Peptídeos ovicidas de Moringa oleifera Lamarck: isolamento, caracterização bioquímica e alvos de ação no modelo biológico Haemonchus contortus

Abstract

Haemonchus contortus is the most dominant endoparasite of sheep and goats, causing huge economic losses. Several anthelmintics have been used to minimize the losses caused by parasitic infection. However, anthelmintic resistance in nematodes has become a major practical problem in many countries and has stimulated the search for alternative and environmentally acceptable control methods, such as bioactive compounds from medicinal plants. The current study reports on the purification and partial characterization of bioactive peptides from Moringa oleifera seeds with ovicidal activity against H. contortus. The purification protocol entailed obtaining an aqueous extract, steps of separations by membranes (cut off 12 and 2 kDa) and ion exchange chromatography. The protein material obtained was subjected to egg hatch assay, flocculant activity, protease inhibition and resistance to proteolysis. Subsequent characterization was performed using SDS-PAGE electrophoresis, high performance liquid chromatography (HPLC) and circular dichroism (CD). The aqueous seed extract from M. oleifera seeds exhibited ovicidal effect inhibiting 96 ± 0.8% of egg hatching rate of H. contortus. Anthelmintics peptides, isolated from the total aqueous extract showed 82 ± 1% ovicidal activity (EC90 of 202.3 µg) and presented a single band of apparent molecular mass of 6.5 kDa under reducing conditions. Furthermore, they showed in vitro flocculant effect (EC90 of 0.812 µg), trypsin inhibitor activity (41 ± 11%), papain inhibitor activity (99 ± 0.2%) and were highly resistant to proteolysis, remaining active even after 24 hours of incubation with pronase E. The peptides were adsorbed on cationic resin and separated in least four isoforms of similar molecular masses (6.5 kDa) distributed by SDS-PAGE, and high purity detected by HPLC. Circular dichroism analysis revealed that three of the isoforms have content of 70% random coil structures, while the fourth isoform found showed α-helix rich structures (69%). Finally, studies involving changes in ionic strength of the medium indicated that electrostatic interactions may be a common mechanism between flocculant and ovicidal activity of the peptides. The results obtained demonstrated the discovery of peptides from M. oleifera with important anthelmintic properties and suggest an affordable and sustainable option for alternative parasite control.