A novel source of the cardiac glycoside digoxin from the endophytic fungus Epicoccum nigrum: isolation, characterization, production enhancement by gamma irradiation mutagenesis and anticancer activity evaluation.

Abstract

Different endophytic fungi were isolated and screened for their digoxin-producing ability. Strain improvement and different culture conditions were studied for more effective production of digoxin. Among the isolated fungi, an isolate produced digoxin in a concentration of 2·07mgl-1 . The digoxin-producing fungal isolate was identified as Epicoccum nigrum Link according to the morphological features and phylogenetic analyses. The potentiality of the fungal strain for production enhancement of digoxin was performed by gamma radiation mutagenesis. Gamma irradiation dose of 1000Gy intensified the digoxin yield by five-fold. Using this dose, a stable mutant strain with improved digoxin productivity was isolated and the stability for digoxin production was followed up across four successive generations. In the effort to increase digoxin magnitude, selection of the proper cultivation medium, addition of some elicitors to the most proper medium and several physical fermentation conditions were tested. Fermentation process carried out in malt extract autolysate medium (pH 6·5) supplemented by methyl jasmonate and inoculated with 2ml of 6-day-old culture and incubated at 25°C for 10days stimulated the highest production of digoxin to attain 50·14mgl-1 . Moreover, cytotoxicity of digoxin separated from the fungal culture was tested against five different cancer cell lines. Based on the MTT assay, digoxin inhibited the proliferation of the five different cancer cell lines and the recorded 50% inhibitory concentration ranged from 10·76 to 35·14μgml-1 . This is the first report on the production and enhancement of digoxin using fungal fermentation as a new and alternate source with high productivity. These findings offer new and alternate sources with excellent biotechnological potential for digoxin production by fungal fermentation. Moreover, digoxin proved to be a promising anticancer agent whose anticancer potential should be assessed in prospective cancer therapy.