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2017

Natural colours of Australia

Rudi Hendra University of Wollongong

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Recommended Citation Hendra, Rudi, Natural colours of Australia, Doctor of Philosophy thesis, School of Chemistry, University of Wollongong, 2017. https://ro.uow.edu.au/theses1/23

Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] NATURAL COLOURS OF AUSTRALIA

A thesis submitted in fulfillment of the requirements for the award of the degree of

DOCTOR OF PHILOSOPHY

UNIVERSITY OF WOLLONGONG

by

Rudi Hendra

S.Si., Apt., University of Padjadjaran (Indonesia) M.Sc, University of Putra Malaysia (Malaysia)

Supervisor: Prof. Paul A. Keller SCHOOL OF CHEMISTRY 2017

Declaration

I, Rudi Hendra hereby declare that this thesis, submitted in fulfilment of the requeirements for the award of the degree of Doctor of Phylosophy, in the School of

Chemistry, University of Wollongong, is fully my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institutions.

i

Abstract

The environment of Australia is rich with colourful plants and animals, which have been used for different purposes. This thesis describes the identification of fifty secondary metabolites from eight separate species Australian, including from flowers, parrot feathers, and moss.

Phytochemical studies on the flowers from four native Australian species (Brachychiton acerifolius, Anigozanthos rufus, Anigozanthos pulcherrimus, and Acacia pycnantha) and additionally, two introduced species (Tibouchina lepidota and Jacaranda mimosafolia) that are commonly grow in Australia, revealed a total of 36 secondary metabolites including five examples which may be responsible for the colouration of the flowers: These were pelargonidin-3-(6-coumaryl-β-glucoside)-5-(6-acetyl-β-glucoside)

47, malvidin 3-(p-coumarylglucoside)-5-acetylxyloside 58, cyanidin-3-rutinoside 62, cyanidin-3-O-(6-O-p-coumaryl)glucoside 65, and isosalipurposide 84) and four new secondary metabolites (2S)-4',5-dihydroxylavanone 7-O-β-D-glucuronide methyl ester

50, 2-amino-6-O-p-coumarylheptanedioic acid 64, chalcone-5'-O-(4-O-p-coumaryl)- glucoside 70, and phenylethanoid glucoside 73. All secondary metabolites isolated in these studies were the first reported from the flowers of the species. Furthermore, the polar extracts from B. acerifolius and T. lepidota, and compounds 46, 49, and 58 showed inhibition of growth of Staphylococcus aureus (ATCC 43300) and

Acinetobacter baumannii (ATCC 19606) at concentration value of 32 µg/mL.

The second part of this thesis used natural product techniques to fully characterize the chemical components from the feathers of Australian parrots. Australian king parrot

(Alisterus scapularis) feathers were initially investigated since this species was the most ii abundant deceased specimen available during the sample collection period. Standard extraction protocols were successfully developed and involved a soxhlet apparatus and methanol in contrast to published methods of acidic pyridine extraction in which our studies showed the generation of unwanted non-natural products. The GC-MS analysis of the hexane fraction indicated four common fatty acid identified as methyl palmitate

(104), linoleic acid (105), methyl oleate (106), and methyl stearate (107). Furthermore, the methanol extract of green feathers of A. scapularis provided five major chemical components, identified as (3,4-dihdroxybenzoic acid (108), butanoic acid (109), 4- hydroxymethyl benzoate (110), p-hydroxybenzaldehyde (111), and genistein (112).

During the isolation of chemical constituents from the green feathers, we were not able to isolate and identifiy any psittacofulvins-typed compounds which have been previously reported to be responsible for the colouration in parrots feathers. In addition, these laboratory protocols can be applied to investigate the chemical constituents from different coloured feathers e.g. red feathers from this species and other species in order to accumulated data from a wide range of Australian parrots.

Our last natural product study was a re-evaluation the NMR spectroscopy data of biflavone compounds (113 and 114) from Australian populations of Ceratodon purpureus by following the same reported method of extraction and separation, then comparing the outcomes with Antarctic populations of this species. This resulted in the isolation and structural reassignment to 120 and 121, and an additional 3 different biflavanones-like compounds (119, 122 and 123). Compounds 120 and 121 were found to have similar spectroscopy data (MS and NMR) to 113 and 114, both were reassigned

5',3''''-dihydroxyamentoflavone and 2,3-dihydro-5',3''''-dihydroxyamentoflavone, respectively. All isolates absorbed in the ultraviolet spectrum and the two biflavones

iii

(120 and 121) showed relatively high antioxidant activities in comparison to the rutin positive control. These properties of 120 and 121 suggest that they are potentially involved in dual photoprotection mechanisms via both direct (UV-screening) and indirect (radical-scavenging) capacities in the moss and contributes to it survival mechanism in the harsh Antarctic environment.

iv

Acknowledments

First, I would like to give my big sincere thanks my supervisor, Prof. Paul A. Keller, for giving me such a great opportunity to study of Australian natural products, guidance and support during the whole of my study in the School of Chemistry University of

Wollongong. I have learnt so many things under your supervison, for helped me in developing scientific writing, and for being there every time I had a question. I would like also to thank to Dr Ari Nugraha for being such a good friend, mentor, and helping me during my early study specialy in elucidation structure. Another special thanks to

Prof. Sharon A. Robinson and Dr Mellinda Waterman who with my supervisor allowed me to involve in the Antarctic moss (Ceratodon purpureus) project.

I am also greatly indebted to to the following people who provided their help in completing this study: Dr Wilford Lie and Dr. Graham E. Ball for the NMR discussion and guideline, Dr Celine Kelso, Dr Alan Maccarone, Karin Maxwell for the Mass spectroscopic support, Mr Joe Daunt for the HPLC support, Dr. Anthony Willis for the crystallography, Australian National Wildlife Collection, CSIRO, Canberra and Cannon

& Ball veterinary surgeons, Wollongong, Australia for supplying the parrots samples,

Mr. Nathan Butterworth for GCMS support, Chemistry society UOW and Monica

Birrento for giving me an experience as one of executive former and current KRG member: Alex, Alireza, Andrew, Aaron, Akash, Ashraf, Adel, Hendris, Yueting,

Sreenu, Phuong, Matt, Stephen Bailey, Stephen Tibben, Steve, Harry, Patrick, Jayden,

Mohammed, you are chemistry rockers. Special thanks to dynamic duo (Mr Jamie

Smyth and Mr Nicholas Butler) for being great friend and time in the lab and office, you

v are real marvel chaps and chemists. I would also like to thank the rest of school of chemistry members for showing me a great time during my study.

Special appreciation to Dr. Glennys O’Brien for giving me an opportunity in the first- year teaching lab which were a valuable experience for my future academi career. I would like to thank to the Indonesian Directorate General Higher Education (DGHE) for providing scholarship and University of Wollongong for International Postgraduate

Tuition Award (IPTA). Extended thanks go to Prof. Adel Zamri, Dr Hilwan Y. Teruna,

Dr Christine Jose, Dr Yum Eryanti, Dr Yuana Nurulita, Yuharmen, Nurbalatif, Yuli

Haryani, Ganis Fia Kartika at the Department of chemistry University of Riau for giving me support and being not only as colleague but also mentor, friend, and family.

Finally thank to my family for infinite love and support, my parents, Rosni and the late

Syamsuddin MY, Afrizal, Meri, Yoel, Budi, Lisa, Dewi, and all my nephews and nieces, I never thank you enough. I would also like to thanks to my extended family: om

Aka, om Elan, om Rizky, om Widi, nenek Homsah, tante Tami, tante Unink, Haning,

Laura, Ajeng, mamah Pipoy, bude Yayuk, bowie Ully for your support and being such a great friend for almost two decades and counting. My Indonesian family in

Wollongong: Mika for always be there when I was grumpy and sharing ideas, Dr

Daniel, Dr Uuf, Dr Handoko, Iftitah, Dhimas, Randu, Mayang, Yani, Lita, Luki for all the great time. Last but not least, thanks to the Indonesia community in Wollongong for sharing experiences and friendship.

vi

Table of Contents

Declaration ...... i

Abstract ...... ii

Acknowledments ...... v

Table of Contents ...... vii

List of Awards and Funding ...... xii

List of Publications ...... xiii

List of Conference Abstracts and Presentations ...... xv

List of Abbreviations ...... xvi

List of Figures ...... xix

List of Tables ...... xxiii

Chapter 1: Introduction ...... 1

1.1. Natural Colours Chemistry ...... 1

1.1.1. Natural Colours: Carotenoids ...... 3

1.1.2 Natural colours: Betalains ...... 8

1.1.3 Natural colours: Anthocyanins ...... 11

1.2 Rationale and Aims of Research ...... 15

CHAPTER 2: Secondary Metabolites from Australian Flowers ...... 19

2.1 Introduction ...... 19

2.2. Phytochemical study of Brachychiton acerifolius flowers ...... 20

vii

2.2.1 Traditional medicinal uses and pharmacological activities of Brachychiton

genus ...... 22

2.2.2 Secondary metabolites from the genus Brachychiton ...... 24

2.2.3 Results and Discussion ...... 25

2.3 Natural product investigations on Tibouchina lepidota flowers ...... 31

2.3.1 Traditional medicinal uses and pharmacological activities of Tibouchina

genus ...... 32

2.3.2 Secondary metabolites from the genus Tibouchina ...... 33

2.3.3 Results and Discussion ...... 35

2.4 Natural products study on Anigozanthos Sp...... 38

2.4.1 Traditional uses of Anigozanthos genus ...... 39

2.4.2 Results and Discussion ...... 41

2.5 Natural product studies on Jacaranda mimosaefolia ...... 50

2.5.1 Medicinal uses of genus Jacaranda ...... 51

2.5.2 Secondary Metabolites from the genus Jacaranda ...... 52

2.5.3 Pharmacological activity studies on the genus Jacaranda ...... 54

2.5.4 Results and Discussion ...... 56

2.6 Natural product studies on Acacia pycnantha ...... 59

2.6.1 Traditional medicinal usage and pharmacological activities of the Acacia

genus ...... 61

2.6.2 Phytochemical studies of Acacia Genus ...... 68

2.6.3 Results and Discussion ...... 70 viii

2.7 Antimicrobial activities of flowers in Australia ...... 75

2.8 Conclusion ...... 79

CHAPTER 3: Exploration of Secondary Metabolites from Alisterus scapularis .... 77

3.1 Introduction ...... 77

3.2 Chemical properties of pigment classes in birds feathers ...... 79

3.2.1 Carotenoids ...... 79

3.2.2 Melanin ...... 83

3.2.3 Porphyrins ...... 85

3.3 Structural colouration ...... 86

3.4 Parrots ...... 87

3.4.1 Mechanisms of Parrot Colouration ...... 87

3.4.2 Australian Parrots ...... 89

3.5 Results and Discussion ...... 91

3.6 Conclusion ...... 101

Chapter 4: Biflavones from Australian populations of Ceratodon purpureus ...... 103

4.1. Introduction ...... 103

4.2 Results and Discussion ...... 107

4.2.1. Isolation and structural elucidation of biflavones ...... 107

4.2.2 Ultraviolet and antioxidant activities of isolated biflavone compounds...... 118

4.3 Conclusion ...... 121

Chapter 5: Conclusions and Future Directions ...... 122

ix

Chapter 6: Experimental ...... 129

6.1 General ...... 129

6.2. Plant and feathers materials ...... 130

6.3. Isolation of constituents of Brachychiton acerifolius flowers ...... 131

6.4 Isolation of constituents of Tibouchina lepidota ...... 135

6.5 Isolation of constituents of Anigozanthos sp flowers ...... 139

6.6 Isolation of constituents of Jacaranda mimosifolia flowers ...... 146

6.7 Isolation of constituents of Acacia pycnantha flowers ...... 153

6.8 Isolation of constituents from Australian King Parrot green feathers ...... 157

6.8.1 Hot acidified pyridine ...... 157

6.8.2 TossueLyser with methanol ...... 157

6.8.3 Soxhlet extraction ...... 157

6.9. Isolation of constituents of Ceratodon purpureus ...... 160

6.9.1. Intracellular sample preparation of Australian C. purpureus ...... 160

6.10. Bioactivity testing...... 163

6.10.1. Antimicrobial ...... 163

6.10.2 Free radical scavenging assays ...... 164

Bibliography ...... 165

Appendix A: New Anthocyanins reported in 1998 – 2003. 43-44 ...... 182

Appendix B: Spectroscopic and spectrometric data of Brachychiton acerifolius constituents ...... 186 x

Appendix C: Spectroscopic and spectrometric data of Anigozanthos sp constituents

...... 188

Appendix D: Spectroscopic and spectrometric data of Jacaranda mimosafolia constituents ...... 194

Appendix D: Spectroscopic and spectrometric data of C. purpureus constituents 197

Appendix E: Crystallography data of Acacia pycnantha constituents ...... 204

xi

List of Awards and Funding

1. Directorate General of Higher Education (DGHE) of Indonesia Postgraduate

Scholarship.

2. A$1,000 for attending ‘RACI National Congress, Adelaide, Australia, 7th – 12th

December, 2014, Awarded by SMAH HDR Student Travel Grant, University of

Wollongong.

3. RACI Young Chemists Encouragement Awards 2014, the School of

Chemistry’s 2014 Annual Conference, University of Wollongong, Australia.

4. A$2,000 for attending ‘The 29th International Symposium on the Chemistry of

Natural Products (ISCNP-20), Izmir, Turkey, 24th – 27th September, 2016,

Awarded by SMAH HDR Student Travel Grant, University of Wollongong.

5. International Postgraduate Tuition Award, 2016, University of Wollongong.

xii

List of Publications

1. Yueting Liu, Rudi Hendra, Aaron J. Oakley, Paul A. Keller, Efficient synthesis

and antioxidant activity of coelenterazine analogues, Tetrahedron Letters, 2014,

55, 6212-6215.

2. Rudi Hendra, Paul A. Keller, Flowers in Australia: Phytochemical Studies on

the Illawarra Flame Tree and Alstonville, Australian Journal of Chemistry, 2016,

69, 925–927. (Journal front cover)

3. Rudi Hendra, Paul A. Keller, Phytochemical Studies on Two Australian

Anigozanthos Plant Species, 2017 (submitted to Journal of Natural Products).

4. Melinda J. Waterman, Ari S. Nugraha, Rudi Hendra, Graham E. Ball, Sharon

A. Robinson, Paul A. Keller, Antarctic moss biflavones show high antioxidant

and ultraviolet screening activity, 2017 (submitted to Journal of Natural

Products).

xiii

xiv

List of Conference Abstracts and Presentations

1. Rudi Hendra, Stephen Tibben., Paul A. Keller. Colours of Australia: Natural

Products from the Feathers of Australian Parrots, RACI NPG One Day

Symposium, University of New South Wales, Australia, 4 October 2013. (Poster

presentation)

2. Rudi Hendra, Paul A. Keller, Phenolic Compounds from Alstonville

(Tibouchina lepidota) Flower, RACI National Congress, Adelaide, Australia, 7 –

12 December 2014. (Poster presentation)

3. Rudi Hendra, Paul A. Keller, COLOURS OF AUSTRALIA: Phytochemical

Study of Illawara Flame Tree Flower (B. acerifolius), RACI NPG One Day

Symposium, University of Western Sydney, Australia, 2 October 2015. (Poster

presentation)

4. Rudi Hendra, Paul A. Keller, Natural Colours of Australia: Australian flowers

and Their Antibacterial Activities, The 29th International Symposium on the

Chemistry of Natural Products, Izmir, Turkey, 24 – 27 September 2016. (Oral

Presentation)

5. Rudi Hendra, Paul A. Keller, Natural Colours of Australia: Australian flowers

and Their Antibacterial Activitiies, RACI NPG One Day Symposium,

University of Wollongong, Australia, 30 September 2016. (Oral Presentation)

xv

List of Abbreviations

13C-NMR Carbon nuclear magnetic resonance

1H-NMR Proton nuclear magnetic resonance

ACN Acetonitrile

AHP Aminohydroxyphenylalanine

AMD Age-related macular degeneration

APT Attach proton test

ATCC American type culture collection

CD Circular dichroism

CHI Chalcone isomerase

CHS Chalcone synthase

CoA Coenzyme A

COSY Correlation spectroscopy

DCM Dichloromethane

DFR Dihroxyflavonol-4-reductase

DMBA 7,12-Dimethylbenz(a)anthracene

DNA Deoxyribonucleic acid

DOXP 1-Deoxyxylulose-5-phospate

DPPH 2,2-Diphenyl-1-picrylhydrazyl

GC Gas chromatography

GC-MS Gas Chromatography-Electronimpact Mass Spectrometry

GGPP Geranylgeranylpyrophosphate gHMBC Heteronuclear multiple bond correlation gHSQC Heteronuclear single quantum coherence

HPLC High performance liquid chromatography

HRESIMS High resolution electrospray mass spectrometry xvi

IGF-I Insulin-like growth factor 1

IOSE Immortalized ovarian epithelium cells

IPP Isopenthenyl pyrophosphate

IR Infrared

LCYB Lycopene β-cyclase

LCYE Lycopene ε-cyclase

L-DOPA L-3,4-Dihydroxyphenylalanine

LRESIMS Low Resolution Electrospray Mass Spectrometry

MHz Megahertz

MIC Minimum inhibition concentration

MIZ Minimum inhibition zone

MRSA Methicillin Resistant Staphylococcus aureus

MS Mass spectroscopy

NMR Nuclear Magnetic Resonance

NSW New South Wales

NSY Neoxanthin synthase

ORAC Oxygen radical absorbance capacity

OVCA420 Ovarian serous adenocarcinoma

P-388 Limphocytic leukaemia cells

PDS Thytoene desaturase

PSY Phytoene synthase

PTCA Pyrrole-2,3,5-tricarboxylic acid

RPE Retinal pigment epithelium

RP-HPLC Reverse phase high performance liquid chromatography

SKOV3 Ovarian carcinoma cells

TFA Trifluoric acid

xvii

TOCSY Total correlation spectroscopy tR Retention time

UV Ultraviolet

ZDS ξ-carotene desaturase

ZEP Zeaxanthin epoxidase

xviii

List of Figures

Figure 1. Chemical structure of mauveine (1) and indigo (2)...... 2

Figure 2. Some common carotenoids...... 4

Figure 3. Biosynthesis of carotenoids in plants...... 6

Figure 4. General structure of betalains...... 8

Figure 5. Biosynthesis pathways of the betalains...... 9

Figure 6. Structures of the major anthocyanins-3-O-glycoside present in plants and

fruits...... 11

Figure 7. Anthocyanins chemical forms depending on pH...... 12

Figure 8. Biosynthetic pathway of anthocyanin...... 14

Figure 9. Plant classification of Brachychiton acerifolius...... 21

Figure 10. Flower of B. acerifolius (a) and the B. acerifolius tree (b)...... 21

Figure 11. Sterculinine I (44) and II (45) isolated from seeds of Sterculia lychnophora.

...... 24

Figure 12. RP-HPLC profile of the ethyl acetate fraction...... 25

Figure 13. Flavonoids and glycosides isolated from B. acerifolius flowers...... 26

Figure 14. Circular dichroism (CD) spectra of compound 49 and 50 were compared

with 2S-(49) and 2R-(50)...... 30

Figure 15. Plant classification of Tibouchina lepidota...... 31

Figure 16. (a) Flowers of T. lepidota, (b) T. lepidota tree...... 32

Figure 17. Isolated phenolic derivative (52) and quercetin-3-glucoside (53) from the

aerial component of T. paratropica...... 33

Figure 18. RP-HPLC profile of the ethyl acetate extract indicating the targets to be

isolated...... 36

xix

Figure 19. Phenolic, flavonoids and glycosides isolated from T. lepidota flowers...... 36

Figure 20. Plant classification of Anigozanthos Sp...... 39

Figure 21. Red kangaroo paw (A. rufus); Yellow kangaroo paw (A. pulcherrimus). .... 39

Figure 22. Phenylphenalenone pigments isolated from whole plant of Anigozanthos

rufus...... 40

Figure 23. HPLC profile of ethyl acetate from A. flavidus (A) and A. pulcherrimus (B)

flowers (zoom)...... 42

Figure 24. Molecular structures isolated from flower of Anigozanthos sp...... 43

Figure 25. Chemical structure of 64 isolated from A. rufus and A. pulcherrimus flowers.

...... 44

Figure 26. Key HMBC and COSY correlations observed for compound 64...... 46

Figure 27. Key HMBC and COSY correlation for compound 70...... 49

Figure 28. Plant classification of Jacaranda mimosifolia...... 51

Figure 29. Jacaranda mimosifolia flowers and leaves...... 51

Figure 30. Jacaranone (72), isolated from the twigs-leaves of J. caucana...... 53

Figure 31. Reverse-phase (RP) HPLC profile of polar extract from J. mimosafolia

flowers...... 56

Figure 32. Isolates obtained from J. mimosafolia flower...... 57

Figure 33. Long range proton-carbon (HMBC) of compound 73...... 58

Figure 34. Acacia pycnantha scientific classification...... 60

Figure 35. A. pycnantha flower, collected from the University of Wollongong...... 61

Figure 36. Reverse-phase (RP) HPLC profile of the ethyl acetate extract from A.

pycnantha flower...... 71

Figure 37. The structure of compounds 80 – 85 isolated from A. pycnantha flower. .... 72

Figure 38. Circular dichroism (CD) spectra of compound 80 and 81...... 73 xx

Figure 39. Absolute configuration of compound 81, as determined by X-Ray

crystallography...... 74

Figure 40. Budgerigar’s head (A) under white light and under UV light (B)...... 78

Figure 41. Eight isoprenoid units (85), common carotenoids compound (4-7, 9-10)

from bird feathers, carotenoids present in parrot blood (5, 6, 86-88)...... 81

Figure 42. Isolated methoxy carotenoids from the feathers of Xipholena punicea...... 83

Figure 43. Degradation of eumelanin (95) and phaeomelanin (97) from bird ferather to

PTCA (96) and AHP (98)...... 84

Figure 44. Turacin (99) from Turaco (Musophagidae)...... 86

Figure 45. Possible chemical structures of psittacofulvins...... 88

Figure 46. The illustration of faunal areas of Australia, proposed by (a) Spencer, (b)

Kikkawa and Pearse...... 90

Figure 47. Examples of specimens obtained from Cannon & Ball veterinary surgeons,

Wollongong, Australia and CSIRO, Canberra...... 92

Figure 48. Australian King Parrot male, obtained from Australian National Wildlife

Collection, CSIRO...... 93

Figure 49. Male Alisterus scapularis under visible and long wave UV light (λ = 365

nm)...... 94

Figure 50. Green feathers before (left) and after (right) extraction with methanol in a

soxhlet apparatus...... 96

Figure 51. RP-HPLC profiles from two different extraction methods with green

feathers...... 97

Figure 52. GC-MS chromatogram of the hexane fraction from green feathers...... 98

Figure 53. Semi-preparative HPLC profile of polar (methanol) fraction...... 99

xxi

Figure 54. Secondary metabolites in the methanol fraction from Australian King Parrot

green feathers...... 99

Figure 55. Ceratodon purpureus scientific classification...... 104

Figure 56. Ceratodon purpureus growing on Antarctic continent...... 104

Figure 57. a) Intracellular isolates and b) cell wall isolates from Australian C.

purpureus...... 105

Figure 58. Typical 13C NMR chemical shifts of hydroxyl substitution in flavone...... 106

Figure 59. Semi-preparative HPLC profile of the methanol extract of C. purpureus. 107

Figure 60. Biflavones isolated from the intracellular space of Australian C. purpureus.

...... 108

Figure 61. A, B, C-rings (top left), D, E, F-rings (bottom left) of proposed flavone unit

of compound 120. Long range proton-carbon correlation (gHMBC) of

compound 120 (right)...... 109

Figure 62. gHMBC spectrum (zoom) for key evidence establishing the position of the

biaryl link in ring B to ring D of 120...... 111

Figure 63. A, B, C-rings (top left), D, E, F-rings (bottom left) of proposed flavone unit

of compound 121. gHMBC of compound 121 (right)...... 114

Figure 64. gHMBC spectrum (zoom) for key evidence establishing the position of the

biaryl link in ring B to ring D of 121...... 115

Figure 65. Australian and Antarctic population of C. purpureus HPLC profiles...... 117

Figure 66. Extinction coefficients in the UV-Vis spectrum (200 – 500 nm) of biflavones

in comparison to the rutin standard……………………………………... 119

xxii

List of Tables

Table 1. Secondary metabolites from genus Tibouchina ...... 34

Table 2. NMR spectroscopic data for compound 64 ...... 44

Table 3. NMR Spectroscopic Data for compound 70...... 47

Table 4. Traditional medicinal uses of Jacaranda species...... 52

Table 5. Secondary metabolites isolated from Jacaranda species...... 53

Table 6. NMR spectroscopic data for compound 73...... 59

Table 7. Ethnopharmacological data of Australia Acacia...... 62

Table 8. Pharmacological activities from Genus of Acacia...... 62

Table 9. Phytochemical compounds isolated from Acacia species...... 68

Table 10. Antibacterial activity of six plant extracts and isolated compounds...... 77

Table 11. 1H and 13C NMR spectroscopic data for compounds 120 and 121 (in

(CD3)2CO)...... 112

Table 12. Concentrations of isolates 119-123 within Australian and Antarctic samples

of C. purpureus based on HPLC analysis...... 118

Table 13. Ultraviolet and antioxidant activities of isolated biflavones compared to rutin

standard for DPPH antioxidant assays………………………………….. 119

xxiii

Chapter 1: Introduction

1.1. Natural Colours Chemistry

Mankind has been using natural products from plants and animals for their survival since the prehistoric era. Plants are one of the most common elements from the biosphere for food, clothing, dyes, medicines, and cosmetics,1-2 with the first written records of their use for medicinal purposes dating back to approximately 2600 BC in

Mesopotamia.3 There has been a growing interest in using natural products, especially those derived from plants, and they have become one of the most important starting points in drug discovery with more than one hundred therapeutics being synthesized or isolated from natural products in the last 20 years.4

Natural products can be classified as one of three main categories - primary, cellular, and secondary metabolites – of which secondary metabolites often attract the greatest interest due to their biological effects.5 They are often non-essential for the organism to survive, but can serve as pheromones, toxins and repellents.5 Natural product-guided drug discovery is typically associated with secondary metabolites and it has been reported that there may be over 10,000 different examples of natural products which possess biological activity in humans.6

Natural products have also been used for dye purposes, e.g. in prehistoric times, man used natural substances from plants and animals to dye furs, textiles, and other objects using a variety of techniques. There is evidence that ancient cave drawings used both inorganic and organic pigments.7 Later, in the Mediterranean civilization, natural pigments such as indigo were utilised for their blue colours, anthraquinone-based chromophores for the reds, 6,6'-dibromoindigo for purple, and carotenoids for yellow.8

The use of natural colours continues to emerge, in particular in the food and textile industries. This trend has accelerated in recent times, as a result of increased consumer demand for natural products.9

Nature expresses itself in a wide spectrum of colours and this spectrum ranging from yellow to black.10 The term natural colourants covers all dyes and pigments derived from plants, insects and minerals.11-12

Pigments are chemical compounds that absorb light in the wavelength range of the visible region.12-13 Before William Perkin synthesised mauveine (1) (Figure 1) in 1856, the Mediterranean people from ancient times extracted mauve/purple colours from marine sea shells and indigo (2) from Indigofera (Figure 1).8

Figure 1. Chemical structure of mauveine (1) and indigo (2).

In addition, colours in plants have drawn attention throughout history with flowers and fruits used not only as artistic ornaments, but they have been found to possess various biological activities.14-15 Anthocyanins and carotenoids are believed to have antioxidant properties which have been extensively studied, as well as anti-inflammatory, and anti- carcinogenic activity.14, 16 In bird colouration, recent reports have suggested that some

2 pigments in bird feathers function not just as colour generators, but also preserve plumage integrity by increasing the resistance of feather keratin to bacterial degradation.17

Natural pigments produced by living organisms can be classified into six major groups, the carotenoids, tetrapyrroles, indolic biochromes, N-heterocyclic biochromes, oxygenous heterocyclic biochromes (flavonoids), and quinones.12 In the following section, the more abundant carotenoids, flavonoids/anthocyanins, betalains, and melanins are described more thoroughly.

1.1.1. Natural Colours: Carotenoids

Carotenoids contain a family of more than 600 pigments which are produced in plants, mosses, algae, bacteria, and fungi.18 They are tetraterpenoids, composed of a central carbon chain of alternating single and double bonds and carry different cyclic or acyclic end groups.19 Carotenoids are classified by their chemical structures as carotenes and oxycarotenoids. They can also be classified as primary carotenoids (β-carotene (4) and violaxanthin), and secondary carotenoids (neoxanthin α-carotene (3), β-cryptoxanthin, zeaxanthin (6), antheraxanthin (8), capsanthin, and capsorubin).13 A selection of carotenoid structures are presented in Figure 2

3

Figure 2. Some common carotenoids.

In higher plants, carotenoids are accumulated in leaves, fruits and flowers but not in the roots, with the exception of carrots and sweet potato which contain β-carotene (4).20-21

Xanthophylls are the main carotenoids present in the flower petals of most plants and

4 these compounds are pale to deep yellow in colour. Astaxanthin (9) was found in large concentrations in the petals of Adonis aestivalis and Adonis annua, resulting in their blood-red colours.22 This carotenoid is rare in plant species but can be found in a number of bacteria, fungi, and algae, where it contributes toward interesting orange-red colours.23 Furthermore, carotenoids such as asthaxanthin (9), cantaxanthin (10), lutein

(5), violaxanthin, β-carotene (4) are found in microalgae such as Haematococcu pluvialis, Chlorella vulgaris, H. pluvialis, Scendedesmus obliquus, and D. salina. 24

Animals such as birds lack the required enzymes to produce carotenoids, and instead rely on dietary sources such as plants, and algae. Lutein (5), α-carotene (3), β-carotene

(4), zeaxanthin (6), and asthaxanthin (9) are found in the bird feathers (e.g. canaries, flamingo, goose, Robin) and parrot blood.25

The biosynthesis of carotenoids occurs in the plastids of plants (Figure 3). Isopentenyl pyrophosphate (IPP) (13) provides the five-carbon building block for carotenoids and is synthesized through the 1-deoxyxylulose-5-phospate (DOXP) pathway.

Geranylgeranylpyrophosphate (GGPP) (14) is formed from the condensation reaction of four IPPs and head-to-head coupling of GGPP, which is then catalysed by phytoene synthase (PSY) to the first C40 carotenoid (phytoene, 15). Phytofluene and ξ-carotene

(16) are formed by a desaturation reaction between thytoene desaturase (PDS) and ξ- carotene desaturase (ZDS) and conjugated double bonds are subsequently created. From this reaction, the pink lycopene (17) is formed from the colourless phytoene (15). The cyclization of lycopene (17) is catalysed by lycopene β-cyclase (LCYB) and lycopene ε- cyclase (LCYE). The pathway in plants typically proceeds only along branches, leading to carotenoids with one β- and one ε-ring (α-carotene (3) and its derivatives) or two β- rings (β-carotene (4)) and its derivatives. The modification of (3) and (4) by

5 hydroxylation or epoxidation, provides various structural analogues of carotenoids. For example, epoxidation at C5-6 and C5'-6' of the β-ring of zeaxanthin (6), catalysed by zeaxanthin epoxidase (ZEP), results in violaxanthin (11) with conversion to neoxanthin

(12) by neoxanthin synthase (NSY).23, 26

Figure 3. Biosynthesis of carotenoids in plants.23

1.1.1.1 Biological effects of carotenoids

The yellow to orange colours produced by carotenoids in some flowers, seeds, fruits, and fungi have important roles in reproduction. It was observed that intracellular 6 accumulation of carotenoids was involved in the later stages of zygospore development by inhibiting the cell-to-cell recognition system in fungus (Phycomyces blakesleanus).27

Carotenoids have also been known to function in photosynthesis as accessory pigments in light harvesting, and photoprotectors against oxidative damage.13

Carotenoids are tolerant to UV radiation and are able to prevent oxidative processes, e.g., lutein (5) and lycopene (17) effectively inhibited hydroperoxide formation of triglycerides.28 Carotenoids show pharmacological effects towards many diseases such as cancer, stroke, and degenerative diseases.13 In vitro studies showed lycopene and tocopherol inhibited the growth of human prostate carcinoma cell lines, due to a delay in cell cycle progression and diminished IGF-I receptor signalling.19 Astaxhantin (9) was reported to show anticancer activity against breast cancer cells in mice, and has also been reported to decrease liver metastasis induced by stress in mice.29

Furthermore, carotenoids play roles in protection against age-related macular degeneration (AMD), which can lead to irreversible blindness. In human or primate retina, the macula lutea (yellow spot) absorbs light in an absorption spectra range similar to the xanthophylls (lutein, zeaxanthin, and meso-zeaxanthin).19, 30 These carotenoids therefore play a role similar to that in plants as protective antioxidants in the eye. Another mechanism for the protective action of the macular carotenoids is the reduction of oxidative stress-induced damage, with this study showing that retinal pigment epithelium (RPE) cells treated with lutein had a reduced phototoxic effect. The presence of lutein and zeaxanthin has also been shown to reduce the amount of lipofuscin formed in cultured RPE cells.30

7

1.1.2 Natural colours: Betalains

The betalains are pigments with a nitrogen containing core structure of betalamic acid

(18) and can be categorized as violet betacyanins (19) and yellow betaxanthins (20)

(Figure 4).31 These pigments are not pH-dependant and are more stable than anthocyanins,23 and can be found in roots, fruits, and flowers in Caryophyllales.

Figure 4. General structure of betalains.14, 32

Red beet (Beta vulgaris subsp. Vulgaris) is the most abundant source of betalains and has been used as a food colourant known as beetroot red. Betalains are also detected in some higher fungi, e.g., the fly agaric (Amanita muscaria), Hygrocybe, and

Hygrosporus.13 To date, 472 genera of plants from eight different families of plants contain betalain compounds and 75 betalains have been studied.

The biosynthesis of betalains and the enzymes involved in this process is less understood compared to flavonoids and other secondary metabolites. It begins with the shikimic acid pathway and tyrosine amino acid pathway (Figure 5), with two molecules of tyrosine (21) forming L-DOPA (22). The next step is the transformation of 22 to

DOPA-quinone (23) which spontaneously converts to cyclo-DOPA (24). A second molecule of 20 reacts with DOPA 4,5-dioxygenase resulting in betalamic acid (18).

8

Furthermore, cyclo-DOPA (24) reacts with betalamic acid (18) followed by glycosylation or condensation of the cyclo-DOPA glycosides with betalamic acid to form betacyanins (19). The biosynthesis of betaxanthin (20) is not well-known but it is suggested the interchange of betalamic acid (18) is one of the main routes, e.g by the spontaneous condensation of betalamic acid (18) with amino acids or amines.13-14

Figure 5. Biosynthesis pathways of the betalains.13

1.1.2.1 Biological effects of betalains

The betalains have several applications in the food industry, including in confectionery, dry mixes, dairy and meat products. Betanin or betanidin 5-O-β-glucoside (isolated from red beet B.vulgaris) is one of the most important betalains and has been approved

9 as an additive for the use in food, drugs, and cosmetic products.33 Although they are useful as natural colourants, these compounds can also be used as plant taxonomic markers, e.g., they can be used to differentiate between species within the family of

Amaranthaceae, Aizoaceae, Basellaceae, Chenopodiaceae, Cactaceae, Nyctaginaceae,

Phytolaccaceae, Portulacaceae, Didieraceae, Caryophyllaceae, and Molluginaceae.13

There is also increasing interest in the antioxidant activities of betalains, e.g. betalain extracts from the hairy root cultures of the red beetroot (B. vulgaris) show high antioxidant activity against the 2,2-diphenyl-1-picrylhydrazyl radical with an EC50 value of 0.11 mg/mL. In addition, isolated betanin and betanidin from B. vulgaris inhibited linoleate peroxidation by cytochrome C with IC50 values of 0.4 and 0.8 µM, respectively. Betanin was found to inhibit H2O2-activated metmyoglobin catalysis of

34-35 low-density lipoprotein oxidation with an IC50 value of <2.5 µM. It was found that the radical scavenging activity of betalains is related to the number of phenolic hydroxyl, and imino groups, and the glycosylation attachment position from betacyanins, e.g. betanidin 6-O-β-glucoside showed high radical scavenging compared to 5-O-β-glucoside.36-37

Further, betalain extracts from cactus pear fruit (Opuntia ficus-indica) were introduced to immortalized ovarian epithelium cells (IOSE), ovarian cancer cell lines (OVCA420,

SKOV3, and bladder cancer cells (UM-UC-6, T24) inducing a significant increase in apoptosis and growth inhibition in all cancer cells. It also affected the cell cycle of cancer cells.38 The studies demonstrated that betanin from B. vulgaris inhibited DMBA-

UV-B, promoted skin carcinogenesis and related splenomegaly as well as the growth of liver cancer in mice treated with DEN as an initiator and phenobarbital as promotor.39

10

1.1.3 Natural colours: Anthocyanins

Anthocyanins are one of the most widespread families of natural pigments in the plant kingdom. They are water-soluble, occur in vascular plants and are responsible for the majority of the orange, red, purple, and blue colours of flowers and fruits.26, 40 The term

‘anthocyanin’ comes from the Greek anthos, a flower, and kyanos, dark blue and they belong to the class of flavonoids.

