Lycorine

Cells viability was also determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma–Aldrich, St. Louis, MO, USA) assay. Lycorine (Sigma–Aldrich, St. Louis, MO, USA) and haemanthamine compounds were evaluated at different concentrations (100, 50, 30, 20, 10, 1 μg/mL) in the cell line with the lowest percentage of cell viability (AGS) and incubated at 37 °C with 5% CO₂ for 48 h. Doxorubicin (Sigma–Aldrich, St. Louis, MO, USA) was used as a positive control (10, 5, 2.5, 1.25, 0.62, 0.31 µg/mL). Subsequently, 50 μL of MTT (1.0 mg/mL buffered saline phosphate) was added. After 4 h of incubation at 37 °C, the MTT culture medium was removed and replaced with 150 μL DMSO (Sigma–Aldrich, St. Louis, MO, USA) to dissolve the formazan crystals. Plates were incubated in dark with agitation for 2 h. Optical density was determined at 540 nm using a microplate reader. All experiments were conducted with three independent trials, each with six replicates.

Aliquots of the extracts of C. orchioides were were diluted 20-fold with deionized water and injected into the HPLC system. HPLC analysis was carried out using an Accela HPLC liquid phase system (Thermo Fisher Scientific, Waltham, MA, USA). An Intersil C₁₈ column (5 mm, 4.6 mm 250 mm; GL Sciences Inc., Tokyo, Japan) was used as the stationary phase. The mobile phase was a mixture of KH₂PO₄ buffer (15 mM, pH 6.35) with methanol (50:50, v/v). The mobile phase was filtered through a 0.45 mm nylon filter membrane and degassed in an ultrasonic bath for 10 min before use. The temperature of the column oven was set at 30 °C, and the flow rate was 0.8 mL/min. The injection volume was 20 mL [30 (link)]. The standard lycorine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Analytical grade methanol was used for the extraction and HPLC grade acetonitrile and methanol used for analysis. Both were purchased from Merck (Darmstadt, Germany).

Standards (gallic acid, lycorine, galanthamine, tyramine, tyrosine, α-glucose, β-glucose, sucrose, alanine, valine) are purchased by Sigma Aldrich (Sigma-Aldrich, St. Quentin Fallavier, France). All solvents used are analytical profile grade, deuterated methanol (CD₃OD), 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (DSS sodium salt), sodium deuteroxide solution (NaOD) 40 wt.% in deuterium oxide (D₂O), 99.5 atom % D, ammonium hydroxide solution (NH₄OH) containing ≥25% NH₃ basis, were purchased from Sigma Aldrich (Sigma-Aldrich, St. Quentin Fallavier, France). All chemicals for in vitro culture are listed in the article Saliba et al. [29 (link)]. SCX cation exchanger column are purchased by Interchim^® (Interchim, Montluçon, France).