor Dr. Jun Lu about his help and concern, as well as the assistance regarding cell culture room management. Thirdly, I would like to express my sincere appreciation to all those who give me a helping hand and a listening ear throughout the duration of this study. I would like to say thanks to the seniors, Kelvin, Joy and Piyush, as well as my friend Cindy. They give me a lot of useful technical guidance and experimental products, as well as invaluable assistance in the process of experiments. Their selfless sacrifices and professional knowledge move me most. Fourthly, I should give my appreciation to all staff in the laboratories of the faculty of health and environmental sciences at Auckland University of Technology who provide us a comfortable environment for study. Finally, I would like to express thanks to my parents for their endless and strong supports. viii