Silica gel 60

20 g of milled poplar bark sample (particle diameter < 0.2 mm) was placed in a cellulose cartridge (30 × 100 mm and 8–15 µm nominal retention; Scharlab, Part number: CT32530100) and placed in the Soxhlet extraction system. The sample was successively extracted with 250 mL of n-hexane, dichloromethane, ethyl acetate, methanol, and water for 24 h with each solvent. Each of the extracts was concentrated by rotary distillation at 40 °C to yield an oily residue.
Oily residues from each of the solvents were gathered and subsequently fractionated by flash chromatography using SiO₂ (Silica Gel 60, 0.04–0.06 mm, 230–400 mesh, Scharlab) as a stationary phase and with an increasing polarity gradient of eluents depending on the extraction solvent polarity (see SI.1, Table S1). TLC for the different fractions obtained by flash chromatography was made using the same eluent in which the fraction was eluted (see SI) on Silica Gel Plates on aluminum foil (Alugram Sil G UV254, Macheray Nagel) and revealed using anisaldehyde and UV light (254 nm).

The UV spectra were obtained with a Shimadzu UV-1800 recording spectrophotometer. The IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl₃ solutions. The NMR spectra were taken on a Bruker Avance III 500 MHz spectrometer (Bruker Biospin) with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in δ values. The HR-ESI-MS was performed on a MicrO-QIITOF mass spectrometer (Bruker Daltonics). The ChE inhibitory reactions were recorded on 96-well microplates using a microplate reader (VersaMax ELISA, USA). Silica gel 60, 0.06–0.2 mm (70–230 mesh ASTM), for column chromatography was purchased from Scharlau (Barcelona, Spain). LiChroprep^® RP-18 (40–63 μm) for liquid chromatography was purchased from Merck KGaA (Germany). Analytical and preparative TLC were carried out on precoated Merck Kieselgel 60F₂₅₄ or RP-18F₂₅₄ plates (0.25 or 0.5 mm thickness).