The use of herbal medicines continues to expand globally, meanwhile, herb-associated hepatotoxicity is becoming a safety issue. As a conventional Chinese medicinal herb, Dioscorea bulbifera rhizome (DBR) has been documented to cause hepatic toxicity. However, the exact underlying mechanism remains largely unexplored. In the present study, we aimed to profile entire endogenous metabolites in a biological system using a multisample integrated metabolomics strategy. Our findings offered additional insights into the molecular mechanism of the DBR-induced hepatotoxicity. We identified different metabolites from rat plasma, urine, and feces by employing gas chromatography-mass spectrometry in combination with multivariate analysis. In total, 55 metabolites distributed in 33 metabolic pathways were identified as being significantly altered in DBR-treated rats. Correlation network analysis revealed that the hub metabolites of hepatotoxicity were mainly associated with amino acid, bile acid, purine, pyrimidine, lipid, and energy metabolism. As such, DBR affected the physiological and biological functions of liver via the regulation of multiple metabolic pathways to an abnormal state. Notably, our findings also demonstrated that the multisample integrated metabolomics strategy has a great potential to identify more biomarkers and pathways in order to elucidate the mechanistic complexity of toxicity of traditional Chinese medicine.

•A new strategy is proposed for precise quality evaluation of herbal medicines.•Fingerprint-activity relationship is modeled by PLSR and BP-ANN.•Principal bioactive markers are rapidly discovered.•Sophora flower-bud and Sophora flower are clearly discriminated.Chromatographic fingerprint has been extensively used as a comprehensive approach for quality evaluation of herbal medicines (HMs). However, similar chemical profiles do not always mean similar efficacies. The present work, taking Sophora flower-bud and Sophora flower as a typical case, attempts to develop a rational strategy based on fingerprint-activity relationship modeling to realize quality evaluation from chemical consistency to effective consistency. A total of 57 batches of Sophora samples were collected and their antioxidant and hyaluronidase inhibitory activities were measured. Chemical fingerprints were established by high performance liquid chromatography (HPLC) coupled with photodiode array (PDA) detector and quadrupole time-of-flight mass spectrometry (Q-TOF MS), and similarity analyses were calculated based on eight common characteristic peaks. Subsequently, three principal bioactive markers were discovered by correlating biological effects with chemical fingerprints via partial least squares regression (PLSR) and back propagation-artificial neural network modeling (BP-ANN). The selected markers were quantified by the ‘single standard to determine multi-components’ method, and then the quantitative data as well as their bioactive properties were subjected to principal component analysis to generate two clear-cut groups. This study not only demonstrates the necessity of effective consistency besides chemical consistency in the quality evaluation of HMs, but also provides an applicable strategy to screen out efficacy-associated markers by fingerprint-activity relationship modeling.

•A dereplication strategy using HPLC–QTOF-MS is proposed.•Two novel triterpenoid saponins are identified and purified from Celosiae Semen.•Their structures are elucidated by spectroscopic and chemical analyses.•Their hepatoprotectivities are evaluated.Although natural products (NPs) from ethnomedical plants have played a vital role in modern drug discovery, separation and purification of bioactive compounds from plant extract is still challenging. In this study, a dereplication strategy using HPLC–QTOF-MS was employed to rapidly discover and highly targeted isolate the novel hepatoprotective triterpenoid saponins from the methanol extract of Celosiae Semen. Firstly, four known saponins, i.e. celosin H, celosin I, celosin J, and pseudoginsenoside RT1 were selected as model compounds, and their fragmentation patterns in ESI-QTOF-MS/MS were characterized. Secondly, an HPLC–QTOF-MS/MS method was applied to chemically screen the saponins of interest, and thereby to guide the subsequent fraction and isolation procedure. Thirdly, the targeted isolation of desired compounds afforded two new triterpenoid saponins namely celosin K (1) and celosin L (2), which were structurally elucidated by combination of extensive NMR spectroscopic and chemical analyses. Finally, the protective effects of compounds 1 and 2 against APAP-induced hepatotoxicity in HepG2 cells were evaluated. These results indicate that the HPLC–QTOF-MS-guided isolation is an efficient methodology for isolating new NPs from medicinal plants through improving selectivity in separation and purification process.

