Anti-inflammatory and Antibacterial Effects of Kinos from Australian and Papua New Guinean ...

178 Pages·2016·2.81 MB·English

by  City East Library

School of Pharmacy

Anti-inflammatory and Antibacterial Effects of Kinos from
Australian and Papua New Guinean Eucalyptus Species

Martha Yahimbu Mungkaje

This thesis is presented for the Degree of
Doctor of Philosophy
of
Curtin University

June 2015
DECLARATION

To the best of my knowledge and belief this thesis contains no material previously
published by any other person except where due acknowledgement has been made.
This thesis contains no material which has been accepted for the award of any other
degree or diploma in any university.

………………………………………………….
June 16, 2015
ii
ACKNOWLEDGEMENTS

I would like to thank all the people who contributed to this Thesis in every way. First
and foremost, I would like to thank my two supervisors, Dr Ricky Lareu and Dr Rima
Caccetta for demanding a high quality of work in all my aspects of my PhD program
and supporting my attendance at conferences. I would like to thank Dr Andrew S.
McWilliam for his initial contributions towards my research. I would also like to thank
Dr Simon Fox for his contributions for ensuring all reagents for my experiments were
in stock at all times. Dr Connie Jackaman is thanked for her patience in training me
on the use of flow cytometer. Ms Irma Larma-Cornwall assisted me with the analysis
of the flow cytometry data for the phagocytosis assay. Dr Chi Ndi of University of
South Australia is thanked for his assistance with the phytochemical screening of
kinos.

I would like to thank my fellow PhD colleagues, Mr. Wolfgang Wimmer and Mr. Alex
Richards for always working together as a team solving problems related to cell
culture, immunology and molecular biology. Ms. Angie Samec and Mr. Giuseppe
Luna assisted me with the Microbiology aspects of my research and sub-fractionation
of kinos work, respectively, in this thesis. I would like to thank Ms. Gaewyn Ellison for
going out of her way from her busy schedule for her experiments for her PhD program
to proof-read different chapters of my thesis. Ms. Sangeetha Mathavan, a PhD
colleague and a very close friend is thanked for always working together to solve
problems for each other‘s research and morally supporting each other.

Without a sponsorship, I would not have studied at Curtin University and finally
produce this thesis. I am very grateful to the Australian Government for giving me the
Australian Leadership Award Scholarship that funded my tuition and living allowances
during my 4 years PhD tenure. I would also like to thank the School of Pharmacy for
giving me a fee waiver for 3 months when my 4 years sponsorship ended.

Finally, I would like to acknowledge family and friends who supported me during my
study. First and foremost, I would like to thank my husband and my two daughters,
iii
my mother, sisters, nephews and nieces for their constant love and support
throughout this long academic journey. I would also like to thank Lydia Warubi, Jossie
Bakri and Ericka Dawanincura for their friendship and support throughout my study
period in Perth, Western Australia.

I would like to dedicate this thesis to my late father, my mother who lived up to see
the final end-product of this thesis despite having arthritis and my two daughters,
Grace Boker Mungkaje and Catherine Autamin Mungkaje.

iv
LIST OF ABBREVIATIONS AND ACRONYMS

Abbreviation/ Full name
Acronym

APH Acid phosphatase
BSA Bovine serum albumin
CLSI Clinical and Laboratory Standards Institute
DCM Dichloromethane
E Escherichia or Eucalyptus
E. coli Escherichia coli
ELISA Enzyme-linked immunosorbent assay
EOs Essential oils
E. calophylla Eucalyptus calophylla
E. confertiflora Eucalyptus confertiflora
ERK Extracellular-signal regulated kinase
FACS Fluorescence-activated cell sorting
FBS Foetal calf serum
AF1 Acidic fraction 1
BF2 Basic fraction 2
DCMF3 Dichloromethane fraction 3
h Hour(s)
IкB Inhibitory kappa beta
IL Interleukin
JAK Janus kinase
JNK C-Jun N-terminal kinase
LPS Lipopolysaccharide
LRP Leukocyte common antigen-related phosphatase
MAPK Mitogen-activated protein kinase
MBC Minimum bactericidal concentration
MHA Mueller-Hinton agar
MHB Mueller-Hinton broth
v
LIST OF ABBREVIATIONS AND ACRONYMS CONTINUED
Abbreviation/ Full name
Acronym

