development of a rapid and robust method to measure antioxidant activity in a lipid system

224 Pages·2016·2.47 MB·English

DEVELOPMENT OF A RAPID AND

ROBUST METHOD TO MEASURE

ANTIOXIDANT ACTIVITY IN A LIPID

SYSTEM

MD AHSAN GHANI
B. Sc. (Chem.), M. Sc. (Chem.)

The thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy

School of Agricultural and Wine Sciences
Charles Sturt University
Australia

March 2016

© Md Ahsan Ghani, 2016
TABLE OF CONTENTS

TABLE OF CONTENTS ......................................................................................................... ii
CERTIFICATE OF AUTHORSHIP ...................................................................................... vii
EDITORIAL NOTE .............................................................................................................. viii
DEDICATION ........................................................................................................................ ix
ACKNOWLEGDEMENTS ..................................................................................................... x
LIST OF PUBLICATIONS/MANUSCRIPTS ARISING FROM THIS THESIS ................ xii
LIST OF TABLES .................................................................................................................xiv
LIST OF FIGURES ............................................................................................................... xv
GLOSSARY AND ABBREVIATIONS ............................................................................... xvii
ABSTRACT ........................................................................................................................... xix
CHAPTER 1. LITERATURE REVIEW ................................................................................. 1
1.1 Introduction: ................................................................................................................... 1
1.2 Lipids ............................................................................................................................. 2
1.3 Lipid oxidation ............................................................................................................... 2
1.4 Oxidisable lipid substrates ............................................................................................. 4
Cell membrane ..................................................................................................................... 4
Oxidation of food lipids ....................................................................................................... 4
1.5 Mechanism of lipid oxidation ........................................................................................ 5
1.6 Lipid oxidation products and toxicity ............................................................................ 7
1.7 Lipid oxidation measurements ..................................................................................... 12
1.7.1 Ferric thiocyanate (FTC) assay ............................................................................. 12
1.7.2 Thiobarbituric acid reactive substances (TBARS) Assay ..................................... 15
1.8 Importance in inhibiting lipid oxidation ...................................................................... 18
1.9 Antioxidants and their activity ..................................................................................... 19
1.10 Roles of plant extracts as natural antioxidants ........................................................... 23
1.11 Australian traditional medicinal plants as antioxidants ............................................. 26
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1.12 Assays for screening antioxidant activity: ................................................................. 32
1.12.1 Ferric thiocyanate (FTC) assay to measure antioxidant activity in lipid systems
....................................................................................................................................... 36
1.12.2 Thiobarbituric acid reactive substances (TBARS) assay to measure antioxidant
activity in lipid systems.................................................................................................. 40
1.13 Current applications of Thiobarbituric acid reactive substances (TBARS) and Ferric
thiocyanate (FTC) assays in antioxidant screening studies ................................................ 44
1.14 Summary of literature review .................................................................................... 51
1.15 Research Question and Objectives ............................................................................. 53
CHAPTER 2. SUBSTRATE AND TBARS VARIABILITY IN A MULTI-PHASE
OXIDATION SYSTEM ........................................................................................................ 55
2.1 Introduction .................................................................................................................. 55
2.2 Materials and methods ................................................................................................. 56
2.2.1 Chemicals and reagents ........................................................................................ 56
2.2.2 Experimental design .............................................................................................. 57
2.2.2.1 Preparation of lipid substrate ......................................................................... 57
2.2.2.2 Measurement of thiobarbituric acid reactive substances (TBARS) ............... 58
2.2.2.3 Measurement of peroxides ............................................................................. 59
2.2.2.4 Removal of pre-formed peroxides and thiobarbituric acid reactive substances
(TBARS) .................................................................................................................... 59
2.3 Statistical analysis ........................................................................................................ 61
2.4 Results and discussion ................................................................................................. 61
2.4.1 Inter- and intra- batch variability of Linoleic acid (LA) in the thiobarbituric acid
reactive substances (TBARS) assay: .............................................................................. 61
2.4.2 Sources of variability: ........................................................................................... 63
2.4.2.1 Effect of order of addition of reagents on variability: .................................... 63
2.4.2.2 Effect of batch to batch Linoleic acid (LA) variability: ................................. 66
2.4.2.3 Effect of different concentrations of Trolox: ................................................. 69
2.4.2.4 Method development to remove pre-formed peroxides ................................. 72
2.4.2.5 Outcomes of oxidation and antioxidant activity using treated Linoleic acid
(LA)............................................................................................................................ 75
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2.5 Conclusions .................................................................................................................. 77
CHAPTER 3. DEVELOPMENT OF A RAPID AND ROBUST METHOD TO MEASURE
ANTIOXIDANT ACTIVITY IN A LINOLEIC ACID EMULSION ................................... 79
3.1 Introduction .................................................................................................................. 79
3.2 Materials and methods ................................................................................................. 81
3.2.1 Chemicals and reagents ........................................................................................ 81
3.2.2 Experimental design .............................................................................................. 82
3.2.2.1 Preparation of solutions ................................................................................. 83
3.2.2.2 Oxidation ........................................................................................................ 86
3.2.2.3 Measurement of peroxides ............................................................................. 87
3.2.2.4 Measurement of thiobarbituric acid reactive substances (TBARS) ............... 88
3.3 Statistical analysis ........................................................................................................ 88
3.4 Results and Discussion ................................................................................................ 89
3.4.1 Effect of Linoleic acid (LA) nano- emulsion ......................................................... 89
