Xdb c8 column

Liquid fungal cultures (1 liter) including fungal tissue and medium were frozen using a dry ice-acetone bath and lyophilized. The combined lyophilized residues were extracted with 500 ml of ethyl acetate-methanol (9:1) for 3.5 h with vigorous stirring. Extracts were filtered over cotton, evaporated to dryness, and stored in 8-ml vials. Crude extracts were fractionated via semipreparative HPLC using an Agilent XDB C₈ column (25 cm by 10 mm, 5-µm particle diameter) with acetonitrile (organic phase) and 0.1% acetic acid in water (aqueous phase) as solvents at a flow rate of 3.2 ml/min. A solvent gradient scheme was used, starting at 5% organic for 3 min, followed by a linear increase to 100% organic over 27 min, holding at 100% organic for 5 min, decreasing back to 5% organic for 0.1 min, and holding at 5% organic for the final 4.9 min, for a total of 40 min. Further purification of fractions containing compounds 7, 9, 10, and 11 was accomplished by semipreparative HPLC using an Agilent XDB C₈ column (25 cm by 10 mm, 5-µm particle diameter) with acetonitrile (organic phase) and 0.1 M (pH 8.0) ammonium acetate in water (aqueous phase) as solvents at a flow rate of 3.2 ml/min with same gradient scheme described above.

Liquid fungal cultures (50 mL × 20), including fungal tissue and medium, were frozen using a dry ice acetone bath and lyophilized. The combined lyophilized residues were extracted with 500 mL of methanol for 3.5 h with vigorous stirring. Extracts were filtered over cotton, evaporated to dryness, and stored in 8 mL vials. Crude extracts were fractionated via semi-preparative HPLC using an Agilent XDB C-8 column (25 cm × 10 mm, 5 μm particle diameter) with acetonitrile (organic phase) and 0.1% acetic acid in water (aqueous phase) as solvents at a flow rate of 3.0 mL/min. A solvent gradient scheme was used, starting at 5% organic for 3 min, followed by a linear increase to 100% organic over 27 min, holding at 100% organic for 5 min, decreasing back to 5% organic for 0.1 min and holding at 5% organic for the final 4.9 min, for a total of 40 min. Further purification of fractions containing 1 and 6 was accomplished by semi-preparative HPLC using the same Agilent XDB C-8 column with acetonitrile (organic phase) and 0.1 M (pH 7.0) ammonium acetate in water (aqueous phase) as solvents at a flow rate of 3.0 mL/min with same gradient scheme as shown above.