Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Abstract

The Aboriginal population of Central Australia use endemic Nicotiana spp. to make a smokeless tobacco product known as pituri that they chew/suck for nicotine absorption. This thesis describes the relative abundance of nicotine alkaloids amongst Australian Nicotiana spp., with special focus on the molecular characteristics of nicotine to nornicotine conversion.The most popular chewed species, N. gossei, is investigated for nicotine release and cytotoxicity in comparison to similar products to gain insight into potential hazards to pituri users.To analyse the alkaloids of Nicotiana leaves, a HPLC-UV method was developed to separate and quantify six closely related alkaloids (nicotine, nornicotine, anatabine, anabasine, myosmine, cotinine). A C18 column with a mobile phase of ammonium formate buffer (pH 10.5) separated the six alkaloids within 13 min with detection at 260 nm. Linearity, precision and reproducibility were satisfactory. The limit of quantification was 2.8 and 4.8 µg/mL for nornicotine and nicotine, respectively, and below 2 µg/mL for other alkaloids. This method quantifies more alkaloids and in less time than previously reported methods.Tobacco alkaloids are responsible in formation of carcinogenic tobacco specific nitrosamines such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To quantify NNN and NNK, a fast LC-MS/MS method was developed using a HILIC column with a triple quadrupole tandem mass spectrometry. The linearity, accuracy, recovery and repeatability for this method were satisfactory, with quantification limit of 2.6 and 4.3 ng/mL for NNN and NNK, respectively. Nornicotine results from demethylation of nicotine, and is associated with negative effects on health. A group of cytochrome P450 genes that are mainly expressed during senescence or drying is involved in this conversion. The conversion loci were amplified in all studied 24 taxa, and sequenced in 6 selected species that contrast in conversion phenotype. Transcript accumulation of the responsible loci in fresh versus dried leaves of low or nonconverter N. gossei, N. excelsior and N. benthamiana maintained a steady level or a slight increase, but increased by 3 fold in cured leaves of the high converter N. goodspeedii, N. velutina and N. cavicola. This indicates the presence of functional loci that are triggered by curing only in high ii converter species and poses a potential risk for chewers of these species due to their greater potential for nornicotine production.The release of nicotine from the leaves of N. gossei was compared to that from a Swedish snus, the CORESTA reference smokeless tobacco (CRP2), and Nicabate chewing gum. A model buccal cavity system was developed and three different chewing conditions, performed manually were tested over 120 minutes: no chewing action, initial chewing and chewing at 15-minute intervals. To simulate the effect of alkaline wood ash, the effect of alkaline pH on the release of nicotine from N. gossei dry leaves was also evaluated. Samples ...

RNA- Seq analysis of Taverniera cuneifolia (Roth) Ali.

“…Nicotine biosynthesis is catalyzed by a series of CYP450 enzymes, including CYP82E4, CYP82E5, and CYP82E10. These enzymes are involved in the conversion of nicotine precursors, such as nornicotine and anatabine, into nicotine (Moghbel, 2016). CYP450 enzymes are also involved in the biosynthesis of avonoids, which are important plant secondary metabolites that play a role in plant pigmentation and protection against UV radiation.…”