Purification of Sea Cucumber Bioactive Compounds

The ethanol extract of the sea cucumbers was purified by standard methodology [15 (link),18 (link)], including column hydrophobic and Si gel chromatography. For the latter stage, the stepwise gradient of solvent systems CHCl3/EtOH/H2O: 100:100:17 → 100:125:25 → 100:150:50 as mobile phase was applied, followed by the additional purification of the obtained fractions with CHCl3/EtOH/H2O (100:125:25) as the mobile phase. Finally, the subfractions: I.0 (22 mg), I.1 (120 mg), II (286 mg), III.1 (66 mg), and III.2 (177 mg) were isolated [15 (link)]. HPLC of the subfraction II on the silica-based column Supelcosil LC-Si (4.6 × 150 mm, 5 µm) with CHCl₃/MeOH/H₂O (55/30/4) as the mobile phase resulted in the isolation of two fractions (II.1 and II.2). The subsequent HPLC of fraction II.2 on the Supelco Ascentis RP-Amide (10 × 250 mm) column with MeOH/H₂O/NH₄OAc (1 M water solution), ratio (60/38.5/1.5), as the mobile phase led to the isolation of four subfractions. The re-chromatography of two of them on a Diasfer 110 C-8 (4.6 × 250 mm) column with MeOH/H₂O/NH₄OAc (1 M water solution) (50/48/2) as the mobile phase applied for the separation of each subfraction, resulted in the isolation of chilensosides E (1) (2.2 mg, R_t 14.12 min) and F (2) (2.8 mg, R_t 17.25 min). The subfraction III.2 was submitted to HPLC on a Supelco Ascentis RP-Amide (10 × 250 mm) column with CH₃CN/H₂O/NH₄OAc (1 M water solution), ratio (30/68/2), as the mobile phase to give two main fractions and some minor ones. The repeated HPLC of one of the main fractions in the same conditions led to the isolation of 11.0 mg of chilensoside G (3) (R_t 16.67 min).