Anthocyanidins are the basic structures of the anthocyanins and consist of an aromatic ring A bonded to heterocyclic ring C containing oxygen, which is also bonded by a carbon–carbon bond to a third aromatic ring B (Figure 6). In nature, the anthocyanidins are found bonded to one or more sugar moieties and are known as anthocyanins (Figure

6).41 Figure 6 shows a selection of common anthocyanins present in plants and their chromatic features, which are significantly affected by the substitution of ring B. The addition of hydroxyl substitutions results in a hypochromic shift from red to violet colour by increasing electron density.40, 42 Over the past decade, more than 50 new anthocyanins have been reported from plants - not only from flowers, but also from leaves, stems, roots, tubers, fruits and seeds (a summary of these is listed in Appendix

A).43-44

Anthocyanidin R 1 R2 Delphinidin (26) OH OH Petunidin (27) OH OCH3 Malvidin (28) OCH3 OCH3 Cyanidin (29) OH H Peonidin (30) OCH3 H Pelargonidin (31) H H

Figure 6. Structures of the major anthocyanins-3-O-glycoside present in plants and fruits.42

11

Unlike the betalains, anthocyanins are highly unstable and are easily degraded. Their stability is affected by factors such as pH, storage temperature, oxygen, and the presence of solvents. In aqueous solution, anthocyanins undergo structural re- arrangements in response to changes in pH and are most stable in acidic solution (pH 1-

3) where they exist primarily as flavylium cations (32) which have a characteristic red colour (Figure 7). The stability of anthocyanins is also influenced by ring B substitution, e.g., pelargonidin (31) is the most stable anthocyanidin due to few hydroxyl or methoxyl group substitutions on ring B. Moreover, anthocyanidin monoglycoside or diglycoside derivatives showed higher stability under neutral pH conditions.41, 45

Figure 7. Anthocyanins chemical forms depending on pH. R1 = H or sugar, R2 = R 3= H or OH or methyl.41

As previously mentioned, anthocyanins are responsible for many colours from scarlet to the blue of flowers, leaves, and fruits. In general, they are biosynthesized in higher plants but seem absent in Marchantiophyta, algae, and other lower plants. Anthocyanins are commonly found in edible fruits such as berries, cherries, peaches, grapes, plums,

12 and pomegranates and also in vegetables such as red onion, black bean, red cabbage, eggplant, corn, and purple sweet potato.13, 16

The biosynthesis pathway of anthocyanins is derived from the flavonoid pathway

(Figure 8). The first step is the formation of tetrahydroxychalcone (34) from the reaction between p-coumaryl-CoA (33) with three molecules of malonyl-CoA in the presence of chalcone synthase (CHS). The C-ring formation from 34 gives the flavanone/narigenin

(35) which is catalysed by chalcone isomerase (CHI). Narigenin (35) is the common precursor to the flavonoids and isoflavonoids. The formation of 36 and 37 arises from the reaction of narigenins (35) in the presence of 3'-hydroxylase or flavanone 3',5'- hydroxylase that substitute hydrogen with hydroxyl group at the 3' and 5' positions of the B-ring. Furthermore, hydroxylation of the C-ring by flavanone 3-hydroxylase provides (38 – 40). Dihydroxykaempferol (39) is converted to leucoanthocyanidin (41 –

43) by dihydroxyflavonol-4-reductase (DFR) and this transformation of leucoanthocyanidin into anthocyanidins involves an oxidation and dehydration step.13, 26

13

Figure 8. Biosynthetic pathway of anthocyanin. F3H: flavanone 3-hydroxylase; F3'H: flavanone 3'-hydroxylase; F3',5'H: flavanone 3',5'-hydroxylase; DFR: dihydroflavonol 4-reductase; LDOX/ANS: leucoanthocyanidin dioxygenase/athocyanidin synthase.26

14

1.1.3.1 Biological effects of anthocyanins

As well as the ability of anthocyanins to produce colours in plants, they also possess biological activities.44 Fourteen anthocyanins (delphinidin, cyanidin, pelargonidin, malvidin, peonidin and their derivatives with different sugar linkages) were studied for their antioxidant activity using an automated oxygen radical absorbance capacity

(ORAC) assay. Cyanidin-3-glucoside (29) showed the highest ORAC activity with values 3.5 times stronger than Trolox (an analogue of Vitamin E) while pelargonidin

(31) had the lowest antioxidant activity. The protective effect of anthocyanins on oxidative stress-induced damage is promising, as demonstrated by in vivo oxidative stress models, with the results showing that cyanidin 3-O-β-D-glucoside (29) significantly suppresses oxidative stress in rat cell lines. 46

Over the past decade, the study of anthocyanins as anticancer agents has been reported both in vitro and in vivo studies. The activities of these compounds were related to their ability to scavenge free radicals, stimulation of phase II detoxifying enzymes, reduced cell proliferation, inflammation, and angiogenesis, leading to apoptosis.16, 47

Anthocyanins have been demonstrated to inhibit malignant cell growth, stimulate apoptosis and modulate oncogenic signalling events in vitro in the range 10-6 to 10-4

M.47

1.2 Rationale and Aims of Research

The finding of palm dates and grass seeds in the Late Middle Paleolithic Era suggested that plants were cultivated and used as dietary source.48 Plants have also been used as medicines, with the earliest records of the use of medicinal plants dating back to 2600

15

BC, and approximately 1000 plant-derived substances in Mesopotamia have since been used therapeutically.49 In modern times, the discovery of new drug leads has relied heavily on medicinal plants, and fungi, from which the first antibiotic (penicillin) was isolated from Penicillum notatum; and marine natural products such as plitidepsin

(isolated from the Mediterranean tunicate Aplidium albicans) which shows substantial efficacy in the treatment of a variety of cancers, including melanoma, small cell and non-small cell lung cancers.

With increasing environmental and health concerns, natural colourants are gaining popularity for their use in the textile, food, cosmetic, and pharmaceutical industries as well as in the arts.50-51 In nature, these natural colourants such carotenoids, anthocyanins or flavonoids, and betalains play important roles in visual attraction for pollinators and seed dispersers, and additionally have diverse biological activities, e.g., antioxidant and antimicrobial properties.13, 52

Avian species are well-known to use their colouration to differentiate between males and females of the same species (sexual dichromatism), and as a communication signals.53 Furthermore, avians such as parrots contain fluorescent pigments, which absorb short-wavelength UV light, and these pigments are used in sexual signalling.54-55

In limited studies, it has also been shown that pigments in some avian feathers such as white leghorn chickens (Gallus domesticus) and scarlet macaw (Ara macao) inhibit the growth of Bacillus licheniformis, however, identification of the pigment composition in feathers has remained largely understudied. Additionally, the state of current research is such that many pigments are identified based on comparative studies of their physical characteristics, e.g. UV-VIS spectrometry, rather than investigation of their absolute chemical structures.56-57

16

A number of plants produce anthocyanins, flavonoids, and phenolic compounds as protectors against UV-radiation and drought,58-59 e.g., mosses are reported to be tolerant to environmental stresses, owing to the production of numerous compounds which possess UV-screening and antioxidant properties.60 Therefore, it is unsurprising that they have a suite of photoprotective strategies and it could be used as indicators for climate change. 60

The study of natural products is an important tool in the discovery of new bioactive species, as it allows for identification of the chemical constituents of particular species.

In combination with ethnopharmacological data, this allows the biologically active constituents of such traditional medicines to be elucidated. Further, it enables identification of the colourants responsible for the striking physical characteristics of a variety of species of both plants and animals.

In addition to their use in both mobility and as attactants, birds use feathers to protect them from harsh conditions such as UV exposure or microbial colonisation. In doing so, chemical protectants may be produced or ingested, then deposited to the feathers, allowing for protection of the individual. By isolating the chemical species from within the feathers, it is therefore possible to gain information about both the chemical components responsible for their colouration, as well as possible modes of action for protective agents.

In an analogous fashion, plant species located in harsh environments survive by the production of particular chemical constituents, e.g. as protectants against harsh UV radiation or other external factors. Isolation and characterisation of these chemical constituents would therefore allow significant insight into the protective modes of action of such species. 17

Therefore, in this dissertation, we aim to use natural product exploration to study the major constituents present in different species of Australian flowers, abundant in the

Illawarra region of New South Wales, Australia, and to explore possible antimicrobial activities of the constituents. We also use this exploration to fully characterize the chemical components from the feathers of Australian parrots. In this study, we are not only focused on the pigments present in the feathers, but rather all of the natural products found within feathers. Furthermore, the UV active compounds present in

Australian population of Ceratodon purpureus were explored and their possible role in survival of the moss against UV-radiation and oxidative stress. These results were then compared to the compounds present in Antarctic populations of this species.

Therefore, this thesis examines these areas above through the following aims:

1. The examination of secondary metabolites of flowers in Australia which was

further narrowed to four species of native Australian flowers (Brachychiton

acerifolius, Anigozanthos rufus, A. pulcherrimus, and Acacia pycnantha) and two

introduced species (Tibiouchina lepidota and Jacaranda mimosafolia).

2. The exploration of secondary metabolites from the green feather of Alisterus

scapularis, focusing on optimization of extraction method, and elucidation of

chemical constituents

3. The examination of secondary metabolites from Australian moss, Ceratodon

purpureus focusing on biflavones and reassignment of published biflavones from

this species including their antioxidant activity.

18

CHAPTER 2: Secondary Metabolites from Australian Flowers

2.1 Introduction

Australia has a hugely variable climate with arid, tropical, desert, alpine, and extreme conditions extending across the country. Australian plants have therefore developed a wide variety of survival methods specific to their environmental challenges, and as such there are many unique morphologies and phenotypes present.61 This includes not only endemic plants, but also the large range of introduced species, some of which are even cultivated.62

Indigenous Australian people have been surviving on the land for at least 40,000 years, with plants providing essential materials including foods, weapons, tools, clothing, natural dyes, and medicine.63 More than one hundred species of Australian plants have been used in traditional medicine, e.g. the Australian Acacia species has been used to treat dysentery, skin diseases, and rheumatism.61 The ethanol extracts from A. kempeana, A. tetragonophylla, A. linarioides, A. brachystachya, A. lineate, A. trineura, and A. olligquinervia leaves show antibacterial activity against B. cereus, E. faecalis, and S. pyogenese.64 1,8-Cineole isolated from E. globulus fruits showed antimicrobial activities against B. subtilis, S. saprophyticus, S. epidermidis, and E. coli with MIC values between 8 – 32 mg/mL.65-66

Even though Australian plants have been traditionally used as medicines, their purported biological effects are largely based on traditional or anecdotal evidence.

Relatively few Australian medicinal plants have undergone scientific investigation, e.g. it has been reported that 20% of Australian plant extracts inhibit bacterial growth, and some have demonstrated antiviral activities.64, 67 For example, ethanol extracts from eight medicinal plants (Acacia implexa, Acacia falcate, Cassytha glabella, Eucalyptus haemastoma, Smilax glyciphylla, Sterculia quadrifida, and Syncarpia glomulifera) used by Indigenous Australian people of New South Wales showed antibacterial activities againts S. aureus with MIC values ranging from 7.81 – 1000 µg/mL.68

As well as some Australian plants being used as medicine, some also possess unique and colourful flowers, with examples such as Anigozanthos (kangaroo paw) species being used as natural dyes by Indigenous populations.69 Despite the vast array of colourful flowers present, relatively few have been subjected to phytochemical studies to establish the chemical structures responsible for both their colour, but also biological activities.

In this chapter, we report the phytochemical studies and the antimicrobial activities of the flowers of four native Australian species which are commonly found in the

Wollongong area including Brachychiton acerifolius (Illawarra flame tree),

Anigozanthos rufus (red kangaroo paw), Anigozanthos pulcherrimus (yellow kangaroo paw), and Acacia pycnantha (Golden wattle). These species were selected due to their abundance, and lack of information regarding the chemical constituents present, including those responsible for their colouration. Additionally, two introduced species which commonly grow in Australia, Tibouchina lepidota (alstonville) and Jacaranda mimosafolia (jacaranda) were used due to their abundance, in order to further probe the natural product constituents within the flowers, including those responsible for their colouration from these species.

2.2. Phytochemical study of Brachychiton acerifolius flowers

The Illawarra flame tree (Brachychiton acerifolius), a member of the Malvaceae family

(Figure 9), is widespread in the subtropical rainforests on the east coast of Australia,

20 stretching north from the Shoalhaven River (NSW) up to Queensland.70 Together with other members of the genus Brachychiton, it is commonly referred to as a kurrajong. B. acerifolius is a temperate climate tree, able to grow to a maximum height of 40 metres.

It flowers in late spring producing scarlet bell-shape-flowers with five partially fused petals. The follicles are dark, brown, wide, and boat-shaped (Figure 10).71

Kingdom : Plantae Division : Spermatophyta Class : Rocids Order : Malves Family : Malvaceae Genus : Brachychiton Species : Brachychiton acerifolius

Figure 9. Plant classification of Brachychiton acerifolius.

Figure 10. Flower of B. acerifolius (a) 63 and the B. acerifolius tree (b).

21

2.2.1 Traditional medicinal uses and pharmacological activities of Brachychiton genus

The Brachychiton genus belongs to the Malvaceae family and this genus was identified by Schoot and Edlicher in 1832. This genus grows predominantly in Australia, and was initially classified under the genus Sterculia, though closer examination of the characteristic of the follicles, seed coats, and embryo strongly supported the retention of

Brachychiton as a separate genus.72 Members of this genus are well-known by a number of common names, including the Illawarra flame-tree, kurrajongs, peanut-tree and the

Queensland bottletree.63

The kurrajong family has been used by the Indigenous population for many purposes.

The fibrous bark from black kurrajongs (Brachychiton populneus) was used to make fishing nets, lines and carry bags in southeast Australia whereas the northern kurrajong

(Brachychiton diversirfolious), together with red-flowered kurrajong (Brachychiton fitzgeraldianus), were used as a source for making armbands, climbing belts, headbands and waist belts in northern Australia.63 They were also used as medicinal plants and dietary sources with the leaves of B. diversirfolious used as a wash for the whole body of patients suffering from fevers, while the gummy exudate was applied to wounds and sores, as an antibacterial remedy.61, 73

There have been some reports of biological or pharmacological activities from this genus, for example, the antibacterial testing of methanol extracts from B. acerifolius flowers which showed activity against B. cereus with an inhibition value of 0.7 cm

22 using the disc diffusion assay. The results showed that the extracts from flowers of B. acerifolius were more active compared to that from the leaves.67

In a different study, twigs from B. diversifolius were investigated for their antibacterial and antioxidant activities. Five different extracts (methanol, ethyl acetate, chloroform, butanol and water) were assayed against plant and human pathogenic bacteria using the agar disc-diffusion method with the methanol and ethyl acetate extracts showing activity against B. subtilis, B. cereus, S. aureus, S. lutea, and E. coli with inhibition zone value of 1-1.5 cm. Further, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method was used to determine the antioxidant activity and the methanol extract exhibited the highest effect with a value of 85.6%.73

Salem et al. investigated the antibacterial, antifungal, and antioxidant activities of fatty acid extracts from different components of B. diversifolius (wood, bark, and leaves).74

Here, all the extracts were moderately effective against B. subtilis (inhibition zone: 9 –

13 mm), and Micrococcus luteus (inhibition zone: 12 – 14 mm) and possessed an antibacterial effect against the growth of E. coli (inhibition zone: 16-22 mm). In these studies, all extracts showed inhibition against Penicillium selerotigenum, Paecilomyces variotti, and Aspergillus niger with values between 60 – 75%. It was found that the total antioxidant activity of the fatty acid extracts from the bark showed 80% inhibition and this value was not significantly different from the positive control (tannic acid) with a value of 85%.

23

2.2.2 Secondary metabolites from the genus Brachychiton

The seeds from different species in the genus Brachychiton have been used as a dietary source by the indigenous people and investigations into the phytochemical compositions has been undertaken. It was found that Brachychiton discolor, B. diversifolius, and B. acerifolius seeds contain essential amino acids used in the diet.75 The fatty acid composition was also determined showing oleic, linoleic, and palmitic acids predominating, with smaller quantities of malvic and sterculic acid also present.75-76 The fatty acids from the wood, bark, and leaves of B. diversifolius were analyzed by gas chromatography (GC) and the results showed the presence of myristic and palmitic acid.

Previous studies on the seeds of Sterculia lychnophora or Brachychiton lychnophora revealed the presence of alkaloids, sterculinine I and sterculinine II (Figure 11) and these seeds have been used in Chinese traditional medicine to treat pharyngitis.77

Chemical studies of the hexane extract of S. acerifolia leaves grown in Bari, Italy showed the presence of vitamin E, germanicol, squalene, α-amyrin, lupeol, and stigmasterol while the methanol extract contained kaempferol (51), quercetin (46), luteolin, rutin (63), and quercetin-3-arabinoside (56).78

Figure 11. Sterculinine I (44) and II (45) isolated from seeds of Sterculia lychnophora.

24

2.2.3 Results and Discussion

This Illawarra flame tree (B. acerifolius) is endemic to east coast of Australia, especially in the Illawarra area. Despite their bright colourful flowers and the reported biological activities of extracts, there are only limited phytochemical studies on this species and no reports on the structures contained within this flower.

The flowers were collected from the University of Wollongong campus, cleaned, freeze-dried, and then extracted with methanol. This crude extract was subjected to solvent-solvent fractionation and produced three fractions: hexane, ethyl acetate and residual fractions. The ethyl acetate and residual fractions were then subjected to reverse-phase (RP) HPLC with optimised conditions determined as the gradient elution of solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in acetonitrile) from 90% to

55% solvent A within 40 minutes (Figure 12). This method was then applied to the fractions on a preparative HPLC scale. The ethyl acetate and residual fractions showed similar HPLC profiles. Therefore, in this study the isolation was focused on the ethyl acetate fraction.

47

49 50 51 46 48

Figure 12. RP-HPLC profile of the ethyl acetate fraction, indicating the targets to be isolated. Peaks were recorded at 254 nm. Peak numbering corresponds to compound numbers.

25

The ethyl acetate fraction was subjected to preparative reverse-phase HPLC which afforded six fractions, and the compounds from each fraction were identified by spectrometric and spectroscopic analysis as secondary metabolites (Figure 13).

Furthermore, the NMR spectroscopic data comparison with published reports used to

1 determine the absolute configuration of the sugar, and measuring the JC1-H1, which is ~

170 Hz for α-anomeric sugar configuration and ~ 160 Hz for β-anomeric sugar

79-80 configuration. The first peak at tR 24 minutes was identified as the known quercetin

81 (46). Two peaks at tR 27 and 30 minutes was identified as kaempferol 3-rutinoside

(48)81 and kaempferol (51)81, respectively.

Figure 13. Flavonoids and glycosides isolated from B. acerifolius flowers.

In this study, compound 47 (Figure 13) at tR 26 minutes was a major constituent from ethyl acetate fraction and was isolated as a red powder. Analysis of the LRESIMS spectrum revealed a peak at m/z 783, assigned to the [M+H]+ ion. Analysis of the 1H

NMR spectrum (500 MHz, CD3OD) of 47 revealed a down-field resonance at δH 9.06 and was assigned to H-4 which is characteristic in anthocyanins82 and via gHMBC

26 spectral analysis showed correlations between this proton and three quaternary carbon peaks at δH 156.7, 157.4 and 164.5, assigned to C5, C8a, and C2, respectively.

1 Furthermore, analysis of H NMR spectrum revealed a set of resonances at δH 8.57 (d, J

= 8.0 Hz, 2H) and 7.03 (d, J = 8.0 Hz, 2H), assigned to H-2',6' and H-3',5' which showed correlations to the quaternary carbon peaks at δC 119.0, 164.5, and 167.8, assigned to C1', C2, and C4', respectively. A set of meta coupled protons were assigned

1 from the H NMR spectrum to the resonances at δH 6.64 (d, J = 2.0 Hz, 1H) and 6.88 (d,

J = 2.0 Hz, 1H), assigned to H6 and H8, respectively. Through gHMBC spectral analysis, this ring was proposed as a 1,2,4,6-tetrasubsituted aromatic ring which contained the quaternary carbons C5, C7, C8a and C4a assigned to the resonances at δC

156.7, 168.8, 157.4, 119.0, respectively.

Further analysis of the 1H NMR spectra revealed a set of ortho coupled proton resonances at δH 7.26 (2H, d, J = 8.9 Hz) and 6.88 (2H, d, J = 8.9 Hz), assigned to H-

5''',9''' and H-6''',8''', respectively. In the 1H NMR spectrum, the olefinic protons with trans configuration showed a large coupling associated to the resonances at 7.38 (1H, d,

J = 12.4 Hz) and 6.23 (1H, d, J = 12.5 Hz) and assigned as H2''' and H3''', respectively.

Therefore, based on this analysis the presence of a coumaroyl acid moiety was proposed.

Analysis of 1H NMR spectrum of 47 revealed the presence of two sugars and the resonances of the two anomeric protons appeared at δH 5.29 (1H, d, J = 7.9 Hz, glucose

A) and δH 5.06 (1H, d, J = 7.9 Hz, glucose B). Based on gHMBC spectral analysis, a correlation of δH 4.45-4.52 with δC 170.1, assigned as H6'' and C1''', respectively indicated that the coumaroyl acid moiety was attached to C6'' of glucose A.

Furthermore, an acetate carbonyl carbon (C7'''') assigned at δC 171.1 showed a long

27 range bond correlation to protons at δH 3.71 (dd, J = 8.4 Hz, 8.0 Hz) and 4.06 (dd, J =

8.4 Hz, 8.0 Hz, H6'''').

In order to confirm the position of the ester linkage between the aglycon and sugars, the gHMBC spectrum was analysed. It revealed a correlation between the resonance at δH

5.29 to a quaternary carbon at δC 145.6, and was assigned as H1'' from glucose A and

C3, respectively. In a similar manner, the resonance at δH 5.06 showed a correlation with a quaternary carbon at δC 156.7 and was assigned as H1'''' from glucose B and C5, respectively. Therefore, based on spectroscopic evidence, compound 47 was proposed as pelargonidin-3-(6-coumarylglucoside)-5-(6-acetylglucoside). This compound has been reported to be present in Hyacinthus orientalis which grows in Japan and was spectroscopically identical to this study.83 Compound 47 is reported for the first time in this species.

Compounds 49 and 50 were isolated as UV-active dark yellow solids. The molecular formulae of C21H20O11 and C22H22O11 of 49 and 50 were confirmed by HREISIMS with an assigned molecular ion [M+Na]+ at m/z 471.3667 and 485.1294, respectively.

25 o Further, both were found to be optically active with values [훼]D -35.9 (c 0.11, MeOH) and +24.57o (c 0.17, MeOH), respectively. The molecular structures of these compounds were elucidated by analysis of the 1D and 2D NMR spectroscopic data followed by comparison with data reported, and the structures revealed as naringenin-7-

O-β-D-glucuronide (49)84 and 4',5-dihydroxyflavanone-7-O-β-D-glucuronide methyl ester (50).84

In order to determine the absolute configuration of these previously-reported compounds, circular dichroism (CD) spectroscopy was carried out and the obtained data compared (Figure 14). Published reports disclosed some CD spectral characteristics of 28 naringenin-7-O-β-glucuronide and 4',5-dihydroxylavanone-7-O-β-D-glucuronide methyl, however the full spectrum was not published. The published CD spectrum of naringenin-7-O-β-glucuronide reported cotton effects at 250 nm (Δε +0.51) and 293 nm

(Δε -3.48) (Figure 14, black diamond) and was assigned as (S) orientation, whereas 4',5- dihydroxylavanone-7-O-β-D-glucuronide methyl ester had reported cotton effects at

249 nm (Δε -5.84) and 290 nm (Δε +2.35) (Figure 14, green triangle), was assigned as

(R) orientation.84

In this study, the CD spectrum from compound 49 showed cotton effects (250 nm (Δε

+0.51) and (293 nm (Δε -1.05), and compound 50 showed a similar pattern to that for 49 with cotton effects (249 nm (Δε +1.45), 290 nm (Δε -5.43)). The CD spectrum of these compounds are shown in Figure 14. Therefore, compound 49 was assigned as (2S) naringenin-7-O-β-glucuronide. However, the CD spectrum of compound 50 indicates opposite cotton effects as reported from (2R)-4',5-dihydroxylavanone 7-O-β-D- glucuronide methyl ester, therefore the stereochemistry was assigned as the opposite 2S isomer. Compound 50, (2S)-4',5-dihydroxylavanone 7-O-β-D-glucuronide methyl ester, has therefore been isolated and identified as a new compound.

29

8 6 4 2 290, 2.35 250, 1.43 0 200 220 240 260 280 300 320 340

CD [mdeg] CD -2 -4 293, -3.48 -6 249, -5.84 -8 Wavelength

50 49 2R-(50) 2S-(49)

Figure 14. Chemical structure of 50 and 2R-(50) and Circular dichroism (CD) spectra of compound 49 and 50 were compared with 2S-(49) and 2R-(50).84

The seeds of B. acerifolius are used as an emollient for its antiseptic properties71 as well as a food source, however there were no laboratory studies to support these claims. The presence of isolated compounds from the ethyl acetate fraction suggest biological activities, for example, kaempferol (48 and 51) and quercetin (46) have been widely reported to have antibacterial activity against Bacillus sp. and other human pathogenic bacteria.85-86 The antibacterial activities of the crude methanol extracts from the leaves and flowers of B. acerifolius have been reported against several pathogenic bacteria with the flowers possessing high antibacterial activity against Bacillus cereus compared to the leaves.67

30

As well as plant colouration, anthocyanins also possess well-known pharmacological activities. Pelargonidin (47) and its glucoside were reported to have strong antioxidant activity.87-88 In this study, two naringenins (49 and 50) were isolated, with naringenin reported to possess chemoprevention potential and inhibition of a breast carcinoma cell line.89

2.3 Natural product investigations on Tibouchina lepidota flowers

Alstonville (Tibouchina lepidota) belongs to the family Melastomataceae (Figure 15) and is a native of South America.90 It was introduced in 1978 in the town Alstonville,

NSW, becoming popular in New South Wales and Queensland.91 It is a large shrub or small tree with a vase-shaped and a spreading, rounded crown. Its leaves are dark green on the upper side and paler on the underside, with large, longitudinal veins. A brilliant display of buttercup-shaped, violet/purple flowers, cloaks the canopy in late Summer and Autumn (Figure 16).

Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Myrtales Family : Melastomataceae Genus : Tibouchina Species : Tibouchina lepidota

Figure 15. Plant classification of Tibouchina lepidota.

31

Figure 16. (a) Flowers of T. lepidota, (b) T. lepidota tree.

2.3.1 Traditional medicinal uses and pharmacological activities of Tibouchina genus

The genus of Tibouchina is predominantly distributed in southern Mexico to northern

Argentina and includes ca. 350 species.90 A characteristic of their flowers is the dark purple colour and they are cultivated as ornamental plants in gardens.92 This genus was also used in traditional medicines, e.g in Brazil, where the leaves from T. grandifolia were used to enhance wound healing and in Ecuador with the leaves of T. laxa used to treat cataract.92-94

Over the past decade, numerous biological activity studies have been reported from this genus, e.g., both the crude extract and isolated compounds from T. semidecandra L. leaves showed strong antixodant activity as analysed using DPPH. Further, this study examined the components for their anti-tyrosinase activity, with quercetin (46) exhibiting strong activity (95% equivalent to kojic acid as positive control).95 Studies on the antimicrobial activities of the ethyl acetate extract from the leaves of T. grandifolia reported weak inhibition of pathogenic fungal Cladosporium cucumerinum at a

32 concentration of 100 µg/mL.93 The isolated phenolic derivative, 2,8-dihydroxy-7H- furo[2,3-f]chromen-7-one (52) (Figure 17), together with quercetin-3-glucoside (53) from the aerial component of T. paratropica, were found to possess antifungal activity against Candida albicans, Fusarium solani and Trichophyton mentagrophytes with MIC values 62.5 – 250 µg/mL.96 The methanolic extract from the aerial component of T. grossa claimed antifungal activity against C. albicans, F. solani, and Aspergillus fumigatus at a concentration of 625 µg/mL.97

Figure 17. Isolated phenolic derivative (52) and quercetin-3-glucoside (53) from the aerial component of T. paratropica.

The methanol extract from the aerial components of T. stenocarpa and T. semidecandra were reported to inhibit the growth of Trypanosoma cruzi and Leishmania donovani with inhibitions of 70.3 and 74.9%, respectively at a concentration of 32 µg/mL.98-99

Compound 52 which was isolated from aerial components of T. paratropica, showed

97% activity against promastigotes of the parasite L. donovani with an IC50 value of

0.809 µg/mL .96

2.3.2 Secondary metabolites from the genus Tibouchina

The genus Tibouchina has approximately 350 species of trees, shrubs, which are mainly distributed in the Americas and have been used traditionally for both medicinal and

33 food colouration.100 Thus far, only eight species have been investigated for their phytochemical content (Table 1).92, 96

Table 1. Secondary metabolites from the genus Tibouchina

Species Country Part of plant Isolates

T. pulchra Brazil Leaves Kaempferol, kaempferol 3-O- galactoside, quercetin, quercetin 3- O-galactoside, quercetin 3-O- rhamnoside, myricetin 3-O- galactoside.101 T. ciliaris Colombia, Leaves Quercetin-3'-O-rhamnoside, kaempferol-7-O-P-cumaroil, kaempferol, ellagic acid.102 T. urvilleana Mexico Leaves, Glutinol, taraxerol, mixture of α- and stems β-amyrins, β-sitosterol, ursolic, oleanolic acids, avucularin, hispidulin 7-O-D-glucopyranoside, aciatic acid, arjunolic acid, quadranoside IV, arjunglucoside T. semidecandra Japan, Leaves Nobotanins C, L, M, and N, methyl Malaysia tri-O-methylgallate, dimethyl hexamethoxydiphenate, trimethyl Leaves, stem octa-O-methylvaloneate.103 bark Quercetin, quercetin 3-O-(2''-O- acetyl)-arabinofuranoside, avicularin, quercitrin, 3,3'-O-dimethyl ellagic acid 4-O-L-rhamnopyranoside.104 T. multiflora Colombia Leaves Nobotanins A, B, C, D, F, G, O, P, M pedunculagin, casuarictin. 105 T. candolleana Brasil Aerial α- and β-amyrins, β-sitosterol, components ursolic acid, oleanolic acid, luteolin, genistein. 21 T. paratropica Argentina Aerial 2,8-dihydroxy-7H-furo[2,3- components f]chromen-7-one, isoquercitrin. 27 T. urvilleana Japan Flowers Malvidin 3-(p-courmarylglucoside)- 5-acetylxyloside. 105

34

2.3.3 Results and Discussion

T. lepidota is an introduced plant in Australia, where it is cultivated. This species is used as a medicinal plant in South America for wound healing, antiseptic, and antimicrobial purposes, 92-94 however, there are no investigation into the chemical constituents contained within the flowers from this species.

Flowers were collected from trees in the University of Wollongong campus area, washed, freeze-dried and extracted with methanol. Liquid-liquid fractionation of the crude methanol extract was used to obtain hexane, ethyl acetate, and residual fractions.

Analysis of the HPLC chromatograms of the ethyl acetate and residue fractions revealed identical profiles. The isolation of secondary metabolites from the ethyl acetate fraction used semi-preparative HPLC with a C-18 silica column, and acetonitrile-water as mobile phase. A small percentage of acid (trifluoroacetic acid/TFA) was incorporated to improve separation resolution and to increase the yield. The optimal conditions for

HPLC separation were determined to be 90% to 55% of solvent A (0.1% TFA in water) within 50 minutes with solvent B (0.1% TFA in acetonitrile) (Figure 18). This method was applied to semi-preparative HPLC and isolated seven secondary metabolites were successfully isolated (Figure 19). Spectroscopic and spectrometric analysis were employed to elucidate molecular structures and the results were compared with published data where appropriate.

35

54 46 58 53 55 56 57

Figure 18. RP-HPLC profile of the ethyl acetate extract indicating the targets to be isolated. Peaks were recorded at 254 nm. Peak labels correspond to compound numbers in this dissertation.

Figure 19. Phenolic, flavonoids and glycosides isolated from T. lepidota flowers.

The peak appearing at tR 9 minutes in the HPLC chromatogram was collected, dried and crystallised producing needles which were found to be gallic acid (54).106 2,3,5-

Trihydroxybenzoic acid (55) 106-107 was isolated as a yellow solid and corresponded to the peak at tR 17 minutes. Four known flavanols and their glycosides were isolated

36 from the ethyl acetate fraction; these were quercetin (46)81, avicularin (56)95, quercetin

3-glucoside (53)81, isorhamnetin 3-rutinoside (57)108 (Figure 19).

In this study, a dark purple solid, compound 58, was also isolated from this fraction (tR

33 minutes) and LRESMS analysis showed peak at m/z 813 assigned to (M+H)+. The 1H

NMR spectra showed downfield resonances at δH 8.70 (s, 1H), 7.95 (s, 2H), 7.02 (s, 1H) and 6.98 (s,1H), assigned to H4, H2'/6', H6, and H8, respectively. An additional resonance at 3.94 (s, 6H) was attributed to the C3' and C5' methoxy protons. These findings align with the structure malvidin aglycone, further supported by LRESMS analysis with a peak at m/z 331 assigned to the malvidin parent structure. Analysis of

1 the H NMR spectra, suggested the presence of two sugar moieties (δH 3.35– 5.45).

Moreover, a set of ortho coupled protons assigned to the resonances at δH 6.79 (d, J =

8.0 Hz, 2H) and 7.44 (d, J = 8.0 Hz, 2H) as well as the olefinic protons to the resonances at δH 6.31 (d, J = 15.0 Hz, 1H) and 7.50 (d, J = 15.0 Hz, 1H) indicated the present of coumaric acid moieties. It was concluded that the major pigment in the purple flowers of T. lepidota was malvidin 3-(coumaryl-glucoside)-5-(acetylxyloside) (58), further supported by HRESMS analysis which showed a peak at m/z 814.2320, assigned

+ as C39H42O19 [M+H] . All the isolated secondary metabolites have been previously reported and their spectroscopy data were compared to that reported. Importantly, they were isolated and identified from T. lepidota for the first time. Compound 58, a purple solid was identified as the anthocyanin responsible for purple colour of the T. lepidota flowers.

Alstonville (T. lepidota) is used mostly as an ornamental plant due to their dark purple flowers with no medicinal usage previously reported, however, other species within the genus have been reported to possess various biological activities such as wound healing

37 and to treat cataract.27 The isolated compounds 46, 56, 57 from our study were found to be present in T. semidecandra L. leaves95 which possess anti-oxidant and anti-tyrosinase activities.95 Furthermore, published data showed that compounds 54 and 55 possess antitumor106 activities and compound 54 was believed to possess high antioxidant activity106 and plays an important role in the prevention of malignant transformation and cancer development.109 T. grandifolia is used for its wound healing properties in Brazil and this could be due to the presence of quercetin 3-glucoside (53) and is reported to have in vivo wound healing properties via linear incision and circular excision wound models.110 It is evident that the flowers of T. lepidota serve not only an ornamental purpose, but also as medicinal plants.

