Phytochemical profiling and anticoccidial activity of Syzygium cumini and Trachyspermum ammi extracts against Eimeria zuernii: Integrated in vitro, in vivo, and in silico approaches

Eimeria zuernii is one of the most pathogenic protozoan parasites causing coccidiosis in livestock. This study evaluated the anticoccidial potential of aqueous and methanolic extracts of herbal mixture of Syzygium cumini and Trachyspermum ammi against E. zuernii. Phytochemical analysis, in vitro sporulation inhibition (SPI), and in vivo fecal oocyst count reduction (FOCR) assays were conducted. Mass spectrometry analysis identified a higher number of phytochemicals in the methanolic extract (28) compared to the aqueous extract (16), including astragalin, malvidin 3-laminaribioside, and myricetin. In the SPI assay, the methanolic extract exhibited superior efficacy, achieving 81.33 % inhibition at 500 µg mL⁻¹ compared to 74 % for the aqueous extract and 87 % for sulphadimidine. In the FOCR trial, 130 naturally infected calves were divided into five groups (n = 26) and treated orally with methanolic extract (500 µg kg⁻¹, 2 mg kg⁻¹, and 4 mg kg⁻¹), sulphadimidine (1.2 mL kg⁻¹), or water. The 4 mg kg⁻¹ methanolic extract dose achieved the highest efficacy, with a 91.3 % reduction in oocyst counts on day 15 post-treatment. Molecular docking of phytochemicals against lactate dehydrogenase (LDH) revealed astragalin as the top binder, showing strong affinity comparable to the natural inhibitor NAD. These findings validate the traditional use of these plant extracts as natural remedies for E. zuernii in calves, particularly in resource-limited settings, and highlight astragalin as a potential lead compound for future drug development. Large-scale field trials are recommended to assess the commercial applicability of these extracts.