Integrated Phytochemical, Computational and In Vitro Evaluation of the Oocysticidal Activity of Pleurotus ostreatus...

Cryptosporidiosis is a significant zoonotic parasite disease caused by Cryptosporidium parvum, impacting both humans and animals, with little therapeutic alternatives available. This work examined the phytochemical composition, antioxidant capacity, in silico antiparasitic potential, and in vitro oocysticidal activity of methanolic Pleurotus ostreatus extract (POE). Fruiting bodies extract was analyzed for phytochemical constituents. The total amounts of phenolics, flavonoids, tannins, and saponins were quantified spectrophotometrically, while antioxidant activity was evaluated by the reducing power test. Gas chromatography-mass spectrometry (GC-MS) chemical profiling revealed 18 identified compounds. Molecular docking was performed on three validated targets of C. parvum: N-myristoyltransferase (NMT), calcium-dependent protein kinase 1 (CDPK1), and lysyl-tRNA synthetase (KRS) to assess possible antiparasitic interactions. Several sterol derivatives produced favorable predicted binding scores, including Ergost-7-en-3-ol (3β) and ergosterol toward NMT (- 9.87 and - 9.28 kcal/mol, respectively), compared with nitazoxanide under the docking conditions. Interaction profiling identified possible ligand-target interactions with proteins involved in protein lipidation, Ca²⁺-dependent signaling, and translation pathways, but these mechanisms remain computational hypotheses pending biochemical validation. ADMET predictions suggested minimal systemic absorption for sterols, which may favor gut-localized activity and prolonged intestinal exposure against C. parvum while minimizing systemic toxicity. In vitro studies revealed a concentration- and time-dependent decrease in C. parvum oocyst numbers and viability. At a concentration of 1000 µg/mL, POE diminished oocyst counts by nearly 71% after 48 h and lowered viability to 37.41%, demonstrating greater reduction of oocyst count and viability than nitazoxanide under comparable conditions with LC₅₀ of 460 µg/mL. These findings collectively underscored P. ostreatus as a potential natural antiparasitic agent, indicating that its sterol-rich extract may demonstrate oocysticidal activity via multi-target molecular interactions and direct impacts on oocyst integrity.