2.5 Purification of compound A
Compound A was extracted by 20 L ethyl acetate. The organic
phase was collected by centrifugation (12,000 g, 4 °C, 10 min) and
evaporated by a rotary evaporator (-0.1Mpa, 40 °C). About 50 ml of a
brown oily liquid crude extract was finally obtained. Then, the crude
extract was diluted three fold with methanol and separated by the
preparative Agilent 1200 LC system (Waldbronn, Germany) equipped with a
preparative Kromasil column (10 mm × 250 mm, Bohus, Sweden). Mobile
phase A was 100% water and mobile phase B was methanol. A linear
gradient of 80% B to 100% B in 30 min at 10 mL/min was chosen.
Collected elutes from 22.4 to 24.4 min was stored at 4 °C overnight for
precipitation. The solid as precipitated from the elutes was collected
by centrifugation (12,000 g, 10 min), re-dissolved with methanol, and
subjected to the preparative Agilent 1200 LC system equipped with a
preparative ODS-BP C18 column (10 mm × 250 mm, Dalian, China). Mobile
phase A was 100% water and mobile phase B was methanol. A linear
gradient of 80% B to 100% B in 30 min at 2.5 mL/min was conducted.
Elutes from 21.0 to 21.6 min was collected, and 2.4 mg of purified
compound A was finally obtained.