Eserine

Imidacloprid (95.6%) was purchased from Dupont. Beta-cypermethrin (95%) was obtained from Suzhou Fumeishi Chemical Co., Ltd. Chlorpyrifos (98%) was obtained from Tianjin Longdeng Chemical Co., Ltd. Chlorfenapyr (98%) was obtained from Jiangsu Academy of Agricultural Sciences. Acetamiprid (90%) was provided by Jiangsu Yangnong Chemical Group Co., Ltd. Azamethiphos (95%) was supplied by Shanghai Yongyuan Chemical Co., Ltd. Piperonyl butoxide (PBO, 90%), s,s,s-tributylphosphorotrithioate (DEF, 98%) and diethyl maleate (DEM, 97%) were purchased from Chem. Service (West Chester, PA). α-Naphthyl acetate (α-NA), β-naphthyl acetate (β-NA), eserine, α-naphthol, β-naphthol, 1-chloro-2,4-dinitrobezene (CDNB), reduced glutathione (GSH), phenylmethylsulfonyl (PMSF), dithiothreitol (DTT), phenylthiourea (PTU), fast blue B salt, sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO) at the highest purity available. The other chemicals were of analytical quality and purchased from commercial suppliers.

Selected hits were obtained from NCI and they were provided as dry powders in variable quantities (5–10 mg). Compounds were initially dissolved in DMSO to give stock solutions of 100 µM. Subsequently, they were diluted to the required concentrations with Tris buffer pH 8.0 for the assay. Enzymatic inhibition assays were performed on BChE from equine serum (Sigma), according to the spectrophotometric Ellman's method³⁷ (link). The experiment was performed in 48-well plates in a final volume of 100 µL. Each well contained 0.22 U/mL eqBChE dissolved in Tris–HCl buffer, pH 8.0. They were preincubated for 20 min at different compound concentrations at 37 °C. Then 0.5 mM butyrylthiocholine iodide (Sigma) and 0.35 mM 5,5′-dithiobis -2-nitrobenzoico (DTNB; Sigma) were added. Colour development was measured spectrophotometrically at 412 nm using microplate reader (BioTek ELx800) at a rate of one measurement per minute over 15 min period. Positive (Eserine, sigma 100 uM) and negative (no inhibitors) controls were tested. All samples were assayed in at least duplicate measurements. In general, the amount of DMSO was kept below 1% in the assay. IC50 values were determined graphically from inhibition curves using Graph Pad prism version 6, Graph Pad Software, Inc..

Acetylcholinesterase (AChE) activity was measured in a 96-well microtiter plate assay based on the method of Ellman et al. (29 (link)), as published previously (30 (link)). For each assay data point, 50 μL of 3 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 50 μL of AChE (1 mg/mL) (Sigma, C3389) or rat brain homogenate (prepared according to previous publications (31 (link), 32 (link))), 35 μL of 50 mM Tris/ HCl pH 8.0, and 40 μL of polyherbal extract were mixed and incubated at 37 °C. The assay was initiated by addition of 25 μL of 15 mM acetylthiocholineiodide (ATCI), with production of 5-thio-2-nitrobenzoate anion read at 412 nm every 30 sec for 10 min using a Spectramax microplate reader (ThermoFisher, UK). Assay reactions with polyherbal extracts were all performed in triplicate at concentrations of 200 μg/mL, 20 μg/mL, 2 μg/mL, 0.2 μg/mL and 0.02 μg/mL. A negative control assay performed in the absence of AChE provided a reagent blank. Eserine (Sigma, E8375) was used as a positive control to inhibit electric eel or rat brain AChE in a dose-dependent manner. The percentage inhibition of AChE by polyherbal extract was calculated relative to inhibition by Eserine, with the herbal extract concentration producing 50% inhibition (IC₅₀) of AChE calculated using GraphPad Prism (version 5.03, Inc., 2010, San Diego California USA) via non-linear regression analysis.

To investigate whether therapeutic drugs (zonisamide, FUJIFILM Wako Pure Chemical Corporation; donepezil HCl, Sanyo Chemical Laboratories Co., Ltd; eserine, Sigma-Aldrich; galantamine, Abcam) have astrocyte proliferative activity, astrocytes were prepared with a density of 6.3 × 10⁴ cells/cm², and zonisamide as another control and three AChE inhibitors were used at the same concentration as Naturido (25 μM). Astrocyte proliferative activity was measured with a BrdU assay (%). On the other hand, AChE that plays a role in the hydrolysis of the neurotrasmitter has proven to be an important therapeutic target for AD and AChE inhibitors have been developed for clinical treatments [21 (link)]. Here, we also tested the relationship between Naturido and AChE inhibition. The inhibitory activity on acetylcholinesterase was measured according to a standard method [22 (link)], and the concentration of donepezil HCl, used for comparative purposes, was determined with a previously described method [21 (link)].