Analysis of cytotoxicity and assessment of α-glucosidase enzyme activity and stability in betaine-based eutectic...

Betaine-based deep eutectic solvents (DES) have gained attention for their potential to enhance protein and enzyme stability, particularly when combined with polyols. In this study, nine novel betaine-based DES-using ethylene glycol or glycerol at various molar ratios-were synthesized and characterized. Their rheological behaviour, cytotoxicity, and effects on the expression of pro-inflammatory genes under cellular stress were evaluated. Based on these assays, a non-cytotoxic concentration was selected for subsequent studies on α-glucosidase activity in the presence of each DES, as well as for evaluating enzyme storage stability at different temperatures and time periods. To further explore enzyme stabilization, the most promising system from each category was chosen to assess the impact of DES concentration and to evaluate residual enzyme activity following exposure to elevated temperatures. All synthesized DES were monophasic, homogeneous, and transparent liquids. Importantly, these systems exhibited low cytotoxicity, improved compared to their individual components. α-Glucosidase activity was enhanced in the presence of all DES compared to conventional buffer, with certain DES promoting greater thermal stability during prolonged storage. Notably, the betaine:glycerol (1:6) system at 15.0 g/L not only provided the highest enzymatic activity but also preserved a slight residual α-glucosidase activity after incubation at 60 °C and 80 °C, while maintaining full activity at 40 °C. These results highlight the potential of betaine:glycerol DES, particularly at optimal concentrations, as effective media for improving the stability and performance of enzymatic systems.