The reliance on synthetic repellents such as N,N-diethyl-meta-toluamide (DEET) has raised health and environmental concerns, prompting the search for safer, plant-based alternatives. Catnip (Nepeta cataria L.), a rich source of iridoid monoterpenes, particularly nepetalactones, known for strong insect-repellent activity. However, their efficient extraction and molecular mechanisms in insect inhibition remains challenging. This study examined the chemical composition, protein–ligand interactions, and safety profiles of nepetalactones in comparison with DEET, with particular focus on mosquito odorant-binding proteins (OBPs) from Anopheles gambiae (AgamOBP), Culex quinquefasciatus (CquiOBP), and Aedes aegypti (AaegOBP). GC–MS/MS analysis identified nepetalactone isomers as the predominant constituents in catnip extracts obtained via steam distillation and olive oil extraction from dried leaves. Molecular docking results indicated that cis,cis-, cis,trans-, and nepetalactone isomers exhibited higher binding affinities toward the target OBPs than DEET. Furthermore, molecular dynamics simulations confirmed that all nepetalactone–OBP complexes exhibited stable conformations characterized by low average RMSD values and persistent hydrogen bond formation. Notably, cis,trans-NL–AaegOBP, NL–AaegOBP, and cis,cis-NL–AgamOBP complexes displayed lower binding free energies (ΔG_MM-PBSA) compared to DEET. These findings suggest that nepetalactones stabilize OBP–ligand interactions while inducing subtle conformational flexibility, potentially disrupting mosquito odorant recognition in a manner distinct from DEET. ADMET predictions indicated that nepetalactones exhibit favorable absorption, distribution, and safety profiles with reduced predicted toxicity compared to DEET. Collectively, these results establish nepetalactones as promising candidates for the development of effective, safe, and sustainable plant-based repellents.

The reliance on synthetic repellents such as N,N-diethyl-meta-toluamide (DEET) has raised health and environmental concerns, prompting the search for safer, plant-derived alternatives. Catnip (Nepeta cataria L.) is a rich source of iridoid monoterpenes, particularly nepetalactones, which are well known for their strong insect-repellent properties. However, the efficient extraction of nepetalactones remains challenging, and their precise mechanisms of action in insect inhibition are not yet fully understood. Thus, this study investigated the chemical composition from various methods, protein–ligand interactions, and pharmacokinetic safety profiles of catnip-derived compounds compared to DEET, with a focus on their interactions with odorant-binding proteins (OBPs) from Anopheles gambiae (AgamOBP), Culex quinquefasciatus (CquiOBP), and Aedes aegypti (AaegOBP). Gas chromatography–tandem mass spectrometry (GC–MS/MS) confirmed the presence of nepetalactone isomers as the major constituents in catnip extracts obtained through stream distillation and dried leaves extracted in olive oil fractions. Molecular docking revealed that cis,cis- and cis,trans-nepetalactones and nepetalactone exhibited high binding affinities, surpassing those of DEET. Molecular dynamics simulations demonstrated that all OBP–ligand complexes achieved stable conformations. Notably, cis,trans-nepetalactone formed a more stable complex with AgamOBP than DEET. These findings suggest that nepetalactones stabilize OBP–ligand interactions while inducing subtle conformational flexibility, potentially disrupting mosquito odorant recognition in a manner distinct from DEET. ADMET predictions indicated that nepetalactones exhibit favorable absorption, distribution, and safety profiles with reduced predicted toxicity compared to DEET. Collectively, these results establish nepetalactones as promising candidates for the development of effective, safe, and sustainable plant-based repellents.