Metabolic profiling of emodin drug-induced liver injury and silybin treatment in rats using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry: A metabolomic and mechanistic approach.

Silybin, an active component in the plant Silybum marianum (L.) Gaertn., is commonly used to protect against liver disease. We investigated silybin's protective potential in rat liver against emodin-induced liver injury 4 weeks. It was found that aspartate aminotransferase and direct bilirubin serum biomarkers for liver toxicity significantly increased, and liver histopathology revealed cholestasis and necrosis in rats administered emodin alone, whereas aspartate aminotransferase and total bile acid levels in rats administered emodin and silybin simultaneously were changed compared to rats administered emodin alone. Liver mRNA and protein levels of Cyp7a1-which plays roles in cholesterol metabolism and bile acid synthesis-and Abcb11 (Bsep)-which facilitates bile salt secretion in hepatocyte canaliculi-were significantly altered with emodin, whereas cotreatment with silybin attenuated emodin's adverse effect. Metabolomic analysis using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry determined eight potential metabolite biomarkers in serum, urine, and liver tissue. Network analysis was conducted to conceptualize the interplay of genes, metabolites, and metabolic pathways for cholesterol metabolism and bile acid synthesis for liver injury. Overall, rats administered only emodin were shown to be a sound model to investigate fat-associated drug-induced hepatoxicity or liver injury and cotreatment of emodin with silybin prevents fatty liver injury. This metabolomic study revealed that emodin-induced fatty liver injury disrupted bile acid synthesis, vitamin B6 , and glycerophospholipid metabolism pathways and that silybin ameliorates liver injury on these compromised pathways.

UPLC-Q-TOF/MS-Based Metabolomics Approach to Reveal the Hepatotoxicity of Emodin and Detoxification of Dihydromyricetin.

Dihydromyricetin (DMY), an important flavanone found in Ampelopsis grossedentata, plays a protective role in liver injury. Our previous research found that DMY protected L02 cells against hepatotoxicity caused by emodin. In this study, serum, urine, and liver samples from rats were systematically used for biochemical analysis, pathological observation, and nontargeted metabolomics to evaluate the toxicity of emodin and DMY intervention. After oral administration of DMY, DMY may alleviate liver injury by improving liver metabolism. Approximately, 8 of 15 metabolites in rat urine and serum were significantly regulated by DMY. Metabolic pathway analysis showed that glutathione metabolism, pyrimidine metabolism, and tryptophan metabolism were the most affected pathways, and 18 proteins were predicted to be potential targets of DMY during the alleviation of liver injury induced by emodin. This research is of great significance in confirming the liver-protective effect of DMY, especially during acute liver injury caused by traditional Chinese medicine.

Identification of the active ingredients from Guangtongxiao decoction in rat bile based on ultra-performance liquid chromatography/Synapt G2 quadrupole time-of-flight tandem mass spectrometry.

OBJECTIVE: To identify the active ingredients and metabolites in rat bile after Guangtongxiao decoction (GTX) had been administered via the rectal route. METHODS: Drug-containing bile samples were collected via a catheter in the bile duct and could be used 5 h after rectal administration. The main active components and their metabolites in rat bile following rectal administration of GTX were identified and analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. RESULTS: Positive and negative modes were applied to analyze and identify the chemical ingredients in the bioactive fractions of GTX. Eight peaks were identified by comparison with the standard compounds: berberine hydrochloride, dehydrocorydaline, tetrahydropalmatine, corydaline, magnoflorine, magnolol, obacunone and albiflorin. Furthermore, 60 metabolites were detected in rat bile based on mass-fragmentation behaviors, and 21 metabolites were reported for the first time. CONCLUSION: Our findings provide a solid basis for further pharmacologic and pharmacokinetic studies of GTX.

Identification and characterization of the structure-activity relationships involved in UGT1A1 inhibition by anthraquinone and dianthrone constituents of Polygonum multiflorum.

The adverse effects of Polygonum (P.) multiflorum, including abnormal bilirubin metabolism, are a serious public health issue. As uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) is the only enzyme responsible for bilirubin metabolism, we investigated the inhibitory effect of a P. multiflorum extract and 10 anthraquinone and dianthrone compounds on UGT1A1 in rat liver microsomes in vitro. The P. multiflorum extract exhibited the strongest inhibitory effect on UGT1A1 activity (inhibition constant [Ki] = 0.3257 µM, 1422 µg of material/mL), followed by cis-emodin dianthrones (Ki = 0.8630 µM), trans-emodin dianthrones (Ki = 1.083 µM), emodin-8-O-glc (Ki = 3.425 µM), and polygonumnolide C2 (Ki = 4.291 µM). Analysis of the structure-activity relationships of these compounds suggested that the spatial orientation of the molecules and the presence of particular functional groups affect UGT1A1 inhibition. A mechanistic analysis showed that all the tested compounds docked into two of the nine active sites of UGT1A1 and suggested that hydrophobic interactions and hydrogen bonds are important for the affinity of the tested compounds for UGT1A1; moreover, their interaction energies were generally in agreement with the Ki values. These findings provide insight into adverse reactions to P. multiflorum and identify the pharmacophores involved in inhibition of UGT1A1.

