The sample (0.2 g) was extracted with 4-ml 100% hexane twice
to remove lipid. The lipid-free residue was extracted with 4-ml
acetone/water/acetic acid (70/29.5/0.5 (v/v/v); AWA) for 2 h with
shaking at room temperature for an AWA-extractable (Free) fraction.
After centrifugation at 10,000g for 20 min at 4 C, the supernatant
was collected and the extraction repeated. Both
supernatants were pooled. A portion of pooled supernatant was
evaporated under reduced pressure (400 mTorr at 130 C) using
a cold trap (Titan™ Vapor Trap, FTS systems Inc., Stone Ridge,
NY), and then reconstituted with 100% DMSO. Another portion
was evaporated and reconstituted with sulphuric acid/methanol
(6.4 N sulphuric acid in methanol). The extraction of bound phenolics
was performed using the method described previously with
slight modification (Adom & Liu, 2002). The residue from the
AWA extraction was mixed with 4-ml 2 N NaOH for 2 h with shaking
under nitrogen gas at room temperature. After adjusting the pH
to 1 with HCl, the mixture was extracted five times with ethyl acetate.
The pooled ethyl acetate fraction was evaporated under reduced
pressure using the cold trap, and then reconstituted with
100% DMSO for a cell-wall bound (Bound) fraction. All reconstituted
solutions were stored at 20 C under nitrogen gas until
analysed.
2.4