1 Introduction
Polygoni Multiflori Radix (PMR), originated from the tuberous root of Polygonum multiflorum Thunb., is a well-known traditional Chinese medicine and has been commonly used in clinical practice in either raw or processed forms for different indications such as detoxification, carbuncle elimination, malaria prevention, bowel relaxation, hair-darkening, nourishment of the liver and kidney, and so on (Chinese Pharmacopoeia Commission, 2015). Although PMR was widely used in clinical practice or as a tonic, a series of cases of hepatic adverse effects associated with PMR or the herbal medicine products containing PMR were constantly reported (But et al., 1996; Park et al., 2001; Cárdenas et al., 2006; Laird et al., 2008; Cho et al., 2009; Jung et al., 2011; Dong et al., 2014). Accordingly, the supervisions of clinical usage of PMR have been conducted by the drug regulatory agencies in Canada, Australia, the United Kingdom, and China (Canadian Adverse Reaction Newsletter, 2003; Medicines and Healthcare products Regulatory Agency, 2006; Complementary Medicines Evaluation Committee, 2008; China Food and Drug Administration, 2014). Therefore, the safe and effective use of PMR in clinical practice has become an important and urgent problem which needed to be resolved.
According to previous reports, more than 100 components from PMR had been isolated and identified including stilbenes, anthraquinones, dianthrones, flavonoids, and phenolic acids (Yang et al., 2016; Wang et al., 2017; Yang et al., 2017; Yang et al., 2018b; Yang et al., 2019). After oral administration in rats with PMR extract, 41 compounds were detected in the rat plasma with stilbenes and quinones as the major components (Wang et al., 2017). The stilbenes were reported to possess anti-oxidative, anti-aging, anti-tumor, and hepatoprotective activities (Lin et al., 2015; Wu et al., 2017; Zhang and Chen, 2018), while the anthraquinones were mainly used for anti-bacterial, anti-fungal, and anti-cancer activities (Lin et al., 2015; Dong et al., 2016; Xun et al., 2019). As for the aspect of toxicity, several anthraquinones including emodin and emodin-8-O-β-D-glucoside, dianthrones such as (Cis)-emodin-emodin dianthrones and (Trans)-emodin-emodin dianthrones as well as stilbenes such as (Trans)-2, 3, 5, 4′-tetrahydroxy stilbene-2-O-β-d-glucopyranoside and (Cis)-2, 3, 5, 4′-tetrahy droxystilbene-2-O-β-d-glucopyranoside have been identified as the potential components for the hepatotoxicity of PMR (Yang et al., 2021). Moreover, among the known anthraquinones, emodin, physcion, and chrysophanol were the most detectable components in the plasma after oral consumption of PMR extract (Li et al., 2020a). These anthraquinones were also reported to induce liver toxicity via multiple mechanisms including reactive oxygen species induced endoplasmic reticulum stress and mitochondrial dysfunction as well as cytochrome c release mediated apoptosis (Lai et al., 2009; Qu et al., 2013). In addition, Zhang et al. (2020a) have also suggested that the stilbene in PMR could synergize the idiosyncratic hepatotoxicity of anthraquinones from PMR.
Bile acids (BAs), the major components of bile, are formed from cholesterol through various enzymatic reactions in the hepatocytes (García-Cañaveras et al., 2012). The homeostasis of BAs plays a crucial role in the metabolism of lipid, glucose, and energy (Verkade et al., 1995; Houten et al., 2006; Kuipers et al., 2014), and the disorder of BAs would cause hepatobiliary diseases (Stanimirov et al., 2015), gallstones (Berr et al., 1992), or gastrointestinal cancers (Bernstein et al., 2005). In recent years, several metabolomic (Dong et al., 2015; Zhang et al., 2015; Xia et al., 2017; Yan et al., 2020; Zhang et al., 2020b) and transcriptome (Jiang et al., 2018) analyses demonstrated that the toxicity mechanisms of PMR-induced liver injury may relate to the disruption of BA homeostasis and the metabolism of energy, amino acids, fatty acids, and lipids. Moreover, cholestasis was also observed after treating the rats with PMR extract for 28 days (Wang et al., 2015). In addition, our group has demonstrated that emodin and physcion, the major components of PMR, could alter BA disposition and induce BA accumulation in the sandwich-cultured rat hepatocytes (Kang et al., 2017). However, whether these anthraquinones have any influence on the BA homeostasis contributing to the PMR-induced liver injury after its long-term exposure in-vivo remains unknown. In this study, to investigate the hepatotoxicity of PMR extract and three individual anthraquinone from PMR, namely, emodin, chrysophanol, and physcion, the ICR mice were consecutively administered with PMR extract or the pure anthraquinones for 14 or 28 days.
