Application of hydrophobic eutectic solvent in efficient biotransformation of total flavonoids of Herba Epimedii

Icaritin, a hydrolysate from total flavonoids of Epimedii (TFE), which has better anti-hepatoma activity than its glycosylated form. In this work, immobilized enzymes 4LP-Tpebgl3@Na-Y and DtRha@ES-107 were used to hydrolyze TFE to prepare icaritin. Five different hydrophobic deep eutectic solvents (HDES) were prepared and the most ideal HDES was successfully selected, which was composed of dodecyl alcohol and thymol with the molar ratio of 2:1. The relative enzyme activity of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 was about 102.4% and 112.5%, respectively. In addition, the thermal and binding stability of 4LP-Tpebgl3@Na-Y and DtRha@ES-107 in HDES was not affected negatively. In the biphasic system composed of 50% (v/v) HDES and Na2HPO4-citric acid buffer (50mM, pH 5.5), 4LP-Tpebgl3@Na-Y (1.0U/mL) and TFE (1g/L) were reacted at 80oC for 1h, and then reacted with DtRha@ES-107 (20U/mL) at 80oC for 2h. Finally, TFE was completely converted to 301.8mg/L icaritin (0.82mM). After 10 cycles, 4LP-Tpebgl3@Na-Y/DtRha@ES-107 still maintained 84.1% original activity. In this study, we developed an efficient methodology for icaritin preparation through the integration of enzymatic catalysis and adsorption separation, presenting a viable approach for large-scale, cost-effective production of icaritin.