3.4.2 Method optimisation ........................................................................................... 94
3.4.3 Oxidation stage at 37 °C ....................................................................................... 95
3.4.3.1 Optimising the concentration of antioxidant .................................................. 95
3.4.4 Oxidation at accelerated conditions ..................................................................... 99
3.4.4.1 Effect of temperature ..................................................................................... 99
3.4.4.2 Effect of catalysts ......................................................................................... 102
3.4.4.3 Effect of oxygen initiator ............................................................................. 106
3.5 Method performance .................................................................................................. 108
3.6 Conclusions .................................................................................................................... 110
CHAPTER 4. SCREENING ANTIOXIDANT ACTIVITY OF SELECTED AUSTRALIAN
NATIVE PLANTS AND IDENTIFICATION OF ACTIVE COMPOUNDS. ................... 112
4.1 Introduction ................................................................................................................ 112
4.2 Materials and methods ............................................................................................... 115
4.2.1 Chemicals and Reagents ..................................................................................... 115
4.2.2 Plant species and materials ................................................................................ 115
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4.2.3 Preparation and fractionation of plant extracts ................................................. 116
4.2.4 Determination of total phenolic contents ............................................................ 119
4.2.5 Free radical scavenging 2,2’-azinobis (3-ethyl-benzothiazolline-6-sulfonic acid)
(ABTS) assay ................................................................................................................ 119
4.2.6 Developed lipid- based ferric thiocyanate (FTC) and thiobarbituric acid reactive
substances (TBARS) assay ........................................................................................... 120
4.2.6.1 Linoleic acid (LA) nano-emulsion and oxidation ........................................ 120
4.2.6.2 Ferric thiocyanate (FTC) assay .................................................................... 121
4.2.6.3 Thiobarbituric acid reactive substances (TBARS) assay ............................. 121
4.2.7 Ultra High Pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)
analysis ........................................................................................................................ 122
4.2.7.1 Crude extracts and fractions sample preparation: ........................................ 122
4.2.7.2 Ultra High Pressure Liquid Chromatography-quadrupole Time of Flight-Mass
Spectrometry (UHPLC-qTOF-MS) analysis............................................................ 122
4.2.7.3 Quality control ............................................................................................. 124
4.3 Statistical analysis ...................................................................................................... 124
4.4 Results and discussion ............................................................................................... 125
4.4.1 Extraction yield ................................................................................................... 125
4.4.2 Determination of total phenolic contents (TPC) ................................................. 126
4.4.3 Screening antioxidant activity ............................................................................. 129
4.4.3.1 2,2’-azinobis (3-ethyl-benzothiazolline-6-sulfonic acid) (ABTS) assay ..... 129
4.4.3.2 Ferric thiocyanate (FTC) assay .................................................................... 132
4.4.3.3 Thiobarbituric acid reactive substances (TBARS) assay ............................. 137
4.4.4 Identification of compounds ................................................................................ 141
4.4.4.1 Acacia implexa bark..................................................................................... 142
4.4.4.2 Acacia implexa leaves .................................................................................. 145
4.4.4.3 Eucalyptus rossii leaves ............................................................................... 147
4.4.4.4 Exocarpos cupressiformis leaves ................................................................. 150
4.5 Conclusions ................................................................................................................ 153
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CHAPTER 5. CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE
RESEARCH ......................................................................................................................... 155
5.1 Conclusions ................................................................................................................ 155
5.1.1 Issues of variability in thiobarbituric acid reactive substances (TBARS) with
multi-phase system ....................................................................................................... 155
5.1.2 Oxidation in linoleic acid (LA) emulsion for thiobarbituric acid reactive
substances (TBARS) or ferric thiocyanate (FTC) (peroxides) assays ......................... 157
5.1.3 Challenges in applying accelerated conditions ................................................... 158
5.1.4 Applicability of method to traditional medicinal plant as real samples .............. 160
5.1.5 Tentative identification of active compounds in traditional medicinal plants .... 161
5.1.6 Research Conclusions ......................................................................................... 162
5.2 Recommendations for Further Research .................................................................... 164
5.2.1 In-depth study of mechanisms of oxidation and antioxidant for thiobarbituric acid
reactive substances (TBARS) and peroxides ............................................................... 164
5.2.2 Specific identification of compounds of Acacia implexa, Eucalyptus rossii,
Exocarpos cupressiformis as antioxidant. .................................................................... 165
5.2.3 Hyphenation of analytical techniques in thiobarbituric acid reactive substances
(TBARS) and ferric thiocyanate (FTC) (peroxides) assays ......................................... 165
5.2.3.1 Flow Injection .............................................................................................. 166
5.2.3.2 Post Column thiobarbituric reactive substances (TBARS)/ ferric thiocyanate
(FTC) (peroxides) assays ......................................................................................... 166
REFERENCES .................................................................................................................... 168
APPENDIX 1 ....................................................................................................................... 193
APPENDIX 2 ....................................................................................................................... 194
APPENDIX 3 ....................................................................................................................... 195
APPENDIX 4 ....................................................................................................................... 196
APPENDIX 5 ....................................................................................................................... 197
APPENDIX 6 ....................................................................................................................... 198
APPENDIX 7 ....................................................................................................................... 199
APPENDIX 8 ....................................................................................................................... 200
APPENDIX 9 ....................................................................................................................... 201
APPENDIX 10 ..................................................................................................................... 202
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CERTIFICATE OF AUTHORSHIP

