The acetone extracts were filtered through a 0.2 μm Minisart SRP 15 filter (Sartorius Stedim Biotech GmbH) and subjected to HPLC gradient analysis (a Spherisorb S5 ODS-2 250×4.6 mm column with an S5 ODS-2 50× 4.6 mm precolumn, Alltech Associates, Inc.), using two solvents: solvent A- acetonitrile/methanol/water (100/10/5, v/v/v); and solvent B- acetone/ethyl acetate (2/1, v/v), at a flow rate of 1 ml min−1.