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Protostane-type triterpenoids are antifibrotic nature components with unique structures in Alismatis Rhizoma. However, the underlying mechanisms of them against liver fibrosis are not well illustrated. The present study aims to study the targets and mechanisms of Alismatis Rhizoma triterpenes responsible for their antifibrotic effects by network pharmacology, molecular docking, and luciferase assay. As a result, six molecular targets responsible for the antifibrotic effects of alisols against liver fibrosis were uncovered by network pharmacology, among which the activation of farnesoid X receptor (FXR/NR1H4) was highlighted and further confirmed by molecular docking and luciferase assay. Our present study provides a scientific basis for treating liver fibrosis by using Alismatis Rhizoma, especially via the FXR activation effects of alisols.

Gynura japonica (also named Tusanqi in Chinese) is used as a folk herbal medicine for treating blood stasis or traumatic injury. However, hundreds of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of preparations made from G. japonica because it contains large amounts of hepatotoxic pyrrolizidine alkaloids (PAs). To date, blood pyrrole-protein adducts (PPAs) are suggested as biomarkers for the diagnosis of PA-induced HSOS in clinics. However, the concentration of PPAs in the blood is greatly affected by several factors including the amount of PA exposure, herb intake period, and blood sampling time after the last exposure. In present study, the kinetic characters of PPAs in serum and liver as well as other potential target organs were studied systematically and comprehensively following multiple exposures of PAs in G. japonica extract (GJE). As results, PPAs content reached to a plateau both in serum and liver after the mice were treated with GJE for 2 weeks on daily basis. PPAs cleared significantly slower in liver (T1/2ke∼184.6 h, ∼7.7 days) than in serum (T1/2ke∼95.8 h, ∼4.0 days). Although more than 90 % PPAs were removed 2 weeks after the last dosing, PPAs still persisted in the liver until the end of the experiment, i.e. 8 weeks after the last dosing. The results would be of great help for understanding the importance of PPAs for PA-induced toxicity and its detoxification.

Pyrrolizidine alkaloids (PAs) are among the most hepatotoxic natural compounds that are widely distributed throughout the world. Most PAs are metabolically activated to trigger toxicity. Exposure to herbal medicine containing PAs and food supplements contaminated by PAs is considered to be one of the two main causes of hepatic sinusoidal obstruction syndrome (HSOS), which is a rare hepatic vascular disease with a high mortality rate. PAs-induced HSOS cases have been reported worldwide. However, there is no clinically effective therapy for PAs-induced HSOS, which is partially because the toxic mechanism is not fully understood. This review focuses on updating the information on the metabolism and the molecular mechanisms of PAs hepatotoxicity, including oxidative stress, apoptosis, and dysfunction of bile acid metabolism, and their interactions. Keywords: Pyrrolizidine alkaloids (PAs), Hepatic sinusoidal obstruction syndrome (HSOS), Metabolism, Toxicity mechanism, Oxidative stress, Apoptosis, Dysfunction of bile acid metabolism

Hepatic sinusoidal obstruction disease (HSOS) is a rare but life-threatening vascular liver disease. However, its underlying mechanism and molecular changes in HSOS are largely unknown, thus greatly hindering the development of its effective treatment. Hepatic sinusoidal endothelial cells (HSECs) are the primary and essential target for HSOS. A tandem mass tag-based shotgun proteomics study was performed using primary cultured HSECs from mice with HSOS induced by senecionine, a representative toxic pyrrolizidine alkaloid (PA). Dynamic changes in proteome were found at the initial period of damage and the essential role of thrombospondin 1 (TSP1) was highlighted in PA-induced HSOS. TSP1 over-expression was further confirmed in human HSECs and liver samples from patients with PA-induced HSOS. LSKL peptide, a known TSP1 inhibitor, protected mice from senecionine-induced HSOS. In addition, TSP1 was found to be covalently modified by dehydropyrrolizidine alkaloids in human HSECs and mouse livers upon senecionine treatment, thus to form the pyrrole-protein adduct. These findings provide useful information on early changes in HSECs upon PA treatment and uncover TSP1 overexpression as a contributor in PA-induced HSOS. Graphical abstract

