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Stock solutions of all graveoline analogs (10 mM) were prepared using DMSO and stored at −80 °C. Further dilutions to working concentrations were made with double-distilled deionized water. AChE (E.C. 3.1.1.7, obtained from electric eel), BuChE (E.C. 3.1.1.8, obtained from equine serum), 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholine chloride (ATC), butylthiocholine chloride, thioflavin T, and tarcine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aβ₄₂ was purchased from Millipore (Billerica, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased formal Aladdin (Shanghai, China). The SH-SY5Y cell line was obtained from the Animal experimental center of Sun Yat-sen University.

HPLC system (Agilent, USA) consisted of a model G1276A pump, model G1367B Autosampler and model G1316A UV detector. The chromatograph was equipped with a reversed-phase C18 column of Grace Alltima (250 mm × 4.6 mm, 5 μm). The QTOF-MS system (Bruker, Germany) with an ESI source was performed. HPLC separations were performed on a Hitachi 655-15 series pumping system equipped with a Hitachi L-2490 refractive index detector using a YMC-Park ODS-A column (250 × 10 mm I.D, S-5 μm, and 12 nm). NMR spectra were performed on a Bruker ARX-300, ARX-400, and ARX-600 spectrometer using trimethylchlorosilane as the internal standard. Column chromatography was performed on a 200–300 mesh silica gel (Qingdao Marine Chemical Factory, People's Republic of China). Column chromatography was performed using YMC ODS-A gel (12 nm S-75 μm, YMC Co., Ltd., Japan) and D-101 macroporous adsorption resin (Shanghai Hualing Resin Factory, People's Republic of China). TLC was performed with precoated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Factory, People's Republic of China). Microplate reader (Thermofisher Scientific, Finland) was used to test the activities. Acetylthiocholine iodide, S-butyrylthiocholine iodide, AChE, and BuChE were bought from Sigma Company.

Cholinesterase inhibitory activity assays were performed according to Ellman et al. [43 (link)] with some modifications [15 (link)]. Fifty microliters of 0.25 U/mL AChE or BuChE (Sigma-Aldrich, St. Louis, MO, USA) in phosphate buffer (8 mM K₂HPO₄, 2.3 mM NaH₂PO₄, 0.15 M NaCl, pH 7.6) and 50 μL of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 min at room temperature before addition of 100 μL of the substrate solution (0.1 M Na₂HPO₄, 0.5 M 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB, Sigma-Aldrich), and 0.6 mM acetylthiocholine iodide (ATCI, Sigma-Aldrich) or butyrylthiocholine iodide (BTCI, Sigma-Aldrich) in Millipore water, pH 7.5). The absorbance at 405 nm was determined using a Thermo Scientific Multiskan FC microplate spectrophotometer (Waltham, MA, 02451, USA) after 5 min. The enzyme-inhibitory activity was calculated as a percentage compared to an assay using a buffer without any inhibitor. The enzyme-inhibitory data were analyzed using the software package Prism (Graph Pad Inc., San Diego, CA, USA). The alkaloid concentrations used to calculate the IC₅₀ values were 15, 30, 60, 120, 160, and 200 μM in both AChE and BuChE assays, while for the alkaloid extract, they were 50, 100, 150, 200, 250, and 300 μg/mL, respectively. The IC₅₀ values are the means ± SD of three individual determinations, each performed in triplicate.