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.