Background The roots of Gynura japonica are used as traditional medicine for treating blood stasis or traumatic injury even though hundreds of hepatic sinusoidal obstruction syndrome cases have been reported after consumption of the roots, which contain large amounts of hepatotoxic pyrrolizidine alkaloids (HPAs). However, no information is available about the toxic compounds in the aerial parts of G. japonica, which are also used as herbal medicines and even vegetables in several areas. Thus, the toxic chemicals in the aerial parts of G. japonica, i.e., HPAs, must be urgently identified. Purpose In this study, we aimed to 1) identify the toxic compounds in different medicinal parts and 2) examine the hepatotoxicity of G. japonica. Study design A total of 35 batches of the roots and aerial parts of G. japonica were collected from different sources and analyzed for HPAs. The hepatotoxicity of different extracts (i.e., total extracts [TE] and total alkaloids [TA]) and a single compound (i.e., senecionine) was evaluated on mice. Methods Qualitative analysis of HPAs was performed using an ultra-performance liquid chromatography (UPLC)-mass spectrometry (MS)-parent ion scan approach, whereas a quantitative assay was performed by a UPLC-MS-selected ion monitoring approach. Male C57BL mice were orally administered the different extracts or the single compound at dosages equivalent to 50 mg HPAs/kg body weight. The sera and the livers were collected at 48 h after treatment and used to evaluate the hepatotoxicity through serum clinical biomarkers assay, liver histology, and bile acid profiling. Results A total of 21 HPAs were identified in the roots and the aerial parts. The roots contained higher levels of HPAs (4.90 mg/g) than did the aerial parts (2.21 mg/g). TE and TA induced similar acute liver injuries, but senecionine was considerably more toxic than these extracts. Mice treated with TE showed significantly impaired bile acid homeostasis in the sera and the livers. Conclusion The roots and aerial parts of G. japonica contained large amounts of HPAs, including senecionine, which were responsible for the hepatotoxicity of G. japonica. Bile acid homeostasis was uniquely impaired after exposure to the plant. Therefore, neither the roots nor the aerial parts of G. japonica should be consumed as medicines or vegetables.

Tumor necrosis factor-α (TNF-α) has different effects on apoptosis depending on activation or inactivation of the nuclear factor-κB (NF-κB) and epidermal growth factor receptor (EGFR) signaling pathways. Helichrysetin, a natural chalcone, inhibits NF-κB nuclear translocation in mouse pancreatic β cells. The present study aimed to identify the effect of helichrysetin on activation of the NF-κB and EGFR signaling pathways induced by TNF-α, and the synergistic effect of helichrysetin and TNF-α on apoptosis of HeLa and T98G cells. Cell proliferation was measured by Cell Counting Kit-8 assay, while apoptosis was measured by Hoechst 33258 and Annexin V/PI staining. NF-κB activity was detected by luciferase assay, protein expression was measured by western blotting and mRNA expression was detected by quantitative PCR assay. The results revealed that in HeLa and T98G cells helichrysetin blocked the increased phosphorylation of NF-κB p65 induced by TNF-α. Although helichrysetin alone decreased cell viability, helichrysetin and TNF-α synergistically decreased cell viability. Helichrysetin, not TNF-α, promoted apoptosis, while the combination of helichrysetin and TNF-α synergistically increased apoptosis. In addition, helichrysetin and TNF-α synergistically enhanced the activation of caspase-3 and poly-(ADP-ribose)-polymerase compared with helichrysetin alone. Helichrysetin inhibited the phosphorylation of transforming growth factor-β activated kinase (TAK1), IκB kinase-α/β (IKK-α/β), NF-κB p65 and EGFR induced by TNF-α. Consistent with the inhibition of NF-κB activation, the increased TNF-α-induced mRNA expression levels of TNF-α, IL-1β, CCL2, CCL5 and CXCL10 were significantly downregulated by helichrysetin. Therefore, helichrysetin and TNF-α synergistically promoted apoptosis by inhibiting TAK1/IKK/NF-κB and TAK1/EGFR signaling pathways in HeLa and T98G cells, indicating a potential therapeutic strategy for cancer.

Background and Aims: The Gynura japonica-induced hepatic sinusoidal obstruction syndrome (HSOS) is closely related to pyrrolizidine alkaloids (PAs), and its prevalence has been increasing worldwide in recent years. However, no effective therapy for PA-induced HSOS in clinics is available, partially due to the failure of quick diagnosis. This study aims to identify blood microRNA (miRNA) signatures as potential biomarkers for PA-induced HSOS in clinics. Methods: The microarray-based miRNA profiling was performed on blood samples of the discovery cohort, which consisted of nine patients with HSOS and nine healthy donors. Differentially expressed miRNAs were further confirmed using a validation cohort, which consisted of 20 independent patients with HSOS. In addition, the rat model was established through the oral administration of the total alkaloid extract from G. japonica to investigate the association of miRNA biomarkers with the progression of HSOS. Bioinformatic analyses, including GO and KEGG enrichment, receiver operating characteristics curve, and correlation analyses were conducted to evaluate the accuracy of the potential miRNA biomarkers. Results: Three miRNAs, namely miR-148a-3p, miR-362-5p, and miR-194-5p, were overexpressed in patients and rats with PA-induced HSOS. These miRNAs were positively related to the severity of liver injury and displayed considerable diagnostic accuracy for patients with HSOS with areas under the curve over 0.87. Conclusion: In summary, this study demonstrated that three miRNAs, hsa-miR-148a-3p, hsa-miR-362-5p, and hsa-miR-194-5p, might serve as potential biomarkers for PA-induced HSOS in clinics.