MIC Minimum inhibitory concentration
Min Minute(s)
μg/mL Microgram per millilitre
mg/mL Milligram per millilitre
μL Microlitre
mL Millilitre
NCCLS National Committee for Clinical and Laboratory
Standards
NF-кB Nuclear factor-kappa beta
NO Nitric oxide
ONOO- Peroxynitrite
P. aeruginosa Pseudomonas aeruginosa
pg/mL Picograms per millilitre
PGN Peptidoglycan
PNG Papua New Guinea(n)
RA Rheumatoid arthritis
RPMI Roswell Park Memorial Institute
ROS Reactive oxygen species
S. aureus Staphylococcus aureus
TE Total kinos extract
TLR Toll-like receptors
TMB 3,5,3',5'-tetramethylbenzidine
TNF-α Tumour necrosis factor alpha
USFDA United States Food and Drug Administration
WA Western Australia(n)
vi
ABSTRACT
Kinos are astringent exudates from Eucalyptus species which are used by the first
Australians to treat inflammatory conditions and bacterial infections. Tannins are the
predominant chemical constituents in kinos as identified by the early investigations of
kinos chemistry. The tannins and cathechins (constitutive of flavonoids), major groups
of compounds are found in plants. Flavonoids are components of nutrition but have
also been extensively investigated for their antimicrobial activities using in vitro and in
vivo study models. However, limited antimicrobial studies have been conducted on
kinos from Eucalyptus species and therefore, this study sets out to validate claims for
their medicinal uses. The main goals were to investigate anti-inflammatory and
antibacterial effects of kinos from Western Australian (WA) and Papua New Guinean
(PNG) Eucalyptus species using in vitro study models.
Kinos from Eucalyptus calophylla (WA) and Eucalyptus confertiflora (PNG) were
solubilized in a water-based buffer. Murine RAW264.7 macrophages were used for
anti-inflammatory and phagocytosis studies. Gram negative and Gram positive
bacteria were tested for their susceptibility to kinos. The level of cytokine secretion by
the kinos treated LPS-stimulated macrophages were measured by ELISA, whilst nitric
oxide (NO) levels were measured by the Griess method. Phagocytosis was evaluated
by fluorescent flow cytometry and antibacterial effects were determined by agar
diffusion and microdilution methods. Phytochemical analyses of kinos were
conducted using thin layer chromatography and qualitative tests.
The RAW264.7 macrophage cells developed vacuoles after being exposed to kinos.
The intensity of the vacuoles increased with increasing concentrations of kinos and
incubation times. The vacuoles were more pronounced in cells exposed to WA kinos
than to the cells exposed to the PNG kinos. The cells were 100% viable for the kinos
concentrations lower than 250 µg/mL and they were 80% viable at this concentration.
The cell viability decreased in a dose-dependent manner. Secretions of interleukin
(IL)-6 and NO were significantly reduced by the kinos in a concentration dependent
manner. In contrast, the secretion of tumour necrosis factor alpha was enhanced by
the kinos whilst IL-10 was also enhanced by WA kinos but it was reduced by the PNG
vii
kinos. The phagocytosis of Gram positive and Gram negative bacteria by
macrophages was enhanced by the kinos. Gram positive bacteria were inhibited by
the kinos with a minimum inhibitory concentration of 1 mg/mL and a minimum
bactericidal concentration of 3 mg/mL. Gram negative bacterial growth was not
affected by kinos.
Phenolics (flavonoids), tannin, and triterpenoid saponins are predominant in the kinos
as evaluated by the phytochemical qualitative (flavonoid and triterpenoids saponin
tests), quantitative (assays of total phenolics and total tannins) and spot tests (kinos
spotted on thin layer chromatography plate). Biological activity was lost for WA kinos
by fractionation whilst some fractions of PNG kinos retained activity. This suggests
that the biological effects of kino on macrophage activity and potentially on growth
and viability of Gram positive bacteria may be multifactorial and possibly synergistic in
nature.
Through these studies we demonstrate that kinos predominantly induce an anti-
inflammatory profile in mouse macrophages and possess antibacterial properties
against Gram positive bacteria. Although limited to the in vitro context we provide
evidence substantiating some of the claims by traditional medicine.

viii
TABLE OF CONTENTS
Anti-inflammatory and Antibacterial Effects of Kinos from Australian and
Papua New Guinean Eucalyptus Species i

DECLARATION ii

ACKNOWLEDGEMENTS iii

LIST OF ABBREVIATIONS AND ACRONYMS v
LIST OF ABBREVIATIONS AND ACRONYMS CONTINUED vi

ABSTRACT vii

TABLE OF CONTENTS ix

CHAPTER 1 1
GENERAL INTRODUCTION 1
1.1. Introduction 2
1.2. Natural products 3
1.3. Link between folk medicine and natural products 5
1.4. First Australian traditional medicine 7
Table 1.1 10
1.4.1. Chemistry of kinos and links between their chemical constituents
and antimicrobial activity 11
1.5. Antimicrobial effects of Eucalyptus species 13
ix
Table 1.2 18
1.6. General methods used in drug discovery research 20
1.7. Innate and adaptive immune systems 22
1.7.1. Innate Immune system 22
Figure 1.1 24
1.7.2. Adaptive Immune system 25
1.7.3. Inflammation and cytokines as the mediators 26
1.8. Pathogenesis of bacteria 29
1.8.1. Factors that enhance pathogenesis of bacteria 30
1.9. Perspective and objectives of this study 34

CHAPTER 2 35
MATERIALS AND METHODS 35
2.1. Introduction 36
2.2. Kinos collection and preparation 37
2.3. Materials 38
Table 2.1 38
Figure 2.1 40
2.4. Cells, cell culture and incubation conditions 41
2.5. Morphology of RAW264.7 cells 41
2.6. Cell viability 41
2.6.1. Acid phosphatase assay 41
2.7. Measurement of cytokines and nitric oxide as mediators of inflammation 42
2.7.1. Tumour necrosis factor alpha (TNF-α) ELISA 42
2.7.2. Interleukin-6 (IL-6) and IL-10 ELISAs 43
2.7.3. Nitric oxide assay 43
x