by jacqui m. mcraeacqui m. mcrae - swinburne … that he always had time for a chat and was ready to...

The Elucidation and Evaluation ofThe Elucidation and Evaluation ofThe Elucidation and Evaluation ofThe Elucidation and Evaluation of

Antibacterial Compounds from the Antibacterial Compounds from the Antibacterial Compounds from the Antibacterial Compounds from the

Australian Traditional Medicinal Plant,Australian Traditional Medicinal Plant,Australian Traditional Medicinal Plant,Australian Traditional Medicinal Plant,

Planchonia careyaPlanchonia careyaPlanchonia careyaPlanchonia careya

Submitted in total fulfillment of the requirements for the degree of

Doctor of PhilosophyDoctor of PhilosophyDoctor of PhilosophyDoctor of Philosophy

By

Jacqui M. McRaeJacqui M. McRaeJacqui M. McRaeJacqui M. McRae

BSc. (Hons.)BSc. (Hons.)BSc. (Hons.)BSc. (Hons.)

Environment and Biotechnology Centre Faculty of Life and Social Sciences Swinburne University of Technology

Hawthorn, Victoria Australia

July 2008

i

AbstractAbstractAbstractAbstract

The leaves of the tropical tree, Planchonia careya (Lecythidaceae), are used

traditionally in the treatment of wounds and ulcers by the indigenous people of northern

Australia. The purpose of this investigation was to isolate and identify some

antibacterial compounds from the leaves of P. careya to validate the use of this species

as a wound healing remedy, and also to evaluate the isolated active compounds for

potential use as chemotherapeutic or topical antibacterial agents. Some of the

comprising flavonoid glycoside constituents were also investigated as potential

chemotaxonomic markers, since morphological similarities of this species to other taxa

has led to many changes in the classification of P. careya.

The comprising compounds of the crude aqueous and methanol leaf extracts were

separated using activity-guided fractionation and this involved the use of various

chromatographic media in conjunction with plate hole diffusion assays. The structures

of the isolated antibacterial compounds were elucidated principally using Nuclear

Magnetic Resonance (NMR) spectroscopy and Mass Spectrometry (MS), and the

antibacterial activity was assessed using Minimum Inhibitory Concentration (MIC)

assays. The cytotoxicity of the isolated compounds was assessed using monkey kidney

epithelial (MA104) cells, to determine the selectivity of the compounds for prokaryotic

cells relative to eukaryotic cells, as well as with human ovarian carcinoma (HeLa) cells

to determine any antitumour activity.

Two known antibacterial compounds, gallocatechin-(48)-gallocatechin (prodel-

phinidin) (1) and (+)-gallocatechin (2), were isolated from the aqueous extract and the

latter exhibited a significant (p < 0.05) effect on the tumour cells relative to the MA104

cells. The benzene triol, pyrogallol (S1) was also obtained from the aqueous extract and

may have been present as a degradation product. Analysis of the comprising flavonol

glycosides from the aqueous extract revealed the known kaempferol 3-O-gentibioside

and quercetin 3-O-glucoside as well as two novel acylated kampferol polyglycosides.

ii

The methanol extract yielded two fatty acids, 9(S)-hydroxyoctadeca-10E,12Z,15Z-

trienoic acid (3), 9(S)-hydroxyoctadeca-10E,12Z-dienoic acid (-dimorphecolic acid)

(4), the triterpene, 2,3,24-trihydroxyolean-12-en-28-oic acid (hyptatic acid-A) (5),

and a mixture of two known acylated triterpenes, 3-O-cis-p-coumaroyl-2,19-

dihydroxyurs-12-en-28-oic acid (3-O-cis-coumaroyl tormentic acid) as well as the trans

isomer (6ab). Compound S2, a mixture of 2,3,19,24-tetrahydroxyurs-12-en-28-oic

acid (24-hydroxyl tormentic acid) and an oleane-type triterpene was also obtained from

the methanol extract. The mixture of 6ab demonstrated the greatest antibacterial activity

and prokaryotic selectivity of the isolated compounds with a MIC against vancomycin-

resistant enterococci (VRE) of 59 g/mL.

The isolation of known antibacterial compounds 1-5 as well as 6ab, S1 and S2 from the

crude leaf extracts of P. careya validates the use of this species as a treatment for sores

and ulcers. Further, the activity of 6ab against the antibiotic-resistant strain, VRE, and

the antitumour activity of 2, 4 and 5, suggests that there is still great potential for the

discovery of potential therapeutic agents from plants. Finally, the presence of novel

acylated kaempferol polyglycosides may assist in taxonomic classification of this and

other Planchonia species.

iii

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

This research would not have been possible without the guidance and support of my

supervisors, Enzo Palombo, Qi Yang, and Russell Crawford. Enzos open door policy

meant that he always had time for a chat and was ready to help whenever it was needed.

Qi was always there with a smile and a word of encouragement. Her attention to detail

always led me in the right direction and I genuinely appreciate the many times that she

went out of her way to help solve a problem. Russell was always willing to assist in any

way that he could and was quick with a kind word and a joke. His support and

encouragement are greatly appreciated. I also gratefully acknowledge time and

assistance provided by my mentor, Noel Hart. Noel generously went out of his way on

many occasions to assist with problems and to teach me many different aspects of

chromatography. His continued friendship is greatly appreciated.

I would also like to express my appreciation for the assistance and advice provided by

the CSIRO instrument specialists, Stuart Littler, Jo Cosgriff, Carl Braybrook and Roger

Mulder. Stuart was eternally patient with my many questions and HPLC demands, and

Jo and Carl were always helpful with my NMR or MS samples. I am especially grateful

for Rogers assistance with my NMR samples, as well as providing me with invaluable

resources, and for the time, patience and guidance that he has generously provided.

Many thanks are also given to the research staff at CSIRO and Swinburne University,

Ngoc Le, Ngan Nguyen, Sheila Curtis, Savitri Galappathie, and Soula Mougos, who

would always point me in the right direction.

The inspiration for this project came from the advice offered by Victoria Gordon of

Ecobiotics and this project would not have been possible without her assistance. I also

acknowledge the assistance of Rebel Eleck and Andrew Ford of the CSIRO Tropical

Forest Research Centre in coordinating and collecting the required plant samples. The

friendship of my fellow postgrads has been invaluable and I greatly appreciate their

moral support. Finally I would like to express my gratitude to my family and friends,

particularly Bogdan Zisu and Rebecca Wise, whose support and encouragement has

been greatly appreciated, especially over the past few years.

iv

DeclarationDeclarationDeclarationDeclaration

I hereby declare, to the best of my knowledge, that the material contained in this thesis

has not been accepted for the award of any other degree or diploma, and has not been

previously published or written by another person, except where due reference is made

in the text. I also declare that where the work is based on joint research or publications,

the relative contributions of the respective workers or authors is disclosed.

---------------------------------------

Jacqui M. McRae

v

List of PublicationsList of PublicationsList of PublicationsList of Publications

Book Chapter

- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2006. Traditional

Knowledge and Indigenous Uses: Ethnobotanical Leads to Drug Discovery, In:

Recent Progress in Medicinal Plants, Volume 13, Govil, J.N. (ed.), Stadium

Press, Houston, pp. 195 -216.

Refereed Journal Articles

- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2008. Acylated

flavonoid tetraglycoside from Planchonia careya leaves, Phytochemistry

Letters, 1 (2008), 99-102.

- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2008. Antibacterial

compounds from Planchonia careya leaf extracts, Journal of

Ethnopharmacology, 116 (2008) 554560.

- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2007. Review of the

methods used for isolating pharmaceutical lead compounds from traditional

medicinal plants, The Environmentalist, 27 (1), 165-174.

Refereed Conference Abstract


proanthocyanidins isolated from the Australian medicinal plant, Planchonia

careya (F. Muell.) R. Knuth (Lecythidaceae), In: 54th Annual Congress for the

Society for Medicinal Plant Research, Helsinki, Finland: Book of abstracts,

Planta medica 2006; 72 (11), DOI: 10.1055/s-2006-949740.

vi

Refereed Papers in Conference Proceedings


methods used for isolating pharmaceutical lead compounds from traditional

medicinal plants, In: Proceedings of the 9th Annual Environmental Research

Event, Tasmania, December 2005, N. Khanna et al. (ed.), RMIT University, CD-

ROM.


current methods involved in extracting and evaluating the antibacterial

compounds of traditional medicinal plants, In: Proceedings of the 8th Annual

Environmental Research Event, New South Wales, December 2004, p. 208-218.

Conference Abstracts


constituents of the Australian traditional medicinal plant, Planchonia careya

(Lecythidaceae), In: Proceedings of the 2007 Bioprocessing Network Annual

Conference, Clayton, Victoria.


constituents of the Australian traditional medicinal plant, Planchonia careya

(Lecythidaceae), In: Proceedings of the 3rd International Congress on

Traditional Medicine & Materia Medica, Kuala Lumpur, Malaysia.

- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2006. Drug discovery

from plants: The limiting factors, In: Proceedings of the 25th Australian Colloid

and Surface Science Student Conference, Beechworth, Victoria.

Table of Contents

vii

Table of ContentsTable of ContentsTable of ContentsTable of Contents

Abstract .................................................................................................................... i

Acknowledgements.................................................................................................. iii

Declaration .............................................................................................................. iv

List of publications .................................................................................................. v

Table of contents ..................................................................................................... vii

List of Tables ........................................................................................................... xvii

List of Figures.......................................................................................................... xxiv

List of Compound Structures................................................................................. xxxvi

Abbreviations .......................................................................................................... xxxix

Chapter 1: Plant Natural Products for Medicinal Applications and

Chemotaxonomy ....................................................................................... 1

1.1 Plants natural products for medicinal applications ........................................ 1

1.1.1. Biodiscovery and the need for new medicines............................................ 1

1.1.2. Infectious diseases ..................................................................................... 5

1.1.3. Cancers ...................................................................................................... 9

1.2 Plants as a source of novel pharmaceuticals ................................................... 11

1.2.1. Secondary metabolites from plants ......................................................... 11

1.2.2. Ethnobotanical leads in natural product discovery ................................ 18

1.2.3. Pharmaceutical lead compounds from plants......................................... 21

1.2.4. Antibacterial agents from plants .............................................................. 23

1.2.5. Anticancer agents from plants ................................................................ 26

1.3 Plant natural products as chemotaxomonic agents ...................................... 30

1.3.1. Biochemical systematics .......................................................................... 30

1.3.2. Biochemistry of flavonoids ..................................................................... 32

1.3.3. Flavonol glycosides and conjugates as chemotaxonomic indicators .... 38

1.4 Summary ........................................................................................................... 40

Table of Contents

viii

Chapter 2: Review of Planchonia careya ...............................................41

2.1 Taxonomy and distribution of Planchonia careya ......................................... 41

2.2 Ethnobotany and phytochemistry of Lecythidaceae species ........................ 45

2.3 Ethnobotany and phytochemistry of Planchonia careya .............................. 47

2.3.1. Traditional uses of Planchonia careya ................................................... 47

2.3.2. Phytochemistry of Planchonia careya ..................................................... 48

2.4 Project aims ....................................................................................................... 50

Chapter 3: Planchonia careya: Small-Scale Optimization of

Extraction Methodology .......................................................................52

3.1 Introduction ...................................................................................................... 52

3.2 Sample processing and extraction .................................................................. 53

3.2.1. Leaf collection and processing ............................................................... 53

3.2.2. Extraction of leaves ................................................................................. 53

3.2.2.1. Trials of different extraction techniques ......................................... 53

3.2.2.2. Optimization of the selected extraction technique .......................... 56

3.3 Assessment of biological activity ..................................................................... 58

3.3.1. Antibacterial assays ................................................................................. 58

3.3.1.1. Investigation of different antibacterial assays ................................ 58

3.3.1.2. Selection of the test organisms for activity-guided fractionation ... 63

3.3.1.3. Assessment of antimycobacterial activity ........................................ 66

3.4 Phytochemical investigations .......................................................................... 68

3.4.1. Thin layer chromatography .................................................................... 68

3.4.1.1. Preliminary investigation of the antibacterial constituents ............ 68

3.4.1.2. Preliminary identification of active constituents using

spray reagents .................................................................................. 69

3.4.2. Column chromatography ........................................................................ 70

3.4.2.1. Comparison of the different chromatographic media ..................... 70

3.4.2.2. Preliminary investigation of the aqueous extract ............................ 72

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ix

3.4.2.2.1. Overview ............................................................................. 72

3.4.2.2.2. Fractionation of the aqueous extract ................................... 73

3.4.2.3. Preliminary investigation of the methanol extract .......................... 78

3.4.2.3.1. Overview ............................................................................. 78

3.4.2.3.2. Fractionation of the methanol extract ................................. 79

3.5 Summary of the optimized methods ............................................................... 85

Chapter 4: Planchonia careya: Large-scale Extraction Methodology ...86

4.1 General experimental ...................................................................................... 86

4.1.1. Chromatography ..................................................................................... 86

4.1.1.1. High Pressure Liquid Chromatography ......................................... 86

4.1.1.2. Preliminary analysis with Thin Layer Chromatography ................ 87

4.1.2. Compound characterization with Nuclear Magnetic Resonance

Spectroscopy ............................................................................................. 88

4.1.2.1. Overview of selected experiments ................................................... 88

4.1.2.2. NMR experimental conditions ......................................................... 89

4.1.3. Further compound characterization ...................................................... 90

4.1.3.1. Mass Spectrometry .......................................................................... 90

4.1.3.2. Optical rotation ............................................................................... 90

4.1.3.3. Melting point ................................................................................... 91

4.1.3.4. IR spectroscopy ............................................................................... 91

4.1.3.5. UV Spectroscopy ............................................................................. 91

4.1.3.6. Calculated Log P ............................................................................ 92

4.1.3.7. Acid hydrolysis of isolated glycosides.............................................. 92

4.1.4. General calculations ................................................................................ 93

4.1.4.1. Calculating the total mass of a sample ........................................... 93

4.1.4.2. Calculating the percent yield .......................................................... 93

4.1.4.3. Calculating the leaf mass per well .................................................. 94

4.2 Plant material .................................................................................................... 95