Pyrrolizidine alkaloids (PAs) are natural toxins found in about 3%-5% of flowering plants. Dehydropyrrolizidine alkaloids contain a double bond in 1, 2-position of the necine bases, including retronecine type PAs (RET-PAs) and their N-oxides (RET N-oxide-PAs), and otonecine type PAs (OTO-PAs), and are known for their significant hepatotoxicity. Most dehydropyrrolizidine alkaloids are metabolically activated by cytochrome P450 (CYP450) enzymes to generate active pyrroles, which further bind to proteins to form pyrrole-protein adducts (PPAs). Methods for predicting PA-induced liver injury are generally performed on in vitro models with extremely low activities of CYP450 enzymes, which is different from the situation in vivo. In this regard, primary cultured mouse hepatocytes, which showed comparable CYP450 activity with the in vivo models, were applied to illustrate the structure-toxicity relationship of 13 dehydropyrrolizidine alkaloids, namely, eight RET-PAs, three RET N-oxide-PAs, and two OTO-PAs. PA-induced cytotoxicity and PA-generated PPAs were analyzed in primary mouse hepatocytes treated with different PAs. Results showed that PA-induced toxicity was correlated with the amount of PA-generated PPAs. RET-PAs and OTO-PAs were generally more toxic than RET N-oxide-PAs and generated higher amount of PPAs. PPAs were utilized to evaluate the efficiency of metabolic activation and predict the toxic potencies of dehydropyrrolizidine alkaloids. The proposed model could be a new approach for toxicity evaluation and risk control of exposure to PAs.

Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with severe liver damage if excessive ingestion. Herein, a novel analytical strategy on utilizing direct analysis in real-time mass spectrometry (DART-MS) was developed, and applied in analysis of six representative PAs. The calibration curves in the range of 10–1000 ng·mL−1 were established, and relative standard deviations (RSDs) were less than 10%. The limits of detection (LODs) and limits of quantitation (LOQs) were 0.55–0.85 ng·mL−1 and 1.83–2.82 ng·mL−1, respectively. The feasibility of method was indicated by analysing real samples including Gynura japonica, drug tablets, granules, and fresh cow’s milk. Moreover, the results of DART-MS were in good agreement with those observed by high performance liquid chromatography mass spectrometry (HPLC-MS), but consumed less time without chromatographic separation. This research provides a facile fashion for safety assessment of herbal and food products containing PAs and presents promising applications in food, pharmaceutical and clinical analysis.

Herbal drug-induced liver injury has been reported worldwide and gained global attention. Thousands of hepatic sinusoidal obstruction syndrome (HSOS) cases have been reported after consumption of herbal medicines and preparations containing pyrrolizidine alkaloids (PAs), which are natural phytotoxins globally distributed. And herbal medicines, such as Gynura japonica, are the current leading cause of PA-induced HSOS. The present study aimed to reveal the mechanism underlying the hepatotoxicity of seneciphylline (Seph), a main PA in G. japonica. Results showed that Seph induced severe liver injury through apoptosis in mice (70 mg/kg Seph, orally) and primary mouse and human hepatocytes (5-50 μM Seph). Further research uncovered that Seph induced apoptosis by disrupting mitochondrial homeostasis, inducing mitochondrial depolarization, mitochondrial membrane potential (MMP) loss, and cytochrome c (Cyt c) release and activating c-Jun N-terminal kinase (JNK). The Seph-induced apoptosis in hepatocytes could be alleviated by Mdivi-1 (50 μM, a dynamin-related protein 1 inhibitor), as well as SP600125 (25 μM, a specific JNK inhibitor) and ZVAD-fmk (50 μM, a general caspase inhibitor). Moreover, the Seph-induced MMP loss in hepatocytes was also rescued by Mdivi-1. In conclusion, Seph induced liver toxicity via activating mitochondrial-mediated apoptosis in mice and primary hepatocytes. Our results provide further information on Seph detoxification and herbal medicines containing Seph such as G. japonica.

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., Graphical abstract fx1, Highlights • Senecionine induces apoptosis in primary mouse and human hepatocytes as well as in mouse livers. • Senecionine induces mitochondrial Drp1 translocation, mitochondrial fragmentation, loss of mitochondrial membrane potential and release of mitochondrial cytochrome c in hepatocytes. • Pharmacological inhibition or genetic deletion of Drp1 protects against senecionine-induced hepatotoxicity. • Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by Pyrrolizidine alkaloids.

