Identification of a glutathione S-transferase inhibitor in onion bulb (Allium cepa L.)

A glutathione S-transferase (GST, EC 2.5.1.18) inhibitor was isolated from the onion bulb by fractionation and purification using Sep-pak Vac C18 column, TLC and HPLC. Five partially purified onion bulb GSTs (GSTa and GSTb as minor GSTs, and GSTc, GSTd and GSTe as dominant GSTs) separated by DEAE column were used to detect the inhibitory substance in onion bulb extract. Among the three fractions of onion bulb extract, the water fraction showed the strongest inhibition on the CDNB conjugating activities of onion bulb GSTs followed by EtOAc faction. The n-hexane fraction had very low or all most no inhibition. Among the GSTs, GSTc and GSTd were found to be highly sensitive to the inhibition of the fractions; where GSTe was poorly sensitive, and GSTa and GSTb were almost insensitive to such inhibition. Substance(s) present in 45% methanol-elution of Sep-pak-Vac C18 column of water fraction was found to strongly inhibit the activity of GSTc. A yellow fraction (Rf = 0.48) on TLC representing 445 mg fresh onion bulb tissue showed 50% inhibition on the activity of GSTc. After purifying by HPLC, the inhibitory substance was identified as quercetin-3,4′-diglucoside by NMR and FAB-MS spectroscopes. The IC50s of quercetin-3,4′-diglucoside were 76.3 μM and 69.3 μM on GSTc and GSTd, respectively. Quercetin-3,4′-diglucoside showed poor inhibitory expression on GSTe, and almost no inhibition on GSTa and GSTb.