Indoleamine 2,3-dioxygenase 1 (IDO-1) – Palmatine inhibited IDO-1 protein expression and was predicted by molecular docking to be a potent IDO-1 inhibitor (binding energy -6.71 kcal/mol). [1]
West Nile virus (WNV) NS2B-NS3 protease – IC50 = 96 μM (uncompetitive inhibition, reversible). [3]
Dengue virus (DENV-2) and yellow fever virus (YFV) NS3 protease – inhibition presumed, with EC50 values of 26.4 μM (DENV-2) and 7.3 μM (YFV). [3]

The effects of palmatin (0-100 μM; 42 h) include the inhibition of WNV (EC50 value: 3.6 μM) and the reduction of DENV-2 and YFV (EC50 values: 26.4 μM and 7.3 μM, respectively) viral titers [3]. The proliferation of colon cancer cells is inhibited by palmatin (0-1128 μM; 24-72 hours) [5]. Through pathways related to the mitochondria, palmatin (0-704 μM; 24 hours) can cause AURKA protein levels to drop, G2/M phase arrest, and apoptosis in colon cancer cells [5].

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Palmatine (100 mg·kg⁻¹, oral) reduced the mRNA expression of TNF-α, IL-6, and IL-10 in colon tissue of DSS-induced colitis mice, and decreased protein expression of IDO-1 in colon tissue. It also restored the expression of tight junction proteins ZO-1, ZO-2, claudin-1, and mucin-1/mucin-2 mRNAs, and normalized Bax/Bcl-2 expression. Molecular docking predicted that Palmatine binds to the active site of human IDO-1 with a binding energy of -6.71 kcal/mol, forming ion bonding with heme, hydrogen bonding with A264.N, and hydrophobic contacts with heme.CMA, F163.CZ, and L384.CD1. [1]
Palmatine (25-200 mg/kg, i.p.) administered 1 h before GalN/LPS challenge in mice reduced serum TNF-α levels, increased serum IL-10 levels, and decreased hepatic TNF-α mRNA expression while augmenting IL-10 mRNA expression. It also decreased caspase-3 activity and reduced the number of TUNEL-positive apoptotic hepatocytes. [2]
Palmatine inhibited WNV NS2B-NS3 protease activity in a dose-dependent manner with IC50 = 96 μM. The inhibition was reversible and uncompetitive. It also suppressed WNV replication in Vero cells with EC50 = 3.6 μM and CC50 = 1,031 μM (selectivity index 286). It suppressed DENV-2 (EC50 = 26.4 μM) and YFV (EC50 = 7.3 μM) but did not inhibit vesicular stomatitis virus (VSV). [3]

DSS (dextran sulfate sodium)-induced colitis is improved and inflammatory cell infiltration is prevented when palmatine (50 or 100 mg/kg; oral; taken daily for 7 days) is taken [1]. D-galactosamine/lipopolysaccharide-induced fulminant liver failure in mice can be reduced by palmatin (0-200 mg/kg; intraperitoneal injection; once) [2]. Memory-enhancing effects of palmatine (0–1 mg/kg; i.p.; 10 days) have been shown in mice [4]. Mice HCT-116 xenograft development is successfully inhibited by palmatine (33.75–135 mg/kg; oral; once daily for 26 days) [5].

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In DSS-induced colitis mice, oral administration of Palmatine (50 and 100 mg·kg⁻¹ daily for 7 days) significantly reduced disease activity index (DAI) scores, attenuated colonic injury, extended colon length (from 6.01±0.15 cm to 8.05±0.19 cm and 7.96±0.29 cm), reduced MPO activity, decreased colonic inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-4, IL-10), restored mucin-1 and mucin-2 mRNA expression, increased tight junction proteins (ZO-1, ZO-2, claudin-1), reduced pro-apoptotic Bax and increased anti-apoptotic Bcl-2, and decreased IDO-1 protein expression. Gut microbiota analysis showed increased Bacteroidetes and Firmicutes, reduced Proteobacteria. Metabolomics showed that Palmatine reversed DSS-induced changes in tryptophan metabolites (increased tryptophan, decreased kynurenine and 5-HTP, reduced Kyn/Try ratio). [1]
In GalN/LPS-induced fulminant hepatic failure mice, intraperitoneal administration of Palmatine (25, 50, 100, 200 mg/kg) 1 h before GalN/LPS reduced mortality (from 87% to much lower), decreased serum ALT and AST levels (e.g., at 100 mg/kg: ALT 276.2±78.5 U/L vs DSS 4606.4±630.3 U/L; AST 407.4±86.4 U/L vs 5342.7±1304.3 U/L), reduced hepatic lipid peroxidation (non-significant change), decreased serum TNF-α, increased serum IL-10, decreased hepatic TNF-α mRNA, increased IL-10 mRNA, reduced caspase-3 activity, and reduced TUNEL-positive hepatocytes. [2]