2.4 Natural products study on Anigozanthos Sp.

Anigozanthos are a member of the Haemodoraceae family and are known commonly as

Kangaroo paws (Figure 20). This genus contains nine species which are endemic in the

South-West Australia and are the floral emblem of Western Australia.111 Flowers from this genus have various colours depending on the species and possess a unique shape with tubular flowers coated with dense hairs which open at the apex with six claw-like structures (Figure 21).112 These plants grow from short, underground, horizontal rhizomes. The sap in the root allows these plants to survive in extreme seasons. In summer, these plants die back to the rhizome and grow back in autumn.112-113

38

Kingdom : Plantae Division : Angiosperms Order : Commelinales Family : Haemodoraceae Subfamily : Conostyloideae Genus : Anigozanthos Species : Anigozanthos Sp.

Figure 20. Plant classification of Anigozanthos Sp.

Figure 21. Red kangaroo paw (A. rufus); Yellow kangaroo paw (A. pulcherrimus).

2.4.1 Traditional uses of Anigozanthos genus

Apart from the unique and attractive flowers, kangaroo paws possess tuberous roots which contain significant levels of starch and is part of the diet for the Indigenous people in the Yellagonga region, Western Australia.114 There is currently no report on

39 the usage of the genus Anigozanthos as a medicinal plant, however, the small red roots from this genus have been used by the Indigenous people in Northern Australia as a dye and these red pigments were reported as phenylphenalenone-type compounds.63, 115

Plants from the family of Hemodoraceae such as Lachnanthes tinctoria, Wachendorfia paniculata, Haemodorium simplex and Anigozanthos species are well-known to contain phenylphenalenones and become chemotaxonomic markers of this family.116-117 Three phenylphenalenone compounds (anigorufone (59), hydroxyanigorufone (60) and dihydroxyanigorufone (61)) were isolated for the first time from the whole plant of

Anigozanthos rufus. 115

Figure 22. Phenylphenalenone pigments isolated from whole plant of Anigozanthos rufus.115

Anigorufone (59) and methoxyanigorufone were isolated from the cultured roots of A. preisii along with hydroxyanigorufone (60), dihydroxyanigorufone (61) (Figure 22).118-

119, and have also been identified from rhizomes and roots extracts of A. flavidus.120

Over the last five decades, the study of the compounds from this genus have been intensively reported from this genus. 115, 116115

The phenylphenalenones are a group of natural products which show diverse and significant biological effects such as antimicrobial, anticancer, and cytotoxic activities,121 e.g. two novel phenalenone compounds from a marine-derived fungus were reported for their inhibitory activity toward DNA polymerase.116 Phenylphenalenones,

40 haemodorone, haemodorol, haemodorose, dilatrin, and xiphidone were isolated from the aerial component of Haemodorum simplex and tested for their cytotoxic activity, with

116 the results displaying moderate cytotoxicity with IC50 values of >26 – 39 µM.

Previous phytochemical studies of the M. fuliginosa flower reported the presence of known phenylphenalenones together with six new compounds. Methyl coumarate, chalcone glucoside and two flavonoids (rutin, p-hydroxycinnamate of salipurposide) were isolated from the same species. The antibacterial activity of the isolated compounds against different pathogenic bacteria revealed the phenylphenalenones with moderate activity, but the p-hydroxycinnamate of salipurposide was more active against

P. aeruginosa than ampicillin.122

2.4.2 Results and Discussion

In this study, the flowers of the endemic red kangaroo paw (A. rufus) flowers and yellow kangaroo paw (A. pulcherrimus) were chosen as there are only limited phytochemical studies reported. The flowers from both species were separately extracted with methanol and the residues subsequently solvent-partitioned between hexane:methanol, resulting in polar and non-polar fractions.

In this study, the chemical composition of polar fractions from both flowers were compared by HPLC analysis followed by isolation of the individual compounds. The polar fractions were initially passed through a short silica C-18 column to separate any remaining less polar components. The isolation of the secondary metabolites from the fractions used reverse phase HPLC with a gradient system. The optimal conditions for

HPLC separation were 90% to 50% solvent A (0.1% trifluoroacetic acid in water) within 35 minutes with solvent B (0.1% trifluoroacetic acid in acetonitrile) (Figure 23).

41

Red 63 68

69 62 67 70 64 65 66 51 48

Yellow

53 71

Figure 23. HPLC profile of ethyl acetate from A. flavidus (A) and A. pulcherrimus (B) flowers (zoom). Peaks labels correspond to compound numbers in this dissertation.

The polar fractions of flowers from both species were subjected to analytical RP-HPLC followed by preparative RP-HPLC and resulted in the isolation of a total of 13 compounds including two new compound (64 and 70) isolated at tR 14.2 and 20.6 minutes, respectively. All the known compounds were identified by comparison of their

NMR spectra data and LRESIMS with those reported in literature, and were revealed as cyanidin-3-rutinoside (62)123, quercetin-3-rutinoside (63)81, cyanidin-3-O-(6-O-p- coumaryl)glucoside (65)124, kaempferol (51)81, kaempferol-3-O-rutinoside (48)81, apigenin 7-β-glucoside (66)125, kaempferol-3-O-(6-O-p-coumaryl)glucoside (67)126, quercetin-3-O-(6-O-p-coumaryl)glucoside (68)127, quercetin-3-glucoside (53)81, luteolin-7-O-β-glucoside (69)128, and dihyroquercetin (71)129 (Figure 24).

42

Figure 24. Molecular structures isolated from flower of Anigozanthos sp. Compound 63, 64, 66-67 were isolated from both species. Compound 62 and 65 were isolated from A. rufus and Compound 71 was isolated from A. pulcherrimus.

The HPLC chromatogram profile of both flowers (Figure 23) showed the presence of similar compounds. These findings are likely due to A. rufus and A. pulcherrimus being closely related with respect to their anatomy and physiology, despite the different

61 colours of the flowers. Compound 63 (tR = 14.07 min) was found to be the major component from the polar extracts of both flowers followed by compound 68 (tR =

17.50 min). Compound 71 was only presence in the flower of A. pulcherrimus in contrast, with compounds 62 and 65 (tR = 13.10, and 14.50 min) which were found only

43 in the A. rufus flower (Figure 23). These anthocyanins (62 and 65) are likely responsible for the red colouration of this flower.

The first new compound (figure 25), 64 was isolated as bright yellow solid and

HRESIMS analysis indicated a peak at m/z 338.1249 ([M+1]), assigned to the molecular

1 13 formula C16H20NO7. The H and C NMR spectral analyses are summarised in Table 2.

Figure 25. Chemical structure of 64 isolated from A. rufus and A. pulcherrimus flowers.

Table 2. NMR spectroscopic data for compound 64

Position Carbon (δC) Proton (δH) 1 171.9 - 2 53.9 3.99 (dd, J = 8.8, 7.5 Hz) 3 31.9 2.10 – 2.00 (m) 4 22.2 1.75 – 1.50 (m) 5 31.3 1.95 – 1.93 (m) 6 72.8 5.10 (dd, J = 7.4, 4.9 Hz) 7 173.8 - 1' 168.6 - 2' 114.5 6.39 (d, J = 15.9 Hz) 3' 145.6 7.68 (d, J = 15.9 Hz) 1'' 127.4 - 2'' 131.1 7.48 (d, J = 8.3 Hz) 3'' 116.9 6.82 (d, J = 8.2 Hz) 4'' 161.4 - 5'' 116.9 6.82 (d, J = 8.2 Hz) 6'' 131.1 7.48 (d, J = 8.3 Hz) 1 13 Note: H and C NMR experiments were performed in CD3OD at 500 MHz and 125 MHz, respectively.

44

13 The C NMR spectrum showed three resonances at δC 168.5, 171.9, and 173.8, assigned to C6, C7, and C1, respectively suggesting the presence of an ester and two carboxylic acids. Analysis of the 1H NMR spectrum revealed two sets of doublets at δ

7.48 (H2''/6'') and 6.82 (H3''/5'') with coupling constants of 8.3 Hz assigned to ortho proton coupling on an aromatic ring, with supporting evidence of a COSY correlation between these protons. Two further doublets at δH 7.68 (H3') and 6.39 (H2'), together with carbon resonances at δC 114.5 (C2') and 145.6 (C3') indicated the presence of pair of trans olefinic protons with a coupling constant of 15.9 Hz. From the analysis of gHMBC data, a doublet at δH 7.48 (H2''/6'') showed long-range correlation with carbon resonances at δC 161.4 and 145.6, assigned as C4'' and C3', respectively (Figure 26).

1 In the upfield region of the H NMR spectrum, two methine protons at δH 5.10 and 3.99, and three methylene groups at δ 2.02, 1.93, and 1.69, were assigned as H6, H2, H3, H5, and H4, respectively. Analysis of gHMBC spectra indicated the carbonyl resonance at

δC 171.9 (C7) correlated with H5 and further, the assigned carbonyl resonance at δC

173.8 (C1) correlated with H3. Moreover, gHMBC spectroscopic analysis showed correlations between H5 and the carbon resonances at δC 168.6 and 22.2, assigned as C1' and C4, respectively. Furthermore, the three bond proton-carbon correlation was observed between H2 and the carbon resonance at δC 22.2, assigned as C4. Therefore, by a combination of above evidence, compound 64 was proposed as the novel compound 2-amino-6-O-p-coumaryl heptanedioic acid.

45

Figure 26. Key HMBC and COSY correlations observed for compound 64.

The second new compound, 70 was isolated as a yellow solid compound with

HRESIMS analysis indicating a peak at m/z 603.1489 [M+Na]+, assigned to the

1 13 molecular formula of C30H28O12Na. The H and C NMR spectral features are summarised in Table 3.

46

Table 3. NMR Spectroscopic Data for compound 70.

Position Carbon (δC) Proton (δH) 1 128.3 2 131.9 7.62 (d, J = 8.0 Hz) 3 117.1 6.87 (d, J = 8.0 Hz) 4 161.8 5 117.1 6.87 (d, J = 8.0 Hz) 6 131.8 7.62 (d, J = 8.0 Hz) 1' 127.2 2' 161.1 3' 165.9 4' 98.7 6.03 (s) 5' 161.4 6' 96.0 6.26 (s) α 125.6 8.02 (d, J = 15.0 Hz) β 144.3 7.68 (d, J = 15.0 Hz) carbonyl 194.8 Glucosyl 1'' 101.7 5.23 (d, J = 7.8 Hz) 2'' 75.3 3.69 (dd, J = 8.9, 7.8 Hz) 3'' 76.3 3.82 (dd, J = 9.5, 9.0 Hz) 4'' 72.1 5.02 (dd, J = 9.5, 8.5 Hz) 5'' 76.7 3.90 (ddd, J = 8.9, 6.5, 12.0 Hz) 6'' 62.3 3.72 (dd, J = 6.5, 1.2 Hz)/3.62 (dd, J = 12.0, 6.5 Hz) Coumaryl 1''' 125.8 2''' 131.4 7.49 (d, J = 8.0 Hz) 3''' 117.0 6.83 (d, J = 8.0 Hz) 4''' 161.2 5''' 117.0 6.83 (d, J = 8.0 Hz) 6''' 131.4 7.49 (d, J = 8.0 Hz) α 114.8 6.36 (d, J = 15.0 Hz) β 147.6 7.70 (d, J = 15.0 Hz) carbonyl 168.7 1 13 Note: H and C NMR experiments were performed in CD3OD at 500 MHz and 125 MHz, respectively.

1 The H NMR spectra analysis of 70 showed resonances at δH 6.03 (s, 1H) and 6.26 (s,

1H) assigned to H4' and H6' of ring A, respectively. gHMBC spectroscopic analysis

47 correlated these protons correlated to four quaternary carbons resonances at δC 165.9,

161.4, 161.1, and 127.2 assigned to C3', C5', C2', and C1', respectively. Furthermore,

1 the H NMR spectrum indicated resonances at δH 7.62 (d, J = 8.0 Hz, 2H) and 6.87 (d, J

= 8.0 Hz, 2H), assigned to H2/6 and H3/5 of ring B, respectively. This 1,4-disubstituted benzene ring contained quaternary carbons C1 and C4 which were assigned using gHMBC spectroscopic analysis to resonances at δC 128.3 and 161.8, respectively.

Analysis of 1H NMR spectra allowed assignment of a set of pair of trans olefinic protons resonances at δH 8.02 (d, J = 15.0 Hz, 1H) and 7.68 (d, J = 15.0 Hz, 1H) assigned to Hα and Hβ, respectively and through gHMBC spectroscopic analysis, the correlation between Hα and carbon resonances at δC 127.2, 128.3, and 194.8 and allowed the assignment of C1', C1, and the ketone carbonyl, respectively (Figure 27).

In addition, analysis of the 1HNMR spectra showed a set of ortho coupled aromatic protons at δH 7.49 (d, J = 8.0 Hz, 2H) and 6.83 (d, J = 8.0 Hz, 2H) assigned to H2'''/H6''' and H2'''/H6''', respectively of ring C. gHMBC spectroscopic analysis showed a correlation between these protons with the quaternary carbons resonances at δC 125.8

(C1''') and 161.2 (C4'''). Two further doublet resonances at δH 7.49 (d, J = 15.0 Hz, 1H) and 6.83 (d, J = 15.0 Hz, 1H) were assigned to Hα' and Hβ', respectively and showed correlations between Hβ'' with carbon resonances at δC 131.4 and 168.7 assigned to

C2'''/6''', and the carbonyl carbon of the ester, respectively (Figure 27).

1H NMR spectral analysis showed the presence of a single sugar moiety, with resonances at δH 5.23 (d, J = 7.8 Hz, 1H), 5.02 (dd, J = 9.8, 5.3 Hz, 1H), 3.90 (t, J = 5.9

Hz,1H), 3.82 (dd, J = 9.8, 9.3 Hz), 3.72/3.62 (dd, J = 12.6, 5.9 Hz), and 3.69 (dd, J =

10.5, 8.6 Hz) which were assigned to H1'', H4'', H5'', H3'', H6'', and H2'', respectively.

The proton sequences were confirmed by gCOSY and TOCSY through bond analysis

48 and suggested a glucose moiety. Further gHMBC analysis indicated a proton-carbon correlation from H1'' to C5', and H4'' to the ester carbonyl carbon, confirming the linkage between ring A to glucose, and glucose to the cinnamoyl moiety, respectively

(Figure 27). Therefore, 70 was proposed as the novel chalcone-5'-O-(4'''-O-p-coumaryl) glucoside.

Figure 27. Key HMBC and COSY correlation for compound 70.

Plants from the family of Haemodoraceae, which reside predominantly in Australia, have been reportedly used by Indigenous people for several purposes. Extracts of the bulbs from the genus Haemodorum have been used as purgative, ointment for snake bites, and as colouring agents.116 Recent studies reported that the methanol and dichloromethane extracts from the flower of the black kangaroo paw (M. fuliginosa) showed potential as antimicrobial agents against Pseudomonas aeruginosa and

Streptococcus pyogenes.122

Quercetin and quercetin glycosides (53, 63, 68) were reported to exhibit antioxidant, anticarcinogenic, anti-inflammatory, and and vasodilating effects.130 Many studies reported the benefit of kaempferol and its glucoside to reduce the risk of chronic

49 disease. Kaempferol and kaempferol with sugar moeities (48, 51) has ability to modulate a number key elements in cellular signal transduction pathways linked to apoptosis, angiogenesis, inflammation, and metastasis.131 In addition, compound 71 is well-known as taxifolin, and is believed to have therapeutic promise against cancer, cardiovascular, and liver disease. 132

2.5 Natural product studies on Jacaranda mimosaefolia

J. mimosaefolia belongs to the Bignoniaceae family, and is native to Brazil (Figure 28).

It has been cultivated in both tropical and subtropical areas of the world, and naturalised in many countries for its beautiful foliage and flowers,133-134 including in Australia where it is cultivated as an ornamental tree. J. mimosaefolia is a tree ranging from 1 to

45 meters tall with blue flowers which bloom in summer and are up to 5 cm long, and grouped in 30 cm panicles. The fruits of this species are oblong capsules, flattened perpendicular to the spetum, and the valves either glabrous or lepidote, possessing numerous seeds (Figure 29). The genus of Jacaranda is commonly known as pioneer trees, because the seeds are easily grown in different climates.135

Past research has revealed diverse biological activities and chemical constituents from different parts of this genus. Jacaranone (72) and phenylethanoids are common compounds presence in this genus, with the former isolated from the leaves of J. mimosaefolia along with scutellarein 7-glucuronide, verbascoside, and phenylacetic β- glucoside.133, 136 In this study, we provide an updated account of the natural products from Australian grown speciemens of this species, including the isolation of a new compound.

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Kingdom : Plantae Division : Angiosperms Order : Lamiales Family : Bignoniaceae Genus : Jacaranda Species : Jacaranda mimosifolia

Figure 28. Plant classification of Jacaranda mimosifolia.

Figure 29. Jacaranda mimosifolia flowers and leaves.

2.5.1 Medicinal uses of genus Jacaranda

The genus Jacaranda consists of 49 species from different regions in Central and South

America and the Caribbean. This genus has been used medicinally in different countries to cure wounds, ulcers, diarrhea, and dysentery, as well as showing antimicrobial properties.133 A summary of the traditional usage of this genus is reported in Table 4.

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Table 4. Traditional medicinal uses of Jacaranda species.

Species Country Part of plant Uses

J. acurifolia Brazil, Argentina Bark Dermatitis, diuretic137; treat syphilis and urinary tract problems.

J. caerulea Cuba, Caribbean Leaves Eczema, pimples, skin cancer.138

J. caroba Brazil Leaves Astringents, diuretic, to treat syphilis, ulcers.139

J. caucana Colombia Leaves Rheumatism, colds, and skin diseases.140

J. copaia Colombia, Brazil, Leaves, bark Skin infection, pneumonia, Bolivia rheumatism, syphilis, snakes bites.141-142

J. cuspidifolia Bolivia Leaves To treat leishmaniasis.143

J. decurrens Brazil Leaves, bark To treat cutaneous disturbances and wound, rheumatism.144 78

J. glabra Bolivia Leaves To treat leishmaniasis, fungal infection.144

J. hesperia Colombia Leaves To treat leishmaniasis. 144

J. mimosifolia Ecuador Bark Purify blood. 144

J. obtusifolia Venezuela, Guyana Bark To promote wound healing. 137

J. puberula Brazil Leaves To treat frostbite. 78

2.5.2 Secondary Metabolites from the genus Jacaranda

A number of phytochemical studies from this genus have been conducted over the past half century. Although the genus of Jacaranda contains 49 species with reports of their ethnobotanical properties, the chemical studies on this genus have been confined to just

52 six species.136, 144 Outcomes include the isolation and identification of jacaranone (72), a phytoquinoid compound, which was reported for the first time from the methanol extract of J. caucana twigs and leaves in 1976,145 and was patented for antitumor activity in 1977.144 Since then, the isolation of this compound, as well as a number of jacaranone-derived glucosidic esters, from different species of this genus has been reported. Additional secondary metabolites isolated from different species of this genus are listed in Table 5 below.

Figure 30. Jacaranone (72), isolated from the twigs-leaves of J. caucana.145

Table 5. Secondary metabolites isolated from Jacaranda species.

Species Part of Isolates plant

J. acutifolia Leaves Jacaranone, methyl-p-hydroxyphenylacetate, phenylacetic acid, 3,4-dihydroxybenzaldehyde, methyl- 3',4'-dihydroxy-trans-cinnamate and a monoterpene glycoside, 3-O-(β-D-quinovosyl)linalol-8-methyl- oate.146 7,2',3',4'-tetrahydroxyflavone 3-O- neohesperidoside. 147

J. caroba Leaves Quercetin-3-O-(6-rhamnosyl)glucoside, kaempferol-3- O-(6-rhamnosyl)hexoside, quercetin-3-O-hexosides,

quercetin-3-O-galactoside, quercetin-3-O-glucoside, kaempferol-3-O-hexoside, isorhamnetin-3-O- rhmanoside-

7,4-di-O-glucoside.148

J. caucana Stem Protocatechuic acid, acteoside, jionoside D, isoacteoside, martynoside, sisymbrifolin, 149 betulinic acid. 150

J. copaia Leaves Ursolic acid. 151

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J. decurrens Leaves Luteolin, 6-hydroxyluteolin 7-O-glucoside, quercetin-3- O-glucoside, quercetin-3-O-galactoside, 152 Verbacosides. 153

J. glabra Leaves Jacaglabroside A, B, C, D, jacaranone, phenylacetic acid. 135

J. Leaves Scutellarein, scutellarein 7-glucoronide, isoquercitrin, mimosifolia isovitexin, apigenin 7-O-β-glucoronopyranoside, luteolin 7-O-β-glucopyranoside methyl ester, jacaranone, jacraninoside A, acetoside, isoacetoside, cistanoside E, 6'-acetylacetoside, campneoside I. 133

Lupeol, betulinaldehyde, terminic acid, betulinic acid, maslinic acid, β-sitosterol glucoside, isoacteoside, benzoic acid, ursolic acid, 1-naphthaleneacetic acid. 134, Stem bark 154

J. oxyphylla Leaves Butyl hexadecanoate, 2-(4-hydroxyphenyl)ethyl triacontanoate, β-sitosterol, sitosterol-3-O-β-D- glucoside, 6'-palmitoyl-sitosterol-3-O-β-D-glucoside, oleanolic acid, ursolic acid. 155

J. puberula Leaves Ursolic acid, oleanolic acid, corosolic acid, and maslinic acid.156

2.5.3 Pharmacological activity studies on the genus Jacaranda

Traditional medicinal usage of the Jacaranda genus includes the treatment of diarrhea and dysentery, and to cure wounds and ulcers,133 and a number of pharmacological studies have been conducted using different species of this genus to assess the validity of these claims. The anticancer activity from this genus was reported for the first time in

1976, whereby jacaranone (72) isolated from the water extract of the twig, leaf and stem bark of J. caucana showed both in vivo and in vitro anticancer activity against P-388 limphocytic leukaemia cells. 145, 150 Five flavonoids, (-)-liquiritigenin, (-)-neoliquiritin, isoliquiritigenin, isoliquiritin, and formononentin were isolated from the twigs of J.

54 obtusifolia, and their anticancer activities were evaluated against NCH-H187 (lung cancer cell line). (-)-Liquiritigenin and isoliquiritigenin were also demonstrated to

157 inhibit growth of cancer cells with IC50 value of 30.1 and 16.6 µg/mL, respectively.

In Brazil, Jacaranda species were used as traditional medicine to treat protozoal infections such as leishmaniasis and malaria. These activities were of interest, and further studies were undertaken in order to elucidate the chemical basis of their traditional medicinal usage. Four phenylethanoid glucosides (jacaglabrosides A – D) from the leaves of J. glabra were found to be active in vitro against the P. falciparum

135 Kl strain with IC50 values ranging from 0.5 – 1.02 µg/mL. The methanolic extract of the leaves of J. puberula also showed activity against Leishmania amazonensis, with

IC50 value 88 µg/mL against promastigote forms, however only moderate activity against amastigote forms.158 Interestingly, ursolic acid, which was isolated from leaves of J. copaia, showed good activity with an IC50 value of 0.02 mM against amastigotes form.151

Jacaranda species found in Central America were traditionally used to treat microbial infection. Studies have demonstrated that the hexane extract from J. mimosifolia leaves possesses high antibacterial activity against B. cereus and E. coli with MIC values of

2.9 and 2.3 µg/mL respectively, compared to gentamycin sulfate,159 however, the stem bark extract of this species showed low antifungal activity against Candida albicans.154

Melanolic acid, ursolic acid, and corosolic acid were isolated from J. oxyphylla leaves, and showed a good antibacterial activities against B. cereus and Salmonella typhimurium with growth inhibition in the range of 84 – 90% at a concentration of 100

µg/mL. 155

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2.5.4 Results and Discussion

In order to study the phytochemical constituents from J. mimosafolia’s flowers grown in

Australia, the flowers were cleaned and freeze-dried to remove water. The dried flowers were extracted with methanol by repeated soaking over 3 days. The combined liquid extracts were concentrated and the residue partitioned between methanol and hexane to remove non-polar compounds and fatty acids.

The polar extract was subjected to reverse-phase HPLC, with optimal conditions using a gradient elution (100% to 60%) of solvent A (0.1% trifluoroacetic acid in water) over

40 minutes with solvent B (0.1% trifluoroacetic acid in acetonitrile) (Figure 31).

Preparative RP-HPLC afforded the separation of nine compounds including five jacarnanone-derived glucosidic esters (76-79135, 160) together with new jacarananone- derived glucosidic ester (73), alongside potential degradation products 72135 and 75.135

The known phenylpropanoid glucoside (74)134 and apigenin 7-β-glucoside (66)125 were also separated.

76 73 66

79 72 75 78 74 77

Figure 31. Reverse-phase (RP) HPLC profile of polar extract from J. mimosafolia flowers. Peaks were recorded at 254 nm. Solvent system: 100 to 60 % water-acetonitrile with 0.1% trifluoroacetic acid within 40 min. Peaks labels correspond to compound numbers in this dissertation.

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Compounds 78 and 79 required additional separation using a modified gradient system of 80 to 60% solvent A in 40 minutes. For both compounds, 1D and 2D NMR spectra showed similar characteristics and their LRESIMS showed the same molecular ion peaks at m/z 733 [M+H]+. The NMR spectra were found to have the same structure as the reported Jacaglabroside C and D isolated from J. glabra.135 Compound 76 - 79 were reported for the first time from the flower of J. mimosafolia.

Figure 32. Isolates obtained from J. mimosafolia flower.

Compound 73 was isolated as a yellow gum. The LRESIMS of 73 showed a peak at m/z

353 corresponding to a monosodiated ion [M+Na]+ and analysis of the HRESIMS

+ showed a peak at m/z 353.0839 assigned to C14H18O9Na (Calcd m/z 353.0849). The analysis of the 1H and HSQC NMR spectra indicated the presence of an aromatic substituted sugar unit, identified as a glucose. Analysis of the 1H NMR spectrum showed a large coupling constant for the anomeric protons at δH 5.42 (J = 8.2 Hz),

57 therefore the sugar was assigned as the β-anomeric sugar configuration.80 Furthermore, a pair of resonances at δH 7.01 (d, J = 8.5 Hz, 2H) and 6.13 (d, J = 8.5 Hz, 2H) were assigned as H2''/6'' and H3''/6'', respectively, which showed gHMBC correlations with resonances at δC 44.3, 67.4, 187.1, which were assigned as C7', C1', and C4', respectively (Figure 33). In addition, 1H NMR spectroscopic analysis showed a resonance at δH 2.84 (s), assigned as the methylene proton H7' which showed gHMBC

13 correlations to correlations to the C NMR resonances at δC 168.8 and 152.5 assigned to C8' and C2'/C6', respectively. This evidence therefore allowed the identification of a jacaranone ester moiety. Further gHMBC spectral anlysis showed a proton-carbon correlation from H1 to C8' confirming the linkage between the sugar with jacaranone acid moiety at the C1 of the sugar (Figure 33). Therefore, compound 73 was proposed as a new phenylethanoid glucoside. The 1H and 13C NMR spectal analysis is summarised in Table 6.

Figure 33. Long range proton-carbon (HMBC) of compound 73.

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Table 6. NMR spectroscopic data for compound 73.

Position Carbon (δC) Proton (δH) β-glucose 1 95.6 5.42 (d, J = 8.2 Hz) 2 73.2 3.30 (dd, J = 8.2, 8.9 Hz) 3 77.1 3.41 (dd, J = 9.0, 9.5 Hz) 4 70.8 3.29 (dd, J = 9.0, 8.0 Hz) 5 75.5 3.52 (ddd, J = 9.0, 6.5, 1.2 Hz ) 6 64.7 4.34 (dd, J = 11.2, 1.2 Hz), 4.09 (dd, J = 11.2, 6.5 Hz) Jacaranone acid 1' 67.4 2'/6' 152.5 7.01 (d, J = 8.5 Hz) 3'/5' 128.1 6.13 (d, J = 8.5 Hz) 4' 187.1 7' 44.3 2.84 (s) 8' 168.8 1 13 Note: H and C NMR experiments were performed in CD3OD at 500 MHz and 125 MHz, respectively.

The leaves and bark of J. mimosafolia have been used in traditional medicine plant in

Brazil, Argentina, and Ecuador to treat sexually transmitted disease and blood purification,134 Previous studies have reported that compounds 77-79 were found to be active in vitro against P. falciparum K1 strain with IC50 value of 0.56, 0.56, and 0.55

µg/ml, respectively. While the majority of isolated compounds in this study are known, this represents the first time that these compounds have been reported from the flowers of this species.

2.6 Natural product studies on Acacia pycnantha

Acacia pycnantha, commonly known as the golden wattle, belongs to the Fabaceae family (Figure 34). Typically, it grows from 3 to 8 meters in height, and is native to

New South Wales, Victoria, and South Australia and has been regarded as Australia’s national flower since 1988.161 The bark of this species is generally dark brown to grey

59 and the mature trees have phyllodes (flat and widened leaf stems) which hang down from the branches. The bright yellow flowers occur in groups of 40 to 80 on 2.5 - 9 cm long raceme, which arise from axillary buds (Figure 35). This species typically flowers between November and May, though they may additionally flower several months later.

161-162 This genus is distributed in Africa, America, Asia, and Australia with more than

1350 species, where 975 species grow in Australia. Several species of this genus are also found in the Middle East and its usage as medicinal plants and house appliances was recorded in the ancient history.163

Kingdom : Plantae Division : Angiosperms Order : Fabales Family : Fabaceae Genus : Acacia Species : Acacia pycnantha

Figure 34. Acacia pycnantha scientific classification.

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Figure 35. A. pycnantha flower, collected from the University of Wollongong.

2.6.1 Traditional medicinal usage and pharmacological activities of the Acacia genus

Acacia have been cultivated in different countries for many purposes such for furniture, and as a source of gum, tannins, perfumes, ink and paint.162 The genus of Acacia has also had roles as traditional medicine, for example, A. confusa, an indigenous species from Taiwan, was used to assist in wound healing and to prevent blood stasis.164

Similarly in Argentina, A. aroma is used as traditional medicine for wound healing as well as for its antiseptic activity and for the treatment of gastrointestinal disorders.165 In

Nigeria, powdered bark from A. nilitica is used for treating acute diarrhea.166

Indigenous Australian people used this genus in traditional medicines, adhesives, fibre craft, and utensils,63, 71, 163 and its seeds also played an important role in the diet, as they

61 are easily grounded to a flour which was mixed with water and eaten either raw or cooked to produce a type of unleavened bread.167 Several species from this genus have been reported to be used to prepare antimicrobial agents. The traditional medicinal usage from the Australian Acacia is described in Table 7.

Table 7. Ethnopharmacological data of Australia Acacia.

Species Part of Preparation Indication (folks) plants

A. auriculiformis leaves Decoction Sores, itchy skin. Soaking leaves and pods in hot water is used externally for leg and body pains.

A. beauverdiana Top Ash Mixed with tobacco and give branches narcotic effect.

A. cuthbertsonii Bark Unknown Toothache, rheumatism.

A. decurrens Bark Decoction Dysentery.

A. estrophiolata Inner bark, Decoction To treat sores, scabies, inflamed young eyes. branches

A. falcata Bark Embrocation Skin diseases.

A. holosericea Roots Infusion Laryngitis.

A. implexa Bark Embrocation Skin diseases.

A. kempeana Leaves Decoction Severe colds of the upper respiratory tract.

A. leptocarpa phyylodes Infusion Sore eyes.

A. legulata Bark infusion Cough, dizziness.

A. lysiphloia Leaves, Decoction Cold, pain killer. twigs

A. melanoxylon Bark Infusion Rheumatic.

A. monticola Branchlets Infusion Coughs, colds.

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A. multisiliqua Leaves Infusion To clear nasal congestion.

A. oncinocarpa Phyllode Decoction Chest infection, fever.

A. pellita Pods, Lather Itchy skin. seeds

A. tetragonophylla Inner bark Infusion Cough, dysentery, antiseptic.

A. translucens Leaves, Soaked Sore eyes. twigs

Note: summarized from 61

Regrettably, most of our understanding of the medicinal usage from Acacia species in

Australia is unreliable, with few species being properly studied for their biological activities. For example, barks from A. implexa and A. falcata showed antibacterial activities against sensitive and multidrug resistant strains of Staphylococcus aureus at

MIC values 7.81 – 100 µg/mL. 68 The biological studies from Acacia species have been reported from different countries, e.g the antibacterial, antioxidant, anti-inflammatory, antifungal, cytotoxicity activities. The know biological activities of various acacia species from around the world are summarized in Table 8.

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Table 8. Pharmacological activities from Genus of Acacia.

Species Country Part of plants Biological activities

A. aroma Argentina Leaves, stem, All ethanol extract showed antibacterial activities against gram-positive bacteria and flowers the MIC value from 0.067 – 0.308 mg/mL.165

A. albida Algeria Leave, bark Ethanol extract from leaves and bark showed good antioxidant activities with IC50 = 27, 29 µg/mL, respectively by using DPPH assay. 168

A. auriculiformis India Bark Ethyl acetate extract showed various antioxidant activities. The extract showed 71.20, 73.66, 83.37, 75.63, and 72.92% inhibition in DPPH, chelating power, lipid peroxidation, site specific and non-site specific deoxyribose scavenging assays.169

Methanol extract showed antifungal activities against Phellinus noxius and Pellinus 170 Australia, Heartwood badius. Japan

A. baileyana Australia Leaves Water extract inhibited 100% larval development and showed anthelmintic activity toward equine cyathostomins in vitro.171

A. confusa Taiwan Bark The ethanolic extract from the bark possessed antioxidant activity based on the DPPH free radical assay with an IC50 = 3.5 µg/mL.

A. cyanophylla Tunisia Flowers Ethyl acetate extract exhibited the highest antioxidant activities (IC50 = 67.3 µg/ml). 172 Isosalipurposide was active against AchE with an IC50 = 52.04 µg/mL.

A. cyclops Tunisia Pods Cyclopside from the pod showed cytotoxic activity against human breast cancer (MCF-

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173 7) cell lines with IC50 50 µg/mL.

A. dealbata India Barks Acetone extract effectively inhibited hydrogen peroxide-induced human hepatoma (HepG2) cells and effectively up-regulate the expressions of SOD, CAT and GPx enzymes.174

A. decurrens Australia Leaves Water extract inhibited 99% larval development and showed anthelmintic activity toward equine cyathostomins in vitro.171

A. etabica Saudi Legumes Methanol extract showed positive results to inhibits K. oxytoca strain MFM with zone Arabia of inhibition between 1.9 cm.175

A. ferruginea India Aerial parts Methanolic extract showed in vivo immunomodulatory and tumor inhibitory.176

Acetone extract effectively inhibited hydrogen peroxide-induced human hepatoma (HepG2) cells and effectively up-regulate the expressions of SOD, CAT and GPx enzymes.174 India Barks

A. iteaphlla Australia Leaves Water extract inhibited 99% larval development and showed anthelmintic activity toward equine cyathostomins in vitro.171

A. kempeana Australia Leaves The ethanolic extract showed positive antibacterial activities against B. cereus, E. faecalis, S. pyogenes at concentration of 1.6 mg/mL. 64

A. leucophloea India Stem barks Ethanol extract showed antidiabetic activities in streptozotoxin-nicotinamide induced type II diabetic Wistar albino male rats at dose of 400 mg/kg for 14 days.177

Acetone extract effectively inhibited hydrogen peroxide-induced human hepatoma

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63

66

India Barks (HepG2) cells and effectively up-regulate the expressions of SOD, CAT and GPx enzymes.174

A. mangium Australia, Heartwood Methanol extract showed antifungal activities against Phellinus noxius and P. badius Japan but lower activities compared to A. auriculiformis.170

A. melanoxylon Australia Leaves Water extract inhibited 100% larval development and showed anthelmintic activity toward equine cyathostomins in vitro.171

A. mellifera Sudan Leaves Ethyl acetate and aqueous fractions showed dose-dependent hepatoprotection in 2,7- dichlorofluorescein-toxicated cells at 48 h.178

A. murrayana Australia Leaves Water extract inhibited 86% larval development and showed anthelmintic activite toward equine cyathostomins in vitro.171

A. nilotica Nigeria, Stem bark, Ethanolic extract showed antibacterial activity. B. subtilis was most susceptible with India leaves the minimum inhibitory concentration 35 mg/mL.166 The methanol extract from the leaves showed highly significant antibacterial activity against Xanthomonas pathovars with MIC 6 µg/mL.179

A. pennata Ivory Leaves Butanolic extract is endowed with analgesic, associated with anti-inflammatory effects coast on acute inflammatory processes.180

A. podalyriifolia Australia Leaves Water extract inhibited 100% larval development and showed anthelmintic activity toward equine cyathostomins in vitro.171

A. pycnantha Australia Leaves Water extract inhibited 97% larval development and showed anthelmintic activity toward equine cyathostomins in vitro.171

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Saudi Leaves Methanol extract showed positive results to inhibits S. aureus strain MFM-10 and K. Arabia pneumonia strain AH-09 with zone of inhibition between 1-1.5 cm.175

A. origena Saudi Legumes Methanol extract showed positive results to inhibit S. aureus strain MFM-10 and K. Arabia pneumonia strain AH-09 with zone of inhibition between 1-1.5 cm.175

A. saligna Australia Leaves Water extract inhibited 93% larval development and showed anthelmintic activite toward equine cyathostomins in vitro.171

Spirostane saponin from the leaves exhibited a potent cytotoxic activity (IC50 = 2.8 Egypt Leaves µg/mL) by using Suphorhodamine B (SRB) cytotoxic assay.181

A. tetragonophylla Australia Leaves The ethanolic extract showed positive antibacterial activities against B. cereus, E. faecalis, S. pyogenes at concentration of 0.8 mg/mL. 64

A. tortilis India Gum The polysaccharide from gum of this species inhibited yeast and mammalian α-D- glucosidase enzyme and significantly reduced postprandial blood glucose level.182

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2.6.2 Phytochemical studies of Acacia Genus

Phytochemical studies from this genus are lacking even though it is widespread in the warm sub-arid and arid parts of the world. Many Acacia species are well-known for their condensed tannins and gums, e.g. catechin and gallocatechin were isolated for the first time from black wattle tree (A. mollissima) in 1959. Gum arabic or gum acacia is one of the more famous gums come from this genus and is isolated mostly from a single

African species, A. senegal.183-184 Alkaloids, terpenes, flavonoids, and saponins arising from phytochemical studies on this genus are summarised in Table 9.

