Malachite green targets various cellular components, including DNA and proteins. It can be photoactivated and is capable of cross-linking DNA, covalently modifying proteins and lipids, and consequently inhibiting cell replication. The compound has a dissociation constant (Kd) of 40.0 nM. It exhibits cytotoxicity against human HepG2 cells with an IC₅₀ of 300.0 nM. Malachite green's antifungal properties are attributed to its ability to disrupt fungal cell membranes and inhibit fungal growth. Its carcinogenic effects are related to its DNA-damaging properties.

In vitro, malachite green inhibits the proliferation of HEp-2 human larynx cells (IC₅₀ = 2.06 µM). It is cytotoxic to HEp-2 cells and Caco-2 human colorectal adenocarcinoma cells (IC₅₀s = 2.03 and 13.8 µM, respectively). It exhibits cytotoxicity against human HepG2 cells with an IC₅₀ of 300.0 nM. Malachite green reduces fungal infection in rainbow trout eggs when used at a concentration of 2 µg/ml for 60 minutes twice a week. It is used as a biochemical indicator dye for titrations, pH indicators, and counterstains in histology applications.

In vivo, malachite green is used in aquaculture as an antifungal agent to reduce fungal infection in fish eggs. It has been shown to reduce fungal infection in, and increase hatching rates of, rainbow trout eggs. However, malachite green is a suspected carcinogen and teratogen. It shows high cytotoxicity and carcinogenicity to mammalian cells and can promote the development of liver tumors. Its use in aquaculture is restricted or banned in many countries due to safety concerns.

In vitro enzyme/receptor binding assays for malachite green are not typical, as the compound is primarily used as a dye and antifungal agent. However, its binding to DNA and proteins can be assessed using spectroscopic methods. The compound's dissociation constant (Kd = 40.0 nM) has been measured. Its cytotoxicity is assessed using standard cell viability assays. The compound's ability to cross-link DNA can be assessed using gel electrophoresis or other DNA damage assays. These assays confirm the compound's mechanism as a DNA-damaging agent.

In vitro cell-based assays for malachite green evaluate its cytotoxicity against various cancer cell lines. HEp-2 human larynx cells, Caco-2 human colorectal adenocarcinoma cells, and HepG2 cells are cultured and treated with serial dilutions of malachite green. Cell viability is assessed using MTT or CellTiter-Glo assays. The IC₅₀ values for cytotoxicity are calculated. The compound's effects on cell proliferation and apoptosis are assessed using flow cytometry or microscopy. These assays confirm the compound's cytotoxic activity.

In vivo animal experiments for malachite green have been conducted in aquaculture settings to evaluate its antifungal efficacy. Rainbow trout eggs are treated with malachite green at 2 µg/ml for 60 minutes twice a week, and fungal infection and hatching rates are assessed. Toxicological studies in rodents have shown that malachite green can promote the development of liver tumors. The compound is a suspected carcinogen and teratogen. Its use in aquaculture is restricted or banned in many countries.

Absorption, Distribution and Excretion
This study determined the distribution of malachite green in channel catfish (Ictalurus punctatus) using intravenous administration (0.8 mg/kg) or water exposure (0.8 mg/L, lasting 1 hour). Following intravenous administration, the mean plasma concentration of the parent compound decreased in three phases, with a terminal elimination half-life of 6.2 hours. After water exposure, malachite green was rapidly absorbed and concentrated in tissues. Its accumulation rate was directly related to the pH of the exposed water. The elimination of the parent compound from plasma after water exposure also followed a three-phase pattern, with a terminal half-life of 4.7 hours. In muscle tissue, the half-life of the parent compound was approximately 67 hours. Malachite green and its metabolites were widely distributed in all tissues. In fish exposed to ¹⁴C-labeled malachite green, the highest total drug equivalent concentration was observed in abdominal fat and the lowest in plasma. Malachite green is rapidly and extensively metabolized to its reduced form, colorless malachite green, which is slowly cleared from tissues. Colorless malachite green is a suitable target analyte for monitoring channel catfish exposure to this drug. Rainbow trout underwent a 6-day therapeutic immersion in a 0.2 mg/L malachite green solution. Malachite green residues were subsequently detected in the muscle, liver, and skin of the test fish. Immediately after immersion, malachite green was detected in the muscle, liver, and skin at concentrations of 0.383, 0.834, and 0.649 mg/kg (including both colored and colorless forms), respectively. After 8 weeks of immersion, a significant decrease in colored malachite green levels was observed, while the colorless form remained detectable in the fish for up to 10 months. Analysis after 12 months of immersion was negative.
Pharmacokinetic (PK) data for malachite green are limited. The compound is absorbed through the skin and gastrointestinal tract. It is metabolized to leucomalachite green, a major metabolite that is more persistent in tissues. Leucomalachite green is a suspected carcinogen. The compound has a molecular weight of 364.91 g/mol. It is soluble in water and organic solvents. Comprehensive pharmacokinetic data are available from toxicological studies.

