Toxicity evaluation of Eleutherine plicata Herb. extracts and possible cell death mechanism

Eleutherine plicata has been shown to be a promising medicinal plant, and its activity has been associated with naphthoquinones. The present study aimed at evaluating the cytotoxicity, genotoxicity, and oral toxicity of the ethanol extract (EEEp), dichloromethane fraction (FDMEp) of E. plicata, and isoeleutherin. For the cytotoxicity evaluation, the viability test (MTT) was used. Genotoxicity was accessed through the Comet assay (alkaline version), acute and subacute oral toxicities were also evaluated. The antioxidant capacity of the samples in the wells where the cells were treated with E. plicata was evaluated. Furthermore, the participation of Caspase-8 in the possible mechanism of action of isoeleutherin, eleutherin, and eleutherol was also investigated through a docking study. FDMEp and isoeleutherin were cytotoxic, with higher rates of DNA fragmentation observed for FDMEp and isoeleutherin, and all samples displayed higher antioxidant potential than the control. In the acute oral toxicity test, EEEp, FDMEp, and isoeleutherin did not cause significant clinical changes. In the subacute toxicity assay, EEEp and FDMEp also did not cause clinical, hematological, or biochemical changes. The three compounds bound similarly to Caspase-8. Despite the results of cytotoxicity, in vitro studies demonstrated that the use of EEEp appears to be safe and cell death may involve its binding to Caspase-8.

ALT, alanine aminotransferase; AST, aspartate aminotransferase; BCRJ, Cell bank of Rio de Janeiro; BFS, bovine fetal serum; Caspase-8; DARP, dopamine releasing protein; DMEM, Dulbecco's Modified Eagle's Medium; DMSO, dimethyl sulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EDTA, ethylenediaminetetraacetic; EEEp, ethanol extract of Eleutherine plicata; Eleutherin; Eleutherine plicata; Eleutherol; FADD, Fas associated death domain; FDMEp, dichloromethane fraction of Eleutherine plicata; FrAE, ethyl acetate fraction of Elutherine plicata; GA, Genetic Algorithm; GOLD, Genetic Optimization for Ligand Docking; HPLC, high performance liquid chromatography; IC50, 50 % cytotoxic concentration; Isoeleutherin; MD, molecular dynamics; MTT, ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]); NMR, nuclear magnetic resonance; NMU, N-methyl-N-nitrosurea; OECD, Organization for Economic Co-Operation and Development; PDB, Protein Data Bank; ROS, reactive oxygen species; RPMI, Roswell Park Memorial Institute medium; RSMD, root mean square deviation; TLC, tin layer chromatography; TNFR, tumour necrosis fator receptor; Toxicity; rpm, rotations per minute.