Ethnopharmacology and 

chemotaxonomy of essential oil 

yielding Australian plants 

Nicholas John Sadgrove 

Thesis Page 1

Ethnopharmacology and chemotaxonomy of novel essential oil 

yielding Australian plants 

Nicholas John Sadgrove 

 

‘As aromatic plants bestow No spicy fragrance while they grow; But crush’d or trodden to the ground, Diffuse their balmy sweets around’ Oliver Goldsmith ‘The captivity, an oratorio (Act 1)’ 1764. 

 

 

‘Who knows but that England may revive in New South Wales when it has sunk in Europe’ Sir Joseph Banks.  

 

 

‘On a feeling and sensitive mind a demolished forest impresses unmingled sadness, whereas its primeval grandeur must inspire anyone to immeasurable delight, who is susceptible to the beauties of nature’ Baron Ferdinand Von Müller. 

 

 

‘Let us regard the forests as an inheritance, given to us by nature, not to be despoiled or devastated, but to be wisely used, reverently honoured and carefully maintained. Let us regard the forests as a gift, entrusted to any of us only for transient care, to be surrendered to posterity as an unimpaired property, increased in riches and augmented in blessings, to pass as a sacred patrimony from generation to generation’ Baron Ferdinand Von Müller.

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Ethnopharmacology and chemotaxonomy of novel essential oil 

yielding Australian plants 

Nicholas Sadgrove 

Bachelor of Environmental Science with Honours (First Class); Southern Cross 

University, Lismore, New South Wales, Australia. 

A thesis submitted for the award of Doctor of Philosophy of the University of New 

England.  

February 2014 

Supervisor: Graham Lloyd Jones 

Co‐supervisors; Ben Greatrex and Kenneth Watson. 

I certify that the substance of this thesis has not already been submitted for any degree and 

is not currently being submitted for any other degree or qualification.  

I certify that any help received in preparing this thesis and all sources used have been 

acknowledged in this thesis.  

_________ _______________    Date _____________ 3/2/14

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Condensed Table of Contents Acknowledgements. …………………………………………………………………….…………………………………………………..……………..5.

Abstract. ………………………………………………………………………………………………………………………………………………………..14.

Chapter 1 – Introduction: essential oils and ethnopharmacology in Australia. ……………………………………………….16.

Journal Articles Part 1: Eremophila longifolia: ethnopharmacology, essential oil chemotypes and

cytogeography. ...................................................................................................................................................55.

Chapter 2 - Characterization and bioactivity of essential oils from novel chemotypes of Eremophila longifolia (F.

Muell) (Myoporaceae): a highly valued traditional Australian medicine. ……………………………………………………….56.

Chapter 3 - A possible role of partially pyrolysed essential oils in Australian Aboriginal traditional ceremonial

and medicinal smoking applications of Eremophila longifolia (R. Br.) F. Muell (Scrophulariaceae). ……………….69.

Chapter 4 – Isolation and characterisation of (-)-genifuranal; the principal antimicrobial component in

traditional smoking applications of Eremophila longifolia (Scrophulariaceae) by Australian Aboriginal peoples.

……………………………………………………………………………………………………………………………………………………………………….80.

Chapter 5 – Cytogeography of essential oil chemotypes of Eremophila longifolia (Schrophulariaceae). ……….93.

Journal Articles Part 2: Ethnopharmacology of medicinal plants used traditionally by Aboriginal Australians.

………………………………………………………………………………………………………………………………………………………………….…105.

Chapter 6 - Chemical and biological characterization of novel essential oils from Eremophila bignoniiflora (F.

Muell) (Myoporaceae): a traditional Aboriginal Australian bush medicine. …………………………………………………106.

Chapter 7 - Chemical and biological characterisation of solvent extracts and essential oils from leaves and fruit

of two Australian species of Pittosporum (Pittosporaceae) used in aboriginal medicinal practice. ………………118.

Chapter 8 - Medicinal compounds, chemically and biologically characterised from smoke, solvent and distilled

extracts from Australian Callitris endlicheri and C. glaucophylla (Cupressaceae): used traditionally in Aboriginal

and colonial pharmacopoeia. …………………………………………………………………………………………….…………………………131.

Chapter 9 - Characterisation and bioactivity of essential oils from Geijera parviflora (Rutaceae): a native bush

medicine from Australia. ……………………………………………………………………………………………………………………………..146.

Chapter 10 - Chemogeography and antimicrobial activity of essential oils from Geijera parviflora and Geijera

salicifolia (Rutaceae): two traditional Australian medicinal plants. ………………………………………………………………159.

Journal Articles Part 3: Phytochemical and chemotaxonomic investigations. ……………………………………………175.

Chapter 11 - Antimicrobial activity of essential oils and solvent extracts from Zieria species (Rutaceae). ……176.

Chapter 12 - Dihydrotagetone, an unusual fruity ketone, is found in enantiopure and enantioenriched forms in

additional Australian native taxa of Phebalium (Rutaceae: Boronieae). ……………………………………………….………189.

Chapter 13 - Composition and antimicrobial activity of the essential oils from the Phebalium squamulosum species complex (Rutaceae) in New South Wales, Australia. ………………………………………………………..………………200.

Chapter 14 - Cineole-rich essential oils from Australian Prostanthera Labill. species (Lamiaceae):

chemotaxonomy and antimicrobial activity. ……………………………………………………………………………….…….…………212.

Part 4: Thesis conclusions and appendices. ……………………………………………………………….……………………….………235.

Chapter 15 – Conclusion. …………………………………………………………………………………………….………………………………236.

Appendix A – Consolidated list of references. ……………………………………………………………………………….……….……245.

Appendix B – Supplementary files chapter 5. ………………………………………………………….……………………………………263.

Appendix C – Supplementary files chapter 10. …………………………………………………………….………………………………270.

