Indoleamine 2,3-dioxygenase (IDO) (Ki = 0.37 μM) [1]

Coptisine has an IC50 value of 6.3 μM and a Ki value of 5.8 μM, making it a highly effective non-competitive IDO inhibitor[1]. The growth of A549, H460, H2170, MDA-MB-231, and HT-29 cells is inhibited by copoxine (0.1-100 μM), with IC50 values of 18.09, 29.50, 21.60, 20.15, and 26.60 μM, in that order. In A549 cells, coptisine (12.5, 25, and 50 μM) concentration-dependently causes G2/M arrest and apoptosis, downregulates the expression of cyclin B1, cdc2, and cdc25C, and increases the expression of pH2AX and p21. In A549 cells, copoposite (12.5, 25, 50 μM) also causes mitochondrial dysfunction and triggers caspase activity. Additionally, ROS levels are raised by coptisine (50 μM) in a time-dependent manner (0.5, 1, 2, 4, 12, and 24 hours) [3].

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- Coptisine (0.1-10 μM) dose-dependently inhibited IDO enzyme activity in IFN-γ-stimulated BV2 microglial cells, reducing the production of kynurenine (KYN) and the KYN/tryptophan (TRP) ratio [1]
- Treatment of IFN-γ-stimulated BV2 cells with Coptisine (1, 5, 10 μM) downregulated the mRNA and protein expression of IDO, as detected by qPCR and Western blot [1]
- Coptisine (5, 10, 20, 40 μM) inhibited the proliferation of non-small-cell lung cancer A549 cells in a dose- and time-dependent manner (IC50 = 18.7 μM at 48 h, detected by MTT assay) [3]
- Coptisine (10, 20, 40 μM) induced G2/M phase cell cycle arrest in A549 cells, as shown by flow cytometry analysis (increased proportion of cells in G2/M phase and decreased proportion in G0/G1 phase) [3]
- Coptisine (10, 20, 40 μM) triggered reactive oxygen species (ROS) generation in A549 cells, leading to mitochondria-mediated apoptosis: increased apoptotic rate (Annexin V-FITC/PI staining), decreased mitochondrial membrane potential (JC-1 staining), upregulated Bax/Bcl-2 ratio, and activated caspase-3, -8, -9 (detected by Western blot) [3]
- Pretreatment with ROS scavenger N-acetylcysteine (NAC) reversed Coptisine-induced ROS production, mitochondrial dysfunction, and apoptosis in A549 cells [3]

Mice's LD50 value for coptisine was 880.18 mg/kg, and its toxicity increased with concentration. The dosage of 154 mg/kg/day for 90 days did not cause toxicity in SD rats. In addition to increasing HDL-c content to varied degrees and slowing down the weight gain brought on by the HFHC diet, copoxine (23.35, 46.7, 70.05 mg/kg, po) increased fecal cholesterol and TBA levels in hamsters in a dose-dependent manner. It also reduced TC, TG, and LDL-c levels in the serum of the animals. Inducing the expression of SREBP-2, LDLR, and CYP7A1 proteins involved in cholesterol metabolism, coptisine (70.05 mg/kg, po) lowers the level of HMGCR protein expression [2].

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- In APP/PS1 double-transgenic Alzheimer's disease (AD) mice (6-month-old), oral administration of Coptisine (20, 40 mg/kg/day) for 3 months significantly improved cognitive impairment. The mice showed increased escape latency in the Morris water maze test, improved discrimination index in the novel object recognition test, and enhanced step-through latency in the passive avoidance test compared with the vehicle group [1]
- Coptisine (20, 40 mg/kg/day) reduced the levels of KYN and KYN/TRP ratio in the serum and brain (hippocampus, cortex) of AD mice, and downregulated IDO mRNA and protein expression in the hippocampus and cortex (detected by qPCR and Western blot) [1]
- Coptisine (20, 40 mg/kg/day) decreased the number of amyloid-beta (Aβ) plaques in the hippocampus and cortex of AD mice (detected by immunohistochemistry) [1]
- In high-fat diet (HFD)-fed Syrian golden hamsters, oral administration of Coptisine (50, 100 mg/kg/day) for 4 weeks significantly reduced serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDL-C) compared with the HFD control group [2]
- Coptisine (50, 100 mg/kg/day) decreased hepatic TC and TG levels, and downregulated the mRNA expression of hepatic cholesterol synthesis-related genes (HMGCR, SREBP-2) and lipid accumulation-related gene (PPARγ) in HFD-fed hamsters (detected by qPCR) [2]