2 Materials and Methods
2.1 Chemicals and Reagents
Emodin, chrysophanol, physcion, and 2,3,4’,5-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) (purities >98%) were obtained from Nanjing Jingzhu Co. Ltd. (Jiangsu, China). The aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), triglyceride (TG), and γ-glutamine transferase (γ-GGT) assay kits were supplied by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime (Jiangsu, China). Individual BA together with deuterium-labelled glycol-chenodeoxycholic acid (d4-GCDCA) were obtained as previously described (Huang et al., 2011). All other chemicals and reagents were of analytical grade and readily available from commercial sources.
2.2 Preparation of the Ethanol Extract of PMR
The raw PMR from Dabashan, Sichuan Province, identified by Prof. Jianping Wang, Huazhong University of Science and Technology, was used in our study. The PMR extract was prepared by crushing the raw materials into powder, followed by refluxing for 48 h with 75% (v/v) ethanol at a solid–liquid ratio of 1:6 and filtration. The extraction was repeated five times and the combined filtrates were concentrated to remove the ethanol and then lyophilized for further use.
2.3 Identification and Determination of Major Anthraquinones From PMR Ethanol Extract
The major components from PMR extract were identified based on the previous study (Li et al., 2021). Briefly, 0.2 g PMR extract was sonicated with 25 ml acetone for 1 h and centrifuged at 1800 g for 10 min. The supernatant was evaporated to dryness, reconstituted with 2 ml methanol (MeOH), and filtered for HPLC analyses. Additionally, the contents of major PMR derived anthraquinones including emodin, chrysophanol, and physcion in the solution were determined via HPLC and the contents of all the analytes were calculated as percentage (%, g/100 g crude material).
2.4 Animals and Treatment
Male ICR mice, weighing 30 ± 5 g, were obtained from the Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology (HUST, Wuhan, China). The mice were acclimatized to the environmentally controlled condition (temperature of 22–27°C, relative humidity of 40%–70%, and 12 h light/dark cycle) for 7 days with free access to food and water before and during the treatment interval. The anthraquinones and PMR extract powder was dissolved in 0.5% (w/v) CMC-Na each day before treatment, and the guideline for the volume of oral administration is 1 ml/100 g body weight in mice. Eighty ICR mice were randomly divided into 10 groups (n = 8): five of the groups were consecutively orally administered with 0.5% (w/v) carboxymethylcellulose sodium (CMC-Na) solution, 100 mg/kg emodin, 100 mg/kg chrysophanol, 100 mg/kg physcion, or 1.5 g/kg PMR extract (equivalent to 30 g crude material/kg) for 14 days; the other five groups received the same doses for 28 days. Twenty-four hours after the last dose, the animals were sacrificed and the blood, bile, and liver tissues were collected for further analyses. All animal experiment procedures were approved by the Institutional Animal Care and Use Committee of Tongji Medical College, HUST.
2.5 Biochemical Analysis and Histopathological Examination
The blood samples were collected and centrifuged at 1,000 g for 10 min, and the supernatant sera were obtained. Biomarkers, including AST, ALT, TG, and γ-GGT in the serum, were determined using the commercially available kits according to the manufacturer’s instructions. Moreover, the livers fixed in 4% formalin were embedded in paraffin and sliced to a thickness of 5 µM. After staining with hematoxylin and eosin (H&E), the slices were examined under a light microscope for structural changes.