Appendix D – Supplementary files chapter 13. …………………………………………………………………………….………………276.

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Acknowledgements

After more than 500 hours of distillation, and travelling distances in excess of 10,000km

searching for plants across the Australian landmass, I have an almost unlimited list of

acknowledgements to make. However, at the risk of forgetting those who are going to come

into contact with my thesis, I would like to commence acknowledgement within my sphere

of supervisors. First and foremost, to my principal supervisor Associate Professor Graham

Lloyd Jones, who may possibly have demonstrated the greatest exhibition of resilience and

perseverance known to academia, in the careful revision and preparation of this thesis. To

my co-supervisors Emeritus Professor Kenneth Watson and Dr Ben William Greatrex, for

their ready response and selfless contribution to this thesis.

To the CSIRO at Chiswick (Uralla, NSW) and their staff, particularly Dr Nicholas Andronicos,

for help with the operation of the flow cytometer and ploidy determination. To Andrew

Wallace at the UNE chemistry department, for his technical expertise and assistance,

generosity with materials and great sense of humour and patience.

For their expertise in plant identification and their generosity in making available the

excellent resource of the N.C.W Beadle Herbarium at the University of New England, I would

like to thank particularly Mr Ian Telford and Professor Jeremy Bruhl. They have always been

prepared to share their great experience and expertise in plant taxonomy, with a novice

such as myself.

I would like to thank the two great phytochemists Ian Southwell and Erich Lassak for their

interest and advice with regard to exploring the various essential oil yielding genera in and

around New South Wales. In this context I would also like to thank Dr David Tucker, for his

useful chemical advice with regard to nuclear magnetic resonance spectroscopy. For his

patience over the email, I thank Robert Chinnock, Australia’s foremost expert on

Eremophila.

I wish to thank the staff and fellow students in the School of Science and Technology, for

their friendship, help and advice. They have put up with many strange smells (not all of

them unpleasant I hope) emanating from our laboratory.

Finally, I wish to thank my family and friends, for what has been a lot of support in this long

and sometimes arduous journey.

Individual further acknowledgements have been given at the end of each experimental

chapter.

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List of publications originating from this thesis.

Sadgrove, N., Gonçalves-Martins, M., & Jones, G. L. (2014). Chemogeography and antimicrobial activity of essential oils from Geijera parviflora and Geijera salicifolia (Rutaceae): Two traditional Australian medicinal plants. Phytochemistry 104: pp. 60-71

Sadgrove, N., Hitchock, M., Watson, K., & Jones, G. L. (2013). Chemical and biological characterization of novel essential oils from Eremophila bignoniiflora (F. Muell) (Myoporaceae): a traditional Aboriginal Australian bush medicine. Phytotherapy Research 27: pp. 1508-1516

Sadgrove, N., & Jones, G. L. (2013). Antimicrobial activity of essential oils and solvent extracts from Zieria species (Rutaceae). Natural Product Communications 8(6): pp. 741-745

Sadgrove, N., & Jones, G. L. (2013). Characterisation and bioactivity of essential oils from Geijera parviflora (Rutaceae): a native bush medicine from Australia. Natural Product Communications 8(6): pp. 747-751

Sadgrove, N., & Jones, G. L. (2013). Chemical and biological characterisation of solvent extracts and essential oils from leaves and fruit of two Australian species of Pittosporum (Pittosporaceae) used in aboriginal medicinal practice. Journal of Ethnopharmacology 145: pp. 813-821

Sadgrove, N., & Jones, G. L. (2013). A possible role of partially pyrolysed essential oils in Australian Aboriginal traditional ceremonial and medicinal smoking applications of Eremophila longifolia (R. Br.) F. Muell (Scrophulariaceae). Journal of Ethnopharmacology 147: pp. 638-644

Sadgrove, N., & Jones, G. L. (2014). Cytogeography of essential oil chemotypes of Eremophila longifolia F. Muell (Schrophulariaceae) Phytochemistry (in press) doi: 10.1016/j.phytochem.2014.05.005:

Sadgrove, N., & Jones, G. L. (2014). Medicinal compounds, chemically and biologically characterised from extracts of Australian Callitris endlicheri and C. glaucophylla (Cupressaceae): used traditionally in Aboriginal and colonial pharmacopoeia. Journal of Ethnopharmacology 153(3): pp. 872-883

Sadgrove, N., Jones, G. L., & Greatrex, B. W. (2014). Isolation and characterisation of (-)-genifuranal: The principal antimicrobial component in traditional smoking applications of Eremophila longifolia (Scrophulariaceae) by Australian Aboriginal peoples. Journal of Ethnopharmacology 154(3): pp. 758-766

Sadgrove, N., Mijajlovic, S., Tucker, D. J., Watson, K., & Jones, G. L. (2011). Characterization and bioactivity of essential oils from novel chemotypes of Eremophila longifolia (F. Muell) (Myoporaceae): a highly valued traditional Australian medicine. Flavour and Fragrance Journal 26(5): pp. 341-350

Sadgrove, N., Telford, I. R. H., Greatrex, B. W., Dowell, A., & Jones, G. L. (2013). Dihydrotagetone, an unusual fruity ketone, is found in enantiopure and enantioenriched forms in additional Australian native taxa of Phebalium (Rutaceae: Boronieae). Natural Product Communications 8(6): pp. 737-740

Sadgrove, N. J., Telford, I. R. H., Greatrex, B. W., & Jones, G. L. (2014). Composition and antimicrobial activity of essential oils from the Phebalium squamulosum species complex (Rutaceae) in New South Wales, Australia. Phytochemistry 97: pp. 38-45