Accumulation of bile acids (BAs) contributes significantly to the pathogenesis of primary biliary cholangitis (PBC). Here, we sought to systematically characterize the serum and fecal BA profiles and the linkage between BAs and gut microbiota in PBC. The serum and fecal BAs were compared between 65 UDCA treatment-naive PBC and 109 healthy controls using UPLC-MS in cross-sectional study. In a prospective study, a subgroup of patients was enrolled for BA and microbiota analysis before and after UDCA therapy. BA compositions in serum and feces significantly differed between treatment-naive PBC and controls. Particularly, PBC was associated with decreased conversions of conjugated to unconjugated, and primary to secondary BAs, indicating impaired microbial metabolism of BAs. PBC patients at advanced stage exhibited a more abnormal BA profile compared with early-stage patients. UDCA treatment led to a decreased level of taurine-conjugated BAs, thereby reversing the conjugated/unconjugated ratio in PBC. Moreover, the level of secondary BAs such as DCA and conjugated DCA inversely correlated with PBC-enriched gut microbes (e.g., Veillonella, Klebsiella), while positively correlated with control-enriched microbes (e.g., Faecalibacterium, Oscillospira). Microbiota analysis also revealed a significant increase of taurine-metabolizing bacteria Bilophila spp. in patients after UDCA, which was strongly correlated with decreased taurine-conjugated BAs. In addition, serum FGF19 was remarkably increased in treatment-naive PBC and decreased after UDCA. Our study established specific alterations of BA compositions in serum and feces of PBC, suggesting the potential for using BAs for diagnosis, and highlighting the possibility of modulating BA profile by altering gut microbiota. Graphical Abstract.

Plantaginis semen, the dried mature seed of Plantago asiatica L. or Plantago deprdssa Willd., has a prominent effect on the treatment of obesity, type 2 diabetes and lipid disorders, however, its clinical application is limited due to inadequate in-depth mechanism exploration and incomplete discussion of action targets of its in vivo. Therefore, an untargeted metabolomics approach was firstly applied to study the serum metabolic differences in mice. Metabolomics analysis was performed using ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) together with multivariate statistical data analysis. The results showed that Plantaginis semen can mainly improve blood lipids, some degree in blood glucose and insulin levels in high-fat mice, in addition, the phenotype of liver and fat stained sections demonstrated remarkable results. A total of 22 metabolites involved in arachidonic acid, glycerophospholipid, glycosphingolipid, linoleate, Omega-3 fatty acid, phosphatidylinositol phosphate and tyrosine metabolisms were identified. In further, it was found that the possible mechanisms of Plantaginis semen on hyperlipidemic mice lied in the biosynthesis of thyroxine, biological effects of enzymes of phospholipase A2 activity, glucosylceramide synthase and inositol essential enzyme 1α, genes expressions of fatty acid metabolism and inflammation. Serum metabolomics revealed that Plantaginis semen could cure the organism disease via regulating multiple metabolic pathways which will be helpful for understanding the mechanism of this herb and providing references for better applications of it in clinic, even researches on other TCMs.

A three-period digoxin-bupropion drug-drug interaction study was performed in cynomolgus monkeys to assess the effect of bupropion and its metabolites on digoxin disposition. Monkeys were administered either an i.v. infusion (0.1 mg/kg) or an oral dose of digoxin (0.2 mg/kg) as control. In single-dosing period, monkeys received an i.v. infusion of bupropion at 1.5 mg/kg together with an infusion or oral dosing of digoxin, respectively. During multiple-dosing period, bupropion was orally administered q.d. at 7.72 mg/kg for 12-day. Then it was co-administered with an i.v. infusion or oral dosing of digoxin, respectively. Renal expression of OATP4C1 and P-gp was examined. Bupropion significantly increased i.v. digoxin CLrenal0-48h by 1 fold in single-dosing period. But it had no effect on the systemic disposition of digoxin. In multiple-dosing period, bupropion significantly increased oral digoxin CLrenal0-48h, CLtotal0-48h, CLnon-renal0-48h and decreased its plasma exposure. Bupropion and its metabolites did not alter creatinine clearance. OATP4C1 was located at the basolateral membrane of proximal tubule cells, while P-gp was on the apical membrane. The effect of multiple dosing with bupropion on the pharmacokinetics of digoxin is more pronounced. The magnitude of increase in digoxin CLrenal0-48h contributed to the decrease in AUC of digoxin in some extent, but certainly is not the major driving force. The lack of systemic exposure after a single dose but a significant decrease in exposure mediated by an increase in the digoxin CLnon-renal0-48h with repeated dosing is likely to be the more clinically relevant.

Background and Aims Hepatic sinusoidal obstruction syndrome (HSOS) is caused by toxic injury to sinusoidal endothelial cells in the liver. The intake of pyrrolizidine alkaloids (PAs) in some Chinese herbal remedies/plants remains the major etiology for HSOS in China. Recently, new diagnostic criteria for PA-induced HSOS (i.e. PA-HSOS) have been developed; however, the efficacy has not been clinically validated. This study aimed to assess the performance of the Nanjing criteria for PA-HSOS. Methods Data obtained from consecutive patients in multiple hospitals, which included 86 PA-HSOS patients and 327 patients with other liver diseases, were retrospectively analyzed. Then, the diagnostic performance of the Nanjing criteria and simplified Nanjing criteria were evaluated and validated. The study is registered in www.chictr.org.cn (ID: ChiCTR1900020784). Results The Nanjing criteria have a sensitivity and specificity of 95.35% and 100%, respectively, while the simplified Nanjing criteria have a sensitivity and specificity of 96.51% and 96.33%, respectively, for the diagnosis of PA-HSOS. Notably, a proportion of patients with Budd-Chiari syndrome (11/49) was misdiagnosed as PA-HSOS on the basis of the simplified Nanjing criteria, and this was mainly due to the overlapping features in the enhanced computed tomography/magnetic resonance imaging examinations. Furthermore, most of these patients (10/11) had occlusion or thrombosis of the hepatic vein, and communicating vessels in the liver were found in 8/11 patients, which were absent in PA-HSOS patients. Conclusions The Nanjing criteria and simplified Nanjing criteria exhibit excellent performance in diagnosing PA-HSOS. Thus, both could be valuable diagnostic tools in clinical practice.