4.3 Extraction of plant material ............................................................................ 95

4.3.1. Aqueous extraction .................................................................................. 95

4.3.2. Methanol extraction ................................................................................ 96

Table of Contents

x

4.4 Compound isolation and purification ............................................................ 96

4.4.1. General methodology .............................................................................. 96

4.4.2. Investigation of bioactive compounds from the aqueous extract .......... 97

4.4.2.1. Stage 1 Separation .......................................................................... 97



4.4.2.4. Isolation of Compound 1 ................................................................. 98


4.4.2.6. Isolation of Compound S1 ............................................................... 99

4.4.3. Investigation of the methanol extract ..................................................... 100

4.4.3.1. Initial separation ............................................................................. 100

4.4.3.2. Investigation of the DCM-soluble fraction ..................................... 100

4.4.3.2.1. Stage 1 Separation of the DCM-soluble fraction ................ 100

4.4.3.2.2. Stage 2 Separation and isolation of Compound 3 ............... 101

4.4.3.2.3. Isolation of Compounds 4, 5 and 6 ab .................................. 101

4.4.3.2.4. Isolation of Compound S2 ................................................. 102

4.4.3.3. Investigation of the methanol water-soluble fraction ..................... 102

4.4.3.3.1. Methanol Water Stage 1 Separation .................................... 102

4.4.3.3.2. Methanol Water Stage 2 Separation .................................... 102

4.4.3.3.3. Re-isolation of Compound 2 .............................................. 103

4.4.4. Investigation of Aqueous Stage 2 Separation Fraction 16 .................... 103



4.4.4.3. Isolation of Compound 8 ................................................................ 104

4.4.5. Investigation of Aqueous Stage 2 Separation Fraction 20 .................... 104



4.4.5.3. Isolation of S3 .................................................................................. 105

4.4.5.4. Isolation of Compound 10 .............................................................. 105

4.4.5.5. Isolation of S4 ................................................................................. 105

4.5 Characterization of isolated compounds ........................................................ 106

4.5.1. Compound 1 ............................................................................................. 106

4.5.2. Compound 2 ............................................................................................ 106

4.5.3. Compound 3 ............................................................................................ 106

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xi

4.5.4. Compound 4 ............................................................................................ 107

4.5.5. Compound 5 ............................................................................................. 107

4.5.6. Compound 6 ............................................................................................ 108

4.5.7. Compound 7 ............................................................................................ 109

4.5.8. Compound 8 ............................................................................................ 109

4.5.9. Compound 9 ............................................................................................ 110

4.5.10. Compound 10 ....................................................................................... 110

4.5.3. Compound S1 .......................................................................................... 111

4.5.3. Compound S2 .......................................................................................... 111

4.6 Evaluation of antibacterial activity ................................................................ 112

4.6.1. Bacteria and media ................................................................................. 112

4.6.1.1. Bacteria ........................................................................................... 112

4.6.1.2. Materials ......................................................................................... 113

4.6.1.3. Standard media ............................................................................... 113

4.6.1.4. Media for fastidious bacteria .......................................................... 113

4.6.1.5. Media for mycobacteria .................................................................. 113

4.6.2. Antibacterial assays ................................................................................. 114

4.6.2.1. Broth culture preparation ............................................................... 114

4.6.2.2. Plate-hole diffusion and minimum inhibitory concentration

assays .................................................................................................. 114

4.6.2.3. Broth dilution assays ....................................................................... 115

4.6.2.4. Bioautography assays ..................................................................... 115

4.7 Evaluation of cytotoxicity ............................................................................... 116

4.7.1. Materials .................................................................................................. 116

4.7.1.1. Cell lines ......................................................................................... 116

4.7.1.2. Media and solutions ........................................................................ 116

4.7.1.3. Equipment ....................................................................................... 116

4.7.2. Cytotoxicity assays ................................................................................... 117

4.7.2.1. Passaging of cell lines...................................................................... 117

4.7.2.2. Determining the optimal cell seeding density for cytotoxicity

assays .................................................................................................. 118

4.7.2.3. Establishing the controls ................................................................. 119

4.7.2.4. MTT cell viability assay .................................................................. 119

4.7.3. Calculations used in the cytotoxicity assays............................................ 119

Table of Contents

xii

4.7.3.1. Calculating the IC50 ....................................................................... 119

4.7.3.2. Calculating errors ........................................................................... 120

4.7.3.3. Statistical analysis of cytotoxicity data ............................................ 121

4.7.3.4. Calculating the therapeutic index .................................................... 121

Chapter 5: Planchonia careya: Activity-guided fractionation ...............123

5.1 Introduction....................................................................................................... 123

5.2 Preparation and fractionation of the aqueous extract................................... 124

5.2.1. Overview ................................................................................................... 124

5.2.2. Preparation of the aqueous extract ......................................................... 125

5.2.3. Fractionation of the aqueous extract ..................................................... 127

5.2.3.1. Aqueous Stage 1 Separation: Separation with

Amberlite XAD-16 resin .................................................................... 127


reverse-phase C18 media.................................................................. 130


Sephadex LH-20 gel .......................................................................... 139

5.2.3.4. Isolation of Compound 1: Prodelphinidin ....................................... 142

5.2.3.5. Isolation of Compound 2: Gallocatechin ......................................... 146

5.2.3.6. Isolation of Compound S1: Pyrogallol ............................................ 149

5.3 Preparation and fractionation of the methanol extract ................................ 152

5.3.1. Overview .................................................................................................. 152

5.3.2. Preparation of the methanol extract........................................................ 153

5.3.3. Fractionation of the methanol extract .................................................... 153

5.3.3.1. Separation using liquid-liquid partitioning ..................................... 153

5.3.3.2. Investigation of the Methanol DCM-Soluble fraction...................... 155

5.3.3.2.1. Methanol DCM Stage 1 Separation:

Separation with silica gel ...................................................... 155

5.3.3.2.2. Methanol DCM Stage 2 Separation and

isolation of Compound 3....................................................... 158

5.3.3.2.3. Isolation of Compounds 4, 5, and 6: -Dimorphecolic acid,

Hyptatic acid-A, and 3-O-coumaroyl tormentic acid........... 162

5.3.3.2.4. Isolation of Compound S2: 24-Hydroxy tormentic acid .... 165

Table of Contents

xiii

5.3.3.3. Investigation of the Methanol Water-soluble fraction ..................... 168

5.3.3.3.1. Methanol Water Stage 1 Separation:

Separation with C18 media ................................................... 168

5.3.3.3.2. Methanol Water Stage 2 Separation:

Separation with MPLC ........................................................ 170

5.3.3.3.3. Re-isolation of Compound 2: Gallocatechin........................ 173

5.4 Summary of compounds isolated from the leaf extracts ............................... 175

Chapter 6: Structural Elucidation and Characterization of Bioactive

Compounds Isolated from Planchonia careya ......................................177

6.1 Introduction....................................................................................................... 177

6.2 Structural elucidation ...................................................................................... 178

6.2.1. Compounds 1 and 2: Proanthocyanidins ............................................... 178

6.2.1.1. Overview ......................................................................................... 178

6.2.1.2. Compound 1: Prodelphinidin........................................................... 178

6.2.1.3 Compound 2: (+)-Gallocatechin ...................................................... 184

6.2.2. Compounds 3 and 4: Hydroxylated fatty acids ...................................... 187

6.2.2.1. Overview .......................................................................................... 187

6.2.2.2. Compound 3: 9-HOTE ..................................................................... 188

6.2.2.3. Compound 4: -Dimorphecolic acid ............................................... 193

6.2.3. Compounds 5 and 6: Pentacyclic triterpenoids ...................................... 198

6.2.3.1. Overview .......................................................................................... 198

6.2.3.2. Compound 5: Hyptatic acid-A ......................................................... 198

6.2.3.3. Compound 6: 3-O-Coumaroyl tormentic acid ................................. 205

6.3 Structural characterization of additional bioactive compounds ................. 213

6.3.1. Overview .................................................................................................. 213

6.3.2 Compound S1: Pyrogallol ....................................................................... 213

6.3.3. Compound S2: 24-Hydroxytormentic acid.............................................. 216

6.4 Summary of the structural elucidation and characterization of

bioactive compounds ......................................................................................... 220