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List of Tables Chapter 1: Table 1 - Essential oil pharmacological types (Schnaubelt, 1999). ……………………………………………………………………………………………………………………………………24 Chapter 2: Table 1 - Quantification of essential oil constituents (% abundance). ……………………………………………………………………………………………………………………………………60 Table 2 - Additional constituents identified but not quantified. ……………………………………………………………………………………………………………………………………61 Table 3 - Results of disc diffusions from oils previously characterized by Smith (2010) showing zones of inhibition measured in mm radius from the outside of the discs to the margin of the clearing zone. ……………………………………………………………………………………………………………………………………62 Table 4 - Mean inhibitory concentrations (MIC) as a percentage of oil v/v in broth ……………………………………………………………………………………………………………………………………62 Table 5 - MIC of the higher oil yielding chemotypes, dominated by ketones such as karahanaenone, isomenthone and menthone. ……………………………………………………………………………………………………………………………………62 Table 6 - Fungal growth inhibition of EOs determined using an agar transplant technique as % in final growth agar. ……………………………………………………………………………………………………………………………………63 Table 7 - Fungal growth inhibition of EOs determined using an agar transplant technique as % in final growth agar. ……………………………………………………………………………………………………………………………………63 Table 8 - Free radical scavenging capacity of essential oil extracted from Eremophila longifolia and reference standards, measured using a DPPH assay in methanol. ……………………………………………………………………………………………………………………………………64 Table 9 - The ferric reducing ability of essential oils. ……………………………………………………………………………………………………………………………………64 Chapter 3: Table 1 - Characterization of essential oil components of non-pyrolysed and pyrodistilled (-πρ) oils in % abundance w/w. ……………………………………………………………………………………………………………………………………74 Table 2 - The average MIC results from broth dilution presented in % oil in broth v/v. Pyrodistilled oils are denoted using the Greek letters for pyro (πρ). Species used here were Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 29213), Klebsiella aurogenes (University of New England Strain) and Candida albicans (ATCC 10231). ……………………………………………………………………………………………………………………………………76 Table 3 - Average MIC results of the thermocycle experiment, showing activity against Staphylococcus aureus (ATCC 29213) presented in % oil in broth v/v. Results in the top row (Incubated) are for normal incubation (as in Table 2), in the second row (@60 1C for 30 s) for an initial thermocycle in increments of 2 1C from 38 1C to 60 1C, holding for 5 s at each increment, and pausing at 60 1C for 30 s before incrementally returning to 38 1C. The other cycle (@60 1C for 2 m) pauses at 60 1C for 2 min. ……………………………………………………………………………………………………………………………………76 Table 4 - Results of a DPPH free radical scavenging assay in MeOH. Results convey the quantity of DPPH quenched in μg relative to 1 mg essential oil or positive control. Positive controls in this experiment were ascorbic acid (Vit. C) and trolox, which quenched DPPH at a stoichiometric ratio (Control+ mg/DPPH mg) of 0.457 and 0.223, respectively. ……………………………………………………………………………………………………………………………………76 Chapter 4: Table 1 - Antibacterial and anti-yeast activity of extracts produced from NJSadgrove6. Extract MIC in mg/ml but control (Tetra or Nyst) in μg/ml. ……………………………………………………………………………………………………………………………………86 Table 2 - Antifungal sporicidal activity of extracts produced from NJSadgrove6 against pathogenic Trichophyton species. Extract MIC in mg/ml but nystatin in μg/ml. ……………………………………………………………………………………………………………………………………86 Table 3 ‐ 1H NMR resonances of 1and 2 and COSY interactions in C6D6. ……………………………………………………………………………………………………………………………………88 Table 4 - Occurrence of compound 1 in essential oil chemotypes of E. longifolia. ……………………………………………………………………………………………………………………………………89 Chapter 5: Table 1 – Essential oil composition of clusters with both diploid and tetraploid populations of E. longifolia. Published arithmetic indices (Pub. AI) are from Adams (2007), shown alongside arithmetic indices (AI) calculated in the present study. Vouchers are listed in shorthand form relative to those lodged at the N.C.W Beadle Herbarium at the University of New England, Armidale NSW Australia 2351. Thus, NS25 is lodged in the herbarium as NJSadgrove25. …………………………………………………………………………………………………………………………...…………96 Table 2 - Essential oil composition of PCA clusters characterised only by tetraploid populations of E. longifolia. Published arithmetic indices (Pub. AI) are from Adams (2007), shown alongside arithmetic indices (AI) calculated in the present study. Vouchers are listed in shorthand form relative to those lodged at the N.C.W Beadle Herbarium at the University of New England, Armidale NSW Australia 2351. Thus, NS25 is lodged in the herbarium as NJSadgrove25. ……………………………………………………………………………………………………………………………………...97 Table 3 - Ploidy and chemotypes of E. longifolia relative to the clusters identified in PCA analysis. ……………………………………………………………………………………………………………………………...………98

