Subhee Azz-Aldeen Azz-Aldeen Noor, Kıyan Hülya Tuba, Percin Ozkorucuklu Sabriye, Yıldız Mehmet Zülfü, İğci Naşit, Karış Mert, Gurel Gurevin Ebru
Department of Biology, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul 34116, Türkiye.
Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskisehir 26470, Türkiye.
ACS Omega. 2025 Jun 12;10(24):25686-25696. doi: 10.1021/acsomega.5c01428. eCollection 2025 Jun 24.
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 of 281.30 μg/mL. The ethyl acetate fraction also showed a reducing effect with an EC 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.
本研究考察了钝尾巨蝰毒液的粗提物及其不同溶剂萃取物的抗氧化性能和酶（乙酰胆碱酯酶、丁酰胆碱酯酶）抑制特性，并采用液相色谱 - 串联质谱（LC - MS/MS）分析研究了其萃取物（乙酸乙酯、甲醇和水）的蛋白质组组成。结果揭示了许多肽和蛋白质家族，表明其具有相当大的生物潜力。在粗毒液中鉴定出了属于八个家族的19种独特蛋白质，在特定萃取物中还有其他鉴定结果。尽管蛇毒中的主要生物活性分子是蛋白质和肽，它们易溶于水基溶剂且可通过色谱法分离，但在此我们也想知道具有不同极性和溶解特性的非蛋白质成分是否也对其活性有贡献。为此，进行了用极性递增的溶剂进行连续萃取。利用金属螯合、还原能力评估、2,2'-联氮双（3 - 乙基苯并噻唑啉 - 6 - 磺酸）（ABTS）以及2,2 - 二苯基 - 1 - 苦基肼（DPPH）的自由基清除能力等技术，确定甲醇和乙酸乙酯萃取物表现出抗氧化活性，而其他萃取物的影响极小。钝尾巨蝰毒液甲醇萃取物表现出显著的金属螯合能力（60.77 ± 0.10%），并在EC为281.30 μg/mL时表现出显著的抗氧化活性（还原能力）。乙酸乙酯萃取物在EC为263.20 μg/mL时也表现出还原作用。在DPPH自由基清除试验和ABTS自由基阳离子脱色试验中，所有萃取物和粗毒液均未表现出抗氧化活性。乙酸乙酯萃取物表现出显著的乙酰胆碱酯酶（AChE）抑制活性（75.39 ± 0.30%），与多奈哌齐（98.60 ± 0.10%）相当；然而，其丁酰胆碱酯酶（BuChE）抑制活性降低（20.65 ± 4.50%）。结果突出了钝尾巨蝰毒液萃取物作为生物活性化学物质来源的潜力以及非多肽成分对其活性的可能贡献。