Table of Contents

xiv

Chapter 7: Biological Evaluation of Bioactive Compounds Isolated

from Planchonia careya..........................................................................221

7.1 Introduction....................................................................................................... 221

7.2 Compounds and crude extracts used in the biological evaluation assays.... 222

7.3 Evaluation of antibacterial activity ................................................................. 223

7.3.1. Minimum inhibitory concentration assays ............................................ 223

7.3.2. Antibacterial activity of the crude extracts and isolated compounds..... 225

7.3.3. Antibacterial activity of compounds isolated from the aqueous

extract ........................................................................................................... 227

7.3.4. Antibacterial activity of compounds isolated from the methanol

extract ........................................................................................................... 229

7.4 Evaluation of antitumour activity ................................................................... 231

7.4.1 Optimization of the cytotoxicity assays..................................................... 231

7.4.1.1. Optimal cell seeding density ............................................................ 231

7.4.1.2. Assessing the potential reduction of MTT by the isolated

compounds ........................................................................................... 234

7.4.2. Cytotoxicity of the crude extracts and isolated compounds.................... 235

7.4.3. Antitumour activity of compounds isolated from the aqueous extract .. 236

7.4.4. Antitumour activity of compounds isolated from the methanol extract. 238

7.5 Summary of the biological evaluation of the isolated bioactive

compounds .................................................................................................. 239

Chapter 8: Isolation of Some Chemotaxonomic Indicators in

Planchonia careya ..................................................................................241

8.1 Introduction....................................................................................................... 241

8.2 HPLC-piloted isolation of flavonol glycosides................................................ 242

8.2.1. Overview .................................................................................................. 242

8.2.2. Separation of fraction AS2:16 with Sephadex LH-20 gel ...................... 243

8.2.3. Isolation of Compound 7: Kaempferol gentiobioside ............................. 245

8.2.4. Isolation of Compound 8: Quercetin glucoside ...................................... 246

8.2.5. Re-isolation of Compound 2: Gallocatechin ......................................... 249

Table of Contents

xv

8.3 HPLC-piloted investigation of flavonol conjugates ....................................... 250

8.3.1. Overview ................................................................................................... 250

8.3.2. Stage 3 Separation of fraction AS2:20 with MPLC................................ 251

8.3.3. Isolation of Compound 9: Acylated kaempferol tetraglycoside.............. 253

8.3.4. Isolation of S3 and re-isolation of Compound 9..................................... 255

8.3.5. Investigation of AS2:20 fraction AG:113-119 ........................................ 257

8.3.6. Isolation of S4 ......................................................................................... 258

8.3.7. Isolation of Compound 10: Acylated kaempferol pentaglycoside .......... 260

8.4 Summary of the isolation of potential chemotaxonomic indicators ............ 262

Chapter 9: Structural Elucidation and Characterization of

Chemotaxonomic Indicators in Planchonia careya................................263

9.1 Introduction....................................................................................................... 263

9.2 Structural elucidation of the isolated flavonol glycosides ............................. 264

9.2.1. Compounds 7 and 8.................................................................................. 264

9.2.1.1. Overview .......................................................................................... 264

9.2.1.2. Compound 7: Kaempferol gentibioside ........................................... 264

9.2.1.3. Compound 8: Quercetin glucoside .................................................. 270

9.3 Structural elucidation of the isolated flavonol conjugates ............................ 275

9.3.1. Compounds 9 and 10................................................................................ 275

9.3.1.1. Overview ......................................................................................... 275

9.3.1.2. Compound 9: Acylated kampferol tetraglycoside ............................ 275

9.3.1.3. Compound 10: Acylated kaempferol pentaglycoside ....................... 287

9.3.2. Preliminary elucidation of S3 and S4 ..................................................... 294

9.3.2.1. Overview ......................................................................................... 294

9.3.2.2. Partial elucidation of S3 ................................................................. 294

9.3.2.3. Partial elucidation of S4 ................................................................. 296

9.4 Summary of the elucidation of the isolated chemotaxonomic indicators .... 298

Table of Contents

xvi

Chapter 10: Conclusions and future research .........................................299

10.1 Conclusions ...................................................................................................... 299

10.1.1. Overview ................................................................................................. 299

10.1.2. Elucidation of antibacterial constituents .............................................. 300

10.1.3. Evaluation of biological activity ............................................................ 301

10.1.4. Identification of potential chemotaxonomic markers........................... 303

10.2 Future research ............................................................................................... 304

10.2.1. Overview ................................................................................................. 304

10.2.2. Further investigation of the isolated compounds ................................. 304

10.2.3. Future studies involving biological activity........................................... 306

10.2.4. Further chemotaxonomic investigations............................................... 308

10.3 Concluding remarks ....................................................................................... 308

Glossary.................................................................................................................... 309

References ................................................................................................................ 317

Appendix 1: Spectral data of the isolated compounds......................................... 337

A1.1. Proanthocyanidins ..................................................................................... 338

A1.2. Hydroxylated fatty acids ........................................................................... 343

A1.3. Pentacyclic triterpenes............................................................................... 348

A1.4. Flavonol glycosides................................................................................... 354

A1.5. Flavonol conjugates .................................................................................. 359

A1.6. Semi-purified bioactive compounds.......................................................... 364

A1.7. Semi-purified chemotaxonomic compounds............................................. 369

Appendix 2: Data for the cell culture calculations............................................... 372

A2.1. Results of the Shapiro-Wilk normality tests for each compound.............. 373

A2.2. The cell inhibitory rates of compounds isolated from the aqueous extract .......... 374

A2.3. The cell inhibitory rates of compounds isolated from the methanol extract......... 375

A2.4. Results of the significance tests for the cytotoxic assays.......................... 376

List of Tables

xvii

List of TablesList of TablesList of TablesList of Tables

Tables from Chapter 1

Table 1.1

The main classes of secondary metabolites and some of their subgroups ................ 13

Table 1.2

Some of the key functions of flavonoids in plants.................................................... 36


Table 2.1

Medicinal uses of some Lecythidaceae species ........................................................ 46


Table 3.1

Comparison of the time required, sample capacity and percent yield of the

different extraction methods ..................................................................................... 55

Table 3.2

The solvent used and resulting percent yields of the immersion extracts................. 57

Table 3.3

The effect of disc thickness on the diameter of the zone of inhibition for the disc

diffusion assays ........................................................................................................ 60

Table 3.4

Comparison of the disc (DDA) and plate-hole (PHDA) diffusion assays ................ 61

Table 3.5

Antibacterial activity of the different extracts against B. cereus determined with

PHDAs ...................................................................................................................... 62

Table 3.6

The antibacterial activity of the crude Methanol Immersion Extract against a

range of bacteria determined with PHDAs ............................................................... 65

List of Tables

xviii

Table 3.7

The antimycobacterial activity of the crude Methanol Immersion Extract

of P. careya leaves determined with PHDAs............................................................ 67

Table 3.8

The TLC solvent systems and Rf values of the active bands determined by

bioautography assays ................................................................................................ 69

Table 3.9

Activity of fractions from the Preliminary Aqueous Stage 1 Separation (PA1)

using XAD-16 resin .................................................................................................. 74

Table 3.10


with C18 media against B. cereus ............................................................................. 75

Table 3.11


with LH-20 gel against B. cereus, determined with PHDAs ................................... 77

