The study focuses on downstream effects such as induction of apoptosis, G2/M arrest, and modulation of proteins like Mcl-1, CDK1, cyclin B1, and AIF translocation. [1]

Chelidonine (5, 10 and 20 μM; 3–4 days) resulted in lesions and ventral curling in Dugesia japonica; at 20 μM, Djmcm2 expression was greatly reduced, but at 5 and 10 μM, no reduction was seen; also, it blocked stem cells' ability to advance through the cell cycle [2]. Melanoma cell lines are susceptible to the cytotoxic effects of chelidonine (0–3 μg/mL; 48 hours) [3]. In A-375 cells, the mitochondrial membrane potential (MMP) was 50% lower at 1 and 1.5 μg/mL and at 3 μg/mL of chelidonine (1, 2, and 3 μg/mL; 24 hours). 62% decrease [3].

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Chelidonine induced apoptosis in T98G human glioblastoma cells in a dose-dependent manner, as indicated by an increase in the sub-G1/0 population (apoptotic cells). At 0.6 µM, the sub-G1/0 population increased approximately 10-fold compared to control.
Chelidonine (1.0 µM for 24 hours) reduced the viability of T98G glioblastoma cells to about 60%, while having little to no effect on the viability of other tested cell lines (A549, MCF7, MDA-MB-231, SW620, HEK293, HUVEC, CCD-25Sk).
Chelidonine treatment led to decreased levels of the anti-apoptotic protein Mcl-1, cleavage (activation) of caspase-3 and caspase-9, and cleavage of PARP, indicating activation of the apoptotic pathway.
Chelidonine induced both caspase-dependent and caspase-independent apoptosis. The pan-caspase inhibitor Z-VAD-FMK partially inhibited apoptosis, and chelidonine treatment also induced the translocation of Apoptosis-Inducing Factor (AIF) from the cytosol to the nucleus, a hallmark of caspase-independent apoptosis.
Chelidonine caused G2/M cell cycle arrest in T98G cells in a dose-dependent manner, particularly at 0.6 µM. This arrest was associated with accumulation of cells in G2/M phase and an eventual increase in the sub-G1/0 apoptotic population.
Prolonged G2/M arrest by chelidonine was associated with the formation of abnormal multipolar spindle assemblies, accumulation of cyclin B1, altered phosphorylation of CDK1 (decreased p-Tyr15, increased p-Thr161), and increased expression of mitotic markers (Aurora A, PLK1, MPM-2).
The CDK1 inhibitor RO-3306 reversed chelidonine-mediated Mcl-1 degradation, mitochondrial depolarization, and apoptosis, suggesting that CDK1 activation plays a key role in linking chelidonine-induced G2/M arrest to apoptosis via Mcl-1 degradation. [1]

Cytotoxicity assay [3]
Cell Types: A-375, A-375-p53DD and A-375-p53sh
Tested Concentrations: 0-3 μg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: demonstrated cytotoxic activity against melanoma cell lines, The values were 0.910±0.017 μg/ml, 0.634±0.009 μg/ml and 0.772±0.045 μg/ml in A-375, A-375-p53DD and A-375-p53sh, respectively.

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Cell Viability Assay: Various cell lines were seeded in 96-well plates and treated with 1.0 µM chelidonine for 24 hours. Cell viability was assessed using the MTS assay by adding the reagent to each well, incubating for 1.5 hours, and measuring absorbance at 490 nm.
Flow Cytometry for Cell Cycle and Apoptosis: For cell cycle analysis, cells were harvested, fixed in cold ethanol, treated with RNase, stained with propidium iodide (PI), and analyzed by flow cytometry. The sub-G1/0 population was used as an indicator of apoptotic cells.
For mitochondrial membrane potential assessment, cells were stained with Mitotracker Red CMXRos and analyzed by flow cytometry.
Western Blot Analysis: Whole-cell lysates were prepared using a protein extraction solution. Protein concentrations were determined, and equal amounts were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and probed with primary antibodies overnight. Membranes were incubated with peroxidase-conjugated secondary antibodies, and bands were visualized using enhanced chemiluminescence.
Immunofluorescence Assay: T98G cells grown on coverslips were fixed, permeabilized, and incubated with primary antibodies (e.g., anti-AIF, anti-α-tubulin, anti-pericentrin) followed by fluorescent secondary antibodies. Nuclei were stained with DAPI. Images were captured using a confocal laser scanning microscope.
Cell Cycle Synchronization: T98G cells were synchronized at the G1/S boundary using a double thymidine block method. Cells were treated with 2 mM thymidine for 12 hours, washed, released into fresh medium for 12 hours, treated again with thymidine for 12 hours, washed, and then released into medium containing chelidonine or vehicle for specified time points. [1]

Metabolism / Metabolites
Paraoxygenase (PON1) is a key enzyme in organophosphate metabolism. PON1 can inactivate certain organophosphates through hydrolysis. PON1 hydrolyzes active metabolites from various organophosphate pesticides and nerve agents (such as soman, sarin, and VX). The existence of PON1 polymorphism leads to differences in the enzyme activity level and catalytic efficiency of this esterase, which in turn suggests that different individuals may be more susceptible to the toxic effects of organophosphate exposure.

