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

Erdal Bedir,Gülten Kuru,Çiğdem Karakoyun,Melis Küçüksolak,Hasan Yusufoğlu,Gamze Doğan

doi:10.3390/molecules26103030

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.

Highlights

Microbial biotransformation is one of the most preferred and powerful procedures in natural product drug discovery and development studies enabling specific biochemical modifications of substrate molecules [1]
We aimed to perform a microbial biotransformation study on gitoxigenin, which was obtained by acid-catalyzed hydrolysis of oleandrin
Compound 7 (3-oxodiginatigenin) exhibited cytotoxic activity against PANC-1 with an IC50 value of 6.24 μM, while 3-epidiginatigenin (8) demonstrated higher bioactivity against PANC-1, A549, and MIA PaCa-2 with 1.95, 8.25 and 3.4 μM IC50 values, respectively. These findings suggested that 12β-monohydroxylated derivatives 7 and 8 of gitoxigenin had higher cytotoxicity especially on PANC-1 cell line compared to the other metabolites

Summary

Introduction

Microbial biotransformation is one of the most preferred and powerful procedures in natural product drug discovery and development studies enabling specific biochemical modifications of substrate molecules [1]. A living cell, or an inactivated microorganism containing several specific enzymes can be used for biotransformation processes leading to the production of new drug candidates with better properties. Microbial enzyme systems are able to catalyze regio- and stereo-specific reactions which are challenging issues in chemical synthesis [2,3,4]. These enzymes are to employ diverse chemical reactions such as oxygenation, oxidation/reduction, cyclization, epoxidation, dehydrogenation, acetylation/deacetylation and epimerization. The use of microbial biotransformation has become one of the essential parts of white biotechnology and green chemistry movements due to requiring less energy and creating less waste [6]

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