Investigating Antibacterial Plant-Derived Compounds from Natural Honey

236 Pages·2015·4.67 MB·English

Investigating Antibacterial Plant-Derived
Compounds from Natural Honey

A thesis submitted for the degree of
Philosophiae Doctor (Ph.D)
by
Jennifer Hawkins
School of Pharmacy and Pharmaceutical
Sciences
Cardiff University
April 2015

1
I
ACKNOWLEDGEMENTS

Firstly, I would like to thank Professor Les Baillie, Dr Natasha de Vere and Dr Alex White for
their insight, guidance and friendship over the last few years.

I would like to pay particular thanks to Natasha for the endless cups of tea, coming to watch me
play against Ireland (despite knowing nothing about rugby!) and for your valued support and
enthusiasm throughout. I would like to thank both Natasha and Col Ford for all their help on the
paper and its analysis too.

Particular thanks goes to Dr Matthew Hegarty and Dr Daniel Smith (IBERS, Aberystwyth
University) for their advice and assistance with the DNA sequencing technologies. Many thanks
to Beverley Adams-Groom (Worchester University) for performing the pollen microscopy
analysis. Furthermore I would like to express my thanks to Anneke Lubben for her expert
guidance and advice on HPLC. Many thanks to Addie Griffiths and the guys in the NBGW for
their insight into DNA barcoding.

I also must thank the Knowledge Economy Skills Scholarships (KESS) group for funding this
project.

I would also like to thank all the members of the microbiology group particularly Bob, Tina, Joon,
Dave, Laura, Harsha and JY for their support, advice and most importantly their friendship - thank
you for the memories and for making these last three years so enjoyable!

Finally I would like to thank my mum and dad - for being there whenever I need you, your
unconditional love and support has made this all possible! Kim, for always being there with a hug
and a smile when I need one, thank you! Andrew, distance means so little when someone means
so much, your daily messages have bought me joy from the other side of the world! Thank you
to the rest of my family and friends especially Sarah, Emma, Leila and the rugby girls for their
continued support and encouragement in everything I do.