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Chapter 6: Table 1 - Disc diffusion using pure essential oils from E. bignoniiflora against a range of bacteria strains. Voucher references are listed in shorthand. Control + constitutes a 10 mg tetracycline disc in the case of bacterial species and a corresponding nystatin disc for the yeast C. albicans. Species used here were Echerichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27703), Staphylococcus epidermidis (ATCC 12228), S. aureus (ATCC 29213), Bacillus subtilis (University of New England Stock), Klebsiella aerogenes (UNE), Salmonella typhimirium (ACM 3598) and Candida albicans (ATCC 10231) ……………………………………………………………………………………………………………………………………110 Table 2 - Broth dilution using E. bignoniiflora essential oils listed under shorthand voucher reference (NJSadgrove41 changed to NS41 et cetera). Control + is tetracycline in mg/ml for the bacterial species and corresponding nystatin for the yeast C. albicans. Species used here were Staphylococcus aureus (ATCC 29213), S. epidermidis (ATCC 12228), Bacillus subtilis (University of New England strain), Klebsiella aurogenes (University of New England strain), Salmonella typhimirium (ACM 3598), Pseudomonas aeruginosa (ATCC 27703) and Candida albicans (ATCC 10231) ……………………………………………………………………………………………………………………………………110 Table 3 - DPPH scavenging ability of E. bignoniiflora essential oils in mg DPPH per ml of essential oil or mg control. Voucher references are listed in shorthand (NJSadgrove41 changed to NS41 et cetera). Positive controls in this experiment were ascorbic acid and trolox, which quenched DPPH at a stoichiometric ratio (Control mg/DPPH mg) of 0.457 and 0.223, respectively ……………………………………………………………………………………………………………………………………111 Table 4 - Characterization and quantification of essential oils from E. bignoniiflora. Voucher references are listed in shorthand (NJSadgrove41 changed to NS41 et cetera). Yield is also presented above quantification data. Temperature programmed retention indices (RI) were calculated and presented alongside published values 1(Adams, 2007) 2(Asuming et al., 2005) 3(Song et al., 2000) 4(Pino et al., 2005) 5(Buchin et al., 2002) 6(Mimica-Dukić et al., 2003) 7(Su et al., 2006) 8(Belsito et al., 2007) 9(Oliveira et al., 2006) 10(Adams, 2000) 11(Flamini et al., 2007) 12(Adams et al., 2006) 13(Kallio et al., 2006) 14(Angioni et al., 2006) 15(Lucero et al., 2003) 16(Adams et al., 2005) 17(Bestmann et al., 1988) ……………………………………………………………………………………………………………………………………112 Chapter 7: Table 1 - Yield (mg) of extract from NS166 using 20 g fruit and 10 g leaf into 40 ml solvent. ……………………………………………………………………………………………………………………………………123 Table 2 - Antimicrobial activity (MIC) of extracts (mg/ml) from NS166. ……………………………………………………………………………………………………………………………………123 Table 3 - Mean inhibitory concentration (MIC) of H2O extracts (% v/v) of NS166 before drying. ……………………………………………………………………………………………………………………………………123 Table 4 - MIC of essential oils from Pittosporum undulatum and Pittosporum angustifolium (% v/v) for three microbial species. ……………………………………………………………………………………………………………………………………123 Table 5 - Chemical composition of the essential oils from leaves and fruit of Pittosporum angustifolium. ……………………………………………………………………………………………………………………………………124 Table 6 - Chemical composition of essential oils from leaf and fruit of Pittosporum undulatum. ……………………………………………………………………………………………………………………………………125 Table 7 - Chemical screening of the extracts from NS166 (Pittosporum angustifolium) using the methods described by Vesoul and Cock (2011). ……………………………………………………………………………………………………………………………………125 Chapter 8: Table 1 - Essential Oils characterised using GC-MS with calculated arithmetic indices (AI) shown alongside published values (Pub. AI) (Adams, 2007). ……………………………………………………………………………………………………………………………………136 Table 2 – GC-MS analysis of the volatile fraction collected in solvent extracts produced using methanol (MeOH). Arithmetic indices (AI) were calculated and are compared here with published values (Pub. AI). The Arithmetic indices references (AI Ref) are as follows; a) (Adams, 2007), b) (Harrison & Priest, 2009), c) (Adams, 1999), d) (Kosyukova & Khorguani, 1989), e) (da Silva et al., 1999), f) (Asuming et al., 2005), g) (Kukic et al., 2006). ……………………………………………………………………………………………………………………………………137 Table 3 - Smoke extracts analysed using GC-MS. Arithmetic indices (AI) were calculated and compared with published values (Pub. AI). The source of the published AI (AI Ref) is as follows; a) (Adams, 2007), b) (Wanakhachornkrai & Lertsiri, 2003), c) (Pino et al., 2001), d) (Mateo et al., 1997), e) (Re-Poppi & Santiago, 2002), f) (Chassagne et al., 1999), g) (Song et al., 2003), h) (Harrison & Priest, 2009), i) (Xu et al., 2003), j) (da Silva et al., 1999), k) (Guyot et al., 1998), l) (Radulovic et al., 2009) ……………………………………………………………………………………………………………………………………138 Table 4- Mass spectral data of unknown compounds in Tables 2 and 3. ……………………………………………………………………………………………………………………………………139 Table 5 – Mean inhibitory concentration (MIC) for sporicidal antifungal activity in mg/ml. Nystatin was in μg/ml. Organisms were from the Trichophyon genus, T. rubrum granular strain and non-granular strain, T. interdigitalis strain 1 and 2, and T. mentagrophytes isolated from a human and kangaroo. ……………………………………………………………………………………………………………………………………139 Table 6 – Mean inhibitory concentrations (MIC) from antimicrobial activity of extracts in mg/ml. The control was either tetracycline for bacterial species of nystatin for yeast (C. albicans) in μg/ml. Bacterial species are Pseudomonas aeruginosa, Klebsiella aerogenes, Escherichea coli, Bacillus subtilis, Salmonella typhimirium, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae and Candida albicans. ……………………………………………………………………………………………………………………………………140 Chapter 9: Table 1 – Geijera parviflora collections listed with corresponding voucher reference and coordinates of collection within NSW. ……………………………………………………………………………………………………………………………………149 Table 2 – Broth dilution (MIC) using G. parviflora essential oils listed under shorthand voucher reference (NJSadgrove14 changed to NS14). Results are presented as percentage essential oil concentration, v/v. For two specimens results from fresh oil is compared with results after the same oil had aged in specified years. ……………………………………………………………………………………………………………………………………150

