Acetylcholinesterase

Protopine hydrochloride (10–40 μM, 24–96 h) suppresses the EMT process, migration, invasion, and viability of liver cancer cells (HepG2, Huh7) [2]. Protopine (10–40 μM, 24 h) hydrochloride inhibits the PI3K/Akt signaling pathway and increases the production of caspase-3 and caspase-9 in HepG2 and Huh7 cells, hence inducing apoptosis [2]. In HepG2 and Huh7 cells, propepine hydrochloride (10–40 μM, 6 hours) causes ROS generation [2]. Protopine hydrochloride (0–10 μg/mL) decreases norepinephrine (NE) absorption in N1 cells and serotonin transporter (SERT) uptake in S6 cells [3].

Mice's memory impairment caused by 1 mg/kg of Scopolamine can be ameliorated by intraperitoneal injection of protopine hydrochloride at doses of 0.1 and 1 mg/kg[1]. Protopine (5–20 mg/kg, intraperitoneal) hydrochloride suppresses tumor growth, PI3K/Akt, and caspase-3 cleavage in xenografted BALB/c mice (injected subcutaneously with Huh-7 or HepG2 cells)[2]. In mouse HTR and TST tests, protopine hydrochloride (5–20 mg/kg, intraperitoneal injection) exhibits effects akin to those of an antidepressant[3]. Rats with focal cerebral ischemia injury respond better to protopine hydrochloride injections intraperitoneally (1-4 mg/kg, once day for 3 days)[4].

Protopine is an isoquinoline alkaloid that possesses various biological activities including the anti-tumour activity. However, the effects of protopine on liver carcinoma cells are still elusive. The aim of this study is to examine the effects of protopine on liver carcinoma cells both in vitro and in vivo.  Methods: MTT assay was performed to measure the cell viability. Wound healing and transwell assays were conducted to assess the motility of cells. Cellular apoptosis and ROS levels were measured by the flow cytometry. Western blotting assay was used to measure the change of proteins. The cytotoxicity of protopine was also evaluated in xenograft mice.  Results: Protopine inhibited viabilities and triggered apoptosis via the intrinsic pathway in a caspase-dependent manner in liver carcinoma cells. Furthermore, protopine also induced accumulation of intracellular ROS which further led to the inhibition of PI3K/Akt signalling pathway. Finally, in vivo study showed that protopine also repressed tumour growth in xenograft mice without noticeable toxicity.  Conclusions: Protopine might be used as a potential therapeutic agent for the treatment of liver carcinoma[2].

Western Blot Analysis[2]
Cell Types: HepG2, Huh7
Tested Concentrations: 10, 20, 40 μM
Incubation Duration: 24 h
Experimental Results: Induced the cleavage of caspase-3 and caspase-9. diminished Bcl-2 and Bcl-xl level. Induced the release of mitochondrial protein cytochrome c into the cytosol.

Animal/Disease Models: 5-Hydroxy-DL-tryptophan (5-HTP)-induced mouse model [3]
Doses: 5, 10, 20 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Increased 5-HTP-induced head Number of hemispheric twitch responses (HTR). Reduce the immobility time tested in the Tail Suspension Test (TST).

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The protopine isolated from a Chinese herb Dactylicapnos scandens Hutch was identified as an inhibitor of both serotonin transporter and noradrenaline transporter in vitro assays. 5-hydroxy-DL-tryptophan(5-HTP)-induced head twitch response (HTR) and tail suspension test were adopted to study whether protopine has anti-depression effect in mice using reference antidepressant fluoxetine and desipramine as positive controls. In HTR test, protopine at doses of 5, 10, 20 mg/kg dose dependently increase the number of 5-HTP-induced HTR. Protopine at doses of 3.75 mg/kg, 7.5 mg/kg and 30 mg/kg also produces a dose-dependent reduction in immobility in the tail suspension test. The present results open up new possibilities for the use of protopine in the treatment of mood disorders, such as mild and moderate states of depression.[3]