Recently, hepatic sinusoidal obstruction syndrome (HSOS) caused by herbal preparations containing pyrrolizidine alkaloids (PAs), such as Gynura Rhizoma (Tusanqi), has gained global attention. However, the lack of a reliable and reproducible animal model has greatly hampered mechanistic studies. Therefore, we aimed to establish a reproducible HSOS mouse model and investigate the hepatotoxic mechanism. The model was established by intragastrical administration of Gynura Rhizoma extract, i.e., 1.0 g extract/kg per day (equal to 16.7 g crude drug/kg per day based on extraction rate and 49.1 mg PA/kg per day based on the total PA content in the extract determined) for 40 successive days. Then, the mice were sacrificed, and their blood samples and livers were collected for analyses. Using hematoxylin–eosin (HE) and Masson staining, scanning electron microscopy imaging, clinical biomarkers, and other assays, we showed that the HSOS was successfully induced in our mouse model. Furthermore, we detected the key factors involved in liver fibrosis in the mice, revealing significantly increased hydroxyproline concentration; elevated expression of α-smooth muscle actin (α-SMA) and fibrosis-related genes such as Collagen-1, Collagen-3, Mmp2, Mmp13, Timp1, Timp3, and Activin, upregulated Smad3 phosphorylation, and increased serum TGF-β levels. Moreover, pro-inflammatory cytokines, including Tnf-α, Il-1β, and Il-6, were also increased in the model. All these results demonstrate the key roles of the TGF-β-Smad3 and inflammatory signaling pathways in this Gynura Rhizoma-induced HSOS mouse model, suggesting that blockade of fibrosis and/or inflammation should be an effective treatment for HSOS.

Picrasma quassioides (D. Don) Benn. is a widely used traditional Chinese medicine for anti-inflammation and antibiosis. Canthinone and β-carboline alkaloids are the main characteristic constituents that possess diverse pharmacological effects, such as anti-inflammatory and anti-infectious properties. In this study, bioautography in thin-layer chromatography indicated that the antiradical activity compound may be alkaloids. Then, a simple, fast, and efficient method was established for the separation and purification of two types of alkaloids from P. quassioides by mass-spectrometry-directed autopurification system. Eight alkaloids were isolated and purified in this one-step methodology. Among them, five compounds (3, 95.1%, 58.8 mg; 4, 98.4%, 71.7 mg; 6, 97.8%, 365.4 mg; 7, 97.7%, 172.7 mg; 8, 98.2%, 180.3 mg) were obtained in large amounts with extremely high purities. Then, the antiradical activities of the isolates showed that 4-methoxy-5-hydroxycanthin-6-one (6) exhibited obvious 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging activity with an IC50 value of 84.037 μM. This study offers a new method for the preparation of targeted bioactive alkaloids in P. quassioides. This work also provides a reference for the separation of other targeted chemical components with potential activities from traditional Chinese herbal medicines.

To investigate the metabolic biomarkers of predicting the transition from pre-diabetes (pre-DM) to normal glucose regulation (NGR) and diabetes (DM) in a longitudinal cohort study. 108 participants with pre-DM were followed up for ten years and divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 regressed to NGR, and 68 remained at pre-DM. Alterations in plasma metabolites in these groups were evaluated by untargeted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Twenty three metabolites related to glycerophospholipid metabolism, oxidation and antioxidation were associated with the process from pre-DM to NGR, while twenty two metabolites related to amino acid metabolism, glycerophospholipid metabolism and mitochondrial β-oxidation played important roles in the progression to DM. Results from stepwise logistic regression analysis showed that five biomarkers (20-Hydroxy-leukotriene E4, Lysopc(20:4), 5-methoxytryptamine, Endomorphin-1, Lysopc(20:3)) were good prediction for the restoration to NGR, and five biomarkers (Iso-valeraldehyde, linoleic acid, Lysopc(18:1), 2-Pyrroloylglycine, Dityrosine) for the development of DM. The findings suggest that the combination of these potential metabolites may be used for the prognosis of pre-DM. Targeting the pathways that involved in these prognostic biomarkers would be beneficial for the regression to NGR and the early prevention of DM among pre-DM.

