Podophyllotoxin

For extractions and solutions, ultrapure water was obtained by a Milli-Q system Millipore (Bedford, MA, USA) and absolute ethanol was purchased from Sigma-Aldrich (St. Louis, MO, USA). The 7% sodium carbonate (Na₂CO₃) solution was obtained by dissolving 7 g of powder in ultrapure water and making up to 100 mL.
The following reagents were supplied by specified suppliers: Gallic acid, Folin reagent, ferric chloride (FeCl₃₎, dimethyl sulfoxide (DMSO) and Na₂CO₃ were purchased from Aladdin (Aladdin Biochemical Technology Co., Ltd., Shanghai, China). Acetylcholinesterase (Electric eel), butyrylcholinesterase (Horse serum), acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5’-dithio bis-(2-nitrobenzoic acid), and Tris (tri (hydroxymethyl) aminomethane) were purchased from Macklin (Macklin Biochemical Co., Ltd., Shanghai, China). DPPH (1,1-diphenyl-2 picrylhydrazyl radical), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation), TPTZ (2,4,6-tri(2-pyridyl)-s-triazine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), acetic acid, podophyllotoxin, and deoxypodophyllotoxin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The murine macrophages, RAW 264.7 cells, were plated into a 96-well cell culture treated flat-bottomed plate (SARSTEDT, Inc., Newton, NC 28658, USA) at a density of 10 × 10⁴ cells/100 μL/well and incubated overnight to enable cell adhesion. A fresh complete medium containing serially diluted concentrations of crude extract, fractions, and compounds was used to replace the previous medium. The final assay concentration values for extracts/fractions and compounds varied from 400 μg/mL to 0.8 μg/mL and 100–0.16 μg/mL, respectively. Podophyllotoxin (Sigma-Aldrich, Munich, Germany) was used as a positive control at a maximum dose of 10 μM. After 48 h of incubation, 10 μL of resazurin solution (0.15 mg/mL dissolved in PBS) was added to each well, and the plates were incubated for an additional 4 h [68 (link)]. Fluorescence data were finally recorded with a Magellan Infinite M200 plate reader (Tecan, Germany) at excitation and emission wavelengths of 530 nm and 590 nm, respectively.

Mueller-Hinton Broth (MHB), Muller-Hinton Agar (MHA), streptomycin, ciprofloxacin, ampicillin, tetracycline, metronidazole, myricetin, podophyllotoxin, dimethyl sulfoxide (DMSO ≥ 99.5%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (≥ 98%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Potato dextrose agar (PDA), methanol, dichloromethane, HPLC-grade acetonitrile, and ethyl acetate were purchased from VWR (Fontenay-sous-Bois, France). Formic acid (FA) was purchased from Merck (Darmstadt, Germany). Water was purified by 0.22 µM membrane filtration and deionization by using a Barnstead Nanopure system from Thermo Scientific (Waltham, MA, USA) or a Milli-Q Plus system (Millipore, Billerica, MA, USA), while the DNeasy UltraClean Microbial kit was purchased from Qiagen (Hilden, Germany) and the GFX PCR DNA and Gel Band Purification Kit were purchased from GE Healthcare (Chicago, IL, USA).

Podophyllotoxin, MTT, propidium iodide (PI), Hoechst 33258 and Rodamine 123 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxycamptothecin (HCPT) was purchased from Harbin Shengtai Pharmaceutical Co., Ltd. (Harbin, China); RPMI 1640 culture medium was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA); fetal bovine serum (FBS) was obtained from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China) and trypsin was purchased from Gibco-BRL (Rockville, MD, USA). Additionally, mouse anti-human β-actin, cytochrome c (cyt-c), caspase-9 and caspase-3 polyclonal antibodies, alkaline phosphatase (AP)-conjugated goat anti mouse polyclonal antibody, SDS-PAGE sample loading buffer, blocking buffer, TBST, buffer5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitroblue tetrazolium (NBT) alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, Haimen, China); detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA).

Pure essential oil compounds in high purity (>95%), reagents, and solvents were obtained from various commercial sources, i.e., Sigma-Aldrich, Fluka, Merck, Frutarom, and Roth. The origin of the standard compounds used in bioassays are as follows: chloroquine diphosphate (Sigma-Aldrich, Buchs, Switzerland), benznidazole (Epichem Ltd., received from DNDi, Geneva), melarsoprol (Arsobal Sanofi-Aventis, received from WHO, Geneva), miltefosine (Sigma-Aldrich, Buchs, Switzerland), podophyllotoxin (Sigma-Aldrich, Buchs, Switzerland), and pentamidine (Sigma-Aldrich, Buchs, Switzerland).

Log-phase hPSCs (days 3–4 of subculture) were treated with metaphase arresting solution (Genial Genetic Solutions Ltd., Cheshire, UK) and 0.02–0.04 μg/ml podophyllotoxin (Sigma-Aldrich) for 90–120 min before chromosome preparation. The number of chromosomes was counted in 20 metaphase cells using a Nikon ECLIPSE Ni microscope and NIS Elements Br Software (Nikon, Tokyo, Japan). mFISH staining was performed using a 24XCyte Human Multicolor FISH Probe kit (MetaSystems GmbH Altlussheim, Germany) and analyzed with a Zeiss Axio Imager up-light microscope and the Isis FISH Imaging System (Carl Zeiss).

The CRC cell lines HT29, DLD1, and Caco2 were purchased from the Korean Cell Line Bank (Seoul, South Korea). Authentications of the cells used in the study were performed by a commercial service (Korean Cell Line Bank) via short tandem repeat profiling. Cells were cultured in either RPMI 1640 or DMEM culture medium (GenDEPOT, Katy, TX, USA) supplemented with 10% fetal bovine serum (GenDEPOT) and 1% penicillin–streptomycin solution. Cells were cultured in a humidified atmosphere at 37 °C in 5% CO₂. Podophyllotoxin and picroPodophyllotoxin were purchased from Sigma-Aldrich (St. Louis, MO, USA). DPT was prepared as previously described and provided by Prof. Goo Yoon [32 ].