In vitro ChE inhibitory activity assay

The inhibitory activities against AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8) of the fungal samples and test compounds were analyzed following Ellman’s method (Ellman et al., 1961) with modifications. AChE (EC 3.1.1.7, from electric eel), BChE (EC 3.1.1.8, from equine serum), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Aladdin (Shanghai, China). AChE and BChE solutions were prepared with water (0.2 units/ml). ATCI and BTCI solutions (10 mM) were prepared with deionized water before using. A DTNB solution (10 mM) was prepared in a 0.1 M, pH 8.0 solution of potassium dihydrogen phosphate (1.36 g, 10 mmol) in 100 ml of water (PBS). The fungal samples for screening were diluted to 1.0 mg/ml with methanol. The test compounds were dissolved in methanol (10 mM) as stock solutions. Subsequently, at least five different concentrations of each compound (double ratio dilution with methanol) were used to determine the half maximal inhibitory concentration (IC₅₀). The tests were performed using 96-well plates. First, 160 μl of PBS, 2 μl of the test sample solution, 20 μl of AChE or BChE, and 10 μl of DTNB were added and mixed in each well, and then preincubated at 37°C for 10 min. After the addition of 10 μl of ATCI or BTCI, the reaction was initiated and incubated at 37°C for another 10 min. Then, the absorbance was measured at 412 nm. The wells with 2 μl of methanol replacing the test sample solution was used to determine 100% of the enzyme activity, and 20 μl of water replacing the enzyme solution was used to determine the blank value. All of the tests for each sample were performed in triplicate. The inhibition curve was fitted by plotting the inhibition rate vs. the logarithm of the test compound concentration. The IC₅₀ values were calculated using Microsoft Excel software, and the data were expressed as the mean ± SD. The inhibition rate was calculated using the formula: IR% = [(A_c-A_s)]/[(A_c-A_b)] × 100%, where A_b denotes the absorbance of the blank control without the enzyme solution, A_c represents the absorbance of the control without the sample solution, and A_s denotes the absorbance of the sample.