Quantitative and qualitative investigation of Duboisia myoporoides R.BR

Scopolamine as the economically most relevant tropane alkaloid (TA) is an important precursor in the pharmaceutical industry. It is obtained from the Australian plant Duboisia. In order to get a better insight into the pathway, three growth stages (6 weeks, 3 months and six months) of wildtype Duboisia myoporoides were studied regarding their qualitative and quantitative distribution of TAs using HPLC-MS and MALDI-imaging MS. Plant extracts of roots, stem and leaves were analysed for each time point by ¹H NMR to investigate the metabolic level. Additionally, mRNA transcripts of the involved genes PMT, TR-1, CYP80F1 [1] and H6 H [2] were determined. The quantitative analysis illustrates that the scopolamine content in the leaves increases significantly over time from 0.55% DW to 1.5% DW, whereas the amount of TAs in the roots does not change (total TA amount 0.5 – 0.8%). This goes hand in hand with the fact that TAs are synthesized in the roots and are stored in the leaves. ¹H NMR measurements show that the composition of the metabolites in the roots remains constant in the different growth stages while the pattern of the stem and leaves differ over time, primarily due to the accumulation of the TAs. In Fig. 1 the qualitative distribution of scopolamine is shown. The ion image of the stem (b) demonstrates a remarkable localisation – the highest signal intensity is found in the outer cortex. Conversely, it is assumed that the transport of alkaloids is performed via the xylem [3]. Our results may indicate that the transport in Duboisia is performed via the outer cortex or, more likely, that TAs accumulates in this tissue. Lastly, the mRNA level reveals that H6 H mRNA is present in all plant organs while i.e. TR-1 mRNA is absent in the leaves, which implies that the transcript levels differ between the tissues.

Fig. 1: Ion images mapping the spatial distribution of scopolamine ([M-H]⁺; m/z 304.15) in Duboisia myoporoides a) leaf, b) stem and c) root of a 6-months old plant. Scale bar 500 µm.

Keywords: Scopolamine, Duboisia myoporoides, HPLC-MS, ¹H NMR, MALDI-imaging MS, mRNA transcripts.

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