I, Md Ahsan Ghani, 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, nor material which to a substantial extent has been
accepted for the award of any other degree or diploma at Charles Sturt University or
any other educational institution, except where due acknowledgment is made in the
thesis. Any contribution made to the research by colleagues with whom I have
worked at Charles Sturt University or elsewhere during my candidature is fully
acknowledged.
I agree that this thesis be accessible for the purpose of study and research in
accordance with the normal conditions established by the Executive Director,
Library Services, Charles Sturt University or nominee, for the care, loan and
reproduction of thesis, subject to confidentiality provisions as approved by the
University.

Name: Md Ahsan Ghani

…………………………… …………………
Signature Date
vii
EDITORIAL NOTE

This thesis has been formatted in accordance with Charles Sturt University’s
academic manual available at the link provided https://policy. csu. edu. au/view.
current. php?id=00309 (section 4: Regulations for the presentation of print theses,
other examinable print works and the written component of examinable multi-media
work). This thesis is written in Australian English.
The published work cited in this thesis was referenced according to the
American Psychological Association (APA) referencing style (6th edition) as
recommended by the Faculty of Science, School of Agricultural and Wine Sciences
which was in line with Charles Sturt University’s referencing style. The APA 6th
edition is available online at http://www. csu. edu.
au/division/library/ereserve/pdf/apa-6ed. pdf.

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DEDICATION

To my parents, wife and son,
Without you all this long journey would not have been possible, love you all so much
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ACKNOWLEGDEMENTS

This project was funded by the Faculty of Science (Compact) scholarship, Charles
Sturt University, Wagga Wagga, NSW, Australia. The project is aligned with the
healthy food initiative of Charles Sturt University and Graham Centre for
Agricultural Innovation. Conference support grant was obtained from Graham
Centre for Agricultural Innovation.

In the first place I would like to express my utmost gratitude to my supervisors,
Associate Professor Paul D Prenzler, Dr Celia Barril, and Dr Danny R Bedgood for
their ongoing supervision, valuable advice, and precise guidance from the very early
stages of this research. I gratefully acknowledge my principal supervisor Associate
Professor Paul D Prenzler for his critical and in-depth thinking about this project,
which provided a backbone for this research and so this thesis. This project would
have not been finished without his tireless contributions throughout the course of
study. Dr Celia’s intelligent guide enhanced the chemistry of this thesis. I was
always benefited and encouraged by Dr Dan’s valuable guide and advice throughout
the research. I acknowledge all my supervisors for continuous assistance in
answering my unintelligent questions about experimental and theoretical study. I
would like to thank them for their precious time to review this thesis and their
constructive comments. Their tireless comments helped me to enhance scientific
inputs in this thesis.
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