Isolamento e caracterização das atividades anticoagulante e anti-inflamatória da crotoxina de crotalus durissus ruruima

Abstract

Crotalus durissus ruruima (C.d.r) is a rattlesnake species found in the northern region of Brazil, whose, among the various toxins present in its venom, crotoxin (CTX) is the most abundant in its venom. Studies have shown that CTX exhibits diverse biological activities, including anti-inflammatory and anticoagulant effects. However, CTX from the Crotalus durissus ruruima subspecies has not yet been isolated or tested for its potential biological properties. Therefore, the main objective of the present study was to evaluate the anti-inflammatory and anticoagulant activities of CTX isolated from C. d. ruruima in an in vitro model. CTX was purified from crude venom through a two-step liquid chromatography process: size-exclusion chromatography (Superdex 75 GL) followed by anion-exchange chromatography (Mono Q GL). Chromatographic procedures were monitored by SDS-PAGE electrophoresis and phospholipase activity assay. To assess the cytotoxic effect of CTX on peripheral blood mononuclear cells (PBMCs), the MTT reduction assay was employed to determine cell viability. The antiinflammatory activity was evaluated by treating PBMCs with CTX followed by stimulation with the pro-inflammatory agent lipopolysaccharide (LPS). After incubation, culture supernatants were collected for quantification of cytokines IL-1β and TNF-α. For anticoagulant activity assessment, platelet-poor plasma was incubated with different concentrations of CTX (3.0; 1.5; 0.75; 0.375; and 0.1875 µg/mL) and tested using the Prothrombin Time assay. Following purification steps, SDS-PAGE analysis revealed bands at approximately 9 kDa and 15 kDa, corresponding to the two subunits of the toxin (CA and CB), along with confirmed phospholipase activity. Cell viability assays indicated that CTX at concentrations of 0.01 µg/mL and 0.05 µg/mL were non-cytotoxic, with 0.01 µg/mL being the most suitable dose. Anti-inflammatory activity assessment showed that both CTX concentrations reduced TNF-α production, while 0.01 µg/mL also reduced IL-1β levels, making it the most effective dose. Regarding anticoagulant activity, the 3 µg/mL dose significantly prolonged clotting time. In conclusion, CTX isolated from C. d. ruruima demonstrated both anti-inflammatory and anticoagulant activities, suggesting its potential as a therapeutic agent for inflammatory disorders. Keywords: Crotoxin; Anti-inflammatory; Anticoagulant; Purification.