Table 9. Phytochemical compounds isolated from Acacia species.

Compound Part of Species Isolates plants

Alkaloids Leaves, A. berlandieri, A. N-Methyl-β-phenethylamine, and amines roots, barks adunca, A. tyramine, N-methyltyramine, cultriformis, A. hordenine, β-phenethylamine, N- greggii. cinnamoylhistamine, methyl- 1,2,3,4-tetrahydro-β-carboline, N,N,-dimethyltryptamine.183, 185-186

Terpenes Leaves A. pennatula β-Sitosterol palmitate, β-sitosterol, lupenone, lupeol, 3-O-β-D- Roots A. jaquemontii glycopyranosylβ-sitosterol, Leaf, stem A. rossei stigmasteryl-β-D- glycopyranosides,187 cassane 1, Pods A. concinna cassane 2,188 sapogenin, acacic acid, acacidiol,189-190 avicin D.191 Aerial parts A. victoriae

Tannins Leaves,Barks A. mollissima (+)-Catechin, (+)-gallocatechin, and epigallocatechin.184, 192 heartwood A. pennatula 1,3-Di-O-galloyl-4,6-(-)- hexahydroxydiphenoyl-

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glucopyranose, 1-O-galloyl-β- glucosylpyranose, 1,6-di-O-galloyl- Leaves β-glucosylpyranose, 1,3,6-tri-O- 193 A. raddiana galloyl-β-glucosylpyranose.

Flavonoids Exudate A. neovernicosa 2',4'-Dihydroxychalcone, 4'- hydroxy-2-methoxychalcone, 2',4'- dihydroxy-3'-methoxychalcone, 7- hydroxyflavanone.194

Leaves, A. cyanophylla Quercetin 3-O-glucoside, flowers isosalipurposide, chalcononaringenin 4-glucoside.195

Heartwood, A. auriculiformis (-)-Teracacidin, (-)-isoteracacidin, aerial parts auriculoside I – V.196

Leaves A. tortilis Apigenin, luteolin, quercetin, 5,7- dihyroxy-4'-p-methyl benzyl isoflavone.197

Leaves A. raddiana Isorhamnetin 3-O-rutinoside, quercetin 3-O-rutinoside, quercetin 3-O-gentiobioside, quercetin 3-O- glucosylgalactoside, quercetin 3-O glucoside, quercetin 3-O galactoside.193

Heartwood A. caffra Oritin-4α-ol, epioritin-4α-ol.198

Leaves A. confusa Myricetin 3-O-(2''-O-galloyl)-α- rhamnopyranoside 7-methyl ether, myricetin 3-O-(3''-O-galloyl)-α- rhamnopyranoside 7-methyl ether, myricetin 3-O-(2'', 3''-di-O-galloyl)- α-rhamnopyranoside, myricitrin, myricitrin 7-methyl ether, myricetin 3-O-(2''-O-galloyl)- α- rhamnopyranoside, myricetin 3-O- (3''-O-galloyl)- α- rhamnopyranoside.199

Leaves A. pennata Quercein 3-O-β-D-glycopyranosyl- 4-O- β-D-glucopyranoside.200

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Heartwood A. giraffae, A. 7,8,4'-Trihydroxymethoxyflavone, galpinii 7,8,4'-tri-O-acetyl-3- methoxyflavone, 3-hydroxy-7,8,4'- trimethoxyflavone, 3-O-acetyl- 7,8,4'-trimethoxyflavone, 3,4'- dihydroxy-7,8-dimethoxyflavone, 3,4'-di-O-acetyl-7,8- dimethoxyflavone, 3,7,8,4'- tetramethoxyflavone.201

2.6.3 Results and Discussion

The golden wattle (A. pycnantha) is an endemic Acacia species which grows in most parts of Australia. Despite their bright colourful flowers, and that it is regarded as

Australia’s national flower, there are no reports on the structures present within the flower. Reported studies showed that water extract from leaves of this species inhibited

97% larval development and showed antihelminthic activity toward equine cyathostomins in vitro at a concentration of 1400 µg/mL, however there are no reports regarding to chemical constituents responsible for this biological activity.202 Therefore, we present the phytochemical constituents in the flower, as well as correlations to their biological activities.

In this study, the flowers were collected from the University of Wollongong campus, freeze-dried and then the dried flower extracted with methanol. The solution was concentrated, and the residue subjected to liquid-liquid extraction to provide hexane, ethyl acetate, and residual fractions.

RP HPLC profiles of both the ethyl acetate and residual fractions produced similar profiles. Thus, the ethyl acetate fraction was selected, and subjected to HPLC separation. The optimized conditions for separation were established as 80% to 40%

70 solvent A (0.1% TFA in water) with solvent B (0.1% TFA in acetonitrile) within 30 minutes, and these conditions were also used in preparative HPLC (Figure 36).

84

83

80,81 53

82 48 85

Figure 36. Reverse-phase (RP) HPLC profile of the ethyl acetate extract from A. pycnantha flower. Peaks were recorded at 254 nm. Solvent system: 80 to 40% water- acetonitrile with 0.1% trifluoroacetic acid within 30 min. Peaks labels correspond to compound numbers in this dissertation.

Utilising the optimized preparative HPLC method, eight known compounds were isolated and identified by comparison of their spectral data (NMR and MS) with those reported in the literature (Figure 37). The mixture containing compounds 80203 and

81204-205, was subjected to recrystallized by slow evaporation of a mixture containing water and acetonitrile at room temperature, and afforded 80 as long white-needle crystals. The crystal and the solution were separated gently using a Pasteur pipette, and the solution concentrated by lyophilisation to obtain compound 81 as an amorphous yellow solid.

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Figure 37. The structure of compounds 80 – 85 isolated from A. pycnantha flower.

Both compounds were found to have similar LRESMS spectra with peaks at m/z 457

[M+Na]+ and 433 [M-H]-. Based on 1D and 2D NMR spectra analysis both compounds were assessed as naringenin with a β-glucose moiety. gHMBC spectral analysis of 80 showed two bond correlation between the assigned anomeric proton at δH 4.79 with the peak at δC 106.1, assigned to C8, which suggested that the naringenin and glucose moiety were linked through a C-C bond. By comparison, compound 81 showed a three- bond correlation between the assigned anomeric proton δH 4.73 with carbon at δC 162.1, assigned to C5, indicating a C-O-C bond link between naringenin to the anomeric proton.

Furthermore, with comparison of their NMR spectra data and LRESIMS with those reported in literature, compounds 80 and 81 were identified as isohemiphloin and naringenin 5-O-glucoside. Both compounds were found to be optically active with

22 o o specific rotation values of [훼]D +29.52 and -97.79 , respectively.

72

In order to determine the absolute configuration of these compounds, circular dichroism

(CD) spectroscopy was performed, and the data compared with reported. The results showed a positive cotton effect at 334 nm (Δε +0.86) and a negative cotton effect at 290 nm (Δε -2.97) (Figure 38). Therefore, compound 80 was determined as (2S)- isohemiphloin, which is in agreement from published reports.203, 206 Configurational assignment at C2 of compound 81 was also possible from CD spectroscopy, which showed positive cotton effects at 334 nm (Δε +2.80) and negative cotton effects at 290 nm (Δε -4.06) (Figure 38), allowing for identification as (2S)-naringenin-5-O-glucoside by comparison with previously-published reports. 206-207 Finally, the absolute configuration of compound 81 was confirmed from the analysis of its single crystal X- ray crystallographic data, which confirmed a (2S) configuration (Figure 39). Compound

85 was naringenin and its spectroscopy data were compared with the publish data.205

6

4

2

0 200 220 240 260 280 300 320 340 360 CD (mdeg) CD -2

-4

-6 Wavelength (nm)

80 81

Figure 38. Circular dichroism (CD) spectra of compound 80 and 81.

73

Figure 39. Absolute configuration of compound 81, as determined by X-Ray crystallography.

During the isolation procedure, compounds 48, 53, and 8281 were also collected as single fraction owing to their close retention time, and therefore required re-separation by preparative RP-HPLC. Optimal separation conditions were found to be 80% to 60% of solvent A (0.1% TFA in water) within 30 minutes with solvent B (0.1% TFA in acetonitrile). Three isolates were collected and spectroscopic analysis revealed these to all be flavanol glycosides, specifically kaempferol 3-rutinoside (48), quercetin 3- glucoside (53), and myricetin 3-rhamnodise (82).

The two major constituents contained within the flowers were isolated at tR 15.0 (83) and 17.8 (84) minutes and were assigned as flavanol-based and chalcone-based.

Compound 83 was assigned as kaempferol-3-rhamnoside based on spectroscopic

74 analysis and data comparison to published reports.208 Compound 84 was isolated as isosalipurposide, a chalcone glycoside,172 and it is responsible for the distinctive colouration of A. cyaophylla flowers. Table 8 (vide supra) shows pharmacological activities from genus of Acacia, e.g., the water extract from the leave of A. baileyana, A. decurrens, A. iteaphlla, A. melanoxylon, A. podalyriifolia, and A. pycnantha grow in

Australia possess anthelmintic activity toward equine cyathostomins. The leaves and stem bark from various species of Acacia also showed antimicrobial activities againsts pathogenic bacteria as well as antoxidant activities.

Although there are limited reports regarding traditional medicinal usage from this species, the isolated compounds from this flowers have various reported activities.

Naringenin and naringenin glucosides (80, 81, 85) are reported to exhibit hydroxyl and superoxide radical scavenger efficiency and also showed effectiveness in the protection against oxidative damage to lipids in a dose-dependent manner.209 Furthermore, chalcone (84) has shown anti-infective, anti-inflammatory and cytotoxic properties.

Ghribia et al reported that compound 84, isolated from A. cyaophylla flowers, was

172 found to be active against acetylcholinesterase with an IC50 value of 52.04 µg/mL.

2.7 Antimicrobial activities of flowers in Australia

Different civilizations have used plants as antimicrobial agents to combat different bacteria, fungi, and viruses. The Indigenous people in Australia have used about 150 species of plants as antiseptic agents,64, 67 including Alocasia brisbanensis, Corymbia intermedia, Crinum asiaticum, and Syncarpia glomulifera to disinfect wounds in northern New South Wales.61, 210 In this study, we justify the antimicrobial activites

75 from four endemic Australian plants and two introduced plants in Australia and correlate with their ethnopharmacological activites.

Methanol extracts from 6 plants species as well as isolated compounds were tested for their antibacterial activity against Staphylococcus aureus (ATCC 43300), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Acinetobacter baumannii

(ATCC 19606), Pseudomonas aeruginosa (ATCC 27853) by whole cell growth inhibition assays with single concentration at 32 µg/mL. The extracts and compounds were found to be active if inhibition was ≥50%. The results of the antibacterial screening against these pathogenic bacteria is shown in Table 10.

76

Table 10. Antibacterial activity of six plant extracts and isolated compounds.

Extract/Compound Antibacterial activity (% inhibition) K. P. A. S. aureus E. coli pneumoniae aeruginosa baumannii A. Pulcherrimus NI NI 7.09 6.45 22.41 A. pycnantha NI NI NI 9.28 NI A. rufus 4.94 NI 9.82 16.77 25.21 B. acerifolius 60.08 23.01 32.69 17.08 55.12 J. mimosafolia 20.43 NI 13.15 NI 12.32 T. lepidota 57.90 32.21 27.70 40.23 53.78 46 56.26 29.45 24.45 19.14 60.59 47 14.03 NI 16.38 17.97 16.32 48 4.07 2.49 15.10 9.83 27.46 49 51.23 NI 13.81 11.61 28.51 50 13.32 NI 10.64 15.43 1.02 57 10.71 NI 9.54 9.31 31.58 58 60.77 11.85 23.73 9.19 51.41 62 8.81 NI 13.93 16.30 35.87 63 7.10 NI 11.99 15.99 21.29 64 10.79 NI 9.56 10.63 38.88 65 11.42 NI 16.91 14.22 11.57 66 24.57 NI 2.40 12.15 14.28 68 6.78 NI 11.34 13.47 5.35 69 4.68 NI 13.91 11.44 NI 70 NI NI 5.30 12.32 0.33 72 8.42 NI 5.70 14.67 24.65 73 3.39 NI 4.48 10.86 27.98 75 NI NI NI 6.42 17.45 76 NI NI 1.41 14.29 NI 77 13.50 NI 1.83 8.49 25.13 78 NI NI 2.12 11.82 45.10 79 NI NI NI 5.96 7.27 80 5.57 NI 15.78 17.70 7.19 81 13.32 NI 21.10 7.73 17.23 82 3.49 NI 9.54 19.03 4.86 84 12.84 NI 10.86 11.71 25.36 Concentration: 32 µg/mL; NI: no inhibition.

The genus of Brachychiton has been traditionally utilised for wound healing and infectious therapy in Indigenous Australian people.3 In modern pathology, the most

77 common bacteria in wound infections have been identified as S. aureus, Enterococcus faecalis, P. aeruginosa, Proteus species.211 Published reports showed the methanol extract of B. acerifolius flowers to inhibit B. cereus, as determined using the disc- diffusion method, however no chemical constituents were indicated as being responsible for this activity in this report.67 In this study, the methanol extract from B. acerifolius flowers, as well as compounds 46 and 49 isolated from this species, showed high antibacterial activity with percent inhibition values of 60.08%, 56.26%, and 51.23%, respectively against S. aureus. Furthermore, Table 10 shows both the methanol extract from B. acerifolius, and compound 46 possess high antibacterial activity against A. baumannii, with percent inhibition values of 55.12% and 60.59%, respectively. Several pathogenic bacteria commonly involved in wound infections such as S. aureus and A. baumannii were sensitive to quercetin (46) and naringenin glucuronide (49) with MIC values range of 2-32 µg/mL.212-214 Overall, the antimicrobial activities from this species maybe due to the present of compound 46 and 49 and within this finding confirmed the use of this species as antimicrobial agents, as used by Indigenous Australians people.

Furthermore, the methanol extract from T. lepidota flowers, as well as including compounds 46 and 58 showed activity high antibacterial activities with percentage of inhibition value 57.90%, 56.26%, and 60.77%, respectively against agianst S. aureus.

The extract and these compounds show high antibacterial activities against A. baumannii with percent inhibition values of 55.12%, 60.59%, and 51.41%, respectively.

The genus of Tibouchina was used to enhance wound healing and previous report that several species from this genus showed antimicrobial activities.93, 96-97 Compound 58 was reported as an anthocyanin and the first time reported to have microbial activities

78 against S. aureus and A. baumannii. It is evident that the flowers of T. lepidota serve not only an ornamental purpose, but also possess antimicrobial activitiy.

2.8 Conclusion

These phytochemical studies successfully isolated the major secondary metabolites contained within the flowers of four native Australian species with an additional two species which commonly grow in Australia. In these studies, thirty-seven isolates were obtained including four new compounds; these were (50) from B. acerifolius, 64 and 70 from Aningozanthos species and 73 from J. mimosafolia flowers. In this study, we were also able to isolate four different anthocyanins; 2 from B. acerifolius, 13 from T. lepidota; and 14, and 17 from A. rufus flowers; these compounds are likely responsible for the colouration of their flowers. Compound 36 was found as a chalcone glucoside, isolated from the A. pycnantha flower, and it is possible that it is responsible for its colouration. In antibacterial study, B. acerifolius and T. lepidota showed active against

S. aureus and A. baumannii as well as compounds 46, 49, and 58 at concentration of 32

µg/mL and it can be concluded that not only these species possess bright colourful flowers, but also show antimicrobial activity.

79

CHAPTER 3: Exploration of Secondary Metabolites from Alisterus scapularis

3.1 Introduction

The colours of bird feather have long captured human interest with the entire visible spectrum found in the plumage of a diverse range of bird species around the world. The mechanism of plumage colouration is divided into two categories: the first uses the chemical properties of pigments such as melanin, carotenoids, and porphyrins to allow the differential absorption and reflection of light to produce colours. The second are

“structural colours”, which rely on the organization and differential refraction properties of nanostructures.215 Structural colouration is reported to be responsible for feather colouration such as blues and purples, while pigments account for most of the red, orange, and yellow feathers, which sometimes arise from dietary sources.25, 216

Unlike humans, bird vision has a wider spectral range to see colours in feathers. As a consequence, the majority of bird species (particularly parrots) possess plumage that reflects ultraviolet (UV) wavelengths and has been shown to be involved in mate choice.217-218 For example, budgerigars (Melopsittacus undulates) show fluorescent plumage under short UV wavelengths (Figure 40). However, the purpose of naturally occurring fluorescent plumage remains a mystery, with suggestions that fluorescence may serve a social signalling functions, or that the fluorescence is merely by-product of highly-conjugated pigment structures.55

A B

Figure 40. Budgerigar’s head (A) under white light and under UV light (B).55

The majority of previous investigations have aimed to identify the mechanism of

colouration in birds feathers, leaving the identification of chemical compounds

responsible for the colouration of feathers as an understudied area. Many reports to

identify these compounds are focussed on their chemical classes rather than the

identification of specific precise structures, and these reports mostly do not fully

characterise chemical compounds but rely on comparisons alone, e.g. the occurance of

carotenoids in 197 birds species was determined using Raman and UV/VIS

spectroscopy, and HPLC by comparison with authentic carotenoids, rather than

complete structural analysis.219

There have been limited studies investigating the biological activities of feather

pigments. In one example, individual feathers from different species of parrots from

Central and South America (Red-tailed black cockatoo (Calyptorhynchus banksii);

Indian ring-necked parakeet (Psittacula krameri); Red-lored Amazon parrot (Amazona

autumnalis) and Scarlet macaw (Ara macao)) were placed in bacterial media containing

B. licheniformis and incubated for 120 hours. The results indicated that all of the

feathers inhibited the growth of B. licheniformis, though the responsible chemical

components for this activity remain unknown.17 Other studies have shown that the

78 uropygial gland in birds such as house sparrows (Passer domesticus) in Spain, secrete an oil as a barrier on plumage, and the mixture of these oils showed antimicrobial and antifungal activities.220 Further studies showed that this gland excretes fatty acids such oleic acid, linoleic acid, and arachidonic acid, as analysed by GCMS, in comparison with authentic fatty acids.221 Nevertheless, these reports lack conclusiveness in terms of both feather pigment identification, as well as most of the additional constituents present in the feathers. Therefore, protocols for extracting and identifying the precise chemical components present in feathers are still lacking.

3.2 Chemical properties of pigment classes in birds feathers

3.2.1 Carotenoids

Carotenoids are the second most prevalent pigment reported in the avian integument after the melanins.25 They are commonly the chemicals responsible for the red, orange and yellow colours, and used in sexual identification in birds, though they also play an important role as free-radical scavengers, protecting the nervous system from oxidative damage, and stimulating immune defence.222-223

Carotenoids are found in almost every form of integument tissue in vertebrates, including the skin, scales, avian feathers, eyes and the skin of mammals, and eggs from fish, amphibians, and reptiles. For example, both the orange colour from Goldfish

(Carassius auratus) and the yellow from Canaries (Serinus canaria) are a product of β- carotene (4), and lutein (5) as identified by comparison of extracts with authentic carotenoids.25

Carotenoids are acyclic 40-carbon molecules, consisting of eight isoprenoid units (85), joined in a head-to-tail pattern and reversed at the centre of the molecule so that the two

79 central methyl groups are in a 1,6 relationship and the remaining non-terminal methyl groups are in a 1,5 relationship (Figure 41 compound 85).224-225

Their linear hydrocarbon skeleton of conjugated double bonds can exist alone or can be cyclized at one or both ends, which are often substituted by different functional groups.

β-Carotene (4) is one of the unsubstituted carotenoids and in the absence of functional groups (Figure 41), it is relatively nonpolar and lipid soluble. The xanthophylls

(hydroxyloxocarotenoids and oxocarotenoids) are carotenoids that contain oxygen and possess polar and water soluble properties.25

80

Figure 41. Eight isoprenoid units (85), common carotenoids compound (4-7, 9-10) from bird feathers, carotenoids present in parrot blood (5, 6, 86-88).

81

Animals are not able to colour their feathers and fur with carotenoids without adequate pigment supplies from the diet. More than 600 carotenoids have been characterized from nature226 but less than 30 have been identified from the feathers of 19 different families of birds, however to date, none have been observed from parrot feathers, however limited studies showed carotenoids present in the blood of different parrot species from Central and South America (Figure 41).25, 56 Further, there are only a few examples of structural elucidation of carotenoids present in bird feathers using MS and

NMR, with most reports using HPLC, Raman or UV/VIS spectroscopy techniques and comparison with limited numbers of authentic carotenoids such as β-carotene (4), lutein

(5), canthaxanthin (10), astaxanthin (9), zeaxantin (6), and lutein-5,6 epoxide (7)

(Figure 41).

In 2010, LaFountain et al. 227 elucidated the carotenoid compounds from male specimens of Pompadour cotinga (Xipholena punicea) from Les Nourages, French

Guiana. These compounds were separated by HPLC, and were characterized by using mass spectroscopy (MS) and NMR, revealing the presence of eight ketocarotenoids, including astaxanthin and canthaxanthin, with six of the ketocarotenoids containing methoxy groups (Figure 42).

82

Figure 42. Isolated methoxy carotenoids from the feathers of Xipholena punicea.227

3.2.2 Melanin

Melanin is a common polymeric pigment found in bird feathers and animal fur. It serves as a basic colouration in the human body, and some animals use it as a sexual signal and social status. It is divided into two major classes: eumelanins (95) and phaeomelanins

(97).228 Melanin colours in birds feathers range from fully black or brown, and charcoal-

83 gray feathers. 229 For example, McGraw et al229-231 studied the presence of melanin in barn swallow (Hirundo rustica erythrogaster) and red-winged blackbird (Agelaius phoeniceus) feathers by comparing the feather extracts with pyrrole-2,3,5-tricarboxylic acid (PTCA) (96) and aminohydroxyphenylalanine (AHP) (99) using HPLC. Eumelanin

(95), when reacted with acidic potassium permanganate, degrades into pyrrole-2,3,5- tricarboxylic acid (PTCA) (96)229 whereas phaeomelanin (97), with its benzothiazine units, can be hydrolyzed with acid into aminohydroxyphenylalanine (AHP) (98).

Compounds 96 and 98 were then compared with authentic samples using HPLC. The results revealed the presence of eumelanin and phaeomelanin with different concentration levels in both species. It is well known that animal melanins can be classified into two major groups: the brown-to-black insoluble eumelanin and the yellow-to-reddish-brown pheomelanin which is soluble in alkali (Figure 43). 231-232

Figure 43. Degradation of eumelanin (95) and phaeomelanin (97) from bird ferathers to PTCA (96) and AHP (98).

Eumelanin is the more predominant form that produces dark black or brown hues in animals. The high degree of conjugation and the large number of indole quinone groups 84 in the irregular eumelanin biopolymer are responsible for the near-uniform absorption across the UV and visible spectrum, resulting in these pigments showing dark black and brown.233 Phaeomelanins are the reddish-brown pigments that are present in red hair, red and yellow fur of mammals and the chestnut feathers of birds. It has a lower molecular-weight and is soluble in alkaline solution, and it has different light- absorbance than eumelanin.

3.2.3 Porphyrins

Natural porphyrins, metalloporphyrins, and bilins are classes of porphyrins that are used by birds as colouration. These pigments are a less common class of feather pigment and can be responsible for certain reds, greens, and rust-browns in feathers and are known to strongly fluoresce red.234

Natural porphyrins, such as coporphyrin, have been found in the 13 orders of birds but most notably from owls. This pigment can form large polymer chains that act much like melanins to absorb strongly in the UV and visible range, thus producing reddish-brown feathers.235

Turacin (Figure 44) is a metalloporphyrin giving a red hue to the feathers from 23 species of turaco (Musophagidae) from Africa.234 It consists of uroporphyrin III, complexed with a Cu (II) ion. UV/Vis spectroscopy is used to analyse the presence of copper, and the porphyrins were identified by comparison with authentic porphyrin samples using chromatography and melting points comparisons. 236-237

85

Figure 44. Turacin (99) from Turaco (Musophagidae).

3.3 Structural colouration

Along with colouration produced by chemical pigments, structural colouration is also present in many birds, with variation in the physical structure of feather tissues underpining white, iridescent, and non-iridescent plumage colouration.238

Non-iridescent feathers produce the same colour from visible light at all angles of reflectance. The structures responsible for this colouration are typically the spongy networks of the inner core, or ‘medullary’ keratin cells of the feather barbs and shafts.

When melanins are present, they tend to absorb transmitted light, rather than reflecting it as iridescent colour.239

Iridescent feathers change colour based on the angle of observation with respect to the angle of incident light. The barbule nanostructures that produce iridescence consist of highly-ordered arrangements of melanin granules, or ‘melanosomes.240 Eumelanin is more commonly used for this purpose than phaeomelanin.241 The colour produced depends on the melanosome thickness, the presence of keratin layers or cavities, and the number and spacing of interfaces.239 Since the arrays occur in layers, changing the angle of incident light or angle of observation will change the path-length travelled by the

86 light, thus producing iridescence. Structural variations on the nanometre-scale have been linked to the observed reflectance spectrum of iridescent feathers at set angles.241

3.4 Parrots

Parrots are referred to as psittacines, and there are approximately 350 species in 86 genera, belonging to the order Psittaciformes. Their diversity is found mostly in Central and South America and Australia. The order Psittaciformes is divided into 3 families which are the Psittacidae (parrots), the Cacatuidae (cockatoos), and the Loriidae

(lorikeets).238 They have a strong and curved bill, strong legs, and clawed zygodactyl

(two toes pointing forward and two backward) feet and are some of the most striking examples of colouration in Nature.238, 242

3.4.1 Mechanisms of Parrot Colouration

Parrots have long captivated naturalists for their brilliant feather colouration, and the uniqueness of their colour system has been known for more than a century. In parrot feathers colouration, it is widely reported that bright reds, oranges, and yellow in the feathers come from a pigments known as psittacofulvins and is a generic label currently given to any non-carotenoid pigment found in parrot feathers.234 Psittacofulvins are lipid-soluble like carotenoids, but have different UV/VIS absorptive properties.234, 243

These compounds were firstly reported in the 19th century (1882) in the red plumage of the Scarlet Macaw (Ara macao).234, 243

In 2001, a more recent analysis of the possible structure of psittacofulvins from the red feathers of the Scarlet Macaw (Ara macao) was conducted.244 The feathers were extracted using methanol in a microniser to release the pigments from the feather keratin, and the solution filtered and evaporated to yield a pigmented residue, which

87 was dissolved in acetone, frozen, and filtered again. This process was repeated and the various extracts were combined before another evaporation, followed by purification on a silica (SiO2) column with a hexane:ethyl acetate (90:10) eluent to obtain the extract.

Four major components of the original pigment mixture were isolated using HPLC and subsequently analysed by MS,244 suggesting chemical constituents with masses of 200,

226, 252, and 278 KDa. Considering the previous suggestion that these compounds were conjugated polyenes, these red psittacofulvins were assigned as polyenic aldehydes based solely on these MS studies. The molecular structures of these four red psittacofulvins were tentatively assigned by their systematic names as tetradecahexenal

(100), hexadecaheptenal (101), octadecaoctenal (102), and eicosanonenal (103) (Figure

45).244

Figure 45. Possible chemical structures of psittacofulvins. 244

Another study set out to analyse the distribution of these compounds in 44 species of parrot from Central and South America.57 This study used a hot acidified pyridine extraction of the feathers followed by HPLC separation with chromatograms compared to identify identical pigments between species. The standard retention times from these compounds were obtained using the pigment extract from the red feathers of the A.

88 macao from South America which these compounds were derived.244 Suprisingly, by comparison of the retention times from previous reports, four compounds were found in varying concentration in all species tested.

3.4.2 Australian Parrots

Since European settlement, there has been a fascination with parrots in Australia where it had been termed as Terra Psittaacorum or the Land of Parrots.245 One-sixth of the world’s parrot species occur in Australia.245

From the dense rainforests, to swept heathlands of southern Tasmania, and to sparsely- vegetated deserts of the arid interior, every region of Australia is inhabited by parrots.

The Galah (Eolophus roseicapilla) and the Cockatiel (Nymphicus hollandicus), are widely distributed throughout of the continent while several parrots such as Eclectus

Parrot (Eclectus roratus) have very restricted ranges.

Kikkawa and Pearse (1969) categorized Australian parrots into several regions (Figure

46). Cockatoos are found in all sub-regions but Lorikeets are absent from the Eyrean sub-region. The Purple-crowned Lorikeet (Glossopitta porphyrocephala) occurs across southernmost Australia. The Platycercini, which include Rosella species, is the dominant parrot group in Australia and is represented in every part of the continent. It is divided into two classes, the first being the “violet-blue cheek-patches” with specimens of this class also defined by having predominantly greenish juvenile feathers. They can be found in the Atherton Tablelands, north-eastern Queensland, south-western New

South Wales, northern Victoria and eastern South Australia. The second class is “cheek-

89 patches are white, or white with some blue”, and can be found in Kosciuskan-

Tasmanian and Timorian division.246

Figure 46. The illustration of faunal areas of Australia, proposed by (a) Spencer, (b) Kikkawa and Pearse.246

Even though Australia is rich with colourful parrots species, the investigation into the chemical consituents from these feathers has remained largely unexplored. The investigation on pigment identification from birds feathers, especially in parrots is based on the comparison between pigment extracts with certain class of chemicals. The pigments have been extracted from feathers and analysed using a selection of techniques such as UV/Vis absorption spectrophotometry or mass spectrometry (MS), but not always in combination with each other. For an example, psittacofulvins are still largely identified based on the UV/Vis absorption spectrum of a crude extract, without attempts to elucidate their structures.57 Since such solutions are mixtures of compounds, it is debatable whether this method of identification is entirely valid. As it stands, the literature provides useful methods for extracting pigments from coloured feathers, but it lacks methods of thorough structural elucidation of all components of these extracts,

90 which is important in natural product chemistry. Furthermore, it is found that certain chemical constituents in the parrot feathers possess antimicrobial activity against B. licheniformis, however the chemical constituents responsible for this activity remain unknown. Therefore, in this study, we explored the natural products found within

Australian parrot feathers, with the intention to fully characterize the isolated chemical components present in the feathers.

3.5 Results and Discussion

In this study, a standard protocol was developed for the isolation and complete identification of chemical constituents from parrot feathers. The focus is not only pigments, but all of the chemical constituents found in the feathers. To achieve this aims, different species of predeceased bird carcasses were obtained from two sources: 1.

Cannon & Ball veterinary surgeons, Wollongong, Australia and 2. The Australian

National Wildlife collection, CSIRO, Canberra (Figure 47). These sources were approved by the University of Wollongong Animal Ethics Committee.

Studies were initiated using the Australian king parrot (A. scapularis) since this species was the most abundant of deceased specimens in the sample collection. Once the chemical constituents were obtained from the Australia king parrot (A. scapularis), these protocols would be used to efficiently accumulate data from a wide range of

Australian parrots.

91

(a) (b) (c)

(d) (e)

Figure 47. Examples of specimens obtained from Cannon & Ball veterinary surgeons, Wollongong, Australia and CSIRO, Canberra. (a) Rainbow lorikeet (Trichoglossus haematodus), (b) Galah (Eolophus roseicapilla), (c) Ausralian king parrot (Alisterus scapularis), (d) Crimson rosella (Platycercus elegans), (e) Eclectus (Eclectus roratus).

Alisterus scaplaris belongs to Psittacidae family or parrot family and is found in the coastal and mountainous regions of the eastern Australian mainland. This species of parrot possesses predominantly red and green plumage colouration and has a length of

42 cm from head-to-tail. The male exhibits a bright red head, chest and underside, with green wings and tail and a small blue section on the back with a light green strip on the wings. The female and juveniles are predominately green, with red undersides, a small blue section on the back, and may have a light green strip on the wings (Figure 48).246

92

Figure 48. Australian King Parrot male, obtained from Australian National Wildlife Collection, CSIRO.

Before the feathers were plucked, the carcasses were observed under visible and UV light to observe any fluorescent pigments present in this species. The result showed that the light green patch on the wing of this species was seen to fluoresce under long wave

UV light (λ = 365 nm) (Figure 49).

93

Visible light

UV light

Figure 49. Male Alisterus scapularis under visible and long wave UV light (λ = 365 nm).

Feathers were plucked from carcasses, separated according to colour and then washed sequentially in ethanol and hexane to remove dirt and lipids from the surface. Prior to extraction, the feathers were cut into small pieces using scissors based on a colour differentiation and ground with a mortar and pestle in the presence of liquid nitrogen to improve extraction efficiency. In this study, the green feather was used as this colour was the most abundant in this species.

To isolate and characterize chemical constituents from parrot feathers, it was necessary to test literature extraction procedures. The first method of extraction was heated acidified pyridine, which has been used previously to extract pigments in animal tissues such as insect wings, crustacean exoskeletons and importantly bird feather such A. macao.57, 247 It was claimed that these conditions weaken the non-covalent hydrogen bonds that bind the pigments to proteins and subsequently release them into solution.247

Therefore, pyridine was acidified by adding HCl (4 drops, 12 N) to pyridine (100 mL)

94 which was added to ground green feathers in a round bottom flask followed by heating at reflux for 4 h, providing an orange extract (17.13%, g/g). The residual feathers were grey after extraction and this observation supports the notion that the green colouration from the feather is due to a combination of yellow or orange pigment and structural colouration.

There was concern that the harsh extraction conditions may induce chemical change, and therefore, in a separate experiment acidified pyridine (100 mL) was heated at reflux in the absence of feathers for 4 h. This resulted in an orange solution containing 73.7 mg of brown/orange solid, presumably the results of polymerization of acidified pyridine, though this was not characterized.248 This result raises doubts about the integrity of samples from previous studies including those that investigated feather pigments that utilised this technique.17, 57, 230, 249-251

Another reported method for pigment extraction was grinding feathers in a microniser

Retsch MM2 equipped with a ZrO container in the presence of methanol for 1 h.244

Unfortunately, due to the lack of access to such machine, an analogous method using a

Quaigen® TissueLyser II was attempted which pulverises samples by rapid shaking with a tungsten bead. Therefore, ground green feathers (50 mg) were frozen in liquid nitrogen and then mixed with methanol in a 1.5 mL centrifuge tube and shaken on the

TissueLyser. This technique however proved ineffectual, with less than 100 mg of sample extracted, and the residual feathers retained their green colour.

In order to obtain the maximum crude extract from parrot feathers without inducing the formation of by-products, we reverted to the soxhlet extraction method.252-253 The advantages of this method include the displacement of transfer equilibrium by repeatedly supplying fresh solvent for contact with the sample, maintaining a relatively

95 high extraction temperature with heat from the distillation flask, and its simplicity and cost effectiveness.253 Therefore, green feathers from the Australian King Parrot were subjected to soxhlet extraction with methanol as the solvent for 72 hours, and the resulting extract concentrated to provide a yellow/orange solid in 4.36% (g/g) mass return. The residual feathers displayed grey appearance and implying that the pigments had been extracted (Figure 50).