II
CONTENTS

DECLARATION ......................................................................... Error! Bookmark not defined.
ACKNOWLEDGEMENTS .......................................................................................................... II
CONTENTS ................................................................................................................................ III
LIST OF FIGURES .................................................................................................................. VIII
LIST OF TABLES ........................................................................................................................ X
ABBREVIATIONS ................................................................................................................... XII
PUBLICATIONS AND COMMUNICATIONS ....................................................................... XV
SUMMARY ............................................................................................................................. XVI
1. GENERAL INTRODUCTION ................................................................................................. 1
1.1 Natural Products as Therapeutics ........................................................................................ 2
1.1.1 The History of Drug Discovery ................................................................................... 2
1.1.2 Plant-derived Natural Products .................................................................................... 4
1.1.3 Antimicrobial Natural Products ................................................................................... 5
1.1.4 The Emergence of Antibiotic Resistant Bacteria ......................................................... 7
1.2 Honey – A ‘Rediscovered’ Therapy ................................................................................... 9
1.2.1 Production of Honey .................................................................................................... 9
1.2.2 History of Honey ........................................................................................................ 10
1.3 Chemical Composition of Honey ...................................................................................... 11
1.3.1 Carbohydrates ............................................................................................................ 11
1.3.2 Proteins and Amino Acids ......................................................................................... 11
1.3.3 Vitamins and Minerals ............................................................................................... 12
1.3.4 Volatile Compounds .................................................................................................. 12
1.3.5 Phenolic Compounds ................................................................................................. 13
1.3.6 Other Compounds ...................................................................................................... 13
1.3.7 Pollen, Propolis and Royal jelly ................................................................................. 14
1.4 Characterisation of Honey ................................................................................................ 15
1.5 Analytical Methods to Determine the Geographical and Botanical Origin of Honey ...... 16
1.5.1 Melissopalynology ..................................................................................................... 16
1.5.2 Biochemical Profile.................................................................................................... 17
1.5.3 DNA Barcoding ......................................................................................................... 18
1.6 The Use of Honey in Modern Medicine ........................................................................... 19
1.6.1 Antibacterial Activity ................................................................................................. 19
1.6.1.1 Sugar ................................................................................................................... 20
1.6.1.2 Hydrogen Peroxide ............................................................................................. 20
1.6.1.3 Low pH ............................................................................................................... 21
1.6.1.4 Bee Derived Antibacterial Peptides .................................................................... 21
1.6.1.5 Plant Derived Antibacterial Phytochemicals ...................................................... 21
1.6.2 Manuka Honey ........................................................................................................... 23
1.6.3 Wound Healing Capabilities ...................................................................................... 25
III
1.7 Microorganisms in Healthcare Settings ............................................................................ 27
1.7.1 Methicillin resistant Staphylococcus aureus (MRSA) ............................................... 27
1.7.2 Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) ................. 28
1.7.3 Bacillus subtilis (B. subtilis) ...................................................................................... 28
1.8 PhD Aims and Objectives ................................................................................................. 29
1.8.1 Aims ........................................................................................................................... 29
1.8.2 Objectives .................................................................................................................. 29
2. GENERAL MATERIALS AND METHODS ........................................................................ 30
2.1 Materials ........................................................................................................................... 31
2.1.1 Honey Sample Collection .......................................................................................... 31
2.1.2 Chemicals and Reagents ............................................................................................ 31
2.1.3 Biological Culture Media ........................................................................................... 34
2.1.4 Bacterial Cultures ....................................................................................................... 34
2.2 Methods............................................................................................................................. 35
2.2.1 Freezer Storage .......................................................................................................... 35
2.2.2 Preparation of Fresh Bacterial Culture Slope ............................................................. 35
2.2.3 Preparation of an Overnight Culture .......................................................................... 35
2.2.4 Miles Misera Viable Bacterial Cell Counting Method .............................................. 35
2.2.5 Streak Plate Method for Isolating Pure Bacterial Colonies........................................ 38
2.2.6 Gram Staining ............................................................................................................ 38
2.3 Statistical Analysis ............................................................................................................ 39
2.3.1 Normal Distribution ................................................................................................... 39
2.3.2 Mann-Whitney U Test ............................................................................................... 39