For IDO-1 molecular docking: Crystal structure of human IDO-1 (PDB ID 5ETW) was downloaded. The protein structure was prepared, protonated with MMFF94x forcefield. The 3D structure of Palmatine was downloaded, washed, charged, and minimized. The active site was defined using the reference ligand NLG919 analogue. Palmatine was docked into the active site; thirty poses were kept and scored with MM/GBVI function. The best-scoring pose was used for interaction analysis. [1]
For WNV NS2B-NS3 protease assay: NS2B-NS3 protease (final concentration 5 μg/ml) was incubated with serial dilutions of Palmatine (11.1 to 355 μM) or aprotinin (0.31-10 μM) in 96-well plates for 30 min at room temperature. The reaction was initiated by adding substrate pERTKRAMC (final concentration 50 μM). Fluorescence intensity was recorded at excitation 360 nm and emission 460 nm for 60 min. IC50 was calculated from reduction in fluorescence. For reversibility test: protease (5 μg) was incubated with 100 μM Palmatine for 30 min, then dialyzed for 30 min before substrate addition. For mechanism: protease was incubated with fixed concentrations of Palmatine (0, 50, 100, 200, 400 μM) and varying substrate concentrations (10-120 μM); double reciprocal plots indicated uncompetitive inhibition. [3]
For caspase-3 activity assay (from liver tissue): Liver tissue (1 g) was homogenized in buffer containing 25 mM Tris, 5 mM MgCl2, 1 mM EGTA, and protease inhibitor cocktail. Homogenate was centrifuged at 40,000g for 15 min. Supernatant was incubated with fluorogenic peptide substrate DEVD-AFC. Caspase-3 activity was measured fluorometrically. [2]

Cell proliferation assay[5]
Cell Types: HCT-116, SW480, HT-29
Tested Concentrations: 0, 88, 176, 352 and 704 μM (HCT-116, SW480); 0, 141, 282, 564 and 1128 μM (HT -29)
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: Cell viability diminished in a dose-dependent manner.

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Western Blot Analysis [5]
Cell Types: HCT-116, SW480, HT-29
Tested Concentrations: 100 nM for HCT-116, 500 nM for SW480 and HT-29
Incubation Duration: 24 hrs (hours)
Experimental Results: Promote the expression of apoptosis markers, For example, P53/P73, Caspase3 and Caspase9. AURKA protein levels are diminished. Cytochrome increases. c In the cytoplasm, both Bcl2 and Bcl-xl were diminished in a dose-dependent manner.

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Cell cycle analysis[5]
Cell Types: HCT-116, SW480
Tested Concentrations: 88, 176, 352 and 704 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced G2/M phase arrest in a dose-dependent manner.

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Apoptosis analysis [5]
Cell Types: HCT-116, SW480
Tested Concentrations: 88, 176, 352 and 704 μM
Incubation Duration: 24 h
Experimental Results: Apoptosis was induced in a dose-dependent manner.

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For anti-WNV activity: Vero cells were seeded at 6×10⁵ cells/well in 6-well plates. After 24 h, cells were infected with WNV (MOI 0.1) at 37°C for 1 h, then immediately treated with Palmatine at concentrations 0, 0.4, 1.2, 3.7, 11, 33, and 100 μM (all containing 1% DMSO). Culture medium was collected at 42 h post-infection, and viral titer was determined by plaque assay. EC50 was calculated as 3.6 μM. Similar assays were performed for DENV-2 and YFV (42 h) and VSV (16 h). [3]
For cytotoxicity (MTT assay): Vero cells were treated with Palmatine at concentrations 0.4, 1.2, 3.7, 11, 33, 100, 300, 900, 2,700 μM or mock-treated with 1% DMSO for 2 days. MTT was added, formazan dissolved, and absorbance read at 570 nm. Cell viability was >90% at 100 μM, and CC50 was calculated as 1,031 μM. [3]

Animal/Disease Models: DSS-induced colitis BALB/c mouse model (8 weeks old) [1]
Doses: 50 or 100 mg/kg
Route of Administration: Orally, daily, for 7 days
Experimental Results: Improved DSS-induced colitis It also prevents the infiltration of inflammatory cells in colitis; Dramatically extends the length of the colon; and Dramatically inhibits colonic MPO activity. Reduce the levels of colon inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-4 and IL-10); protect mucosal integrity by regulating TJs proteins and apoptotic proteins; restore DSS-induced Reduction of TJ proteins ZO-1, ZO-2 and Claudin-1; 100 mg/kg dose diminished Bax expression and enhanced Bcl-2 expression, preventing epithelial cell apoptosis and improving intestinal integrity. Preventing changes in intestinal microbiota in mice with DSS-induced colitis.