Figure 50. Green feathers before (left) and after (right) extraction with methanol in a soxhlet apparatus.

In order to compare the 3 different extraction protocols (acidified pyridine, microniser grinding in methanol, and soxhlet extraction), the extracts were subjected to reverse- phase (RP) HPLC and profiles compared. A gradient of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.1% trifluoroacetic acid (TFA) in acetonitrile (solvent B) was used as eluent in a gradient from 100% to 0% solvent A in 20 minutes chromatographic runs (Figure 51). A comparison of the HPLC profiles showed that acidified pyridine with and without feathers were similar indicating the presence of unwanted products, likely to arise from products generated during the harsh extraction process. Although the profile of the sample from soxhlet extraction showed less compounds present, this is likely due to the lack of materials arising from either product degradation and/or pyridinium-based products.

96

1a. Acidified pyridine

1b. Acidified pyridine (no feathers)

2. Methanol in Soxhlet apparatus

Figure 51. RP-HPLC profiles from two different extraction methods with green feathers. 1a acidified pyridine with feathers, 1b. acidified pyridine in the absence of feather. 2. The feathers in methanol in soxhlet apparatus. The chromatograms were recorded at 254 nm wavelength.

The crude extract from the soxhlet extraction was then subjected to liquid-liquid extraction with methanol and hexane. The hexane fraction was analysed using GC-MS with the major constituents appearing at TR 8.41; 9.06; 9.09; 9.18 min (Figure 52), which were characterised as common fatty acids, including methyl palmitate (104), linoleic acid (105), methyl oleate (106), and methyl stearate (107). This marks the first isolation these compounds from this species.

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Figure 52. GC-MS chromatogram of the hexane fraction from green feathers.

Studies have reported these compounds (104–107) show several antimicrobial activities, e.g. synthesised unsaturated fatty acids (linoleic acid, palmitic acid, oleic acid) showed various inhibition against Staphylococcus aureus and Staphylococcus pyogens with IC50 values between 0.1 – 0.4 mM.254 These compounds in the feathers might arise from uropygial gland secretions, which occur in many birds including parrots; this gland is responsible for the secretion of preen oil.255 There is also speculation that this oil may have anti-parasitic, and antibacterial properties, or help with the production of Vitamin

D .220, 256-257 The relative composition of preen oil can be vary between bird species depending on age, sex, and diet and the chemical constituents from this oil can be determined by hydrolysing the esters into their respectively alcohols before their quantification by gas chromatography (GC).258

The polar (methanol) fraction was analysed using HPLC. Optimal conditions for separation were a C-18 column with 0.1% trifluoroacetic acid (TFA) in water (solvent

A) and acetonitrile (solvent B) in gradient of 100% to 50% of solvent A to 50% of solvent A in 60 minutes. Five compounds were successfully isolated at tR of 15, 24, 26,

27, and 36 minutes (Figure 53).

98

109 112 108

110 111

Figure 53. Semi-preparative HPLC profile of polar (methanol) fraction. Peaks were recorded at 254 nm. Solvent system: 100 to 50 % water-acetonitrile with trifluoroacetic acid (TFA) within 60 minutes. Peaks labels correspond to compound numbers in this dissertation.

Figure 54. Secondary metabolites in the methanol fraction from Australian King Parrot green feathers.

3,4-Dihydroxybenzoic acid (108) was isolated as a pale brown solid. The LREIMS indicated peak at m/z: 154 (M+) and matched the ion fragmentation pattern in the MS library system (NIST MS 135616). The 1H NMR spectroscopic data of this compound agreed with this structure reported in the literature.259 With subsequent analyses with

NMR spectroscopy and MS, compounds at tR of 24, 26, 27 minutes were assigned as

99 butanoic acid (109)260, 4-hydroxymethyl benzoate (110)261, p-hydroxybenzaldehyde

(111)262 (Figure 54).

The isolated compound (112) at tR 36 minutes appeared as a UV active yellow solid.

Analysis of the LRESMS showed a peak at m/z 271 assigned to a [M+H]+, and

HRESIMS analysis showed peaks at at m/z 271.0606 [M+1]+ and was assigned as molecular formula of C15H11O5. 1D and 2D NMR spectroscopic data analysis revealed

112 as genistein or 4',5,7-trihydroxyisoflavone, with the 1H and 13C NMR spectroscopic data in agreement with that reported in the literature.263 Interestingly, genestein is reported to possess various biological activites such as cancer prevention, tyrosine kinase inhibition, and antimicrobial activites.264 Reported studies showed that genestein moderate antimicrobial activities against Staphylococcus pasteurianus, Bacillus cereus, and Staphylococcus aureus with MIC value at 100 µM.265

The presence of either flavonoids or isoflavonoids has not previously been reported in bird feathers. Animal such as birds lack the necessary enzymes to produce secondary metabolites such as flavonoids and carotenoids, thus, the sources of these compounds is through diet (leaves, flowers, seed, algae).25 For instance, the European songbird (Sylvia atricapilla) showed the presence of flavonoids circulated in the blood stream.266

During the isolation of chemical constituents from the green feathers, we were not able to isolate and identifiy any psittacofulvins-typed compounds or carotenoids which have been previously reported to be responsible for the colouration in parrots feathers, as well as carotenoids.57, 234 Further, genistein was isolated as a yellow solid and the analysis of the HPLC chromatogram (Figure 53) indicated that it is the major peak presence in the polar extract. Thus, the presence of this compound maybe responsible for the yellow colouration of the extract from the feathers.

100

In an ongoing study, ten isolates were obtained from polar extract of the green feather using the same extraction and isolation methods. The isolates were obtained from the major and minor peaks from the preparative HPLC, however due to the time constraints and the poor quality, quantity, and purity of the isolates, we were unable to fully elucidate the structures. Of the ten isolates, six were UV active compounds and showed the presence of aromatic protons based on 1H NMR spectroscopic analysis. Suprisingly, all the results displayed different characterisation data and it means that there is an unique number of distinct constituents present in the feather of this species.267

3.6 Conclusion

This study successfully developed a reliable protocol for the extraction of the chemical constituents from green feathers of A. scapularis. It required grinding the feather using liquid nitrogen prior to extraction in a soxhlet apparatus, which was found to improve mass return and minimised compound degradation.

During this study, the hexane extract was analysed by GCMS which showed the presence of common fatty acids, including methyl palmitate (104), linoleic acid (105), methyl oleate (106), and methyl stearate (107). The presence of these fatty acids in the feathers might arise from secretions from the uropygial gland in birds. Furthermore, in this study, a HPLC protocol was optimised for the separation from methanol extract of green feather of A. scapularis, with five major chemical components 3,4- dihdroxybenzoic acid (108), butanoic acid (109), 4-hydroxymethyl benzoate (110), p- hydroxybenzaldehyde (111), and genistein (112), were isolated and identified even though the biosynthesis of these compounds in the Australian king parrot’s feather is unclear.

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The established protocol including extraction and isolation of chemical constituents from the feathers in this studies can be applied to other colours in the Australian king parrot and also others species to accumulated in Australian parrots. These chemical constituents might be possess links to their biological activities such bacterial resistance in the feathers or evolutionary lineage.

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Chapter 4: Biflavones from Australian populations of Ceratodon purpureus

This research is a collaborative project between Prof. Paul A. Keller from the School of Chemistry and Prof. Sharon Robinson from School of Biological Sciences University of Wollongong. The research is reported and discussed here as a single comprehensive story, however the work components were previously divided into three different PhD projects and separately completed by Rudi Hendra (PhD student from School of Chemistry), Ari Satia Nugraha (PhD student from School of Chemistry) and Melinda Waterman (PhD student from School of Biological Sciences). The work components are described as follow:

Melinda : - Moss cultivation in green house Waterman - Extraction and isolation of cell wall constituents - Comparisons between Australian and Antarctic C. purpureus - Anti-oxidant activity test of cell walls constituents Ari Satia : - Moss cultivation in Gwynneville Nugraha - Initial extraction of intracellular constituents - Initial isolation and characterisation of two biflavones from intracellular constituents - Anti-oxidant activity test of intracellular constituents Rudi Hendra : - Extraction of intracellular constituents - Isolation and re-examination of structural elucidation of the two biflavones from intracellular constituents. - Isolation and characterization of an additional three biflavanones from intracellular constituents. - Comparisons between Australian and Antarctic C. purpureus. - Anti-oxidant activity testing of 5 biflavones.

4.1. Introduction

Ceratodon purpureus, also known as fire moss, is a cosmopolitan moss belonging to the briopsidae family (Figure 55). The stems are erect, usually about 1.3 cm long and sometimes become 7–8 cm long in shaded places. The leaves are short and hairlike, spreading when moist and twisted when dry (Figure 56).268 This species is able to live on land, walls, roofs, and growth from tropical into polar habitats. It was reported that C. purpureus is an endemic moss in East Antarctica, where it is regularly exposed to intense UV light.269-270 Previous studies showed that in spite of the harsh environmental conditions, this species possesses the ability to maintain photosynthesis during desiccation with the moss accumulating less DNA-damage during desiccation despite elevated UV radiation.271-272 Different studies on endemic mosses in the Antarctic, e.g. C. purpureus, Bryum pesudotriquetrum, and Schistidium anatartici, showed a positive correlation between the accumulation of UV absorbing compounds and exposure to UV-B radiation however the chemical constituents responsible for this activity remain unknown.270 Further studies showed that C. purpureus to be the most UV tolerant compared to the other mosses in Anatarctica. 273

Kingdom : Plantae Division : Bryophyta

Order : Dicranales Family : Ditrichace/Bryopsidaae Genus : Ceratodon Species : Ceratodon purpureus

Figure 55. Ceratodon purpureus scientific classification.

Figure 56. Ceratodon purpureus growing on Antarctic continent, taken by Prof. Paul A. Keller (left) and Dr. Melinda Waterman (right).274

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Our previous study on the Australian population of C. purpureus reported the presence of p-coumaric acid (115), vanillin (116), p-hydroxybenzaldehyde (117), and trans- ferulic acid (118) from methanol extract (Figure 57b). This study also purported the isolation and structural elucidation of biflavone (113) and biflavone ether (114) (Figure

57a). 274 60

a. Initial proposed structures of the Intracellular isolates from Australian C. purpureus.

b. Cell wall isolates from Australian C. purpureus.

Figure 57. a) Intracellular isolates and b) cell wall isolates from Australian C. purpureus.

However, a thorough re-evaluation of the NMR spectroscopy data of both biflavone compounds, identified inconsistencies in the proposed structures of both 113 and 114.

For example analysis of the 13C NMR spectroscopic data of 113 showed carbon resonances at δC 163.1 and 163.2, however there were likely incorrectly assigned as ortho-substitution in ring A (C6 and C7) (Figure 57a). It is known that the substituent

105 effects of hydroxyl and methoxy groups can affect the 13C NMR chemical shift of flavanol derivatives, whereby the substitution of hydroxyl affects the ortho- and meta- position of flavonol to move the downfield (Figure 58), e.g., analysis of the 13C NMR spectroscopic data from tamarixetin (5,7,3'-trihydroxy-4'-methoxyflavonol) showed a set of carbons resonances at δC 160.6 and 163.8 assigned as meta hydroxyl substitution in ring A.275

Figure 58. Typical 13C NMR chemical shifts of hydroxyl substitution in flavone.275

Compound 114 was also likely incorrectly assigned, e.g. carbons resonances at δC

145.69 and 145.67 were assigned as meta-substitution in ring E (Figure 57a). Based on the reported studies, these chemical shift indicated an ortho-substitution. 275

Furthermore, from structure 113, it was reported that the C5 (ring A) connected to C4''' from ring E to form a biaryl link between two flavones (Figure 57a), however gHMBC spectral analysis showed no long-range proton-carbon correlation between H2'''/6''' with carbon at δC 105.3 assigned as C4'''. Consequently, the proposed biaryl link between two flavones via these carbons is doubtful and the link between two flavones is likely incorrect. Therefore, in this chapter, we aimed to reinvestigate the structural elucidation of both biflavones by following the same method of extraction and separation.

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4.2 Results and Discussion

4.2.1. Isolation and structural elucidation of biflavones

In order to reassess of the biflavanone structures, samples of Australian population of C. purpureus were collected from Dapto, New South Wales, Australia (34°29'36.5"S,

150°47'47"E) in May 2015. The moss was cleaned, washed and then extracted using the same method as previously reported.274 The methanol extracts were then subjected to semi-preparative HPLC separation with a 40 minutes gradient elution of 80% to 30% solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile (Figure 59).274

This resulted in the reisolation of 120 and 121, as well as three additional biflavanones

(119, 122 and 123) not previously identified from this species (Figure 60).

121 120 122 119 123

Figure 59. Semi-preparative HPLC profile of the methanol extract of C. purpureus. Peaks were recorded at 254 nm. Peaks labels correspond to compound numbers in this dissertation.

In order to obtain better resolution in NMR spectroscopy, all the isolated constituents were run at 600 MHz NMR using a cryoprobe in the School of Chemistry, the

University of New South Wales. NMR analysis of the constituents were performed initially in DMSO-d6, as most biflavones have been reported using this solvent. More

107 recent evidence however suggests that numerous errors in the structural assignment of isolated compounds from natural products have occurred, specifically in the assignment of 3,5-dioxygenated aromatic rings, when d6-DMSO was used, including for

276 flavones. Therefore, this study additionally analysed isolated compounds using d6- acetone.

Figure 60. Biflavones isolated from the intracellular space of Australian C. purpureus. Compound 122 is likely to also be a biflavone but its precise structure is unknown.

Compound 120 was isolated as a UV active yellow solid. The IR spectrum showed stretches at 1,653 and 3,213 cm-1, assigned to carbonyl and hydroxyl stretches, respectively. The molecular formula of C30H18O12 of compound 120 was confirmed by

HRESIMS with an assigned molecular ion [M-H]- to the peak at m/z 569.0732. A comparison of HPLC, MS, and NMR spectroscopic data indicated that 120 was the same as the originally isolated 113 .

108

1 The H NMR spectrum of 120 showed a set of meta coupled signals at δH 6.52 (1H, d, J

= 1.5 Hz) and 6.46 (1H, d, J = 1.5 Hz) which were assigned to H6 and H8 of the A-ring, respectively. From gHMBC data analysis, ring A was proposed as a 1,2,4,6- tetrasubstituted aromatic ring which contained the quaternary carbons C5, C7, C8a, and

C4a assigned to carbons resonances at δC 162.5, 165.2, 159.5, and 105.5. One singlet at

δH 6.59 was assigned to H3 of the ring C and gHMBC analysis showed correlated between this proton with C1' in the B ring and C4a in the ring A (Figure 61, right).

Figure 61. A, B, C-rings (top left), D, E, F-rings (bottom left) of proposed flavone unit of compound 120. Long range proton-carbon correlation (gHMBC) of compound 120 (right). Arrow indicate long range proton-carbon correlation.

Furthermore, the 1H NMR analysis showed a second set of meta coupled systems with resonances at δH 7.18 (1H, d, J = 2.0 Hz) and 7.28 (1H, d, J = 2.0 Hz), assigned to H2' and H6' of the ring B, respectively. Additional gHMBC data analysis allowed assignment of ring B as a 1,3,4,5-tetrasubstituted aromatic ring with the four quaternary carbons C1', C5', C4', C3' assigned to resonances at δC 124.1, 120.2, 150.4, and 146.3, respectively. gHMBC spectroscopic analysis indicated a strong correlation from H2' to

109

C4', and a weak 2-bond correlation from H2' to downfield C3'. Therefore, compound

120 contained a tetrahydroxyflavone unit, illustrated as the A, B, C rings (Figure 61).

1 Further analysis of the H NMR spectrum showed a set of signals at δH 7.63 (1H, d, J =

2.0 Hz,), 7.62 (1H, dd, J = 2.0 Hz, J = 8.0 Hz) and 6.81 (1H, d, J = 8.0 Hz), assigned to

H6''', H2''' and H5''' of ring E, respectively. The presence of a 1,3,4-trisubstituted aromatic ring was supported by gHMBC spectral analysis which showed correlations between H2'''/ H6''' with quaternary carbons at δC 123.8, 146.9, and 150.2 which were assigned as C1''', C3''', and C4''', respectively. A singlet at δH 6.66 was assigned to H3'' of the F-ring, gHMBC data analysis correlated with C1''' in the E-ring and C4a'' in the

D-ring (Figure 61, bottom left).

1 A singlet resonance at δH 6.24 was observed in the H NMR spectrum and assigned to

H6'' of the ring D (1,2,3,4,6-pentasubstituted aromatic ring). gHMBC spectral analysis showed four correlations from H6'' to quaternary carbon peaks at δC 104.7, 105.7, 163.2, and 164.9 which were assigned to C8'', C4a'', C8a'', and C7'', respectively (Figure 61, right). Two weak bond correlations from H6'' to more downfield C7'' or C8a'' suggested they were attached to electronegative atoms whereas a strong correlation to upfield C8'' suggested it is attached to another carbon atom. Therefore, another tetrahydroxyflavone unit was constructed by the D, E and F-rings.

NMR spectral analysis of compound 120 showed upfield quaternary carbons at δC 120.2 and 104.7, assigned to C5' (ring B) and C8'' (ring D) - this suggested no electronegative functional group substitution on these carbons. gHMBC spectral analysis showed strong long-range correlations between both H6' from ring B, and H6'' from ring D, the carbon resonance at δC 104.7, assigned as C8'' from ring D (Figure 63). This is key evidence in establishing the position of the biaryl axis connecting the two flavones unit. Thus, the

110 link between two flavone units (ABC and DEF) is via a C-C bridge connecting C5' and

C8'' forming a biflavone. Therefore, this compound can be reliably reassigned from the original 113 structure to 120, the biflavone 8-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2- yl)-2,3-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one or 5',3'''-dihyroxyamentoflavone. The complete 1H and 13C NMR data are presented in table 11.

H6''-C8'' H6'-C8''

Figure 62. gHMBC spectrum (zoom) for key evidence establishing the position of the biaryl link in ring B to ring D of 120.

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Table 11. 1H and 13C NMR spectroscopic data for compounds 120 and 121 (in (CD3)2CO). 120 121 δH (J in Hz) δC δH (J in Hz) δC 2 165.3 5.46 (dd, J = 13.2, 2.9 80.4

Hz) 3 6.66 (s) 104.6 3.23/3.27 (dd, J = 17.2, 43.8/43.9 13.2 Hz)

2.78 (dd, J = 17.2, 2.9 Hz) 4 183.1 197.3/197.4 5 OH – 13.02 163.1 OH - 12.17/12.18 165.1 6 6.24 (d, J = 2.1 Hz) 99.7 5.91/5.92 (br d J = 2.2 96.8

Hz) 7 164.9 167.6/167.7 8 6.52 (d, J = 2.1 Hz) 94.9 6.06/6.11 (br d J = 2.2 96.1

Hz) 8a 159.0 164.5/164.6 4a 105.4 103.1/103.2 1' 123.3 131.1/131.2 2' 7.62 (d, J = 2.3 Hz) 113.8 7.15/7.17 (d, J = Hz) 114.7/114.3 3' 147.0 146.5/146.6 4' 148.9 145.1 5' 120.7 119.8/119.9 6' 7.64 (d, J = 2.3 Hz) 123.7 7.04/7.06 (d, J = Hz) 123.2/122.9 2'' 165.5 165.3/165.4 3'' 6.59 (s) 104.1 6.58/6.59 (s) 103.9/103.8 4'' 183.5 183.5 5'' OH – 13.21 162.5 OH – 13.17 162.3 6'' 6.46 (s) 99.8 6.42 (s) 99.7 7'' 162.5 162.4 8'' 104.7 105.3 8a'' 156.2 156.1 4a'' 105.6 - 105.6 1''' 123.9 - 123.7/123.8 2''' 7.28 (d, J = 2.2 Hz) 114.5 7.29 (d, J = 2.2 Hz) 114.4/114.5 3''' 146.4 - 146.4/146.5 4''' 150.1 - 150.2 5''' 6.81 (d, J = 8.4 Hz) 116.5 6.91/6.92 (d, J = 8.4 Hz) 116.5 6''' 7.19 (dd, J = 2.2, 120.2 7.17/7.18 (dd, J = 2.2, 120.2/120.3

8.4 Hz) 8.4 Hz) Note: Two diastereomers are apparent in the spectra of compound 121, presumably due to one chiral centre being present at C2 combined with atropisomerism associated with restricted rotation around the C5'-C8'' axis. Where separate resonances have been resolved, both chemical shifts are given in the table.

112

Compound 121 was isolated as an UV active yellow solid and analysis of the IR spectrum showed stretches at 1,653 and 3,330 cm-1, assigned to carbonyl and hydroxyl stretches, respectively. The proposed molecular formula of C30H20O12 was confirmed by the assignment of the molecular ion [M+H]+ at the peak m/z 573.1044 observed in the

HRESIMS spectrum.

NMR analysis of 121 showed two equal intensity sets of signals as well as from the original isolated 114. It assumed that this because there are two sets of diastereomers.

The structure of 121 contains both a stereogenic atom in the ring C and three ortho hydroxy substituents about the B-D axis, which could provide sufficient rotational hindrance to lead to atropisomerism around the B-D linkage, and observation of peak doubling due to diastereomers. Although biflavone atropisomerism was reported in the

1960s, it has been recently noted 277-278 that the probable presence of biflavone atropisomerism is largely and undeservedly not recognized.

1 H NMR spectral analysis showed a set of meta coupled proton resonances at δH 5.92

(1H, d, 4J = 1.5 Hz) and 6.08 (1H, d, 4J = 1.5 Hz), which were assigned to H6 and H8 of ring A, respectively. This 1,2,4,6-tetrasubstituted aromatic ring contained four quaternary carbons C4a, C5, C7, and C8a which were assigned using gHMBC spectral

1 analysis to resonances at δC 103.6, 164.6, 165.2, and 164.6, respectively. Additional H

NMR spectral analysis revealed another set of meta coupled protons at δH 6.97 (1H, d,

J = 1.5 Hz) and 7.16 (1H, d, J = 1.5 Hz) assigned to H6' and H2' of ring B. gCOSY spectral analysis showed a weak correlation between H6' and H2'. gHMBC spectral analysis showed correlation between these protons with carbon resonance at δC 145.3, assinged as C4' (Figure 63, top left).

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Figure 63. A, B, C-rings (top left), D, E, F-rings (bottom left) of proposed flavone unit of compound 121. gHMBC of compound 121 (right). Arrow indicate long range proton- carbon correlation.

1H NMR spectral analysis suggested the presence of a 2,3-dihydrogenated tetrahydroxyflavone with H2, H3a and H3b (ring C) assigned at δH 5.47 (1H, dd, J =

13.5, 3.1), 3.24 (dd, J = 17.5 Hz, J = 13.5 Hz) and 2.79 (dd, J = 17.5 Hz, J =13.5 Hz), respectively. gCOSY spectral analysis, confirmed that H2 and H3 are vicinally positioned (Figure 63, top left).

Another flavonoid unit of compound 121 was identified through an analogues analysis

1 4 process. The H NMR resonances at δH 7.29 (1H, s), 7.18 (1H, d, J= 7.5 Hz) and 6.93

(1H, d, 4J= 7.5 Hz) were assigned to H2''', H6''' and H5''' of ring B, respectively and contained quaternary carbon C1''', C3''', and C4''' which were assigned using gHMBC spectral analysis to peaks at δC 123.9, 146.6, 150.3, respectively. A singlet resonance at

δH 6.59 (1H, s) was assigned to H3'' (ring F) which showed a correlation with the quaternary carbon C1'' (ring F) and C4a'' (ring D). A singlet resonance at δH 6.25 (1H, s) was assigned to H6'' ring D and gHMBC analysis showed 2-bonds correlations to C7'' to

C5'' at δC 162.5, 162.3, respectively. Furthermore, a strong correlation from H6'' to C8''

114 at δC 105.3 suggested that this quaternary carbon is upfield and is attached to an additional carbon. Therefore, this compound contained a trihydroxyflavone unit (ring D,

E, F) (Figure 63, bottom left).

From the NMR spectral analysis above, the resonances assigned to C5' from ring B and

C8'' from ring D were present in a relative upfield position indicating that these carbons were not attached to an electronegative functional group such as OH. gHMBC spectra analysis showed strong correlation between H6' from ring B, H6'' from ring D with carbon resonance at δC 105.3 assigned as C8'' from ring D (Figure 64) and this finding is key evidence in establishing the position of the biaryl axis connecting the two flavones.

This compound was elucidated as biflavone 8-(5-(5,7-dihydroxy-4-oxochroman-2-yl)-

2,3-dihydroxyphenyl)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one or

2,3-dihydro-5',3'''-dihydroxyamentoflavone

H6'-C8'' H6''-C8''

Figure 64. gHMBC spectrum (zoom) for key evidence establishing the position of the biaryl link in ring B to ring D of 121.

115

These two isolated biflavones have been previously reported from the moss

Plagiomnium cuspidatum but only the 1D NMR spectra were reported.279-280

Surprisingly, despite the reportings of the structure of biflavone 120 and 121, there have yet to be a full NMR characterization, including the assessment of all protons and carbons (Table 11) to their resonances, and importantly, there was no evidence supporting the positioning of the biaryl axis connecting the two flavone moieties.

Therefore, we report here for the first time, the full NMR assignment including evidence for the precise location of the biaryl axis.

In addition to the two major constituents 120 and 121, an additional three compounds were isolated in significantly smaller quantities. It is likely that these are also biflavones, based on analysis from mass spectrometric data, UV spectroscopic data, and partial NMR assignments. The precise structure of compound 122 was unable to be determined, whereas compound 123 is likely to be a dihydrobiflavone of the type illustrated in Figure 60. Although the moieties defined by rings A-C and D-F-E could be reasonably identified, the connections to ring B could not be determined due to both the weak signal to noise ratio in the NMR analysis, and sample contamination. Similarly, the complete structure of compound 119 was not possible to finalize, but is likely to be a structural isomer of 120. The moiety defined by rings D-F-E could be determined as could the flavone subunit A-C, the latter connected through C2, however, the bonds to ring B were also not possible to define (Figure 60).

Australian populations of C. purpureus were the initial focus for the bulk extraction and isolation of compounds from this species due to the high conservation status of

Antarctic flora. Both Australian and Antarctic C. purpureus are genetically similar281-282 and are highly tolerant to UV radiation283 which suggests that they produce similar UV-

116 active secondary metabolites. Unsurprisingly, 119 - 123 were present in the Antarctic population, confirmed by comparison of analytical HPLC (Figure 65), which also showed biflavones 120 and 121 as some of the most abundant UV-active compounds within Antarctic intracellular extracts.

Australian population 121

120 122 119 123

Antarctic population 121

120 123 119 122

Figure 65. Australian and Antarctic population of C. purpureus HPLC profiles.

The relative abundance of the two biflavones varied between the populations (Table

12). The isolated yields of these compounds from Australian C. purpureus were 6.0

(120) and 9.7 mg.g-1 dry wt (121), which were calculated from the integration of their corresponding peaks in HPLC traces. Applying this calculation showed how variable the biflavone concentrations can be between populations; however, isolate 121 was consistently more abundant than 120. More specifically for the Antarctic samples, biflavone 121 was on average 1.3-fold (ranging from one- to three-fold) more abundant than biflavone 120 in the crude intracellular extract. In contrast, biflavone 121 was 1.8- fold more abundant than 120 in the Australian population.

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Table 12. Concentrations (means ± s.d.) of isolates 119-123 within Australian (n = 7) and Antarctic (n = 8) samples of C. purpureus based on HPLC analysis.

Population Concentration (mg.g-1dry wt)

119 120 121 122 123

Australian 0.05 ± 0.01 5.95 ± 2.71 9.68 ± 5.42 0.34 ± 0.17 0.37 ± 0.14

Antarctic 0.05 ± 0.02 3.60 ± 2.09 4.76 ± 2.93 0.44 ± 0.23 1.89 ± 1.03

It is possible that the varied abundance of the biflavones across different populations might be related to different environmental stresses and seasonal differences between the two climates. It is suggested that the biflavones have a predominant role in photoprotection against UV damage either via screening-out harmful UV radiation or as scavengers of UV-induced radicals.274

4.2.2 Ultraviolet and antioxidant activities of isolated biflavone compounds.

Bryophytes (mosses and liverworts) such as C. purpureus produce photoprotective secondary metabolites, e.g. flavonoids, which reduce the impacts of damaging UV radiation penetrating their tissues, including the prevention of DNA damage by countering reactive oxygen species (ROS).60, 274

In order to investigate the ability of isolated biflavones to absorb UV radiation and the ability to scavenge free radicals, all the compounds were analysed by using UV/VIS spectroscopy and DPPH antioxidant assay. The absorbance spectra and antioxidant activities of 119-123 were compared with the high performing standard, rutin (Table

13). All isolates absorbed UV radiation (200 – 400 nm) relatively strongly compared to the standard (Figure. 66).

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Table 13. Ultraviolet and antioxidant activities of isolated biflavones compared to rutin standard for DPPH antioxidant assays. IC50 values represent means SD (n=3).

-1 -1 Compound Ɛ (M cm ) AA (IC50, µM)

λmax 1 λmax 2

119 350 (350 nm) 3290 (264 nm) > 400

120 12,298 (353 nm) 13,105 (255 nm) 33.5±1.6

121 7,028 (349 nm) 8,508 (275 nm) 127.8±3.8

122 380 (332 nm) 1490 (287 nm) > 400

123 703 (332 nm) 2550 (289 nm) > 400

Rutin 6,917 (359 nm) 8,297 (258 nm) 38.6 ±0.3

*Wavelengths specified in brackets; AA: antioxidant activity.

20000.00 18000.00 16000.00 14000.00 12000.00 10000.00 8000.00 6000.00 4000.00

2000.00 Extinction cooefficient (M/cm) 0.00 200 250 300 350 400 450 500 Wavelength (nm)

119 120 121 122 123 Rutin

Figure 66. Extinction coefficients in the UV-Vis spectrum (200 – 500 nm) of biflavones in comparison to the rutin standard. All samples were diluted in MeOH.

Biflavones 120 and 121 showed relatively high absorbance across a range of UV wavelengths, exhibiting peaks at 353 (Ɛ = 12,298 M-1cm-1) and 255 nm for 120 and 349

119

(Ɛ = 7,028 M-1cm-1) and 287 nm for 122. However, compound 119, 123, and 124 displayed lower absorbance over the entire UV-B range (280 – 315 nm).

Further analysis using a DPPH assay proved their potential as antioxidants (Table 13).

Biflavone 120 demonstrated considerably lower IC50 values (33.5 µM) relative to the strongly-antioxidant standard, rutin (38.6 µM). Additionally, isolate 121 showed radical-scavenging activity with IC50 values of 127.8 µM, respectively. The remaining isolates 119, 123, and 124 showed much weaker antioxidant values with IC50 concentrations in the mM range.

Biflavone 120 was approximately 13% stronger than rutin, a widely-used as a positive control for antioxidant assays. The model flavonoid rutin has displayed greater radical- scavenging efficiencies compared to that of the commercially available antioxidant butylated hydroxytoluene (BHT).284-285 We report here for the first time the testing of isolate 120 as an antioxidant and our findings show that it could be as antioxidant agent.

Compounds 120-121 thus combine great potential as radical-scavengers with good UV absorbing capacity particularly over the UV-A wavelengths (315 – 400 nm). This suggests that these biflavones have dual functions in photoprotection: firstly by directly reducing UV radiation transmission to susceptible intracellular components and secondly by quenching UV-induced ROS. Generally, it has been reported that biflavones have relatively impressive capacities as antioxidants.286 Perhaps 120 - 121 are constitutively produced and accumulate in the cell to function predominantly as antioxidants but when exposed to more stressful environmental conditions such as high light or UV stresses, these highly abundant intracellular compounds are deposited in the cell wall where their UV-absorbing abilities also offer direct photoprotection. Overall,

120 the UV photoprotective compounds 120 – 121 are likely to contribute to the survival of

C. purpureus in the harsh environments (e.g. Antarctic and Australian desert.

4.3 Conclusion

The previous study proposed two isolated biflavones (113 and 114) from Ceratodon purpureus. However, inconsistencies in the original NMR spectroscopic analysis of these compounds led to an incorrect assignment of NMR spectral data, including the location of biaryl axis. Therefore, in this study we reassigned the chemical structures of biflavones 113 and 114 using samples from the Australian population of Ceratodon purpureus, by following the same extraction and isolation procedure. Compound 120 and 121 were found to have similar spectroscopy data (MS and NMR) with 113 and

114, assigned as 5',3''''-dihydroxyamentoflavone and 2,3-dihydro-5',3''''- dihydroxyamentoflavone, respectively. This study additionally isolated three different biflavanones (119, 122, and 123), based on analysis from mass spectrometric data, UV spectroscopic data, their anti-oxidative measurements and partial NMR assignments.

However, The precise structure of compounds were unable to be determined. All compounds were found in both Australian and Antarctic C. purpureus with compound

120 and 121 as the major biflavanones from both populations. The different relative abundances of these isolated biflavanones might be due to variations in conditions and climate.

All biflavanones compounds absorbed in the ultraviolet spectrum, with compounds 120 and 121 showing high absorbance of UV radiation in the UVA. These compounds also showed relatively high antioxidant activity in comparison to the rutin positive control.

121

These compounds are reported for the first time in C. purpureus and additionally exhaustive structural elucidation of 120 and 121 was established.

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Chapter 5: Conclusions and Future Directions

The study of natural products is an important tool in the discovery of new bioactive species, as it allows for identification of the chemical constituents of particular species and allows active components of traditional medicines to be elucidated. Further, natural products study can be used as a tool in the identification of the colourants responsible for the striking physical characteristics of a variety of species of both plants and animals. Certain animals such as birds use chemical constituents are not only for protection from hars conditions but also used them as attractants, sex determination, and protect them from microbial colonisation. In the same manner, plants species located in harsh environments survive by the production of certain chemical constituents, as protectants against UV radiation or other external factors. Isolation and characterisation of these chemical constituents therefore would allow significant insight into the protective modes of action of such species.

The environment of Australia is rich with colourful plants and animals, where some of these species have historically been used as medicines and natural dyes. Studies reported here successfully established laboratory protocols for the isolation of secondary metabolites from plants and bird feathers from Australia using a natural products chemistry approach to investigate individual questions.

Firstly, phytochemical studies were undertaken on the flowers from four species of native Australian plants (Brachychiton acerifolius, Anigozanthos rufus, Anigozanthos pulcherrimus, and Acacia pycnantha) which are commonly found in the Wollongong area, and additionally, two introduced species (Tibouchina lepidota and Jacaranda mimosafolia) which commonly grow in Australia. A total of 36 compounds were isolated from the various polar fractions from the six species, including four anthocyanins, five flavanones, one flavanonol, two flavones, eleven flavonols, two chalcones, two benzoic acid derivatives, one quinone, seven phenylethanoid glucosydes, and one heptanedioic acid derivative, including four new compounds.

From B. acerifolius (described in section 2.2), six major compounds (46 – 51) were isolated, with 47 being the major compound, identified as pelargonidin-3-(6- coumarylglucoside)-5-(6-acetylglucoside), an anthocyanin that is responsible for the colouration of this flower. In addition, a new compound 50 was isolated and identified as (2S)-4',5-dihydroxylavanone 7-O-β-D-glucuronide methyl ester, with the absolute configuration of the 2-position confirmed using CD spectroscopy. Furthermore, seven compounds (53 – 58), and 46, earlier reported from B. acerifolius, were isolated from T. lepidota (described in section 2.2), with 46, 54, 58 as the major constituents. Compound

58 was identified as malvidin 3-(coumaryl glucoside)-5-(acetylxyloside), a purple compound that is responsible for the colouration of the flower from this species.

