Inhibitory effects of 6-O-palmitoyl-L-ascorbic acid (ASC16) on the enzymatic activity of Bothrops alternatus venom

In the anti-venom production process, it is essential to use whole venom during immunization to generate specific antibodies capable of neutralizing all venom components. However, efforts are being made to improve the traditional immunization process by reducing the toxic effects of venom components on the immunized animals while ensuring an optimal immune response in accordance with good manufacturing practices. In this context, we aimed to evaluate the capacity of ASC16 to inhibit the enzymatic activity of the main components of B. alternatus venom. In vitro studies were conducted to evaluate the inhibitory effect of ASC16 on metalloproteinases (SVMP), serine proteinases (SVSP) and phospholipases A₂ (PLA₂) using specific substrates. In vivo assays measured edema-forming activity, and histological analysis with hematoxylin and eosin staining were performed. Additionally, hemorrhage inhibition was tested using a murine model. In silico studies were also carried out using bothropasin as a model. The results of enzyme inhibition showed that ASC16 significantly inhibited SVMP (49.31% ± 0.62), SVSP (63.11 ± 3.86%) and PLA₂ activity (36.52% ± 0.09). ASC16 did not reduce edema but it significantly inhibited hemorrhage (66.32%). In silico analysis suggested that ASC16's hydrophobic portion binds to critical residues involved in catalysis of the SVMP (Glu146 and His145), while the hydrophilic fraction also interacts with the protein (Pro109, Thr110, Gly112, and Tyr132). These findings position ASC16 as a promising candidate for use as an additive inhibitor in adjuvant formulations in antivenom immunization schemes for B. alternatus.