Human cytochrome P450 enzymes: CYP1A1 (IC50 = 6.2 μM), CYP1A2 (IC50 = 10.0 μM), CYP1B1 (IC50 = 2.7 μM; Ki = 0.5 μM, competitive inhibition).[1]
The targets of Alizarin include cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP1B1). It strongly inhibits these P450 isoforms with IC50 values of 6.2 μM, 10.0 μM, and 2.7 μM, respectively. Alizarin is also used as a staining agent that binds to free calcium and certain calcium compounds, staining them a red or light purple color.

Alizarin weakly inhibits CYP2A6 and CYP2E1. Alizarin shows competitive inhibition against CYP1B1 with Ki of 0.5 μM. Alizarin reduces the mutagenicity of MeIQx, which induced by each CYP1A2 or CYP1B1, while does not effectively reduce the mutation induced by B[a]P. Alizarin exhibits antioxidants against iodophenol-derived phenoxyl radicals, superoxide anion radicals and lipid peroxidation in rat liver microsomes.
Cell Assay: Alizarin is proved to have anti-tumor efficacy. It suppresses the cell growth of the prostate cancer, breast cancer and osteosarcoma cell lines in vitro. Among these, the osteosarcoma cells appear to be most sensitive. The IC50 values of Alizarin against three osteosarcoma cell lines Saos-2, MG-63 and U-2 OS are 27.5, 29 and 69.9μg/ml, respectively. Alizarin inhibits the cell growth through cell proliferation blockade rather than induction of apoptosis. It inhibits the phosphorylation of ERK. In addition, Alizarin is also found to induce S-phase arrest as well as a decrease of the G0/G1 and G2/M phases.

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Alizarin strongly inhibited the activities of human recombinant CYP1A1, CYP1A2 and CYP1B1 in a dose-dependent manner, and weakly suppressed CYP2A6 and CYP2E1, but did not inhibit CYP2C19, CYP3A4 or CYP3A5. Carminic acid showed no inhibition.[1]
For CYP1B1-mediated 7-ethoxycoumarin O-deethylation, alizarin exhibited competitive inhibition with a Ki value of 0.5 μM. The Lineweaver-Burk plots showed that k values (slopes) were proportional to alizarin concentration, suggesting one molecule of alizarin interacts with one molecule of CYP1B1. The Km of CYP1B1 was 11 μM.[1]
In the Ames test using S. typhimurium TA1538 co-expressing human CYP and NADPH-cytochrome P450 reductase, alizarin suppressed the mutagenicity of MeIQx (activated by CYP1A2 or CYP1B1) in a dose-dependent manner, but did not effectively reduce the mutagenicity of B[a]P (activated by CYP1A1 or CYP1B1). Alizarin did not suppress AFB1 mutagenicity (activated by CYP3A4).[1]
In vitro, Alizarin is a potent inhibitor of CYP1A1, CYP1A2, and CYP1B1 with IC50 values of 6.2 μM, 10.0 μM, and 2.7 μM, respectively. It is also used as a staining agent in biological research because it stains free calcium and certain calcium compounds a red or light purple color. These in vitro activities have been characterized in enzymatic and histochemical assays.

Alizarin also reduces the hepatic content of thiobarbituric acid-reactive substances and the serum level of alanine aminotransferase in poisoned animals

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In vivo, Alizarin is not used as a therapeutic agent. It is primarily used as a staining agent in biomedical research involving bone growth, osteoporosis, bone marrow, calcium deposits in the vascular system, cellular signaling, gene expression, tissue engineering, and mesenchymal stem cells. It stains calcium deposits, allowing for visualization and quantification.

Enzyme activities of human recombinant CYPs (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C19, CYP2E1, CYP3A4, CYP3A5) co-expressed with NADPH-cytochrome P450 reductase in E. coli membrane fractions were measured. For CYP1A1 and CYP1B1, activity was determined by 7-ethoxycoumarin O-deethylation using 40 μM and 20 μM substrate respectively, and fluorescence quantification. For CYP1A2, activity was measured by 7-ethoxyresorufin O-deethylation. For CYP2A6, activity was measured by coumarin 7-hydroxylation. For CYP2C19, CYP2E1, CYP3A4 and CYP3A5, metabolite quantification was performed by HPLC with UV detection: CYP2C19 by 4'-hydroxylation of (S)-mephenytoin, CYP2E1 by 6-hydroxylation of chlorzoxazone, and CYP3A4/3A5 by 1'-hydroxylation of midazolam (20 μM for CYP3A4, 7 μM for CYP3A5) using diazepam as internal standard. To evaluate Ki of alizarin against CYP1B1, enzyme activities were measured in the presence of 0.2-1.6 μM alizarin at substrate doses from 2.5 to 40 μM.[1]
Non-cellular enzyme assays for Alizarin involve measuring its inhibition of CYP450 isoforms using purified enzymes and appropriate substrates. IC50 values for CYP1A1 (6.2 μM), CYP1A2 (10.0 μM), and CYP1B1 (2.7 μM) are determined in these assays. These assays confirm the compound's activity as a P450 inhibitor.