The toxicity of malachite green is significant. It is a suspected carcinogen and teratogen. It shows high cytotoxicity and carcinogenicity to mammalian cells and can promote the development of liver tumors. The compound causes skin and eye irritation. Its use in aquaculture is restricted or banned in many countries due to safety concerns. For research use only, not for human or veterinary use.

[1]. Sea shell powder as a new adsorbent to remove Basic Green 4 (Malachite Green) from aqueous solutions: Equilibrium, kinetic and thermodynamic studies. Chemical Engineering Journal, 164(1), 168–177.

Basic Green 4 is a green crystal with a metallic luster. Its aqueous solution is blue-green. It is used for dyeing silk and wool, and for biological staining. Malachite Green is an organochloride salt, a monochloride salt of the malachite green cation. It can be used as a green dye, a histological counterstain, and in aquaculture due to its antifungal properties. It has various uses, including fluorescent dye, histological dye, antifungal drug, carcinogen, teratogen, environmental pollutant, and antibacterial agent. It contains the malachite green cation. See also: Malachite green cation (with active moiety); glutaraldehyde; Malachite green (component); Malachite green; Sodium chloride (component)... See more...

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Mechanism of Action
... The mitogen-activated protein kinase (MAP) signaling pathway in MG-induced precancerous cells was studied. Western blot analysis of MG (malachite green)-induced precancerous cells showed that ERK1 was not phosphorylated, while phosphorylated ERK2 increased, and phosphorylated JNK2 expression decreased. However, the total expression levels of ERK, JNK, and p38 kinases were similar in control and precancerous SHE (Syrian hamster embryo) cells. Indirect immunofluorescence studies showed that phosphorylated ERK in MG-induced precancerous cells had distinct nuclear localization. Flow cytometry analysis showed an increase in the number of S-phase cells in precancerous cells compared to control SHE cells. To understand the mechanisms by which MY (Metanil yellow) and MG (malachite green) promote tumorigenesis, we investigated the levels of PCNA (cell proliferation marker) and cell cycle regulatory proteins, cyclin D1 and its associated kinase cdk4, and cyclin B1 and its associated kinase cdc2. Immunohistochemical staining showed elevated PCNA levels in animals treated with DEN (N-nitrosodiethylamine) and then given MY and MG. Western blot and Northern blot hybridization results showed that, compared to the untreated group or the DEN control group, the expression of cyclin D1 and its associated kinase cdk4, as well as cyclin B1 and its associated kinase cdc2, was increased in the livers of rats treated with DEN and then given MY and MG. Transcriptional elongation experiments revealed that the increase in mRNA levels was due to the increased transcription rate of these genes.
Malachite green (Basic Green 4) is a triphenylmethane dye used as a green dye, histological counterstain, and in aquaculture due to its antifungal properties. It inhibits the proliferation of HEp-2 cells (IC₅₀ = 2.06 µM) and is cytotoxic to HEp-2 and Caco-2 cells. Malachite green reduces fungal infection in rainbow trout eggs. It is a suspected carcinogen and teratogen. Its use in aquaculture is restricted or banned in many countries.

C23H25N2CL

364.91

364.17

569-64-2

10309-95-2 (Parent)

11294

Green to black solid powder

1.0448 (rough estimate)

520.91°C (rough estimate)

158-160ºC

1.5940 (estimate)

1.397

0

2

3

26

516

0

FDZZZRQASAIRJF-UHFFFAOYSA-M

InChI=1S/C23H25N2.ClH/c1-24(2)21-14-10-19(11-15-21)23(18-8-6-5-7-9-18)20-12-16-22(17-13-20)25(3)4;/h5-17H,1-4H3;1H/q+1;/p-1

[4-[[4-(dimethylamino)phenyl]-phenylmethylidene]cyclohexa-2,5-dien-1-ylidene]-dimethylazanium;chloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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.)