Studies On Metabolism Of Indirubin In Vivo And In Vitro

Indirubin is the major anti-carcinoma active ingredient of Indigo naturalis of Traditional Chinese drug. It has significant inhibitory effect on chronic myeloid leukemia. The inhibitory effect of indirubin and its derivatives on cell growth was found in some human tumor cell lines relating with cyclin dependent kinase(CDK) or Src kinase which widely exists in human bodies. Indirubin and its derivatives may become the new anti-tumor medicine for therapy of cancers that have CDK, Src kinase.Indirubin and tanshinone IIA serve as adjuvant components to intensify tetraarsenic tetrasulfate-induced ubiquitination and degradation of PML-RARα, strengthen reprogramming of myeloid differentiation regulators and enhance G1/G0 arrest in APL cells. Indirubin and tanshinone IIA increase cellular uptake of arsenic by inducing up-regulation of Aquaglyceroporin 9 (AQP9) which is a transmembrane protein governing arsenic uptake and cellular sensitivity of arsenic.In recent years, indirubin has attracted increasing interest due to its various beneficial biological activities to human health, which made it necessary to further study the metabolism of indirubin to elucidate its biological effects. It is important for rational evaluation of animal data as well as extrapolation to humans, and for guiding clinical use. Our study focused on the in vivo and in vitro metabolism of indirubin to explain the in vivo and in vitro correlation of indirubin metabolism, and evaluate the security and rationality of co-administration of indirubin with western medicine and traditional Chinese medicine.1. Study On The Metabolism Of Indirubin In Rat Liver MicrosomesRat liver microsomes were used for the research of metabolism of indirubin in vitro. It is helpful in the further understanding of indirubin metabolic mechanism, the prediction of pharmacokinetic drug-drug interactions and interpatient variability in drug exposure. Liver microsomes of male S.D. rats were prepared using calcium ion precipitation method. The in vitro metabolism of indirubin was studied by incubation with rat liver microsomes. Three phase I metabolites of indirubin were detected in rat liver microsomal incubates with 100ng/mL of indirubin and 3h of incubation. These metabolites were identified as hydrolysis, oxidization and di-oxidization metabolites of indirubin.This article describes a sensitive and selective method for the determination of indirubin in microsomal incubates. A liquid–liquid extraction procedure was used. The extracts were analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry using ketoconazole as an internal standard. The excellent sensitivity and selectivity of the liquid chromatography–tandem mass spectrometry method allowed quantitation and identification of indirubin at low levels with a run time of 5.0 minutes. A linear calibration range spanned 1-100 ng/mL. The relative standard deviations for the intra-day and inter-day precision were less than 3.6% and 13.4%, respectively. The average recoveries varied between 94.0% and 104.4%. The limit of detection was 0.5 ng/mL. Because of its simplicity and accuracy, the established method is suitable for the application in the determination of indirubin in microsomal incubates.2. Study on the Metabolisms of Indirubin in RatsTo investigate the metabolism of indirubin in rats. Urine, fence samples at 4.5,8.5,12,24,36,48h and bile samples at 4,8,12,24h were collected after a single dose of 200 mg/kg indirubin was administered to rats. The urine, fence and bile samples were extracted by liquid-liquid Extraction and then analyzed with LC/MS-MS. 13 metabolites were identified in rats'urine, fence and bile, that is, Reduction, Methylation, Hydrolysis, Oxidation, Di-Oxidation, Di-Sulfation, Glucuronidation, Glutathione conjugation - 2H, Oxidation + Glutathione - 2H, Oxidation+Sulfonation, Glucuronidation + Di-Oxidation, Di-Oxidation + Glutathione - 2H, Di-Oxidation + Di-Glucuronidation.