Table 3.12

Activity of fractions from the Preliminary Methanol Stage 1 Separation (PM1)

with silica gel against B. cereus, determined with PHDAs....................................... 79

Table 3.13

Solvent systems of Preliminary Methanol Stage 2 Separation (PM2) with silica

gel and the activity of each fraction against B. cereus, determined with PHDAs .... 82

Table 3.14


with C18 media against B. cereus, determined with PHDAs ................................... 82

Table 3.15


with preparative HPLC against B. cereus, determined with PHDAs........................ 84


Table 5.1

Antibacterial activity of extracts from the large scale collection compared with

those of the small-scale collection ............................................................................ 126

List of Tables

xix

Table 5.2

The antibacterial activity and concentrations of fractions from the Aqueous Stage 1

Separation using XAD-16 resin ................................................................................ 129

Table 5.3


Separation (AS2) using C18 media........................................................................... 132

Table 5.4


Separation with Sephadex LH-20 gel ....................................................................... 140

Table 5.5

The antibacterial activity of fractions from the preparative HPLC separation of

AS3:38-45 (C1)......................................................................................................... 144

Table 5.6

The antibacterial activity of fractions from the preparative HPLC separation of

AS3:22-26 (C2)......................................................................................................... 147

Table 5.7

The antibacterial activity of fractions from the preparative reversed phase

HPLC separation of AS3:14 (C3) ............................................................................ 150

Table 5.8

The antibacterial activity and concentrations of fractions from the separation of the

methanol extract with liquid-liquid partitioning ...................................................... 155

Table 5.9

The different solvent systems trialled using TLC with silica plates ......................... 156

Table 5.10

The antibacterial activity and elution solvent of fractions from the Methanol DCM

Stage 1 Separation (MD1) with silica gel ................................................................. 157

Table 5.11

The antibacterial activity of fractions from the Methanol DCM Stage 2 Separation

(MD2) with MPLC and the retention times of the main peaks in

the HPLC chromatogram .......................................................................................... 160

Table 5.12

The antibacterial activity and concentrations of fractions from the preparative HPLC

separation of Methanol DCM Stage 2 Separation fractions 23-26 (C4).................. 164

List of Tables

xx

Table 5.13

The antibacterial activity and concentrations of fractions from the preparative

HPLC separation of MD2:4-6 (C5) .......................................................................... 166

Table 5.14

The antibacterial activity and elution solvent of fractions from the Methanol Water

Stage 1 Separation (MW1), determined by plate-hole diffusion assays .................. 169

Table 5.15

The antibacterial activity and elution solvent of fractions from the Methanol Water

Stage 2 Separation (MW2), determined by plate-hole diffusion assays ................... 171


Table 6.1

The 1H and 13C NMR chemical shifts for 1 in D2O at 400 MHz.............................. 181

Table 6.2

The COSY and HMBC correlations of 1, in D2O at 400 MHz ................................ 182

Table 6.3

The chemical shifts of the carbons and protons of 2, in D2O at 400 MHz,

compared with those given in the literature .............................................................. 186

Table 6.4

The chemical shifts of the carbons and protons of 3 in CD3OD at 400 MHz,


Table 6.5

The COSY and HMBC correlations of 3, in CD3OD at 400 MHz .......................... 192

Table 6.6

The chemical shifts of the carbons and protons of 4 in CD3OD at 500 MHz,


Table 6.7

The 1H and 13C NMR chemical shifts of 5 in CD3OD at 500 MHz.......................... 201

Table 6.8

The COSY and HMBC correlations of 5, in CD3OD at 400 MHz .......................... 202

Table 6.9

The 1H and 13C NMR chemical shifts of the triterpene core structure of 6a ............ 207

List of Tables

xxi

Table 6.10

The 1H NMR chemical shifts of the triterpene core structure of 6b ........................ 208

Table 6.11

The chemical shifts of the carbons and protons of the acyl group of 6a in CD3OD

at 500 MHz................................................................................................................ 210

Table 6.12

The chemical shifts of the carbons and protons of the acyl group of 6b in CD3OD

at 500 MHz ............................................................................................................... 210

Table 6.13

The ROESY correlations of 6a, in CD3OD at 500 MHz ......................................... 211

Table 6.14

The chemical shifts of the carbons and protons of S1 in CD3OD (500 MHz),

compared with those given in the literature ............................................................. 215

Table 6.15

The chemical shifts of the carbons and protons of S2 in CD3OD (500 MHz) ......... 219


Table 7.1

The physical properties of the isolated compounds and concentrations prepared

for the biological evaluation...................................................................................... 222

Table 7.2

Comparison of the MICs produced by the PHDA and BDA methods (mg/mL) for

the crude fractions of compounds 1 and 2, as well as tetracycline ........................... 224

Table 7.3

MICs for the isolated compounds and crude extracts against Gram positive and

Gram negative bacteria (mg/mL) as well as the cytotoxicity (IC50) against

MA104 cells (g/mL) for comparison ...................................................................... 226


Table 9.1

The 1H and 13C NMR data for 7, in CD3OD at 400 MHz......................................... 267

List of Tables

xxii

Table 9.2

The chemical shifts of the carbons and protons of 8, quercetin 3-O-glucoside,

in CD3OD at 400 MHz, compared with the 13C chemical shifts given in the

literature for the glucoside (Glc) and galactoside (Gal)............................................ 273

Table 9.3

The 1H and 13C NMR data of the aglycone and coumaroyl groups of 9, in CD3OD

at 400 MHz................................................................................................................ 278

Table 9.4

The 1H and 13C NMR data of the sugar moieties of 9, in CD3OD at 400 MHz........ 279

Table 9.5

The COSY and HMBC correlations for the protons of the aglycone and

coumaroyl groups of 9, in CD3OD at 400 MHz ....................................................... 282

Table 9.6

The COSY and HMBC correlations for the protons of the sugar moieties of 9,

in CD3OD at 400 MHz ............................................................................................. 283

Table 9.7

The ROESY interactions for the protons of the sugar moieties of 9,

in CD3OD at 400 MHz ............................................................................................. 285

Table 9.8

The ROESY interactions for the protons of the aglycone and coumaroyl

groups of 9, in CD3OD at 400 MHz ......................................................................... 286

Table 9.9

The 1H NMR and COSY data of the aglycone, coumaroyl and glucose moieties

of 10 in CD3OD at 500 MHz .................................................................................... 289

Table 9.10

The 1H NMR and COSY data of the rhamnose moieties of 10 in CD3OD

at 500 MHz................................................................................................................ 291

Table 9.11

The 1H NMR chemical shifts of the aglycone and coumaroyl moieties of the

major and minor compounds present in S3 in CD3OD at 500 MHz ........................ 296

List of Tables

xxiii

Tables from Appendix 2

Table A2.1

The determined Shapiro-Wilk normality test significance (generated by SPSS

v14.0) of the cell and solvent blanks for each compound against MA104 cells....... 373

Table A2.2

The determined Shapiro-Wilk normality test significance (generated by SPSS

v14.0) of the cell and solvent blanks for each compound against HeLa cells .......... 373

Table A2.3

Statistical data for each compound including the t value calculated using the

one-sample t-test ....................................................................................................... 376

List of Figures

xxiv

List of FiguresList of FiguresList of FiguresList of Figures

Figures from Chapter 1

Figure 1.1

Origin of new prescription drugs from 1981-2002 ................................................... 4


Figure 2.1

The distribution of some Planchonia species ........................................................... 42