2.3.3 One-way Analysis of Variance (ANOVA) ................................................................ 40
2.3.4 Kruskal Wallis Test .................................................................................................... 40
2.3.5 Pearson’s Correlation ................................................................................................. 40
2.3.6 Spearman’s Correlation.............................................................................................. 41
3. THE ANTIBACTERIAL ACTIVITY OF HONEY ............................................................... 42
3.1 Introduction ....................................................................................................................... 43
3.1.1 The Antibacterial Activity of Honey ......................................................................... 43
3.1.2 Methods for Determining the Activity of Honey ....................................................... 43
3.2 Chapter Aims and Objectives ........................................................................................... 45
3.3 Materials and Methods ...................................................................................................... 46
3.3.1 Honey Samples .......................................................................................................... 46
3.3.2 Physiochemical Honey Properties.............................................................................. 46
3.3.2.1 pH Analysis ......................................................................................................... 46
3.3.2.2 Water Content ..................................................................................................... 46
3.3.3 Antimicrobial Assay Optimisation ............................................................................. 46
3.3.3.1 Bacterial Selection .............................................................................................. 46
3.3.3.2 Agar Diffusion Optimisation Assay .................................................................... 47
3.3.3.3 Agar Diffusion Assay.......................................................................................... 48
IV
3.3.3.4 Broth Microdilution Optimisation Assay ............................................................ 48
3.3.3.5 Broth Microdilution Assay .................................................................................. 49
3.3.4 Standardisation of Positive Controls .......................................................................... 49
3.3.4.1 Phenol Control .................................................................................................... 49
3.3.4.2 Thymol Control ................................................................................................... 50
3.3.4.3 Sugar Control ...................................................................................................... 50
3.3.5 Characterisation of Antibacterial Activity in Honey ................................................. 50
3.3.5.2 Neutralisation of Hydrogen Peroxide.................................................................. 51
3.3.5.3 Neutralisation of Methylglyoxal (MGO) ............................................................ 52
3.3.5.4 Neutralisation of Bee Defensin-1 ........................................................................ 52
3.3.5.5 Neutralisation of Free Acidity ............................................................................. 52
3.4 Results ............................................................................................................................... 52
3.4.1 Characterisation of Honey ......................................................................................... 52
3.4.1.1 pH Analysis ......................................................................................................... 52
3.4.1.2 Water Content ..................................................................................................... 52
3.4.2 Development of Controls for Honey Screening Assay .............................................. 53
3.4.2.1 Standardisation of Phenol Positive Control ........................................................ 53
3.4.2.2 Standardisation of Thymol Control ..................................................................... 54
3.4.2.3 Standardisation of Artificial Sugar Control ........................................................ 55
3.4.3 Development of an Antimicrobial Screening Assay for Honey ................................. 56
3.4.3.1 Optimisation of the Agar Diffusion Assay.......................................................... 56
3.4.3.2 Media Selection for Agar Diffusion Assay - MRSA .......................................... 56
3.4.3.3 Media Selection for Agar Diffusion Assay – B. subtilis ..................................... 57
3.4.3.4 Optimisation of Broth Dilution Assay ................................................................ 59
3.4.4. Determination of the Antibacterial Activity of Honey Samples ............................... 60
3.4.4.1 Agar Diffusion Assay.......................................................................................... 60
3.4.4.2 Broth based dilution assay .................................................................................. 61
3.4.5 The Antibacterial Activity of Hydrogen Peroxide in Honey ..................................... 62
3.4.6 Neutralisation of Known Antibacterial Factors ......................................................... 64
3.4.6.1 Neutralisation of Hydrogen Peroxide.................................................................. 64
3.4.6.2 Neutralisation of Methylglyoxal ......................................................................... 66
3.4.6.3 Neutralisation of Bee Derived Defensin-1 Antibacterial Activity ...................... 68
3.4.6.4 Neutralisation of Acidity ..................................................................................... 68
3.5 Discussion ......................................................................................................................... 70
4. IDENTIFYING THE BOTANICAL CONSTITUENTS OF HONEY – A DNA
METABARCODING APPROACH ........................................................................................... 75
4.1 Chapter Introduction ......................................................................................................... 76
4.1.1 Traditional Approaches of Pollen Analysis ............................................................... 76
4.1.2 Molecular Biology for Honey Characterisation ......................................................... 77
4.2 Chapter Aims and Objectives ........................................................................................... 80
4.3 Methods and materials ...................................................................................................... 81
V
4.3.1 Honey Samples Analysed .......................................................................................... 81
4.3.2 Traditional Pollen Analysis ........................................................................................ 81
4.3.2.1 Pollen Counts ...................................................................................................... 82
4.3.2.2 Melissopalynology .............................................................................................. 82
4.3.3 DNA Extraction Protocol ........................................................................................... 82
4.3.4 Next Generation Sequencing (NGS) - 454 Analysis .................................................. 83
4.3.4.1 Honey Samples Analysed - 454 .......................................................................... 83
