Larvicidal and antioxidant potential of Iranian Propolis against Anisakis simplex L3: A comparative in vitro study with chemical and safety profiling

Background and Aim: Anisakis simplex is a zoonotic nematode responsible for anisakidosis in humans, mainly transmitted through the consumption of raw or undercooked marine fish. Increasing resistance to synthetic anthelmintics and concerns about their safety have driven the search for natural bioactive compounds with antiparasitic potential. Propolis, a resinous substance produced by honeybees, contains various biologically active constituents with known antimicrobial, antioxidant, and antiparasitic properties. However, information on the larvicidal activity of Propolis against Anisakis simplex larvae remains limited. Therefore, this study aimed to evaluate the larvicidal efficacy, antioxidant activity, chemical composition, and cytotoxic safety of Propolis extracts collected from different regions of Iran against A. simplex third-stage larvae (L3) under in vitro conditions. Materials and Methods: Propolis samples were collected from four geographically distinct regions of Iran, including Tehran, Kermanshah, Neyshabour, and South Khorasan. Extracts were prepared using an organic solvent extraction method. Chemical composition was determined using gas chromatography–mass spectrometry (GC–MS). Larvicidal activity against A. simplex L3 larvae was assessed in vitro at different concentrations (0.25–2.0 mg/mL) and exposure times (24 and 48 h). Median inhibitory concentration (IC₅₀) values were calculated using probit analysis. Antioxidant capacity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Cytotoxicity and safety were evaluated using the methyl thiazolyl tetrazolium assay on Vero cell lines. Results: GC–MS analysis revealed that n-hexane derivatives were the predominant compounds, followed by cyclopentanemethyl and hexadecanoic acid, with variations among regions. Propolis extracts exhibited strong dose- and time-dependent larvicidal activity against A. simplex L3 larvae. Complete larval mortality (100%) was observed at 2 mg/mL after 24 h, whereas lower concentrations required longer exposure, with all concentrations achieving ≥99% mortality after 48 h. Significant regional variation in potency was observed, with IC₅₀ values ranging from 20.65 ± 1.2 μg/mL in Tehran samples to 111.23 ± 5.8 μg/mL in South Khorasan samples. Antioxidant analysis demonstrated concentration-dependent radical scavenging activity. Cytotoxicity testing showed high cell viability (>85%) at all tested concentrations, indicating good safety of the extracts. Conclusion: Iranian Propolis extracts showed potent in vitro larvicidal activity against A. simplex L3 larvae, along with strong antioxidant properties and low cytotoxicity. These findings suggest that Propolis may be a promising natural source of antiparasitic compounds and warrant further development as an alternative to synthetic anthelmintics, although additional in vivo and mechanistic studies are required. Keywords: Anisakis simplex, antioxidant activity, cytotoxicity, gas chromatography–mass spectrometry, in vitro, larvicidal activity, Propolis, zoonotic nematode.