Purification of antioxidants in PLx
PLx (30 ml) was applied to a Sephadex G-10 column (i.d.
30824 mm, 582 ml) pre-equibrated with distilled water, and
eluted with distilled water at a flow rate of 0.4 ml/min. The
eluted fractions (10 ml each) were assayed for antioxidant
activity, and active fractions (zone A and B in Figure 1) were
collected, lyophilized, and dissolved in 30 ml of distilled water.
Reverse-phase (RP)-HPLC was performed on a DELTA
3000 apparatus (Waters, USA) equipped with an UV detector
operated at 280 nm. Two ml of each sample was applied to an
ODS Mightysil RP-18 GP column (5 particle size, i.d.
10250 mm, 19.6 ml, Cica MERCK, Japan) and eluted with
an acetonitrile/water linear gradient system (5–30 per cent
acetonitrile for 250 min) at a flow rate of 1.0 ml/min.