Mechanism of Chinese Medicine Herbs Effects on Chronic Heart Failure Based on Metabolic Profiling.

Chronic heart failure (CHF) is a major public health problem in huge population worldwide. The detailed understanding of CHF mechanism is still limited. Zheng (syndrome) is the criterion of diagnosis and therapeutic in Traditional Chinese Medicine (TCM). Syndrome prediction may be a better approach for understanding of CHF mechanism basis and its treatment. The authors studied disturbed metabolic biomarkers to construct a predicting mode to assess the diagnostic value of different syndrome of CHF and explore the Chinese herbal medicine (CHM) efficacy on CHF patients. A cohort of 110 patients from 11 independent centers was studied and all patients were divided into 3 groups according to TCM syndrome differentiation: group of Qi deficiency syndrome, group of Qi deficiency and Blood stasis syndrome, and group of Qi deficiency and Blood stasis and Water retention syndrome. Plasma metabolomic profiles were determined by UPLC-TOF/MS and analyzed by multivariate statistics. About 6 representative metabolites were highly possible to be associated with CHF, 4, 7, and 5 metabolites with Qi deficiency syndrome, Qi deficiency and Blood stasis syndrome, and Qi deficiency and Blood stasis and Water retention syndrome (VIP > 1, p < 0.05). The diagnostic model was further constructed based on the metabolites to diagnose other CHF patients with satisfying sensitivity and specificity (sensitivity and specificity are 97.1 and 80.6% for CHF group vs. NH group; 97.1 and 80.0% for QD group vs. NH group; 97.1 and 79.5% for QB group vs. NH group; 97.1 and 88.9% for QBW group vs. NH group), validating the robustness of plasma metabolic profiling to diagnostic strategy. By comparison of the metabolic profiles, 9 biomarkers, 2-arachidonoylglycerophosphocholine, LysoPE 16:0, PS 21:0, LysoPE 20:4, LysoPE 18:0, linoleic acid, LysoPE 18:2, 4-hydroxybenzenesulfonic acid, and LysoPE 22:6, may be especially for the effect of CHM granules. A predicting model was attempted to construct and predict patient based on the related symptoms of CHF and the potential biomarkers regulated by CHM were explored. This trial was registered with NCT01939236 (https://clinicaltrials.gov/).

[Pharmacokinetics of 8-O-acetylharpagide and harpagide after oral administration of Ajuga decumbens extract in beagle dog].

8-O-acetylharpagide and harpagide are two kinds of effective component of Ajuga decumbens extract. A sensitive LC-MS/MS method has been established for pharmacokinetics of 8-O-acetylharpagide and harpagide in beagle dog after oral administration of from A. decumbens extract. Female beagle dogs received orally 12.9, 25.7 mg x kg(-1) p. o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartment analysis. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), which was run at a flow rate of 0.3 mL x min(-1). Chromatographic separation was achieved on an Agilent ZORBAX XDB-C18 column (2.1 mm x 50 mm, 3.5 microm) using a gradient elution of 5% B at 0-2 min, 95% B at 2. 1-5 min and 5% B at 5. 1-10 min. All analytes, including the IS, were monitored under positive ionization conditions and quantified in MRM mode with transitions of m/z 429.2-369.2 for 8-O-acetylharpagide, m/z 387.2-207.2 for harpagide, and m/z 149.2-103.1 for IS. High purity nitrogen was employed as both the nebulizing and drying gas. Other parameters of the mass spectrometer were optimized as follows: drying gas flow 10.0 L x min(-1); drying gas temperature 300 degrees C; capillary voltage 4 000 V. Results showed that 8-O-acetylharpagide and harpagide showed a dose-dependence profile. T(max) of 8-O-acetylharpagide is 1.7 h, and T(max) of harpagide is 1.57 h, which was higher than T(max) of 8-O-acetylharpagide and harpagide after oral administration of from A. decumbens extract in rats. The different pharmacokinetic parameters may be due to the species differences of rat and beagle dog.

Pharmacokinetics of 8-O-acetylharpagide and harpagide after oral administration of Ajuga decumbens Thunb extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga decumbens Thunb is a medicinal plant native to China popularly used to treat chronic pelvic inflammation and hysteromyoma. Its main bioactive components are iridoid glycosides, such as 8-O-acetylharpagide and harpagide that had presented antibacterial, anti-inflammatory, and antiviral activities. AIM OF THE STUDY: To establish a sensitive LC-MS/MS method and compare the pharmacokinetics of 8-O-acetylharpagide and harpagide in rats after oral administration of their pure forms and from compounds obtained from Ajuga decumbens extract. MATERIALS AND METHODS: Rats received orally 15 mg/kg (equivalent of 6 mg/kg 8-O-acetylharpagide and 1.5mg/kg harpagide), 30 mg/kg and 60 mg/kg of Ajuga decumbens Thunb extract and were compared to animals that received 12 mg/kg of 8-O-acetylharpagide or 3mg/kg of harpagide p.o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartmental analysis. RESULTS: Results showed that the iridoid glycosides were quickly absorbed by oral route and showed a dose-dependence profile. Pharmacokinetic parameters of both glycosides were essentially the same except Tmax when dosed as the extract or pure forms. CONCLUSION: 8-O-acetylharpagide was metabolized to harpagide, which affected the pharmacokinetic profiles of harpagide when dosed as the extract. This pharmacokinetic study seems to be useful for a further clinical study of Ajuga decumbens Thunb extract.

