Wild Nicotiana: Profiling the Alkaloids of Australian Species

Abstract

Nicotiana is a genus within the family Solanaceae indigenous to North America, South America and Australia that include the plants commonly referred to as ’tobacco’. Tobacco refers to the dried, cured and fermented leaves of Nicotiana species, favoured as a drug due its high concentrations of psychoactive pyridine alkaloids, namely nicotine and its derivatives. The ability to thrive in Australia’s arid climate make the Nicotiana species unique within the genus and currently little is understood about them, though indigenous use and popularity suggests the presence of potent entheogenic compounds. The last formal revision of Nicotiana recorded a total of 21 Australian species; however, recent investigations across Australia led to the discovery of speciation events resulting in the description of many novel species. This thesis contributes to the body of knowledge regarding the presence, quantity, and proportions of dominant pyridine alkaloids (nicotine, myosmine, anabasine and cotinine) within novel and known Australian Nicotiana species found growing wild. In addition to this, it was hypothesised that claims regarding the entheogenic use of Nicotiana as a hallucinogen may be due to the presence of psychedelic -carboline alkaloids that are detected in tobacco smoke. Therefore the presence, quantity and proportions of the -carbolines harmane and norharmane were also investigated in this work. Wild, naturally-occurring Nicotiana specimens grown without human intervention were collected in this study to reflect the impact of their native Australian climate and conditions. Fresh collection methods were prioritised with specimens subsequently being frozen in liquid nitrogen; however, specimens were also Air-Dried, Preserved in aqueous methylated spirits, Press-Dried and Silica-Dried. The separated organs of each specimen were then macerated in methanol and the alkaloids of the crude extract concentrated by acid-base Liquid-Liquid Extraction. Directed quantitative analysis of nicotine, mysomine, anabasine, cotinine, harmane and norharmane within the extracts was conducted by Gas Chromatography Mass Spectrometry with an error of less than 5% across each of the analytes for all of the 178 extracts analysed within this study. Results from this thesis validate the hypothesis that -carbolines are present within Nicotiana. Harmane and norharmane were detected consistently in all but one of the species analysed in this work. Additionally, this work contributes the first analysis of recently described Australian Nicotiana species: N. faucicola, N. sp. ’Maralinga’, N. pauliniana and N. yandinga. In total, 39 specimens of 17 different Nicotiana species were analysed and compared to the alkaloid proportions and quantities reported in previous studies. Overall, lower total alkaloid quantities were observed in the wild Nicotiana species compared to the cultivated species of previous studies, and the data collected in this study confirmed the variability noted specifically within Nicotiana species endemic to Australia. The mild variability observed overall was unsurprising; however, some species did demonstrate remarkable and unexpected similarities within the alkaloid distributions that were detected across the species. Comparisons between the different collection methods used in this study confirmed their impact on the secondary alkaloid profile subsequently detected. Overall, it was noted that the methods that involved drying the plant specimen prior to analysis achieved higher yields than the specimens that were fresh. Similar evaluation of the impact that analysing different plant organs can have on the alkaloid profile showed that while higher quantities appeared in the leaves and stems of the plant, as expected, there was no indication that specific alkaloids were linked to specific plant organs; all six of the reference alkaloids analysed in this study were found in the leaves, stems, flowers and roots of the Nicotiana specimens collected. Finally, evaluation of the influence of climate on the presence of alkaloids in wild-grown Nicotiana species indicated that higher quantities of alkaloids were detected in species exposed to higher daily temperatures.