Nowadays, more and more attention has been paid to osteoporosis caused by diabetes mellitus. Elevated levels of pro-inflammatory cytokines in diabetic patients activate the activity of osteoclasts through the RANKL/OPG pathway. The nuclear transcription factor SREBP2, a master regulator of cholesterol metabolism, has been found involved in osteoclastogenesis. In our previous study, we have identified anhydroicaritin as a potent inhibitor of transcription factor SREBPs, which improves dyslipidemia and insulin resistance. In this study, we demonstrated that anhydroicaritin could also decrease the level of SREBP2 and its target genes in osteoclasts induced by RANKL without significant cytotoxicity. Moreover, anhydroicaritin suppressed RANKL-induced osteoclasts differentiation. In STZ-induced diabetic mice model, we found that the osteoclasts were largely increased accompanied with deterioration of bone structure. Anhydroicaritin decreased the level of blood glucose and alleviated insulin resistance. More importantly, anhydroicaritin inhibited osteoclast differentiation and rescued diabetes-induced bone loss in vivo. In conclusion, anhydroicaritin, a potent SREBP2 inhibitor, inhibits the osteoclasts formation and improves diabetes-induced bone loss.

Polygoni Multiflori Radix (PMR) has been commonly used as a tonic in China for centuries. However, PMR-associated hepatotoxicity is becoming a safety issue. In our previous in vivo study, an interaction between stilbenes and anthraquinones has been discovered and a hypothesis is proposed that the interaction between stilbene glucoside-enriching fraction and emodin may contribute to the side effects of PMR. To further support our previous in vivo results in rats, the present in vitro study was designed to evaluate the effects of 2, 3, 5, 4′-tetrahydroxystilbene-2-O-β-D-glucopyranoside (TSG) on the cellular absorption and human liver microsome metabolism of emodin. The obtained results indicated that the absorption of emodin in Caco-2 cells was enhanced and the metabolism of emodin in human liver microsomes was inhibited after TSG treatment. The effects of the transport inhibitors on the cellular emodin accumulation were also examined. Western blot assay suggested that the depressed metabolism of emodin could be attributed to the down-regulation of UDP-glucuronosyltransferases (UGTs) 1A8, 1A10, and 2B7. These findings definitively demonstrated the existence of interaction between TSG and emodin, which provide a basis for a better understanding of the underlying mechanism for PMR-induced liver injury.

•A targeted metabolomics of bile acids was applied to characterize the process of PMR-induced hepatotoxicity.•Hyodeoxycholic acid in serum and tauro-β-muricholic acid in urine were identified as potential biomarkers.•The expression of Bsep and Ntcp in the liver indicated that hepatotoxicity was related to the disorders of BAs metabolism.Polygoni Multiflori Radix (PMR) has been widely used as a tonic for centuries. However, hepatotoxicity cases linked to PMR have been frequently reported and appropriate biomarkers for clinical diagnosis are currently lacking. Here, an approach using UPLC-QqQ/MS-based targeted metabolomics of bile acids (BAs) complemented with biochemistry and histopathology was applied to characterize the development and recovery processes of PMR-induced hepatotoxicity in rats and to identify biomarkers. The expression of bile salt export pump (Bsep) and sodium taurocholate cotransporting polypeptide (Ntcp) were evaluated to investigate the underlying mechanism. Steatosis and inflammatory cell infiltration were observed in PMR-treated rats, which were accompanied by the elevation of serum biochemistry. The metabolic profiles of BAs were analyzed by Principal Component Analysis, hyodeoxycholic acid (HDCA) in serum and tauro-β-muricholic acid (TβMCA) in urine were identified as potential biomarkers for PMR-induced hepatotoxicity. The elevated expression of Bsep and decreased expression of Ntcp in the liver of PMRtreated rats indicated that hepatotoxicity was related to the disorders of BAs metabolism. Our study demonstrated that BAs may be used for clinical diagnosis of PMR-induced hepatotoxicity. Urine TβMCA was identified as a promising biomarker to facilitate the clinical monitoring of PMR-induced hepatotoxicity and may serve as potential therapeutic target.Download high-res image (293KB)Download full-size image

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues (AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I (AA-I) and aristolochic acid II (AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-QqQ-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts.

•A strategy is proposed for screening and targeted isolation of cyclopamine analogs.•Combination of LC-Q-TOF-MS and LC-QqQ-MS is employed.•Twenty four cyclopamine analogs are verified from Fritillaria and Veratrum plants.•Five target compounds are isolated and their activities are evaluated.Cyclopamine, an inhibitor of the Hedgehog (Hh) signaling pathway, has been paid much attention in treating a wide variety of tumors. However, isolation and purification of cyclopamine analogs from medicinal plants remain challengeable. We herein proposed an efficient strategy using liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS) and liquid chromatography triple-quadrupole mass spectrometry (LC-QqQ-MS) for rapid screening and targeted isolation of cyclopamine analogs in Fritillaria and Veratrum plants. Firstly, fifteen reference compounds were characterized by LC-Q-TOF-MS and their characteristic fragment ions were summarized. Secondly, according to the characteristic fragment ions at m/z 67.1, 84.1, 109.1 and 114.1, rapid chemical screening of plant extracts was carried out by LC-QqQ-MS using precursor ion scan mode and 69 pre-target compounds were screened out. Thirdly, 24 real target compounds were verified by LC-Q-TOF-MS based on relative abundances (over 20%) of characteristic fragment ions. Fourthly, the targeted isolation of Fritillaria ussuriensis bulb and Veratrum dahuricum rhizome afforded a novel cyclopamine analog namely 15β-hydroxy-23-isopengbeisine B as well as four known ones, whose structures were determined by nuclear magnetic resonance (NMR) analysis. Additionally, these five analogs were evaluated for the inhibitory activity of Hh signaling pathway in NIH/3T3 cell and cytotoxicity in PANC-1 and HepG2 cells. These results indicated that the proposed strategy was reliable for rapid discovery and targeted isolation of important natural products from chemically complex plant matrices.Download high-res image (126KB)Download full-size image

