-
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
- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2006. Antibacterial
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
- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2005. Review of the
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.
- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2004. Review of the
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
- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2007. Antibacterial
constituents of the Australian traditional medicinal plant, Planchonia careya
(Lecythidaceae), In: Proceedings of the 2007 Bioprocessing Network Annual
Conference, Clayton, Victoria.
- McRae, J.M., Yang, Q., Crawford, R.J., Palombo, E.A., 2007. Antibacterial
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
-
Table of Contents
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
4.4.2.2. Stage 2 Separation .......................................................................... 98
4.4.2.3. Stage 3 Separation .......................................................................... 98
4.4.2.4. Isolation of Compound 1 ................................................................. 98
4.4.2.5. Isolation of Compound 2 ................................................................. 99
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
4.4.4.1. Initial separation ............................................................................. 103
4.4.4.2. Isolation of Compound 7 ................................................................. 103
4.4.4.3. Isolation of Compound 8 ................................................................ 104
4.4.5. Investigation of Aqueous Stage 2 Separation Fraction 20 .................... 104
4.4.5.1. Initial separation ............................................................................. 104
4.4.5.2. Isolation of Compound 9 ................................................................. 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
-
Table of Contents
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
5.2.3.2. Aqueous Stage 2 Separation: Separation with
reverse-phase C18 media.................................................................. 130
5.2.3.3. Aqueous Stage 3 Separation: Separation with
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
Tables from Chapter 2
Table 2.1
Medicinal uses of some Lecythidaceae species ........................................................ 46
Tables from Chapter 3
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
Activity of fractions from the Preliminary Aqueous Stage 2 Separation (PA2)
with C18 media against B. cereus ............................................................................. 75
Table 3.11
Activity of fractions from the Preliminary Aqueous Stage 3 Separation (PA3)
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
Activity of fractions from the Preliminary Methanol Stage 3 Separation (PM3)
with C18 media against B. cereus, determined with PHDAs ................................... 82
Table 3.15
Activity of fractions from the Preliminary Methanol Stage 4 Separation (PM4)
with preparative HPLC against B. cereus, determined with PHDAs........................ 84
Tables from Chapter 5
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
The antibacterial activity and concentrations of fractions from the Aqueous Stage 2
Separation (AS2) using C18 media........................................................................... 132
Table 5.4
The antibacterial activity and concentrations of fractions from the Aqueous Stage 3
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
Tables from Chapter 6
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,
compared with those given in the literature .............................................................. 190
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,
compared with those given in the literature .............................................................. 196
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
Tables from Chapter 7
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
Tables from Chapter 9
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
Figures from Chapter 2
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
Figures from Chapter 3
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
Figures from Chapter 5
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
(a) Fractions AS2:13 and 14 in 30% MeOH//H2O at 210 nm; and (b) Fractions
AS2:15 and 16 in 40% MeOH//H2O at 210 nm .................................................... 137
Figure 5.6
(a) Fractions AS2:17 and 18 in 50% MeOH//H2O at 210 nm; and (b) Fractions
AS2:19 and 20 in 65% MeOH//H2O at 210 nm .................................................... 138
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
Figures from Chapter 6
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
Figures from Chapter 7
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
Figures from Chapter 8
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
Figures from Chapter 9
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
The COSY (top) and HSQC (bottom) NMR spectra for compound 4 in
CD3OD at 400 MHz .................................................................................................. 346
Figure A1.10
The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound 4 .... 347
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
The COSY (top) and HSQC (bottom) NMR spectra for compound 5 in
CD3OD at 400 MHz .................................................................................................. 349
Figure A1.13
The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound 5 .... 350
Figure A1.14
The COSY (top) and HSQC (bottom) NMR spectra for compound 6 in
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
The HMBC in CD3OD at 400 MHz (top) and ESIMS (bottom) for compound 7 .... 356
-
List of Figures
xxxiv
Figure A1.20
The COSY (top) and HSQC (bottom) NMR spectra for compound 8 in CD3OD
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
The COSY (top) and HSQC (bottom) NMR spectra for compound 9 in CD3OD
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
-
List of Compound Structures
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)
-
List of Compound Structures
xxxviii
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