Biotechnology Progress 2014-01-01

Production, HPLC analysis, and in situ apoptotic activities of swainsonine toward lepidopteran, Sf-21 cell line.

Digar Singh, Gurvinder Kaur

Index: Biotechnol. Prog. 30(5) , 1196-205, (2014)

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Abstract

Swainsonine, a secondary metabolite from Metarhizium anisopliae has been extensively studied in the complementary areas of therapeutics and toxicology. This work aims to develop a simple UV-HPLC method of analyses for swainsonine in Metarhizium fermentation broth and to explore its in situ entomotoxic activities. The partially purified broth was quantitatively analyzed using middle UV (205 nm)-reverse phase HPLC method with different mobile phases and gradient programmes. Swainsonine was eluted as single peak at (te ) 6.0-6.9 min with average concentration of 4.04 ± 0.52 μg/mL using optimal mobile phase (0.1% trifluoroacetic acid in water and acetonitrile). The mass spectrometry analysis further indicated the characteristic MS1 species for swainsonine, [M+H](+) 174.30 in corresponding HPLC peaks. The antiproliferative effects of swainsonine on lepidopteran, Sf-21 cells were determined through 3-(4, 5-dimethylthia-zol-2-yl)-2, 5-diphenyl tetrazolium bromide (IC50 standard  = 3.90 μM and IC50 purified  = 5.27 μM) and trypan blue dye exclusion (IC50 standard  = 6.91 μM and IC50 purified  = 8.67 μM) assays. The fluorescence activated cell sorting evaluation of Sf-21 cells showed nearly 35% and 42% of population in various apoptotic stages at 36 h, when treated with standard and purified swainsonine, respectively. The morphodimensional field emission scanning electron and atomic force microscopic analyses further confirmed the characteristic apoptotic features like membrane blebbings, ruptures and volume shrinkage in the lepidopteran cells after 24-36 h of post-treatment incubation. The study describes the potential entomotoxic activities of swainsonine and its role in the virulence of Metarhizium spp.© 2014 American Institute of Chemical Engineers.