Evaluation of cytotoxic potential of phytochemicals identified from Gymnostachyum febrifugum roots and deciphering the molecular mechanism using in vitro and in silico analysis

Gymnostachyum febrifugum , an ethnomedicinal plant with antioxidant, hepatoprotective, anti-inflammatory and cytotoxic potential against breast and lung cancers. The present study was planned to identify and characterise cytotoxic phytochemicals from the G. febrifugum root ethyl acetate extract and its fractions to understand the possible molecular mechanisms of action of these phytochemicals using in vitro and in silico analyses against breast cancer. The study involves the bioassay-guided fractionation of cytotoxic phytochemicals from the G. febrifugum root ethyl acetate extract using column chromatography. Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, clonogenic and scratch assays. Additionally, induction of cell death, ROS production, and regulation of the cell cycle and apoptosis were assessed by fluorescent staining, flow cytometry, and immunoblotting. The phytochemicals in the cytotoxic fractions were characterised spectroscopically, and their molecular targets were identified from network pharmacology. The cytotoxic effect of the ethyl acetate fractions (GF1-GF4) evaluated against MCF7 cells showed IC 50 values ranging from 54.64 ± 1.99 to 84.47 ± 1.0 µg/mL. Clonogenic, scratch, and live-dead assay also confirmed the significant ( p < 0.001) cytotoxic potential of GF2 and GF3 fractions. The identification and characterisation of the phytochemicals in the cytotoxic GF2 and GF3 fractions using GC-MS/MS and HR-LCMS/MS analysis revealed the presence of 2,4-di-tert-butylphenol (2,4-DTBP) (GF2) and kalopanax saponin C (GF3), which might have contributed to generating cytosolic ROS, G1 phase arrest and enhanced level of caspase 3 as observed in the molecular mechanism-based studies. Further, the in silico analysis using a network pharmacology approach identified HSD11B1, NRIH3, PTGS1, UGT2B7, AR, BCL2L1, GLI1, JUN and F2 as the major protein targets for 2,4-DTBP and kalopanax saponin C, all of which require further experimental validation.