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Protopine, an isoquinoline alkaloidis, is known to produce many effects such as vasodilation, down-regulation of glutamate levels in brain and decrease of intracellular calcium. However, so far there is no report on the effect of protopine in cerebral ischaemia. In this study, the effect of protopine on the focal cerebral ischaemia was investigated in rats. Male Sprague-Dawley rats were divided into five groups: sham-operated group, vehicle-treated group and three doses of protopine-treated groups (0.98, 1.96 and 3.92 mg/kg). Protopine was intraperitoneally administered to rats once daily for 3 days prior to the ischaemia and 0.9% normal saline to rats in the vehicle-treated group in the same pattern. Rats in the sham-operated group were given 0.9% normal saline without the ischaemia. The focal cerebral ischaemia was induced by the middle cerebral artery occlusion for 24 hr via the intraluminal filament technique. The results showed that pre-treatment with protopine reduced the cerebral infarction ratio and serum lactate dehydrogenase activity, and improved the ischaemia-induced neurological deficit score and histological changes of brain in a dose-dependent manner. The further studies demonstrated that protopine increased superoxide dismutase activity in serum, and decreased total calcium and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive cells in the ischaemic brain tissue in the middle cerebral artery occlusion rats. The results indicate that protopine is able to produce an effective protection on the injury caused by the focal cerebral ischaemia in rats possibly through the multiple effects of calcium antagonism, antioxidation and depression of cell apoptosis.[4]

Absorption, Distribution, and Excretion
Xanfu Siwu Decoction (XFSWD) has been widely used clinically for the treatment of primary dysmenorrhea for hundreds of years with significant efficacy. One component of XFSWD, namely the eluent obtained by eluting the 60% ethanol-water extract through a macroporous adsorption resin (XFSWE), has a significant analgesic effect. This study aimed to investigate the pharmacokinetics and tissue distribution of four main active components (berberine, protopine, tetrahydroberberine, and tetrahydropalmatine) in dysmenorrhea rats after oral administration of XFSWE, and to compare the differences between normal rats and dysmenorrhea rats. A dysmenorrhea rat model was established using estradiol benzoate and oxytocin, with an experimental period of 7 days. On the last day of the experimental period, both normal rats and dysmenorrhea rats were orally administered XFSWE, and blood and tissue samples were collected at different time points. The contents of berberine, protopine, tetrahydroberberine, and tetrahydropalmatine in blood and tissue samples were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were calculated based on plasma concentration-time data using a non-compartmental model. One-way ANOVA was used to test differences in pharmacokinetic parameters among groups. Results showed statistically significant differences in Cmax, Tmax, AUC(0-t), AUC(0-∞), MRT(0-t), MRT(0-∞), and CL/F between normal rats and dysmenorrhea rats administered the same oral dose of XFSWE (P<0.05). Tissue distribution analysis showed the following overall trend: spleen > liver > kidney > uterus > heart > lung > ovary > brain > thymus; M-60 min > M-120 min > M-30 min > C-60 min > C-120 min > C-30 min. The protoporphyrin content in the liver, spleen, and uterus of dysmenorrhea rats was higher than in other tissues. Compared with normal rats, the compound content in different tissues of dysmenorrhea rats differed significantly at different time points (P<0.05). This study reports for the first time the pharmacokinetics and tissue distribution of dysmenorrhea-related animals. Results showed that berberine, protopine, tetrahydroberberine, and tetrahydropalmatine were absorbed at higher rates and eliminated more slowly in rats with dysmenorrhea syndrome, suggesting altered drug metabolism rates and extent. The results also showed significant differences in the concentrations of berberine, protopine, and tetrahydropalmatine in certain organs and at different time points between normal and dysmenorrhea-related rats, suggesting altered organ blood flow and perfusion rates in these animals. PMID:24837303

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Metabolites/Metabolites
California poppy preparations are used as herbal medicines and medicinal materials. This article describes the metabolic and toxicological analysis of the California poppy alkaloids papaverine and protopine in rat urine using gas chromatography-mass spectrometry. …Protopine…first undergoes extensive 2,3-methylenedioxy demethylation, followed by catechol-O-methylation. All phenolic hydroxyl metabolites are partially bound. The authors established a systematic toxicological analysis method using acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation, combined with full-scan gas chromatography-mass spectrometry. This method can detect the main metabolites of papaverine and protopine in rat urine, and the dosage used should be equivalent to that of drug users. Therefore, according to the authors' systematic toxicological analysis procedure, the use of papaverine preparations should also be detectable in human urine.