2.6 Sample Preparation
The sample preparation procedure was based on our previous method for the analysis of BAs with slight modification (Huang et al., 2011). For the plasma samples, the d4-GCDCA (internal standard, IS) solution (10 μl, 1 μg/ml) was spiked into 100 μl plasma, followed by the addition of 1 ml ice-cold alkaline acetonitrile (ACN) containing 5% (v/v) NH4OH, vortexed for 10 min and centrifuged at 16,000 × g for 10 min. The supernatant was aspirated, evaporated, and reconstituted in 100 μl of 50% MeOH in water. For the liver samples, after homogenizing approximately 100 mg of liver with 2 volumes of H2O, 10 μl IS solution was spiked into 300 μl liver homogenate and 2 ml of ice-cold 5% NH4OH in ACN was added. After vortexing for 10 min, the samples were centrifuged at 16,000 × g for 10 min. The supernatant was aspirated, and the pellet was extracted with another 1 ml of ice-cold 5% NH4OH in ACN. The supernatants were pooled, evaporated to dryness, and reconstituted with 100 μl 50% MeOH in water. As for the bile samples, the Supelclean™ LC-18 SPE cartridges (Sigma–Aldrich, St. Louis, MO) were used for sample extraction. First, the bile samples were diluted 20- and 2000-fold with deionized water. Then, 100 μl of diluted bile samples spiked with 10 μl IS was loaded onto SPE cartridges which were preconditioned with 2 ml MeOH, followed by 2 ml H2O. Thereafter, the loaded cartridges were washed with 2 ml H2O and eluted with 4 ml MeOH. Finally, the eluted solution was evaporated to dryness and reconstituted with 100 μl 50% MeOH in water.
2.7 LC-MS/MS Analysis
Simultaneous determination of individual endogenous BAs in the bile, liver homogenate, and plasma was performed according to our previously published liquid chromatography with the tandem mass spectrometry (LC-MS/MS) method with slight modifications (Huang et al., 2011). Chromatographic separation was achieved on a Waters ACQUITY HSS T3 column (2.1 × 100 mm, 1.8 μm; Waters, United States). The mobile phase consisted of 7.5 mM ammonium acetate, adjusted to pH 7.0 using 10 M ammonium hydroxide (mobile phase A) and MeOH (mobile phase B) and the flow rate was 0.3 ml/min. The elution gradient profile started from 50% mobile phase B, linearly increased to 90% mobile phase B in 20 min, held at 90% for 2 min, and brought back to 50% in 0.01 min followed by 4 min re-equilibration. The BA analysis was performed on a Shimadzu Prominence UFLC system (Shimadzu Corporation, Japan) and an API 4000 QTrap® triple quadrupole mass spectrometer (AB SCIEX, United States) with an electrospray ionization (ESI) source. All the BAs were measured in negative ionization mode using mass transitions listed in our previous report. The main working parameters for the MS were set as: capillary voltage, −4000 V; source temperature, 600°C; curtain gas flow, 20 psi; nebulizer gas flow, 35 psi; and collision energy, high.
2.8 Quantitative Real-Time PCR
Total RNA was extracted from liver using TRIzol reagent according to the manufacture’s instruction (Invitrogen, United States). The RNA samples were reversed-transcribed into complementary DNA (cDNA) via Fermentas RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, United States) according to the manufacturer’s protocol. The cDNA was amplified using an ABI Step-One Sequence Detection System (Applied Biosystems, United States) with SYBR Green PCR Master Mix (Bio-Rad, United States). The gene expression was determined by normalization with control gene Gapdh using the ΔΔCt method and the primer sequences are set as: Bsep (Gene ID: 27413, forward: 5′-GCTGCCAAGGATGCTAATGC-3′ and reverse: 5′-TTGGGTTTCCGTATGAGGGC-3′), Mrp2 (Gene ID: 12780, forward: 5′- CCTTGGGCTTTCTTTGGCTC-3′ and reverse: 5′-ACACAACGAACACCTGCTTG-3′), and Gapdh (Gene ID: 14433, forward: 5′-GTCGGTGTGAACGGATTTGG-3′ and reverse: 5′-TCAGATGCCTGCTTCCCATTC-3′).
2.9 Western Blotting
Total protein in the liver was extracted with RIPA Lysis Buffer (Beyotime) containing 1 mM PMSF, and the total protein concentration was determined using the BCA protein assay kit. An equal amount of proteins (50 μg) was separated by electrophoresis on sodium dodecyl sulfate/8.75% polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Millipore, United States) for 2 h through wet transfer method using Mini Trans-Blot (Bio-Rad, United States). Subsequently, the membranes were blocked with 5% nonfat milk dissolving in 0.5% TBST buffer for 1.5 h and then incubated overnight at 4°C with specific primary antibodies including Bsep (goat, Santa Cruz Biotech), Mrp2 (rabbit, Abcam), and β-actin (mouse, Proteintech). After washing the membrane with 0.5% TBST buffer for three times, the membranes were further incubated with corresponding secondary antibodies at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence detection system (Millipore, United States).
