Briefly, 0.2 g of lyophilized kale sample was extracted three
times with boiling methanol (2 mL, 70%). The known amount of
glucotropaeolin (0.2 mL, 5 mM; 99%, AppliChem, Germany) was
added to each sample just before the first extraction as an internal
standard for the HPLC analysis. The extract portions were combined
to give a final volume of 6 mL. The extracted GLS were purified on
column filled with 0.5 mL of DEAE Sephadex A-25 anion-exchange
resin (Sigma Aldrich, Germany). The column was washed with 2 mL
imidazole formate (6 M) and twice with 1 mL Millipore water and
then loaded with 6 mL of each extract. Afterward sulphatase water
solution (1.67 mg/mL, 250 mL) (Helix pomatia type H1 Sigma, Germany)
was introduced onto the column and the columns were
incubated for 12 h at room temperature. Next day, the desulfoeGLS
were eluted with deionized water (2 0.75 mL) and injected
(50 mL) into HPLC.