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Table 3 – Disc diffusion using essential oils from G. parviflora. Results are presented as mm zones of inhibition measured from the perimeter of the disc. Voucher references are listed in shorthand. A comparison is also made between aged and fresh oil for NS48. ……………………………………………………………………………………………………………………………………150 Table 4 – Results of characterization and quantification of essential oils from G. parviflora. ……………………………………………………………………………………………………………………………………151 Table 5 – Comparison of essential oils from two specimens of G. parviflora. Data show differences between oils when fresh and aged. Aged oils were characterized before and after aging in the number of years specified below. ……………………………………………………………………………………………………………………………………151 Chapter 10: Table 1 – Chemical character of essential oils hydrodistilled from Geijera salicifolia. Arithmetic indices (AI) are listed alongside published values (Pub. AI) from Adams (2007). Numerical clusters are included with respect to those determined in principal component analysis. ……………………………………………………………………………………………………………………………………163 Table 2 – Chemical character of essential oils from Geijera parviflora. Arithmetic indices (AI) are listed alongside published values (Pub. AI) from Adams (2007). Numerical clusters are included with respect to those determined in principal component analysis. ……………………………………………………………………………………………………………………………………164 Table 3 – Chemical character of essential oils from G. parviflora specimens collected from within close geographical proximity south from Gundawindi, northern New South Wales. A dichloromethane partition of the hydrosol of NS374 is also included, which demonstrates the occurrence of bioactive coumarins. ……………………………………………………………………………………………………………………………………165 Table 4 - Vouchers corresponding to numerical clusters depicted in Figure 1 (PCA) ……………………………………………………………………………………………………………………………………166 Table 5 – Mean inhibitory concentrations of essential oils against pathogenic Trichophyton spp. rubrum (granular and non-granular strains); mentagrophytes (strain collected from a kangaroo and a human); interdigitalis (strain 1 and 2). MIC units are % v/v of essential oil in agar and units for nystatin are ug/ml of agar. Where the symbol ‘>’ is used the concentration required to achieve inhibition was higher than used in this assay. ……………………………………………………………………………………………………………………………………166 Table 6 – Mean inhibitory concentrations of essential oils against bacterial species; Staphylococcus aureus, S. epidermidis, Salmonella typhimurium, Escherechia coli, Bacillus subtilis, Klebsiella aerogenes, Pseudomonas aeruginosa and the yeast Candida albicans. Essential oil concentrations are presented as % v/v and the controls are μg/ml. Tetracycline was used for bacterial species and nystatin for the yeast C. albicans. Where the symbol ‘>’ is used the concentration required to achieve inhibition was higher than used in this assay. ……………………………………………………………………………………………………………………………………167 Table 7 - Free radical scavenging activity of essential oils against the radical DPPH dissolved in Methanol. The postive controls used were Ascorbic acid and Trolox. ……………………………………………………………………………………………………………………………………167 Table 8 – Geijera species, coordinates and locations of collections made in the current study. ……………………………………………………………………………………………………………………………………167 Chapter 11: Table 1 – Chemical types produced by comprehensive essential oil analysis of the Zieria genus. ……………………………………………………………………………………………………………………………………180 Table 2 – Name and location of vouchers reference numbers deposited at the UNE Beadle Herbarium. ……………………………………………………………………………………………………………………………………180 Table 3 – MIC results from broth dilutions against bacterial strains (and yeast) using essential oils, %, v/v (Tetracycline and Nystatin were the positive controls with units of μg/mL). Microbial species described in full in experimental section. ……………………………………………………………………………………………………………………………………180 Table 4 – MIC results from broth dilution against pathogenic Trichophyton species, using essential oils in %, v/v (Nystatin was the positive control with units in μg/mL). ……………………………………………………………………………………………………………………………………180 Table 5 – MIC results using solvent extracts with units mg/mL, against bacterial strains (and yeast) (Tetracycline and nystatin were the positive control with units μg/mL). ……………………………………………………………………………………………………………………………………180 Table 6 – MIC results using solvent extracts with units mg/mL, against pathogenic Trichophyton species (Nystatin as the positive control with units μg/mL). ……………………………………………………………………………………………………………………………………180 Table 7 – Chemical composition of essential oils from Zieria species. Arithmetic indices are compared with published values (Pub. AI) from Adams (2007). ……………………………………………………………………………………………………………………………………181 Chapter 12: Table 1 – Voucher identifiers for essential oil of populations currently assigned to Phebalium glandulosum subspecies and P. squamulosum subsp. verrucosum. ……………………………………………………………………………………………………………………………………193 Table 2 – Characterization and quantification of essential oils from Phebalium species (published arithmetic indices are from Adams (2007). ……………………………………………………………………………………………………………………………………193 Table 3 – Results of polarimetry, showing two enantiotypes in P. squamulosum subsp. verrucosum (polarimetry was performed on whole essential oils as well as dihydrotagetone, fractionated from the same essential oil). ……………………………………………………………………………………………………………………………………193 Table 4 – Characterization and quantification of essential oils distilled from P. glandulosum subsp. angustifolium with a yield of 0.4%, w/w, of wet weight leaves (published arithmetic indices (Pub. AI) were sourced from Adams (2007)). ……………………………………………………………………………………………………………………………………193