4.3.4.2 Polymerase Chain Reaction (PCR) Amplification – 454 .................................... 83
4.3.3.3 High Throughput Sequencing - 454 .................................................................... 85
4.3.3.4 Data Analysis - 454 ............................................................................................. 85
4.3.5 Next Generation Sequencing (NGS) - Illumina Analysis .......................................... 85
4.3.5.1 Illumina Analysis Trial Run ................................................................................ 85
4.3.5.2 Honey Samples Analysed – Illumina .................................................................. 85
4.3.5.3 Polymerase Chain Reaction (PCR) Amplification – Illumina ............................ 86
4.3.5.4 High Throughput Sequencing - Illumina ............................................................ 87
4.3.5.5 Data Analysis - Illumina ..................................................................................... 87
4.4 Results ............................................................................................................................... 89
4.4.1 Traditional Pollen Analysis ........................................................................................ 89
4.4.1.1 Pollen Counts ...................................................................................................... 89
4.4.1.2 Melissopalynology .............................................................................................. 89
4.4.2 Next Generation Sequencing - 454 analysis .............................................................. 93
4.4.3 Next Generation Sequencing - Illumina Results ........................................................ 95
4.4.4 Comparison of Melissopalynology, 454 and Illumina ............................................... 97
4.4.4.1 The Taxa Similarity across the Melissopalynology, 454 and Illumina ............... 98
4.4.4.2 Discrimination between Melissopalynology, 454 and Illumina Techniques .... 102
4.4.4.3 Repeat Sampling Using DNA Metabarcoding and Melissopalynology ............ 103
4.5 Discussion ....................................................................................................................... 106
5. CHARACTERISATION OF ANTIBACTERIAL COMPONENTS OF HONEY AND THE
PLANTS WHICH CONTRIBUTE TO ITS PRODUCTION ................................................... 110
5.1 Chapter Introduction ....................................................................................................... 111
5.1.1 Extraction Techniques for Polyphenols ................................................................... 112
5.1.2 Antibacterial Activity Guided Separation Techniques............................................. 113
5.1.3 Advanced Chromatographic and Electrophoretic Analysis of Polyphenols ............ 114
5.2 Chapter Aims and Objectives ......................................................................................... 116
5.3 Methods and materials .................................................................................................... 117
5.3.1 Selection of Honey Samples .................................................................................... 117
5.3.2 Selection of Plant Material ....................................................................................... 117
5.3.3 Bacterial Cultures ..................................................................................................... 117
5.3.4 Organic Extractions.................................................................................................. 118
5.3.5 Phenolic Compounds Extraction .............................................................................. 119
5.3.6 Antimicrobial Activity of Honey and Plant Extracts ............................................... 119
VI
5.3.7 Thin Layer Chromatography Optimisation .............................................................. 120
5.3.8 Bioautographic Method Direct-Variant (Chromatogram Overlay) .......................... 121
5.3.9 TLC/MS Interface Analysis ..................................................................................... 122
5.3.10 Extraction of Compounds from a TLC Plate to Recover Compounds ................... 122
5.3.11 HPLC Analysis of Honey Extractions ................................................................... 123
5.3.11.1 Sample Preparation and Standards .................................................................. 123
5.3.11.2 HPLC Analysis ............................................................................................... 123
5.4 Results ............................................................................................................................. 124
5.4.1 Solvent Extractions .................................................................................................. 124
5.4.1.1 Extraction of Crude Material ............................................................................ 124
5.4.1.2 Extraction Efficacy of Honey and Plant Compounds ....................................... 124
5.4.2 Antibacterial Activity of Honey and Plant Extracts ................................................. 125
5.4.2.1 Antibacterial Analysis of Honey Extract .......................................................... 125
5.4.2.2 Antibacterial Analysis of Plant Extracts ........................................................... 129
5.4.2.3 Comparison of Plant and Honey Antibacterial Activity ................................... 130
5.4.3 Thin Layer Chromatography (TLC) Assay .............................................................. 132
5.4.4 Bioautographic Method (Chromatogram Overlay) and TLC/MS Analysis ............. 134
5.4.4.1 Standardisation of Thymol Positive Control ..................................................... 134
5.4.4.2 Screening Crude Extracts for Antibacterial Activity – Honey .......................... 134
5.4.4.3 Screening Crude Extracts For Antibacterial Activity – Plants .......................... 141
5.4.4.4 Comparing the Antibacterial Activity of the Extracts ....................................... 145
5.4.4.5 Comparison of the Antibacterial Phenolic Compounds Detected in Honey and
Plants ............................................................................................................................. 147
5.4.5 Isolation of Antibacterial Compounds ..................................................................... 149
5.4.6 High Performance Liquid Chromatography (HPLC) ............................................... 150
5.4.6.1 HPLC Analysis ................................................................................................. 150
5.4.6.2 HPLC Analysis –Honey and Plant Extracts ...................................................... 153
5.5 Discussion ....................................................................................................................... 159
6. GENERAL DISCUSSION ................................................................................................... 165
6.1 General Discussion ......................................................................................................... 166
6.2 Limitations and Future Work .......................................................................................... 170
6.3 Concluding Remarks ....................................................................................................... 172
7. REFERENCES ..................................................................................................................... 173
8. APPENDIX ........................................................................................................................... 197