Phytochemical investigation of two species from the genus Anigozanthos (A. flavidus and A. pulcherrimus) flowers resulted in the isolation of twelve compounds from A. flavidus (62 – 70) and eleven compounds from A. pulcherrimus (63, 64, 66 – 71)

(described in section 2.3) including 48 and 51, with these later two compounds reported earlier from B. acerifolius and additionally 53 from T. lepidota. Compounds 62 and 64 were isolated as red solid compounds from A. rufus, and identified as cyanidin-3- rutinoside and cyanidin-3-O-(6-O-p-coumaryl) glucoside, respectively. They may be responsible for the colouration of this flower with both being reported for the first time from this species. Compounds 64 and 70 were isolated from both species as new natural

123 product compounds, and were identified as 2-amino-6-O-p-coumaryl heptanedioic acid and chalcone-5'-O-(4'''-O-p-coumaryl) glucoside, respectively.

From the methanol extract of the J. mimosafolia flower (described in section 2.4), one quinone (72) and six known phenylethanoid glucosides (74 – 79) were isolated along with one new phenylethanoid glucoside (73). In addition, 66 was isolated and identified as a flavone glucoside, reported previously from Anigozanthos sp. The phytochemical investigation of the flowers of Acacia pycnantha resulted in the isolation of eight compounds (80 – 85) including 48 and 53 which were also isolated from B. acerifolius and T. lepidota, respectively (described in section 2.5). Compounds 80, 81, and 85 were identified as (+)-(2S)-isohermiphloin and (-)-(2S)-naringenin 5-O-glucoside, and (2S) naringenin, respectively, with their absolute configuration of the 2-position confirmed by CD spectroscopy. The structure of 81 was also confirmed by X-ray crystal structural analysis. Compound 84 was identified as isosalipurposide, a chalcone glycoside, and was found in A. cyaophylla flowers. It is responsible for the colouration of flowers from

Acacia genus and is a known as a dye and giving its golden or orange colour.

The crude methanol extracts of all six flowers investigated here, alongside several isolated compounds, were investigated for their antimicrobial activities against various pathogenic bacteria as described in section 2.6. Among the six extracts, those from B. acerifolius and T. lepidota showed inhibition of growth of Staphylococcus aureus

(ATCC 43300) and Acinetobacter baumannii (ATCC 19606) at concentration value of

32 µg/mL. Of the number of isolated compounds from six different species of flowers, compounds 46, 49, and 58 showed similar inhibition in growth of S. aureus and A. baumannii at the same concentration. These compounds were isolated from B. acerifolius (46 and 49) and T. lepidota (46 and 58) flowers.

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In general, the major compounds, including the coloured, examples were isolated from six different species from endemic and introduced flowers in Australia. Since compounds 46, 49, and 58 showed activity against S. aureus (ATCC 43300) and A. baumannii (ATCC 19606), further testing for the determination of minimum inhibition concentration (MIC) and mode of action would be worthwhile. It would be also interesting to carry out the antimicrobial testing on some crude fractions. The phytochemical and biological studies may then be used to develop monographs for

Australian plants.

We also used natural product exploration to fully characterize the chemical components from the feathers of Australian parrots. In this study, we not only focused on the pigments present in the feathers, but rather all of the natural products found within feathers. In this study, we used Australian king parrot (Alisterus scapularis) feathers, since this species was the most abundant among the deceased specimens obtained during the sample collection period (described in Chapter 3).

We successfully developed a standard extraction protocol of chemical constituents from feather. The extraction in a soxhlet apparatus using methanol was found to improve mass return and minimised compound degradation compared to heating in acidified pyridine, where previous studies (including those that investigated feather pigments) had utilised this technique, and microniser grinding in methanol. The green feathers from A. scapularis in methanol were extracted using a soxhlet apparatus and resulted in

1.85 g of crude material which was liquid-liquid partitioned with hexane to produce a hexane fraction (1.22 g) and a methanol fraction (630 mg).

The GC-MS analysis of the hexane fraction indicated four common fatty acid identified as methyl palmitate (104), linoleic acid (105), methyl oleate (106), and methyl stearate 125

(107). Furthermore, the methanol extract of green feather of A. scapularis, five major chemical components were isolated and identified as 3,4-dihdroxybenzoic acid (108), butanoic acid (109), 4-hydroxymethyl benzoate (110), p-hydroxybenzaldehyde (111), and genistein (112).

Due to time constraints and the isolation of compounds in smaller quantities, we were only able to isolate and fully characterize the major compounds from the green feathers of A. scaplaris. In the long term, the laboratory protocol in this study can be applied to different coloured e.g. red feathers from this species and other species in order to accumulated data from a wide range of Australian parrots. This data set would be a great asset to many different fields such evolutionary lineage in birds feathers or linkage between bacterial resistance and classes of chemical in the feathers by using this standard extraction and isolation protocols from the feathers.

Our final natural product study was re-evaluation of the NMR spectroscopy data from biflavone compounds (114 and 113) from Australian population of Ceratodon purpureus by following the same method of extraction and separation (described in

Chapter 4), followed by comparison with Antarctic populations of this species. This resulted in the isolation of 120 and 121, and an additional 3 different biflavanones (119,

122 and 123) not previously identified from this species.

Compound 120 and 121 were found to have similar spectroscopy data (MS and NMR) to 113 and 114, and were reassigned 5',3''''-dihydroxyamentoflavone and 2,3-dihydro-

5',3''''-dihydroxyamentoflavone, respectively. The detailed spectroscopic analysis (1D and 2D NMR spectral analysis) of these structures was reported for the first time in this study. Along with the reassignment of these compounds, we isolated three additional biflavanones-like compounds (119, 122, and 123), however we are not able to 126 determine precise structures. We have found that biflavones 120 and 121 are the major compounds within both Australian and Antarctic C. purpureus, with differing relative abundances of these compounds observed between populations.

All isolates absorbed in the ultraviolet spectrum and the two biflavones 120 and 121 showed relatively high antioxidant activities in comparison to the rutin positive control.

These properties of 120 and 121 suggest that they are potentially involved in dual photoprotection mechanisms via both direct (UV-screening) and indirect (radical- scavenging) capacities. Overall, UV photoprotective compounds 120 - 121 are probable to contribute to the harsh nature of C. purpureus.

In general, by using natural product studies approach, we successfully isolated and identified of fifty secondary metabolites from eight separate species from Australian plants and birds feathers. Four anthocyanins and one chalcone were isolated from four different Australian flowers species and these compounds may arise responsible for the colouration of this species. In addition, four new compounds were isolated from three different Australian flowers species. The antimicrobial activities of the crude extracts of six different Australian flowers species and the selected pure compounds that we have studied were in alignment with their ethnopharmacological uses.

In addition, we successfully established extraction and isolation of the chemical constituents from the green feathers of the Australian king parrot. GCMS analysis identified common fatty acids which might arise from the uropygial gland. We also isolated and fully characterized different classes of chemical constituents from the feathers including genistein (112) which might be responsible for the colouration of the feathers. In the same manner using natural product studies, we successfully to isolate five biflavones from C. purpureus and additionally exhaustive structural elucidation of 127 two chemical structures of biflavones from previous studies. All the compounds absorbed in the ultraviolet spectrum and possess various antioxidant activity.

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Chapter 6: Experimental

6.1 General

Analytical HPLC was performed on either a Waters (Waters 1525 pump, Waters 2487 detector, controlled by Breeze software v3.30) with a Symmetry C-18 column (5 μm,

4.9 x 150 mm) or a Shimadzu HPLC system (SOD-M10AVP diode array detector, CTO-

20AC column oven, LC-10ATVP pump, SIL-10Ai auto-injector, SC-10AVP system controller, DGU-20ALVP degasser, controlled by Shimadzu Class-VP software v6.12

SP3) with a Wakosil C-18 RS column (5 µm, 4.6 x 250 mm). Preparative HPLC was performed on a Waters prep-LC system (LC-600 controller, 2489 detector, LC150

Pump, PD1 degasser) with a Waters reverse-phase OBD SunfireTM C-18 column (5 μm,

TM 19 x 150 mm) protected with a Waters Sunfire C-18 guard column (5 μm, 19 x 10 mm). All analytical HPLC samples were filtered through Whatman syringe filter PTFE

0.45 μm, 4 mm and preparative HPLC samples were filtered through Whatman syringe filter 0.45 μm, 30 mm. A Büchi Rotary Evaporator (R-114/200) with a high vacuum pump was used for evaporation of solvents under reduced pressure at 40 ºC. Nitrogen gas was used for evaporating smaller quantities of solvents. A high vacuum system with a liquid nitrogen trap was used for the drying of compounds. Gas Chromatography

Mass Spectrometry (GC-MS) was performed on a Varian GC (fused silica 0.25 μm, BP-

5 capillary column 0.25 x 30,000 mm) with hydrogen gas under temperature ranging

40-280 ºC. Direct insertion electron impact mass spectra (DI-MS) were obtained on a

Shimadzu QP-5050 spectrometer with the DI probe heated from 40-200 ºC at 50

ºC/minute. HREIMS were run on a VG Autospec Mass Spectrometer. Electron spray

(ESI) mass spectra were obtained on a LCMS-2010 EV (Shimadzu). Samples were injected as a solution in methanol (HPLC grade). HRESIMS were run on Waters QTof Ultima mass spectrometer equipped with Lockspray electrospray ionisation source controlled by MassLynx software (version 4.0). One and two dimensional NMR experiments including 1H-NMR, 13C-NMR, APT, DEPT, NOE, gCOSY, TOCSY,

NOESY, gHSQC, gHMBC were obtained on a Varian Unity Inova-500 MHz NMR spectrometer, controlled by Varian VNMR software (version 6.1 revision C).

Deuterated chloroform was used as NMR solvent (unless mentioned). 13C-NMR spectra were also obtained from APT spectrum through absolute value mode conversion. In addition, 1D and 2D NMR analysis (including gCOSY, gHSQC, gHMBC) was performed using a Bruker Avance III 600 MHz instrument fitted with a 5mm TCI cryoprobe in the School of Chemistry, the University of New South Wales. Optical rotations were measured on a Jasco P-2000 polarimeter. UV-visible spectra of samples in MeOH were obtained using a Shimadzu UV-VIS Spectrophotometer UV-1601. CD spectra were recorded on a JASCO J-810 spectropolarimeter with a pathlength of 0.1 cm and concentration between 50-100 µM in methanol.

All the solvents were AR (analytical reagent) grade or distilled except HPLC grade for the HPLC. Solvents mixtures are stated as volume to volume (v/v) proportion.

6.2. Plant and feathers materials

Flowers from six different species (Brachychiton acerifolius, Tibouchina lepidota,

Anigozanthos rufus, A. pulcherrimus, Jacaranda mimosaefolia, and Acacia pycnantha) were collected from Wollongong, NSW. T. lepidota and J. mimosaefolia flowers were collected in April 2013 and October 2014 from Aschroft Place, Wollongong, NSW

(34°24'08.3"S 150°52'36.4"E). B. acerifolius, A. rufus, A. pulcherrimus, and A. pycnantha flowers were collected on the University of Wollongong, NSW 34°24'16.9"S

130

150°52'38.2"E) in October- November 2013. The flowers were cleaned, washed, freeze- dried and were then powdered and stored in sealed-plastic zip lock bags. Furthermore, twelve male Australian King Parrot (Alisterus scapularis) specimens were obtained from Australian National Wildlife Collection, CSIRO, Canberra on December 2013. In addition, various species of birds were collected from Cannon & Ball veterinary surgeons, Wollongong, Australia between 2013-2014. The specimens were separated based on species, stored at freezer before further analysis. The feathers were plucked and then separated according to colours, stored in sealed-plastic zip lock bags.

6.3. Isolation of constituents of Brachychiton acerifolius flowers

To flower powder (300 g), methanol (2.0 L) was added and the solution was stirred for

48 hours, filtrated and the supernatant vacuum dried to produce extract (13.54 g). A suspension of this extract in 20% (v/v) methanol was sequentially fractioned with hexane, dichloromethane, and ethyl acetate produced four fractions hexane (4.21 g), dichloromethane (0.728 g), EtOAc (3.17 g), and residual (3.52 g) fractions.

The ethyl acetate fraction (500 mg) was applied a short silica column (2 cm x 5 cm) and elution with acetonitrile:methanol:H2O (8:1:1) produced 100 mL of solution, which was reduced to 10 mL by evaporation. This sample was then injected in eight blocks into preparative HPLC with a gradient elution from 90% to 50% solvent A (0.1 % TFA in

H2O) in 40 minutes with solvent B (0.1 % TFA in ACN), separating isolates 46 to 51.

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Quercetin 46

A UV active pale yellow crystal needle (0.016 mg/g dried

plant sample); UVmax (MeOH) (nm): 229; 255; 371; 1H-

81 NMR (CDCl3, 500 MHz), 7.63 (dd, J = 8.5, 2.2 Hz, 1H,

H6'), 7.73 (d, J = 2.1 Hz, 1H, H2'), 6.89 (d, J = 8.5 Hz, 1H, H5'), 6.39 (d, J = 2.1 Hz,

13 81 1H, H8), 6.18 (d, J = 2.1 Hz, 1H, H6); C-NMR (CDCl3, 125 MHz), 178.5 (C4),

163.1 (C5), 165.3 (C7), 159.1 (C8a), 158.6 (C2), 149.8 (C3'), 145.9 (C4'), 135.1 (C3),

122.2 (C1'), 120.5 (C6'), 114.6 (C5'), 114.5 (C2'), 105.7 (C4a), 99.0 (C6), 94.0 (C8);

LREIMS: 303 [M+1]+.

Pelargonidin-3-(6-coumaryl-β-glucoside)-5-(6-acetyl-β-glucoside) 47

A UV active red solid (0.094 mg/g dried plant

sample); UVmax (MeOH) (nm): 233; 344; 508; 1H-

83 NMR (0.1% CF3COOD in CD3OD, 500 MHz) ,

9.05 (s, 1H, H4), 8.56 (d, J = 8.3 Hz, 2H, H2'/6' ),

7.38 (d, J = 12.4 Hz, H2'''), 7.26 (d, J = 8.3 Hz, 2H,

H2''''/6''''), 7.05 (d, J = 8.3Hz, 2H, H3'/5'), 6.88 (d, J

= 8.3Hz, 2H, H3''''/5''''), 6.23 (d, J = 12.5 Hz, 1H,

H3'''), 6.19 (s, 1H, H6), 6.17 (s, 1H, H8), 5.29 (d, J = 8.0 Hz, 1H, H1''), 5.06 (d, J = 8.0

Hz, 1H, H1''''), 4.45-4.52 (m, 1H, H6''), 4.06 (dd, J = 8.4 Hz, 8.0 Hz, H6'''''a), 3.80 (dd, J

= 8.4 Hz, 8.0 Hz, 1H, H5''), 3.79 (dd, J = 9.0, 8.0 Hz, 1H, H2'''''), 3.73 (dd, J = 9.0, 8.0

Hz, 1H, H2''), 3.71 (dd, J = 8.4 Hz, 8.0 Hz, H6'''''b), 3.69 (dd, J = 9.0, 9.5 Hz, H3''), 3.68

(dd, J = 9.5, 6.0 Hz, 1H, H5'''''), 3.60 (dd, J = 9.0, 8.0 Hz, 1H, H3'''''), 3.47 (dd, J = 9.0,

8.0 Hz, 1H, H4'''''), 3.40 (dd, J = 9.0, 8.0 Hz, 1H, H4''), 1.99 (s, 3H, H8'''''); 13C-NMR

83 (0.1% CF3COOD in CD3OD, 125 MHz) , 171.1 (C7'''''), 170.1 (C1'''), 168.8 (C7),

132

167.8 (C4'), 164.5 (C2), 160.3 (C4''''), 157.4 (C8a), 156.7 (C5), 149.8 (C2'''), 145.6

(C3), 135.8 (C4), 134.57 (C2'/6'), 132.4 (C2''''/6''''), 127.9 (C1''''), 119 (C1'), 116.5

(C3'/5'), 115.1 (C3'''), 114.5 (C3''''/5''''), 111.5 (C4a), 103.7 (C1''), 100.2 (C1'''''), 98.6

(C6), 98.1 (C8), 78.6 (C3''), 77.5 (C3'''''), 76.4 (C5'''''), 75.4 (C5''), 74.9 (C2''), 74.2

(C2'''''), 72.3 (C4''), 71.3 (C4'''''), 64.5 (C6'''''), 64.2 (C6''), 19.2 (C8'''''); LREIMS m/z:

783 [M+1]+.

Kaempferol-3-rutinoside 48

A UV active yellow solid (0.021 mg/g dried

plant sample); UVmax (MeOH) (nm): 225;

1 81 268; 349; H-NMR (CD3OD, 500 MHz) ,

8.06 (d, J = 8.4 Hz, 2H, H2'/6'), 6.89 (d, J =

8.5 Hz, 2H, H3'/5'), 6.39 (d, J = 2.1 Hz, 1H,

H8), 6.20 (d, J = 2.1 Hz, 1H, H6), 5.12 (d, J = 7.0 Hz, 1H, H1''), 4.52 (d, J = 1.5 Hz,

1H, H1'''), 3.80 (dd, J = 8.4, 8.0 Hz, 1H, H6''a), 3.62 (dd, J = 8.5, 1.5 Hz, 1H, H2'''),

3.51 (dd, J = 8.4, 8.0 Hz, 1H, H3'''), 3.44 (dd, J = 9.5, 6.0 Hz, 1H, H5'''), 3.43-3.41 (m,

1H, H2''), 3.40-3.38 (m, 1H, H3''), 3.37 (dd, J = 8.4, 8.0 Hz, 1H, H6''b), 3.32-3.30 (m,

1H, H5''), 3.27 (dd, J = 9.5, 9.0 Hz, 1H, H4'''), 3.24 (dd, J = 8.4, 8.0 Hz, 1H, H4''), 1.11

13 81 (s, 3H, H6'''); C-NMR (CD3OD, 125 MHz) , 179.5 (C4), 166.1 (C7), 163.1 (C5),

161.6 (C2), 159.5 (C4'), 158.6 (C8a), 135.3 (C3), 132.6 (C2'/6'), 122.7 (C1'), 116.4

(C3'/5'), 105.7 (C4a), 104.8 (C1''), 102.5 (C1'''), 99.8 (C6), 94.4 (C8), 78.3 (C3''), 77.3

(C5''), 75.9 (C2''), 74.0 (C4'''), 72.4 (C3'''), 72.2 (C2'''), 71.6 (C4''), 69.9 (C5'''), 68.7

+ (C6''), 18.1 (C6'''); LREIMS, m/z: 595 [M+1] . HRESIMS: calculated for C27H30O15Na

[M+Na]+: 617.1482, found 617.1484.

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Naringenin-7-O-glucoronide 49

A UV active yellow solid (0.020 mg/g of dried

plant sample); UVmax (MeOH) (nm): 227; 268;

348. CD (nm, Δε): 217 (+6.17), 248 (+1,49), 287 (-

25 o 1 84 6.07) (100µM, MeOH); [훼]D -35.9 (c 0.11, MeOH). H-NMR (CD3OD, 500 MHz) ,

7.33 (d, J = 8.2 Hz, 2H, H2'/6'), 6.82 (d, J = 8.2 Hz, 2H, H3'/5'), 6.47 (s, 1H, H8), 6.12

(s, 1H, H6), 5.40 (dd, J = 13.0, 3.1 Hz, 1H, H2), 5.06 (d, J = 7.5 Hz, H1''), 4.03 (d, J =

9.6 Hz, 1H, H5''), 3.60 (dd, J = 8.9, 8.0 Hz, 1H, H4''), 3.49 (dd, J = 7.5, 8.9 Hz, H2''),

3.47 (dd, J = 8.9, 8.0 Hz, H3''), 3.17 (dd, J = 17.2, 13.0 Hz, 1H, H3a), 2.75 (dd, J =

13 84 17.2, 3.0 Hz, 1H, H3b); C-NMR (CD3OD, 125 MHz) , 198.8 (C4), 169.4 (C6''),

166.8 (C7), 165.1 (C5), 164.8 (C8a), 159.3 (C4'), 131.0 (C1'), 129.0 (C2'/6'), 116.5

(C3'/5'), 105.2 (C4a), 101.2 (C1''), 98.1 (C6), 97.1 (C8), 80.9 (C2), 77.3 (C2''), 76.1

(C5''), 73.8 (C3''), 72.6 (C4''), 44.3 (C3); LRESMS, m/z: 471 [M+Na]+. HRESIMS:

+ calculated for C21H20O11Na [M+Na] : 471.3667, found 471.3675

(2S)-4ˡ,5-Dihydroxyflavanone-7-O-β-D-glucuronide methyl ester 50

A UV active pale yellow crystal needle (0.032

mg/g of dried plant sample); UVmax (MeOH)

(nm): 227; 268; 348. CD (nm, Δε): 217 (+1.7), 248

25 o (+0.59), 287 (-1.60) (30µM, MeOH); [훼]D +24.57

1 84 (c 0.17, MeOH). H-NMR (CD3OD, 500 MHz) , 7.33 (d, J = 8.2 Hz, 2H, H2'/6'), 6.82

(d, J = 8.2 Hz, 2H, H3'/5'), 6.47 (s, 1H, H8), 6.12 (s, 1H, H6), 5.40 (dd, J = 13.0, 3.1 Hz,

1H, H2), 5.06 (d, J = 7.5, Hz, H1''), 4.03 (t, J = 9.6 Hz, 1H, H5''), 3.78 (s, 3H, H7''),

3.60 (dd, J = 8.9, 8.0 Hz, 1H, H4''), 3.49 (dd, J = 6.5, 8.0 Hz, H2''), 3.47 (dd, J = 8.9, 8.0

Hz, H3''), 3.17 (dd, J = 17.2, 13.0 Hz, 1H, H3a), 2.75 (dd, J = 17.2, 3.0 Hz, 1H, H3b);

134

13 84 C-NMR (CD3OD, 125 MHz) , 198.8 (C4), 169.4 (C6''), 166.8 (C7), 165.1 (C5),

164.8 (C8a), 159.3 (C4'), 131.0 (C1'), 129 (C2'/6'), 116.5 (C3'/5'), 105.2 (C4a), 101.2

(C1''), 98.1 (C6), 97.1 (C8), 80.9 (C2), 77.3 (C2''), 76.1 (C5''), 73.8 (C3''), 72.6 (C4''),

52.3 (C7''), 44.3 (C3); LRESMS, m/z: 487 [M+Na]+.

Kaempferol 51

A UV active yellow solid (0.025 mg/g dried plant sample);

1 UVmax (MeOH) (nm): 228; 267; 366. H-NMR (CD3OD, 500

MHz)81, 8.06 (d, J = 8.4 Hz, 2H, H2'/6'), 6.89 (d, J = 8.5 Hz,

2H, H3'/5'), 6.39 (d, J = 2.1 Hz, 1H, H8), 6.20 (d, J = 2.1 Hz, 1H, H6); 13C-NMR

81 (CD3OD, 125 MHz) , 179.5 (4), 166.1 (C7), 163.1 (C5), 161.6 (C2), 159.5 (C4'), 158.6

(C8a), 135.3 (C3), 132.6 (C2'/6'), 122.7 (C1'), 116.4 (C3'/5'), 105.7 (C4a), 99.8 (C6),

94.4 (C8); LREIMS, m/z: 309 [M+Na]+

6.4 Isolation of constituents of Tibouchina lepidota

A suspension of powdered flowers (250 g) in methanol (2.0 L) was stirred for 48 hours, then filtered and the supernatant vacuum dried to produce a dried extract (14.35 g). This extract was further fractionated into different solvents of polarity producing fractions from hexane (5.12 g), dichloromethane (0.81 g), ethyl acetate (4.33 g) and a residue

(3.04 g).

The ethyl acetate fraction (500 mg) was prepared for semi preparative HPLC by dissolution in methanol:acetonitrile (1:1, 50 mL), filtration through a Whatman syringe filter PTFE 0.45 μm and the final volume was reduced to 25 mL. Preparative HPLC employed a gradient elution from 90% to 50% solvent A within 50 minutes and

135 produced six compounds (54-58). The sample at retention time 22 minutes as 46 (0.76 mg/g dried plant sample) and structural elucidation is described in section 6.3.

Gallic acid 54

A UV active yellow crystal (0.72 mg/g dried plant sample); UVmax

1 106 (MeOH) (nm): 254. H-NMR (CD3OD, 500 MHz) , 7.06 (s, 2H, H2/6);

13 106 C-NMR (CD3OD, 125 MHz) , 170.7 (COOH), 146.4 (C3/5), 139.7

(C4), 122.0 (C1), 110.6 (C2/6). LRESMS, m/z 171 [M+1]+.

2,3,5-Trihydroxybenzoic acid 55

A UV active white solid (0.72 mg/g dried plant sample); UVmax

1 106 (MeOH) (nm): 254. H-NMR (CD3OD, 500 MHz) , 7.48 (s, 1H, H6),

13 106 6.97 (s, 1H, H4); C-NMR (CD3OD, 125 MHz) , 169.6 (COOH),

152.3 (C5), 145.3 (C3), 139.0 (C2), 122.3 (C1), 111.5 (C6), 102.6 (C4). LRESMS, m/z

171 [M+1]+.

Avicularin 56

A UV active yellow solid (0.38 mg/g dried plant

sample); UVmax (MeOH) (nm): 226; 259; 353. 1H-

95 NMR (CD3OD, 500 MHz) , 7.72 (s, 1H, H2'), 7.60

(d, J = 8.0 Hz, 1H, H6'), 6.89 (d, J = 8.0 Hz, 1H, H5'),

6.42 (s, 1H, H8), 6.22 (s, 1H, H6), 5.14 (d, J = 7.5 Hz, 1H, H1''), 3.91 (dd, J = 9.0, 7.5

Hz, 1H, H2''), 3.84 (dd, J = 9.0, 9.5 Hz, 1H, H3''), 3.66 (dd, J = 9.5, 8.0 Hz, 1H, H4''),

13 95 3.44 (d, J = 8.0 Hz 1H, H5'') C-NMR (CDCl3, 125 MHz) , 179.5 (C4), 166.3 (C7),

164.7 (C5), 158.9 (C8a), 158.7 (C2), 149.7 (C4'), 146.3 (C3'), 135.6 (C2), 122.95 (C1'),

136

122.90 (C6'), 117.4 (C2'), 116.0 (C5'), 105.3 (C2), 104.1 (C1''), 99.7 (C2), 94.5 (C2),

73.5 (C4''), 72.2 (C2''), 67.1 (C3''), 66.3 (C5''); LREIMS, m/z: 435 [M+H]+

Quercetin 3-glucoside 53

A UV active pale yellow solid (0.64 mg/g dried plant

sample); UVmax (MeOH) (nm): 228; 259; 359. 1H-

95 NMR (CD3OD, 500 MHz) , 7.72 (s, 1H, H2'), 7.60 (d,

J = 8.0 Hz, 1H, H6'), 6.89 (d, J = 8.0 Hz, 1H, H5'), 6.42

(s, 1H, H8), 6.22 (s, 1H, H6), 5.24 (d, J = 7.5 Hz, 1H, H1''), 3.70 (d, J = 12.5 Hz, 1H,

H6''a), 3.58 (dd, J = 12.5, 7.5 Hz, 1H, H6''b), 3.48 (dd, J = 7.0, 8.0 Hz, 1H, H2''), 3.42

(dd, J = 8.0, 8.0 Hz, 1H, H5''), 3.35 (d, J = 8.0 Hz, 1H, H4''), 3.22 (d, J = 8.0 Hz, 1H,

13 H3''); C-NMR (CD3OD, 125 MHz), 179.5 (C4), 166.3 (C7), 164.7 (C5), 158.9 (C8a),

158.7 (C2), 149.7 (C4'), 146.3 (C3'), 135.6 (C3), 122.95 (C1'), 122.90 (C6'), 117.4

(C2'), 116.0 (C5'), 105.3 (C2), 104.1 (C1''), 99.7 (C6), 94.5 (C8), 78.1 (C3''), 77.8 (C5''),

75.4 (C2''), 71.0 (C4''), 62.2 (C6''); LRESMS: m/z 465 (M+H)+

Isorhamnetin 3-rutinoside 57

A UV active yellow solid (0.53 mg/g dried plant

sample); UVmax (MeOH) (nm): 225; 268; 359.

1 108 H-NMR (CD3OD, 500 MHz) , 7.94 (s, 1H,

H2'), 7.62 (d, J = 8.5 Hz, 1H, H6'), 6.91 (d, J =

8.5 Hz, 1H, H5'), 6.41 (s, 1H, H8), 6.21 (s, 1H, H6), 5.23 (d, J = 7.0 Hz, 1H, H1''), 4.53

(d, J = 1.5 Hz, 1H, H1'''), 3.95 (s, 3H, OCH3), 3.82 (dd, J = 12.5, 7.5 Hz, 1H, H6b''),

3.62 (dd, J = 9.0, 1.5 Hz, 1H, H2'''), 3.49 (dd, J = 9.0, 6.5 Hz, 1H, H2''), 3.49 (dd, J =

9.0, 2.5 Hz, 1H, H3'''), 3.46 (dd, J = 9.0, 9.5 Hz, 1H, H3''), 3.42-3.41 (m, 1H, H6a''),

3.40 (dd, J = 9.5, 6.0 Hz, 1H, H5'''), 3.38 (dd, J = 10.0, 6.0 Hz, 1H, H5''), 3.26 (dd, J =

137

9.5, 9.5 Hz, 1H, H4'''), 3.26 (dd, J = 9.5, 7.0 Hz, 1H, H4''), 1.10 (d, J = 6.0 Hz, 3H,

13 105, 108 H6'''); C-NMR (CD3OD, 125 MHz) , 179.3 (C4), 166.0 (C7), 163.0 (C5), 158.9

(C2), 158.5 (C8a), 150.8 (C4'), 148.3 (C3'), 135.5 (C3), 124.0 (C6'), 123.4 (C1'), 116.1

(C5'), 114.6 (C2'), 105.7 (C4a), 104.4 (C1''), 102.5 (C1'''), 100.0 (C6), 94.9 (C8), 78.2

(C3''), 77.3 (C5''), 75.9 (C2''), 73.8 (C4'''), 72.3 (C3'''), 72.1 (C2'''), 71.6 (C4''), 69.8

+ (C5'''), 68.5 (C6''), 56.8 (C (OCH3), 17.9 (C6'''); LRESMS m/z 625 (M+H) .

Malvidin 3-(p-coumarylglucoside)-5-acetylxyloside 58

A UV active dark purple solid (0.82 mg/g dried plant

sample); UVmax (MeOH) (nm): 210; 291; 311; 537.

1 105 H-NMR (CD3OD, 500 MHz) , 8.70 (s, 1H, H4), 7.96

(s, 2H, H2'/6'), 7.45 (d, J = 15.8 Hz, 1H, H3'''), 7.28 (d,

J = 8.1 Hz, 2H, H2''''/6''''), 7.02 (s, 1H, H8), 6.84 (s, 1H,

H6), 6.73 (d, J = 8.1 Hz, 2H, H3''''/5''''), 6.27 (d, J =

15.9 Hz, 1H, H2'''), 5.48 (d, J = 7.7 Hz, 1H, H1''), 5.29

(d, J = 7.5 Hz, 1H, H1'''''), 5.13 (dd, J = 7.5 Hz, 8.0 Hz, 1H, H2'''''), 4.63 (1H, dd, J =

10.5, 6.5 Hz, 1H, H6''a), 4.21 (1H, dd, J = 10.5, 6.5 Hz, 1H, H6''b), 4.38 (dd, J = 7.5 Hz,

8.0 Hz, 1H, H5''), 3.99 (s, 6H, OCH3), 3.79 (dd, J = 7.5 Hz, 8.0 Hz, 1H, H2''), 3.75 (dd,

J = 8.5 Hz, 8.0 Hz, H3'''''), 3.66 (dd, J = 7.5 Hz, 8.0 Hz, 1H, H4'''''), 3.63 (dd, J = 7.5 Hz,

8.0 Hz, 1H, H4''), 3.51 (1H, dd, J = 10.5, 6.5 Hz, 1H, H5'''''a), 3.91 (1H, dd, J = 10.5,

6.5 Hz, 1H, H5'''''b), 3.48 (dd, J = 8.5 Hz, 8.0 Hz, 1H, H3''), 2.15 (s, 3H, H7''''); 13C-

105 NMR (CD3OD, 125 MHz) , 171.1 (C6'''''), 167.9 (C1'''), 167.8 (C7), 162.5 (C2), 159.8

(C4''''), 157.7 (C5), 157.4 (C8a), 148.4 (C3'/5'), 145.8 (C3), 145.5 (C3'''), 144.9 (C4'),

135.8 (C4), 130 (C2''''/9''''), 125.3, (C1''''), 118 (C1'), 115.1 (C3''''/5''''), 113.4 (C2'''),

111.5 (C4a), 109.9 (C2'/6'), 102.9 (C6), 99.9 (C1'''''), 99.8 (C1''), 95.8 (C8), 76.9 (C4'''''),

138

74.1 (C5''), 73.7 (C2'''''), 73.24 (C2''), 73.20 (C3'''''), 70.5 (C3''), 69.1 (C4''), 65.5 (C5'''''),

+ 63.2 (C6''), 55.8 (2x OCH3), 19.8 (C7'''''). ESIMS, m/z: 813 [M+1] , HRESIMS:

+ calculated for C39H42O19 [M+1] : 814.2338, found 814.2320.

6.5 Isolation of constituents of Anigozanthos sp flowers

Dried red kangaroo paw (Anigozanthos rufus) flowers (100 g) and yellow red kangaroo paw (Anigozanthos pulcherrimus) flowers (80 g) were ground to a powder under liquid nitrogen in a mortar and pestle. Both samples were extracted separately by stirring in

MeOH (1 L, in 2.0 L conical flask) for 24 h, filtered and the supernatants were concentrated to produce crude extract (36.4 g (A. rufus); 28.7g (A. pulcherrimus)). Each crude extract was fractionated into solvents of different polarity and produced methanol fractions (A. rufus (9.7 g); A. pulcherrimus (6.8 g)) and hexane fractions (A. rufus (26.3 g); A. pulcherrimus (20.5 g).

Each methanol fraction (300 mg) in methanol (50 mL) was filtered through a Whatman syringe filter 0.45 μm and the volume was reduced to 20 mL. Preparative HPLC employed a gradient elution from 95% of solvent A (0.1% TFA in water) to 50% of solvent B (0.1% TFA in ACN) within 35 minutes. A methanol fraction from A. rufus produced twelve compounds (48, 51, 53, 62 – 70) with A. pucherrimus producing eleven compounds (48, 51, 53, 63, 64, 66 – 71). The compounds at retention time at

12.3, 13.5 and 14.5 minutes were identified as 51, 48 and 53, respectively and the structural elucidation are described in section 6.3 and 6.4.