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Chapter 13: Table 1 – Vouchers for this study of collections of taxa currently assigned to Phebalium squamulosum. In the text essential oils are cited in shortened form using the collector code, for example, N.J.Sadgrove 186 becomes NJS186 in the text. ……………………………………………………………………………………………………………………………………203 Table 2 – Chemical composition of essential oils from Phebalium squamulosum subspecies. Quantification data in relative abundance (%) is presented here as a range. Original data is included as supplementary material. Published arithmetic indices (Pub. AI) are from Adams (2007) or Singh et al. (2003) for dihydrotagetone. Calculated arithmetic indices (AI) were produced by the authors. Data related to the essential oil from P. squamulosum subsp. verrucosum was published earlier by Sadgrove et al. (2013) with further details of the enantiomeric composition of dihydrotagetone. ……………………………………………………………………………………………………………………………………205 Table 3 – Mean inhibitory concentrations of essential oils from subspecies of the squamulosum complex. Additional microbial species examined were Salmonella typhimirium and Escherechia coli but no inhibition was observed up to 4% v/v essential oil concentration. The control was tetracycline in lg/ml or nystatin where C. albicans was used. Essential oils MICs are presented as percentages calculated in volume of oil relative to volume of media. ……………………………………………………………………………………………………………………………………208 Chapter 14: Table 1 – Vouchers, coordinates and locations of wild collections of Prostanthera species. ……………………………………………………………………………………………………………………………………228 Table 2 – The mean inhibitory concentrations from essential oils hydrodistilled from cultivated Prostanthera specimens, presented as % v/v of essential oil in agar. The control is presented as μg/ml, which was either tetracycline or for C. albicans nystatin was used. ……………………………………………………………………………………………………………………………………229 Table 3 – The mean inhibitory concentrations from essential oils hydrodistilled from field collected Prostanthera specimens, presented as % v/v of essential oil in agar. The control is presented as μg/ml, which was either tetracycline or for C. albicans nystatin was used. ……………………………………………………………………………………………………………………………………229 Table 4 – DPPH scavenging ability of Prostanthera essential oils in μg DPPH per one mg of essential oil or positive control. Positive controls in this experiment were ascorbic acid and Trolox. ……………………………………………………………………………………………………………………………………229 Table 5 - Chemical character of essential oils from cultivated species of Prostanthera. ……………………………………………………………………………………………………………………………………230 Table 6 - Chemical character P. ovalifolia field collections. ……………………………………………………………………………………………………………………………………231 Table 7 - Chemical character of essential oils from other field collections of Prostanthera. ……………………………………………………………………………………………………………………………………232 Chapter 15: Table 1 - Possible commercial scale applications from essential oil yielding flora in Australia ……………………………………………………………………………………………………………………………………242 Appendix B Table 4 – Chemical character of essential oils from Eremophila longifolia (Regional New South Wales part 1). ……………………………………………………………………………………………………………………………………264 Table 5 - Chemical character of essential oils from Eremophila longifolia (Regional New South Wales part 2). Published arithmetic indices (Pub. AI) are from Adams (2007), shown alongside arithmetic indices (AI) calculated in the present study. Vouchers are listed in shorthand form relative to those lodged at the N.C.W Beadle Herbarium at the University of New England, Armidale NSW Australia 2351. Thus, NS25 is lodged in the herbarium as NJSadgrove25. ……………………………………………………………………………………………………………………………………266 Table 6 - Chemical character of essential oils from Eremophila longifolia (Specimens from Mutawintji National Park near Broken Hill New South Wales). Published arithmetic indices (Pub. AI) are from Adams (2007), shown alongside arithmetic indices (AI) calculated in the present study. Vouchers are listed in shorthand form relative to those lodged at the N.C.W Beadle Herbarium at the University of New England, Armidale NSW Australia 2351. Thus, NS25 is lodged in the herbarium as NJSadgrove25. ……………………………………………………………………………………………………………………………………267 Table 7 - Chemical character of essential oils from Eremophila longifolia (Western Australian specimens). Published arithmetic indices (Pub. AI) are from Adams (2007), shown alongside arithmetic indices (AI) calculated in the present study. Vouchers are listed in shorthand form relative to those lodged at the N.C.W Beadle Herbarium at the University of New England, Armidale NSW Australia 2351. Thus, NS25 is lodged in the herbarium as NJSadgrove25. ……………………………………………………………………………………………………………………………………268 Table 8 - Chemical character of essential oils from Eremophila longifolia (Northern Territory specimens). Published arithmetic indices (Pub. AI) are from Adams (2007), shown alongside arithmetic indices (AI) calculated in the present study. Vouchers are listed in shorthand form relative to those lodged at the N.C.W Beadle Herbarium at the University of New England, Armidale NSW Australia 2351. Thus, NS25 is lodged in the herbarium as NJSadgrove25. ……………………………………………………………………………………………………………………………………269 Appendix C: Table 8 – Chemical character of essential oils from Geijera parviflora specimens. Arithmetic indices (AI) are included alongside published values from Adams (2007). ……………………………………………………………………………………………………………………………………273 Table 9 – Chemical character of essential oils from Geijera parviflora specimens. Arithmetic indices (AI) are included alongside published values from Adams (2007). ……………………………………………………………………………………………………………………………………274

