3.2.1. Crude Extract Preparation and Effective Compound Isolation

One gram of *A. keiskei* roots extracts was dissolved in acetone and coated in silica gel (1.5 g). The sample was subjected to medium-pressure liquid chromatography (MPLC, Isolera ONE, Uppsala, Sweden) on a silica SP column (Daiso gel, 50 μm, 10 g, column volume: 25 mL) with a fixed flow rate of 10 mL/min. The step-gradient of purification method was used to obtain the 0%, 20%, 30%, 40%, 60%, 80% and 100% fraction ethyl acetate solvents. A total volume of 100 mL was collected for each of the 6 ethyl acetate (EA) fractions (20 mL/tube). Tube numbers 12 to 15 were loaded on the same silica column for HPLC (250 × 10 mm, Luna 5μ Silica (2) 100 Å). The eluting solvent was ethyl acetate-hexane with a ratio of 20:80. At the flow rate of 4.0 mL/min, we were able to isolate 4-hydroxyderricin (**7**), xanthoangelol (**1**), laserpitin (**16**) and isolaserpitin (**17**). Similarly, HPLC was performed on tube numbers 18 to 23 on the same silica column. EA-hexane with a ratio of 40:60 was used as the eluting solvent. With the flow rate of 4.0 mL/min, we successfully isolated xanthoangelol D (**3**) and xanthoangelol H (**6**). All of the compounds were identified through HR-MS and NMR and their identities were confirmed via comparison with the literature values.