VII
LIST OF FIGURES

Figure 1. 1: Plant-derived anticancer agent Paclitaxel (A) and its analogues Docetaxel (B) ....... 5
Figure 1. 2: Alexander Fleming’s original culture plate ............................................................... 6
Figure 1. 3: The evolution of antibiotic resistance of Methicillin resistant S. aureus (MRSA) .... 8
Figure 1. 4: Structure of selected volatile components found in honey ...................................... 12
Figure 1. 5: Glucose oxidase chemical reaction in honey ........................................................... 14
Figure 1.6: International Honey Commission (IHC) compositional criteria described for
standardised analysis ................................................................................................................... 15
Figure 1. 7: Antibacterial components of honey ......................................................................... 20
Figure 1. 8: Manuka bush from which bees gather nectar to produce antibacterial mono-floral
Manuka honey ............................................................................................................................. 23

Figure 2. 1: MRSA (NCTC 11939) standard curve .................................................................... 36
Figure 2. 2: B. subtilis (ATCC 6633) standard curve ................................................................. 37
Figure 2. 3: B. subtilis (ATCC 39090) standard curve ............................................................... 37
Figure 2. 4: Streak plate method ................................................................................................. 38

Figure 3. 1: Methodologies for the neutralisation of known antibacterial compounds in honey 51
Figure 3. 2: Phenol MRSA (NCTC 11939), B. subtilis (ATCC 6633) and B. subtilis (ATCC
39090) standard curve ................................................................................................................. 53
Figure 3. 3: Thymol MRSA (NCTC 11939) standard curve ...................................................... 54
Figure 3. 4: Effect of 100% sugar solution on the growth of MRSA and B. subtilis .................. 55
Figure 3. 5: The effect of media composition on the antibacterial activity of honey samples against
MRSA (NCTC 11939) ................................................................................................................ 56
Figure 3. 6: The effect of agar composition on the antibacterial activity of H53 (Manuka) against
B. subtilis (ATCC 6633). ............................................................................................................ 58
Figure 3. 7: The effect of agar composition on the antibacterial of honey against B. subtilis (ATCC
39090). ........................................................................................................................................ 59
Figure 3. 8: The sensitivity of MRSA and B. subtilis (ATCC 6633) to hydrogen peroxide ....... 63
Figure 3. 9: Catalase treated hydrogen peroxide at a range of concentrations on an agar diffusion
assay. ........................................................................................................................................... 64

Figure 4. 1: Floral markers for the characterisation of honey. .................................................... 76
Figure 4. 2: Pyrosequencing (454) process overview ................................................................. 78
Figure 4. 3: Total number of pollen grains identified in honey samples .................................... 89
Figure 4. 4: SEM images of H24 ................................................................................................ 91
Figure 4. 5: Summary of plant taxa detected through pollen analysis in eleven honey samples. 92
Figure 4. 6: Summary of plant taxa detected through 454 analysis in ten honey samples.......... 94
Figure 4. 7: Summary of plant taxa detected through Illumina analysis in ten honey samples. . 97
Figure 4.8: Summary of plant taxa detected through melissopalynology (M), 454 (R) and Illumina
(I) analysis in nine honey samples .............................................................................................. 99
Figure 4. 9: List of taxa detected for replicate analyses of honey sample H24 and H25 using DNA
metabarcoding and melissopalynology. .................................................................................... 105

Figure 5. 1: Flavonoid and phenolic acids commonly found in honey ..................................... 111
Figure 5. 2: Elution head-based HPTLC-MS: TLC-MS Interface (CAMAG 2009) and solvent
pathway in the elution head ...................................................................................................... 115
VIII
Figure 5. 3 Comparison of the total yield (mg) of honey and plant extracts following organic
solvent extraction ...................................................................................................................... 125
Figure 5. 4: MIC in a 96 well plate against MRSA .................................................................. 127
Figure 5. 5: MIC in a 96 well plate against E.coli .................................................................... 128
Figure 5. 6: MIC in a 96 well plate against P. aeruginosa ....................................................... 128
Figure 5. 7: Visualisation of TLC plate of a honey extraction .................................................. 133
Figure 5. 8: The visualisation of positive control ..................................................................... 133
Figure 5. 9: The overlay assay of MRSA and thymol visualised using 2 mg/ml INT chloride. 134
Figure 5. 10: Overlay assay of honey extracts against MRSA. ................................................ 135
Figure 5. 11: Overlay assay of honey extracts against E. coli. ................................................. 140
Figure 5. 12: Overlay assay of honey extracts against P. aeruginosa. .................................... 141
Figure 5. 13: Overlay assay of plant extracts against MRSA. .................................................. 142
Figure 5. 14: Summary of the number of zones of clearing detected for the honey and plant
extracts on the overlay assay ..................................................................................................... 146
Figure 5. 15: Mass spectrum of compounds extracted from E17 and E05/E06 after separation.
.................................................................................................................................................. 149
Figure 5. 16: HPLC-MS chromatogram of standard solutions, the best peak obtained from either
positive of negative mode was included: continues overleaf .................................................... 152
Figure 5. 17: Identification of Naringenin using HPLC in honey and plant extracts (negative
mode) ........................................................................................................................................ 154
Figure 5. 18: Identification of Kaempferol using HPLC in honey and plant extracts (negative
mode) ........................................................................................................................................ 155
Figure 5. 19: Identification of Chrysin using HPLC in honey and plant extracts (negative mode)
.................................................................................................................................................. 156
Figure 5. 20: Chemical structure of pinobanksin (m/z329) and the fragmentation pattern of the
two pinobanksin derivatives attributed to an m/z of 328. ......................................................... 163

IX