Desenvolvimento e avaliação de antídotos para envenenamento pela serpente Bothrops brazili: investigação do potencial antiofídico de ácidos fenólicos e obtenção de um sistema nanoparticulado de quitosana para produção de antissoros

Abstract

Ophidism causes high mortality and morbidity in many regions of the world. Currently, the main treatment is serum therapy, which has some limitations, such as: high production cost, low effectiveness in neutralizing local effects, difficult access in some regions and adverse reactions. Considering these factors, this study has the general objective of presenting new alternatives to improve the treatment of snakebite. For this purpose, Bothrops brazili snake venom was used, a little studied species that is found mainly in the Amazon region. Thus, the present study has as specific objectives (1) to analyze the inhibitory potential of chlorogenic and rosmarinic acids against the local and systemic effects induced by B. brazili envenomation, (2) to obtain and characterize chitosan nanoparticles associated with the venom of this species through two modes (incorporation and adsorption), in order to be evaluated in relation to the immunoadjuvant potential for the production of a new serum. The evaluation of the antiophidic potential of chlorogenic and rosmarinic acids was carried out through in vitro, in vivo and in silico assays. In in vitro assays, the acids were able to inhibit the activity of proteases and phospholipases of the venom, in addition to reducing the pro-coagulant effect in human plasma caused by the venom. In vivo, these compounds inhibited local effects such as edema, hemorrhage, increased myeloperoxidase enzyme and myotoxicity. In silico the acids were shown to be able to interact with phospholipases. In addition, the acids mitigated the systemic effects resulting from the envenomation, which were: kidney, liver, muscle damage, hemostatic changes (partially activated thromboplastin time, prothrombin time and platelet count), hematological (erythrogram and leukogram) and lipid peroxidation. The nanoparticles were obtained by ionic gelation and presented a size between 150 and 190 nm, a potential of approximately +30 mV and a polydispersion index of approximately 0.400. The nanoparticles showed 97% efficiency to incorporate and adsorb the venom and have a homogeneous shape and size. The spectroscopy analysis of the infrared region by Fourier transform revealed that the venom showed bands at 1543 cm-1 and 1651 cm-1 referring to amide groups. The nanoparticles showed bands at 916, 1087, 1259 and 1340 cm-1 that underwent deviation after incorporation and adsorption of the venom. In conclusion, these results show that chlorogenic and rosmarinic acids have the potential to inhibit the local and systemic effects induced by B. brazili envenomation, which makes them possible alternatives for the complementation of serum therapy. Additionally, chitosan nanoparticles were efficient in incorporating and adsorbing the venom, showing great potential to be used as an adjuvant in the production of a new antivenom.