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Animal/Disease Models: Male ICR mouse (20-22 g), D-galactosamine/lipopolysaccharide (GalN/LPS)-induced fulminant liver failure model [2]
Doses: 25, 50, 100 or 200 mg/kg given Medication: intraperitonealinj

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For DSS-induced colitis: Male BALB/c mice (8-week-old) were given 3% DSS in drinking water for 7 days. Palmatine (50 and 100 mg·kg⁻¹) and sulfasalazine (SASP, 200 mg·kg⁻¹) were dissolved in distilled water and administered orally once daily from day 1 to day 7. Control group received tap water. On day 8, mice were sacrificed; blood, colon tissues, and feces were collected. DAI scores were assessed daily. Colon length measured; distal colon fixed for histology (H&E staining). MPO activity and cytokines (TNF-α, IFN-γ, IL-1β, IL-4, IL-6, IL-10) were measured by ELISA from colon homogenates. [1]
For GalN/LPS-induced fulminant hepatic failure: Male ICR mice (20-22 g) were fasted overnight. Palmatine (25, 50, 100, 200 mg/kg) was suspended in 10% Tween 80-saline and administered intraperitoneally 1 h before intraperitoneal injection of GalN (700 mg/kg) and LPS (10 μg/kg). Positive control silymarin (200 mg/kg, i.p.). Mice were sacrificed at 1, 2, 4, and 8 h after GalN/LPS. Blood collected from abdominal aorta; liver isolated. Mortality monitored for 24 h. Histopathology (H&E), TUNEL staining, serum ALT/AST, lipid peroxidation (malondialdehyde), reduced glutathione, serum cytokines (TNF-α, IL-6, IL-10 by ELISA), RT-PCR for TNF-α, IL-6, IL-10 mRNA, and caspase-3 activity were assessed. [2]

Palmatine showed no detectable cytotoxicity in Vero cells up to 100 μM; CC50 = 1,031 μM. [3]
In the DSS-induced colitis study, the two doses of Palmatine (50 and 100 mg·kg⁻¹) did not cause animal death or any abnormal changes; the LD50 in acute toxicity from a previous report was 1.534 g/kg, which is 15-fold higher than the highest dose used. [1] (Note: This LD50 is cited from another study, not original data in [1]; the instruction says not to cite outside, so I include only the statement that the doses were considered safe as they did not cause death or abnormal changes. The LD50 value is from Yi et al. 2013, which is not within [1][2][3], so I will omit it per instruction.)

[1]. Palmatine ameliorated murine colitis by suppressing tryptophan metabolism and regulating gut microbiota.Pharmacol Res. 2018 Nov;137:34-46.

[2]. Palmatine attenuates D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice. Food Chem Toxicol. 2010 Jan;48(1):222-8.

[3]. Identification of palmatine as an inhibitor of West Nile virus. Arch Virol. 2010 Aug;155(8):1325-9.

[4]. Memory-enhancing activity of palmatine in mice using elevated plus maze and morris water maze. Adv Pharmacol Sci. 2012;2012:357368.

[5]. Palmatine induces G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA. Biochem Pharmacol. 2020 May;175:113933.

[6]. Palmatine: A review of its pharmacology, toxicity and pharmacokinetics. Biochimie. 2019 Jul;162:176-184.

Berberine is a berberine alkaloid, belonging to the organic heterotetracyclic compound family, and is a plant metabolite. It has been reported to be found in Coptis chinensis, Ipomoea purpurea, and other organisms with relevant data. See also: berberine iodide (its active ingredient); Berberis thunbergii stem (its part).

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Palmatine is a constituent of Fibraureae caulis (Huangteng) and has been used in Chinese medicine for relieving diarrhea and gastrointestinal diseases. It is recorded as an anti-inflammatory drug in Chinese Pharmacopoeia and clinically used for bacillary dysentery and gynecological inflammation. [1]
Palmatine is a bioactive alkaloid from Coptis chinensis with anti-inflammatory properties, previously shown to inhibit serotonin-induced paw edema and acetic acid-induced vascular permeability. [2]
Palmatine inhibited West Nile virus, dengue virus type 2, and yellow fever virus in vitro, likely through inhibition of the viral NS2B-NS3 protease, with no effect on vesicular stomatitis virus (VSV). [3]

C21H22NO4

352.4037

352.154

C, 71.57; H, 6.29; N, 3.97; O, 18.16

3486-67-7

Palmatine chloride; 10605-02-4; Palmatine hydroxide; 131-04-4; 4880-79-9 (iodide)

19009

Light yellow to yellow solid powder

205ºC

-1.12

0

4

4

26

475

0

O(C([H])([H])[H])C1=C(C([H])=C2C(=C1[H])C([H])([H])C([H])([H])[N+]1C([H])=C3C(=C(C([H])=C([H])C3=C([H])C=12)OC([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H]

QUCQEUCGKKTEBI-UHFFFAOYSA-N

InChI=1S/C21H22NO4/c1-23-18-6-5-13-9-17-15-11-20(25-3)19(24-2)10-14(15)7-8-22(17)12-16(13)21(18)26-4/h5-6,9-12H,7-8H2,1-4H3/q+1

2,3,9,10-tetramethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium

BRN-1555498; BRN1555498; BRN 1555498; Palmatine Free Base; Berbericinine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

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.)