ISSN print edition: 0366-6352 ISSN electronic edition: 1336-9075 Registr. No.: MK SR 9/7 Published monthly

Optimized purification and detection of PLA2 enzymes in snake venom and snake wine: a cost-effective approach for bioactive compound analysis

Wanita Donwang, Thitima Rujiralai, and Wilairat Cheewasedtham

Analytical Chemistry and Environment Research Unit, Division of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani, Thailand

E-mail: wilairat.c@psu.ac.th

Received: 19 March 2025  Accepted: 18 July 2025

Abstract:

Snake wine and other venom-infused alcoholic beverages are sold across Asia, where many people believe in their health benefits despite occasional cases of poisoning. However, regulating these products poses a challenge for authorities, as existing toxin detection methods are costly and time-consuming. This study presents a simple and low-cost method for detecting and measuring phospholipase A2 enzymes (PLA2s), key toxic enzymes found in snake venom. PLA2s were extracted from monocled cobra (Naja kaouthia) venom and commercial snake wine using a three-phase partitioning (TPP) method. The extracted PLA2s were analyzed by high-performance liquid chromatography. Optimization revealed that trifluoroacetic acid was the most effective purification solvent and the TPP conditions involved 60% (w/v) ammonium sulfate with tertiary-butanol, followed by centrifugation at 600 g and separation in an Apollo C18 column with detection at 220 nm. The calibration curve was linear in the range of 50–500 µg mL⁻¹ (y = 11.836x – 112.48, R² = 0.9978). Recoveries and coefficient of variation ranged from 89.4 to 98.3 and 0.3 – 5.9%, respectively. The limit of detection was 5.276 µg mL⁻¹. This validated method enables rapid, accurate monitoring of PLA2s in snake venom-infused alcoholic products, supporting efforts to improve public safety and product regulation.

Keywords: Snake venom; Phospholipases A2; Three-phase partitioning; Ethanolic extract; Energy drink; Halal

Full paper is available at www.springerlink.com.

DOI: 10.1007/s11696-025-04262-7

Chemical Papers 79 (11) 7437–7446 (2025)