Putative binding of Piper betle L. phytochemicals to mannitol pathway enzymes during sporulation of Eimeria tenella

Coccidiosis, caused by Eimeria species, remains a major challenge to the poultry industry. The parasite transmission relies on the environmental sporogony stage during which the parasite transforms from non-infectious to infectious oocysts. This host-independent metabolism is driven by the mannitol pathway, which operates in oocysts of Eimeria, Toxoplasma , and Cryptosporidium . Our previous in vitro study demonstrated that Piper betle L. essential oil (PBEO) exhibits anticoccidial activity through oocysticidal effects and sporulation inhibition (IC₅₀ 1.31% at 72 h). Here, we performed systematic in silico molecular docking to evaluate ten major PBEO phytochemicals against three key E. tenella mannitol pathway enzymes: mannitol-1-phosphate dehydrogenase (M1PDH), mannitol-1-phosphate phosphatase (M1Pase), and hexokinase. In hexokinase enzyme, 3-allyl-6-methoxyphenyl acetate showed the strongest binding affinity (-6.2 kcal/mol), stabilized by a conventional hydrogen bond with CYS161 and a pi-sigma interaction with Leu58. For M1Pase, β-selinene and β-caryophyllene exhibited the highest affinities (-5.9 kcal/mol each) through hydrophobic alkyl networks. Next, the M1PDH receptor, β-selinene, achieved the most favourable binding (-7.4 kcal/mol), closely followed by the bicyclic sesquiterpene derivative (-7.1 kcal/mol). PBEO constituents potentially exert anti-sporulation effects by simultaneous inhibition of multiple mannitol pathway enzymes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping and analysis (map00051) confirms that these enzymes form a non-redundant metabolic unit essential for sporulation, with no bypass reactions annotated in the parasite genome. These findings support further bio-guided isolation and development of PBEO-derived compounds as anticoccidial agents targeting the environmental stage of coccidian parasites.