Dual electrophoretically-mediated microanalysis in multiple injection mode for the simultaneous determination of acetylcholinesterase and α-glucosidase activity applied to selected polyphenols.

ABSTRACT

Acetylcholinesterase (AChE) and α-glucosidase (α-glu) are key target enzymes in the search for novel strategies in the treatment of Alzheimer's disease and type II diabetes. Therefore, methods to assess the enzyme inhibition are of great value in the research field. Here is proposed a novel a dual electrophoretically-mediated microanalysis for the simultaneous determination of both enzymes' activity. In order to do so, the various solutions required for both assays were introduced in the capillary electrophoresis system using the multiple injections approach. Enzymatic kinetic parameters were tested, Km for AChE and α-glu were 3.81 and 0.43 mmol L-1. Ki values were 4.27 µmol L-1 for neostigmine (an AChE inhibitor) and 0.40 mmol L-1 for acarbose (an α-glu inhibitor). Results of IC50 (concentration for 50% of inhibition) were 5.11 ± 0.47 µmol L-1 and 0.58 ± 0,02 mmol L-1 for neostigmine and acarbose, respectively. All parameters (except for Ki of neostigmine) were comparable with the literature, indicating a good reliability of the proposed method to evaluate these enzymes activity. Total time analysis was approximately 10 min, being possible to perform around 12 enzymatic assays per hour, with low sample and reagent consumption, thus satisfying some of the principles of green chemistry. The method was applied to evaluate 10 phenolic compounds, of wich p-coumaric acid showed the best inhibitory activity for AChE (40.14 ± 4.75% at 10 mg L-1); and quercetin for α-glu (46.53 ± 4.90% at 10 mg L-1).