*3.3. Extraction and Isolation*

The fresh animals (2.2 kg) were chopped into small pieces and extracted four times by MeOH with heating. The MeOH extract was evaporated *in vacuo*, and the residue (37 g) was dissolved in H2O (1.3 L). The H2O-soluble fraction was passed in two portions through an Amberlite XAD-2 column (7.5 × 28 cm) and eluted with distilled H2O until a negative chloride ion reaction was obtained, followed by elution with EtOH. The combined EtOH eluate was evaporated to give a brownish material (4.4 g). The resulting total fraction was chromatographed on a Si gel column (6.5× 15 cm) using CH3Cl-EtOH (stepwise gradient, 3:1→1:2, *v*/*v*), EtOH, and EtOH-H2O (stepwise gradient, 20:1→9:1, *v*/*v*) to give ten main fractions (1−10). Fractions 4 and 5 mainly contained the mixtures of polyhydroxylated steroids based on TLC data on Si gel plates in the eluent system toluene-EtOH (9:5, *v*/*v*). HPLC separation of fraction 4 (110 mg) on a Discovery C18 column with 55% aq. EtOH (1.5 mL/min) as an eluent system yielded pure **5** (4.1 mg, tR 48.8 min) and subfraction 4.1 and 4.2 that were further purified on a YMC-Pack Pro C18 column with 78% aq. MeOH (0.9 mL/min) as an eluent system to give pure **2** (0.9 mg, tR 15.3 min), **3** (0.9 mg, tR 14.1 min), and **4** (1.1 mg, tR 10.4 min). HPLC separation of fraction 5 (182 mg) on a Discovery C18 column with 55% aq. EtOH (1.5 mL/min) as an eluent system gave pure **1** (49.6 mg, tR 21.0 min) and **6** (23.6 mg, tR 26.1 min).
