Influence of Phenological Stage on the Phytochemical Composition and In Vitro Ovicidal Activity of Tithonia diversifolia (Hemsl.) A. Gray Ethyl Acetate Extracts Against Haemonchus contortus

Haemonchus contortus is a pathogenic gastrointestinal nematode that impairs ruminant productivity, and whose control is hindered by widespread anthelmintic resistance. Tithonia diversifolia has been proposed as a sustainable forage source rich in plant secondary metabolites (PSMs). This study evaluated the in vitro ovicidal activity and phytochemical composition of T. diversifolia hydroalcoholic extracts (HAE) across three phenological stages (28, 42, and 56 days of regrowth). Leaf material collected from Tlapacoyan, Veracruz, was extracted by sonication and maceration, followed by liquid–liquid partitioning into ethyl acetate (EtOAc–F) and aqueous (Aq–F) fractions, and the resulting samples were characterized by HPLC-PDA. Egg hatch assays were used to determine half-maximal inhibitory concentration (IC50) values against H. contortus. The hydroalcoholic extract exhibited significant ovicidal activity, with IC50 values of 6.23, 10.40, and 4.02 mg mL−1 for the 28 d, 42 d, and 56 d cuts, respectively. After partitioning, the Aq–F showed a total loss of bioactivity, whereas the EtOAc–F showed marked bioactivity enrichment, with IC50 values reduced to 0.66, 0.64, and 0.51 mg mL−1. Chromatographic analysis showed that ethyl acetate fractions retained hydroxycinnamic acids, phenolic compounds and lignans. Although the 42-day stage exhibited the greatest phytocomplexity, the 56-day stage displayed the highest ovicidal activity. This pattern suggests that, for the T. diversifolia ETOAc-F, bioactivity is more closely related to the relative abundance and identity of specific phenolic compounds and lignans, rather than phytocomplexity alone. These findings support the future standardization of T. diversifolia extracts and help define an optimal harvest window for subsequent in vivo assessments within integrated parasite management frameworks.