- Recombinant human IDO protein was incubated with different concentrations of Coptisine (0.01-10 μM) and L-tryptophan (substrate) in reaction buffer. After incubation at 37°C for 1 hour, the reaction was terminated, and the concentration of KYN (product of IDO-catalyzed reaction) was measured by high-performance liquid chromatography (HPLC) to calculate IDO enzyme activity inhibition rate and Ki value [1]

- BV2 microglial cell experiment: Cells were seeded and stimulated with IFN-γ (20 ng/mL) for 24 hours to induce IDO expression, then treated with Coptisine (0.1-10 μM) for another 24 hours. qPCR was used to detect IDO mRNA expression (with GAPDH as internal control), Western blot to detect IDO protein level, and HPLC to measure KYN and TRP concentrations in cell supernatants [1]
- A549 cell proliferation assay: Cells were seeded in 96-well plates and treated with Coptisine (5-40 μM) for 24, 48, 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability [3]
- A549 cell cycle assay: Cells were treated with Coptisine (10-40 μM) for 24 hours, harvested, fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [3]
- A549 cell apoptosis assay: Cells were treated with Coptisine (10-40 μM) for 24 hours, stained with Annexin V-FITC/PI, and apoptotic rate was detected by flow cytometry; mitochondrial membrane potential was measured by JC-1 staining and flow cytometry [3]
- Western blot for A549 cells: Cells were treated with Coptisine (10-40 μM) for 24 hours, lysed to extract proteins, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against Bax, Bcl-2, caspase-3, -8, -9, and β-actin. Bands were visualized and quantified [3]
- ROS detection in A549 cells: Cells were loaded with DCFH-DA probe, treated with Coptisine (10-40 μM) for 24 hours (some cells pretreated with NAC for 1 hour), and ROS level was detected by flow cytometry [3]

- AD mouse model (APP/PS1 double-transgenic mice): 6-month-old male mice were randomly divided into vehicle group and Coptisine treatment groups (20, 40 mg/kg/day, n=10 per group). Coptisine was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) solution. Mice received daily oral gavage for 3 months. Cognitive function tests (Morris water maze, novel object recognition, passive avoidance) were performed before sacrifice. Serum, hippocampus, and cortex tissues were collected for KYN/TRP ratio detection, qPCR, Western blot, and immunohistochemistry [1]
- Hypercholesterolemic hamster model: Male Syrian golden hamsters were fed a high-fat diet (HFD) for 2 weeks to induce hypercholesterolemia, then randomly divided into HFD control group and Coptisine treatment groups (50, 100 mg/kg/day, n=8 per group). Normal diet group was set as control. Coptisine was dissolved in distilled water and administered by daily oral gavage for 4 weeks. Body weight was recorded weekly. At the end of treatment, serum was collected to detect lipid profiles (TC, TG, LDL-C, HDL-C), and liver tissue was collected for TC/TG content measurement and qPCR analysis [2]

