Phytochemical study and anticancer potential of high antioxidant Australian native plants

452 Pages·2016·7.28 MB·English

by  Joseph P Sirdaarta

Griffith University

Phytochemical study and anticancer potential of high

antioxidant Australian native plants
Principal supervisor: Dr Ian Edwin Cock
Co-supervisor: Dr Sarah Ashmore

By
Joseph P Sirdaarta
B. Food Science and Nutrition (Hons)

School of Natural Sciences
Griffith University

Submitted in fulfilment of the requirements of the degree of
Doctor of Philosophy
On the April 11th 2016

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Statement of Originality

This work, entitled Phytochemical study and anticancer potential of high antioxidant
Australian native plants, has not previously been submitted for a degree in any university.
To the best of my knowledge and belief, the thesis contains no material previously published
or written by another person except where due reference is made in the thesis itself.

Joseph P Sirdaarta
April 11th, 2016

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Acknowledgements
I would like to take this opportunity to thank the Lord Jesus Christ for giving me this
opportunity to pursue my PhD. He has all my gratitude for his provisions and support through
trying times.
Furthermore, I would like to thank Griffith University for offering me the opportunity
through scholarships to complete the program. I would like to thank my supervisors Dr Ian
Cock and Dr Sarah Ashmore for their help and guidance.
Specially, I would like to thank Dr Ian Cock for his invaluable help, mentoring, guidance and
belief in me even during the most trying of times. I would say that without him, this PhD may
not have been possible to accomplish.
I would like to thank Ben Matthews from Smart waters (GC) for his guidance with the QTOF
analysis. Also, I would like to thank Alan White for the use of his GC-MS head space
equipment and his technical expertise during the analysis of results.
I would like to thank Dr Giovanna (Jenny) Di Trapani and Professor Franck Clarke for the
gift of JEG-3/JAR cells and to A/Professor Ann McDonnell for the gift of MG-63 and
MC3T3-E1 cells. I would like to thank Dr Paran Rayan for her initial help in getting me
started with the cell lines. I would like to thank Raja Vadivelu for his help with cell-imaging
and the subsequent analysis. I am grateful to Tak Kim and A/Professor Qin Li for their
collaboration with our group. Their financial help was timely during our crisis.
I would like to thank my parents and my brother John for their financial assistance and
support during this period of study.

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Publications Arising From This Thesis

1. Sirdaarta, J., Matthews B., I.E. Cock. : Kakadu plum fruit extracts inhibit growth of the
bacterial triggers of rheumatoid arthritis: Identification of stilbene and tannin components.
Journal of Functional Foods. 06/2015; 17:610-620. doi:10.1016/j.jff.2015.06.019.

2. Sirdaarta, J., Matthews, B., White, A., Cock, I.E.: The potential of selected Australian
medicinal plants with anti Proteus activity for the treatment and prevention of rheumatoid
arthritis. Pharmacognosy Magazine 05/2015; 11(42 (Supp 1)):S190-S208. doi:10.4103/0973-
1296.157734.

3. Sirdaarta, J., Cock, I.E.: The anti-proliferative properties of Australian plants with high
antioxidant capacities against cancer cell lines. Pharmacognosy Communications 10/2014;
4(4):71-82. doi10.5530/pc.2014.4.8.

4. Sirdaarta, J., McDonnell, A.M., Rayan, P., Matthews, B., Cock, I.E.: High performance liquid
chromatography-mass spectrometry analysis of high antioxidant Australian fruits with
antiproliferative activity against cancer cells. Pharmacognosy Magazine 01/2016; doi:
10.4103/0973-1296.179678.

5. Winnett, V., Boyer, H., Sirdaarta, J., Cock. I.E.: The potential of Tasmannia lanceolata as a
natural preservative and medicinal agent: Antimicrobial activity and toxicity. Pharmacognosy
Communications 01/2014; 4(1):42-52. doi:10.5530/pc.2014.1.7.

Conference Proceedings

1. Sirdaarta, J. Cock, I.E.: Antioxidant and anticancer activities of Terminalia ferdinandiana
(Kakadu Plum). 3rd International Symposium on Medicinal Plants (ISMP), Petra, Jordan;
01/2012.

