Serum metabolomics strategy for investigating the hepatotoxicity induced by different exposure times and doses of Gynura segetum (Lour.) Merr. in rats based on GC-MS.

Gynura segetum (Lour.) Merr. (GS), has been widely used in Chinese folk medicine and can promote circulation, relieve pain and remove stasis. In recent years, the hepatotoxicity caused by GS has been reported, however its mechanism is not fully elucidated. Metabolomic techniques are powerful means to explore the toxicological mechanism and therapeutic effects of traditional Chinese medicine. The purpose of this study was to establish a serum metabolomics method based on Gas Chromatography-Mass Spectrometry (GC-MS) to explore the hepatotoxicity mechanism of different exposure times and doses of GS in rats. Sprague Dawley (SD) rats were administered daily with distilled water, 7.5 g kg-1 GS, or 15 g kg-1 GS by intragastrical gavage for either 10 or 21 days. The methods adopted included enzyme-linked immunosorbent assay (ELISA), Hematoxylin and Eosin (H&E) staining and GC-MS-based serum metabolomics. Serum biochemistry analysis showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total bilirubin (TBIL) and total bile acid (TBA) significantly (P < 0.05) increased while the levels of albumin (ALB) and high-density lipoprotein (HDL) significantly (P < 0.05) decreased in GS-treated groups, compared with the control group. Interestingly, the ALT, AST, TG and ALB levels changed in a time- and dose-dependent manner. The results of H&E staining showed the degree of liver damage after administration of GS gradually deepened with the extension of administration time and the increase of the dose. According to the results of metabolomics analysis, 26 differential metabolites were identified, which were involved in 8 metabolic pathways including phenylalanine metabolism, glyoxylic acid and dicarboxylic acid metabolism and so on. Meanwhile, the number of differential metabolites in different GS-treated groups was associated with GS exposure time and dose. Therefore, we concluded that GS might induce hepatotoxicity depending on the exposure time and dose.

Determination and tissue distribution comparisons of five xanthones after orally administering crude and processed gamboge.

Caged polyprenylated xanthones are the main active ingredients isolated from the resin of Garcinia hanburyi, which has been reported to exhibit potential anticancer and anti-inflammatory activities. This study aimed to develop sensitive and specific ultra-performance liquid chromatography coupled with the triple quadrupole mass spectrometry method for investigating the tissue distribution of five xanthones in rats: ß-morellic acid, isogambogenic acid, gambogenic acid, R-gambogic acid and S-gambogic acid. All tissue samples were prepared using the liquid-liquid extraction method and separated on a C8 column with a gradient system. Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction monitoring using positive ionization. The method established in this assay was successfully applied to the tissue distribution study of the five selected xanthones after orally administering crude and processed gamboge in rat tissues. The results indicated that these five xanthones were distributed to rat tissues rapidly and could be detected in all of the selected tissues after oral administration. After processing, the contents of R-gambogic acid and S-gambogic acid in the gastrointestinal tract were significantly reduced. The findings of this study might be helpful in further understanding the processing mechanism of gamboge and providing references for its reasonable clinical application.

[Simultaneous determination of 11 neurotransmitters in brain microdialysis samples from rats by UPLC-MS/MS].

