Abstract
Cortex Dictamni is a commonly-used traditional Chinese herbal medicine for the treatment of skin inflammation, tinea, and eczema. Recently, some studies reported that Cortex Dictamni might induce liver injury, suggesting more attention to its safety. The current study was designed to investigate subchronic toxicity of Cortex Dictamni aqueous extract (CDAE) and ethanol extract (CDEE) in mice and the potential hepatotoxicity mechanisms in vitro. Firstly, CDAE or CDEE groups were administrated with varying dosages (2.3, 4.6, or 9.2 g/kg/day, p.o.) in mice for 28 days in subchronic toxicity studies. General clinical signs and biochemical parameters were examined, and morphological analyses were conducted. Secondly, we identified the different constituents of CDAE and CDEE using HPLC-MS/MS and chose major components for further study. In order to determine the toxic components, we investigated the cytotoxicity of extracts and chosen components using CCK-8 assay in HepG2 cells. Furthermore, we explored the possible hepatotoxicity mechanisms of Cortex Dictamni using a high content analysis (HCA). The results showed that no significant differences of general clinical signs were observed in mice. Aspartate alanine aminotransferase (ALT) and aminotransferase (AST) were significantly increased in the high-dose CDAE and CDEE groups compared to the control group. Meanwhile, the absolute and relative liver weights and liver/brain ratio were significantly elevated, and histological examination of liver demonstrated cellular enlargement or nuclear shrinkage. In UPLC analysis, we compared the chemical constituents between CDAE and CDEE, and chose dictamnine, obakunone, and fraxinellone for hepatotoxicity evaluation in the in vitro studies. In the CCK-8 assay, CDAE, CDEE, dictamnine, obakunone, and fraxinellone decreased the cell viability in a dose-dependent manner after treatment for 48 h. Furthermore, the cell number decreased, while the nuclear intensity, cell membrane permeability, and concentration of reactive oxygen species were shown to increase, meanwhile, mitochondrial membrane potential was also changed in HepG2 cells following 48 h of compounds treatment using HCA. Our studies suggested that CDAE and CDEE have potential hepatotoxicity, and that the alcohol extraction process could increase toxicity. Dictamnine, obakunone, and fraxinellone may be the possible toxic components in Cortex Dictamni with dictamnine as the most potentially hepatotoxic component, whose potential hepatotoxicity mechanism may be associated with cell apoptosis. Moreover, this study could provide valuable data for clinical drug safety research of Cortex Dictamni and a good example for safety study of other Chinese herbal medicines.
Highlights
Drug-induced liver injury (DILI) often results in the termination of drug development at the clinical stage or withdrawal of a drug from the market [1]
ALB, GLB and total protein (TP) increased in the Cortex Dictamni aqueous extract (CDAE) and CDEE-treated group with 4.6 g/kg and 9.2 g/kg dose compared to the control group (p < 0.01, p < 0.05)
The cell number decreased, while the nuclear intensity, mitochondria membrane potential, cell membrane permeability, and concentration of reactive oxygen species increased after CDAE and CDEE exposure, suggesting that hepatotoxicity induced by CDAE and CDEE may lead to cell apoptosis
Summary
Drug-induced liver injury (DILI) often results in the termination of drug development at the clinical stage or withdrawal of a drug from the market [1]. Cortex Dictamni is widely used, some studies have reported that some Chinese patented medicines or compounds derived from Cortex Dictamni induce liver toxicity in recent years, such as Compound. There were some studies related to the toxicity of Cortex Dictamni. We conducted research on possible drug-related factors and concluded that Cortex Dictamni could be the main contributor to liver damage. Here we conducted the study in vivo and in vitro hepatotoxicity of CDAE and CDEE, which could provide valuable data for clinical drug safety research of Cortex Dictamni. These research results will provide new data on the clinical safety of Cortex Dictamni and help guide rational drug use in clinical applications and successfully develop new preparations
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