Chelidonine

Chelidonine (54274, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) (D2650, Sigma-Aldrich, St. Louis, MO, USA). Before use, the stock solution was serially diluted with the solvent so that the final concentration of DMSO was the same in all related samples (5 µL/mL). Cells were seeded in 6-well-plates (~5 × 10⁵ cells/well in 2 mL medium). Cells grown to ~80–90% confluency (approx. 24 h after seeding) were treated with Chelidonine (1 or 4 µg/mL) and cultured for the indicated duration afterwards. Control cells were treated with the same amount of DMSO and kept at the same experimental conditions. After alkaloid treatment, cells were harvested and prepared for further investigations as described later. Due to alkaloid treatment, a fraction of cells detached from the surface of the culture plate and floated in the medium. In order to avoid the loss of detached cells, the supernatant was collected before trypsinization. Cells retrieved from the supernatant by centrifugation were pooled with the trypsinized ones thereafter.

Chelidonine purchased from Sigma-Aldrich was used in this study. To inhibit apoptosis, the pan-caspase inhibitor Z-VAD-FMK (Enzo Life Sciences, Farmingdale, NY, USA) was used and dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used as a vehicle. For western blot analysis, the following antibodies were used: anticaspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP), Mcl-1, BAK, p-CDK1(Thr161), aurora A and p-PLK1 from Cell Signaling Technology (Beverly, MA, USA); anticyclin B1, CDK1, p-CDK1(Tyr15), and PLK1 from Santa Cruz Biotechnology (CA, USA); anti-BAX from BD Biosciences; anti-p-Ser/Thr-MPM-2 from Millipore; and anti-α-tubulin from Sigma-Aldrich. Anti-mouse IgG peroxidase conjugate and anti-rabbit IgG peroxidase conjugate from Sigma-Aldrich were used as secondary antibodies. For immunofluorescence assay, the following primary antibodies were used: anti-AIF from Santa Cruz, anti-α-tubulin from Sigma-Aldrich, and antipericentrin from abcam (Cambridge, MA, USA), and anti-mouse-FITC and anti-mouse-TRITC antibody were purchased from Sigma-Aldrich. To stain nucleus, we used DAPI from Sigma-Aldrich.

Acetonitrile (MeCN), methanol (MeOH), and 1-butyl-3-methylimidazolium tetrafluoroborate of chromatographic quality were obtained from E. Merck (Darmstadt, Germany), dimethyl sulfoxide (DMSO) was from Sigma-Aldrich (Saint Louis, MO, USA).
Alkaloid standards (berberine, boldine, chelidonine, and papaverine) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Columbamine, magnoflorine, palmatine, sanguinarine, chelerythrine hernandezine, and tetrandrine, were purchased from Chem Faces Biochemical Co. Ltd. (Wuhan, China).
Plant material was collected and identified in the Botanical Garden of Maria Curie-Skłodowska University in Lublin (Poland) in spring and summer 2016. Chelidonium majus was collected in May of 2017.
The plants were divided into roots and aboveground parts. Plants organs were cut into pieces and dried at ambient temperature for 1–2 weeks. Branches of B. vulgaris were decorticate and dried for 1–2 weeks under the same conditions.

Stylopine and chelidonine were purchased from ChromaDex Inc. (Los Angeles, CA, USA), sanguinarine chloride hydrate and chelidonine from Sigma-Aldrich (Darmstadt, Germany), protopine from Extrasynthese (Genay, France) and bicuculline from Cayman Chemical Company (Ann Arbor, Michigan, USA). All references were of analytical standard. Trifluoroacetic acid for HPLC, HPLC grade acetonitrile and methanol were purchased from Sigma-Aldrich (Darmstadt, Germany). The ultra-pure water used for HPLC analysis was obtained from a Millipore system (Milli-Q RG) (Millipore, France). Acetycholine for the antispastic assays was obtained from Sigma-Aldrich (Darmstadt, Germany). Ferric chloride, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (97%); diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) (>98%), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazine (DPPH), 2,4,6-tris (2-pyridyl)-striazine (TPTZ) for antioxidant assays, acetylthiocholine iodide (ATCI), acetylcholinesterase from Electrophorus electricus (electric eel) (AchE), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and galantamine hydrobromide for the AchE inhibition assay were purchased from Sigma-Aldrich (Darmstadt, Germany).