Kinetic study of acetylcholinesterase inhibitory activity of Echinophora cinerea

Acetylcholinesterase inhibitors are used for the treatment of Alzheimer’s disease. Alzheimer’s disease arises as a result of malfunction of different biochemical pathways. Multiple pathogenic factors, including aggregated amyloid-β-peptide and tau protein, excessive transition metals, oxidative stress and reduced acetylcholine levels have been implicated in this disease pathology [1]. Acetylcholinesterase inhibitors may interact with the central cholinergic system function to improve memory and cognitive disorders in the patients by decreasing the breakdown of acetylcholine in brain synapses [2]. The anti-cholinesterase activity of some plants in the world has been approved [2, 3]. In this study we use extract oil and methanolic exteract of Echinophora cinerea and test their acetylcholinesterase inhibitory activity. The enzyme was assayed according to the method of Ellman et al. [4] with acetylthiocholine as substrate. The product of thiocholine reaction with 5,5 -dithiobis(2-nitrobenzoic acid) was determined on a Unicom spectrophotometer. The absorbance at 412 nm was recorded as a function of time. The change rate of absorbance was linear over a period of 2 min for all concentrations of substrate and the slope was used to calculate the initial velocity. Enzyme activity was determined at 37º C in a 1.0 ml reaction mixture containing 0.10 M phosphate buffer, pH 7.4, 0.01 M 5,5’-dithiobis(2-nitrobenzoic acid) containing sodium bicarbonate (1.5 mg per ml), acetylthiocholine, and inhibitors as indicated, with all reagents except acetylthiocholine adjusted to pH 7.4. All determinations were performed a minimum of four times. In order to determinate the kinetic parameters and elucidate the type of inhibition, the enzyme activity was measured in the presence of an increasing concentration of acetylthiocholine iodide (ACTI) (2-20 mM), and in the absence or presence of each extract. Results showed that Vmax and Km values of the enzyme are 0.196 μg/min and 1.381 mg/ml, respectively. However, in the presence of extract oil and methanolic extract of Echinophora cinerea Vmax values are 0.116 and 0.074 μg/min and Km values are 1.173 and 0.956 mg/ml, respectively. The concentration required for 50% enzyme inhibition (IC50 value) was 4.45 and 3.93 mg/ml for the extract oil and methanolic extract of Echinophora cinerea, respectively.