In the present work, AChE from Electrophorus electricus (Sigma C2888) was purchased from Sigma‐Aldrich Chemie GmbH. In vitro effects on AChE activity of the newly synthesized N-substituted sulfonyl amides (6a–j ) incorporating 1,3,4-oxadiazol structural motif and reference compound, THA, were evaluated by the method of Ellman et al. [42 (link), 43 (link)]. Analysis results were obtained spectrophotometrically at 412 nm using acetylthiocholine iodide (PubChem CID: 74629, Sigma 01480) as a substrate as in our previous assays [44 (link), 45 (link)]. Also, hCAs (hCA I and II) were purified from human erythrocytes by Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. The inhibition effects of these N-substituted sulfonyl amide derivatives (6a–j ) and reference compound, AAZ versus the esterase activity of the hCAs were determined by following the change in absorbance at 348 nm according to the assay defined by Verporte et al. [46 (link)–48 (link)]. hCAs activities were measured using 4-nitrophenyl acetate (PubChem CID: 13,243, Sigma N8130). All the measurements were repeated thrice.
Electrophorus electricus
Manufactured by Merck Group
Sourced in United States, Germany
Electrophorus electricus is a type of laboratory equipment used for the generation and study of electric currents. It is a device that can produce high-voltage electrical discharges, which can be used for various scientific experiments and demonstrations. The core function of Electrophorus electricus is to generate and demonstrate static electricity.
Automatically generated - may contain errors
Lab products found in correlation
V 570 spectrophotometer, by Jasco (2 mentions)
Elisa el10a, by Biobase (2 mentions)
Prism 9, by GraphPad (2 mentions)
C3389, by Merck Group (1 mentions)
Pherastarfs detection system, by BMG Labtech (1 mentions)
Sre020, by Merck Group (1 mentions)
Thermo multiscan ex, by Thermo Fisher Scientific (1 mentions)
Acetylcholine iodide, by Merck Group (1 mentions)
Essentia lc 16, by Shimadzu (1 mentions)
Mcp 200, by Anton Paar (1 mentions)
Chirascan plus circular dichroism spectrometer, by Applied Photophysics (1 mentions)
Equinox 55, by Bruker (1 mentions)
Agilent 6530 accurate mass q tof lc ms spectrometer, by Agilent Technologies (1 mentions)
Avance 3 hd 400 mhz nmr spectrometer, by Bruker (1 mentions)
Lambda 950 uv vis nir spectrophotometer, by PerkinElmer (1 mentions)
Acetylthiocholine iodide, by Merck Group (1 mentions)
Milliq integral, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
K2hpo4, by Merck Group (1 mentions)
Nah2po4, by Merck Group (1 mentions)
15 protocols using electrophorus electricus
1
Inhibition of AChE and hCAs by N-Substituted Sulfonyl Amides
2
Cholinesterase Inhibition Assay of Essential Oil
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cholinesterase (ChEs) inhibition of EO was determined for the enzymes (i) acetylcholinesterase (AChE) and (ii) butyrylcholinesterase (BuChE). The procedure was followed as described by Ellman et al. [62 (link)] and Calva et al. [57 (link)]. Phosphate buffered saline (pH = 7.4), DTNB (5,5′-dithiobis-(2-nitrobenzoic acid) ion (1.5 mM) a reagent that reacts with thiocholine to give the yellow coloration and the EO sample in DMSO (1% v/v) were prepared. The reaction of DTNB is monitored by measuring its absorption at 412 nm. AChE, from Electrophorus electricus (Sigma-Aldrich, C3389, St. Louis, MO, USA) and BuChE, from horse serum, (Sigma-Aldrich, SRE020, St. Louis, MO, USA) are dissolved in PBS (pH = 7.4) at 24 mU/mL. Preincubation was carried out for 10 min and acetylcholine iodide (1.5 mM) is added to initiate the reaction. The reaction is monitored for 30 min at 30 °C in a PherastarFS detection system (BMG Labtech). Inhibitory concentration (IC50) values were calculated in the online package GNUPLOT (www.ic50.tk , www.gnuplot.info) (accessed on 1 March 2022). Measurements were performed by triplicate. The reference drug inhibitor of ChEs was Donepezil, for AChE and BuChE with an IC50 value of 100 nM and 8500 nM, respectively. False positives are not excluded for high concentrations (>100 ug/mL) of amine or aldehyde compounds [59 (link)].
