Double beam spectrophotometer

Ellman assay [33 (link)] was used to assess the F. ammoniacum aerial parts methanolic extract/fractions for their cholinesterase inhibition potentials. About 205 µL extract/fractions and 5 µL of AChE (0.03 U/mL)/BChE (0.01 U/mL) along with 5 µL DTNB were taken in a cuvette and incubated at 30 °C for 15 min. After incubation, 5 µL acetylthiocholine iodide or butyrylthiocholine iodide (substrate) were added to the mixture that resulted in yellow coloration due to formation of 5-Thio-2-nitro benzoate anion. Then, the absorbance of the resulting mixture was measured at 412 nm through double beam spectrophotometer (Thermo electron-corporation, Waltham, MA, USA). As a negative control, a solution containing all the above-mentioned components except plant extracts/fractions were mixed together. The same procedure mentioned above was used to constitute the reaction mixture of positive control galantamine and absorbance was measured at 412 nm. For each sample, absorbance was recorded for 4 min. Percent enzyme activity and inhibition potential of both enzymes were measured using the following formulae: $V=\Delta Abs/\Delta t$
$\% \mathrm{Enzyme} \mathrm{activity}=\frac{V}{Vmax}\times 100$
$\% \mathrm{Enzyme} \mathrm{inhibition}=100-\% \mathrm{enzyme} \mathrm{activity}$
where: V shows the rate of reaction in the presence of inhibitor and Vmax, the rate of reaction in its absence.

Anticholinesterase inhibition potential of essential oil of E. umbellata were determined spectrophotometrically using the reported standard Ellman assay [25 (link)]. Acetyl choline iodide and butyrylcholine iodide were used as substrates. About 205 μL of essential oil having concentration in the range of 31.05–1000 μg/mL were added to a cuvette containing 5 μL of AChE (0.03 U/mL) and BChE (0.01 U/mL), through micropipette. Then 5 μL of DTNB was added to mixture kept in a water bath at 30 °C. After incubation for 15 min, 5 μL of substrates (acetylthiocholine iodide or butyrylthiocholine iodide) were added to the mixtures that resulted in yellow coloration (5-Thio-2-nitro benzoate anion color). Then the absorbance was recorded at 412 nm using double beam spectrophotometer (Thermo electron corporation, USA). A blank solution was prepared containing only essential oil but no enzyme. Galantamine was used as a positive control for which same procedure mentioned above was used. The absorption of each sample was recorded for 4 min. Percent enzyme activity and percent inhibition was calculated using the following equations:
$$V=∆\mathit{Abs}/∆t.$$ $$\%\mathit{\text{enzyme activity}}=\frac{V}{\mathit{\text{Vmax}}}\times 100.$$ $$\%\mathit{\text{enzyme inhibition}}=100-\%\mathit{\text{enzyme activity}}.$$ Where: V is rate of reaction in presence of inhibitor while Vmax is the rate of reaction in absence of inhibitor.