To investigate the metabolic biomarkers of predicting the transition from pre-diabetes (pre-DM) to normal glucose regulation (NGR) and diabetes (DM) in a longitudinal cohort study. 108 participants with pre-DM were followed up for ten years and divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 regressed to NGR, and 68 remained at pre-DM. Alterations in plasma metabolites in these groups were evaluated by untargeted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Twenty three metabolites related to glycerophospholipid metabolism, oxidation and antioxidation were associated with the process from pre-DM to NGR, while twenty two metabolites related to amino acid metabolism, glycerophospholipid metabolism and mitochondrial β-oxidation played important roles in the progression to DM. Results from stepwise logistic regression analysis showed that five biomarkers (20-Hydroxy-leukotriene E4, Lysopc(20:4), 5-methoxytryptamine, Endomorphin-1, Lysopc(20:3)) were good prediction for the restoration to NGR, and five biomarkers (Iso-valeraldehyde, linoleic acid, Lysopc(18:1), 2-Pyrroloylglycine, Dityrosine) for the development of DM. The findings suggest that the combination of these potential metabolites may be used for the prognosis of pre-DM. Targeting the pathways that involved in these prognostic biomarkers would be beneficial for the regression to NGR and the early prevention of DM among pre-DM.

The toxicity assessment of herbal medicines is important for human health and appropriate utilization of these medicines. However, challenges have to be overcome because of the complexity of coexisting multiple components in herbal medicines and the highly interconnected organismal system. In this study, a target profiling approach was established by combining the characteristic fingerprint analysis of herbal chemicals with potential toxicity through a precursor ion scan-based mass spectroscopy and the target profiling analysis of biomarkers responsible for the toxicity. Through this newly developed approach, the comparative hepatotoxicity assessment of two herbal medicines from the same genus, Senecio vulgaris L. and Senecio scandens Buch.-Ham, was performed. Significant differences were found between the two species in their chemical markers (i.e., pyrrolizidine alkaloids) and biomarkers (i.e., bile acids) responsible for their toxicities. This result was consistent with the conventional toxicity assessment conducted by histopathological examination and clinical serum index assay on experimental animal models. In conclusion, this study provided a new approach for the hepatotoxicity assessment of herbal medicines containing pyrrolizidine alkaloids, which are widely distributed in various herbal medicines. The target profiling approach may shed light on the toxicity assessment of other herbal medicines with potential toxicity.

Different members of Lagochilus genus have been used in folkloric medicine to treat hemorrhages and inflammation. However, only a few species of them have received scientific attention supporting their efficacy. Here, the hemostatic and antiinflammatory activities of five Lagochilus species were determined and compared by using in vivo assays. The results showed that the extracts of Lagochilus lanatonodus and Lagochilus diacanthophyllus showed better hemostatic activities among five species. The high doses of L. lanatonodus extracts were able to shorten the values of thrombin time, activated partial thromboplastin time and prothrombin time in a rat model. Moreover, the extracts of L. lanatonodus and L. diacanthophyllus showed strong inhibitory effects on the acute phase of inflammation in both xylene-induced ear edema mouse model and carrageenan-induced paw edema rat model. In parallel, the treatment of these extracts modulated the expressions of those inflammatory parameters, that is, nitric oxide, prostaglandin E2 , inducible nitric oxide synthase, malondialdehyde and superoxide dismutase. L. lanatonodus and L. diacanthophyllus showed better hemostatic and antiinflammatory activities in several test models: these results therefore supported the folkloric utilization. L. lanatonodus was found to be the most active Lagochilus species.

The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement worldwide. To study the role of jujube in brain benefits, the expression of neurotrophic factors and anti-oxidant enzymes in the jujube-treated cultured astrocytes was determined. Application of a chemical standardized water extract of jujube in cultured astrocytes for 24 h stimulated the expressions of nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in a concentration-dependent manner. The pre-treatment with H89, a protein kinase A inhibitor, attenuated the jujube-induced expression of neurotrophic factors. In parallel, the treatment of jujube water extract induced the transcriptional expressions of the enzymes responsible for anti-oxidation, i.e. NAD(P)H: quinine oxidoreductase 1, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit and glutathione S-transferase, in a concentration-dependent manner. These results proposed the benefits of jujube in regulating expressions of neurotrophic factors and anti-oxidant enzymes in cultured astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

Pyrrolizidine alkaloids are highly hepatotoxic natural chemicals that produce irreversible chronic and acute hepatotoxic effects on human beings. Purification of large amounts of pyrrolizidine alkaloids is necessary for toxicity studies. In this study, an efficient method for targeted analysis and purification of pyrrolizidine alkaloid cis/trans isomers from herbal materials was developed for the first time. Targeted analysis of the hepatotoxic pyrrolizidine alkaloids was performed by liquid chromatography with tandem mass spectrometry (precursor ion scan and daughter ion scan), and the purification of pyrrolizidine alkaloids was achieved with a mass-directed auto purification system. The extraction and preparative liquid chromatography conditions were optimized. The developed method was applied to analysis of Gynura japonica (Thunb.) Juel., a herbal medicine traditionally used for detumescence and relieving pain but is potentially hepatotoxic as it contains pyrrolizidine alkaloids. Twelve pyrrolizidine alkaloids (six cis/trans isomer pairs) were identified with reference compounds or characterized by liquid chromatography with tandem mass spectrometry, and five individual pyrrolizidine alkaloids, including (E)-seneciphylline, seneciphylline, integerrimine, senecionine, and seneciphyllinine, were prepared from G. japonica roots with high efficiency. The results of this work provide a new technique for the preparation of large amounts of pyrrolizidine alkaloid reference substances, which will also benefit toxicological studies of pyrrolizidine alkaloids and treatments for pyrrolizidine alkaloid-induced toxicity.

