Thesis Page 235 Part 4: Thesis conclusions and appendices ‘If you want to know the end, look at the beginning’ African Proverb ‘The rule which forbids ending a sentence with a preposition is the kind of nonsense up with which I will not put’ Sir Winston Churchill ‘If your knees aren’t green by the end of the day, you ought to seriously re- examine your life’ Bill Watterson ‘The will to overcome a passion is in the end merely the will of another or several other passions’ Friedrich Nietzsche Thesis Page 236 Chapter 15 – Conclusion Thesis Page 237 15.1.0 Summary 15.1.1 Part 1 – Eremophila longifolia: ethnopharmacology, essential oil chemotypes and cytogeography With regard to the identification and delineation of essential oil chemotypes of Eremophila longifolia, it is now clear that the first such chemotype identified in 1971 by Della and Jefferies, with an essential oil made up predominantly of the potentially hepatotoxic carcinogenic phenyopropanoids safrole and methyl eugenol, is confined to a small geographic region in Australia’s far west, in central-west Western Australia. This is important since although E. longifolia has a widespread distribution throughout the Australian landmass perceptions still prevail that this single chemotype reflects the constituents of all individuals of the species. This is simply not true. Further clarification reveals that this chemotype is an unusual biotype with diploid cytology. In all, a total of three diploid populations were identified in Australia, the other two being geographically clustered in western New South Wales and producing terpenoid based essential oils via the mevalonate pathway. These ketone rich chemotypes, as is the case for the phenylpropanoid type, produce significantly high yields of essential oils, making them potentially suitable for commercial development. The first of these types is the isomenthone/menthone type (CT.A), which produces an essential oil yield at an impressive range of 3-8% g/g wet weight of leaves. The second is the karahanaenone type (CT.B), yielding at a range of 1-5% for diploid specimens, or 0.3- 0.7% for tetraploid specimens. Both these high yielding diploid types are good candidates as cultivars for commercial plantations. Should such plantations be established and developed this would make a significant contribution to Australia’s essential oil industry. Essential oils and or/extracts from the high yielding CT.A isomenthone/menthone type could be used to make ointments and lotions suitable for topical, antifungal, aromatherapeutic and cosmeceutical/aesthetic applications (Table 1). At present it is unclear how CT.B could be utilised, but karahanaenone is already in demand as a feedstock in the flavour and fragrance industry and may also be useful as a chemical scaffold for further drug development. In addition to the five essential oil chemotypes of E. longifolia identified prior to the current study, four new types have now been characterised. One of these new essential oils, with dominant components of bornyl- and fenchyl-acetate, is similar in composition to the antimicrobial essential oil produced from Eremophila bignoniiflora. Traditional ethnomedicinal use of E. bignoniiflora by Australian Aboriginal people involved applications consistent with antispasmodic activity and headache therapy. Because essential oils rich in esters are often associated with antispasmodic and nervous calming activity, the essential oils from E. bignoniiflora may have contributed to this effect. The same essential oil produced from the new chemotype of E. longifolia, in significantly higher yields, could be marketed for treatment of headaches, nervous tension or gastrointestinal disorders (Table 1). Interestingly, another of the newly characterised chemotypes of E. longifolia produces an essential oil comprised predominantly of fenchone and camphor (2-bornanone), which are analogues of the previous mentioned fenchyl- and bornyl acetate respectively, after removal of the acetate groups. In the case of fenchone and Thesis Page 238 camphor, a ketone is in the place of the ester; however, in the case of the other known chemotype, dominated by fenchol and borneol, an alcohol functional group is in the place of the ester. Clearly, the oils produced by these three chemotypes are of very similar biosynthetic provenance. The essential oils dominated by the alcohols, fenchol and borneol, demonstrated high antimicrobial activity against the yeast C. albicans, bacterial species, such as Staphylococcus aureus, S. epidermidis, and the human pathogenic fungal species Trichophyton rubrum, T. mentagrophytes and T. interdigitalis. Similar activity was demonstrated by the fenchyl- and bornyl acetate oils against C. albicans and S. epidermidis. The fenchone rich