Molecular interaction between andrographolide and glutathione follows second order kinetics

The intracellular level of glutathione (GSH) was significantly decreased after the addition of andrographolide (1) to cell cultures of HepG2. When the molecular interaction between andrographolide and GSH was investigated under a condition mimicking the in vivo environment, we observed that the level of GSH dropped in the presence of andrographolide. Stoichiometric analysis indicates that the reaction between these two reactants was 1 to 1 at pH 7 and followed second order kinetics. The activation energy of the overall reaction was 41.9±10 kJ·mol^-1 according to the Arrhenius equation. Using a micro-liquid-liquid extraction method followed by micellar electrokinetic chromatographic separation, two major products were isolated and identified, and their chemical structures were determined as 14-deoxy-12-(glutathione-amino)-andrographolide (2) and 14-deoxy-12- (glutathione-S-yl)-andrographolide (3). Based on these structural findings, a hypothetical mechanism of reaction between glutathione and andrographolide was proposed. It is concluded that the α,β-unsaturated lactone moiety of andrographolide reacts with GSH through a Michael addition followed by dehydration of the adduct. © 2008 Pharmaceutical Society of Japan.