Isolation and Purification of Natural Compounds

The frozen animals (260.5 g, dry weight after extraction) were cut into pieces and extracted exhaustively with acetone at room temperature (2.0 L × 4). The organic extract was evaporated to give a brown residue, which was then partitioned between Et₂O and H₂O. The Et₂O solution was evaporated to give a dark brown residue (4.0 g). The obtained residue was subjected to gradient silica gel (200–300 mesh) column chromatography (CC) [Et₂O/petroleum ether (PE) 0→100%] and yielded nine fractions (Fr. 1–9). Fr. 2 was divided into three subfractions (Fr. 2A–2C) by Sephadex LH-20 CC (PE/CH₂Cl₂/MeOH, 2:1:1). Following two-stage purification including Sephadex LH-20 CC (CH₂Cl₂, 100%) and silica gel CC (300–400 mesh, PE/Et₂O 100:1→10:1), the subfraction Fr. 2C13 was purified by semi-preparative HPLC (MeCN, 100%, 3.0 mL/min) and analytical HPLC (MeOH/H₂O, 80:20, 1.0 mL/min) to yield compounds 1 (2.0 mg, t_R = 16.4 min), 2 (0.3 mg, t_R = 18.4 min) and 4 (1.0 mg, t_R = 15.3 min), respectively. Fr. 6 was split by Sephadex LH-20 CC (PE/CH₂Cl₂/MeOH, 2:1:1) to give two subfractions (Fr. 6A and Fr. 6B). Next, Fr. 6B was purified by Sephadex LH-20 CC (CH₂Cl₂, 100%), yielding subfraction Fr. 6BA. Final purification of Fra. 6BA was achieved by semi-preparative HPLC (MeOH/H₂O, 80:20, 2.8 mL/min) to afford compounds 7 (0.5 mg, t_R = 24.5 min) and 6 (1.0 mg, t_R = 22.0 min) and the mixture of compounds 3 and 5. The mixture was further purified by analytical HPLC (MeCN/H₂O, 60:40, 1.0 mL/min) to yield pure 3 (1.1 mg, t_R = 13.0 min) and 5 (1.0 mg, t_R = 14.6 min), respectively. Fr. 9 was subjected to a column of Sephadex LH-20 eluted with CH₂Cl₂/MeOH, 1:1, to yield two subfractions (Fr. 9A and 9B). Fr. 9A was first split by Sephadex LH-20 column chromatography (PE/CH₂Cl₂/MeOH, 2:1:1) to give five subfractions (Fr. 9AA–Fr. 9AE). Fr. 9AE was purified by RP-HPLC (MeCN/H₂O, 60:40, 3.0 mL/min), yielding a subfraction (Fr. 9AEC, t_R = 8.0 min). Since there are two points observed on the thin-layer chromatography (TLC), Fr. 9AEC was purified by silica gel CC (300–400 mesh, CH₂Cl₂/MeOH, 96:4) to give compounds 12 (1.0 mg) and 14 (0.5 mg). Fr. 9AD was purified with silica gel CC (300–400 mesh, Et₂O/PE, 1:1), followed by semi-preparative HPLC (MeCN/H₂O, 60:40, 3.0 mL/min) to afford 13 (5.4mg, t_R = 2.1 min), 11 (3.2mg, t_R = 8.8 min) and fraction 9ADFG (t_R = 13.7min). In a similar manner, Fr. 9ADFG was purified by silica gel CC (300–400 mesh, CH₂Cl₂/MeOH, 98:2) to give compound 10 (2.6 mg). Moreover, compound 9 (2.7 mg, t_R = 7.3 min) was obtained from Fr. 9B through Sephadex LH-20 CC (PE/CH₂Cl₂/MeOH, 2:1:1) followed by RP-HPLC (MeCN/H₂O, 60:40, 3.0 mL/min), while compound 15 (2.7 mg, t_R = 7.3 min) was obtained from the Fr. 9B through Sephadex LH-20 CC (PE/CH₂Cl₂/MeOH, 2:1:1), silica gel CC (200–300 mesh, Et₂O/PE 50%→100%), followed by RP-HPLC (MeCN/H₂O, 60:40, 3.0 mL/min).