2.10 Data Processing and Statistical Analysis
All values were indicated as mean ± standard deviation (mean ± SD). Statistical analyses were carried out using GraphPad Prism 7.0 (GraphPad Software Inc., United States). Differences between the two groups were analyzed by Student’s t-test, while multiple comparisons were carried out by one-way ANOVA followed by Dunnett’s post hoc test. Differences with a probability value (P) < 0.05 were statistically significant. MetaboAnalyst 5.0 was used to perform the Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) for the evaluation of the differences of the BAs in the liver, bile, and plasma between the control and treatment groups.
4 Discussion
BA homeostasis is strictly maintained by a series of uptake and efflux transporters as well as synthesis and metabolism enzymes (Rodrigues et al., 2014). Experimental toxicology studies have identified the disturbed metabolism of BAs as a common and early event of drug-induced liver injury by diverse hepatotoxins (Yamazaki et al., 2013). As indicated by the previous studies, PMR induced liver injury was closely associated with BA homeostasis disruption. In the current work, we explored the hepatotoxic effect of three major PMR-derived anthraquinones including emodin, chrysophanol, and physcion as well as the PMR ethanol extract based on BA disposition and complemented with serum biochemical indicators and histopathological examination in the mice. All these three anthraquinones and the PMR extract were found to induce liver injury as evidenced by the histopathological examination and the hepatotoxic biomarker levels in the serum. Additionally, it was found that all the anthraquinones of interest and PMR extract could alter the BA homeostasis.
To uncover any potential toxicity of the PMR extract or the anthraquinones, the doses were set at high levels and the dose of PMR was around 25-fold the highest recommended human dose obtained from the 2015 edition of the Chinese Pharmacopoeia (the dose of human was converted to mice based on the body surface area conversion (US Food and Drug Administration, 2005). According to the previous reports, there is an interaction between the co-occurring components in PMR. Xing et al. (2019) have evidenced that the Cmax of emodin in the rat plasma could be increased by 3–7 times in the presence of TSG. In our current study, the dose of PMR was selected as 30 g/kg, which was equivalent to 14.1 mg/kg emodin. To mimic the maximum exposure of emodin after administration with PMR extract, the dose of emodin was selected as 100 mg/kg (14.1 mg/kg × 7 = 98.7 mg/kg). In order to compare the impact of these anthraquinones parallelly, the dose of other two anthraquinones, chrysophanol and physcion, was also selected as 100 mg/kg.
Currently, the serum biochemical parameters such as ALT, AST, and ALP are used as conventional biomarkers for the clinical diagnosis (Park et al., 2001; Jung et al., 2011; Dong et al., 2014). In our study, the AST levels were significantly increased after treating for 28 days with the PMR extract or the pure anthraquinones, which correlated well with the histopathological findings. However, neither anthraquinones nor the PMR extract altered the biomarkers tested in our current study including ALT, AST, TG, and γ-GGT after 14-day treatment, while the distinct results were observed that physcion and PMR caused liver damage after 14-day treatment evidenced by the liver histopathological changes. Such discrepancy may be resulted from the limited sensitivity and specificity of ALT and AST in the serum for early detection of liver injury (McGill, 2016). Although, ALT was suggested to be a more sensitive biomarker for liver injury in clinical practice (Senior, 2009), the significant elevation of AST rather than ALT was observed in all the treatment groups in our current study and the previous report in which the rats were treated with the PMR extract (equivalence to 20 g/kg crude material) for 21 days (Ma et al., 2015). Such discrepancy could be due to the species difference between the rodents and humans or AST was suggested to be a more sensitive biomarker for PMR-induced liver injury. McGill reviewed the mechanisms and interpretation of the plasma levels of ALT and AST elevation in liver injury and stated that ALT and AST have poor prognostic utility in acute liver injury and liver failure (McGill, 2016). In addition, physcion was reported to be cytotoxic to red blood cells by triggering hemolysis and programmed cell death (Akiel et al., 2021). As a result, unpredictable response to the different degree of hemolysis might also affect the accurate measures of such biochemical testing (Lippi et al., 2006).
