*2.11. Acetylcholinesterase (AChE) Activity Assay*

The effects of L-dopa and *M. pruriens* seed extract on the acetylcholinesterase activity were measured using a colorimetric assay based on an Ellman method. Briefly, samples (50 μL) were mixed with acetylcholinesterase enzyme (50 μL) in a 96-well plate. The assay buffer (50 μL) was used as a blank. The reaction was initiated by the addition of 1x substrate mix solution to the sample and blank wells. The hydrolysis of this substrate was immediately monitored by measuring the absorbance at a maximum wavelength of 412 nm in kinetic mode at 1 min-interval for 20 min. The formation of yellow 5-thio-2 nitrobenzoate anion was detected as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide. The enzymatic activity was calculated as a percentage of the velocity of the reaction with and without the extract. The velocity of the reaction was determined by constructing a kinetic curve between the absorbance and the incubation time. The slope was in units of O.D./min. The absorbance was calculated from the following equation.

$$A = \mathfrak{e} \times I \times \mathfrak{c}$$

where *A* is the absorbance (in O.D), *ε* is the extinction coefficient (13,600 L/molxcm), *I* is the path length of a 96-well plate, and *c* is the AChE activity in units (μmole.min).

To calculate the enzymatic activity (units/liter), the following equation was used.

$$\text{AChE activity} = \frac{Slope\left(\frac{OD}{\text{min}}\right) \times 10^{-4} (liter) \times 10^6 \left(\mu \frac{molc}{molc}\right)}{13,600 \left(\frac{liter}{molc} \times cm\right) \times 0.3 \times 5 \times 10^{-5} \left(liter\right)}$$