Absorption, Distribution and Excretion
Corydalis saxicola Bunting is an important component of many traditional Chinese medicine formulas. It has been shown to possess various pharmacological activities, including antibacterial, antiviral, and anticancer activities. Its active components include dehydrocarbamide, Coptisine, dehydroapocarbamide, and tetradehydroscoline. This study aimed to investigate the pharmacokinetics and tissue distribution of Corydalis saxicola in rats using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The systemic clearance of the four active alkaloids in plasma exceeded 93% of hepatic blood flow, indicating that they are likely rapidly cleared by the liver. After intravenous and oral administration, less than 10% of the drug was excreted in the urine, suggesting that these four alkaloids may undergo significant metabolism in vivo, or that the drug may be excreted via routes other than urine. The excretion of these four alkaloids after oral administration was significantly lower than that after intravenous administration, suggesting a significant first-pass effect after oral administration. The high apparent volume of distribution indicates that these four alkaloids are widely distributed in rats. Our results also indicate that these four alkaloids can be absorbed after oral administration, although less than 15% of the drug is absorbed into the systemic circulation. In summary, the good oral bioavailability of these four active alkaloids in rats makes further research on the extract of Coptis chinensis worthy of improvement in its oral bioavailability.
This study investigated the absorption of Coptisine chloride (COP) and Coptisine erythrin (BRB) as certain traditional Chinese medicine chemical components in human intestinal epithelial cells. Using a Caco-2 (human colon adenocarcinoma cell line) cell monolayer as an intestinal epithelial cell model, the permeability of COP and BRB from the apical (AP side) to the basal lateral (BL side) and from the BL side to the AP side was examined. The concentrations of these two alkaloids were determined using reversed-phase high-performance liquid chromatography-ultraviolet detection. Transport parameters and apparent permeability coefficients (P_app) were calculated and compared with the corresponding parameters for propranolol and atenolol. The P(app) values were also compared with those of previously reported model compounds (propranolol and atenolol). The P(app) values for COP and BRB were (1.103 ± 0.162) × 10⁻⁵ and (1.309 ± 0.102) × 10⁻⁵ cm·s⁻¹ (from AP side to BL side), and (0.300 ± 0.041) × 10⁻⁵ and (1.955 ± 0.055) × 10⁻⁵ cm·s⁻¹ (from BL side to AP side). Their P(app) values were the same as those for propranolol [(2.23 ± 0.10) × 10⁻⁵ cm·s⁻¹] (a transcellular transport marker, used as a high-permeability control). On the other hand, the efflux transport of BRB was 1.49 times higher than the influx transport, with a P(app) value of 0.67. However, the P(app A-->B)/P(app B-->A) value for COP was 3.67, indicating that efflux transport did not participate in its absorption mechanism in the Caco-2 cell monolayer model. Both COP and BRB can be absorbed by intestinal epithelial cells and are completely absorbed compounds. BRB may be involved in the efflux mechanism from the basal side to the apex in the Caco-2 cell monolayer model. To determine the pharmacokinetics, distribution, and interconversion of total alkaloids, Coptisine, coptisine, Coptisine, and palmatine in rats, the total alkaloids and Coptisine were fed to rats, and their contents in plasma, tissues, and the gastrointestinal tract were determined by reversed-phase high-performance liquid chromatography. The peak time of Coptisine in blood was 2.0. The maximum plasma concentrations of Coptisine in rats were 5.0 hours (Cmax 3.7 mg·L⁻¹) and 5.0 hours (Cmax 2.8 mg·L⁻¹), respectively. Coptisine in rat blood can be converted to tetrandrine. After gavage administration of total alkaloids to rats, the gastric Coptisine content decreased monotonically, while the contents of Coptisine, palmatine, and tetrandrine gradually increased, suggesting that Coptisine may be converted to tetrandrine in the stomach. Animal experiments showed that Coptisine and palmatine are mainly distributed in the lungs, followed by the liver; while tetrandrine and Coptisine are mainly distributed in the liver, followed by the lungs. Coptisine can be converted to tetrandrine. The mechanism by which Coptisine reaches two maximum plasma concentrations in blood may be partly related to gastrointestinal propulsion.
Absorption and Transport Mechanisms This study used a Caco-2 cell uptake and transport model to investigate the uptake and transport of Coptisine, palmatine, lutein, and Coptisine. Cyclosporine A and verapamil were added as P-glycoprotein (P-gp) inhibitors, and MK-571 as a multidrug resistance-associated protein 2 (MRP2) inhibitor, respectively. In the uptake experiment, Coptisine, palmatine, lutein, and Coptisine were all uptaken by Caco-2 cells, and their uptake increased in the presence of cyclosporine A or verapamil. In the transport experiment, the apparent P(AP-BL) values of Coptisine, palmatine, lutein, and Coptisine ranged from 0.1 to 1.0 × 10⁶ cm/s, lower than the apparent P(BL-AB) value. The ER values of all compounds were greater than 2. Cyclosporine A and verapamil both increased the uptake of these compounds. P(app) (AP-BL) increased, but P(app) (BL-AB) of Coptisine, palmatine, myristine, and Coptisine decreased; ER value decreased by >50%. MK-571 had no effect on the transmembrane transport of Coptisine, palmatine, myristine, and Coptisine. In the concentration range of 1-100 μM, Coptisine, palmatine, myristine, and Coptisine had no significant effect on the bidirectional transport of Rho123. Coptisine, palmatine, myristine, and Coptisine are all substrates of P-gp; in the concentration range of 1-100 μM, Coptisine, palmatine, myristine, and Coptisine had no inhibitory effect on P-gp. Jiaotai Wan (JTW) is an important traditional Chinese medicine formula, composed of Coptis chinensis and Cortex cinnamomi powder. It is a famous traditional Chinese medicine formula for treating insomnia and has been used for hundreds of years. This study aimed to compare the pharmacokinetic characteristics of five protoCoptisine alkaloids (Coptisine, palmatine, Coptisine, epiCoptisine, and palmatine) – the main active components of cinnamon powder – in normal and insomnia-prone rats. We also investigated the pharmacokinetic differences of these five protoCoptisine alkaloids after single and multiple administrations. An insomnia-prone rat model was established by intraperitoneal injection of a single dose of p-chlorophenylalanine (PCPA). The concentrations of the five protoCoptisine alkaloids in rat plasma were quantitatively analyzed using rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples were collected at different time points, drug concentration-time curves were plotted, pharmacokinetic curves were constructed, and pharmacokinetic parameters were estimated. Student's t-tests were performed using SPSS 17.0 software. In the single-dose normal control group, the absorption of the five protoCoptisine alkaloids was slow, bioavailability was low, and the time to peak concentration was delayed. Following a single oral dose, the Cmax and Tmax of the five components in insomnia rats were significantly different from those in normal rats. After multiple oral doses, the pharmacokinetic parameters of the five proCoptisine alkaloids in insomnia rats changed considerably. Significant differences (p<0.05) were observed in the main pharmacokinetic parameters (such as Cmax and Tmax) between single and multiple oral doses in normal rats. The absorption of the five components in the multiple-dose group was superior to that in the single-dose group in insomnia rats. Notably, the plasma concentration curve in the multiple-dose model group showed three peaks. The pharmacokinetic behavior of the five components was described in this paper. In both the normal and model groups, the pharmacokinetic behavior of multiple doses was significantly different from that of a single dose; regardless of whether a single or multiple dose was administered, the pharmacokinetic behavior of insomnia rats was significantly different from that of normal rats. Multiple doses may increase the absorption of JTW in insomnia rats, thereby improving its bioavailability and exerting a more active therapeutic effect.

