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The genus Digitalis: Molecular taxonomy, preservation, active constituents and therapeutic applications

Final Report Summary - DIGITALIS (The genus Digitalis: Molecular taxonomy, preservation, active constituents and therapeutic applications)

The topics of this joint exchange program are all related to a specific plant genus of commercial interest and its taxonomy (Taxonomy of the Genus Digitalis), the propagation and investigation of endangered species of this plant genus (Plant Tissue Culture), the natural products used in medicine occurring in Digitalis plants (Therapeutic Use of Cardenolides), and the isolation, structure elucidation and biological testing of some of the rare but pharmacological relevant cardenolides (Reference and Bulk Substances).

The objectives aim at intensifying circumstantial ongoing co-operations and exchange of materials and people in a more reliable and coordinated way. In a long-term view and on the basis of “merging” of the partners we expect to offer joint botanical excursions for students, since all of the partners organize excursions in their area. This issue is, however, not part of this proposal. The expected spin-off and accidental input (e.g. research on other plants or natural products) will also be beneficial for more specific research topics of the individual program partners.

During the 2nd period one workshop was organized at FAU (Friedrich-Alexander-University) in Erlangen, Germany, in December 2014. Several project partners were able to attend that meeting (two from AIBU, three from UMFG/UFSC as well as two secondees from Brazil and three (former) secondees from FAU. During the 2nd period two secondees were completing their secondments in the respective host intitution and nine were seconded to address 15 work packages. All packages contained both, training and research activities. Work packages realized in the 2nd period again focused on structure isolation and elucidation at UFMG and molecular biology and production of therapeutic cardenolides at FAU. Tissue culture work was mainly carried out at AIBU (Abant Izzet Baysal University) in Bolu, Turkey and pharmacological testing was carried out at FFCUL (Faculdade de Ciências da Universidade de Lisboa ), UFMG (Universidade Federal de Minas Gerais) in Belo Horizonte, Brazil and UFSC (Universidade Federal de Santa Catarina) in Florianopolis, Brazil. At the end of the project UFLA (Universidade Federal de Lavras ) in Lavras, Brazil was identified as a partner for tissue culture and agricultural aspects of the project.

Eleven joint publications demonstrate the successful collaboration in all areas identified in the proposal. Secondees contributing to this work came from the partner institutions mentioned in parentheses.

a) Chemistry (UFMG/FAU): The formation of the butenolide ring was studied starting from pregnane 21-O-malonyl hemiesters with and without a hydroxyl group in position C-14β. Efficient lactonization was only achieved in the case of the 14β-hydroxylated derivative. We provided chemical and mechanistic evidence that 14β-hydroxylation would precede lactonization in cardenolide biosynthesis (Pádua et al. 2016). Moreover, we demonstrated that the catalytic mechanism of the 3-ketosteroid isomerase of Digitalis lanata involves an intramolelcular proton transfer and the activity is not associated with the 3β-hydroxysteroid dehydrogenase activity (Meitinger et al. 2016)

b) Plant Tissue Culture (AIBU/FAU/UFLA/UFMG/FFCUL): Several Digitalis species endogenous to Turkey or Portugal were investigated. Digitalis ferruginea subsp. schischkinii was collected from different locations along the Black Sea region of Turkey an its cardenolide pattern analyzed. Plants were also obtained by in vitro regeneration and the regenerants analyzed (Yücesan et al. 2016). The somaclonal variation of cardenolide content in Heywood's foxglove was demonstrated (Kreis et al. 2015) as well as the cardenoldie content in callus-mediated regenerants of Digitalis lamarckii (dwarf foxglove). The regeneration protocol described in that study was also used for the in vitro propagation of D. lamarckii (Yücesan et al. 2014). Another foxglove endemic to Turkey, Digitalis cariensis (Aegean Foxglove) was also propagated from in vitro cultures and the cardenolide profiles of the regenerants determined (Mohammed et al., 2015). Finally the the biotransformation of 21-O-acetyl-deoxycorticosterone by cell suspension cultures of Digitalis lanata was investigated (Pádua et al 2012).

c) Therapeutic use of cardenolides (UFSC/UFMG/FAU): It was demonstrated that cell proliferation, invasion and migration in human lung cancer cell line was inhibited by the cardenolides digitoxigenin monodigitoxoside and convallatoxin. This work provided the first insights into the effects of these cardenolides on key steps of lung cancer metastasis (Schneider et al. 2016). Moreover, it was shown that a new semisynthetic cardenolide analog 3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin (AMANTADIG) affects G2/M cell cycle arrest and miRNA expression profiles and enhances pro-apoptotic survivin-2B expression in renal cell carcinoma cell lines. Treatment with cardenoldies induced G2/M cell cycle arrest. Because cells in the G2/M phase are radio- and chemotherapy sensitive, cardiac glycosides like AMANTADIG could potentially improve the efficacy of radio- and/or chemotherapy (Nolte et al., submitted).

d) Taxonomy (AIBU/FAU/UFMG): One joint study aimed to define progesterone 5β-reductases (P5βR) as function-associated molecular markers at the plant family level. Therefore, cDNAs were isolated from 25 Brassicaceae species and the sequences were used in a molecular phylogeny study. It is concluded that P5βR genes or P5βR proteins can be used as valuable function-associated molecular markers to infer taxonomic relationship and evolutionary diversification from a metabolic/catalytic perspective (Munkert et al. 2015). In another approach the Phylogeny of Anatolian (Turkey) species in the Digitalis sect. Globiflorae (Plantaginaceae) was investigated This study presented a new understanding of the speciation patterns of endemic and non-endemic eastern Digitalis species distributed in Anatolia, Turkey, underlying molecular, biogeographical and comparative morphological analyses (Eker et al. 2016).

The yet unpublished work can mainly be categorized in two of the general objective of the project, namely 1) Therapeutic use of cardenolides focusing on a) the effects of natural cardenolides and the semi-synthetic AMANTADIG on prostate cancer cell lines, where a manuscript is about to be finalized, as well as b) anti-inflammatory property of plant extracts, with an emphasis on the essential oil of Digitalis species, and 2) Plant tissue culture and plant regeneration focusing on a) induction, analysis and maintenance of hairy root cultures, b) effects of illumination on cardenolide production of Turkish foxgloves in a Brazilian environment, c) tissue culture studies of Digitalis lanata collected from Thrace region of Turkey and d) Organic fertilization quail effect on vegetative growth and antioxidant capacity of Digitalis mariana ssp. heywoodii. These studies are in different phases of data compilation and manuscript preparation, mainly Turkish and Brazilan secondees were involved in this part of the project. A chemical concern was the establishment of the chemical synthesis of the antiviral and cytostatic cardenolide glucoevatromonoside, summarized in a manuscript to which several Brazilian and German secondees contributed with their data. Related to this work was the evaluation of biotransformation process and a molecular biology approaches for the production of glucoevatromonoside, a promising candidate for drug development. At the end of the project the complementing expertise of the groups released enormous synergies and lots of data await there publication in peer-reviewed journals. At the end a tendency is seen to bundle the research activities in focusing on the anti-cancer potential of cardenolide structures and their production.

In summary, many of the results obtained during the joint research activities has already been published in peer-reviewed international journals. Several lines of research are still ongoing or the data are about to be compiled for publication. Hence the impact on basic science is obvious. Tissue culture and and pharmacological work always have a potential applied aspect which, however, can not be evaluated at the moment. The story of the Eastern Digitalis species seems to be quite complete, the potential applications of natural and semi-synthetic cardenolides in tumor therapy are obvious.