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Table 10 - Chemical character of essential oils from Geijera parviflora specimens. Arithmetic indices (AI) are included alongside published values from Adams (2007). ……………………………………………………………………………………………………………………………………275 Appendix D: Table 4 – Composition of essential oils from New South Wales populations currently assigned to P. squamulosum subsp. squamulosum, collected from Gloucester Tops (NJS246), Bluff Rock Tenterfield (NJS282) and Donnybrook State Forest Tenterfield (NJS303) and P. squamulosum subsp. lineare (NJS314 and 334), from Wingen Maid Nature Reserve Scone. Published arithmetic indices (Pub. AI) are from Adams (2007) and calculated arithmetic indices (AI) were produced by the authors. Essential oil components are displayed in relative abundance (%) by mass of component compared to the whole essential oil. ……………………………………………………………………………………………………………………………………279 Table 5 – Composition of essential oils from P. squamulosum subsp. gracile. Published arithmetic indices (Pub. AI) are from Adams (2007) and calculated arithmetic indices (AI) were produced by the authors. Essential oil components are displayed in relative abundance (%) by mass of component compared to the whole essential oil. ……………………………………………………………………………………………………………………………………280 Table 6 – Comparison of composition of essential oils from P. squamulosum subsp. ozothamnoides collected from a cultivated plant ex Tinderry Mountains (IRT ), and from wild populations in the Blue Mountains (NS273) and a putative new species here referred to as subsp. aff. Ozothamnoides (NS289 and NS300). Published arithmetic indices (Pub. AI) are from Adams (2007) and calculated arithmetic indices (AI) were produced by the authors. Essential oil components are displayed in relative abundance (%) by mass of component compared to the whole essential oil. ……………………………………………………………………………………………………………………………………281 Table 7 – Composition of essential oils from P. squamulosum subsp. coriaceum from Warrambungles National Park. Published arithmetic indices (Pub. AI) are from Adams (2007) and calculated arithmetic indices (AI) were produced by the authors. Essential oil components are displayed in relative abundance (%) by mass of component compared to the whole essential oil. ……………………………………………………………………………………………………………………………………282 Table 8 – Composition of essential oils from P. squamulosum subsp. verrucosum. Published arithmetic indices (Pub. AI) are from Adams (2007) or Singh (2003) for dihydrotagetone. Calculated arithmetic indices (AI) were produced by the authors. Essential oil components are displayed in relative abundance (%) by mass of component compared to the whole essential oil. This table was published earlier by Sadgrove et al. (2013) with further details of the enantiomeric composition of dihydrotagetone. ……………………………………………………………………………………………………………………………………283

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List of Figures or Schemes Chapter 1: Figure 1 - Isothiocyanates contain both sulphur and nitrogen; a) Phenylethyl, b) 3-methylpropyl and c) Benzyl isothiocyanate (Hüsnü Can Baser & Demirci, 2007). ……………………………………………………………………………………………………………………………………32 Chapter 2: Figure 1 - Map of New South Wales showing the trail of collection sites travelling westward. ……………………………………………………………………………………………………………………………………58 Figure 2 - Result of bioautography: (A) borneol, (B) α-terpineol, (C) isomenthone, (D) terpinolene, (E) α-terpinene, (F) karahanaenone, (G) sabinene, (H) p-cymene, (I) mixture, (J) D-limonene. ……………………………………………………………………………………………………………………………………63 Chapter 3: Figure 1 - HPLC-PAD of pure essential oil (left) contrasted with the equivalent partially pyrolysed oil (right). ……………………………………………………………………………………………………………………………………75 Chapter 4: Figure 1 – Smoke extraction from E. longifolia. ……………………………………………………………………………………………………………………………………84 Figure 2 - Chromatograms of DCM extract (A), and the smoke extract partitioned into chloroform (B), produced using Method B (Figure 1) after 10 min ……………………………………………………………………………………………………………………………………87 Scheme 1 - Genifuranal 1 and derivitisation reactions for structure elucidation. ……………………………………………………………………………………………………………………………………87 Scheme 2 - Proposed scheme for the formation of 1 from Geniposidic acid ……………………………………………………………………………………………………………………………………88 Chapter 5: Figure 1 – Scatter plots from the three PCA performed in this study. ………………………………………………………………………………………………………………………………...……98 Figure 2 – Flow cytometric histograms showing A) diploidy and B) tetraploidy. The logarithmic scale of the x axis demonstrates that gate P4 is approximately half the fluorescence intensity of gate P1, which cooperates with Barlow’s cytogeography (Barlow, 1971). ………………………………………………………………………………………………………………………...……………98 Figure 3 – Distribution of essential oil chemotypes of E. longifolia and respective ploidy in Australia. Hollow symbols represent diploid specimens and the others represent tetraploid. More detail of essential oil types are provided in Table 3. …………………………………………………………………………………………………………………………...…………99 Figure 4 - Contrast of morphology of E. longifolia. The isomenthone diploid was photographed in Mutawintji National Park outside of Broken Hill (NSW), the fenchol tetraploid was photographed at Mt Magnet in Western Australia (Murchison District) and the safrole diploid was photographed at Sandstone airstrip, also from Western Australia’s Murchison District. …………………………………………………………………………………………………………….………………………101 Chapter 6: Figure 1. Location of collections of E. bignoniiflora. Map was produced using the google maps applications …………………………………………………………………………………………………………….………………………109 Figure 2. Bioautography of E. bignoniiflora essential oil with inhibition zones corresponding to principal active constituents for antimicrobial activity. The test organism overlays were Staphylococcus aureus (ATCC 29213) and S. epidermidis (ATCC 12228). Voucher references are listed in shorthand (NJSadgrove41 changed to 41 et cetera). ………………………………………………………………………………………………………………………….…………111 Chapter 7: Figure 1 – Map of the Australian continent showing locations of wild Pittosporum collections. Collections of Pittosporum angustifolium are symbolically indicated by a circle and collections of Pittosporum undulatum are symbolically indicated by a square. ……………………………………………………………………………………………………………………………………121 Chapter 8: Figure 1 – Methods for smoke extraction of Callitris needles ……………………………………………………………………………………………………………………………………135 Chapter 9: Figure 1 – Location of G. parviflora collections in NSW (Voucher in Shorthand). ……………………………………………………………………………………………………………………………………149 Figure 2 – Apparatus and configuration of experiment aimed at condensing the smoke produced from heated leaves of G. parviflora. ……………………………………………………………………………………………………………………………………150 Figure 3 – DPPH scavenging ability of G. parviflora essential oils in μg per μL of essential oil. All oils were fresh except NS14 and NS48, which were aged two and three years respectively. Positive controls in this experiment were ascorbic acid and trolox, which quenched DPPH at a stoichiometric ratio (Control mg/DPPH mg) of 0.46 and 0.22, respectively. ……………………………………………………………………………………………………………………………………151 Chapter 10: Figure 1 – Numerical clusters produced in principal component analysis (PCA) ……………………………………………………………………………………………………………………………………165 Figure 2 - Geographical distribution of numerical clusters produced in principal component analysis. ……………………………………………………………………………………………………………………………………166 Figure 3 - The 'Yellow Wilga', which produced an essentil oil with an unusually high quantity of

