Extraction and isolation of compounds
A sequential extraction process was used to prepare an extract of Paulownia leaves. The ground plant material was extracted with 5% methanol (v / v) in an ultrasonic bath at room temperature for 30 minutes and macerated with a magnetic stirrer at room temperature for half an hour. The contents were centrifuged at 4000 G for 10 min and filtered. The pellet was re-extracted with 30% methanol (v / v) and the third extraction was done with 70% methanol. The supernatants were combined and concentrated under reduced pressure and lyophilized. The extraction yield was 41.7%.
The crude methanol extract was purified stepwise using various chromatographic methods. First, the extract was applied to a pre-conditioned RP-C18 column (80 × 70 mm, 140 µm; Cosmosil C18-PREP; Nacalai Tesque, INC., Japan) which was then washed with 1% methanol (v / v) to remove sugars. Active metabolites were eluted with 80% methanol (v / v) to give fraction A. The yield at this stage was 44.1%.
In the next step fraction A was separated by flash chromatography on a reverse phase column (140 x 12 mm, 40 µm; Cosmosil C18-PREP, Nacalai Tesque, INC., Japan) connected to a Gilson HPLC apparatus. A linear gradient of an aqueous acetonitrile solution (2-30% v / v) containing 0.1% formic acid over 140 min was used as the mobile phase at a flow rate of 8 ml min-1 at room temperature. Subsequently, from fraction A, sub-fractions B, C and D were obtained, which respectively accounted for 23.4%, 33.6% and 36.3% of fraction A.
Isolation of compounds from subfractions was made on column Atlantis Prep T3, C18 5µm, 10x250 mm (Waters) by preparation chromatography using isocratically different concentration of methanol. From subfraction B two compounds were purified, from C three compounds, while from D one compound.