3
Anticholinesterase Activity of Hop Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anticholinesterase activity of hop extracts was investigated [8 (link),21 (link)] by detecting AChE activity photometrically (λ = 412 nm, 25 °C) by means of a Jasco V-570 spectrophotometer (Tokyo, Japan). Briefly, about 0.01 EU of enzyme (EC 3.1.1.7, from Electrophorus electricus, Sigma–Aldrich, Milan, Italy) were incubated in phosphate buffer (0.1 M, pH 8.00) plus 5,5′dithiobis(2-nitrobenzoic) acid (DTNB, 0.2 mM) either in the absence or in the presence of different aliquots of hop extracts. For n-hexane extract, the addition of Tween-20 (0.4%, v/v) allowed its re-suspension in phosphate buffer. Reaction was started by the addition of saturating concentration (2.5 mM) of acetylthiocholine iodide and the rate of absorbance change was obtained as tangent to the initial part of the progress curve. IC50 values were calculated by means of Grafit 4.0 (Erithacus Software Ltd., East Grinstead, UK). Results were expressed as % of the control (reaction rate measured in the absence of plant extract). Data were submitted to ANOVA followed by Tukey’s HSD test for mean comparisons.
4
Acetylcholinesterase Inhibition Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The AChE inhibitory activity was carried out according to Ellman et al. 1961. [28 (link)], with the modifications incorporated by López et al. 2002 [29 (link)]. A stock solution of 518U of AChE from Electrophorus electricus (Sigma, Schnelldorf, Bayern, Germany) was prepared and kept at −20 °C. Acetylcholine iodide and DTNB were also obtained from Sigma, Schnelldorf, Bayern, Germany.
To perform the analysis, 50 µL of AChE in phosphate buffer (8 mM K2HPO4, 2.3 mM NaHPO4, and 0.15 M NaCl, at pH 7.5) was incorporated into 50 µL of the sample dissolved in that same buffer, in flat-bottom 96-well plates. The plates were subsequently incubated for 30 min at 21–25 °C. Immediately afterwards, 100 µL of substrate solution was added (0.1 M Na2HPO4, 0.5 M DTNB, and 0.6 mM ACh iodide in Millipore water at a pH of 7.5). After 5 min, the absorbance was read on an ELISA plate reader (Multiscan EX Thermo Scientific®, Waltham, MA, USA). Galanthamine was used as a positive control (employing a 1:10 dilution bank from 10−3 to 10−7 M). Phosphate buffer was used as blank.
To determine the inhibitory potential of each extract, their respective IC50 was established. In order to do so, a semilogarithmic linear regression study was carried out from the concentrations analyzed and the inhibitory values obtained.
To perform the analysis, 50 µL of AChE in phosphate buffer (8 mM K2HPO4, 2.3 mM NaHPO4, and 0.15 M NaCl, at pH 7.5) was incorporated into 50 µL of the sample dissolved in that same buffer, in flat-bottom 96-well plates. The plates were subsequently incubated for 30 min at 21–25 °C. Immediately afterwards, 100 µL of substrate solution was added (0.1 M Na2HPO4, 0.5 M DTNB, and 0.6 mM ACh iodide in Millipore water at a pH of 7.5). After 5 min, the absorbance was read on an ELISA plate reader (Multiscan EX Thermo Scientific®, Waltham, MA, USA). Galanthamine was used as a positive control (employing a 1:10 dilution bank from 10−3 to 10−7 M). Phosphate buffer was used as blank.
To determine the inhibitory potential of each extract, their respective IC50 was established. In order to do so, a semilogarithmic linear regression study was carried out from the concentrations analyzed and the inhibitory values obtained.
5
Analytical Characterization of Natural Products
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optical rotations were carried out on an MCP 200 (Anton Paar, Graz, Austria) polarimeter. UV spectra were measured at a Lambda 950 UV-Vis-NIR spectrophotometer (PerkinElmer, Akron, OH, USA). A Chirascan-plus Circular Dichroism Spectrometer (Applied Photophysics Ltd., Leatherhead, UK) was used to obtain experimental ECD data. A Fourier transformation infra-red spectrometer (FTIR) coupled with an infra-red microscope EQUINOX 55 (Bruker, Wissembourg, France) recorded the FTIR spectrum. NMR spectra were tested by a BRUKER AVANCE III HD (400 MHz) NMR spectrometer with tetramethylsilane (TMS) as the internal standard. HR-ESIMS data were determined using an Agilent 6530 accurate-Mass Q-TOF LC-MS spectrometer. Column chromatography (CC) was used using silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China). The semi-preparative HPLC was performed on an Essentia LC-16 (Shimadzu, Jiangsu, China). Acetylcholine esterase (AChE) was from Electrophorus electricus (product number: C3389-2KU, Sigma-Aldrich, Saint Louis, MO, USA).
6
Acetylcholinesterase Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analytical reagents used were dichloromethane (ACS grade, Alfa Aesar, Ward Hill, MA, USA), acetone (AR/GR grade, Merck, Rahway, NJ, USA), dimethyl sulfoxide (LR grade, Merck), type I water (milli-Q® Integral, Merck, Billerica, MA, USA), NaCl (≥99.5%, Merck), NaH2PO4 (99–102%, Merck), K2HPO4 (≥99%, Merck), Na2HPO4 (≥99.5%, Merck), tween® 20 (polysorbate 20, Sigma-Aldrich, St. Louis, MO, USA), DTNB (5,5′-dithiobis(2-nitrobenzoic acid) ≥98%, Sigma-Aldrich), ATChI (acetylthiocholine iodide ≥98%, Sigma-Aldrich), and acetylcholinesterase enzyme (AChE) from Electrophorus electricus (1000 U/mg, Sigma-Aldrich).