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sabinene. Image taken by Maximilien Gonçalves-Martins. ……………………………………………………………………………………………………………………………………169 Chapter 13: Figure 1 – Leaf morphology of the Phebalium squamulosum complex (Wilson, 1970), with molecular structures of the main chemotypically significant essential oil components. ……………………………………………………………………………………………………………………………………203 Figure 2 – Sampling sites for Phebalium squamulosum: d, subsp. squamulosum; j, subsp. coriaceum; +, subsp. gracile; , subsp. lineare; N, subsp. ozothamnoides; ., subsp. aff. ozothamnoides; ⁄, subsp. verrucosum. ……………………………………………………………………………………………………………………………………204 Figure 3 – Scoring plot from principal component analysis, showing the five species, with PC1 plotted against PC3. ……………………………………………………………………………………………………………………………………204 Chapter 14: Figure 1 – Morphological variants of specimens collected for this study, currently assigned to P. rotundifolia. ……………………………………………………………………………………………………………………………………215 Figure 2 – Morphological variation in specimens collected for this study, currently assigned to P. ovalifolia. ……………………………………………………………………………………………………………………………………216 Figure 3 – 1,8-cineole and the main sesquiterpenoids characterised from P. ovalifolia and P. rotundifolia varieties. ……………………………………………………………………………………………………………………………………221 Appendix C: Figure 4 – Mass spectrum of Unknown (A). ……………………………………………………………………………………………………………………………………271 Figure 5 – Mass spectrum of Unknown (B). ……………………………………………………………………………………………………………………………………272 Appendix D: Figure 4 - Loading plot from principal component analysis showing PC1 plotted against PC3. ……………………………………………………………………………………………………………………………………277 Figure 5 – Mass spectrum of squamulosone. ……………………………………………………………………………………………………………………………………278

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Abstract

The first of three main focal points of this study was to uncover commercially viable natural

products, particularly essential oils, complemented with information related to cultivar

requirements and biological activity. Emphasis was subsequently placed on Eremophila

longifolia, which produced a range of geographically specific chemotypes. To investigate

potential factors related to this variability a ploidy analysis was performed, which revealed

that the high yielding isomenthone diploid chemotype of E. longifolia from western New

South Wales is genetically different from all other chemotypes in Australia, which are

normally tetraploid, except of course the diploid phenylpropanoid chemotype occupying a

small geographic range on the north-west coast of Western Australia. With moderate to

high antimicrobial activity of essential oils the isomenthone chemotype is therefore judged

to be the best choice for cultivation.

The second focal point of this study was to investigate ethnopharmacological activity of

significant medicinal plants used by Australian Aboriginal people. Again E. longifolia was

involved in this investigation along with Callitris species, Geijera species, Pittosporum

species and Eremophila bignoniiflora. In all investigations the character of medicinal

compounds was determined using GC-MS and in some investigations 1D and 2D NMR was

used. Thus, volatile compounds were the emphasis of all ethnopharmacological

investigations. Using novel smoke extraction experiments partially pyrolysed essential oils

were produced from E. longifolia and various smoke extracts from the three species E.

longifolia, Callitris endlicheri and C. glaucophylla. Smoke extracts were demonstrated to

have significantly enhanced antimicrobial activity. With regard to E. longifolia activity was

largely linked to a novel heat derivative, Genifuranal. With regard to Callitris species, using

comparative solvent extractions activity was largely linked to abietane diterpenes, most

importantly pisiferal and another fixed hydrophilic compound that remained unidentified.

With regard to E. bignoniiflora, Geijera parviflora and Pittosporum angustifolium, intact

volatile compounds were presumed to be involved in medicinal activity. Fenchyl and bornyl

acetate dominated in essential oils from E. bignoniiflora and the well-known bioactive

coumarins osthole, xanthylitin and isopsoralen were characterized from a distinct specimen

of G. parviflora. In essential oils from P. angustifolium compounds such as acetic acid, decyl

ester, or 1-dodecanol were characterized, which are structurally similar to chemosemiotic

signaling compounds involved in mother-infant communication.

The third focal point of this study was use essential oil character to investigate

phytochemical differences between morphologically different populations of the same plant

species, or phytochemical correlations between morphologically similar species in the same

family. The objective here was to complement studies aimed at taxonomic species revision.

In conjunction with investigated bioactivities of essential oils and extracts chemical

character of Zieria floydii essential oils was demonstrated to be approximately the same as

essential oils from Z. furfuracea and Z. granulata, which complements the morphological

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similarities first described by A. G. Floyd when the species was discovered. With regard to

Phebalium species dihydrotagetone was found to be a chemical marker for Phebalium

glandulosum subspecies, which influenced re-classification of Phebalium squamulosum

subsp. verrucosum. In addition, Phebalium squamulosum subspecies were demonstrated to

be divided into squamulosone groups (southern populations) verses elemol groups

(northern populations), as components in the essential oil. Again this influenced species

delimitation.

In our preliminary study which was aimed at highlighting the need to undertake subsequent

comprehensive chemotaxonomic studies on Prostanthera, several essential oil variants of P.

ovalifolia, P. rotundifolia and P. lasianthos were demonstrated. In general the former two of

these species produce essential oils dominated by 1,8-cineole together with tricyclic

sesquiterpene alcohols or furans on the backbone of decahydro-napthalene or –azulene

structures; with a cyclopropane moiety on alcohols or a furan on the others. Essential oil

composition was demonstrated to be in congruence with morphological and geographical

variations. This qualifies chemotaxonomy as a useful complement to morphological and

phylogenetic studies in future studies aimed at species revision.

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