2. Sirdaarta J., Cock, I.E.: Anticancer activity and phytochemical profiling of Terminalia
ferdinandiana (Kakadu Plum) fruit extracts. The 15th International Congress of The
International Society for Ethnopharmacology, Petra Jordan; 05/2015

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Table of Contents
ABSTRACT ............................................................................................................................................ 18
1.1 Natural Therapeutics: Historical background ........................................................................... 22
1.2 Plants and phytochemicals of therapeutic significance ............................................................. 23
1.3 Carcinogenesis .......................................................................................................................... 28
1.3.1 Cancer and its relationship to oxidative stress ...................................................................... 31
1.3.2 Cellular antioxidant systems and the relation to redox stress ........................................... 35
1.3.2.1 Glutathione system ........................................................................................................ 35
1.3.2.2 Other protein sulfhydryls .............................................................................................. 36
1.3.2.3 Thiols and disulphides .................................................................................................. 37
1.3.2.4 Thioredoxin system ....................................................................................................... 40
1.3.3 Cell processes governed by cellular redox state ............................................................... 41
1.3.3.1 Proliferation and differentiation .................................................................................... 41
1.3.3.2 Death ............................................................................................................................. 42
1.3.3.3 Regulated cell death (apoptosis) ................................................................................... 42
1.4 Therapeutical intervention in cancer: current cancer targets and chemotherapies .................... 45
1.4.1 Polyphenols as antioxidants .............................................................................................. 48
1.5 Current research into plants as a therapeutic agents ................................................................. 50
1.5.1 Neglect of Australian flora and medical significance ....................................................... 51
1.5.2 Genus Terminalia .............................................................................................................. 53
1.5.2.1 Australia Terminalia species ......................................................................................... 54

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1.5.2.2 Phytochemistry and anti-oxidant capacity of genus Terminalia ................................... 56
1.5.2.3 T. ferdinandiana phytochemistry and antioxidant properties ....................................... 62
1.5.3 Genus Tasmannia family Winteraceae ............................................................................. 65
1.5.3.1. Tasmannia lanceolata ................................................................................................... 66
1.5.4 Genus Syzygium ................................................................................................................ 70
1.5.4.1 Australian species: Syzygium australe (brush cherry) and Syzygium leuhmannii
(riberry) 72
1.5.5 Genus Davidsonia ............................................................................................................. 72
1.5.6 Genus Elaeocarpus ........................................................................................................... 73
1.5.7 Genus Kunzea ................................................................................................................... 74
1.5.8 Genus Podocarpus ............................................................................................................ 75
1.5.9 Genus Acronychia ............................................................................................................. 76
1.6 Aims .......................................................................................................................................... 77
CHAPTER 2: MATERIALS AND METHODS ........................................................................................... 79
2.1 Plant material ............................................................................................................................ 79
2.2 Solvent extraction ..................................................................................................................... 79
2.3 Qualitative phytochemical studies ............................................................................................ 80
2.3.1 Alkaloids ........................................................................................................................... 80
2.3.2 Saponins ............................................................................................................................ 81
2.3.3 Tannins .............................................................................................................................. 82
2.3.4 Phenolic compounds ......................................................................................................... 82
2.3.5 Terpenoids ......................................................................................................................... 82
2.3.6 Cardiac glycosides ............................................................................................................ 83

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2.3.7 Anthraquinones ................................................................................................................. 83
2.3.8 Flavonoids ......................................................................................................................... 84
2.3.9 Phytosteroids ..................................................................................................................... 84
2.4 Toxicity screening ..................................................................................................................... 85
2.4.1 Reference toxins for toxicity assay ................................................................................... 85
2.4.2 Evaluation of toxicity ........................................................................................................ 85
2.5 Antioxidative Assays ................................................................................................................ 87
2.5.1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay ................................... 87
2.6 Cell proliferation assays ............................................................................................................ 88
2.6.1 Cell lines ........................................................................................................................... 88
2.6.2 Culture and passaging of cell lines.................................................................................... 89
2.6.3 Cryopreservation and cryoretrieval of cell lines ............................................................... 90
2.6.4 MTS based assays for cell proliferation ............................................................................ 91
2.7 Statistics .................................................................................................................................... 92
2.8 Metabolomics profiling studies ................................................................................................. 92
2.8.1 Quadrupole time of flight mass spectrometry (QTOF MS/MS) ....................................... 92
2.8.2 Non-Targeted GC-MS Head Space Analysis .................................................................... 94
2.9 Cell Imaging Studies ................................................................................................................. 95
CHAPTER 3: SCREENING HIGH ANTIOXIDANT AUSTRALIAN PLANTS FOR ANTIPROLIFERATIVE
ACTIVITY .............................................................................................................................................. 96
3.1 Extraction yields and qualitative phytochemical studies .......................................................... 97
3.1.1 Extraction yields ............................................................................................................... 97

