Antiparasitic activity of Colombian Amazon palm extracts against Giardia lamblia trophozoites: Insights into cellular death mechanisms

Juan Javier García-Bustos ^1,2 Gabriel Luna Pizarro ³ Rocío G Patolsky ³ Mariana Belén Joray ⁴ Vivian Villalba-Vizcaino ² Paula Galeano ¹ Fabián Espitia-Almeida ^5,6 Marco Correa Múnera ¹ Mehmet Oztürk ⁷ Andrea Ropolo ³ Constanza Feliziani ³ MARIA Carolina TOUZ ^3* Jeronimo Laiolo ^3,8*

• ¹ Universidad de La Amazonia, Florencia, Caquetá, Colombia
• ² Universidad del Magdalena SUE-Caribe, Santa Marta, Magdalena, Colombia
• ³ Instituto de Investigación Médica Mercedes y Martín Ferreyra, Consejo Nacional de Investigaciones Científicas y Técnicas (INIMEC-CONICET), Universidad Nacional de Córdoba, Córdoba 5016, Argentina., Córdoba, Córdoba, Argentina
• ⁴ Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas, Consejo Nacional de Investigaciones Científicas y Técnicas (CIDIE-CONICET-UCC), Universidad Católica de Córdoba, Córdoba, Córdoba, Argentina
• ⁵ Universidad Simón Bolívar, Barranquilla, Colombia
• ⁶ Universidad del Atlántico, Puerto Colombia, Colombia
• ⁷ Mugla Sitki Koçman University, Mugla, Türkiye
• ⁸ Universidad Católica de Córdoba, Córdoba, Córdoba, Argentina

Colombian plants have a long history of use in traditional medicine and ethnopharmacology, particularly for treating stomach pain, digestive issues, diarrhea, and other gastrointestinal disorders. Recent studies have renewed interest in their potential therapeutic properties. This study evaluated the giardicidal activity of 15 crude extracts from plants native to the Colombian Amazon against Giardia lamblia, the protozoan responsible for giardiasis-a prevalent enteric disease affecting populations worldwide. The MTT colorimetric assay was employed to evaluate the effectiveness of these extracts at a concentration of 500 µg/mL against Giardia WB/1267 trophozoites (genotype A). Extracts demonstrating significant activity were further analyzed to determine their half-maximal inhibitory concentration (IC50). Among the tested extracts, the Attalea butyracea fruit extract (P-2) exhibited the highest activity against WB/1267 (IC50 = 62.10 ± 6.57 µg/mL) and demonstrated giardicidal activity against GS/M (IC50 = 100.90 ± 3.40 µg/mL, genotype B) human infecting strains. These results prompted a detailed investigation into its mechanism of action using the WB/1267 strain as a model. At its IC50 concentration, P-2 primarily exerted its antiproliferative effect by induction of early apoptosis. A notable increase in late apoptosis and necrosis was observed at 2xIC50. Immunofluorescence assay (IFA) and confocal microscopy revealed chromatin condensation in treated trophozoites, while flow cytometry indicated G1/S cell cycle arrest. Furthermore, exposure to P-2 led to oxidative stress, evidenced by a significant increase in reactive oxygen species (ROS). The extract's ability to disrupt various structural components of the parasite was confirmed through IFA and transmission electron microscopy. Interestingly, the P-2 extract effectively synergized with the first-line drug metronidazole against Giardia WB/1267 trophozoites. These findings underscore the therapeutic potential of Colombian plant extracts in treating giardiasis, particularly highlighting the novel giardicidal activity of Attalea butyracea fruit extract and its promise for further therapeutic development.