Pyrrolizidine alkaloids (PAs) are among the most hepatotoxic natural products that produce irreversible injury to humans via the consumption of herbal medicine and honey, and through tea preparation. Toxicity and death caused by PA exposure have been reported worldwide. Metabolomics and genomics provide scientific and systematic views of a living organism and have become powerful techniques for toxicology research. In this study, senecionine hepatotoxicity on rats was determined via a combination of metabolomic and genomic analyses. From the global analysis generated from two omics data, the compromised bile acid homeostasis in vivo was innovatively demonstrated and confirmed. Serum profiling of bile acids was altered with significantly elevated conjugated bile acids after senecionine exposure, which was in accordance with toxicity. Similarly, the hepatic mRNA levels of several key genes associated with bile acid metabolism were significantly changed. This process included cholesterol 7-α hydroxylase, bile acid CoA-amino acid N-acetyltransferase, sodium taurocholate cotransporting polypeptide, organic anion-transporting polypeptides, and multidrug-resistance-associated protein 3. In conclusion, a cross-omics study provides a comprehensive analysis method for studying the toxicity caused by senecionine, which is a hepatotoxic PA. Moreover, the change in bile acid metabolism and the respective transporters may provide a new PA toxicity mechanism.

The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed "MS-NMR-MS" method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.

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.

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.

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 CCl(4)-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 CCl(4) 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.

To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms. Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR. RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression. Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.目的：研究钙蛋白酶(calpain)在低氧诱导肺动脉高压肺血管重构中的作用及机制。方法：SD大鼠随机分为低氧模型组和常氧对照组。插管法测定大鼠右心室收缩压及平均肺动脉压，右心室/(左心室+室间隔)比值评价右心肥厚指数，HE染色检测血管重构情况，分别采用实时荧光定量PCR和Western印迹检测肺动脉calpain-1，-2和-4 mRNA和蛋白的表达。原代培养的大鼠肺动脉平滑肌细胞分为4组：常氧对照组、常氧对照+ MDL28170(calpain抑制剂)组、低氧模型组、低氧模型+MDL28170组。MTS及流式细胞术观察细胞增殖情况，实时荧光定量PCR检测Ki-67及增殖细胞核抗原(proliferating cell nuclear antigen，PCNA) mRNA表达情况。结果：与常氧对照组大鼠比较，低氧模型组大鼠右心室收缩压、平均肺动脉压及右心肥厚指数显著增加，肺动脉血管显著重构；同时，低氧模型组大鼠肺动脉中calpain-1，-2，-4 mRNA和蛋白表达也显著上调。与常氧对照组细胞比较，低氧模型组细胞显著增殖，MDL28170可显著抑制低氧诱导的肺动脉平滑肌细胞增殖，同时逆转低氧诱导的Ki-67和PCNA mRNA表达上调。结论：calpain介导低氧诱导的肺动脉高压肺血管重构，其机制可能与介导低氧诱导的肺动脉平滑肌细胞增殖有关。.

XiaoyanLidan tablets (XYLDTs) are traditional Chinese medicines frequently used for syndromes of the liver and gallbladder, cholecystitis and cholangitis. To evaluate the consistency of the quality of commercial XYLDT preparations, we established a simple and reliable ultra-performance liquid chromatography (UPLC) method with a photodiode array (PDA) detector and mass spectrometry (MS), including a fingerprint analysis and quantification of the main pharmacologically-active markers. In the UPLC-PDA detection-based fingerprint analysis of XYLDTs, approximately 39 peaks were found in the XYLDT chromatogram, 26 of which were attributed to Picrasmaquassioides, nine to Andrographis and four to Isodonserra. Subsequently, the structures of these bioactive markers were identified through ESI-MS analyses. Using the chemometricmethods of similarity analysis and principal component analysis, the five significant herbal componentswere determined as 4-methoxy-5-hydroxycanthin-6-one, andrographolide, dehydroandrographolide, neoandrographolide and rosmarinic acid, and these components were qualitatively assessed. Our experimental results demonstrated that combining the fingerprint analysis with UPLC-MS and multi-ingredient determination is useful for rapid pharmaceutical quality evaluation. Moreover, the combined approach can potentially differentiate the origin, determine the authenticity and assess the overall quality of the formulae.

