Two in vitro anthelmintic assays of four Paraguayan medicinal plants for proof of concept of the role of polyphenols in their biological activities and LC-HRMS analysis

Ethnopharmacological relevance: Gastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Hae-monchus spp. , and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. The aims of our study: Four medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occi-dentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. Materials and methods: To evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without poly-vinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). Results: C. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42-41.80 mu g/mL) and A. absinthium on egg hatching processes (EC50 = 121.70-137.34 mu g/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus , and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20-67.00% of larvae exsheathment, (p < 0.05) and 40.51-24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. Conclusions: The present study demonstrated that C. occidentalis, A. absinthium, and K. odorata , which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.