Larvicidal potential of Spinifex longifolius against mosquito vectors: Insights from bioactivity, molecular docking, and in silico toxicity assessment.

Mosquito-borne diseases remain a major global health challenge. Their impact is intensified by widespread insecticide resistance, creating the need for novel alternatives. This study characterized Spinifex longifolius R.Br. (Poales: Poaceae) extracts based on their chemical composition and larvicidal activity against Aedes aegypti (L., 1762), Anopheles stephensi Liston, 1901, and Culex quinquefasciatus Say, 1823. The potential mechanism of action and ecotoxicity predictions for non-target organisms was investigated through molecular docking, and ADMET-based properties. Plant material was extracted using a Soxhlet apparatus with solvents of varying polarity (aqueous, methanol, ethanol, chloroform, n-Hexane). Gas chromatography-mass spectrometry (GC-MS) analysis revealed a diverse phytochemical profile with stigmasterol (40.07%), campesterol (17.5%), and γ-sitosterol (9.7%) as the major constituents. Larvicidal bioassays at concentrations of 50-400 ppm showed that ethanol and methanol extracts were the most effective. The ethanol extract caused 100% mortality of Aedes aegypti larvae at 250 ppm. The lowest LC₅₀ values for the ethanol extract were 124.79 ppm for A. aegypti, 99.98 ppm for A. stephensi, and 137.05 ppm for Culex quinquefasciatus. Molecular docking studies identified β-Amyrin as the compound with the highest binding affinity toward the acetylcholinesterase enzyme (PDB ID: 6ARY), forming a key hydrogen bond with Arg599 and multiple hydrophobic interactions. Normal mode analysis confirmed the structural stability of the 6ARY-β-Amyrin complex and elucidated flexible regions involved in enzyme inhibition. Multivariate toxicity profiling using hierarchical clustering, K-means analysis, and principal component analysis identified three distinct toxicity classes. The protozoa and invertebrates were the most sensitive endpoints while several compounds exhibited reduced predicted toxicity toward fish. Despite the promising larvicidal activity, further investigations are required to minimize potential environmental risks and ensure the safe application of the extract.