Phenylethanoid glycosides are a group of phenolic compounds with diverse biological activities such as hypotensive, diuretic, and hypoglycemic effects. In this study, a target profiling analysis approach using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry (MS) was established on the basis of parent ion scanning for m/z 161, the characteristic product ion for phenylethanoid glycosides. It was successfully employed to discriminate the chemical composition of phenylethanoid glycosides between Plantaginis Herba and Plantaginis Semen, two medicinal parts of Plantago plants, which are widely used as herbal medicine in China. Totally, 34 phenylethanoid glycosides were characterized and tentatively identified by their retention times, MS, and tandem quadrupole MS (MS/MS) data. Combined with chemometrics analysis of principal component analysis and orthogonal projection to latent structural discriminate analysis, eight of them, especially acteoside and plantamajoside, were picked out and contributed to the chemical distinction between Plantaginis Herba and Plantaginis Semen, which might be responsible for the differences in diuretic and hypotensive effects between the two medicinal parts. This new approach for target profiling provides not only a novel idea for specific analysis of active chemical constituents in the same type, but also a promising and reference method for quality evaluation of traditional Chinese medicines.

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, 57.3 mg/kg; IC50, 5.41 μM) than that of adonifonine (LD50, 163.3 mg/kg; IC50, 49.91 μM). 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.

Pyrrolizidine 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 (C SEN-NO/C SEN) was significantly larger than that for adonifoline N-oxide (ADO-NO) and ADO (C ADO-NO/C ADO) (P

Pyrrolizidine alkaloids (PAs) possess significant hepatotoxicity to humans and animals after metabolic activation by liver P450 enzymes. Metabolism pathways of PAs have been studied for several decades, including metabolic activation, hydroxylation, N-oxidation, and hydrolysis. However, the glucuronidation of intact PAs has not been investigated, although glucuronidation plays an important role in the elimination and detoxication of xenobiotics. In this study, PAs glucuronidation was investigated, and three important points were found. First, we demonstrated that senecionine (SEN)-a representative hepatotoxic PA-could be conjugated by glucuronic acid via an N-glucuronidation reaction catalyzed by uridine diphosphate glucuronosyl transferase in human liver microsomes. Second, glucuronidation of SEN was catalyzed not only by human but also other animal species and showed significant species differences. Rabbits, cattle, sheep, pigs, and humans showed the significantly higher glucuronidation activity than mice, rats, dogs, and guinea pigs on SEN. Kinetics of SEN glucuronidation in humans, pigs, and rabbits followed the one-site binding model of the Michaelis-Menten equation, while cattle and sheep followed the two-sites binding model of the Michaelis-Menten equation. Third, besides SEN, other hepatotoxic PAs including monocrotaline, adonifoline, and isoline also underwent N-glucuronidation in humans and several animal species such as rabbits, cattle, sheep, and pigs.

Senecionine (SEN) is a representative of the hepatotoxic pyrrolizidine alkaloids. Although phase I metabolism for cytochrome P450-mediated metabolic activation of SEN was investigated extensively, phase II metabolism for glucuronidation of this compound has not been investigated until now. In our present study, one unique glucuronidation product of SEN in human liver microsomes (HLMs) was identified as SEN N-glucuronide using an authentically synthesized product for which the structure was identified via (1)H and (13)C NMR analysis. Subsequently, kinetics indicated that SEN N-glucuronidation followed the typical Michaelis-Menten model and only one major isozyme participated in it. Finally, this isozyme was demonstrated to be UDP-glucuronosyltransferase (UGT) 1A4, with the direct evidence that recombinant UGT1A4 exhibited predominant and exclusive activity on SEN N-glucuronidation. This result was confirmed by other experiments including chemical inhibition by selective inhibitors and a correlation study between activities of SEN N-glucuronidation and various UGT isozymes. The exclusive role of UGT1A4 on SEN N-glucuronidation was strengthened additionally by its inhibitory kinetic study in which the selective inhibitor of UGT1A4 showed a similar inhibition pattern and K(i) values in both HLM and recombinant UGT1A4 systems. Because UGT2B10 activity failed to correlate with SEN N-glucuronidation in HLMs from 10 individuals, it was impossible for UGT2B10 to play an important role in this metabolism.

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.

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-MSn) (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.

A pre-column derivatization high-performance liquid chromatography method with diode array detection was developed and validated to determine the total retronecine esters-type hepatotoxic pyrrolizidine alkaloids (RET-HPAs) in herbs. The RET-HPAs reacted with o-chloranil in methanolic solution heated for 3 h, and an oxidative derivative was produced that could be detected at a maximal absorption of 223 nm. The analysis was performed using a C18 column with an isocratic elution of methanol and aqueous 0.01% triethylamine (adjusted to pH 4 with formic acid), and the detection was carried out with DAD at 223 nm. The validation of the method included linearity, sensitivity, recovery and stability. It showed a good linear regression (r(2) > 0.9900) in the range of 2.5-250 microM with a limit of detection (S/N = 3) of 0.5 microM. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.6%. The obtained recoveries for both of the extraction and derivatization process were between 94.6 and 100.7% (n = 3).