Molecular Interaction between Andrographolide and Glutathione Follows Second Order Kinetics

Abstract

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 a a,b b-unsaturated lactone moiety of andrographolide reacts with GSH through a Michael addition followed by dehydration of the adduct.

Taxifolin Enhances Andrographolide-Induced Mitotic Arrest and Apoptosis in Human Prostate Cancer Cells via Spindle Assembly Checkpoint Activation

“…This observation suggested that Andro alone at this concentration (20 µM) may disturb the proper formation of mitotic spindles in the cells. We have shown that Andro alone can induce DNA damage by altering the redox states in cells [16], [31]. The formation of small dot-shaped chromosomes may be due to limited DNA damage induced by Andro, which is consistent with the finding that during apoptosis, DU145 cells do not form DNA ladders typical of other apoptotic cells but instead form DNA fragments that are 50 kb long (51).…”

Intracellular glutathione regulates Andrographolide-induced cytotoxicity on hepatoma Hep3B cells

“…There are reports that ANDRO may directly react with thiol of GSH in vitro, and the assumed hypothesis is that the α,β-unsaturated lactone moiety of ANDRO can react with GSH through the Michael addition reaction. 16,22 The results illustrated in Figure 2B also confirmed the potential existence of the direct reaction of ANDRO with GSH in vitro. BSO is a well-known inhibitor of GSH synthesis, and it has been reported that pretreatment with BSO augments the hepatotoxicity of many chemicals, which can be detoxified by GSH.…”

Targeting Nrf2-Mediated Gene Transcription by Extremely Potent Synthetic Triterpenoids Attenuate Dopaminergic Neurotoxicity in the MPTP Mouse Model of Parkinson's Disease

“…S1 and S2B-D). Auranofin is a bulky molecule, known to alkylate active protein thiols (or selenols such as in thioredoxin reductase) by a ligand-exchange mechanism, the so-called sulfhydryl shuttle (29), while andrographolide, a labdane diterpenoid recently patented for the treatment of Amyotrophic Lateral Sclerosis and other neurodegenerative disorders (UK Patent application GB2464813), has an a,b-unsaturated lactone moiety alkylating protein or peptide-active -SH or -NH2 groups by Michael addition followed by dehydration (53). The observed lag-periods for TP may be interpreted as a switch in the ratelimiting step, meaning that there is a step preceding their direct alkylation of Keap1 reactive cysteines.…”