This study established a method for simultaneous determination of 11 neurotransmitters, such as acetylcholine, glutamic acid, glycine, and norepinephrine from rat brain microdialysis samples using UPLC-MS/MS. A total of 20 µL of rat brain dialysate was diluted with 60 µL of acetonitrile-water(4∶1) and centrifuged for 10 min at 10 000 r·min~(-1),and 5 µL was injected into UPLC-MS/MS system for assay. Chromatographic separation was performed on a Waters ACQUITY BEH amide column(2.1 mm×100 mm, 1.7 µm) with gradient elution using acetonitrile/0.2% formic acid-water as mobile phases with a flow rate of 0.35 mL·min~(-1) and column temperature of 35 ℃. The eluate was detected by multiple-reaction monitoring(MRM) scanning with an electrospray ionization source operating in the positive ionization mode with an analysis duration of 3.5 min. The relationship between the recovery rate of 11 neurotransmitters and the perfusion rate or the concentration of neurotransmitters was investigated. Furthermore, the effects of puerarin alone or combined with borneol on the content of 11 neurotransmitters in the striatum of rats were investigated. The results showed the calibration curves displayed good linear regression with coefficients all greater than 0.99 and the lower limit of quantification(LLOQ) less than 12.5 nmol·L~(-1) for each analyte. The within-run and between-run precision(RSD) of the 11 neurotransmitters at low, medium, and high levels was less than 9.3%, and the relative error of the accuracy ranged from-8.4% to 9.5%. The stability, recovery, and matrix effects were in line with the biological sample analysis requirements. As revealed by experimental results, the levels of most neurotransmitters in the brain striatum changed significantly after rats were treated with puerarin as compared with the conditions in the blank group. Except for dopamine and norepinephrine, the degree of changes of other neurotransmitters in the combination group(borneol and puerarin) was less than that of the puerarin group. The established UPLC-MS/MS method could be applied to the quantitative determination of 11 neurotransmitters in microdialysis samples, providing an efficient and useful tool to study neurotransmitter changes in animal models of health and diseases.

[Analysis of metabolites of 4,5-dicaffeoylquinic acid in rat plasma and urine based on LC-MS].

Ultra high performance liquid chromatography tandem high field orbital trap mass spectrometry(UPLC-Orbitrap Elite-MS/MS) method was applied in this paper to analyze the metabolites of 4,5-dicaffeoylquinic acid in rat plasma and urine after oral administration. A gradient elution was performed by using Thermo C_(18) column(2.1 mm×100 mm, 1.9 µm), with 0.1% formic acid solution-acetonitrile as the mobile phase. Mass spectral data of biological samples were collected in negative ion mode. The data were extracted by Compound Discovery 2.1 software. Then the blank group samples and the drug samples were compared for exact molecular weight and the mass fragmentation information, and the secondary fragment fitting ratio was calculated to finally attribute the metabolites. As a result, 15 metabolites were detected in rat plasma, and 16 metabolites were detected in urine. The involving metabolic reactions included methylation, hydration, dehydration, reduction, glucuronide conjugation, and sulfation reaction. The metabolites in plasma and urine complemented each other and initially revealed the migration and excretion patterns of this compound in the body. A method for pre-processing biological samples, high-resolution LC-MS instrumentation data, and qualitative software was established in this study to identify metabolite structures, laying the foundation for the study of the active ingredients and in vivo pharmacodynamics forms of Chinese medicines.

Protective effect of 7,3',4'-flavon-3-ol (fisetin) on acetaminophen-induced hepatotoxicity in vitro and in vivo.

BACKGROUND: Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in clinic. Fisetin (FST) is a phenolic compound that has been isolated from many natural products. PURPOSE: Our aim is to study the protection effect and mechanisms of FST on APAP-induced hepatotoxicity in endogenous metabolism and metabolomics in vitro and in vivo. METHODS: FST was i.g. administered to mice at 10, 20 and 40 mg/kg for 7 days and a single dose of APAP (400 mg/kg) was given on the last day. Serum and tissue were collected for biochemical analysis. L-02 cells were used to assess cell viability. LC-MS was used to study the metabolic fingerprinting in vivo and vitro. PCA and OPLS-DA were used to search the potential biomarkers (VIP > 1, p < 0.05). The pathway analysis was conducted on Metaboanalyst 4.0. Then liver oxidative stress indices and glutathione markers were examined using PCR and kits. RESULTS: ALT, AST, liver histological observation and cell viability results showed that FST could reverse APAP induced toxicology in mice and L-02 cells. In metabolomics study, 26 metabolites in vitro and 60 metabolites in vivo were identified by searching in the library and most of them decreased to normal level in FST treatment. It is observed in pathway analysis that the most significant pathway was glutathione metabolism. Furthermore, the results of mRNA and immunofluorescence showed that FST suppressed ROS formation in liver tissue and L-02 cells, as well as restored the expression of GPX1, GST and other antioxidative enzymes genes. CONCLUSION: Our results indicate that FST prevented APAP-induced hepatotoxicity by regulating glutathione metabolism and the expression of related antioxidative signals.