Toxicity Summary
Chelidonine is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Nerve gases and substances in many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thereby completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. Inhibition of acetylcholinesterase results in the accumulation and sustained action of acetylcholine, leading to the continuous transmission of nerve impulses and the inability to stop muscle contractions. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds designed to bind to the enzyme's active site. Its structural requirements include a phosphorus atom with two lipophilic groups, a leaving group (e.g., a halide or thiocyanate), and a terminal oxygen atom. Chelidonine exhibits inhibitory activity against acetylcholinesterase and butyrylcholinesterase. (Wikipedia) Generally, some alkaloid extracts from Chelidonium majus, such as those containing protoberberine and benzo[c]phenanthridine alkaloids (e.g., chelidonine), can intercalate into DNA, thereby inhibiting the biosynthesis of DNA and RNA polymerases, topoisomerases, telomerases, and even ribosomal proteins, or binding to tubulin/microtubules to act as a spindle toxin. Chelidonine can overcome multidrug resistance (MDR) in different cancer cell lines by interacting with ABC transporters, CYP3A4, and GST to induce apoptosis and produce cytotoxic effects. It can induce apoptosis in MDR cells, accompanied by activation of caspase-3, -8, and -6/9 and exposure of phosphatidylserine (PS). (A15442) Chelidonine is known to cause mitotic arrest and interact weakly with tubulin. Chelidonine has been shown to have a weak inhibitory effect on cell growth in two normal cell lines (monkey kidney cells and Hs27 cells), two transformed cell lines (Vero cells and Graham 293 cells), and two malignant cell lines (WHCO5 cells and HeLa cells). (A15443)

[1]. Chelidonine Induces Caspase-Dependent and Caspase-Independent Cell Death through G2/M Arrest in the T98G Human Glioblastoma Cell Line. Evid Based Complement Alternat Med. 2019 May 26;2019:6318179.

[2]. The in vivo effect of chelidonine on the stem cell system of planarians. Eur J Pharmacol. 2012 Jul 5;686(1-3):1-7.

[3]. Benzo[c]phenanthridine alkaloids exhibit strong anti-proliferative activity in malignant melanoma cells regardless of their p53 status. J Dermatol Sci. 2011 Apr;62(1):22-35.

Chelidonine is a basic alkaloid belonging to the benzophenanthridine class of alkaloids and alkaloid antibiotics. It has been reported to exist in Stylophorum lasiocarpum, Chelidonium majus, and other organisms with relevant data. Chelidonine is isolated from poppy plants and possesses inhibitory activity against acetylcholinesterase and butyrylcholinesterase. See also: Chelidonium majus inflorescence (partial); chelidonine (+) (note moved here). Chelidonine is the main isoquinoline alkaloid extracted from Chelidonium majus L. In this study, chelidonine exhibited selective cytotoxicity against T98G human glioblastoma cells. The proposed mechanism involves inducing G2/M phase cell cycle arrest through the formation of abnormal multipolar spindles and dysregulation of the cyclin B1/CDK1 complex. This blockade, along with the CDK1-mediated degradation of the anti-apoptotic protein Mcl-1, leads to mitochondrial dysfunction and activates both caspase-dependent (via caspase-3/9) and caspase-independent (via AIF translocation) apoptosis pathways. This study suggests that chelidonine is a potential lead compound for anticancer chemotherapy, particularly for the treatment of aggressive glioblastoma. [1]

C20H19NO5

353.37

353.126

476-32-4

4312-31-6 (hydrochloride);63937-19-9 (sulfate)

197810

White to off-white solid powder

1.4±0.1 g/cm3

507.4±50.0 °C at 760 mmHg

135-140ºC

260.7±30.1 °C

0.0±1.4 mmHg at 25°C

1.667

2.75

1

6

0

26

560

3

CN1CC2=C(C=CC3=C2OCO3)[C@@H]4[C@H]1C5=CC6=C(C=C5C[C@@H]4O)OCO6

GHKISGDRQRSCII-ZOCIIQOWSA-N

InChI=1S/C20H19NO5/c1-21-7-13-11(2-3-15-20(13)26-9-23-15)18-14(22)4-10-5-16-17(25-8-24-16)6-12(10)19(18)21/h2-3,5-6,14,18-19,22H,4,7-9H2,1H3/t14-,18-,19+/m0/s1

(1S,12S,13R)-24-methyl-5,7,18,20-tetraoxa-24-azahexacyclo[11.11.0.02,10.04,8.014,22.017,21]tetracosa-2,4(8),9,14(22),15,17(21)-hexaen-12-ol

Chelidonine Khelidonin Stylophorin Stylophorin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~282.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)