Mechanisms of inhibiting proliferation of vascular smooth muscle cells by serum of rats treated with Dahuang Zhechong pill.

UNLABELLED: Dahuang Zhechong pill (DHZCP), a famous and classical Chinese herbal prescription, consists of twelve traditional Chinese drugs: Eupolyphaga sinensis Walker., Rheum officinale Baill., Scutellaria baicalensis Georgi., Glycyrrhiza uralensis Fisch., Prunus persica Batsch., Prunus armeniaca L., Paeonia lactiflora Pall., Rehmannia glutinosa Libosch., Toxicodendron vernicifluum F.A. Barkl., Tabanus bivittatus Mats., Hirudo nipponica Whitman. and Holotrichia diomphalia Bates., and is clinically used to treat hepatic diseases, gynecopathy and atherosclerosis in China. Our previous studies confirm that DHZCP is able to significantly inhibit proliferation of vascular smooth muscle cells (VSMCs) in vivo and in vitro. AIM OF THE STUDY: To investigate the mechanisms of inhibition of VSMCs proliferation by DHZCP with the method of Serum Pharmacology. MATERIALS AND METHODS: VSMCs proliferation of rat was assayed by measuring the cell viability with the MTT method, and platelet-derived growth factor (PDGF) expression in VSMCs was examined by the immunocytochemical method. Cycle and apoptosis of VSMCs were evaluated with flow cytometry. RESULTS: The serum of DHZCP-treated rats not only inhibited endothelin-1 (ET-1) stimulated cell proliferation and PDGF expression in VSMCs, but also promoted apoptosis of the proliferated VSMCs. Meanwhile, the serum of rats containing DHZCP interfered with the cycle of PDGF-stimulated VSMCs, increasing proportion of the cells in G(0)/G(1) phases and decreasing proportion of the cells in S and G(2)/M phases. CONCLUSION: These suggest that the inhibitory effect of DHZCP on VSMCs proliferation is partially attributed to depressing PDGF expression in VSMCs, retarding the cell cycle and to promoting apoptosis of VSMCs.

[Metabolic kinetic of puerarin in beagle liver microsomal by HPLC-ESI-MS].

OBJECTIVE: To develop a HPLC-ESI-MS method for the determination of puerarin and its metabolite and study the metabolic kinetics in beagle dog liver microsomes. METHOD: Beagle dog liver microsomes were prepared by using ultracentrifugation method. Chromatography was performed on a Shimadzu C18 column (2.0 mm x 150 mm, 5 microm). Amethanol-water gradient system was used. ESI interface was applied in the positive, and SIM m/z 417 was puerarin and m/z 531 was daidzein. RESULT: The puerarin was metabolized by NADPH regenerating system in beagle dog microsomes. The Michaelis-Menten parameters Km and Vmax in beagle dog microsomes were initially estimated by analyzing Lineweave-Brurk plot. The Vmax Km of puerarin were (0.047 +/- 0.006) mg x min(-1) x g(-1), (1.22 +/- 0.53) mg x L(-1). CONCLUSION: The puerarin and daidzein can be rapidly determined by HPLC-MS in beagle dog microsomes and the puerarin was metabolized to daidzein by CY P450. The study can give help for Baige capsule.

In vitro inhibition of proliferation of vascular smooth muscle cells by serum of rats treated with Dahuang Zhechong pill.

Dahuang Zhechong pill (DHZCP) is a famous and classical Chinese herbal prescription, which is clinically used to treat hepatic, gynecological and cardiovascular diseases in China. The aim of this study was to observe the effects of the serum of rats treated with DHZCP on the proliferation of cultured rat vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF), oxidized low density lipoprotein (ox-LDL) and hyperlipidemic serum (HLS), and on DNA, protein and collagen syntheses of VSMCs induced by PDGF in vitro. VSMCs proliferation was assayed by measuring the cell viability with MTT method, and syntheses of DNA, protein and collagen were evaluated by detecting [(3)H]-thymidine, [(3)H]-leucine and [(3)H]-proline incorporations, respectively. The results showed that PDGF, ox-LDL and HLS stimulated the proliferation of rat VSMCs in vitro. The serum of rats treated with DHZCP significantly inhibited the proliferation of rat VSMCs induced by the above stimulants and the incorporations of [(3)H]-thymidine, [(3)H]-leucine and [(3)H]-proline into rat VSMCs induced by PDGF in comparison with the model control group (P<0.01). The data suggest that DHZCP is able to obviously inhibit VSMCs proliferation via interfering with syntheses of DNA and protein, and to decrease production of extracellular matrix by VSMCs through antagonizing collagen synthesis.