In hamsters fed a high-fat diet, oral administration of Coptisine (50, 100 mg/kg/day) for 4 consecutive weeks did not cause significant changes in body weight, food intake, or organ coefficients (liver, kidney, spleen). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) levels were all within the normal range, indicating that no obvious hepatotoxicity or nephrotoxicity was observed [2]. Coptisine did not induce significant cytotoxicity in BV2 cells at concentrations up to 10 μM (as detected by MTT assay) [1].

[1]. The IDO inhibitor coptisine ameliorates cognitive impairment in a mouse model of Alzheimer's disease. J Alzheimers Dis. 2015;43(1):291-302.

[2]. The safety and anti-hypercholesterolemic effect of coptisine in Syrian golden hamsters. Lipids. 2015 Feb;50(2):185-94.

[3]. Coptisine-induced cell cycle arrest at G2/M phase and reactive oxygen species-dependent mitochondria-mediated apoptosis in non-small-cell lung cancer A549 cells. Tumour Biol. 2017 Mar;39(3):1010428317694565.

Coptisine is an alkaloid and a metabolite. It has been reported that Coptisine is found in Coptis chinensis, Corydalis yanhusuo, and other organisms with relevant data. See also: Saussurea indica (partial); Chelidonium majus inflorescence apex (partial). - Coptisine is a natural isoquinoline alkaloid extracted from Coptis chinensis and other Chinese medicinal herbs [1][2][3] - The mechanism by which Coptisine improves cognitive impairment associated with Alzheimer's disease includes inhibiting IDO activity and expression, reducing kynurenine (KYN) production, and alleviating Aβ deposition [1] - Coptisine exerts its cholesterol-lowering effect by downregulating the expression of hepatic cholesterol synthesis genes (HMGCR, SREBP-2) and lipid accumulation genes (PPARγ) [2] - The antitumor effect of Coptisine on A549 cells is mediated by ROS-dependent mitochondrial apoptosis pathway and G2/M phase cell cycle arrest [3]

C19H14NO4

320.3188

320.092

3486-66-6

Coptisine Sulfate;1198398-71-8;Coptisine chloride;6020-18-4

72322

Typically exists as solid at room temperature

212-217℃

-0.87

0

4

0

24

502

0

O1C([H])([H])OC2=C1C([H])=C1C(=C2[H])C([H])([H])C([H])([H])[N+]2C([H])=C3C4=C(C([H])=C([H])C3=C([H])C=21)OC([H])([H])O4

XYHOBCMEDLZUMP-UHFFFAOYSA-N

InChI=1S/C19H14NO4/c1-2-16-19(24-10-21-16)14-8-20-4-3-12-6-17-18(23-9-22-17)7-13(12)15(20)5-11(1)14/h1-2,5-8H,3-4,9-10H2/q+1

5,7,17,19-tetraoxa-13-azoniahexacyclo[11.11.0.02,10.04,8.015,23.016,20]tetracosa-1(13),2,4(8),9,14,16(20),21,23-octaene

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.)