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3.1.2 Syzygium species (Syzygium leuhmannii, Syzygium australe) (fruit and leaf)
phytochemical analysis ..................................................................................................................... 99
3.1.3 Davidsonia pruriens (fruit and leaf) phytochemical analysis ......................................... 101
3.1.4 Elaeocarpus angustifolius (fruit) phytochemical analysis .............................................. 102
3.1.5 Kunzea pomifera (muntries) fruit phytochemical analysis ............................................. 102
3.1.6 Podocarpus elatus fruit phytochemical analysis ............................................................. 103
3.1.7 Acronychia acidula fruit phytochemical analysis ........................................................... 103
3.1.8 Terminalia ferdinandiana (fruit and leaf) phytochemical analysis ................................. 104
3.1.9 Tasmannia lanceolata (fruit and leaf) phytochemical analysis ...................................... 105
3.2 DPPH assays for the determination of antioxidant content..................................................... 113
3.3 Antiproliferative MTS assays of Australian plant extracts against cancer cell lines. ............. 114
3.3.1 Antiproliferative potential of Syzygium species extracts................................................. 114
3.3.2 Antiproliferative potential of Davidsonia pruriens extracts. .......................................... 120
3.3.3 Antiproliferative potential of Elaeocarpus angustifolius extracts. ................................. 123
3.3.4 Antiproliferative potential of Kunzea pomifera extracts. ................................................ 124
3.3.5 Antiproliferative potential of Podocarpus elatus extracts. ............................................. 128
3.3.6 Antiproliferative potential of Acronychia acidula extracts. ............................................ 129
3.3.7 Antiproliferative potential of Terminalia ferdinandiana extracts. .................................. 132
3.3.8 Antiproliferative potential of Tasmannia lanceolata extracts. ........................................ 135
3.3.9 Quantification of antiproliferative efficacy by IC determination ................................. 140
50
3.4 A. franciscana nauplii toxicity assay ...................................................................................... 146
3.4.1 Toxicity of Syzygium species extracts. ............................................................................ 146
3.4.2 Toxicity of Davidsonia pruriens extracts ....................................................................... 151

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3.4.3 Toxicity of Elaeocarpus angustifolius extracts ............................................................... 153
3.4.4 Toxicity of Kunzea pomifera extracts ............................................................................. 155
3.4.5 Toxicity of Podocarpus elatus extracts........................................................................... 157
3.4.6 Toxicity of Acronychia acidula extracts ......................................................................... 159
3.4.7 Toxicity of Terminalia ferdinandiana extracts ............................................................... 160
3.4.8 Toxicity of Tasmannia lanceolata extracts ..................................................................... 162
3.5 Discussion ............................................................................................................................... 165
CHAPTER 4: CELL LINE STUDIES – EXTENDED AND DIAGNOSTIC SCREENING OF SELECTED HIGH
ANTIOXIDANT PLANTS. ...................................................................................................................... 170
4.1 Extended cell line studies: Testing of T. ferdinandiana and T. lanceolata extracts against
diagnostic cell lines. ............................................................................................................................ 171
4.1.1 Antiproliferative potential of T. ferdinandiana extracts against JEG-3 and JAR cells ... 173
4.1.2 Antiproliferative potential of T. ferdinandiana extracts against MC3T3-E1 and MG-63
cells 177
4.1.3 Antiproliferative potential of T. lanceolata extracts against JEG-3 and JAR. ................ 182
4.1.4 Antiproliferative potential of T. lanceolata extracts against MC3T3-E1 and MG-63 cells
187
4.1.5 Quantification of antiproliferative efficacy by IC determination ................................. 192
50
4.2 Cell Imaging Studies ............................................................................................................... 193
4.3 Discussion ............................................................................................................................... 197
CHAPTER 5: COMPARATIVE METABOLOMIC PROFILE ANALYSIS OF TERMINALIA FERDINANDIANA
FRUIT EXTRACTS ................................................................................................................................ 204
5.1 Introduction ............................................................................................................................. 204

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5.2 Materials and Methods ............................................................................................................ 208
5.2.1 Preparation of samples for metabolomic analysis ........................................................... 208
5.2.2 Non-targeted HPLC-MS QTOF analysis ........................................................................ 208
5.2.3 Non-targeted GC-MS head space analysis ...................................................................... 209
5.3 Results ..................................................................................................................................... 209
5.3.1 Qualitative HPLC-MS analysis ....................................................................................... 209
5.3.1.1 Qualitative mass spectral analysis of the T. ferdinandiana fruit extracts ................... 217
5.3.1.2 GC-MS Head space analysis of the T. ferdinandiana fruit extracts ............................ 284
5.4 Discussion ............................................................................................................................... 301
CHAPTER 6: COMPARATIVE METABOLOMIC PROFILE ANALYSIS OF TASMANNIA LANCEOLATA
BERRY EXTRACTS .............................................................................................................................. 313
6.1 Material and Methods ............................................................................................................. 313
6.1.1 Preparation of samples for metabolomic analysis ........................................................... 313
6.2 Non-targeted HPLC-MS QTOF analysis ................................................................................ 313
6.3 Non-targeted GC-MS head space analysis .............................................................................. 314
6.4 Results ..................................................................................................................................... 314
6.4.1 HPLC-MS/MS analysis................................................................................................... 314
6.4.1.1 Qualitative mass spectral analysis of the T. lanceolata berry extracts ........................ 323
6.4.1.2 GC-MS head space analysis ........................................................................................ 378
6.5 Discussion ............................................................................................................................... 400
CHAPTER 7: DISCUSSION ................................................................................................................... 413
REFERENCES ...................................................................................................................................... 429

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