Studies On In Vivo And In Vitro Metabolism Of Kurarinone, A Hepatotoxic Component From Sophora Flavescens

Zhixue capsule was recalled by the State Food and Drug Administration in 2008 because of severe adverse hepatic reactions. Zhixue capsule is composed of ethanol extracts of Cortex Dictamni (ECD) and Sophora flavescens (ESF). In our preliminary study, we observed the hepatotoxic effects of ESF on rat primary hepatocytes. However, ECD did not exhibit hepatotoxicity at the same concentration range. Radix Sophora flavescentis derived from the dried roots of Sophora flavescens. It is commonly used traditional Chinese medicine with function of detoxification, dissipating heat, drying the damp and dispelling wind. It was first recorded in Shennong Bencao Jing and was classified as middle type of medicine. The main active ingredients of the roots of Sophora flavescens are alkaloids and flavonoids. However, people only put a lot of in-depth study on the alkaloids and took the content of alkaloids as its quality standards in the past. Obviously, it's difficult to reflect the quality of Sophora flavescens. Meanwhile, with the development of the pharmacology study on Sophora flavescens, these pharmacological effects of the flavonoids have been shown, such as anti-inflammation, anti-bacteria, anti-cancer and so on. It has become hot spots of current research of traditional Chinese medicine due to its active ingredients and broad prospects of development and application. In our preliminary study, kurarinone (Kur) and sophoraflavanone G (SFG) were identified as hepatotoxic components using bioassay-guided isolation combined with primary rat hepatocyte model. For further study the toxic effect of these two compounds on human hepatocytes and the relationship between metabolism and toxicity, a semi-preparative HPLC was used to isolate the active compounds. The activity of these two compounds against human liver cells was tested after the structure elucidation. Then in vitro metabolism studies of Kur on human liver microsomes and rat liver microsomes were taken to explore the relationship between metabolism and toxicity. Finally, the toxicokinetics study of Kur in rat was performed. All these studies will provide useful information on the rational use of Sophora flavescens and experimental basis of quality control.1. Isolation of hepatotoxic compounds and in vitro toxicy testAIM:To isolate the hepatotoxic compounds based on Semi-preparative HPLC and measure their toxic activity against different cell lines.Method:The crushed dried roots of Sophora flavescens were extracted three times by refluxing with 95% ethanol for 2 h each time. The combined extract was filtered and evaporated to dryness by rotary evaporation under reduced pressure. The solid residue was dissolved in water as crude aqueous extracts, and then extracted with ethyl acetate three times. The ethyl acetate phases were combined and evaporated to dryness and isolation was performed using semi-preparative HPLC. The isolation conditions were the same as early experiment and the peaks with the same retention time were collected. The structures of these compounds were elucidated based on mass spectrometry (MS), proton nuclear magnetic resonance(1H-NMR), and carbon-13 NMR (13C-NMR) analyses. Then, the toxicity of the compounds against 3T3 cell line and HL-7702 cell line were evaluated by MTT assay.Results:Compared with early experiment (70% ethanol), we used lower polarity^ solvent (95% ethanol) and ethyl acetate extraction was added after this step. Hepatotoxic compounds were concentrated and were confirmed as Kur and SFG. IC50 of Kur and SFG on 3T3 cell were 37.17 (35.81-38.58) ?g/mL and 33.91(31.28-36.76) ?g/mL, and human HL-7702 liver cells were 21.13 (20.09-22.23) ?g/mL and 17.1 (15.82-18.49) ?g/mL, respectively.Conclusion:The results suggest that low polarity solvent extraction of Sophora flavescens may increase its hepatotoxicity. Kur and SFG possess strong toxicity toward 3T3 cells and HL-7702 cells. The content of Kur in ESF is 3.3 times of SFG2. In vitro metabolism study of KurAIM:To explore the metabolites of Kur after incubation with rat liver microsomes (RLMs) and human liver microsomes (HLMs), character the relationship between metabolism and its hepatotoxicity and identify the CYP450 phenotype involved. Method:After pre-incubation of Kur with RLMs or HLMs for 5 minutes, metabolic enzymes cofactors of NADPH and UDPGA individually or simultaneously were added for cytochromes P450-mediated and UGT-mediated Kur biotransformations. Then the samples were sent to LC-MSn for analysis. The RLMs was used for metabolism-toxicity study. The test group was consist of Kur at various concentrations, RLMs and NADPH. The control group consists of the same ingredients except the RLMs was inactivated. Both group were co-incubated with 3T3 cell for 24h before MTT was added to evaluate the cell viability. The CYP450 phenotype identification was performed on HLMs by chemical inhibitors. The percentage of Kur remaining was used for quantitative analysis.Results:The main metabolites in RLMs phase ? metabolism was hydroxylated metabolites M1 (m/z 453) and a small amount of M2 (m/z 451), formed by dihydroxylation and then eliminated one molecule of water. M4-6(m/z 613) were phase ? metabolites, formed by glucuronidation. M1, M2 were phase ? plus phase ? metabolites in RLMs. The main metabolites in HLMs phase ? metabolism was hydroxylated metabolites M1 (m/z 453) and little isomer M3(m/z 453). Phase ? metabolites were the same as RLMs. Ml, M3, M4-6 were phase ? plus phase ? metabolites in HLMs. Compared with control, the 3T3 cell viability in test group was significant higher at Kur 6.25,25,50 ?g/mL. The chemical inhibitor omeprazole,4-methylpyrozole, quinidine and ketoconazole showed significant inhibition on Kur elimination (P< 0.01). ?-Naphthoflavone and sulf-aphenazole also showed some extent of inhibition(P< 0.05).Conclusion:M1 and M4-6 were the mutual metabolites in phase I and phase ? metabolism in RLMs and HLMs. M2 was specific metabolite in RLMs phase I metabolism. The phase ? metabolites were the main metabolites in RLMs, while in HLMs, phase ? metabolites were the main metabolites. The CYP450 mediated metabolism of Kur in RLMs lead to the detoxification of Kur and CYP2C19?CYP2D6?CYP2E1 and CYP3A4 might be the key CYPs responsible for the detoxification of Kur in HLMs.3. Toxicokinetics of Kur in ratsAIM:To study the toxicokinetics of Kur in Rats and evaluate the relationship between exposure levels and toxic dosages.Method:SD rats were assigned randomly to three groups, one vehicle control and two treatment groups with six rats each. ESF was administered orally twice daily for 3 d. Blood samples were collected from the rats in all groups via the retro-orbital plexus at a designated time (0,0.5,1,2,3,4,6,8,12 and 24 h after treatment). The Kur in rat plasma was determined by LC-MS after centrifuged at 4500 RPM for 10 minutes. Blood samples collected at 0 h,8 h and 3h after final administration were used for measurement of serum aminotransferases levels. The livers, hearts and kidneys were dissected and used. for histological examination.Results:The administration of ESF 1.25 g/kg and 2.5 g/kg significantly elevated ALT and AST contents in serum after 8 h (p< 0.05) compared with the vehicle control group. A dose-dependent increase of both ALT and AST levels occurred after 3 d. The histopathological studies of the liver showed remarkable fatty degeneration with numerous micro and macro vesicles in ESF1.25 g/kg and 2.5 g/kg treated rats compared with the control group. No obvious changes were found in hearts and kidneys. The Tmax of ESF1.25 g/kg and 2.5 g/kg was 1h and 6 h. T1/2 was between 1 h and 6 h.Conclusion:The toxic target organ of ESF in rats mainly focused on liver and it's characterized by increase of serum transaminases levels and fatty degeneration of liver.