GC-MS phytochemical screening, molecular docking, MD simulation, and in vitro antioxidant assay of Euphorbia...

Euphorbia thymifolia L., a medicinal herb traditionally recognized for its many medicinal properties, was investigated in the present study to evaluate the antioxidant potential of its hexane fraction and to elucidate its possible mechanism of action through Keap1-Nrf2 pathway modulation using in silico analyses, along with validation through in vitro assays. The plant material was initially extracted using ethanol, followed by fractionation with hexane. The obtained hexane fraction was subjected to GC-MS analysis to identify its phytoconstituents, which were further evaluated through in silico molecular docking and molecular dynamics (MD) simulations against Keap1 to predict their potential to activate Nrf2 signaling. The antioxidant activity of the fraction was subsequently validated using DPPH, nitric oxide scavenging, and reducing power assays. Phytochemical screening and GC-MS analysis confirmed the presence of bioactive fatty acid constituents such as palmitic acid, linoleic acid, and linolenic acid. Antioxidant evaluation using DPPH and nitric oxide radical scavenging assays demonstrated concentration-dependent activity, with IC₅₀ values of approximately 204 µg/mL and 169.40 µg/mL, respectively, which were moderately comparable to the standard. Molecular docking studies revealed notable interactions of the key phytoconstituents with the Keap1 protein, particularly Taraxerol, which exhibited a binding affinity of - 11.6 kcal/mol. Furthermore, a 100 ns molecular dynamics simulation, along with post-simulation analyses, confirmed the stability of the ligand-protein complex. The present study shows that Euphorbia thymifolia L. includes bioactive compounds with significant antioxidant activity, as evidenced by in vitro experiments and Keap1-Nrf2 pathway interactions discovered in silico. The stable ligand-protein interaction shows that it has potential as a natural source for antioxidant therapy development and should be investigated further.