Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique

Abstract

An electrophoretically mediated microanalysis (EMMA) method for screening acetylcholinesterase (AChE) inhibitors in natural extracts is described. In this method, solutions of AChE and the mixture of the substrate and the natural extract were successively injected into the capillary, and mixed electrophoretically by applying a voltage for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at UV 230 nm represents the enzyme activity. Since the extract is mixed with the substrate, there is no need to separate the components before testing the inhibition. The inhibitory activity of the natural extract as a whole can be easily found if the peak area of the product is reduced. This makes the present method suitable for screening inhibitors in complex mixtures, such as natural extracts. Compared to the commonly used spectrometric method for screening of AChE inhibitors, the major advantage of the present method is the elimination of Ellman reagent, which is essential for the spectrometric method. This not only simplifies the experimental procedure but also minimizes false-positive results. Moreover, it is an obvious advantage of combining the separation power with the on-column enzyme assay for further investigating which compound(s) is/are responsible for the inhibition. The method was validated using a commercially available AChE inhibitor tacrine and a small chemical library containing four AChE inhibitors and 32 natural extracts. Inhibitors in natural extracts were identified with the present method.

Immobilized capillary enzyme reactor based on layer‐by‐layer assembling acetylcholinesterase for inhibitor screening by CE

“…This is consistent with the literature [42]. In the present experiment, the K i value of tacrine was determined as 10 mM, which is comparable with 9 mM determined in our previous work measured with EMMA [43], but it is higher than 0.15 mM, determined with the Ellman method [42]. This difference may be due to the different measurement approaches.…”

“…This could be due to the fact that the amount of the immobilized enzyme is much lower than that of the enzyme solution in EMMA. The IC 50 value was determined as 1.97 mM, which was higher than 0.3 mM obtained with EMMA in our previous work [43] and 0.25 mM obtained in literature by the Ellman method [42]. The difference could be due to the different mixing and measurement approach.…”

Screening of acetylcholinesterase inhibitors by CE after enzymatic reaction at capillary inlet

“…For the screening of AChE inhibitors, the classical plug -plug mode [20], as well as the use of an immobilized capillary AChE reactor based on a layer-by-layer assembly [21] has been proposed.…”

Study of acetylcholinesterase inhibitors using CE with contactless conductivity detection

“…Heleg-Shabtai et al [16] presented a sensitive on-chip AChE assay, in which the resulting labeled product was detected by laser-induced fluorescence. An EMMA technique using UV detection was reported by Tang et al [17] to screen AChE inhibitors in natural extracts. Thiocholine, as a product from enzymatic reaction, was measured directly as a peak in the electrophoretic separation by its UV absorbance at 230 nm.…”