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. 2021 May 19;26(10):3030.
doi: 10.3390/molecules26103030.

New Cardenolides from Biotransformation of Gitoxigenin by the Endophytic Fungus Alternaria eureka 1E1BL1: Characterization and Cytotoxic Activities

Erdal Bedir  1 , Çiğdem Karakoyun  2 , Gamze Doğan  1 , Gülten Kuru  1 , Melis Küçüksolak  1 , Hasan Yusufoğlu  3
Affiliations

New Cardenolides from Biotransformation of Gitoxigenin by the Endophytic Fungus Alternaria eureka 1E1BL1: Characterization and Cytotoxic Activities

Erdal Bedir et al. Molecules. .

Abstract

Microbial biotransformation is an important tool in drug discovery and for metabolism studies. To expand our bioactive natural product library via modification and to identify possible mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endophytic fungus Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin (yield; ~25%). After 21 days of incubation, five new cardenolides 1, 3, 4, 6, and 8 and three previously- identified compounds 2, 5 and 7 were isolated using chromatographic methods. Structural elucidations were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate. Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity than the substrate. The new metabolite 8 exhibited the highest activity with IC50 values of 8.25, 1.95 and 3.4 µM against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 µM. Our results suggest that A. eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.

Keywords: Alternaria eureka 1E1BL1; Nerium oleander L.; biotransformation; cardenolides; cytotoxicity; endophytic fungus; gitoxigenin; oleandrin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Acid-catalysed hydrolysis of cardiac glycoside oleandrin and structures of oleandrin, gitoxigenin and L-oleandrose (TFA: trifluoroacetic acid).
Figure 2
Figure 2
Chemical structures of gitoxigenin (substrate) and eight biotransformation products transformed by A. eureka 1E1BL1.
Figure 3
Figure 3
Key HMBC and COSY correlations of the new metabolites (compounds 1, 3, 4, 6 and 8) obtained from the microbial biotransformation of gitoxigenin by A. eureka 1E1BL1.
Figure 4
Figure 4
Key HMBC and COSY correlations of the known metabolites (compounds 2, 5 and 7) obtained from the microbial biotransformation of gitoxigenin by A. eureka 1E1BL1.
Figure 5
Figure 5
Structures and yields of the metabolites obtained via microbial biotransformation of gitoxigenin by A. eureka 1E1BL1.
Figure 6
Figure 6
A plausible path for the formation of acetal group in 4.
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