7
AChE Inhibition Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The AChE assay was conducted according to the method reported by Marston et al. [16 (link)] The enzyme AChE (500 U), from Electrophorus electric us (electric eel, Sigma Aldrich, Missouri, EUA, E.C. 3.1.1.7), was dissolved in tris-hydrochloric acid buffer (pH 7.8) and stabilized by the addition of bovine serum albumin fraction V (0.1%). Thymol, thymol acetate, and the essential oil of L. thymoides fresh leaves were applied to thin-layer chromatography (TLC) plates to obtain spots with concentrations from 0.01 to 1000 ng/spot. Physostigmine was used as a positive control. The plates were sprayed with the AChE solution (3.33 U/mL), dried, and incubated at 37°C for 20 min. The enzyme activity was detected by spraying with a solution of 0.25% of 1-naphtyl acetate in ethanol and a 0.25% aqueous solution of Fast Blue B salt (20 mL). Potential acetylcholinesterase inhibitors appeared as clear zones on a purple background.
8
Acetylcholinesterase Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ellman’s
method was used
to assess the inhibition of AChE.30 (link) Briefly,
5,5′-dithiobis[2-nitrobenzoic acid] (3 mM, 125 μL), acetylthiocholine
(1.5 mM, 25 μL), Tris–HCl buffer (pH 8.0, 50 mM, 50 μL),
and the sample solution (10 mg/mL, 5 μL) were mixed in a 96-well
microtiter plate, and then, AChE from the electric eel Electrophorus electricus (0.28 U/mL, Sigma-Aldrich)
was added to the mixture. Absorption with the sample (A), with control water (B), and without AChE (C) at 412 nm was measured after 5 min of incubation at room
temperature. Inhibition was calculated using the following equation Concentration–inhibition
curves (n = 3 at each data point) were generated,
and IC50 values were calculated using the software GraphPad
Prism.
method was used
to assess the inhibition of AChE.30 (link) Briefly,
5,5′-dithiobis[2-nitrobenzoic acid] (3 mM, 125 μL), acetylthiocholine
(1.5 mM, 25 μL), Tris–HCl buffer (pH 8.0, 50 mM, 50 μL),
and the sample solution (10 mg/mL, 5 μL) were mixed in a 96-well
microtiter plate, and then, AChE from the electric eel Electrophorus electricus (0.28 U/mL, Sigma-Aldrich)
was added to the mixture. Absorption with the sample (A), with control water (B), and without AChE (C) at 412 nm was measured after 5 min of incubation at room
temperature. Inhibition was calculated using the following equation Concentration–inhibition
curves (n = 3 at each data point) were generated,
and IC50 values were calculated using the software GraphPad
Prism.
9
Acetylcholinesterase Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatographic grade methanol and acetonitrile were obtained from (Concord Technology (Tianjin) Co., Ltd.). Analytical grade petroleum ether, dichloromethane, ethyl acetate, methanol, trifluoroacetic acid, formic acid, and dimethyl sulfoxide were obtained from (Concord Technology (Tianjin) Co., Ltd.). 5,5-dithiobis [2-nitrobenzoic acid] (DTNB, CAS: 69-78-3), acetylthiocholine iodide (ATCI, CAS: 2260-50-6), and acetylcholinesterase (CAS: 9000-81-1) from Electrophorus electricus (Sigma Aldrich). Brain–Heart Infusion Agar and Broth were obtained from Qingdao Hope Bio-Technology Co., Ltd. Gentamicin sulfate (CAS: 1405-41-0) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd.
10
Fluorometric Acetylcholinesterase Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Acetylcholinesterase (Type VI-S, EC 3.1.1.7, from Electrophorus electricus, lyophilized powder, 217 U/mg) and choline oxidase (EC 1.1.3.17, from Alcaligenes sp., lyophilized powder, 15 U/mg), acetylcholine chloride (ACh), bovine serum albumin (BSA) and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich. Certified reference pesticide standards were all purchased from Dr. Ehrensdorfer (Augsburg, Germany). Water-soluble CdSe/ZnS core/shell quantum dots (QDs) were obtained from Ocean Nanotech (Springdale, AR, USA). Phosphate buffer solution (PBS) (pH 8.0, 10 mM) and Milli-Q ultrapure water (Millipore, ≥18 MΩ·cm) were used throughout. All chemicals were used without further purification. Individual stock solutions of the pesticides were prepared in acetonitrile and stored at 4 °C. A series of working solutions were prepared daily by an appropriate dilution by PBS (pH 8.0, 10 mM) which contains 0.5 mg/mL of BSA.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!