In preclinical studies, the change of BAs was also used as an important physiological indicator of liver damage (Casini et al., 1997) and several reports have also recommended the GDCA in the bile, hyodeoxycholic acid (HDCA) in the plasma, or TMCA in the urine as potential biomarkers for PMR induced liver injury in the SD rats (Dong et al., 2015; Zhao et al., 2017). In the current study, the total BAs in the liver were significantly increased accompanied with the significant decrease in the bile after treating with physcion and PMR for 14 days. These results correlated well with the histopathological results that the liver obtained from these two groups showed typical inflammatory cell infiltration. After 28-day treatment, it was noted that the accumulation of the total BAs in the liver was decreased in the physcion treatment group, which may be related to the adaptive protective mechanisms (Zollner et al., 2006). However, such a reduction of total BAs in the liver did not prevent the occurrence of liver damage evidenced by the histopathological findings. A plausible explanation for such hepatic toxicity was that the accumulation of physcion in the liver resulted from long-term treatment leading to hepatocellular damage. It was found that physcion could induce significant cytotoxic effects against the human liver cell line L02, HepG2 cells, rat primary hepatocytes, and sandwich-cultured rat hepatocytes in a dose dependent manner (Xun et al., 2019).
The toxicity of the BAs is related to their structures and the hydrophobic BAs is more toxic than the hydrophilic BAs (LCA ≥ DCA ≥ CDCA >> CA) (Trottier et al., 2006; Schadt et al., 2016). The accumulation of hydrophobic BAs in the liver can result in mitochondrial dysfunction by generating reactive oxygen species (ROS), which in turn causes liver injury (Casini et al., 1997; Perez and Briz, 2009). In our study, it was worth noting that after 14-day or 28-day administration with anthraquinones or PMR extract, the hydrophobic BAs (DCA, CDCA, and CA) in the liver were significantly accumulated. Moreover, the significant accumulation of the total LCA was also observed in the liver after treating the mice for 28 days. BA conjugation with glycine or taurine could increase the solubility and decrease the toxicity of the corresponding BAs (Trottier et al., 2006). The significantly decreased proportion of the conjugated BAs in the liver, at least in part, indicated that toxic BA detoxification could be altered by PMR and typical anthraquinones. Taken together, the accumulation of these hydrophobic BAs and inhibition of BA conjugation were considered to contribute to the liver toxicity induced by PMR and anthraquinones of interest. This might also be a possible explanation for the discrepancy between the attenuation of liver total BA accumulation in the physcion treatment group and the liver damage evidenced by histological examination after treating for 28 days.
In human, BAs undergo sulphation to reduce their cytotoxicity (Agellon and Torchia, 2000; Zwicker and Agellon, 2013). Sulfotransferase-2A1(SULT2A1) is the enzyme catalyzing the BAs into BA-sulfates, which are more hydrophilic and readily excreted into feces and urine (Alnouti, 2009). According to previous study, the activity and expression of SULT2A1 could be significantly inhibited by emodin or rhein in the HepG2 cells (Maiti et al., 2014). The inhibition of SULT2A1 might directly contribute to the decrease of liver BA-sulfates level in our current study. Moreover, hepatic transporters also play critical roles in maintaining the BA homeostasis (Agellon and Torchia, 2000). BAs were synthesized from cholesterol in the hepatocytes and secreted across the canalicular membrane in an ATP-dependent fashion via BSEP/Bsep and MRP2/Mrp2 (Akita et al., 2001). Since canalicular secretion is considered as the rate-limiting step of the vectorial export of toxic BAs from hepatocytes into bile, impaired BA biliary transport may directly result in hepatic BA accumulation. In addition, basolateral BA efflux transporters, Mrp3 and Mrp4, are also involved in the BA homeostasis and the inhibition of both canalicular and basolateral BA efflux transporters may worsen liver injury (Corsini and Bortolini, 2013; Köck et al., 2014). In our current study, the significant decrease of gene and protein expression of Bsep in the physcion and PMR groups was supposed to be the major factor of the hepatic total BA accumulation after treating for 14 days. After 28 days of treatment, hepatic total BA accumulation was attenuated due to the elevated mRNA and protein expression of Bsep in the physcion group. However, the increased accumulation of hepatic total BAs was contradictory with the significant elevation of Bsep after treatment with PMR extract for 28 days. Such discrepancy could be explained by the