Figure 2.2

(a) A herbarium specimen of P. careya leaves (CSIRO Tropical Research

Centre Herbarium, Yarrumburra, Queensland, Australia); and (b) a live specimen

from Cairns, Queensland........................................................................................... 44

Figure 2.3

The flowers of (a) P. careya; (b) Careya sp.; and (c) B. racemosa .......................... 44


Figure 3.1

An overview of the activity-guided fractionation of the crude aqueous extract

(Extracts 3a and 3b) showing the media used and fractions produced at each

stage of separation .................................................................................................... 72

Figure 3.2

HPLC chromatograms of the Preliminary Aqueous Stage 2 Separation (PA2)

fraction PA2:6 with PA3 active fractions, PA3:6-12 and PA3:20-23,

in 10% MeOH/H2O at 210 nm .............................................................................. 77

Figure 3.3

An overview of the activity-guided fractionation of the Methanol Immersion

Extract showing the media used and fractions produced at each

stage of separation .................................................................................................... 78

List of Figures

xxv

Figure 3.4

HPLC chromatograms of Preliminary Methanol Stage 2 Separation (PM2)

fractions 6 and 7 in 85% MeOH at 250 nm .......................................................... 81

Figure 3.5

HPLC chromatogram of the active Preliminary Methanol Stage 3 Separation

fraction 20 (PM3:20) with PM4 fractions PM4:7 and PM4:10 in 70% MeOH/H2O

at 210 nm................................................................................................................ 84


Figure 5.1

An overview of the activity-guided fractionation of the aqueous extract showing

the media used and fractions produced at each stage of separation.......................... 125

Figure 5.2

HPLC chromatograms of the main fractions from the Aqueous Stage 1 Separation

in 85% MeOH/H2O at 210 nm............................................................................... 128

Figure 5.3

(a) Fractions AS2:4 and 5 in 5% MeOH//H2O at 210 nm; and (b) Fractions

AS2:7, 8 and 9 with compounds 1 and 2 isolated from Fraction AS2:6

in 5% MeOH//H2O at 210 nm ................................................................................ 134

Figure 5.4

(a) Fractions AS2:8, 9 and 10 in 15% MeOH//H2O at 210 nm; and (b) Fractions

AS2:11 and 12 in 20% MeOH//H2O at 210 nm .................................................... 135

Figure 5.5



Figure 5.6



Figure 5.7

The retention times of the range of compounds from Aqueous Stage 3 Separation

with Sephadex LH-20 gel, demonstrated with fractions AS3:14, AS3:24,

AS3:42, AS3:67, AS3:84, and AS3:110 in 5% MeOH/H2O at 210 nm ................ 141

List of Figures

xxvi

Figure 5.8

(a) HPLC chromatograms of compound 1 (C1:17-25) with the combined Aqueous

Stage 3 Separation fractions 38-45 (C1) in 3% MeOH/H2O at 210 nm; and (b)

the UV profile of 1 .................................................................................................... 145

Figure 5.9

(a) HPLC chromatograms of 2 (C2:61-65) with Aqueous Stage 3 Separation

fractions 22-26 (C2) in 3% MeOH/H2O at 210 nm; and (b) the UV profile of 2 .. 148

Figure 5.10

(a) HPLC chromatograms of S1 (C3:13-15) with Aqueous Stage 3 Separation

(AS3) fraction 14 (C3) in 5% MeOH/H2O at 210 nm; and (b) the UV

profile of S1 .............................................................................................................. 151

Figure 5.11

An overview of the activity-guided fractionation of the aqueous extract showing

the media used and fractions produced at each stage of separation .......................... 152

Figure 5.12

HPLC chromatograms of the Methanol DCM Stage 1 Separation (MD1) fractions

6 to 8 in 65% MeOH/H2O at 210 nm (a gradient to 100% MeOH has been used

to shorten the elution time of the later eluting peaks in fraction MD1:6) ................. 158

Figure 5.13

(a) HPLC chromatograms of the Methanol DCM Stage 1 Separation fraction 7

(MD1:7) with combined Methanol DCM Stage 2 Separation (MD2) fractions 4-6,

14-19 (Compound 3) and 23-26, in 70% MeOH/H2O at 210 nm; and

(b) the corresponding UV profile of 3 ..................................................................... 161

Figure 5.14

(a) HPLC chromatograms of compounds 4 6 in 75% MeOH/H2O at 210 nm;

and (b) the corresponding UV profiles...................................................................... 163

Figure 5.15

(a) HPLC chromatograms of compound S2 (C5:7-9) with Methanol DCM Stage 2

Separation fractions 4-6 (C5) in 70% MeOH/H2O at 210 nm; and (b) the UV

profile of S2 ............................................................................................................. 167

Figure 5.16

HPLC chromatograms of the Methanol Water Stage 1 (MW1) fractions 3-8

in 15% MeOH/H2O at 210 nm .............................................................................. 170

List of Figures

xxvii

Figure 5.17

HPLC chromatograms of the Methanol Water Stage 2 (MW2) fractions 6, 9-11,

14 and 20 in 5% MeOH/H2O at 210 nm ............................................................... 172

Figure 5.18

A 3D HPLC chromatogram of Methanol Water Stage 2 Separation fraction 10

(MW2:10 or MP) in 5% MeOH/H2O showing the UV profiles of each

comprising peak ........................................................................................................ 172

Figure 5.19

(a) HPLC chromatograms of compound 2 isolated from the aqueous extract and

MP:21 in 10% MeOH/H2O at 210 nm; and (b) the corresponding UV profile

of MP:21 (refer to Figure 5.9 for the UV profile of 2) ............................................. 174


Figure 6.1

The elucidated structure of 1, with some key HMBC relationships ......................... 179

Figure 6.2

The 1H (top) and 13C (bottom) NMR spectra for 1 in D2O at 400 MHz .................. 180

Figure 6.3

The elucidated structure of 2 with the 1H NMR spectrum CD3OD at 400 MHz 185

Figure 6.4

The elucidated structure of 3 .................................................................................... 188

Figure 6.5

The 1H (top) and JMOD (bottom) NMR spectra for compound 3 in CD3OD

at 400 MHz ............................................................................................................... 189

Figure 6.6

The elucidated structure of 4..................................................................................... 193

Figure 6.7

The 1H (top) and 13C (bottom) NMR spectra for 4 in CD3OD at 500 MHz ............ 194

Figure 6.8

The elucidated structure of 5.................................................................................... 199

Figure 6.9

The 1H NMR spectrum in CD3OD at 400 MHz (top) and 13C JMOD NMR

spectrum in d5-pyridine at 500 MHz (bottom) for compound 5 .............................. 200

List of Figures

xxviii

Figure 6.10

The structure of 5, with some key HMBC correlations ........................................... 203

Figure 6.11

Elucidated structure of 6a, with some key HMBC correlations and the 1H NMR

spectrum in CD3OD at 500 MHz .............................................................................. 206

Figure 6.12

The key ROESY correlations of the core structure of 6ab ...................................... 212

Figure 6.13

The elucidated structure of S1 with the 1H (top) and 13C (bottom) NMR spectra

in CD3OD at 400 MHz ............................................................................................ 214

Figure 6.14

The elucidated structure of the main component in S2, with some key HMBC

correlations ............................................................................................................... 216

Figure 6.15

The 1H (top) and 13C (bottom) NMR spectra for S2 in CD3OD at 400 MHz .......... 218


Figure 7.1

The average optical density (OD) after 72 hours incubation of wells seeded with

solutions containing a serial dilution of cell concentrations. The optimal seeding

concentration is shown for MA104 cells and HeLa cells. Absorbance units

(AU) have been corrected for the media blank ......................................................... 233