The Ames test was performed using S. typhimurium TA1538 strains co-expressing human CYP and NADPH-cytochrome P450 reductase (TA1538 1A1/OR, 1A2/OR, 1B1/OR, or 3A4/OR). Bacteria were cultured in modified Terrific Broth at 30°C for 8 h (except TA1538 1A2/OR at 25°C), then induced with 1.5 mM IPTG for 18 h. Cells were collected by centrifugation and resuspended in 0.1 M sodium phosphate buffer (pH 7.4) to a concentration of 1-2×10^9 cells/ml. Bacterial suspension (~10^8 cells) was incubated with mutagen (0.1 μM MeIQx for CYP1A2, 0.5 μM MeIQx for CYP1B1, 1.5 μM B[a]P for CYP1A1/CYP1B1, or 25 μM AFB1 for CYP3A4) in the absence or presence of alizarin (10^-3 to 10^-7 M) at 37°C for 20 min without S9 mix. After two days, His+ revertant colonies were counted.[1]
In vitro cell-based assays for Alizarin are not typically performed for its P450 inhibitory activity. However, its staining properties are used in cell-based assays to detect calcium deposits. Cells are cultured and stained with Alizarin Red S, and calcium deposits are visualized and quantified.

In vivo animal studies for Alizarin are not typically performed as it is not a therapeutic agent. However, it is used in ex vivo studies to stain calcium deposits in tissues. Tissues are stained with Alizarin Red S to visualize mineralization.

Alizarin is a small molecule with a molecular weight of 240.21 g/mol. It is insoluble in water but dissolves in alkalis, alcohol, and ether. It is used as a pH indicator and as a histochemical reagent for calcium, thallium, titanium, and zirconium.

Alizarin is not a drug and has no established therapeutic safety profile. It is generally considered safe for use as a laboratory reagent and dye. Appropriate safety precautions should be taken when handling Alizarin as a research chemical.

Mutat Res.2002 Oct 31;508(1-2):147-56.

Alizarin is a dihydroxyanthraquinone, namely anthracene-9,10-dione, with the two hydroxyl groups located at positions 1 and 2, respectively. It can be used as a chromophore, dye, and plant metabolite. Alizarin has been reported in Rubia lanceolata, Rubia argyi, and other organisms with relevant data. See also: Root (part) of Rubia tinctorum.

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Alizarin has one fewer hydroxyl group than purpurin (lacking the 4-position hydroxy group), which may account for differences in antigenotoxic effects and CYP inhibition potency. Alizarin was less effective than purpurin at inhibiting CYP1A2 and at reducing B[a]P mutagenicity. It is a competitive-type inhibitor of CYP1B1, whereas purpurin shows mixed-type inhibition. The antigenotoxic activity of alizarin is at least partially attributable to suppression of CYP-mediated bioactivation of mutagens. These anthraquinone compounds may be beneficial chemopreventive agents at appropriate doses.[1]
Alizarin (1,2-dihydroxyanthraquinone) is an organic compound historically used as a red dye. It is a potent inhibitor of CYP1A1, CYP1A2, and CYP1B1 with IC50 values of 6.2 μM, 10.0 μM, and 2.7 μM, respectively. It is commonly used in biomedical research as a staining agent for calcium. Alizarin is not a drug and is available as a research reagent.

C14H8O4

240.21

240.042

72-48-0

6293

Yellow to brown solid powder

1.5±0.1 g/cm3

430.0±40.0 °C at 760 mmHg

287 °C

228.0±23.8 °C

0.0±1.1 mmHg at 25°C

1.733

4.09

2

4

0

18

378

0

RGCKGOZRHPZPFP-UHFFFAOYSA-N

InChI=1S/C14H8O4/c15-10-6-5-9-11(14(10)18)13(17)8-4-2-1-3-7(8)12(9)16/h1-6,15,18H

1,2-dihydroxyanthracene-9,10-dione

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)

Solubility in Formulation 1: 2.5 mg/mL (10.41 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (10.41 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)