concurrent inhibition of the basolateral BA efflux induced by these three anthraquinones in PMR as demonstrated in our previous study (Kang et al., 2017). Unlike Bsep, the inhibition of Mrp2 may not induce the accumulation of hepatic BAs (Iyanagi et al., 1998), which was consistent with our results that the altered protein expression of Mrp2 showed no impact on the hepatic total BA levels in the emodin and chrysophanol treated groups. Furthermore, the inconsistency of the changed expression of Bsep and Mrp2 with the individual hepatic BAs could be due to their distinct affinity to such efflux transporters (Noe et al., 2001). Compared with the control group, the total BAs in the bile were decreased by around 86% and 85%, while the total hepatic BA level was increased by 3.1- and 1.7-fold in the physcion- and PMR-treated groups, respectively. However, in agreement with the previous reports (Wang et al., 2015; Zhao et al., 2017), the expression of BA efflux transporters, Bsep and Mrp2, was increased with the administration prolonging to 28 days, which was in accordance with the disposition of BAs that the percentage of amidated BAs in the liver, the major substrates of Bsep, was significantly decreased. Since the individual BAs such as CDCA, DCA, CA, and LCA are the agonists of Farnesoid X receptor (FXR/Fxr) with CDCA as the most potent one (Stofan and Guo, 2020), the accumulated BAs could activate FXR/Fxr and therefore inhibit the synthesis of BAs and induce the expression of Bsep to attenuate the overload of hepatic BAs (Schaap et al., 2014). Moreover, CA and UDCA were reported to induce the expression Mrp2 with a Fxr-independent manner in the mice (Zollner et al., 2003). Therefore, the regulation of the expression of the two transporters in our current study could be explained as the adaptive response for limiting the hepatic BA overload (Zollner et al., 2006). Additionally, the decrease of part of the BAs in the plasma in our study may result from the decreased reabsorption of BAs due to the inhibition of the excretion of BAs into the bile as well as the inhibition of the bile acid efflux transporters Mrp3 and Mrp4. Also, the plasma BA concentrations could be affected by the hepatocellular uptake from the blood evidenced by the downregulated expression of major hepatic BA uptake transporter, sodium taurocholate cotransporting polypeptide (Ntcp) in our previous study (Kang et al., 2017). In addition, renal elimination, hepatic synthesis and metabolism, and intestinal metabolism and elimination from feces of BAs have also been recognized as another vital factor for the BA levels in the plasma. Therefore, the complex regulation processes might be a plausible reason for the relatively minor alteration of BAs in the plasma.
Taken together, the present study comprehensively elucidated the role of PMR or its major anthraquinones in BA disposition. It was found that PMR or physcion could induce hepatic BA accumulation after treating for 14 days, which were potentially associated with the inhibition of the function and expression of hepatic BA canalicular efflux transporters, Bsep and Mrp2, as well as the basolateral efflux transporters, Mrp3 and Mrp4. Such cholestasis could be attenuated via regulating the expression of BA transporters with a prolonging administration duration up to 28 days. This study, for the first time, investigated the hepatotoxicity of the major anthraquinones from PMR based on BA homeostasis and found that all the tested anthraquinones of interest could change the disposition of the BAs to a different extent in mice. However, in addition to anthraquinones, there are many other components such as stilbene, tannins, dianthrones, and flavonoids in the PMR and the stilbene was reported to increase the systemic exposure of emodin via increasing its absorption and inhibiting its metabolism (Li et al., 2020b). That could also be a possible explanation that the hepatoxicity of other traditional Chinese medicine containing anthraquinones was less reported than PMR. It is also worth noting that dianthrones derivatives, such as (Cis)-emodin-emodin dianthrones and (Trans)-emodin-emodin, have recently been reported to be associated with PMR hepatotoxicity (Yang et al., 2018a; Li et al., 2020). Furthermore, the mutual transformation between the dianthrones derivatives and anthraquinones results in more complicated potential toxic source analysis for PMR induced liver injury (Yang et al., 2018a). Therefore, the influence of the interaction between anthraquinones and other components from PMR on the BA steady-state should be taken into further consideration for the quality control and safe use of PMR in practice. In addition, due to the species difference between the mice and human, the translation of the current results to humans warrants further investigation.