139

Cyanidin-3-rutinoside 62

A dark red solid (0.042 mg/g dried plant

sample); UV (MeOH) λmax: 280 (7451); 519

1 123 (8905) nm. H-NMR (CD3OD, 500 MHz) ,

8.90 (s, 1H, H4), 8.22 (d, J = 8.3 Hz, 1H, H6'),

8.00 (s, 1H, H2'), 7.00 (d, J = 8.7 Hz, 1H, H5'),

6.86 (s, 1H, H8), 6.67 (s, 1H, H6), 5.25 (d, J = 8.3 Hz, 1H, H1''), 4.68 (d, J = 1.5 Hz,

1H, H1'''), 3.80 (dd, J = 8.4, 8.0 Hz, 1H, H6''a), 3.62 (dd, J = 8.4, 1.5 Hz, 1H, H2'''),

3.51 (dd, J = 8.4, 8.0 Hz, 1H, H3'''), 3.44 (dd, J = 8.4, 8.0 Hz, 1H, H5'''), 3.43-3.41 (m,

1H, H2''), 3.40-3.38 (m, 1H, H3''), 3.37 (dd, J = 8.4, 8.0 Hz, 1H, H6''b), 3.32-3.30 (m,

1H, H5''), 3.27 (dd, J = 8.4, 8.0 Hz, 1H, H4'''), 3.24 (dd, J = 8.4, 8.0 Hz, 1H, H4''), 1.11

13 123 (s, 3H, H6'''); C-NMR (CD3OD, 125 MHz) , 168.7 (C7), 163.5 (C2), 157.9 (C5),

156.4 (C8a), 154.3 (C4'), 145.9 (C3'), 143.8 (C3), 136.3 (C4), 128.4 (C6'), 120.1 (C1'),

118.6 (C2'), 117.4 (C5'), 111.8 (C4a), 103.7 (C6), 103.6 (C1''), 102.4 (C1'''), 95.4 (C8),

78.1 (C5''), 77.5 (C3''), 74.8 (C2''), 74.3 (C4'''), 72.5 (C3'''), 72.0 (C2'''), 71.3 (C4''), 69.8

(C5'''), 67.3 (C6''), 18.2 (C6'''). ESIMS, m/z: 595 [M+1]+. HRESIMS calculated for

+ C27H31O15 [M+H] : 595.1752, found 595.1781

Quercetin-3-rutinoside 63

A UV active pale yellow solid (0.23 mg/g dried

plant sample); UVmax (MeOH) (nm): 225; 268;

1 81 349. H-NMR (CD3OD, 500 MHz) , 7.67 (s,

1H, H2'), 7.62 (d, J = 8.5 Hz, 1H, H6'), 6.88 (d,

J = 8.5 Hz, 1H, H5'), 6.40 (d, J = 2.0 Hz, 1H,

H8), 6.21 (d, J = 2.0 Hz, 1H, H6), 5.10 (d, J = 7.5 Hz, 1H, H1''), 4.52 (d, J = 1.5 Hz,

140

1H, H1'''), 3.80 (dd, J = 8.4, 8.0 Hz, 1H, H6''a), 3.62 (dd, J = 8.4, 1.5 Hz, 1H, H2'''),

3.51 (dd, J = 8.4, 8.0 Hz, 1H, H3'''), 3.44 (dd, J = 8.4, 8.0 Hz, 1H, H5'''), 3.43-3.41 (m,

1H, H2''), 3.40-3.38 (m, 1H, H3''), 3.37 (dd, J = 8.4, 8.0 Hz, 1H, H6''b), 3.32-3.30 (m,

1H, H5''), 3.27 (dd, J = 8.4, 8.0 Hz, 1H, H4'''), 3.24 (dd, J = 8.4, 8.0 Hz, 1H, H4''), 1.11

13 81 (s, 3H, H6'''); C-NMR (CD3OD, 125 MHz) , 179.4 (C4), 166.0 (C7), 162.9 (C5),

159.3 (C2), 158.5 (C9), 149.8 (C4'), 145.8 (C3'), 135.6 (C3), 123.6 (C6'), 123.1 (C1'),

117.7 (C2'), 116.0 (C5'), 105.6 (C10), 104.7 (C1''), 102.6 (C1'''), 99.9 (C6), 94.7 (C8),

78.1 (C5''), 77.2 (C3''), 75.7 (C2''), 73.9 (C4'''), 72.2 (C3'''), 72.1 (C2'''), 71.4 (C4''), 69.7

(C5'''), 68.5 (C6''), 17.9 (C6'''); ESIMS, m/z 611 (M+H)+; HESIMS: calculated for

+ C27H30O16Na (M+Na) : 633.1432, found 633.1443.

2-Amino-6-O-p-coumaryl heptanedioic acid 64

A UV active pale yellow oily solid (0.40 mg/g dried

plant sample); UVmax (MeOH) (nm): 225; 292; 325.

25 o -1 [훼]D 0 (c 0.13, MeOH); IR [cm ]: 3365 (br, m),

1 2944 (s), s831 (s), 1717 (s); HNMR (CD3OD, 500 MHz), 7.68 (d, J = 15.9 Hz, 1H,

H3'), 7.48 (d, J = 8.3 Hz, 2H, H2''/6''), 6.82 (d, J = 8.2 Hz, 2H, H3''/5''), 6.39 (d, J = 15.9

Hz, 1H, H2'), 5.10 (t, J = 7.4 Hz, 1H, H6), 3.99 (t, J = 8.8, 7.5 Hz, 1H, H2), 2.10-2.00

13 (m, 2H, H3), 1.95-1.93 (m, 2H, H5), 1.75-1.5 (m, 2H, H4); CNMR (CD3OD, 125

MHz), 173.8 (C7), 171.9 (C1), 168.6 (C1'), 161.4 (C4''), 145.6 (C3'), 131.1 (C2''/6''),

127.4 (C1''), 116.9 (C3''/5''), 114.5 (C2'), 72.8 (C6), 53.9 (C2), 31.9 (C3), 31.3 (C5),

22.2 (C4); ESIMS, m/z: 338 [M+1]; HRESIMS: calculated for C16H20NO7 m/z:

338.1215, found 338.1240 [M+H]+.

141

Cyanidin-3-O-(6-O-p-coumaryl)glucoside 65

A UV active dark red solid (0.040 mg/g dried plant

sample); UV (MeOH) λmax: 282 (8302); 313 (7052); 523

1 124 (6981) nm. H-NMR (CD3OD, 500 MHz) , 8.91 (s,

1H, H4), 8.24 (dd, J = 8.7, 2.2 Hz, 1H, H6'), 8.00 (d, J =

2.2 Hz, 1H, H2'), 7.44 (d, J = 16.0 Hz, 1H, H3'''), 7.30

(d, J = 8.2 Hz, 2H, H2''''/6''''), 7.01 (d, J = 8.7 Hz, 1H,

H5'), 6.82 (s, 1H, H8), 6.79 (d, J = 8.2 Hz, 2H, H3''''/5''''), 6.53 (s, 1H, H6), 6.22 (d, J =

16.0 Hz, 1H, H2'''), 5.32 (d, J = 7.6 Hz, 1H, H1''), 4.53 (dd, J = 12.0, 7.5 Hz, 1H, H6''a),

4.37 (dd, J = 12.0, 7.5 Hz, 1H, H6''b), 3.48 (dd, J = 7.0, 8.0 Hz, 1H, H2''), 3.42 (dd, J =

8.0, 8.0 Hz, 1H, H5''), 3.35 (d, J = 8.0 Hz, 1H, H4''), 3.22 (d, J = 8.0 Hz, 1H, H3''); 13C-

124 NMR (CD3OD, 125 MHz) , 170.9 (C7), 169.1 (C1'''), 164.5 (C2), 161.0 (C7'''), 159.1

(C5), 157.8 (C8a), 155.8 (C4'), 147.6 (C3'), 146.5 (C3'''), 145.1 (C3), 136.8 (C4), 131.4

(C2''''/6''''), 128.5 (C1'), 128.3 (C6'), 126.1 (C4'''), 118.5 (C2'), 117.4 (C5'), 116.8

(C3''''/5''''), 114.6 (C2'''), 113 (C4a), 103.3 (C6), 103.1 (C1''), 95.2 (C8), 78.0 (C2''), 76.1

(C3''), 74.8 (C5''), 71.8 (C4''), 64.5 (C6''). ESIMS, m/z: 595 [M+1]+. HRESIMS

+ calculated for C30H27O13 m/z: 595.1452, found 595.1471 [M+H] .

Apigenin-7-O-β-glucuronide 66

A UV active yellow solid (0.025 mg/g dried plant

sample); UVmax (MeOH) (nm): 225; 268; 349.

1 125 HNMR (CD3OD, 500 MHz) , 7.86 (d, J = 8.5

Hz, 2H, H2'/6'), 6.79 (d, J = 2.2 Hz, 1H, H8), 6.63 (s, 1H, H3), 6.47 (d, J = 2.1 Hz, 1H,

H6), 5.14 (d, J = 7.0 Hz, 1H, H1''), 4.03 (d, J = 9.6 Hz, 1H, H5''), 3.60 (dd, J = 8.9, 8.0

Hz, 1H, H4''), 3.49 (dd, J = 7.5, 8.9 Hz, H2''), 3.47 (dd, J = 8.9, 8.0 Hz, H3''). 13CNMR

142

125 (CD3OD, 125 MHz) , 184.2 (C4), 172.2 (C6''), 166.9 (C2), 164.6 (C7), 163.0 (C5),

162.8 (C4'), 158.9 (C8a), 129.8 (C2'/6'), 123.1 (C1'), 117.2 (C3'/5'), 104.3 (C4a), 103.9

(C3), 101.5 (C1''), 101.3 (C6), 96.1 (C8), 77.3 (C3''), 76.7 (C5''), 74.5 (C2''), 73.0 (C4'');

LREIMS, m/z: 469 [M+Na]+.

Kaempferol-3-O-(6-O-p-coumaryl) glucoside 67

A UV active yellow solid (0.030 mg/g

dried plant sample); UVmax (MeOH)

1 (nm): 225; 255; 352. H-NMR (CD3OD,

500 MHz)126, 8.06 (d, J = 8.3 Hz, 2H,

H2'/6'), 7.40 (d, J = 15.8 Hz, 1H, H3'''), 7.31 (d, J = 8.1 Hz, 2H, H2''''/6''''), 6.84 (d, J =

8.3 Hz, 2H, H3''''/5''''), 6.81 (d, J = 2.4 Hz, 2H, H3'/5'), 6.80 (d, J = 8.1 Hz, 1H, H2'),

6.32 (s, 1H, H8), 6.14 (s, 1H, H6), 6.07 (d, J = 15.9 Hz, 1H, H2'''), 5.24 (d, J = 6.7 Hz,

1H, H1''), 4.18 (dd, J = 11.5, 1.2 Hz, 1H, H6''a), 4.30 (dd, J = 11.9, 6.1 Hz, 1H, H6''b),

3.48 (dd, J = 7.0, 8.9 Hz, 1H, H2''), 3.42 (ddd, J = 8.9, 6.1, 1.2 Hz, 1H, H5''), 3.35 (dd, J

13 = 9.0, 8.0 Hz, 1H, H4''), 3.22 (dd, J = 9.0, 9.5 Hz, 1H, H3''); C-NMR (CD3OD, 125

MHz)126, 179.7 (C4), 168.8 (C1'''), 165.9 (C7), 163.8 (C5), 161.6 (C2), 158.4 (C8a),

146.8 (C4'), 146.6 (C3'''), 142.2 (C4''''), 132.4 (C3), 132.3 (C2'/6'), 131.2 (C2''''/6''''),

126.1 (C1''''), 123.0 (C1'), 116.6 (C3''''/C8'''), 116.3 (C3'/5'), 114.9 (C2'''), 105.7 (C4a),

104.1 (C1''), 100.2 (C6), 95.0 (C8), 78.2 (C3''), 75.9 (C2''), 75.8 (C5''), 71.9 (C4''), 64.5

- + (C6''); LREIMS, m/z: 593 [M-H] , 617 [M+Na] .

143

Quercetin-3-O-(6-O-p-coumaryl)glucoside 68

A UV active yellow solid (0.16 mg/g dried

plant sample); UVmax (MeOH) (nm): 225;

1 127 255; 365. H-NMR (CD3OD, 500 MHz) ,

7.99 (d, J = 8.3 Hz, 1H, H6'), 7.40 (d, J =

15.8 Hz, 1H, H3'''), 7.31 (d, J = 8.1 Hz, 2H,

H2''''/6''''), 6.84 (d, J = 8.3 Hz, 2H, H3''''/5''''), 6.81 (d, J = 8.0 Hz, 1H, H5'), 6.80 (d, J =

2.4 Hz, 1H, H2'), 6.32 (s, 1H, H8), 6.14 (s, 1H, H6), 6.07 (d, J = 15.9 Hz, 1H, H2'''),

5.24 (d, J = 7.0 Hz, 1H, H1''), 4.18 (dd, J = 11.5, 1.2 Hz,1H, H6''a), 4.30 (dd, J = 11.9,

6.1 Hz, 1H, H6''b), 3.48 (dd, J = 7.0, 8.9 Hz, 1H, H2''), 3.42 (ddd, J = 8.9, 6.1, 1.2 Hz,

1H, H5''), 3.35 (dd, J = 9.0, 8.0 Hz, 1H, H4''), 3.22 (dd, J = 9.0, 9.5 Hz, 1H, H3''); 13C-

127 NMR (CD3OD, 125 MHz) , 179.7 (C4), 168.8 (C1'''), 165.9 (C7), 163.8 (C5), 161.6

(C2 ), 158.4 (C8a), 147.1 (C3'), 146.8 (C4'), 146.6 (C3'''), 142.2 (C4''''), 136.5 (C3),

132.4 (C5'), 132.3 (C6'), 131.2 (C2''''/6''''), 126.1 (C1''''), 123.0 (C1'), 116.6 (C3''''/5''''),

116.2 (C2'), 114.9 (C2'''), 105.7 (C4a), 104.1 (C1''), 100.2 (C6), 95.0 (C8), 78.2 (C3''),

75.9 (C2''), 75.8 (C5''), 71.9 (C4''), 64.5 (C6''); LREIMS, m/z: 609 [M-H]-, 633

+ [M+Na] .

Luteolin 7-O-β-glucoside 69

A UV active yellow solid (0.08 mg/g dried plant

sample); UV (MeOH) λmax: 228 (13855); 259

1 (13084); 359 (11830) nm. H-NMR (CD3OD, 500

MHz)128, 7.84 (d, J = 2.2 Hz, 1H, H2'), 7.59 (dd, J

= 8.5, 2.3 Hz, 1H, H6'), 6.87 (d, J = 8.6 Hz, 1H, H5'), 6.63 (s, 1H, H3), 6.41 (s, 1H, H8),

6.21 (s, 1H, H6), 5.14 (d, J = 6.5 Hz, 1H, H1''), 3.70 (d, J = 12.5 Hz, 1H, H6''a), 3.58

144

(dd, J = 12.5, 7.5 Hz, 1H, H6''b), 3.48 (dd, J = 7.0, 8.0 Hz, 1H, H2''), 3.42 (dd, J = 8.0,

8.0 Hz, 1H, H5''), 3.35 (dd, J = 8.0, 7.0 Hz, 1H, H4''), 3.22 (dd, J = 8.0, 7.0 Hz, 1H,

13 128 H3''); C-NMR (CD3OD, 125 MHz) , 178.9 (C4), 165.0 (C7), 163.1 (C5), 159.1

(C8a), 158.5 (C2), 149.1 (C3'), 146.8 (C4'), 122.2 (C1'), 122.0 (C6'), 116.0 (C2'), 115.7

(C5'), 106.0 (C4a), 104.4 (C1''), 103.8 (C3), 99.2 (C6), 95.2 (C8), 73.5 (C4''), 72.3

(C2''), 67.2 (C3''), 66.3 (C5''), 62.6 (C6''); LREIMS, m/z: 449 [M+1].

Chalcone-5'-O-(4-O-p-coumaryl)glucoside 70

A UV active yellow solid (0.08 mg/g dried

plant sample); UV (MeOH) λmax: 225 (15455);

22 o 254 (13384); 298 (11830) nm. [훼]D +12.8 (c

0.15, MeOH); IR [cm-1]: 3566 (br, m), 2360

1 (s), 1683 (s); H-NMR (CD3OD, 500 MHz),

8.02 (d, J = 15.0 Hz, 1H, H2), 7.70 (d, J =

15.0 Hz, 1H, H3'''), 7.68 (d, J = 15.0 Hz, 1H, H3), 7.62 (d, J = 8.0 Hz, 2H, H2'/6'), 7.49

(d, J = 8.0 Hz, 2H, H2'''''/6'''''), 6.87 (d, J = 8.0 Hz, H3'/5'), 6.83 (d, J = 8.0 Hz, 2H,

H3'''''/5'''''), 6.36 (d, J = 15.0 Hz, 1H, H2''''), 6.26 (s, 1H, H6''), 6.03 (s, 1H, H4''), 5.23

(d, J = 7.8 Hz, 1H, H1'''), 5.02 (dd, J = 9.5, 8.5 Hz, 1H, H4'''), 3.90 (ddd, J = 8.9, 6.5,

1.2 Hz, 1H, H5'''), 3.82 (dd, J = 9.5, 9.0 Hz, 1H, H3'''), 3.72 (dd, J = 6.5 , 1.2 Hz, 2H,

H6'''a), 3.62 (dd, J = 11.0, 6.1 Hz, 2H, H6'''b), 3.69 (dd, J = 8.9, 7.8 Hz, 1H, H2'''); 13C-

NMR (CD3OD, 125 MHz), 194.8 (C1), 168.7 (C1''''), 165.9 (C3''), 161.8 (C4'), 161.4

(C5''), 161.2 (C4'''''), 161.1 (C2''), 147.6 (C3''''), 144.3 (C3), 131.9 (C2'/ C6'), 131.4

(C2'''''/6'''''), 128.3 (C1'), 127.2 (C1''), 125.8 (C1'''''), 125.6 (C2), 117.1 (C3'/5'), 117.0

(C3'''''/5'''''), 114.8 (C2''''), 101.7 (C1'''), 98.7 (C4''), 96.0 (C6''), 76.7 (C5'''), 76.3 (C3'''),

145

75.3 (C2'''), 72.1 (C4'''), 62.3 (C6'''); LREIMS, m/z: 603 [M+Na]+ HRESIMS

+ calculated for C30H28O12Na [M+Na] : 603.1611, found 603.1589.

Dihydroquercetin 71

A UV active yellow solid (0.0023 mg/g dried plant sample);

25 o UVmax (MeOH) (nm): 225; 255; 352. [훼]D +46 (c 0.15,

1 129 MeOH); H-NMR (CD3OD, 500 MHz) , 6.97 (d, J = 2.1

Hz, 1H, H2'), 6.85 (dd, J = 8.1, 2.1 Hz, 1H, H6'), 6.81 (d, J = 8.1 Hz, 1H, H5'), 5.93 (d,

J = 2.1 Hz, 1H, H6), 5.89 (d, J = 2.1 Hz, H8), 4.92 (d, J = 11.5 Hz, 1H, H2), 4.51 (d, J =

13 129 11.5 Hz, 1H, H3); C-NMR (CD3OD, 125 MHz) , 198.5 (C4), 169.6 (C7), 167.1

(C5), 163.6 (C8a), 147.9 (C4'), 146.5 (C3'), 129.8 (C1'), 121.0 (C6'), 116.2 (C5'), 116.0

(C2'), 102.2 (C4a), 97.4 (C6), 96.39 (C8), 85.2 (C2), 73.7 (C3); LREIMS, m/z: 305 (M-

H)+

6.6 Isolation of constituents of Jacaranda mimosifolia flowers

The powder from the flowers of J. mimosifolia (85 g) was suspended in MeOH (1 L, in

2.0 L conical flask) and stirred for 24 h, filtered and the supernatant vacuum dried to produce a crude extract (22.6 g). A suspension of the extract in methanol was sequentially fractionated with hexane, and provided 2 fractions (hexane (12.9 g) and methanol (8.8 g)). The methanol fraction (400 mg) was applied to short C-18 silica column (1.5 x 5 cm) and eluted with methanol (50 mL) which was then concentrated to

20 mL and filtered through a HPLC sample filter (0.45 μm). This sample was then injected in 10 blocks into preparative HPLC with a gradient elution from 100% of

146 solvent A (0.1% TFA in water) to 60% of solvent A within 60 minutes (solvent B, 0.1%

TFA in ACN) and produced nine compounds. The sample at retention time 32.5 minutes was identified as 66 (2.45 mg/g dried plant sample) and structural elucidation is described in section 6.5.

Jacaranone 72

A UV active yellow gum (0.98 mg/g dried plant sample); UV

1 135 (CH3OH) λmax (log ε) 225. H-NMR (CD3OD, 500 MHz) , 7.07 (d,

J = 9.7 Hz, 2H, H3/5), 6.15 (d, J = 9.7 Hz, 2H, H2/6), 3.65 (s, 3H, H9), 2.76 (s, 2H,

13 135 H7); C-NMR (CD3OD, 125 MHz) , 186.1 (C4), 169.6 (C8), 151.4 (C2/6), 127.2

(C3/5), 66.7 (C1) , 50.97 (C9), 44.35 (C7). ESMS, m/z 183 (M+H)+

Phenylethanoid glucoside 73

A UV active yellow solid (1.6 mg/g dried plant sample); UV

25 (CH3OH) λmax (log ε) 220 (4.4) nm; [훼]D : +1.5 (c 0.15,

-1 CH3OH); IR [cm ]: 3364 (br, m), 1668 (s). H-NMR

(CD3OD, 500 MHz), 7.02 (d, J = 9.7, 2H, H2/6), 6.12 (d, J = 9.7 Hz, 2H, H3/5), 5.40 (d,

J = 8.2 Hz, 1H, H1'), 4.34 (dd, J = 1.2, 11.2 Hz, 1H, H6'a), 4.09 (dd, J = 6.5, 11.2 Hz,

1H, H6'b), 3.52 (ddd, J = 9.0, 6.5, 1.2 Hz, 1H, H5'), 3.41 (dd, J = 9.0, 9.5 Hz, 1H, H3'),

3.30 (dd, J = 8.2, 8.9 Hz, 1H, H2'), 3.29 (dd, J = 9.0, 8.0 Hz, 1H, H2'), 2.83 (s, 2H, H7);

13 C-NMR (CD3OD, 125 MHz), 186.1 (C4), 169.6 (C8) , 151.4 (C2/6), 127.2 (C3/5),

95.6 (C1'), 77.1 (C3'), 75.5 (C5'), 73.2 (C2'), 70.8 (C4'), 66.7 (C1), 64.7 (C6') , 44.35

+ + (C7). ESMS, m/z 353 (M+Na) HRESIMS calculated for C14H18O9Na [M+Na] :

353.0849, found 353.0839.

147

Isoacteoside 74

A UV active yellow solid (0.4 mg/g dried plant

sample); UV MeOH max : 340 and 225 nm; IR

max: 3500 (OH), 2931 (CH), 1710, 1602 and

1520 (aromatic), 1446, 1155, 810 and 648 cm-1;

1 134 H-NMR (CD3OD), 500 MHz) , 7.68 (d, J =

15.5 Hz, 1H, H7''), 7.23 (s, 1H, H2''), 7.09 (d, J = 8.10 Hz, H6''), 6.73 (d, J = 8.0 Hz,

1H, H5''), 6.56 (s, 1H, H2'), 6.51 (d, J = 8.0 Hz, 1H, H5'), 6.28 (d, J = 8.0 Hz, 1H, H6'),

5.88 (d, J = 15.5 Hz, 1H, H8''), 5.00 (d, J = 8.0 Hz, 1H, H1'''), 4.29 (d, J = 8.8 Hz, 1H,

H6a), 4.27 (d, J = 8.8, 1H, H6b), 4.18 (d, J = 7.0 Hz, 1H, H1), 4.09-4.00 (m, 1H, H5'''),

3.94 (t, J = 10.0 Hz, 1H, H8'a), 3.69 (t, J = 10.0 Hz, 1H, H8'b), 3.90-3.80 (m, 1H, H2'''),

3.67-3.60 (m, 1H, H3'''), 3.49-3.46 (m, 1H, H3), 3.44-3.40 (m, 1H, H5), 3.37-3.35 (m,

1H, H4), 3.30-3.20 (m, 1H, H4'''), 3.25-3.15 (m, 1H, H2), 2.50 (t, J = 10.0 Hz, 2H, H7'),

13 134 1.14 (s, 3H, H6'''); C-NMR (CD3OD, 125 MHz) , 168.8 (C9''), 147.4 (C4''), 146.1

(C7''), 144.2 (C3''), 143.7 (C3'), 142.1 (C4'), 130.5 (C1'), 126.3 (C1''), 122.7 (C6''),

120.9 (C6'), 116.5 (C2'), 116.3 (C5''), 115.9 (C5'), 114.8 (C2''), 113.5 (C8''), 102.0 (C1),

101.1 (C1'''), 82.4 (C3), 73.5 (C2), 73.3 (C5), 71.9 (C4'''), 71.3 (C8'), 70.3 (C2'''), 70.1

(C3'''), 68.8 (C4), 68.7 (C5'''), 63.5 (C6), 34.5 (C7'), 16.52 (C6'''). ESMS, m/z 625

(M+Na)+.

Hydroxyphenylacetic acid 75

A UV active yellow solid (0.81 mg/g dried plant sample); UV

1 135 (CH3OH) λmax (log ε) 225 nm. H-NMR (CD3OD, 500 MHz) ,

148

7.08 (d, J = 9.7 Hz, 2H, H2/6), 6.72 (d, J = 9.7 Hz, 2H, H3/5), 3.48 (s, 2H, H7); 13C-

135 NMR (CD3OD, 125 MHz) , 176.4 (C8), 157.6 (C4), 131.5 (C2/6), 127.0 (C1), 116.4

(C3/5), 41.1 (C7). ESMS, m/z 153 (M+H)+.

1,6-Bis(1-hydroxy-4-oxo-2,5-cyclohexadiene-1-acetyl)-3-(p-hydroxybenzeneacetyl)- β-glucopyranoside 76

A UV active yellow solid (1.8 mg/g dried plant

sample); UV MeOH max : 340 and 225 nm;

25 [훼]D : +6.5 (c 0.05, MeOH); IR: 3396.7,

3440.2, 1673.1, 1633.5 cm-1. 1H-NMR

135 ((CD3)2CO, 500 MHz) , 7.01 (d, J = 8.0 Hz, 2H, H2'''/6'''), 6.98 (d, J = 8.0 Hz, 2H,

H2''/6''), 6.86 (d, J = 8.0 Hz, 2H, H2'/6'), 6.62 (d, J = 8.0 Hz, 2H, H3'''/5'''), 6.07 (d, J =

8.0 Hz, 2H, H3''/5''), 6.03 (d, J = 8.0 Hz, 2H, H3'/5'), 5.48 (d, J = 7.5 Hz, 1H, H1), 4.93

(dd, J = 7.9, 9.0 Hz, 1H, H2), 4.43 (d, J = 10.8 Hz, 1H, H6a), 4.19 (dd, J = 10.8; 5.4,

1H, H6b), 3.74-3.71 (m, 1H, H3), 3.70-3.68 (m, 1H, H5), 3.67 (s, 2H, H7'''a), 3.51 (dd,

J = 9.0, 8.5 Hz, 1H, H4), 2.79 (s, 1H, H7'''b), 2.67 (d, J = 15.0 Hz, 1H, H7'a), 2.55 (d, J

13 135 = 15.0 Hz, 1H, H7'b); C-NMR ((CD3)2CO, 125 MHz) , 186.6 (C4''), 186.1 (C4'),

170.7 (C8'''), 169.4 (C8''), 168.0 (C8'), 156.5 (C4'''), 152.8 (C2''/6''), 152.7 (C2'/6'),

131.5 (C2'''/6'''), 128.7 (C3''/5''), 128.6 (C3'/5'), 125.5 (C1'''), 116.5 (C3'''/5'''), 93.4 (C1),

75.7 (C5), 75.3 (C3), 73.6 (C), 71.0 (C4), 67.8 (C1''), 67.5 (C1'), 64.1 (C6), 45.7 (C7''),

45.0 (C7'), 41.2 (C7'''). ESMS, m/z 637 (M+Na)+.

149

Jacaglabroside B 77

A UV active yellow solid (0.45 mg/g dried

plant sample); UV (CH3CN) λmax (log ε) 220

25 (4.6) nm; [훼]D : +2.2 (c 0.20, CH3OH); IR

νmax 3421, 1746, 1671, 1625, 1517, 1398,

-1 1 135 1230, 1152, 1076, 1014, 862, 714 cm ; H-NMR ((CD3)2CO, 500 MHz) , 7.28 (d, J

= 8.0 Hz, 2H, H3'''/5'''), 7.26 (d, J = 8.0 Hz, 2H, H2'''/6'''), 7.24 (d, J = 8.0 Hz, 1H, H4'''),

6.98 (d, J = 8.0 Hz, 2H, H2''/6''), 6.86 (d, J = 8.0 Hz, 2H, H2'/6'), 6.07 (d, J = 8.0 Hz,

2H, H3''/5''), 6.03 (d, J = 8.0 Hz, 2H, H3'/5'), 5.48 (d, J = 7.5 Hz, 1H, H1), 4.93 (dd, J =

9.0, 8.5 Hz, 1H, H2), 4.43 (d, J = 10.8 Hz, 1H, H6a), 4.19 (dd, J = 10.8, 5.4 Hz, 1H,

H6b), 3.74-3.72 (m, 1H, H3), 3.70-3.68 (m, 1H, H5), 3.67 (s, 2H, H7'''a), 3.51 (dd, J =

9.0, 8.5 Hz, 1H, H4), 2.79 (s, 1H, H7'''b), 2.67 (d, J = 15.0 Hz, 1H, H7'a), 2.55 (d, J =

13 135 15.0 Hz, 1H, H7'b); C-NMR ((CD3)2CO, 125 MHz) , 186.6 (C4''), 186.1 (C4'),

170.7 (C8'''), 169.4 (C8''), 168.0 (C8'), 152.8 (C2''/6''), 152.7 (C2'/6'), 135.5 (C1'''),

130.5 (C2'''/6'''), 129.4 (C3'''/5'''), 128.7 (C3''/5''), 128.6 (C3'/5'), 126.7 (C4'''), 93.4 (C1),

75.7 (C5), 75.3 (C3), 73.6 (C), 71.0 (C4), 67.8 (C1''), 67.5 (C1'), 64.1 (C6), 45.7 (C7''),

45.0 (C7'), 41.2 (C7'''). ESMS, m/z 599 (M+H)+.

Jacaglabroside C 78

A UV active yellow solid (0.90 mg/g dried plant

sample); UV (CH3CN) λmax (log ε) 223 (4.6)

25 nm; [훼]D : +3.5 (c 0.16, CH3OH); IR (KBr)

νmax 3423, 1750, 1671, 1625, 1517, 1232,

150

-1 1 1152, 1076, 1018, 862, 714 cm . H-NMR ((CD3)2CO, 500 MHz), ((CD3)2CO, 500

MHz)135, 7.30 (d, J = 8.0 Hz, 2H, H3''''/5''''), 7.25 (d, J = 8.0 Hz, 2H, H2''''/6''''), 7.23 (d,

J = 8.0 Hz, 1H, H4''''), 7.01 (d, J = 8.0 Hz, 2H, H2'''/6'''), 6.98 (d, J = 8.0 Hz, 2H,

H2''/6''), 6.86 (d, J = 8.0 Hz, 2H, H2'/6'), 6.62 (d, J = 8.0 Hz, 2H, H3'''/5'''), 6.07 (d, J =

8.0 Hz, 2H, H3''/5''), 6.03 (d, J = 8.0 Hz, 2H, H3'/5'), 5.48 (d, J = 7.5 Hz, 1H, H1), 5.27

(dd, J = 9.0, 7.9 Hz, 1H, H3), 4.93 (dd, J = 7.9, 7.5 Hz, 1H, H2), 4.43 (dd, J = 10.8, 5.4

Hz, 1H, H6a), 4.19 (dd, J = 10.8, 5.4, 1H, H6b), 3.70-3.60 (m, 1H, H5), 3.58 (d, J =

16.2 Hz, 1H, H7''''a), 3.54 (d, J = 16.2 Hz, 1H, H7'''b), 3.51 (dd, J = 9.0, 5.4 Hz, 1H,

H4), 3.49 (d, J = 16.2 Hz, 1H, H7'''a), 3.40 (d, J = 16.2 Hz, 1H, H7'''b), 2.67 (d, J = 15.0

13 135 Hz, 1H, H7'a), 2.55 (d, J = 15.0 Hz, 1H, H7'b); C-NMR ((CD3)2CO, 125 MHz) ,

186.6 (C4''), 186.1 (C4'), 170.7 (C8'''), 170.1 (C8''''), 169.4 (C8''), 168.0 (C8'), 156.5

(C4'''), 152.8 (C2''/6''), 152.7 (C2'/6'), 134.9 (C1''''), 131.5 (C2'''/6'''), 130.7 (C2''''/6''''),

129.1 (C3''''/5''''), 128.7 (C3''/5''), 128.6 (C3'/5'), 127.5 (C4''''), 125.5 (C1'''), 116.5

(C3'''/5'''), 93.4 (C1), 75.7 (C5), 75.3 (C3), 73.6 (C), 71.0 (C4), 67.8 (C1''), 67.5 (C1'),

64.1 (C6), 45.7 (C7''), 45.0 (C7'), 41.8 (C7''''), 41.2 (C7'''). ESMS, m/z 733 (M+H)+.

151

Jacaglabroside D 79

A UV active yellow solid (1.10 mg/g dried

plant sample); UV (CH3CN) λmax (log ε) 220

25 (4.4) nm; [훼]D : +1.9 (c 0.16, CH3OH); UV

(CH3CN) λmax (log ε) 220 (4.4) nm; IR (KBr)

νmax 3422, 1738, 1671, 1626, 1398, 1261,

1152, 1079, 1012, 863, 715 cm-1 1H-NMR

135 ((CD3)2CO, 500 MHz) , ((CD3)2CO, 500 MHz), 7.30 (J = 8.0 Hz, 2H, H3'''/5'''), 7.25

(J = 8.0 Hz, 2H, H2'''/6'''), 7.23 (d, J = 8.0 Hz, 1H, H4'''), 7.01 (d, J = 8.0 Hz, 2H,

H2''''/6''''), 6.98 (d, J = 8.0 Hz, 2H, H2''/6''), 6.86 (d, J = 8.0 Hz, 2H, H2'/6'), 6.62 (d, J =

8.0 Hz, 2H, H3''''/5''''), 6.07 (d, J = 8.0 Hz, 2H, H3''/5''), 6.03 (d, J = 8.0 Hz, 2H, H3'/5'),

5.48 (d, J = 7.5 Hz, 1H, H1), 5.27 (dd, J = 9.0, 7.9 Hz, 1H, H3), 4.93 (dd, J = 7.9, 7.5

Hz, 1H, H2), 4.43 (d, J = 10.8 Hz, 1H, H6a), 4.19 (dd, J = 10.8; 5.4, 1H, H6b), 3.70-

3.60 (m, 1H, H5), 3.58-3.57 (d, J = 16.2 Hz, 1H, H7'''a), 3.56-3.54 (m, 1H, H7''''a), 3.51

(dd, J = 9.0, 7.5 Hz, 1H, H4), 3.46 (m, 1H, H7''''b), 3.49 (d, J = 16.2 Hz, 1H, H7'''b),

13 2.67 (d, J = 15.0 Hz, 1H, H7'a), 2.55 (d, J = 15.0 Hz, 1H, H7'b); C-NMR ((CD3)2CO,

125 MHz)135, 186.6 (C4''), 186.1 (C4'), 170.7 (C8''''), 170.1 (C8'''), 169.4 (C8''), 168.0

(C8'), 156.5 (C4''''), 152.8 (C2''/6''), 152.7 (C2'/6'), 134.9 (C1'''), 131.5 (C2''''/6''''), 130.7

(C2'''/6'''), 129.1 (C3'''/5'''), 128.7 (C3''/5''), 128.6 (C3'/5'), 127.5 (C4'''), 125.5 (C1''''),

116.5 (C3''''/5''''), 93.4 (C1), 75.7 (C5), 75.3 (C3), 73.6 (C), 71.0 (C4), 67.8 (C1''), 67.5

(C1'), 64.1 (C6), 45.7 (C7''), 45.0 (C7'), 41.8 (C7'''), 41.2 (C7''''). ESMS, m/z 733

(M+H)+.

152

6.7 Isolation of constituents of Acacia pycnantha flowers

The powder from the flowers of A. pycnantha (100 g) was suspended in methanol (1 L, in 2.0 L conical flask) and then stirred for 24 h, filtered and the supernatants vacuum dried to produce a crude extract (35.6 g). A suspension of the extract in methanol was sequently fractionated with hexane, providing 2 fractions (hexane (21.9 g) and methanol fraction (11.7 g)).

The methanol fraction (400 mg) was applied to short C-18 silica column (1.5 x 5 cm) and eluted with methanol (50 mL) which was then concentrated to 20 mL and filtered through a HPLC sample filter (0.45 μm). This sample was then injected in 10 blocks into preparative HPLC with a gradient elution from 80% of solvent A (0.1% TFA in water) to 60% of solvent A within 35 minutes (solvent B, 0.1% TFA in ACN) and produced eight compounds. Compound 80 and 81 were collected as one fraction at retention time 11.25 minutes. The fraction was recrystallized from water and acetonitrile at room temperature with slow evaporation, and produced 81 as long white needle crystals. The crystals and solution were seperated gently using a Pasteur pipette, and the solution was concentrated to obtain compound 80 as yellow solid compound.

The samples at retention times 12 and 12.5 minutes were identified as 48 and 53, respectively and their spectroscopy data were presented at section 6.5.

