1.The supernatant liquid was then loaded onto an Amberlite XAD-2 column (sigma, 30x7 cm )and eluted with 2.0 liters of the following gradient: (1) water, (2) 10% aqueous MeOH, and (3) 30% aqueous MeOH, (4) 50%queous MeOH, (5) 70% aqueous MeOH, (6) 90% aqueous MeOH, (7) MeOH and (8) 氯仿
2.All of the fractions were concentrated under reduced pressure to provide the crude material. As a result of activities observed in fractions 5 through 8, these fractions were combined and loaded onto a YMC-ODs-120A (reverse-phase silica gel, C18; 20 X 2.5 cm)(50微米) eluting with the following gradient, the fraction number in the table being indicated with the same numbers: (1.1) 20% aqueous MeOH, (2.1) 40% aqueous MeOH, (3.1) 60% aqueous MeOH, (4.1) 80% aqueous MeOH. and (5.1) MeOH. 3.Further separation of the combined fractions of 2.1 and 3.1 by 5 HPLC reverse-phase silica gel (C8,250 x 10 mm,10微米0, 40% MeOH: 50% MeOH: 10% MeCN: H2O: MeOH, at a flow rate of 2.0 milliliter per minute and UV detection at 220 nrn) provided five additional major fractions 1.2-5.2.