Qualitative and quantitative determination of Atractylodes rhizome using ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry with data-dependent processing.

A quick and effective workflow based on ultra-performance liquid chromatography coupled with electron spray ionization and LTQ-Orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) was established for compositional analysis and screening of the characteristic compounds of three species of Atractylodes rhizome for quality evaluation. This technique was employed to determine the seven main components in Atractylodes rhizome samples. Ultimately, 78 constituents were identified; of these, seven characteristic compounds were selected for species discrimination, comprising atractylodin (63), atractylenolide I (43), atractylenolide II (49), atractylenolide III (53), atractylon (69), methyl-atractylenolide II (54) and (4E,6E,12E)-tetradecadecatriene-8,10-diyne-1,3-diacetate (59). The seven main compounds, including six characteristic compounds, were simultaneously determined in 29 batches of Atractylodes rhizome samples. Thus, the method validation showed acceptable results. Quantitative analysis showed significantly different contents of the seven main components among the three species of Atractylodes rhizome, which indicates possible distinctions in the pharmacological effects. This established method can simultaneously provide qualitative and quantitative results for compositional characterization of Atractylodes rhizomes and for quality control.

Naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption.

Pharmacological activities of some natural products diminish and even disappear after purification. In this study, we explored the mechanisms underlying the decrease of acute oral toxicity of Coptidis Rhizoma extract after purification. The water solubility, in vitro absorption, and plasma exposure of berberine (the major active compound) in the Coptidis Rhizoma extract were much better than those of pure berberine. Scanning electron microscopy, laser scanning confocal microscopy (LSCM), and dynamic light scattering experiments confirmed that nanoparticles attached to very fine precipitates existed in the aqueous extract solution. The LSCM experiment showed that the precipitates were absorbed with the particles by the mouse intestine. High-speed centrifugation of the extract could not remove the nanoparticles and did not influence plasma exposure or acute oral toxicity. However, after extract dilution, the attached precipitates vanished, although the nanoparticles were preserved, and there were no differences in the acute oral toxicity and plasma exposure between the extract and pure berberine. The nanoparticles were then purified and identified as proteinaceous. Furthermore, they could absorb co-dissolved berberine. Our results indicate that naturally occurring proteinaceous nanoparticles in Coptidis Rhizoma extract act as concentration-dependent carriers that facilitate berberine absorption. These findings should inspire related studies in other natural products.

Identification and pharmacokinetics of multiple constituents in rat plasma after oral administration of Yinchenzhufu decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Yinchenzhufu decoction (YCZFD) is a classical Chinese herbal formula and has been used to treat severe jaundice in chronic liver injuries since the Qing Dynasty (18th century CE). To identify the components absorbed into the blood in YCZFD and explore their pharmacokinetic profile for understanding the effective ingredients of YCZFD. MATERIALS AND METHODS: After rats were given YCZFD by intragastric administration, the plasma was processed by precipitation of protein. The compounds in YCZFD extract and the plasma were identified by using high-resolution mass spectrometry with a database-directed strategy. The pharmacokinetics of multiple compounds from YCZFD in rat plasma was studied by using the established UPLC-MS/MS method. RESULTS: Forty compounds in YCZFD extract and 21 prototype compounds with 11 metabolites in rat plasma were detected after oral administration. The pharmacokinetic parameters of glycyrrhizic acid, glycyrrhetic acid, cinnamic acid, ononin, atractylenolide III, and liquiritin from YCZFD were obtained in rats. CONCLUSIONS: The identified constituents and the pharmacokinetic features of YCZFD are helpful for understanding the material bases of its therapeutic effects.