153

Isohemiphloin 80

A UV active yellow solid (0.50 mg/g of dried plant sample);

25 o UVmax (MeOH) (nm): 216; 232; 334. [훼]D +29.52 (c 0.25,

1 203 MeOH). H-NMR (CD3OD, 500 MHz) , 7.30 (d, J = 8.2

Hz, 2H, H2'/6'), 6.81 (d, J = 8.2 Hz, 2H, H3'/5'), 5.96 (s, 1H,

H6), 5.34 (dd, J = 13.0, 3.1 Hz, 1H, H2), 4.81 (d, J = 7.2, Hz, H1''), 4.03 (ddd, J = 1.2,

6.5, 9.0 Hz, 1H, H5''), 3.70 (dd, J = 1.2, 6.5 Hz, H6a''), 3.60 (dd, J = 9.0, 8.0 Hz, 1H,

H4''), 3.58 (dd, J = 6.5, 11.5 Hz, 1H, H6''b), 3.49 (dd, J = 9.0, 7.0 Hz, H2''), 3.47 (dd, J

= 9.0, 9.5 Hz, H3''), 2.97 (dd, J = 17.2, 13.0 Hz,1H, H3a), 2.75 (dd, J = 17.2, 3.0 Hz,

13 203 1H, H3b); C-NMR (CD3OD, 125 MHz) , 198.4 (C4), 167.4 (C6''), 164.4 (C7),

164.3 (C5), 159.9 (C8a), 159.3 (C4'), 131.0 (C1'), 129.1 (C2'/6'), 116.5 (C3'/5'), 105.8

(C8), 103.8 (C4a), 96.5 (C6), 80.2 (C2), 73.37 (C1''), 77.3 (C2''), 76.1 (C5''), 73.8 (C3''),

72.6 (C4''), 46.3 (C3); LRESMS, m/z: 435 [M+H]+.

Naringenin-5-O-glucoside 81

A UV active white crystal (0.50 mg/g of dried plant

25 sample); UVmax (MeOH) (nm): 216; 237; 334; [훼]D -

o 1 97.79 (c 0.20, MeOH). H-NMR (CD3OD, 500

MHz)204, 7.28 (d, J = 8.2 Hz, 2H, H2'/6'), 6.81 (d, J =

8.2 Hz, 2H, H3'/5'), 6.12 (s, 1H, H6), 6.47 (s, 1H, H8), 5.29 (dd, J = 13.0, 3.1 Hz, 1H,

H2), 4.75 (d, J = 7.2 Hz, H1''), 4.03 (ddd, J = 1.2, 6.5, 9.0 Hz, 1H, H5''), 3.70 (dd, J =

1.2, 6.5 Hz, H6a''), 3.60 (dd, J = 9.0, 8.0 Hz, 1H, H4''), 3.58 (dd, J = 6.5, 11.5 Hz, 1H,

H6''b), 3.49 (dd, J = 9.0, 7.0 Hz, H2''), 3.47 (dd, J = 9.0, 9.5 Hz, H3''), 2.97 (dd, J =

13 17.2, 13.0 Hz,1H, H3a), 2.75 (dd, J = 17.2, 3.0 Hz, 1H, H3b); C-NMR (CD3OD, 125

MHz)204, 193.1 (C4), 167.1 (C6''), 166.6 (C7), 162.4 (C5), 159.9 (C8a), 159.3 (C4'),

154

131.0 (C1'), 128.9 (C2'/6'), 116.5 (C3'/5'), 105.2 (C4a), 104.8 (C1''), 100.4 (C6), 99.6

(C8), 80.2 (C2), 77.3 (C2''), 76.1 (C5''), 73.8 (C3''), 72.6 (C4''), 46.3 (C3); LRESMS, m/z: 435 [M+H]+.

Myricetin 3-rhamnoside 82

A UV active pale yellow solid (0.84 mg/g dried plant

sample); UVmax (MeOH) (nm): 229; 255; 371. 1H-

81 NMR (CD3OD, 500 MHz) , 6.95 (s, 2H, H2'/6'),

6.39 (s, 1H, H8), 6.23 (s, 1H, H6), 5.34 (d, J = 7.5 Hz,

1H, H1''), 4.22 (dd, 7.5, 9.0 Hz, 1H, H2''), 3.78 (dd, J = 9.5, 9.0 Hz, 1H, H3''), 3.52 (dd,

J = 9.0, 6.5 Hz, 1H, H5''), 3.37 (dd, J = 9.5, 9.0 Hz, 1H, H4''), 0.97 (d, 6.5 Hz, 3H, H6'');

13 81 C-NMR (CD3OD, 125 MHz) , 179.5 (C4), 164.1 (C7), 163.7 (C5), 158.9 (C8a),

158.7 (C2), 149.7 (C3'/5'), 146.3 (C4'), 135.6 (C3), 122.95 (C1'), 108.9 (C2'/6'), 105.3

(C4a), 102.0 (C1''), 98.5 (C6), 93.0 (C8), 73.2 (C4''), 72.1 (C3''), 71.5 (C5''), 71.7 (C2'')

18.5 (C6''); LRESMS m/z 487 (M+Na)+.

Kaempferol 3-rhamnoside 83

A UV active pale yellow solid (1.5 mg/g dried plant

sample); UVmax (MeOH) (nm): 229; 257; 341. 1H-

81 NMR (CD3OD, 500 MHz) , 8.06 (d, J = 8.4 Hz, 2H,

H2'/6'), 6.89 (d, J = 8.5 Hz, 2H, H3'/5'), 6.39 (d, J = 2.1 Hz, 1H, H8), 6.20 (d, J = 2.1

Hz, 1H, H6), 5.34 (d, J = 7.5 Hz, 1H, H1''), 4.24 (dd, 7.5, 9.0 Hz, 1H, H2''), 3.76 (dd, J =

9.5, 9.0 Hz, 1H, H3''), 3.50 (dd, J = 9.0, 6.5 Hz, 1H, H5''), 3.37 (dd, J = 9.5, 9.0 Hz, 1H,

13 81 H4''), 0.97 (d, 6.5 dHz, 3H, H6''); C-NMR (CD3OD, 125 MHz) , 179.5 (4), 166.1

(C7), 163.1 (C5), 161.6 (C2), 159.5 (C4'), 158.6 (C8a), 135.3 (C3), 132.6 (C2'/6'), 122.7

155

(C1'), 116.4 (C3'/5'), 105.7 (C4a), 102.4 (C1''), 99.8 (C6), 94.4 (C8), 73.2 (C4''), 72.1

(C3''), 71.5 (C5''), 71.7 (C2'') 18.5 (C6'') ; LRESMS m/z 431 (M+Na)- .

Isosalipurposide 84

A UV active yellow solid (0.08 mg/g dried plant

sample); UVmax (MeOH) (nm): 219; 269; 338. 1H-

172 NMR (CD3OD, 500 MHz) , 8.02 (d, J = 15.0 Hz,

1H, H2), 7.68 (d, J = 15.0 Hz, 1H, H3), 7.62 (d, J =

8.0 Hz, 2H, H2'/6'), 6.87 (d, J = 8.0 Hz, H3'/5'), 6.26 (s, 1H, H5''), 6.03 (s, 1H, H3''),

5.15 (d, J = 7.8 Hz, 1H, H1'''), 3.94 (dd, J = 12.6, 1.5 Hz, 2H, H6'''a), 3.76 (dd, J = 12.6,

1.5 Hz, 2H, H6'''b), 3.60 (dd, J = 10.5, 7.5 Hz, 1H, H3'''), 3.57-3.54 (m, 1H, H5'''), 3.50-

13 172 3.47 (m, 1H, H4'''), 3.46-3.39 (m, 1H, H2'''); C-NMR (CD3OD, 125 MHz) , 194.8

(C1), 165.9 (C5), 161.8 (C4'), 161.4 (C7), 161.1 (C3), 144.3 (C2), 131.9 (C2'/ C6'),

128.3 (C1'), 127.2 (C1''), 125.6 (C2), 117.1 (C3'/5'), 101.9 (C'''), 98.6 (C3''), 96.0 (C5''),

76.7 (C5'''), 76.3 (C3'''), 75.3 (C2'''), 72.1 (C4'''), 62.3 (C6'''); LREIMS, m/z: 435

[M+H]+.

Naringenin 85

A UV active yellow solid (0.55 mg/g of dried plant sample);

25 o UVmax (MeOH) (nm): 226; 237; 334; [훼]D -35.8 (c 0.25,

1 205 MeOH). H-NMR (CD3OD, 500 MHz) , 7.30 (d, J = 8.2 Hz,

2H, H2'/6'), 6.81 (d, J = 8.2 Hz, 2H, H3'/5'), 5.96 (s, 1H, H6), 5.34 (dd, J = 13.0, 3.1 Hz,

1H, H2), 2.97 (dd, J = 17.2, 13.0 Hz,1H, H3a), 2.75 (dd, J = 17.2, 3.0 Hz, 1H, H3b);

13 205 C-NMR (CD3OD, 125 MHz) , 198.4 (C4), 164.4 (C7), 164.3 (C5), 159.9 (C8a),

156

159.3 (C4'), 131.0 (C1'), 129.1 (C2'/6'), 116.5 (C3'/5'), 103.8 (C4a), 96.5 (C6), 98.8

(C8), 80.2 (C2), 46.3 (C3); LRESMS, m/z: 289 [M+H]+.

6.8 Isolation of constituents from Australian King Parrot green feathers

Feathers were hand-plucked from frozen specimens, and separated according to colour.

They were stirred in excess absolute ethanol (15 mins), gravity filtered, then stirred in excess hexane (15 mins) before again being gravity filtered and dried. Their quills, were then removed and the feathers were separated according their colour before they were alternately chopped into small pieces according their colour and ground under liquid nitrogen. In order to obtain optimal crude extract from the green feather, several extraction methods were applied.

6.8.1 Hot acidified pyridine

Acidified pyridine was prepared by adding HCl (4 drops) to pyridine (100 mL). It was then added to processed feathers (0.5 g) and heated at reflux for 4 h yielding a brown/orange extract (72.5 mg).

6.8.2 TossueLyser with methanol

In this extraction process, A Qiagen TissueLyser II was used to pulverise processed the feathers. Processed feathers (0.05 g) were frozen with liquid nitrogen (5 mL) and added to methanol (0.5 mL) in an Eppendorf tube. A tungsten bead was inserted prior to shaking on the TissueLyser for 15 mins. A negligible quantity of extract was obtained.

6.8.3 Soxhlet extraction

Processed feathers (0.4 g) were placed in a cellulose thimble, stoppered with cotton wool, and inserted into a Soxhlet apparatus. Methanol (200 mL) was heated at reflux

157 within the apparatus for 72 h, yielding a yellow extract (174.4 mg) and this extraction method was repeated for 10 times with fresh processed feathers and resulting 1.85 g crude methanol extract. A suspension of crude extract in methanol was sequential fractionation with methanol and hexane to provide two fractions (hexane (1.22 g) and methanol (630 mg)).

The hexane fraction was prepared for GC-MS and the resulting spectrum were compared to samples within the NIST08 database. The data showed six major peaks, assigned as methyl palmitate (104), linoleic acid (105), methyl oleate (106), and methyl stearate (107).

The methanol extract was eluted with methanol (50 mL) and filtered through a

Whatman syringe filter 0.45 μm and then reduced to 10 mL by evaporation. This extract was then injected in 10 blocks into preparative HPLC with a gradient elution from

100% of solvent A (0.1% TFA in water) to 50% of solvent A within 55 minutes (solvent

B, 0.1% TFA in ACN) to produce compounds 108 – 109.

3,4-Dihydroxybenzoic acid 108

An UV pale brown solid (0.05 mg/g dried plant sample); UVmax (MeOH)

1 259 (nm): 268; 295; H-NMR (CD3OD, 500 MHz) , 7.42 (s, 1H, H2), 7.41 (d,

13 J = 8.0, 1H, H6), 6.78 (d, J = 8.0, 1H, H5); C-NMR (CD3OD, 125

MHz)259, 170.3 (COOH), 151.5 (C4), 146.6 (C3), 123.8 (C6), 123.1 (C1), 117.7 (C2),

115.7 (C5); ESMS, m/z 155 (M+1)+.

Butanoic acid 109

A UV active colourless oil (0.06 mg/g dried plant sample); UVmax

158

1 260 (MeOH) (nm): 225; 268; H-NMR (CD3OD, 500 MHz) , 2.28 (t, J = 7.4 Hz, 2H, H2),

13 260 1.64 (m, 2H, H3), 1.35 (d, 8.0 Hz, 3H, H4); C-NMR (CD3OD, 125 MHz) , 177.3

(COOH), 34.5 (C2), 28.0 (C3), 18.3 (C4); EIMS, m/z 88 (M)+.

4-Hydroxymethyl benzoate 110

A UV active white solid (0.018 mg/g dried plant sample); UVmax

1 261 (MeOH) (nm): 225; 259; H-NMR (CD3OD, 500 MHz) , 7.86 (d, J =

13 8.0, 2H, H2/6), 6.82 (d, J = 8.0, 2H, H3/5), 3.85 (OCH3); C-NMR (CD3OD, 125

MHz)261, 168.8 (COO-), 163.0 (C4), 132.9 (C2/6), 121.9 (C1), 115.8 (C3/5), 51.8

+ (OCH3); LREIMS: m/z: 154 (M ).

p-Hydroxybenzaldehyde 111

A UV active pale yellow solid (0.025 mg/g dried plant sample);

1 262 UVmax (MeOH) (nm): 221; 284; H-NMR (CD3OD, 500 MHz) ,

13 7.76 (d, J = 8.0, 2H, H2/6), 6.72 (d, J = 8.0, 2H, H3/5); C-NMR (CD3OD, 125

MHz)262, 170.8 (COOH), 162.1 (C4), 131.9 (C2/6), 120.9 (C1), 116.8 (C3/5);

LREIMS: m/z: 122 (M+).

Genistein 112

A UV active yellow solid (0.08 mg/g dried plant

sample); UVmax (MeOH) (nm): 258; 330; 1H-NMR

263 (CD3OD, 500 MHz) , 8.06 (s, 1H, H2), 7.38 (d, J =

8.0, 2H, H2'/6'), 6.87 (d, J = 8.0, 2H, H3'/5'), 6.33 (s, 1H, H8), 6.22 (s, 1H, H6); 13C-

263 NMR (CD3OD, 125 MHz) , 181.9 (C4), 165.3 (C7), 164.3 (C5), 159.8 (C8a), 158.8

(C4'), 154.3 (C2), 131.4 (C2'/6'), 124.8 (C1'), 116.1 (C3'/5'), 106.2 (C4a), 99.67 (C6),

159

+ 94.8 (C8); LRESMS: m/z: 271 [M+H] . HRESMS calculated for C15H10O5: 271.0509 found 271.0606 [M+H]+.

6.9. Isolation of constituents of Ceratodon purpureus

6.9.1. Intracellular sample preparation of Australian C. purpureus

Moss (30.5 g dried) were ground to a powder under liquid nitrogen in a mortar and pestle. Intracellular compounds were extracted from this powder by stirring in MeOH

(400 mL, in 1.0 L conical flask) for 24 h. The resultant extract was filtered and the residue was extracted ten times. The supernatants were pooled and concentrated in vacuo to produce crude extract (1.97 g).

The extract (500 mg) was dissolved in EtOAC–MeOH–ACN–H2O (2:2:2:0.5, 6.5 mL).

This solution was loaded into a syringe (10 mL) filled with silica (5 mL) and flushed with EtOAc (10 mL), ACN (20 mL), ACN–MeOH–H2O (9:0.5:0.5, 20 mL) to produce a filtrate (50 mL). This solution was then passed through a HPLC sample filter

(0.45 μm). This procedure was applied to the remaining extract. A gradient elution from

80% to 30% of solvent A (0.1% TFA in H2O) within 40 min (solvent B, 0.1% TFA in

ACN) was used for the semi-preparative HPLC separation and produced compounds

119 - 123.

5'-Hydroxyamentoflavone 119

An UV active pale yellow solid (0.52 mg /g dried

1 287 plant sample); H-NMR (DMSO-d6, 600 MHz) ,

7.48 (6'), 7.46 (H2'), 7.07 (d, J = 2.0 Hz, H2'''),7.02

(dd, J = 8.0; 2.0 Hz, H6'''), 6.68 (d, J = 8.0 Hz, H5'''),

160

13 1H, H6'), 6.64 (s, H3), 6.45 (s, H8), 6.40 (s, H6''), 6.18 (s, H6),. C-NMR (DMSO-d6,

150 MHz), 122.3 (C2'), 118.6 (C6'''), 115.5 (C5'''), 113.7 (C2'''), 112.1 (C6'), 102.9

(C3''), 102.5 (C3), 98.8 (C6), 98.1 (C6''), 93.9 (C8). LRESMS, m/z 569 [M-H]+. The complete structure of compound 119 was not possible to finalize, but is likely to be a structural isomer of 120.

5',3'''-dihydroxyamentaflavone 120

An UV active pale yellow solid (2.52 mg /g dried

plant sample); m.p: burnt at 295 ºC; λmax 255

(16,053) 350 (14,146); (IR [cm-1]: 3213 (m), 1653 (s),

1430 (s), 1339 (s), 1253 (s), 1165 (s), 836 (s); 1H-

279 NMR ((CD3)2CO, 600 MHz) , 7.63 (dd, J = 2.2 Hz, 8.4 Hz, 1H, H6'''), 7.62 (d, J = 2.2

Hz, 1H, H2'''), 7.28 (d, J = 2.3 Hz,1H, H2'), 7.18 (d, J = 2.3 Hz 1H, H6'), 6.81 (d, J =

8.4 Hz, 1H, H5'''), 6.66 (s, 1H, H3''), 6.59 (s, 1H, H3), 6.52 (d, J = 2.1 Hz, 1H, H6), 6.46

13 279 (s, 1H, H6''), 6.24 (d, J = 2.1 Hz, 1H, H8). C-NMR ((CD3)2CO, 150 MHz) , 183.7

(C4), 183.1 (C4''), 165.7 (C2), 165.4 (C2''), 165.7 (C7), 165.2 (C5''), 164.9 (C7''), 163.2

(C8a''), 162.5 (C5), 159.5 (C8a), 150.4 (C4'), 150.2 (C4'''), 146.9 (C3'''), 146.3 (C3'),

124.1 (C1'), 123.8 (C6'''), 123.3 (C1'''), 120.2 (C5'), 119.8 (C2'), 116.2 (C5'''), 114.09

(C6'), 113.5 (C2'''), 105.7 (C4a''), 105.5 (C4a), 104.8 (C8''), 104.2 (C3''), 103.7 (C3),

99.5 (C6''), 99.2 (C8), 94.6 (C6) LRESMS, m/z 569 (M-H)-. HRESMS: calculated for

- C30H17O12 (M-H) : 569.0720, found 569.0732.

2,3-Dihydro-5',3''''-dihydroxyrobustaflavone 121

An UV active pale yellow solid (3.35 mg /g dried

plant sample); m.p: burnt at 295 ºC; λmax 211

161

(10,047), 274 (5,208), 287 (5,000), 347 (4,320); IR [cm-1]: 3330 (m), 2956 (s), 1653 (s),

1 279 1194 (s), 1189 (s), 1128 (s), 800 (s), 725 (s); H-NMR ((CD3)2CO, 600 MHz) , 7.30

(d, J = 2.0 Hz 1H, H2'), 7.182 (d, J = 2.0 Hz, 1H, H6'), 7.180 (d, 2.0 Hz, 1H, H2'''), 6.92

(d, J = 7.5 Hz, 1H, H5'''), 6.79 (dd, J = 2.0, 7.5 Hz, 1H, H6'''), 6.59 (s, 1H, H3''), 6.44 (s,

1H, H6''), 6.08 (s, 1H, H6), 5.94 (s, 1H, H8), 5.47 (dd, J = 13.5, 3.1 Hz, 1H, H2), 3.24

(dd, J = 17.5, 13.5 Hz, 1H, H3a), 2.79 (dd, J = 17.5, 3.1 Hz, 1H, H3b); 13C-NMR

279 ((CD3)2CO, 150 MHz) , 196.5 (C4), 182.7 (C4''), 166.8 (C2), 164.3 (C7), 163.8 (C7''),

162.3 (C5''), 161.7 (C5), 161.4 (C9''), 155.4 (C9), 149.4 (C3'), 145.8 (C4'), 145.7 (C3'''),

144.3 (C5'''), 122.9 (C1'), 122.8 (C6'''), 120.4 (C1'''), 119.9 (C6'), 116.3 (C5'), 115.37

(C4'''), 114.2 (C2'''), 114.1 (C2'), 104.8 (C10), 104.6 (C10''), 103.6 (C3), 105.3 (C8''),

95.83 (C8), 95.81 (C6''), 80.0 (C2''), 43.1 (C3''); LRESMS, m/z 573 (M+H)+.

+ HRESMS: calculated for C30H21O12 (M+H) :573.1033 found 573.1044.

2,3-Dihydroxyamentoflavone 123

An UV active pale yellow solid (0.50 mg /g dried plant

1 sample); H-NMR (DMSO-d6, 600 MHz), 12.78 (s, OH,

H5), 12.60 (s, OH, H5''), 8.60 (s, OH, H4'''), 7.42

(H2'''/H6'''), 6.92 (H3'''/H5'''), 6.63 (s, H3), 6.40 (s, H8),

13 6.27 (s, H6), 6.15 (s, H8''), 5.51 (H2''), 3.24 (H3a), 2.79 (H3b). C-NMR (DMSO-d6,

150 MHz), 166.6 (C5), 165.1 (C2), 163.1 (C5''), 158.9 (C4'''), 130.8 (C1'''), 129.4

(C2'''/6'''), 116.2 (C3'''/5'''), 110.8 (C3), 105.8 (C4a), 105.5 (C6''), 103.3 (C4a''), 100.0

(C6), 95.9 (C8''), 94.9 (C8), 80.3 (C2''). LRESMS, m/z 539 [M-H]-. The precise structure of this compound was unable to be determined, however it is likely to be dihydrobiflavone.

162

6.10. Bioactivity testing

6.10.1. Antimicrobial

Antimicrobial activities were conducted by the Community for Open Antimicrobial

Drug Discovery, Queensland, Australia. All the methanol extracts from both species and isolated compounds were evaluated for their antimicrobial activities against seven microorganisms (Staphylococcus aureus ATCC 43300 (MRSA), Eschericia coli ATCC

25922 (FDA control strain), Klebsiella penumoniae ATCC 700603 (MDA),

Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606,

Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 208821). All the samples were dissolved in DMSO with concentration of 10 mg/mL. Each sample (5 µL) was plated in duplicate into a 384-well non-binding surface plate for each strain. All bacteria were cultured in Cation-adjusted Mueller Hinton broth at 37 oC overnight, then

45 µL was added to each well of the compound containing plates, giving a cell density of 5x105 CFU/mL and give a final compound concentration of 32 µg/mL. All the plates were covered and incubated at 37 oC for 18 h. For the fungi strains, all the fungi were cultured for 3 days on Yeast Extract-Peptone Dextrose agar at 30 oC and these stock suspensions were diluted with Yeast Nitrogen Base broth to a final concentration of

2.5x105 CFU/mL. Then, 45 µL was added to each well of the compound containing plates, giving a final compound concentration of 32 µg/mL and incubated at 35 oC for

24 h. Inhibition of bacterial growth was determined measuring absorbance at 600 nm using a Tecan M1000 Pro mocochromator plate reader while growth inhibition of the fungi was determined measuring absorbance at 530 nm after the addition of resazurin

(0.001% final concentration) and incubation at 35 oC for additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and

163 positive control (microorganisms without inhibitors) on the same plate. The significance of the value was determined by Z-scores, calculated using the average and standard deviation of the sample wells (no controls). Samples with inhibition value above 80% and Z-Score above 2.5 for either replicate were classed as actives.

6.10.2 Free radical scavenging assays

The ability of the isolated compounds to scavenge free radicals was determined using

DPPH microplate assay adapted from a range of published methods reviewed in Brand-

Williams et al.288 Sample solutions (50 µL) were loaded into a 96-well plate (in replicates of four) and serially diluted. A separate set of wells were loaded with 50 µL of either MeOH or the model flavonoid, rutin (820 µM) for the negative and positive controls, respectively. To the first three rows of sample solution, DPPH (200 µM, 100

µL) was added and thoroughly mixed, allowing the last row to be used to correct for background absorbance (Absblank). Plates were incubated for 30 min in the absence of light before the absorbance at 517 nm was measured using a microplate reader. Lower absorbances signified higher scavenging activities. Free radical-scavenging activities were determined using Equation 1. These percentage activities were graphed against logarithmic sample concentrations and the sample concentrations that sequestered 50% of the DPPH free radicals (i.e. loss of purple colour) were interpolated and are presented as IC50 mean ± s.d. values. All antioxidant activity data were tested for significant differences between each pair using Student’s t-tests (α = 0.05).

(Abs −Abs )x100 AA% = 100 − sample blank ……………………..equation 1 Absnegative control

164

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Appendix A: New Anthocyanins reported in 1998 – 2003. 43-44

Anthocyanins Plants source [6'''-(Dp 3-(6''-p-coumarylglucoside)-7-glucoside] Agapanthus [6'''-Km 3-glucoside-7-xyloside-4'-glucoside] succinate praecox (Liliaceae) [6'-(Cy 3-glucoside)] [6'''-Km 3-sophoroside-7-glucuronide] Allium malonate schoenoprasum [6'-(Cy 3-(3-acetylglucoside)] [6'''-Km 3-sophoroside-7- (Liliaceae) glucuronide] malonate Cy 3-(2-glucosylgalactoside) Cornus suecica (Cornaceae) Dp 3-glucoside-5-(6-malonylglucoside) Crocus antalyensis (Iridaceae) Pt 3,7-diglucoside

Pt 3-(6-malonylglucoside-7-(6-malonylglucoside), Mv 3-(6- Crocus malonylglucoside)-7-(6-malonylglucoside) chrysanthus (Iridaceae) Pn 3-neohesperidoside Cyclamen persicum (Primulaceae) Pg 3,5-diglucoside methyl diester Dianthus caryophyllus (Caryophyllaceae) Dp 3-neohesperidoside-7-(6-malonylglucoside) Felicia amelloides (Compositae) Cy 3-[6-(6-ferulylglucosyl)-2-xylosylglucoside] Glennia littoralis (Umbelliferae) Pt 3-(6-p-coumarylglucoside)-5-(6-malonylglucoside) Hyacinthus Pg 3-(6-p-coumarylglucoside)-5-(6-acetylglucoside) orientalis Pg 3-(6-ferulylglucoside)-5-(6-malonylglucoside) (Liliaceae) Pg 3-glucoside-5-(6-malonylglucoside) Pg 3-(6-p-coumarylglucoside)-5-(4-malonylglucoside) Cy 3-[2-(6-caffeylglucosyl)-6-(caffeylglucoside)]-5-glucoside, Ipomoea asarifolia Cy 3-[2-(6-p-coumarylglucosyl)-6-(caffeylglucoside)]-5- (Convolvulaceae) glucoside Cy 3-[(6-caffeyl)-2-(6-caffeylglucoside)]-5-glucoside Ipomoea batatas Pn 3-[(6-caffeyl)-2-(6-caffeylglucoside)]-5-glucoside (Convolvulaceae) Cy 3-[4-(4-glucosyl)-6-caffeylglucosyl-6-caffeylsophoroside] Ipomoea purpurea Cy 3-rutinoside-7-glucoside Lilium cv. Holean

182

(Liliaceae) Dp 3'-(2-galloyl-6-acetylgalactoside) Nymphaea Dp 3'-(2-galloylgalactoside) caerulea (Nympheaceae) Cy 3-(2-galloyl-6-acetylgalactoside) Nymphaea x marliaca (Nympheaceae) Mv 3-(4'''-p-coumarylrutinoside)-5-glucoside Petunia hybrida (Solanaceae) Pt 3-[4''-(4-glucosyl-p-coumaryl)-rutinoside] Petunia occidentalis (Solanaceae) Dp 3-(6-p-coumarylglucoside)-5-(4-acetyl-6- Salvia uliginosa malonylglucoside) (Labiatae) Cy 3-(6-malonylglucoside)-3''-glucoside-7-(6- Sophronitis caffeylglucoside) coccinea Cy 3-(6-malonylglucoside-3'-glucoside-7-(6-ferulylglucoside) (Orchidaceae) Dp 3-(4-glucosyl-p-coumarylglucoside)-5-(6- Triteleia bridgesii malonylglucoside) (Liliaceae) Pg 3-(2'''-acetylrutinoside) Tulipa cv. Queen Cy 3-(2''-acetylrutinoside) Wilhelmina Dp 3-(2'''-acetylrutinoside) (Liliaceae) Dp 3-(3''-acetylrutinoside) Tulipa gesneriana (Liliaceae) Pn 3-sambubioside Vaccinium Pt 3-sambubioside pedifolium Mv 3-sambubioside (Ericaceae) 6,7,3'-Trihydroxy-5,4_-dimethoxyflavylium Arrabidaea chica 6,7,3',4'-Tetrahydroxy-5-methoxyflavylium leaves (Bignoniaceae) 5-CarboxypyranoCy 3-glucoside Allium cepa scales 5-CarboxypyranoCy 3-(6-malonylglucoside) (Alliaceae) Pyranocyanins C and D Ribes nigra seeds Pyranodelphinins C and D (Saxifragaceae) Luteolinidin 5-(3-glucosyl-2-acetylglucoside) Blechnum novae- zelandiae fronds (Pteridophyta) 8-C-GlucosylCy 3-(6-malonylglucoside) Tricyrtis formosana flowers (Liliaceae) 6-OH Pg 3-glucoside Alstroemeria 6-OH Pg 3-(6-rhamnosylglucoside) flowers 6-OH Cy 3-(6-malonylglucoside) (Alstroemeriaceae)

183

6-OH Dp 3-glucoside 6-OH Dp 3-(6-malonylglucoside) Pg 3-[2-(6-feruloylglucosyl)glucoside]-5-glucoside Raphanus sativus Pg 3-[2-(6-feruloylglucosyl)-6-caffeoylglucoside]-5-glucoside roots (Cruciferae) Pg3-[2-(6-caffeoylglucosyl)-6-p-coumaroylglucoside]-5- glucoside Pg 3-[2-(6-caffeoylglucosyl)-6-feruloylglucoside]-5-glucoside Pg3-[2-(6-feruloylglucosyl)-6-p-coumaroylglucoside]-5- glucoside Pg 3-[2-(2-feruloylglucosyl)-6-feruloylglucoside]-5-glucoside Pg 3-(2-xylosyl-6-malonylgalactoside) Anemone Pg 3-(2-xylosyl-6-methylmalonylgalactoside) coronaria flowers 12{[4-(glucosyl)caffeoyl]-tartarylmalonate (Ranunculaceae) Cy-3-[2-(2-caffeoylglucosyl)-6-malonylgalactoside]-7-(6- caffeoylglucoside)-3'- glucuronide Dp-3-[2-(2-caffeoylglucosyl)-6-malonylgalactoside]-7-(6- caffeoylglucoside)-3'- glucuronide Dp 3-[2-(2-caffeoylglucosyl)galactoside]-7-(6- caffeoylglucoside)-3'- glucuronide Cy 3-(2-galloyl-6-rhamnosylgalactoside) Acalypha hispida flowers (Euphorbiaceae) Cy-3-[2-(2-sinapoylxylosyl)-6-(4-glucosyl-p- Arabidopsis coumaroyl)glucoside]-5-(6-malonylglucoside) thaliana leaves and stems (Cruciferae) Cy 3-(3-glucosyl-6-malonylglucoside)-4'-glucoside Allium cepa scales (Alliaceae) Cy 7-(3-glucosyl-6-malonylglucoside)-4'-glucoside Cy-3-[2-(6-caffeoylglucosyl)-6-4-(6-3,5- Ipomoea asarifolia dihydroxycinnamoylglucosyl) caffeoylglucoside]-5-glucoside flowers Cy 3-[2-(6-p-coumaroylglucosyl)-6-4-(6-pcoumaroylglucosyl) (Convolvulaceae) Cy 3-(6-acetylgalactoside) Nymphaea alba leaves (Nymphaeaceae) Cy 3-(2-xylosyl-6-malonylglucoside)-7-glucoside Meconopsis grandis flowers (Papaveraceae) Dp 3,5-di-(6-malonylglucoside) Cichorium intybus flowers (Compositae) Dp 3-(6-malonylglucoside)-5-glucoside Dp 3-(6-p-coumaroylgalactoside) Camellia sinensis

184

flowers and leaves (Theaceae) Dp 3-(2-rhamnosyl-6-malonylglucoside) Clitoria ternatea

flowers

(Leguminosae) Dp 3-[2-(6-feruloylglucosyl)-6-p-coumaroylglucoside]-5-(6- Ajuga reptans malonylglucoside) flowers and flower Dp 3-[2-(6-feruloylglucosyl)-6-feruloylglucoside]-5-(6- cultures (Labiatae) malonylglucoside) Cy 3-[2-(6-p-coumaroylglucosyl)-6-p-coumaroylglucoside]-5- glucoside

Dp 3,7-di-glucoside-3ˡ,5ˡ-di-(6-p-coumaroylglucoside) Dianella nigra and Dp 3-glucoside-7,3ˡ,5ˡ-tri-(6-p-coumaroylglucoside) Dianella Dp 3,7,3ˡ,5ˡ-tetra-(6-p-coumaroylglucoside) tasmanica berries (Liliaceae)

Dp 3-(6-p-coumaroylglucoside)-5-(4-rhamnosyl-6- Muscari malonylglucoside) armeniacum flowers (Liliaceae) Mv 3-[6-(4-4-(6-feruloylglucosyl)-p-coumaroyl Petunia hybrida rhamnosyl)glucoside]-5- glucoside flowers (Solanaceae) Pn 3-[6-(4-caffeoylrhamnosyl)glucoside]-5-glucoside Solanum

Pt 3-[6-(4-caffeoylrhamnosyl)glucoside]-5-glucoside tuberosum tubers

and sprouts (Solanaceae) Pn 3-(6-malonylglucoside)-5-glucoside Allium cepa scales Pn 3-(6-malonylglucoside) (Alliaceae)

Pg: pelargonidin; Cy: cyanidin; Pn: peonidin; Dp: delphinidin; Pt; petudnidin; Mv: malvidin; Km: kaempferol. Sugar link for sugar, galactose, xylose, rhamnose and all in the pyranose form. 43-44

185

Appendix B: Spectroscopic and spectrometric data of Brachychiton acerifolius constituents

1 H-NMR spectrum of compound 50 (CD3OD)

gHSQC spectrum of compound 50 (CD3OD)

186 gHMBC spectrum of compound 50 (CD3OD)

gCOSY spectrum of compound 50 (CD3OD)

187

Appendix C: Spectroscopic and spectrometric data of Anigozanthos sp constituents

1 H-NMR spectrum of compound 64 (CD3OD)

gCOSY spectrum of compound 64 (CD3OD)

188 gHSQC spectrum of compound 64 (CD3OD)

gHMBC spectrum of compound 64 (CD3OD)

189

APT spectrum of compound 64 (in absolute mode, CD3OD)

HRESI mass spectrum of 64

190

1 H-NMR spectrum of compound 70 (CD3OD)

gHSQC spectrum of compound 70 (CD3OD)

191

gCOSY spectrum of compound 70 (CD3OD)

gHMBC spectrum of compound 70 (CD3OD)

192

APT spectrum of compound 70 (in absolute mode, CD3OD)

HRESI mass spectrum of 70

193

Appendix D: Spectroscopic and spectrometric data of Jacaranda mimosafolia constituents

1 H-NMR spectrum of 73 (CD3OD)

13 C-NMR spectrum of 73 (CD3OD)

194 gCOSY spectrum of 73 (CD3OD)

gHSQC spectrum of 73 (CD3OD)

195 gHMBC spectrum of 73 (CD3OD)

HRESI mass spectrum of 73

196

Appendix D: Spectroscopic and spectrometric data of C. purpureus constituents

1H-NMR spectrum of biflavone 120 (Acetone-d6)

13APT-NMR spectrum of biflavone 120 (in absolute mode, Acetone-d6)

197 gCOSY spectrum of biflavone 120 (600 MHz, Acetone-d6)

198 gHSQC spectrum of biflavone 120 (Acetone-d6)

199 gHMBC spectrum of biflavone 120 (Acetone-d6)

HRESIMS spectrum of biflavone 74

200

1H-NMR spectrum of 121 (Acetone-d6)

13C-NMR spectrum of 121 (Acetone-d6)

201 gCOSY spectrum of 121 (Acetone-d6)

gHSQC spectrum of 121 (Acetone-d6)

202 gHMBC spectrum of 121 (Acetone-d6)

HRESIMS spectrum of 121

203

Appendix E: Crystallography data of Acacia pycnantha constituents

Compound 81 (Naringenin-5-O-glucoside)

204

205

206

207

208

209

210