It is vital to monitor the holistic toxicokinetics of toxic Chinese herbal medicines (CHMs) for safety. Although an integrated strategy based on the area under the curve (AUC) has been proposed to characterize the pharmacokinetic/toxicokinetic properties of CHMs, improvement is still needed. This study attempted to use 50% inhibitory concentration (IC50) as weighting coefficient to investigate holistic toxicokinetics of the major diosbulbins i.e. diosbulbin A (DA), diosbulbin B (DB), and diosbulbin C (DC) after oral administration of Dioscorea bulbifera rhizome (DBR) extract. Firstly, the cytotoxicities of the three diosbulbins on human hepatic L02 cells were evaluated and the IC50 values were calculated. Then, integrated toxicokinetics of multiple diosbulbins based on AUC and IC50 were determined. Finally, correlations between integrated plasma concentrations and hepatic injury biomarkers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bile acid (TBA) were analyzed. As a result, integrated plasma concentrations were correlated well with TBA and the correlation between TBA and IC50-weighting integrated plasma concentrations was better than that of AUC-weighting integrated plasma concentrations. In conclusion, the newly developed IC50-weighting method is expected to generate more reasonable integrated toxicokinetic parameters, which will help to guide the safe usage of DBR in clinical settings.

The emodin-involved hepatotoxicity has been gaining increasing attention. The purpose of the present study was to evaluate the cytotoxicity of emodin on cultured human liver cells (L-02) and predict the possible relation between its cytotoxicity and cellular toxicokinetics. Cell viability and cell damage were assessed by Cell Counting Kit-8 (CCK-8) assay and phase-contrast microscopy, respectively. Cytotoxicity tests demonstrated a concentration- and time-dependent toxic effect of emodin on L-02 cells. Furthermore, emodin at concentration of 30 μM led to a significant apoptosis in a time-dependent manner supported by the morphological changes of drug-treated cells. In addition, to elucidate the toxicokinetic characteristics of emodin, a highly sensitive and selective liquid chromatography–mass spectrometry (LC–MS) method was employed and validated for detecting the dynamic alteration of emodin in cells and cell culture media. The proposed method appeared to be suitable for the analysis of emodin with desirable linearity (r2 > 0.99), and satisfying precision being less than 8.7%. The range of recoveries of this method was 90.2–101.9%. The preliminary cellular toxicokinetic study revealed a time-dependent intracellular accumulation of emodin, which was consistent with its in vitro toxic effects. These findings confirmed the cytotoxicity of emodin against L-02 cells and displayed the cytotoxic manner of emodin in terms of its cellular uptake and accumulation in L-02 cells.

Baccharane glycosides represent a group of rare saponins in plant kingdom and have been regarded as chemical marker for quality control of Impatientis Semen. Based on the structural skeleton, the baccharane glycosides were classified into three types: hosenkol A, hosenkol B and hosenkol C type. In this study, a rapid-resolution liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (RRLC/ESI-Q-TOF MS/MS) was performed to investigate the fragmentation behaviours of baccharane glycosides from Impatientis Semen. In full scan mass spectrum, the accurate determination of molecular formula was obtained by the predominant ion [M+COO]− in negative mode. In the MS/MS spectrum, fragmentation reactions of the [M+H]+ acquired in positive mode were recorded to provide abundant structural information on the aglycone and glycosyl moieties. The characteristic ion for hosenkol A and hosenkol B type glycosides was at m/z 399, while for hosenkol C type glycosides the diagnostic ion was at m/z 381. Neutral losses of monosaccharide, disaccharide, H2O and C3H4 were observed for stepwise structural characterization. As a result, 19 compounds including 9 target saponins and 10 non-target saponins were rapidly screened out in ethanol extract of Impatientis Semen, and 5 of them were found to be novel baccharane glycosides.

