Discrimination of wild types and hybrids of Duboisia myoporoides and Duboisia leichhardtii at different growth stages using 1H NMR-based metabolite profiling and tropane alkaloids-targeted HPLC-MS analysis

Abstract

Duboisia species, which belong to the family of Solanaceae, are commercially cultivated in large scale, as they are main source of the pharmaceutically-used active compound scopolamine. In this study, 1H NMR-based metabolite profiling linking primary with secondary metabolism and additional quantification via HPCL-MS with special focus on the tropane alkaloids were applied to compare leaf and root extracts of three wild types and two hybrids of Duboisia myoporoides and D. leichhardtii at different developmental stages grown under controlled conditions in climate chambers and under agricultural field plantation. Based on the leaf extracts, a clear distinction between the Duboisia hybrids and the wild types Duboisia myoporoides and D. leichhardtii using principal component analysis of 1H NMR data was observed. The average content in scopolamine in the hybrids of Duboisia cultivated in climate chambers increased significantly from month 3–6 after potting of the rooted cuttings, however not so for the examined wild types. The Duboisia hybrids grown in climate chambers showed higher growth and contained more sugars and amino acids than Duboisia hybrids grown in the field, which in contrast showed an enhanced flux towards tropane alkaloids as well as flavonoids. For a more detailed analysis of tropane alkaloids, an appropriate HPLC-MS method was developed and validated. The measurements revealed large differences in the alkaloid pattern within the different genotypes under investigation, especially regarding the last enzymatic step, the conversion from hyoscamine to scopolamine by the hyoscyamine 6β–hydroxylase. Scopolamine was found in highest concentrations in Duboisia hybrids (20.04 ± 4.05 and 17.82 ± 3.52 mg/g dry wt) followed by Duboisia myoporoides (12.71 ± 2.55 mg/g dry wt), both showing a high selectivity for scopolamine in contrast to Duboisia leichhardtii (3.38 ± 0.59 and 5.09 ± 1.24 mg/g dry wt) with hyoscyamine being the predominant alkaloid.