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Research Article | 29 Oct 2026

Anthelmintic activity and mechanistic insights into Piper betle L. essential oil against adult Ascaridia galli: An integrated in vitro, ultrastructural, histopathological, and in silico study

Nur Mayazah Churin'in1, Penny Humaidah Hamid1,2, Herjuno Ari Nugroho3, Pamungkas Rizki Ferdian4, Syahputra Wibowo5, and Nur Balqis Maulydia5 Show more
VETERINARY WORLD | Article No. 36 | pg no. 4888-4919 | Vol. 19, Issue 10 | DOI: 10.14202/vetworld.2026.4888-4919
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ABSTRACT

                      
Background and Aim: Ascaridia galli is an economically important intestinal nematode of poultry, and reliance on conventional anthelmintics raises concerns regarding reduced drug sensitivity and residues. Piper betle L. essential oil (PBEO) contains diverse bioactive constituents, but its activity against avian nematodes remains unexplored. This study evaluated the in vitro anthelmintic activity of PBEO against adult A. galli and investigated associated structural damage, chemical composition, and potential molecular targets.
Materials and Methods: Adult A. galli were collected from naturally infected chickens slaughtered at a traditional poultry slaughterhouse in Boyolali Regency, Central Java, Indonesia. Worms were exposed to PBEO at 10, 1, 0.1, 0.01, and 0.001% (v/v), with concentration-matched vehicle controls. Mortality was assessed at 10–160 min and analyzed using a generalized linear mixed model, while the median lethal concentration (LC₅₀) was estimated by probit regression. Cuticular and internal tissue alterations were evaluated using scanning electron microscopy and hematoxylin and eosin histopathology, respectively. PBEO constituents were characterized by gas chromatography–mass spectrometry (GC–MS). Ten selected constituents were molecularly docked against aquaporin (AQP), glutamate-gated chloride channel, nicotinic acetylcholine receptor, and Na⁺/K⁺-ATPase.
Results: PBEO induced significant concentration- and time-dependent mortality (p < 0.05). The 10% concentration increased mortality from 26.67 ± 37.23% at 10 min to 100% at 160 min, whereas 1% PBEO produced 56.67 ± 34.44% mortality at 160 min. The estimated LC₅₀ was 0.71% (95% confidence interval 0.47–1.06%). Electron microscopy revealed lip abrasion, anterior cuticular distortion, irregular folding, and surface disintegration. Histopathology demonstrated uterine disorganization, egg deformation, cuticular disruption, and muscle fragmentation. GC–MS resolved 50 peaks; 49 were identified, with eugenol as the most abundant constituent (8.48%). Docking predicted multi-target interactions with binding affinities ranging from −7.2 to −4.4 kcal/mol; the strongest interactions involved 4-allylphenyl acetate and β-caryophyllene with AQP.
Conclusion: PBEO exhibits measurable in vitro anthelmintic activity against adult A. galli, accompanied by extensive cuticular, reproductive, and muscular damage. Its chemically diverse constituents show predicted multi-target interactions that may contribute to the observed activity. Bio-guided fractionation, target-based validation, and in vivo poultry studies are required to establish efficacy, safety, and translational potential.
                      
Keywords: Ascaridia galli, anthelmintic activity, cuticular damage, essential oil, molecular docking, Piper betle, poultry nematode, scanning electron microscopy.