*4.10. Anticholinesterase Activity*

The AChE inhibitory effect was measured based on the methodology designed by Ellman, et al. [45], with slight modifications as suggested by Rhee, et al. [46], as previously described by Valarezo et al. [33]. The inhibition of AChE was detected after the addition of Acetylthiocholine as the enzyme substrate and several concentrations of EO dissolved in MeOH. The enzyme reaction was monitored in a microplate reader (EPOCH 2, BioTek, Winooski, VT, USA) at 405 nm for 60 min. Final concentrations of 1000, 100, and 10 μg/mL of the EO in MeOH were prepared to assess the enzyme inhibition. The assay was carried out by triplicate in 96-well microplates. Donepezil was used as positive control. The IC50 value was calculated from the progression curve with the Graph Pad Prism software (v8.0.1.5., Graph Pad, San Diego, CA, USA)

#### *4.11. Statistical Analysis*

All procedures were repeated three times, except for the biological activity, which was repeated nine times. Microsoft Excel was used to collect the data and Minitab 17 (Version 17.1.0., Minitab LLC., State College, PA, USA) was used to calculate the measures of central tendency. The results are expressed as mean values.
