IntroductionThere is an evident need for novel ophthalmic therapeutics that operate outside the conventional antibiotic and chemically synthetized antiseptic paradigms. Medicinal plants represent a chemically rich and cost-effective source of bioactive compounds, though their use requires careful toxicological assessment.MethodsIn this study, extracts from four Papaveraceae species - Chelidonium majus (roots), Corydalis cheilanthifolia (aerial parts and roots), Glaucium flavum (aerial parts and roots), and Fumaria officinalis (herb) - were investigated across three experimental levels: in silico, in vitro, and in vivo, using the Galleria mellonella larval model.ResultsChemical profiling identified several isoquinoline alkaloids, including berberine, coptisine, protopine, chelidonine, allocryptopine, glaucine, and tetrahydropalmatine. In silico assessment suggested low systemic toxicity but possible blood-brain barrier permeability for some compounds. In vitro antimicrobial testing against Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 15442, performed using broth microdilution and bacterial-cellulose-based diffusion assays, demonstrated moderate but reproducible activity of extracts, particularly in Ch. majus roots (MIC equal 1.56 mg/mL for S. aureus and 6.25 mg/mL for P. aeruginosa) and in C. cheilanthifolia, where depending on the type of extract (herb_2, herb_1, root), range of MIC was 0,78–1.56 mg/mL for S. aureus and 3,12–6,25 mg/mL for P. aeruginosa. Cytotoxicity testing on fibroblast cell lines showed no significant toxicity in applied concentrations. In vivo testing showed that all extracts, except C. cheilanthifolia (herb_1) and Ch. majus (root) were virtually non-lethal to G. mellonella larvae, indicating a favorable safety profile in 0.05 mg/mL to 0.002 mg/mL concentrations. To evaluate their suitability for ocular application, wettability tests were performed using commercial tear substitute formulations. The incorporation of the C. cheilanthifolia herb_2 extract at bactericidal concentrations did not alter these physicochemical properties.DiscussionOverall, the Papaveraceae extracts demonstrated moderate antibacterial activity and low cytotoxicity in applied concentrations, supporting their potential use as adjuvant components in eye drop formulations - enhancing therapeutic efficacy, reducing required antiseptic dosage, and potentially lowering the risk of resistance development.