4.5. Enzymatic Deglycosidation of Cyclic Diarylheptanoid Glycosides

Enzymatic hydrolysis was done using β-glucosidase (almond) to obtain the aglycone by deglycosidation of cyclic diarylheptanoid glycosides [20] with an exception for compounds 12 and 15. The enzyme hydrolysis was conducted as follows: 21.0 mg (0.040 mmol) of a compound was dissolved in 3.0 mL of 0.2 M acetate buffer (0.2 M acetic acid + 0.2 M sodium acetate, pH 4.4). The solution was treated with 40 mg of β-glucosidase and stirred. The mixture was incubated while stirring in the ultrasonic water bath at 38 °C for 2 days. After incubation, the reaction mixture was mixed with 10 mL of absolute EtOH and evaporated to dryness by using a vacuum rotary evaporator at 40 °C. The residues were dissolved in CHCl₃/H₂O (1 + 1), thoroughly mixed, left to settle and finally the two phases were separated. The obtained upper and lower phase were dried and separately purified by semi-preparative HPLC method eluted with water (A) and MeOH (B), flow rate: 2 mL/min, gradient: 60% B (10 min) 60–100% B (1 min) → 100% B (4 min). The collected peaks from HPLC purification of the upper and lower phase were dried under nitrogen stream and subjected to ¹H-NMR measurement. Prior to ¹H-NMR measurement, the samples were dissolved in 0.6 mL deuterated methanol, filled in the NMR tubes followed by measurement at 300 MHz, 298 K. The recorded ¹H-NMR spectra data of the obtained peaks were compared with the existing data to identify which peak stands particularly for the aglycone.