Galanthamine hydrobromide

The acetyl-cholinesterase inhibition assay was determined by Ellman colorimetric method [36] (link) as modified by Albano et al. [31] . In a total volume of 1 ml, 415 μl of Tris–HCl buffer 0.1 M (pH 8), 10 μl of solution of essential oils in methanol with different concentrations and 25 μl of enzyme (electric eel acetyl-cholinesterase, type-VI-S, EC 3.1.1.7, Sigma–Aldrich, St. Louis, USA) solution containing 0.5 U/ml were incubated for 15 min at room temperature. 75 μl of a solution of AChI (acetyl-thiocholine) (Sigma–Aldrich, Steinheim, Germany) 1.83 mM and 475 μl of DTNB (5,5-dithiobis-2-nitrobenzoic acid), 3 mM (Sigma–Aldrich, Steinheim, Germany) were added and the final mixture incubated for 30 min, at room temperature. Absorbance of the mixture was measured at 412 nm in a UV-Visible 752 spectrophotometer (Techmel and Techmel, USA). Galanthamine hydrobromide (Sigma–Aldrich, Steinheim, Germany) was used as positive control. The percentage inhibition of enzyme activity was calculated by comparison with the negative control: $\text{Inhibition}\%=\left(\frac{A_0-A_1}{A_0}\right)\times 100$ where A₀ was the absorbance of the negative control (enzyme + methanol) and A₁ was the absorbance of the sample (enzyme + solution of EO). Tests were carried out in triplicate and data were analyzed using descriptive statistics.

NMR spectra were recorded using a Bruker Avance 500 spectrometer (500 MHz for ¹H NMR, 125 MHz for ¹³C NMR) (Bruker, Billerica, MA) with TMS as an internal reference. Column chromatography was performed with silica gel (60 N, spherical, neutral, 40–50 μm, Kanto Chemical Co., Inc., Tokyo, Japan), YMC RP-18 (Fuji Silysia Chemical Ltd., Kasugai, Japan) and Sephadex LH-20 (Dowex^® 50WX2-100, Sigma-Aldrich, St. Louis, MO). Analytical TLC separations were performed on pre-coated silica gel 60F254 and RP-18 F254 plates (0.25 or 0.50 mm thickness, Merck KGaA, Darmstadt, Germany). Acetylcholinesterase, acetylthiocholine (ACTI), 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) and galanthamine hydrobromide were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulphoxide (DMSO) was purchased from Merck (Darmstadt, Germany). All chemicals used were of the highest grade available.