The Metabolism Study Of Sophoraflavanone G, A Hepatotoxic Component From Sophora Flavescens

Radix Sophora flavescent is derived from the dried roots of Sophora flavescenes Ait. It is commonly used traditional Chinese medicine with function of detoxification, dissipating heat, drying the damp and dispelling wind and used to treat abscess, dysentery, eczema and skin itch. Our previous study found Radix Sophora flavescenti was a hepatotoxic ingredient of zhixue capsule which was recalled by the State Food and Drug Administration in 2008 because of severe adverse hepatic reactions. In our preliminary study, kurarinone (Kur) and sophoraflavanone G (SFG) (prenylated flavanones) were identified as hepatotoxic components using bioassay-guided isolation combined with primary rat hepatocyte model. The content of SFG was lower than that of Kur in Sophora flavescenes Ait. However, SFG presented stronger hepatotoxicity compared with Kur. Metabolism of components may be associated with hepatotoxicity. So far no studies have reported the metabolites, the cytochrome P450 and UDP-glucuronosyltransferase isoforms involved in the metabolism of SFG. The role of metabolism in cytotoxic effects of SFG on cell viability of 3T3 cells has not been investigated.In this paper, we mainly investigated time and dose-dependent cytotoxic effects of SFG on cell viability of HL-7702 cells. After the study on the major metabolites of SFG in RLMs, HLMs and primary rat hepatocytes, the phase I and II metabolism of SFG in both RLMs and HLMs were studied, including the enzyme kinetics, key CYPs and UGTs isoforms involved. Besides, we evaluated the effect of metabolism on the hepatotoxicity of SFG by 3T3 cells. The study is expected to promote further research of the new drug and guide the clinical medication.The research includes four parts as following:1. In vitro hepatotoxic study of SFGAIM:To explore cytotoxic effects of SFG on cell viability of human HL-7702 liver cellsMethod:HL-7702 cells under log phase of growth (5×10/mL) were seeded in a 96-well plate and incubated overnight. SFG was then added to the seeded cells within a concentration range of 5-160μM and incubated for 24 h,48 h and 72 h. Then, the cell viability of HL-7702 cell line was evaluated by MTT assay.Results:IC50 of SFG on human HL-7702 liver cells for 24 h,48 h and 72 h was 45.7 (34.19-52.47)μM,31.18 (28.19-34.50)μM,24.11 (21.98-26.44)μM, respectively.Conclusion:The results of the current study suggested that SFG display hepatotoxic effects on HL-7702 cell in a dose-and time-dependent manner.2. Identification of metabolites of SFGAIM:To investigate metabolites of SFG in RLMs, HLMs and primary rat hepatocytes, find main metabolits based on relative peak area quantification and compare the species difference between human and rats.Method:Freshly isolated hepatocyte cells (2×104/mL) were seeded in a 24-well plate coated with rat tail collagen and incubated in CO2 incubator at 37℃ for 2 h. SFG was then added to the seeded cells. After 4 h of incubation and centrifugation, the supernatant was sent to LC-MSn for analysis. After pre-incubation of SFG 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 SFG biotransformations. Then the samples were sent to LC-MSn for analysis.Results:SFG was metabolized to three phase Ⅰ metabolites, di-hydroxylated SFG (M1), mono-hydroxylated SFG (M2), dehydrogenated product of mono-hydroxylated SFG (M3) and three SFG glucuronides (M4, M5 and M6) by RLMs. One more phase Ⅰ plus phase Ⅱ metabolite M7 (glucuronide of M2) was produced compared to those of phase Ⅱ metabolites in RLMs. SFG was metabolized to only one phase Ⅰ metabolite, mono-hydroxylated SFG (M2) and two SFG glucuronides (M4 and M5). One more phase Ⅰ plus phase Ⅱ metabolite M7 (glucuronide of M2) was produced compared to those of phase Ⅱ metabolites in HLMs. M2, M4 and M5 were major metabolites based on relative peak area quantification.Conclusion:Metabolism of SFG in HLMs and RLMs presented species difference. Mono-hydroxylation was the mian pathway of phase Ⅰ metabolism. The major metabolic pathway of SFG was phase Ⅱ metabolism.3. Identification of metabolic pathway of SFGAIM:To study main metabolic pathways of SFG in RLMs and HLMs by chemical inhibition study, exploring the enzyme kinetics and the roles of cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferase isoforms (UGTs) involved in the metabolism of SFG in RLMs and HLMs.Method:1. To set up the HPLC-UV analyse method of SFG in rat and human liver microsomal incubations.2. To investigate the metabolic characteristics of SFG in RLMs and HLMs from incubation time, liver microsomal concentration and substrate concentration.3. Chemical inhibitors of CYPs and UGTs were used to investigate the effects on the metabolism of SFG.Results:1. A high sensitive HPLC-UV analyse method of SFG in liver microsomal incubations was established.2. The formation kinetics of M2 (mono-hydroxylated SFG) conformed to biphasic kinetics and the formation kinetics of M4 and M5 (SFG glucuronides) best fitted the Hill equation kinetics in RLMs. The formation kinetics of SFG glucuronides (M4 and M5) conformed to Hill equation kinetics and Michaelis-Menten model, respectively in HLMs.3. Furafylline and diethyldithiocarbamate can obviously inhibit the formation of M2 in RLMs. Both phenylbutazone and 1-naphthol can inhibit the formation of M4 and M5, the formation of M4 can also be inhibited by β-estradiol in RLMs.1-naphthol and β-estradiol presented prominent inhibition effect on the formation of M4, and the formation of M5 was only inhibited by 1-naphthol in HLMs.Conclusion:CYP1A2 and CYP2E1 played key roles in the mono-hydroxylation of SFG in RLMs. The glucuronidation of SFG may be mediated by multiple UGT1A isoforms in RLMs and HLMs.4. The effect of metabolism on cytotoxicity of SFGAIM:To investigate the role of metabolism in cytotoxic effects of SFG on cell viability of 3T3 cells after incubation with RLMs and HLMs.Method:After incubation with RLMs and HLMs, SFG supernatants were added to the 96-well plate seeded with 3T3 cells and co-incubated for 24 h before MTT was added to evaluate the cell viability and investigate the effect of metabolism on cytotoxicity of 3T3 cells.Results:Compared with control, the 3T3 cell viability in test group was significant higher. The difference had statistic significance.Conclusion:After incubation with RLMs and HLMs, the cytotoxic effects of SFG on cell viability of 3T3 cells decreased. The result suggested that metabolism lead to detoxication.