Radix Polygoni Multiflori, Rhizoma et Radix Polygoni Cuspidati, and Radix et Rhizoma Rhei are the most frequently used traditional Chinese medicines in the family Polygonaceae. The three herbal medicines (HMs) contain similar types of compounds. In Chinese Pharmacopoeia, five high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection methods have been employed for their quality control. The aim of this study was to develop a simple and conventional strategy, segmental monitoring based on variable wavelength detection (VWD), for simultaneous quantification of phenolic acids, flavonoids, stilbenes and anthraquinones in the three chemically analogous HMs. Compared with the commonly used HPLC-diode array detection (DAD), the proposed method afforded desirable performance on linearity, precision and sensitivity. Additionally, a quadrupole time-of-flight mass spectrometry (QTOF-MS) was applied to the structural confirmation of analytes from complex matrices. Based on the chemical profiles and contents of the analyses, 27 samples from three HMs were well classified using the principal component analysis. The results of this study demonstrated the potential and applicability of segmental monitoring strategy based on VWD for comprehensive quality control of HMs.

We present herein a novel bioseparation/chemical analysis strategy for protein–ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography–mass spectrometry (LC–MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC–MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5 s, and on-line prepare the “clean” sample to be directly compatible with the LC–MS analysis. The improvement in performance of this 2D-TFC/LC–MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC–MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

Steroidal alkaloids are naturally occurring nitrogen-containing compounds in many edible or medicinal plants, such as potato, tomato, Fritillaria and American hellebore, which possess a variety of toxicological and pharmacological effects on humans. The aim of this study is to explore the potential of liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) method in the determination of these important alkaloids in plant matrices. The application of this method has been proven through 26 naturally occurring steroidal alkaloids in Fritillaria species. Accurate mass measurements within 4 ppm error were obtained for all the alkaloids detected out of various plant matrices, which allowed an unequivocal identification of the target steroidal alkaloids. The bunching factor for mass spectrometer, an important parameter significantly affecting the precision and accuracy of quantitative method, was firstly optimized in this work and satisfactory precision and linearity were achieved by the optimization of that parameter. The ranges of RSD values of intra-day and inter-day variability for all alkaloids were decreased remarkably from 41.8–159% and 13.2–140% to 0.32–7.98% and 2.37–16.1%, respectively, when the value of bunching factor was optimized from 1 to 3. Linearity of response more than two orders of magnitude was also demonstrated (regression coefficient >0.99). The LC/TOF-MS detection method offered improvements to the sensitivity, compared with previously applied LC (or GC) methods, with limits of detection down to 0.0014–0.0335 μg/ml. The results in this paper illustrate the robustness and applicability of LC/TOF-MS for steroidal alkaloids analysis in plant samples. In addition, relative quantitative determination of steroidal alkaloid with one popular analyte verticinone which is commercially available was also investigated in order to break through the choke point of lack of standards in phytochemical analysis. The accuracies of relative quantitative method for steroidal alkaloids determinations with verticinone were 90.6–110.0% (average 98.5%) suggesting that it is feasible to quantify steroidal alkaloids by the proposed relative quantitative determination method within acceptable errors.

Ethnopharmacological relevanceRhododendri Mollis Flos (RMF), termed as Naoyanghua in Chinese, is a traditional anti-rheumatoid arthritis and bruises herb with associated cardiotoxicity. The predominant rhodojaponins occurring in RMF are responsible for its efficacy and toxicity. The narrow therapeutic window of rhodojaponins necessitates monitoring the pharmacokinetics and pharmacodynamics so as to ensure the safety in practical applications of RMF.Materials and methodsFifty-four male Sprague-Dawley rats were divided into a control group, a low-dose group and a high-dose group. After oral administration of RMF extract, the cardiotoxicity of RMF was evaluated by assessing ventricular function and by measuring the plasma levels of LDH, CK-MB and AST. Then, an LC–MS method was established to determine the rat plasma concentrations of three major rhodojaponins including rhodojaponin I, II and III (R-I, II and III) and was applied to pharmacokinetic study. Finally, based on an AUC-weighting approach, the integrated pharmacokinetics of three rhodojaponins was determined.ResultsCompared with control group, cardiotoxicity was observed in RMF-treated rats with left ventricular dysfunction and with the continuously increased levels of LDH and CK-MB in a dose-dependent manner. The pharmacokinetic parameters (AUC0−t, AUC0−∞, t1/2, Tmax and Cmax) for R-I, II and III were markedly different, and the integrated pharmacokinetics was therefore converted to describe the holistic pharmacokinetic profiles of R-I, II and III, which correlated pretty well with cardiotoxicity.ConclusionsIt was found that myocardial damage was elicited by RMF extract in a dose-dependent manner and the plasma levels of LDH and CK-MB could reveal the severity of myocardial injury as potential markers. This study also highlighted the potential of integrated pharmacokinetics to provid a more comprehensive understanding of the relationship between the pharmacokinetic behaviors of traditional Chinese herbal medicine and its efficacy.Download high-res image (159KB)Download full-size image