essential oil is yet to be tested for antimicrobial activity. The third new essential oil chemotype of E. longifolia is rich in α-pinene, sabinene, limonene and α-terpinolene. At first this essential oil appeared to be consistent with an earlier type reported in individual E. longifolia from Alice Springs, in the Northern Territory. However, it’s unusually high concentration of α-terpinolene, makes this new essential oil unique. The fourth new chemotype is dominated by p-cymen-8-ol, along with a host of other unidentified compounds. Currently then, at least nine chemotypes of E. longifolia have been characterised but preliminary results suggest that others wait to be confirmed. All essential oil chemotypes occurring outside the small regions of the safrole/methyl eugenol diploid type, the isomenthone/menthone diploid type and the karahanaenone diploid type show tetraploid cytology. The karahanaenone and isomenthone/menthone types also exist as tetraploid forms but produce relatively low essential oil yields by comparison with the diploid varieties. Such tetraploid types appear as randomly emerging individuals in isolated patches throughout the range of E. longifolia, probably emerging as a result of sexual reproduction and assortment of recessive allelic traits related to biosynthesis. Consideration within the context of proposals to cultivate commercial scale crops of E. longifolia species, quality control of plantations of tetraploid chemotypes may involve the elimination of karahanaenone and isomenthone/menthone chemotypes emerging in plantations from sexual reproduction. However, in any case, this is not expected to occur with any great frequency since this species has a preference for reproduction by root suckers. With regard to the emergence of unintended chemotypes in populations of known chemotypes, one may consider the emergence of the safrole/methyl eugenol type a potential risk in a commercial scale plantation, particularly since safrole and methyl eugenol have been red flagged as potential hepatotoxic carcinogens. Our research indicates that the risk of this occurring is vanishingly small. Thus far the safrole/methyl eugenol type has not been demonstrated to occur in tetraploid form. However, even if this did occur, the parent chemotype would produce essential oils via the shikimic acid pathway, because emergent chemotypes may not contradict the biosynthetic origins of the parent chemotype. With regard to the role of volatiles in the medicinal efficacy of smoke or steam fumigation rituals, using E. longifolia, both partially pyrolysed essential oils and the Thesis Page 239 more hydrophilic component ‘genifuranal’ are involved. Most of the essential oil components are present in the leaf tissue before heating, but are accompanied with other derived artefacts in the steamy smoke that is produced when the leaves are placed on hot embers for use in medicinal applications consistent with antibacterial or antifungal applications, as well as lactagogue activity. The smoking procedure was also used to prepare surgical tools, no doubt for sterilization but conceptualised as a type of exorcism ritual. The essential oils and artefacts were also accompanied by pyrolysed derivatives including radical essential oil fragments and other phenolic or benzoid constituents; together producing significantly enhanced antimicrobial activity, as demonstrated in our microtitre plate broth dilution assays. ‘Genifuranal’ itself exhibited significant antimicrobial activities, with mean inhibitory concentrations as low as 100 μg/ml against some species. As we were not able to detect this compound in the leaves of E. longifolia without heating it into a gaseous state, we hypothesise that ‘genifuranal’ is the product of heat induced cleavage of a glycosidic bond. The proposed glycoside is geniposidic acid, already demonstrated to occur in the leaves of E. longifolia in previous studies, and demonstrated to exhibit cardioactivity. In traditional fumigation rituals, ‘genifuranal’ and partially pyrolysed essential oils are delivered in warm air to the patient. Although the transdermal absorption of components such as ‘genifuranal’ are expected to produce significant biological activity, the first application with warm air is itself expected to have enhanced activity, relative to cooler applications. In this regard, antimicrobial assays produced in vitro have limited capability of capturing this enhanced activity from warm air delivery. Thus, antimicrobial activities produced in smoke fumigation rituals are expected to be higher than those demonstrated in the laboratory. 15.1.2 Part 2 – Ethnopharmacology of medicinal plants used traditionally by Aboriginal Australians The medicinal potential of the essential oil of E. bignoniiflora has