Anthelmintic-like activity of polyphenolic compounds and their interactions against the cattle nematode Cooperia...

Polyphenolic compounds (PCs) have been proposed as one of the most bioactive group of secondary metabolites occurring in nature and have been associated to anthelmintic (AH)-like activity of plants against cattle nematodes. However, little is known regarding their synergetic / antagonistic interactions. This study assessed the in vitro AH-like activity of commercial PCs: quercetin, caffeic acid, rutin and coumarin, and their combinations against the egg hatching and larval exsheathment of Cooperia punctata; one of the most prevalent nematodes affecting grazing cattle in tropical regions. The molecules selected for the in vitro analysis were identified as bioactive phytochemicals of plants through bio-guided fractionation in previous studies. To estimate mean effective concentrations (EC₅₀) five increasing concentrations were used for both Egg hatching inhibition assay (EHIA) and larval exsheathment inhibition assay (LEIA) (0.6-9.8 mg mL^-1 and 0.15-2.4 mg mL^-1, respectively). From the four molecules, only rutin did not affect egg hatching; while quercetin, showed no bioactivity against eggs or larvae (P > 0.766 and P > 0.621, respectively). Best-fit EC₅₀ estimated through the EHIA was considered for PCs classification as bioactive (coumarin and caffeic acid) and non-bioactive (quercetin and rutin). Phytochemical interactions were subsequently assessed combining bioactive:non-bioactive PCs (8:2 ratio), and the nature of their interaction was classified using the fractional inhibitory concentration index (FIC_index). Combinations had a highly synergistic interaction against larval exsheathment (FIC_index < 0.5) except for coumarin:rutin against egg hatching (FIC_index> 0.5). Quercetin and rutin acted as PCs AH-like activity enhancers, reducing EC₅₀ of bioactive molecules in a range of 43%-64% and 68%-83% for EHIA and LEIA, respectively. A linear relationship between low molecular weight of molecules and ovicidal activity was observed; where, molecules with lower molecular weight displayed better-fit EC₅₀ for ovicidal activity. Furthermore, coumarin and caffeic acid bioactivity against free-living stages of C. punctata makes them suitable candidates as markers for anthelmintic-like activity in bioactive forages. Combinations used through this investigation showed a potent anthelmintic-like activity against free-living forms of C. punctata, representing a first step towards the identification of promising alternatives for nematode control.