Alizarin

240, 21

Alizarin weakly inhibits CYP2A6 and CYP2E1. Alizarin shows competitive inhibition against CYP1B1 with Ki of 0.5 uM. Alizarin deduces the mutagenicity of MeIQx, which induced by each CYP1A2 or CYP1B1, while does not effectively reduce the mutation induced by B[a]P. [1] Alizarin exhibits antioxidants against iodophenol-derived phenoxyl radicals, superoxide anion radicals and lipid peroxidation in rat liver microsomes. [2]

Measurement of enzyme activities of CYPs, Enzyme activities are measured by fluorometric quantification of each metabolite produced by O-deethylation of 7-ethoxycoumarin by CYP1A1 and CYP1B1, O-deethylation of 7-ethoxyresorufin by CYP1A2 and 7-hydroxylation of coumarin by CYP2A6, using Hitachi F-2000 fluorescence spectrophotometer. The fluorescence of metabolite is not influenced by the presence of anthraquinone pigments at the doses tested in these experiments. 7-Ethoxycoumarin is used at 40 uM for CYP1A1 and at 20 uM for CYP1B1. In the cases of CYP2C19, CYP2E1, CYP3A4 and CYP3A5, the amount of each metabolite derived from the catalytic reaction by individual CYPs is determined by HPLC equipped with a UV detector. The activities of CYP2C19 and CYP2E1 are determined by 4 -hydroxylation of (S)-mephenytoin and by 6-hydroxylation of chlorzoxazone, respectively. In the case of CYP3As, their activities are determined by 1 -hydroxylation of midazolam with a modification in which the metabolites are extracted from the incubation mixture before injection to HPLC. Briefly, 1 uM diazepam is added to the reaction mixture as an internal standard with an excess amount of ammonium sulfate and 5 volumes of ethyl acetate immediately after the incubation. After shaking for 10 min, 1 -hydroxymidazolam and diazepam are extracted into the ethyl acetate layer by centrifugation at 2000xg for 10 min, at 4 C. Then ethyl acetate is removed by evaporation and the residue is used for HPLC after dissolving in methanol. A volume of 20 uM of midazolam is used for the detection of CYP3A4 activity and 7 uM for CYP3A5. To evaluate the Km value of CYP1B1, the measurement of activity is carried out at substrate doses from 2.5 to 40 uM. Ki value of Alizarin against the activity of CYP1B1 is calculated from the enzyme activities in the presence of 0.2-1.6 uM Alizarin.

Alizarin is a red coloring mordant dye extracted from the roots of madder plants (Rubia tinctorum L). Alizarin strongly inhibits P450 isoform CYP1A1, CYP1A2, and CYP1B1 with IC50 of 6.2 μM, 10.0 μM, and 2.7 μM, respectively in an enzymatic assay. There are also literature reports Alizarin increases the CYP1A1 enzymatic activity and acts as an agonist to the AHR receptor in HepG2 cells.