Subchronic Toxicity Studies of Cortex Dictamni Extracts in Mice and Its Potential Hepatotoxicity Mechanisms in Vitro

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.

Selective and reliable determination of obacunone in rat plasma using solid‐phase extraction by liquid chromatography tandem mass spectrometry: Application to a pharmacokinetic study

“…It has been prescribed for the treatment of rheumatism, coronary atherosclerosis, jaundice and skin disorders (Chang, Lan, Yang, & Heinrich, 2016; Lv et al, 2015). However, the safety of this herb medicine has been questioned because some reports indicated that the oral administration of Dictamni cortex resulted in severe liver injury (Efferth et al, 2017; Fan et al, 2018; Wang et al, 2014; Wang, Xu, & Wang, 2007). Obacunone, one of the triterpenoids from Dictamni cortex, was the major bioactive constituent of this herb (Fan et al, 2018; Nam et al, 2005), which has been demonstrated to have anti‐cancer activity (Chidambara Murthy, Jayaprakasha, & Patil, 2011; Murthy, Jayaprakasha, & Patil, 2015; Tanaka et al, 2000, 2001; Xie et al, 2019).…”

“…Obacunone, one of the triterpenoids from Dictamni cortex, was the major bioactive constituent of this herb (Fan et al, 2018; Nam et al, 2005), which has been demonstrated to have anti‐cancer activity (Chidambara Murthy, Jayaprakasha, & Patil, 2011; Murthy, Jayaprakasha, & Patil, 2015; Tanaka et al, 2000, 2001; Xie et al, 2019). However, obacunone was also suggested to be one of the potentially hepatotoxic ingredients (Fan et al, 2018; Lang, Zhang, Wang, & Zhou, 2020). Lang et al indicated that a single oral dose of 50 mg/kg of obacunone resulted in the increase in alanine aminotransferase and aspartate aminotransferase in mice serum and cytochrome P3A4‐mediated bioactivation would be involved in obacunone‐caused liver injury (Lang, Zhang, Wang, & Zhou, 2020).…”

Multi-Omics Integration to Reveal the Mechanism of Hepatotoxicity Induced by Dictamnine

“…Intrinsic hepatotoxicity refers to the direct toxic stress leading to hepatic cells death associated with a predictable liver injury that can be reproduced in animal models in a dose-dependent manner. Water and ethanol extracts of DC have potential hepatotoxicity in sub-chronic toxicity studies, with the alcohol extraction process increasing the toxicity level ( Fan et al, 2018 ). In our in vitro and in vivo study, dictamnine was a possible toxic component by high content analysis.…”

“…The preliminary findings in our study revealed that aqueous and ethanol extracts of DC have potential hepatotoxicity. The hepatotoxic component in DC has been confirmed as dictamnine by liquid chromatography with tandem mass spectrometry (LC-MS/MS) ( Fan et al, 2018 ). However, the precise pathogenesis mechanism of dictamnine-induced hepatotoxicity remains largely unexplored.…”

In Vitro and In Vivo Metabolic Activation of Obacunone, A Bioactive and Potentially Hepatotoxic Constituent of Dictamni Cortex

“…Dictamni cortex, a widely used herbal medicine in China, was reported to cause liver injury [3][4][5]. However, the mechanism behind this has not been adequately investigated.…”