Figure 7.2

The reduction of MTT by different concentrations of the crude samples of isolated

compounds 1 and 2. Absorbance units (AU) have been corrected for the

media blank .............................................................................................................. 235

Figure 7.3

The 50% inhibitory concentrations (g/mL) of the isolated compounds against

MA104 and HeLa cells with the standard errors of each result ................................ 236

List of Figures

xxix


Figure 8.1

An overview of the HPLC-piloted fractionation of Aqueous Stage 2 Separation

fraction 16 (AS2:16) showing the media used and fractions produced at each

stage of separation..................................................................................................... 243

Figure 8.2

A 3D HPLC profile showing the retention times and corresponding UV profiles

of the comprising compounds of Aqueous Stage 2 Separation fraction 16

(AS2:16) in 35% MeOH/H2O ................................................................................... 244

Figure 8.3

HPLC chromatograms of AS2:16 (FG) with the main fractions produced from

the separation with Sephadex LH-20 gel in 35% MeOH/H2O at 254 nm ............. 245

Figure 8.4

(a) HPLC chromatograms of the supernatant and precipitate (compound 7) of

AS2:16 fraction FG:16 in 40% MeOH/H2O at 210 nm; and (b) the UV profile

of the isolated compound .......................................................................................... 247

Figure 8.5

(a) HPLC chromatograms of isolated compound 8 with AS2:16 fraction FG:21 in

35% MeOH/H2O at 254 nm; and (b) the UV profile of the isolated compound.... 248

Figure 8.6

HPLC chromatograms of the original isolated compound 2 (from AS2:6) with

the re-isolated compound from AS2:16 in 10% MeOH/H2O at 210 nm ............... 249

Figure 8.7

An overview of the HPLC-piloted fractionation of Aqueous Stage 2 Separation

fraction 20 (AS2:20) showing the media used and fractions produced at each

stage of separation..................................................................................................... 250

Figure 8.8

A 3D HPLC chromatogram showing the retention times and corresponding UV

profiles of the comprising compounds of Aqueous Stage 2 Separation fraction 20

(AS2:20) in 30% ACN/H2O...................................................................................... 252

Figure 8.9

HPLC chromatograms of Aqueous Stage 2 Separation fraction 20 (AS2:20) in

65% MeOH/H2O and 40% ACN/H2O at 210 nm .................................................. 252

List of Figures

xxx

Figure 8.10

HPLC chromatograms of a range of fractions from the separation of AS2:20 (AG)

with MPLC in 35% ACN/H2O at 210 nm ............................................................. 253

Figure 8.11

(a) HPLC chromatograms of AS2:20 separation fraction AG:48-70 (AG1) with

AG1:8, 9 and 15 as well as compound 9 (AG1:13-14) in 35% ACN/H2O at

210 nm; and (b) the UV profile of the isolated compound ................................... 254

Figure 8.12

(a) HPLC chromatogram of isolated compound 9 with AS2:20 separation fraction

AG:48-70 in 35% ACN/H2O at 210 nm; and (b) the UV profile of the

isolated compound .................................................................................................... 255

Figure 8.13

(a) HPLC chromatograms of AS2:20 Separation fraction AG:71-94 (AG2)

with fractions AG2:5-13, 15-19, 20, and 21-32 in 35% ACN/H2O at 210 nm;

and (b) the UV profile of the re-isolated compound 9 (AG2:5-13) and

S3 (AG2:21-32) ....................................................................................................... 256

Figure 8.14

HPLC chromatograms of Aqueous Stage 2 Separation fraction 20 (AS2:20) and

the combined AS2:20 fraction AFG:113-119 in 35% ACN/H2O at 210 nm......... 257

Figure 8.15

(a) HPLC chromatograms of AG:113-119 (AG3) fraction AG3:10-16 with

AG3:10-16:9-13 and 16-23 in 50% ACN/H2O at 210 nm; and (b) the UV

profile of the AG3:10-16:16-23 ............................................................................... 259

Figure 8.16

HPLC chromatograms of AG:113-119 (AG3) fraction AG3:17-21 with

AG3:17-21:16, 17-19, 20-22, 23, and 24-26 (compound 10) in 45% ACN/H2O

at 210 nm .............................................................................................................. 260

Figure 8.17

(a) HPLC chromatograms of isolated compound 10 with fraction AG3:17-21

in 40% ACN/H2O at 210 nm; and (b) the UV profile of the isolated compound.. 261

List of Figures

xxxi


Figure 9.1

The elucidated structure of 7, Kaempferol 3-O-gentibioside, with some key

HMBC correlations ................................................................................................... 265

Figure 9.2

The 1H (top) and 13C (bottom) NMR spectra of 7 in CD3OD at 400 MHz ............ 266

Figure 9.3

TLC plate showing the Rf values of authentic glucose and galactose, as well as

those of the hydrolysis products of 7 ....................................................................... 269

Figure 9.4

The elucidated structure of 8, Quercetin glucoside, with some key HMBC

correlations and the 1H NMR spectra in CD3OD at 500 MHz ................................. 271

Figure 9.5

TLC plate showing the Rf values of authentic glucose and galactose, as well as

those of the hydrolysis products of 8 ....................................................................... 274

Figure 9.6

The elucidated structure of 9, an acylated kaempferol tetraglycoside ...................... 276

Figure 9.7

The 1H (top) and 13C (bottom) NMR spectra for compound 9 in CD3OD

at 500 MHz .............................................................................................................. 277

Figure 9.8

Acylated kaempferol hexaglycoside isolated by Crublet et al. (2003) .................... 280

Figure 9.9

The Rf values of SD1 (rhamnose/glucose 3:1), SD2 (rhamnose/galactose 3:1)

and the hydrolysis products of crude fraction AS2:20.............................................. 281

Figure 9.10

Some key HMBC relationships of 9 ......................................................................... 282

Figure 9.11

Some key ROESY interactions of 9......................................................................... 284

Figure 9.12

The elucidated structure of 10, an acylated kaempferol pentaglycoside with the 1H NMR spectra in CD3OD at 400 MHz .................................................................. 290

List of Figures

xxxii

Figure 9.13

Diagram of the reference compound used in Tables 9.9 and 9.10

(Crublet et al., 2003). R refers to a rhamnose unit ................................................... 290

Figure 9.14

Comparison of the original 1H NMR spectra of 10 in CD3OD at 400 MHz (top)

and at 500 MHz (bottom) where a transition to the trans configuration is evident .. 293

Figure 9.15

Comparison of the 1H spectra of 10 after long-term storage (top) and 9 (bottom)

in CD3OD at 500 MHz ............................................................................................. 293

Figure 9.16

1H spectrum of S3 in CD3OD at 500 MHz (top) and an expansion of the aromatic

region of the spectrum (bottom) showing the presence of two

configurational isomers ............................................................................................ 295

Figure 9.17

1H spectrum of S4 in CD3OD at 400 MHz (top) and an expansion of the aromatic

region of the spectrum (middle). After short-term storage, the cis configuration

becomes less predominant (bottom) ........................................................................ 297

Figures from Appendix 1

Figure A1.1

Elucidated structures of compounds 1, prodelphinidin (top) and 2, gallocatechin

(bottom) .................................................................................................................... 338

Figure A1.2

The COSY (top) and HSQC (bottom) for compound 1 in D2O at 400 MHz............ 339

