Ellman’s colorimetric method was used [77 (link)] with modifications described previously [78 (link)]. Tested sample (10 μL) at concentration 5 mg/mL was mixed with 20 μL of AChE (or BChE) solution (0.28 U/mL) and completed after 5 min with 35 μL of ATChI (or BTCh) (1.5 mmol/L), 175 μL of 0.3 mmol/L DTNB (containing 10 mmol/L NaCl and 2 mmol/L MgCl2) and 110 μL with Tris-HCl buffer (50 mmol/L, pH 8.0). Samples containing 10 μL of Tris-HCl buffer instead of the studied sample were run in the same way (“blank” samples). The increase in the absorbance due to the spontaneous hydrolysis of the substrate was monitored using “blank” samples containing ATCh (or BTCh) and DTNB completed to 350 μL with Tris-HCl buffer. All samples were incubated at 22 °C (30 min; incubation time was determined after optimization experiments, details not shown), and the absorbance was measured (405 nm, 96-well microplate reader, Tecan Sunrise, Grödig, Austria). The “false-positive” effect of studied compounds was measured according to Rhee et al. [79 (link)] with minor modifications, as described previously [78 (link)]: after mixing of the substrate with the enzyme and buffer, the “false-positive” sample was left for incubation. Then, a studied sample and DTNB were added, followed by an immediate measurement of the absorbance.
Reference cholinesterase inhibitors were used for the calculations of results (eserine, neostigmine, magniflorine, rivastigmine and donepezil). For this purpose, for each compound, 16 dilutions in pure DMSO were prepared (2.57–41.14 μg/mL). These solutions (10 μL) were tested as described above and calibration curves were produced.
Each sample was analyzed in at least eight repeats, and all solutions used in a set of analyses were prepared in the same buffer. For calculations, the background of the sample (10 μL mixed with 340 μL of Tris buffer) was measured at 405 nm and subtracted during calculations. Then, the absorbance of the test sample was subtracted from the absorbance of the “blank” sample.
Reference cholinesterase inhibitors were used for the calculations of results (eserine, neostigmine, magniflorine, rivastigmine and donepezil). For this purpose, for each compound, 16 dilutions in pure DMSO were prepared (2.57–41.14 μg/mL). These solutions (10 μL) were tested as described above and calibration curves were produced.
Each sample was analyzed in at least eight repeats, and all solutions used in a set of analyses were prepared in the same buffer. For calculations, the background of the sample (10 μL mixed with 340 μL of Tris buffer) was measured at 405 nm and subtracted during calculations. Then, the absorbance of the test sample was subtracted from the absorbance of the “blank” sample.
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