Influence of abiotic factors on growth and biosynthesis of secondary plant components in Duboisia species

Die in Australien beheimatete Pflanzengattung Duboisia, die historisch schon von den Aborigines verwendet wurde, ist bis heute die Hauptquelle der medizinisch genutzten Tropanalkaloide. Davon werden Scopolamin und dessen Derivate häufig als anticholinerge Wirkstoffe in der Behandlung von postoperativer Übelkeit und Erbrechen, Reiseübelkeit sowie gastrointestinalen, renalen und biliären Krämpfen angewandt. Bis heute wird die globale Marktversorgung mit Scopolamin hauptsächlich über den großflächigen Freilandanbau von Hybriden aus Duboisia myoporoides und Duboisia leichhardtii gewährleistet. Das Ziel dieser Arbeit war die Untersuchung des Einflusses von abiotischen Faktoren auf Wuchs und Biosynthese sekundärer Pflanzeninhaltsstoffe, insbesondere des Scopolamins, in Duboisia. Dabei wurde 1H NMR Metabolite Profiling für einen allgemeinen Vergleich des Primär- und Sekundärstoffwechsels verschiedener Genotypen, Pflanzenorgane, Altersgruppen und Anbaubedingungen eingesetzt. Darüber hinaus wurde eine passende HPLC-MS Methode für die Gehaltsbestimmung der enthaltenen Tropanalkaloide entwickelt und validiert. Die Ergebnisse zeigen, dass die Menge an Tropanalkaloiden in Duboisia stark von genetischen Merkmalen sowie von Umwelteinflüssen abhängt, während der Entwicklungszustand eine untergeordnete Rolle spielt. Um den Einfluss der Umweltfaktoren Temperatur, Licht und Makronährstoffe auf die Scopolaminbiosynthese und den Wuchs systematisch zu untersuchen, wurden Pflanzen dreier Genotypen unter kontrollierten Bedingungen in Klimakammern angebaut. Die Datenanalyse zeigt einen hohen Einfluss von Lichtintensität und Stickstoffversorgung auf die Scopolaminproduktion und das Pflanzenwachstum; diese Parameter können gezielt zur Steigerung von Scopolamin- und Biomasseproduktion genutzt werden. Zusammenfassend trägt diese Forschungsarbeit zu einem besseren Verständnis der Interaktion von abiotischen Faktoren mit dem Alkaloidstoffwechsel und dem Pflanzenwachstum bei. Zukünftig werden die in dieser Arbeit entwickelten Methoden dazu beitragen, vielversprechende Genotypen in der Züchtung zu selektieren und geeignete Anbaumethoden für eine optimierte Scopolaminproduktion auszuwählen.The Australian plant genus Duboisia has a long history of use dating back to the Aborigines and still today, it is main source of the medicinally used tropane alkaloids. Thereof scopolamine and its derivatives are often applied as anticholinergic agents in the treatment of postoperative nausea and vomiting, motion sickness as well as gastrointestinal, renal and biliary spasms. Until today, the global supply in scopolamine is mainly covered by large scale field cultivation of hybrids of Duboisia myoporoides and Duboisia leichhardtii. The objective of this work was to evaluate the impact of abiotic elicitors on growth and biosynthesis of secondary plant components in Duboisia species with special focus on scopolamine. Thereby, 1H NMR-based metabolite profiling was applied for a global analysis of primary and secondary metabolism comparing different genotypes, plant organs, growth stages and cultivation conditions. An appropriate HPLC-MS method was developed and validated for a detailed analysis of tropane alkaloids. The results demonstrate that the abundance of tropane alkaloids in Duboisia species is largely influenced by genetic characteristics as well as by environmental conditions, whereas the plant age plays an inferior role. In order to systematically analyse the influence of temperature, light and macronutrients on scopolamine biosynthesis and plant growth, plants of three different genotypes were grown in climate chambers under controlled conditions. The data analysis hereby reveals that especially the light intensity as well as the nitrogen supply have a major impact on the scopolamine and biomass production. All in all, this research contributes to a better understanding of the interaction of abiotic factors with alkaloid metabolism and plant growth. Prospectively, the employment of the methods established within this work will help to select promising genotypes in breeding as well as suitable cultivation conditions for an optimised production of scopolamine

1H NMR- based metabolite profiling of tropane alkaloids in Duboisia spec.

Duboisia R.Br. (Solanaceae) is the main source of the tropane alkaloid scopolamine, which is an important precursor of various active pharmaceutical ingredients due to its anticholinergic properties. As only little is known about the metabolite composition among the different species, NMRbased metabolic profiling was done in order to elucidate primary and secondary metabolism in Duboisia especially focusing on the tropane alkaloid pathway. For this purpose, plants of five different genotypes (Duboisia myoporoides, D. leichardtii and hybrids of D. myoporoides and D. leichhardtii) were cultivated under strictly controlled conditions in climate chambers, leaf and root extracts were prepared and measured via 1H NMR. 14 different metabolites could be identified using 1D- and 2D-NMR techniques. Principal component analysis of the NMR data allowed a clear distinction between Duboisia hybrids and the wild types, which could be again subgrouped in D. myoporoides and D. leichhardtii, based on the metabolites identified

Einfluss von abiotischen Faktoren auf Wuchs und Scopolamin-Biosynthese in Duboisia myoporoides.

Duboisia ist eine in Australien beheimatete Pflanze aus der Familie der Nachtschattengewächse, die Tropanalkaloide als sekundäre Pflanzeninhaltsstoffe enthält, deren quantitativ und ökonomisch bedeutsamstes Scopolamin ist.Um neue Erkenntnisse hinsichtlich des Einflusses abiotischer Faktoren auf Wuchs und Scopolamin-Biosynthese zu gewinnen, wurden Pflanzen der Art Duboisia myoporoides in Hydrokultur in Klimakammern unter streng kontrollierten Bedingungen angebaut (kalibrierte Einstellungen für Beleuchtungsdauer, Lichtintensität und Temperatur). Wichtige Messgrößen stellten dabei Biomasse und Alkaloidgehalt dar, letzterer gemessen mittels UHPLC und LC-MS.Stichwörter: Duboisia, LC-MS, Scopolamin, TropanalkaloideInfluence of abiotic factors on growth and biosynthesis of scopolamine in Duboisia myoporoides.Duboisia, a native Australian plant belonging to the family of Solanaceae, contains tropane alkaloids as secondary plant components, thereof quantitatively as well as economically most important scopolamine. In order to obtain new findings regarding the effect of abiotic factors on biomass and biosynthesis of scopolamine, plants of the species Duboisia myoporoides were grown in climate chambers using hydroponics under strictly controlled conditions (calibrated settings regarding lighting period, light intensity and temperature). Essential measurement variables were biomass and alkaloid content, the latter analyzed via UHPLC and LC-MS. Keywords: Duboisia, LC-MS, scopolamine, tropane alkaloid

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

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, H 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.\ua0leichhardtii 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.\ua0leichhardtii using principal component analysis of H 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\ua0±\ua04.05 and 17.82\ua0±\ua03.52\ua0mg/g\ua0dry\ua0wt) followed by Duboisia myoporoides (12.71\ua0±\ua02.55\ua0mg/g\ua0dry\ua0wt), both showing a high selectivity for scopolamine in contrast to Duboisia leichhardtii (3.38\ua0±\ua00.59 and 5.09\ua0±\ua01.24\ua0mg/g\ua0dry\ua0wt) with hyoscyamine being the predominant alkaloid