Experimental Study On Hepatotoxicity Of Medicinal Ingredients Of Zhixue Capsule

Zhixue Capsule, composed of ethanolic extracts of Cortex Dictamni and Radix Sophorae flavescentis, is a Chinese herbal compound for the treatment of hematochezia, straining feeling and pain in anus caused by internal hemorrhoid at stageⅠandⅡ. As a new medicine, Zhixue Capsule came into market in 2004. Both crude drugs, Radix Sophora flavescentis derived from the dried roots of Sophora flavescens and Cortex Dictanmi from the dried root barks from Dictamnus dasycarpus Turcaz., are commonly used traditional Chinese medicine with function of detoxification, dissipating heat, drying the damp and dispelling wind. Cortex Dictamni is the principal drug while Radix Sophorae improves the effect of dissipating heat and drying the damp.However, in the recent years the state ADR monitoring center has received a number of cases of hepatotoxicity associated with Zhixue Capsule and the clinical reporting rate of its hepatotoxicity reached a peak in 2008, leading to the recall of relevant drugs from the market in Nov,2008. The drug has not been withdrawn and its future depends on the causality assessment of hepatotoxicity. Both herbs contain liver-protective constituents and their hepatotoxicity remains to be clarified.In order to clarify causal association between Zhixue Capsule and liver injury and to investigate potential components responsible for hepatotoxicity, the study was to determine possible hepatotoxic herb of Zhixue Capsule by in vitro model at first step. In vivo experiments were then conducted to assess the effects of hepatotoxic herb on liver functions and hepatic histomorphology in mice and rats. Furthermore, semi-preparative HPLC technique was carried out to explore hepatotoxic constituent and to determine its chemical structure.1. Determination of the hepatotoxic herb of Zhixue CapsuleAIM:To determinate the hepatotoxic herb of Zhixue Capsule based on primary rat hepatocyte model Method:Experimental drugs were prepared according to the patent of Zhixue Capsule. Ethanolic extracts of Radix Sophorae Flavescentis (ESF,yield 10.7%), Cortex Dictamni (ECD, yield 20.0%) and compound formula (ECF, yield 12.5%), matrine and oxymatrine (both were bought from the market). Hepatocytes were isolated by the two-step perfusion. Drugs mentioned above were exposed to hepatocytes and tested for effect on cell viability by MTT method.Results:Hepatocytes were not affected by 3 h exposure to ECD or ECF ranged from 74 to 250μg/ml. Cell viability of hepatocytes treated with 111μg/ml,167μg/ml, 250μg/ml ESF was 101.6%,25.9%,9.7% compared with vehicle group, respectively. IC50 for 3 h,6 h,12 h exposure at ESF were 162.9 (155.8-170.4),144.5 (141.9-147.1),143.0 (140.9-145.3)μg/ml. Matrine and oxymatrine, as main alkaloids of ESF, did not affect cell viability at concentration range from 0.05 to 5 mM.Conclusion:ESF of Zhixue Capsule displayed hepatotoxic effects in primary rat hepatocytes in a dose-and time-dependent manner. ECD did not exhibit hepatotoxicity at same concentration range. Matrine and oxymatrine did not injury cell viability at high concentration.2. In vivo study on hepatotoxicity of ESFAim:To study the effect of ESF on liver by different administration routeMethod:Mice were randomly assigned into control and ESF treated group. Mice were administered with ESF 50 mg/kg intravenously for 7 days, and liver injury was assessed by measuring the level of aminotransferase (ALT) and aspartate aminotransferase (AST). Rats were randomly assigned into control and ESF treated group. ESF 2.5 g/kg and 5 g/kg and were administrated orally to rats for 7 days and the level of ALT and AST was measured. HE staining was used to assess the histomorphology of rat liver slices.Result:After intravenous injection for 3 days, the level of ALT in ESF treated mice was 1.33 times higher than that of control group. After oral administration for 3 days, the levels of ALT in ESF 2.5 g/kg and 5 g/kg treated rats were 1.48 times and 1.53 times higher than that of control group, respectively. After oral administration for 7 days, the levels of ALT in ESF 2.5 g/kg and 5 g/kg treated rats were 1.43 times and 1.60 times higher than that of control group, respectively. HE staining showed liver of rat administrated with 5 g/kg displayed vacuolar degeneration and hydropic degeneration.Conclusion:ESF could cause liver injury in mice and rats by intravenous and oral administration. Increased level of ALT was concomitant with degeneration of hepatocytes as observed by HE staining. Severity of liver injury did not worsen with increasing administration time.3. Determination and characterization of hepatotoxic components of ESFAIM:To isolate and identify the hepatotoxic components of ESFMethod:Semi-preparative HPLC method was used to isolate sub-fractions with according to their retention time. Eight fractions were collected with the retention time of 12-17 min,20-25 min,26.5-31 min,33-37 min,38.5-40 min 43-51 min, 52-56.5 min and 60-64.5 min. Eight fractions were incubated with rat primary hepatocytes for 3 h and cell viability was assessed by MTT method. Fractions that exhibited strong cytotoxicity were further isolated to obtain main toxic components. The chemical structures of hepatotoxic components were confirmed by MS, 1H-NMR,13C-NMR technique.Result:The polarity of FR1 to FR8 decreased progressively with increasing retention time. Cell viability of hepatocytes treated with eight fractions at the concentration of 50μg/ml were 96.0%,102.3%,99.9%,86.1%,77.7%,112.5%,7.5%,21.1% compared with vehicle group, respectively. FR7 and FR8 displayed stronger hepatotoxicity than other fractions. After further isolation of FR7 and FR8, two hepatotoxic components, kurarinone from Fraction 7 and sophoraflavanone G from Fraction 8, were obtained. Both compounds belong to prenylated flavonoids. IC50 of Kur and SFG were 13.12 (12.02-14.32) and 6.99 (6.63-7.38)μg/ml, respectively. Conclusion:Hepatotoxic components of ESF existed in low-polarity fractions. Kur isolated from FR7 and SFG isolated from FR8 were main constituents responsible for hepatotoxicity of ESF.