Abstract
This study examines the antioxidant and enzyme (acetylcholinesterase, butyrylcholinesterase) inhibitory properties of crude and different solvent fractions of Macrovipera lebetinus obtusa venom and investigates the proteomic composition of its fractions (ethyl acetate, methanol, and water) employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The results revealed many peptide and protein families, suggesting considerable biological potential. Nineteen unique protein identifications belonging to eight families were achieved in the crude venom, along with additional identifications in particular fractions. Although major bioactive molecules in snake venoms are proteins and peptides, which dissolve readily in water-based solvents and can be separated by chromatography, here we also wondered if nonprotein components with different polarities and solubilization characteristics may also contribute to their activities. To this end, sequential fractionation with solvents of increasing polarity was performed. Utilizing techniques such as metal chelation, reducing power assessment, 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and free radical scavenging capabilities of 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was determined that the methanol and ethyl acetate fractions demonstrated antioxidant activity, whereas other fractions displayed minimal effects. The methanol fraction of M. lebetinus obtusa venom exhibited significant metal chelation (60.77 ± 0.10%) and showed substantial antioxidant activity (reducing power) with an EC(50) of 281.30 μg/mL. The ethyl acetate fraction also showed a reducing effect with an EC(50) of 263.20 μg/mL. None of the fractions and crude venom showed antioxidant activity in the DPPH free radical scavenging assay and the ABTS(·+) radical cation decolorization test. The ethyl acetate fraction exhibited significant acetylcholinesterase (AChE) inhibitory activity (75.39 ± 0.30%) comparable to donepezil (98.60 ± 0.10%); however, it displayed reduced butyrylcholinesterase (BuChE) inhibitory activity (20.65 ± 4.50%). The results highlight the potential of M. lebetinus obtusa venom fractions as sources of bioactive chemicals and the possible contribution of nonpolypeptide content to its activities.