*3.3. Extraction and Isolation*

The sponge material (dry weight 216 g) was chopped into pieces and extracted with EtOH at room temperature. The ethanol soluble materials (21.0 g) were obtained after concentration of the extract was dissolved in distilled water (300 mL) and partitioned in turn with CHCl3 (200 mL × 3). Evaporation of the chloroform extracts gave dark green gum (12.7 g) that was further separated on a silica gel column (7 cm × 10 cm) using the mixtures of CHCl3/EtOH as stepwise eluent systems to yield the fractions A–F (Figure S1).

Fraction D (301.4 mg), eluted with CHCl3/EtOH (15:1), was separated by column chromatography (CC) (YMC ODS-A, 15 mm × 100 mm, MeOH/H2O, 90–100%) to yield three subfractions (D1–D3). The subfraction D1 (128.5 mg) was further subjected to silica gel CC (hexane/EtOAc stepwise systems) to obtain three subfractions. The subfraction D1.2 (17.0 mg) was purified by normal-phase HPLC (Ultrasphere-Si, hexane/EtOAc, 1:1, 2 mL/min) to yield compound **13** (6.3 mg, 0.003%). Subfraction D1.3 (6.6 mg) was separated by reverse-phase HPLC (YMC-Pack ODS-A, 10 mm × 250 mm, 95% MeOH, 2 mL/min) to obtain compounds **1** (0.8 mg, 0.0004%), **3** (0.4 mg, 0.003%) and **7** (1.8 mg, 0.012%).

Fraction E (904.4 mg), eluted with CHCl3/EtOH (10:1), was fractionated by silica gel CC (25 mm × 60 mm, hexane/EtOAc, 1:1) to yield four smaller fractions. Fraction E2 (227.4 mg) was subjected to normal-phase HPLC (Ultrasphere-Si, hexane/EtOAc, 1:1, 2 mL/min) resulting in three subfractions. The first subfraction E2.1 (4.0 mg) was purified using reverse-phase HPLC (YMC-Pack ODS-A, 4.6 mm × 250 mm, 90% EtOH, 0.6 mL/min) to afford compound **2** (0.8 mg, 0.0004%) and to re-isolate metabolite **13** (1.1 mg). As a result of the process of E2.2 (22.9 mg) reverse-phase separation (YMC-Pack ODS-A, 10 mm × 250 mm, 2 mL/min) in 95% MeOH, the compounds **4** (1.0 mg, 0.0005%), **6** (2.4 mg, 0.001%), **8** (6.3 mg, 0.003%), **10** (2.0 mg, 0.0009%) and **12** (1.7 mg, 0.0008%) were obtained. The last mentioned HPLC procedures and 94% MeOH as eluent were used for the subfraction E2.3 that allowed us to isolate the individual steroids **5** (6.8 mg, 0.003%), **9** (3.3 mg, 0.002%), **11** (8.0 mg, 0.004%), **14** (1.3 mg, 0.0006%) and to gain new portions of the metabolites **3** (5.8 mg) and **7** (24.3 mg).