Figure A1.3

The HMBC in D2O at 400 MHz (top) and the ESIMS (bottom) for compound 1.... 340

Figure A1.4

The 13C NMR spectra for compound 2 in CD3OD at 400 MHz ............................... 341

Figure A1.5

The COSY in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound 2 342

Figure A1.6

Elucidated structures of compounds 3, 9-HOTE (top) and 4, 9-HODE (bottom) .... 343

List of Figures

xxxiii

Figure A1.7

The COSY (top) and HSQC (bottom) NMR spectra for compound 3 in

CD3OD at 400 MHz .................................................................................................. 344

Figure A1.8

The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound 3 .... 345

Figure A1.9


CD3OD at 400 MHz .................................................................................................. 346

Figure A1.10


Figure A1.11

Elucidated structures of compounds 5, Hyptatic acid -A (top) and 6a, 3-O-cis-p-

coumaroyl tormentic acid (bottom). Compound 6b is the trans isomer................... 348

Figure A1.12


CD3OD at 400 MHz .................................................................................................. 349

Figure A1.13


Figure A1.14


CD3OD at 400 MHz .................................................................................................. 351

Figure A1.15

The HMBC in CD3OD at 400 MHz (top) and ROESY in CD3OD at 500 MHz

(bottom) for compound 6 ......................................................................................... 352

Figure A1.16

The ESIMS for compound 6 ..................................................................................... 353

Figure A1.17

Elucidated structures of compounds 7, kaempferol gentiobioside (top) and 8,

Quercetin glucoside (bottom) ................................................................................... 354

Figure A1.18

The COSY (top) and HSQC (bottom) NMR spectra for compound 7 in CD3OD

at 400 MHz .............................................................................................................. 355

Figure A1.19


List of Figures

xxxiv

Figure A1.20


at 400 MHz................................................................................................................ 357

Figure A1.21

The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound 8 ... 358

Figure A1.22

Elucidated structures of compounds 9, Acylated kaempferol tetraglycoside (top)

and 10, acylated kaempferol pentaglycoside (bottom) ............................................. 359

Figure A1.23


at 400 MHz................................................................................................................ 360

Figure A1.24

The HMBC in CD3OD at 400 MHz (top) and ROESY in CD3OD at 500 MHz

(bottom) for compound 9 ......................................................................................... 361

Figure A1.25

The HRESIMS (top) and the IR spectrum (bottom) for compound 9....................... 362

Figure A1.26

The ESIMS (top) and COSY in CD3OD at 500 MHz (bottom) for compound 10 ... 363

Figure A1.27

Elucidated structures of compounds S1, Pyrogallol (top) and S2,

24-hydroxytormentic acid (bottom) ..... .................................................................... 364

Figure A1.28

The COSY (top) and HSQC (bottom) NMR spectra for compound S1 in CD3OD

at 400 MHz .............................................................................................................. 365

Figure A1.29

The HMBC in CD3OD at 400 MHz (top) and GC-MS library match (bottom) for

compound S1............................................................................................................. 366

Figure A1.30

The COSY (top) and HSQC (bottom) NMR spectra for compound S2 in CD3OD

at 400 MHz................................................................................................................ 367

Figure A1.31

The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound S2.. 368

Figure A1.32

The COSY in CD3OD at 400 MHz (top) and ESIMS (bottom) for S3..................... 369

List of Figures

xxxv

Figure A1.33

The COSY (top) and HSQC (bottom) NMR spectra for S4 in CD3OD at

400 MHz .................................................................................................................. 370

Figure A1.34

The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for S4.................... 371

Figures from Appendix 2

Figure A2.1

The percent inhibition of MA104 cells and HeLa cells per concentration of each

compound isolated from the aqueous extract. The concentration of each compound

closest to the IC50 is given......................................................................................... 374

Figure A2.2

The percent inhibition of MA104 cells and HeLa cells per concentration of each

compound isolated from the methanol extract. The concentration of each

compound closest to the IC50 is given....................................................................... 375

List of Compound Structures

xxxvi

List of Compound StructuresList of Compound StructuresList of Compound StructuresList of Compound Structures

Compounds described in Chapter 1

1.1 Morphine ....................................................................................................... 2

1.2 Quinine.......................................................................................................... 2

1.3 Berberine ....................................................................................................... 12

1.4 Strychnine ..................................................................................................... 12

1.5 Scopoletin...................................................................................................... 14

1.6 Suksdorfin ..................................................................................................... 14

1.7 Benzoquinone................................................................................................ 14

1.8 Anthroquinone .............................................................................................. 14

1.9 Plumbagin ..................................................................................................... 14

1.10 Menthol ......................................................................................................... 15

1.11 Thymol .......................................................................................................... 15

1.12 1,8-Cineole .................................................................................................... 15

1.13 Artemisinin.................................................................................................... 16

1.14 -Carotene ..................................................................................................... 16

1.15 Squalene ........................................................................................................ 16

1.16 Ergosterol-5,8-endoperoxide......................................................................... 17

1.17 Oleanolic acid................................................................................................ 17

1.18 Ursolic acid ................................................................................................... 17

1.19 Jujubogenin saponins .................................................................................... 17

1.20 Steroidal saponins ......................................................................................... 17

1.21 Salicin............................................................................................................ 20

1.22 Castanospermine ........................................................................................... 20

1.23 Tubocurarine ................................................................................................. 20

1.24 Sanguarine..................................................................................................... 24

1.25 Epicatechin gallate ........................................................................................ 24

1.26 Carbazole alkaloid......................................................................................... 25

1.27 Rubraxanthone .............................................................................................. 25


xxxvii

1.28 Dehydroxycostuslactone ............................................................................... 25

1.29 Vincristine ..................................................................................................... 27

1.30 Vinblastine .................................................................................................... 27

1.31 Paclitaxel ....................................................................................................... 28

1.32 Camptothecin ................................................................................................ 29

1.33 Combretastatin A-4 ....................................................................................... 29

1.34 Standard numbering of flavonoids ................................................................ 33

1.35 2,2'-Dihydroxychalcone ................................................................................ 33

1.36 Apigenin ........................................................................................................ 33

1.37 Hesperetin ..................................................................................................... 33

1.38 Delphinidin.................................................................................................... 33

1.39 Daidzein ........................................................................................................ 33

1.40 -D-Glucose .................................................................................................. 34

1.41 -L-Rhamnose ............................................................................................... 34

1.42 Xylose ........................................................................................................... 34

1.43 -Arabinose................................................................................................... 34

1.44 Cinnamic acid................................................................................................ 35

1.45 p-Coumaric acid ............................................................................................ 35

1.46 Caffeic acid ................................................................................................... 35

1.47 Tiglic acid...................................................................................................... 35

1.48 Malonic acid.................................................................................................. 35

1.49 Galangin ........................................................................................................ 38

1.50 Datiscetin....................................................................................................... 38

1.51 Kaempferol.................................................................................................... 38

1.52 Morin............................................................................................................. 38

1.53 Quercetin ....................................................................................................... 38

1.54 Myricetin ....................................................................................................... 38

(Structures obtained from Wishart D.S., 2007, unless otherwise specified in the text)


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Compounds described in Chapter 2

2.1 Barringtogenol C........................................................................................... 49

2.2 16-Deoxy-barringtogenol C .......................................................................... 49

2.3 16,21,22,28-Tetrahydroxyolean-12-en-3-one......................................... 49

2.4 21,22-Ditigloylbarringtogenol C-28-O

by jacqui m. mcraeacqui m. mcrae - swinburne … that he always had time for a chat and was ready to...

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