Supernatants were filtered using Sep-pak C18 filters(Waters) and 10 ml were passed through a cation exchange column
(4 g, Dowex 50W-X8, 50–100 mesh,H+form), washed with water to a total volume of 25 ml
and filtered through a 0.45-mm membrane, sealed in
vials and stored at -20C until analyzed. Solutions were filtered again through a 0.45-mm Millipore filter prior to HPLC analysis (Medlicott et al., 1985).