Aizhen Xiong

Evaluation of the protective effect of Rhei Radix et Rhizoma against α-naphthylisothiocyanate induced liver injury based on metabolic profile of bile acids.

ETHNOPHARMACOLOGICAL RELEVANCEnTo evaluate the hepatoprotective effect of the root and rhizome of Rhubarb (Rhei Radix et Rhizoma) against α-naphthylisothiocyanate (ANIT)-induced liver injury using metabolic profile of bile acids (BAs) along with biochemical parameters and histological alterations.nnnMATERIALS AND METHODSnUltra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determinate the concentration of BAs, which was followed by multivariate statistical analysis of Principal Component Analysis (PCA) and Partial Least Squares Discriminate Analysis (PLS-DA).nnnRESULTSnBased on PCA results, three groups (Vehicle group, ANIT group and RhO+ANIT group) were clearly distinguished. Tauro-cholic acid (TCA), tauro-hyodesoxycholic acid (THDCA), glyco-cholic acid (GCA), and glyco-chenodeoxycholic acid (GCDCA) were proved to be the most important markers corresponding to ANIT-induced liver injury and protection provided by Rhubarb, which is further confirmed by PLS-DA. A correlation was found between the foregoing BAs and biochemical parameters including serum aspartate aminotransferase (ALT) and aspartate aminotransferase (AST), which confirmed that TCA, THDAC, GCA, and GCDCA could be considered as sensitive biomarkers.nnnCONCLUSIONnThe variance of the BAs contents can be used to evaluate ANIT-induced hepatotoxicity caused by ANIT and protective effects of Rhubarb. It also lays the foundation for the further research on the mechanisms of cholestasis as well as the therapeutic effect of Rhubarb.

Identification of acteoside and its major metabolites in rat urine by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

In this study, metabolites in the urine samples of rats orally administered with acteoside, a phenylethanoid glycoside compound, were detected and identified using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS) combined with an automated MS(E) technique. Up to 35 metabolites (19 metabolites of the parent drug and 16 metabolites of the degradation products) were observed, including processes of oxidization, glucuronidation, sulfation, and methyl conjugation. According to the metabolic pathways, acteoside mainly functioned as a prodrug and underwent hydrolysis before being absorbed into the blood. The degradation products, especially caffeic acid and hydroxytyrosol, were involved in further metabolism which was responsible for the low oral bioavailability but obvious pharmacological activities of acteoside. In summary, this work provided valuable information on acteoside metabolism through the rapid and reliable UPLC/ESI-QTOF-MS technique, which could be widely used for the investigation of natural product metabolites.

A strategy for screening and identifying mycotoxins in herbal medicine using ultra‐performance liquid chromatography with tandem quadrupole time‐of‐flight mass spectrometry

The objective of this study was to develop an effective strategy for screening and identifying mycotoxins in herbal medicine (HM). Here, Imperatae Rhizoma, a commonly used Chinese herb, was selected as a model HM. A crude drug contaminated with fungi was analyzed by comparing with uncontaminated ones. Ultra-performance LC coupled to tandem quadrupole TOF-MS (UPLC-Q-TOF-MS) with collision energy function was applied to analyze different samples from Imperatae Rhizoma. Then, MarkerLynx(TM) software was employed to screen the excess components in analytes, compared with control samples, and those selected markers were likely to be the metabolites of fungi. Furthermore, each of the accurate masses of the markers obtained from MarkerLynx(TM) was then searched in a mycotoxins/fungal metabolites database established in advance. The molecular formulas with relative mass error between the measured and theoretical mass within 5 ppm were chosen and then applied to MassFragment(TM) analysis for further confirmation of their structures. With the use of this approach, five mycotoxins that have never been reported in HM were identified in contaminated Imperatae Rhizoma. The results demonstrate the potential of UPLC-Q-TOF-MS coupled with the MarkerLynx(TM) software and MassFragment(TM) tool as an efficient and convenient method to screen and identify mycotoxins in herbal materials and aid in the quality control of HM.