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XFSWD (Xiangfu Siwu Decoction) has been widely used clinically for the treatment of primary dysmenorrhea for hundreds of years and has shown good efficacy. One component of XFSWD, namely the product eluted with a macroporous adsorption resin using a 60% ethanol-water extract (XFSWE), shows significant analgesic effects. This study aims to investigate the pharmacokinetics and tissue distribution of four main bioactive components (berberine, protopine, tetrahydroberberine, and tetrahydropalmatine) in rats with dysmenorrhea symptoms after oral administration of XFSWE, and to compare the differences between normal rats and rats with dysmenorrhea symptoms. This study used estradiol benzoate and oxytocin to construct a rat model of dysmenorrhea symptoms. The experiment lasted for 7 days. On the last day of the experiment, normal rats and rats with dysmenorrhea symptoms were orally administered XFSWE, and blood and tissue samples were collected at different time points. The levels of berberine, protopine, tetrahydroberberine, and tetrahydropalmatine in the blood and tissue samples were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters were calculated based on plasma concentration-time data using a non-compartmental model. Differences in pharmacokinetic parameters among the groups were tested using one-way ANOVA. The results showed statistically significant differences in Cmax, Tmax, AUC(0-t), AUC(0-∞), MRT(0-t), MRT(0-∞), and CL/F between normal rats and rats with dysmenorrhea symptoms who were orally administered the same dose of XFSWE (P<0.05). Tissue distribution studies showed an overall trend of: spleen > liver > kidney > uterus > heart > lung > ovary > brain > thymus, with M-60 min > M-120 min > M-30 min > C-60 min > C-120 min > C-30 min. The protoporphyrin content in the liver, spleen, and uterus of rats exhibiting dysmenorrhea symptoms was higher than in other tissues. Compared with normal rats, the content of compounds in different tissues at different time points was significantly different in rats with dysmenorrhea symptoms (P<0.05). This study is the first to report the pharmacokinetics and tissue distribution in animals with dysmenorrhea symptoms. The results indicate that berberine, protoporphyrin, tetrahydroberberine, and tetrahydropalmatine were absorbed more readily and eliminated more slowly in rats with dysmenorrhea symptoms, suggesting that the rate and extent of drug metabolism in these rats were altered. The results also showed significant differences in the concentrations of berberine, protopine, and tetrahydropalmatine in certain organs and at specific time points between normal and dysmenorrhea rats, suggesting altered blood flow and perfusion rates in related organs of dysmenorrhea rats. California poppy preparations are used as herbal medicines and medicinal materials. This paper uses gas chromatography-mass spectrometry (GC-MS) to study the metabolism and toxicology of the California poppy alkaloids papaverine and protopine in rat urine. Protopine first undergoes extensive 2,3-methylenedioxy demethylation, followed by catechol-O-methylation. All phenolic hydroxyl metabolites are partially bound. The authors established a systematic toxicological analysis method using acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation, combined with full-scan GC-MS, which can detect the major metabolites of papaverine and protopine in rat urine at doses comparable to those in drug users. Therefore, the authors' systematic toxicological analysis method should also be able to detect the use of California poppy preparations in human urine.

Toxicity Overview
Identification and Uses: Protopine is a solid used as a drug. Human Exposure and Toxicity: Gene reporter assays using transiently transfected HepG2 cells confirmed that protopine-induced CYP1A1 expression was associated with slight or negligible activation of aryl hydrocarbon receptors. Protopine-induced CYP1A mRNA levels in HepG2 cells and human hepatocytes did not lead to increased CYP1A protein or activity levels. Animal Studies: In vitro radiolabeling studies showed that protopine can enhance the binding of γ-aminobutyric acid to rat brain synaptic membrane receptors. Protopine has antiarrhythmic effects and may directly inhibit rapid electrical activity in cardiomyocytes.
Studies have found that protopine can inhibit histamine H1 receptors and platelet aggregation, and has analgesic effects. Protopine is a compound with opioid-like biological activity, acting on opioid receptors. Its properties include inducing analgesia or anesthesia. Protopeptides selectively bind to histamine H1 receptors without activating them, thus blocking the effects of endogenous histamine. Classic antihistamines primarily antagonize or prevent the effects of histamine in immediate-type hypersensitivity reactions. They act on the bronchi, capillaries, and other smooth muscles to prevent or alleviate motion sickness, seasonal rhinitis, and allergic dermatitis, as well as to induce drowsiness. Protopeptides can also act as platelet aggregation inhibitors, antagonizing or inhibiting any mechanism leading to platelet aggregation, whether during activation and morphological changes, or after dense granule release and prostaglandin-thromboxane system stimulation. Protopeptides can inhibit the contraction of isolated myocardial papillary muscles and endothelin-induced proliferation of vascular smooth muscle cells. They can also shorten the action potential duration and prolong the effective refractory period of guinea pig myocardial papillary muscles. The protective effect of protopeptides against myocardial ischemia-reperfusion injury in rats and their induced relaxation of the rat thoracic aorta are related to the inhibition of calcium ion influx into voltage-gated and receptor-gated calcium ion channels. Protopine has long been a focus of biological research. Studies have shown that it possesses antiparasitic activity and exhibits weaker cytotoxicity compared to other isoquinoline alkaloids. In vitro experiments have revealed that protopine has a cytoprotective effect against oxidative stress-induced cell death. Animal model studies have demonstrated that this alkaloid possesses antiarrhythmic, antithrombotic, anti-inflammatory, and hepatoprotective effects. The biological activity of protopine may be related to its ability to inhibit calcium, sodium, and potassium channels. (PMID:15588728; PMID:21419197; L2104)

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Interactions
This study investigated the antiarrhythmic effects of protopine on experimental arrhythmias in various animal models. Protopine increased the aconitine dose required to induce ventricular tachycardia (VP), ventricular tachycardia (VT), and ventricular fibrillation (VF) in rats, and increased the strophanthin (strophanthin K) dose required to induce VP in guinea pigs. Furthermore, protopine shortened the duration of aconitine-induced central arrhythmias and phenylephrine-induced arrhythmias in rats. It also prevented arrhythmias induced by intravenous calcium chloride and inhaled chloroform in rats and mice, respectively. In rabbits, the drug increased the incidence of ventricular fibrillation (VFT). These findings suggest that protopine has antiarrhythmic effects and may directly inhibit the rapid electrical activity of cardiomyocytes. Lu Z et al.; Chinese Pharmaceutical Journal; 30: 81-84 (Reference 9) (1995)

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Antidote and First Aid Treatment
/SRP:/ Immediate First Aid: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask, or simple breathing mask, and follow the training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, tilt the patient forward or place them in the left lateral decubitus position (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek medical attention.

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Non-human Toxicity Values
Guinea Pig Intraperitoneal LD50: 116 mg/kg
Guinea Pig Oral LD50: 237 mg/kg
Mouse Intraperitoneal LD50: 482 mg/kg

[1]. Protopine from Corydalis ternata has anticholinesterase and antiamnesic activities. Planta Med. 1999 Apr;65(3):218-21.

[2]. Protopine triggers apoptosis via the intrinsic pathway and regulation of ROS/PI3K/Akt signalling pathway in liver carcinoma. Cancer Cell Int. 2021 Jul 27;21(1):396.

[3]. Protopine inhibits serotonin transporter and noradrenaline transporter and has the antidepressant-like effect in mice models. Neuropharmacology. 2006 Jun;50(8):934-40.

[4]. Protective effect of protopine on the focal cerebral ischaemic injury in rats. Basic Clin Pharmacol Toxicol. 2007 Aug;101(2):85-9.

Mechanism of Action
Calcium chloride (0.2 g/kg, intravenous injection) induced ventricular fibrillation in rats for 2 seconds, leading to death. Protopilogic hydrochloride (10 mg/kg) prolonged ventricular fibrillation to 186 seconds. Furthermore, in all treated animals, sinus rhythm was restored within 3 minutes of administration.

C20H20CLNO5

389.8295

389.103

6164-47-2

Protopine;130-86-9

22543

PRISMS FROM ALC

547.5ºC at 760 mmHg

208ºC

284.9ºC

3.297

1

6

0

27

542

0

Cl.CN1CCC2=CC3OCOC=3C=C2C(=O)CC2=C(C3OCOC=3C=C2)C1

NWNVDSJZGYDVQW-UHFFFAOYSA-N

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

15-methyl-7,9,19,21-tetraoxa-15-azapentacyclo[15.7.0.04,12.06,10.018,22]tetracosa-1(17),4,6(10),11,18(22),23-hexaen-3-one;hydrochloride

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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