Authentication of Senecio scandens and S. vulgaris based on the comprehensive secondary metabolic patterns gained by UPLC–DAD/ESI-MS

A secondary metabolic pattern using ultra-performance liquid chromatography (UPLC)-DAD/ESI-MS was constructed to gain chemical information for authentication of Senecio scandens (SS) and Senecio vulgaris (SV), the two representative species containing hepatotoxic pyrrolizidine alkaloids (HPAs). The metabolic pattern showed three groups of bioactive constituents: phenolic/aromatic acids, flavonoid glycosides and the HPAs. 47 peaks were identified including 19 phenolic/aromatic acids, 10 flavonoid glycosides and 18 PAs by direct comparison with the available reference compounds or deduced from the UV absorption and their ESI-MS fragmentation patterns. The two species could be authenticated diagnostically by their metabolic profiling of the three chromatographic fingerprints. Although both SS and SV contain PAs as the characteristic constituents, only 2 PAs, adonifoline and adonifoline N-oxide were detected in SS, while other 16 PAs were detected in SV, including the highly toxic senecionine, retrorsine, seneciphylline and their corresponding N-oxides. The concentration of PAs in SV is also higher than that in SS. The number and concentration of the phenolic compounds in SS were higher than in SV. Jacaranone derivatives were only detected in SS and jacaranone ethyl ester was detected as the predominant constituent. In the fingerprint of the n-butanol extracts, 10 quercetin and kaempferol glycosides derivatives were detected. 9 were found in SS and only 2 in SV. PAs, jacaranone derivatives and flavonoid glycosides can serve as the metabolic markers to distinguish the Senecio plants from each other, and provide evidence for their clinical application in the consideration of safety and efficacy.

Identification of metabolites of adonifoline, a hepatotoxic pyrrolizidine alkaloid, by liquid chromatography/tandem and high‐resolution mass spectrometry

Hepatotoxic pyrrolizidine alkaloid (HPA)-containing plants have always been a threat to human and livestock health worldwide. Adonifoline, a main HPA in Senecio scandens Buch.-Ham. ex D. Don (Qianli guang), was used officially as an infusion in cases of oral and pharyngeal infections in China. In this study in vivo metabolism of adonifoline was studied for the first time by identifying the metabolites of adonifoline present in bile, urine and feces of rats using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS(n)) (ion trap) as well as liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) (quadrupole-time of flight). In total 19 metabolites were identified and, among them, retronecine-N-oxides were confirmed by matching their fragmentation patterns with their fully characterized synthetic compounds. These metabolites are all involved in both phase I and phase II metabolic processes and the principal in vivo metabolism pathways of adonifoline were proposed.

Radix Paeoniae Rubra and Radix Paeoniae Alba Attenuate CCl4-Induced Acute Liver Injury: An Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) Based Metabolomic Approach for the Pharmacodynamic Study of Traditional Chinese Medicines (TCMs)

Metabolomics has been frequently used in pharmacodynamic studies, especially those on traditional Chinese medicine (TCM). Radix Paeoniae Alba and Radix Paeoniae Rubra are popularly used in TCM, and both have hepatoprotective effects. In this study, a CCl4-induced acute liver injury rat model was established and confirmed by the observed serum aminotransferase activities. The metabolomics approach was applied to study the influence of Radix Paeoniae Alba and Radix Paeoniae Rubra on the metabolic changes in rats with acute liver injury. The partial least-squares-discriminant analysis (PLS-DA) of rat serum and their ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) fingerprints allowed discrimination of controlled, acute liver injury-model rats after administration of the two types of TCMs. The time-dependent PLS-DA plots showed that the changes in the metabolic patterns of the rats, which were administered with the TCMs, had stabilized within 2 h after they received the intraperitoneal CCl4 injection. The results indicated the protective effect of TCMs against liver injury. Several potential biomarkers were detected and identified, which included creatine, deoxycholic acid, choline, 5-methylenetetrahydrofolate, folic acid, and glycocholic acid. The physiological significance of these metabolic changes was discussed.

The comparative pharmacokinetics of two pyrrolizidine alkaloids, senecionine and adonifoline, and their main metabolites in rats after intravenous and oral administration by UPLC/ESIMS

AbstractPyrrolizidine alkaloids (PAs) are considered to be one of the most hepatotoxic groups of compounds of plant origin and are present in about 3% of the world’s flowering plants. Most PAs represent a considerable health hazard to both livestock and humans through the consumption of plants and PA-contaminated products such as milk, honey, herbal teas, and medicines. This study determined the differences in the in vivo pharmacokinetic behavior of senecionine (SEN), adonifoline (ADO), and their main metabolites in rats after intravenous administration and oral administration by ultraperformance liquid chromatography/electrospray ionization mass spectrometry. Upon intravenous administration and oral administration of SEN and ADO, significant differences in pharmacokinetics were observed, with the SEN and ADO being absorbed fast with lower bioavailability and being quickly metabolized to PA N-oxides and hydroxylation products of PAs or their N-oxides. It could be seen that the plasma concentration ratio of senecionine N-oxide (SEN-NO) to SEN (CSEN-NO/CSEN) was significantly larger than that for adonifoline N-oxide (ADO-NO) and ADO (CADO-NO/CADO) (Pu2009<u20090.001) for both dosing routes in rats. The high N-oxygenation activity and extensive toxicity of SEN, compared with ADO, in rats raised the question of whether or not the higher metabolic rate of SEN in rats in vivo was related to its potent toxicity. The toxicity of SEN-NO and ADO-NO needs to be evaluated further and compared in vitro/in vivo. This study was most helpful for interpreting the metabolism of metabolic bioactivation and detoxication, and toxicity differences among SEN, ADO and other PAs.n FigureThe comparative of senecionine and adonifoline metabolized to N-oxide in rats after intravenous and oral administration. (SEN: senecionine, ADO: adonifoline, SEN-NO: senecionine N-oxide, ADO-NO: adonifoline N-oxide)

UPLC-MS based metabolomics study on Senecio scandens and S. vulgaris: an approach for the differentiation of two Senecio herbs with similar morphology but different toxicity

Pyrrolizidine alkaloids show significant hepatotoxicity as they can bind to DNA or proteins after being activated in liver. Senecio vulgaris L., like many Compositae herbs containing pyrrolizidine alkaloids, was reported to have great hepatotoxicity. However, Senicio scandens Buch.-Ham., from the same genus, which was also used as a herb and documented in China Pharmacopoeia published in 2010, hardly showed any side effects or relevant toxicity. In the present study, we conducted the metabolomics study using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to obtain the different metabolic profiles of the two Senecio herbs. In addition, principle component analysis (PCA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA) were introduced for the multivariate analysis, and MS/MS was applied to the identification of target alkaloid markers which contributed most to the established models. As a result, ten pyrrolizidine alkaloids, including adonifoline, senecionine, senecionine N-oxide, retrorsine, retrorsine N-oxide and seneciphylline, were selected and identified. Among them, adonifoline was found to be a specific marker for S. scandens while senecionine and its N-oxidative were characteristic markers for S. vulgaris. Furthermore, the hepatotoxicity studies in vivo and in vitro showed that senecionine had more potent toxicity (LD50, 57.3xa0mg/kg; IC50, 5.41xa0μM) than that of adonifonine (LD50, 163.3xa0mg/kg; IC50, 49.91xa0μM). Taken together, the present study provides not only better understanding of the different toxicity between the two Senecio herbs containing pyrrolizidine alkaloids but also a reference method, which can be applied to other genetically closed species with similar morphology but different toxicity.

Simultaneous determination of senecionine, adonifoline and their metabolites in rat serum by UPLC–ESIMS and its application in pharmacokinetic studies

A rapid, selective and sensitive ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC-ESIMS) method was firstly developed and validated for the simultaneous determination of two hepatotoxic pyrrolizidine alkaloids (HPAs), senecionine (SEN), adonifoline (ADO), and their N-oxides (SENNOX and ADONOX), the main metabolites in rat serum. The whole analysis was achieved within 4.5 min by gradient elution on an ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, i.d. 1.7 microm) following a solid phase extraction for serum samples. Good linearity was achieved using weighted (1/x2) least squares linear regression over a 1600-fold dynamic range for SEN and ADO (LLOQ was about 0.006 microg/ml) and 800-fold dynamic range for SENNOX and ADONOX (LLOQ was about 0.012 microg/ml). The R.S.D. of intra- and inter-day precision was below 4.91% and 11.15% respectively, while the R.E. of accuracy was within 4.52%, 6.81%, 2.69%, and 7.12% for SEN, SENNOX, ADO, and ADONOX, respectively. The developed method was successfully applied to the in vivo pharmacokinetic study in rats after intravenous administration of SEN and ADO.

Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs.