The clinical population pharmacokinetics, metabolomics and therapeutic analysis of alkaloids from Alstonia scholaris leaves in acute bronchitis patients.

BACKGROUND: Capsule of alkaloids from leaf of Alstonia scholaris (CALAS) is a new investigational botanical drug (No. 2011L01436) for respiratory disease. Clinical population pharmacokinetics (PK), metabolomics and therapeutic data are essential to guide dosing in patients. Previous research has demonstrated the potential therapeutic effect of CALAS on acute bronchitis. Further clinical trial data are needed to verify its clinical efficacy, pharmacokinetics behavior, and influence of dosage and other factors. PURPOSE: To verify the clinical efficacy and explore the potential biomarkers related to CALAS treatment for acute bronchitis. MATERIALS AND METHODS: Oral CALAS was assessed in a randomized, double-blind, placebo-controlled trial. Fifty-five eligible patients were randomly assigned to four cohorts to receive 20, 40 or 80 mg, of CALAS three times daily for seven days, or placebo. Each CALAS cohort included 15 subjects, and the placebo group included 10 subjects. A population PK model of CALAS was developed using plasma with four major alkaloid components. Metabolomics analysis was performed to identify biomarkers correlated with the therapeutic effect of CALAS, and efficacy and safety were assessed based on clinical symptoms and adverse events. RESULTS: The symptoms of acute bronchitis were alleviated by CALAS treatment without serious adverse events or clinically significant changes in vital signs, electrocardiography or upper abdominal Doppler ultrasonography. Moreover, one compartment model with first-order absorption showed that an increase in aspartate transaminase will reduce the clearance (CL) of scholaricine, and picrinine CL was inversely proportional to body mass index, while 19-epischolaricine and vallesamine CL increased with aging. The serum samples from acute bronchitis patients at different time points were analyzed using UPLC-QTOF in combination with the orthogonal projection to latent structures-discriminant analysis, which indicated higher levels of lysophosphatidylcholines, lysophosphatidylethanolamines and amino acids with CALAS treatment than with placebo. CONCLUSION: This is the first study to evaluate the clinical efficacy and explored the potential biomarkers related to CALAS therapeutic mechanism of acute bronchitis by means of clinical trial combined the metabolomics study. This exploratory study provides a basis for further research on clinical efficacy and optimal dosing regimens based on pharmacokinetics behavior. Additional acute bronchitis patients and CALAS PK samples collected in future studies may be used to improve model performance and maximize its clinical value.

Discovery of quality markers in Rubus Chingii Hu using UPLC-ESI-QTOF-MS.

Raspberry, the fruit of Rubus Chingii Hu, has been used as a traditional Chinese medicine (TCM) to nourish kidney and strengthen Yang-qi. In order to determine the quality of raspberry, the quality markers (Q-markers) of raspberry that can improve renal function were investigated using UPLC-ESI-QTOF-MS in this study. The results of serum pharmacochemistry indicated that six components rutin, ellagic acid, kaempferol-3-rutinoside, astragalin, tiliroside, and goshonoside F5 in raspberry were absorbed into rat blood. The HEK293 cells treated with cisplatin were used to evaluate the kidney-protecting activity of these absorbed components. All these components could markedly inhibit cell damage induced by cisplatin and restore the levels of malondialdehyde (MDA) and catalase (CAT) in the cells, suggesting that these components may be the Q-markers of raspberry. More importantly, except for ellagic acid, other five Q-markers in raspberries from Dexing of Jiangxi province were higher than those from most of other areas. It is well known that Dexing raspberry is the Dao-di herbs raspberry used in the clinic of Chinese Medicine, demonstrating that these components could be used as Q-markers of raspberry. This study provides a reliable and valuable method for quality evaluation of raspberry.

A strategy combining solid-phase extraction, multiple mass defect filtering and molecular networking for rapid structural classification and annotation of natural products: characterization of chemical diversity in Citrus aurantium as a case study.

Medicinal plants are complex chemical systems containing thousands of secondary metabolites. The rapid classification and characterization of the components in medicinal plants using mass spectrometry (MS) remains an immense challenge. Herein, a novel strategy is presented for MS through the combination of solid-phase extraction (SPE), multiple mass defect filtering (MMDF) and molecular networking (MN). This strategy enables efficient classification and annotation of natural products. When combined with SPE and MMDF, the improved analytical method of MN can perform the rapid annotation of diverse natural products in Citrus aurantium according to the tandem mass spectrometry (MS/MS) fragments. In MN, MS2LDA can be initially applied to recognize substructures of natural products, according to the common fragmentation patterns and neutral losses in multiple MS/MS spectra. MolNetEnhancer was adopted here to obtain chemical classifications provided by ClassyFire. The results suggest that the integrated SPE-MMDF-MN method was capable of rapidly annotating a greater number of natural products from Citrus aurantium than the classical MN strategy alone. Moreover, SPE and MMDF enhanced the effectiveness of MN for annotating, classifying and distinguishing different types of natural products. Our workflow provides the foundation for the automated, high-throughput structural classification and annotation of secondary metabolites with various chemical structures. The developed approach can be widely applied in the analysis of constituents in natural products.

[UPLC-Q-TOF-MS-based metabolomics study of celastrol].

The mass spectrometry-based metabolomics method was used to systematically investigate the formation of celastrol metabolites,and the effect of celastrol on endogenous metabolites. The mice plasma,urine and feces samples were collected after oral administration of celastrol. Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS) was applied to analyze the exogenous metabolites of celastrol and its altered endogenous metabolites. Mass defect filtering was adopted to screen for the exogenous metabolites of celastrol. Multivariate statistical analysis was used to identify the endogenous metabolites affected by celastrol. Celastrol and its eight metabolites were detected in urine and feces of mice,and 5 metabolites of them were reported for the first time. The hydroxylated metabolites were observed in the metabolism of both human liver microsomes and mouse liver microsomes. Further recombinant enzyme experiments revealed CYP3 A4 was the major metabolic enzyme involved in the formation of hydroxylated metabolites. Urinary metabolomics revealed that celastrol can affect the excretion of intestinal bacteria-related endogenous metabolites,including hippuric acid,phenylacetylglycine,5-hydroxyindoleacetic acid,urocanic acid,cinnamoylglycine,phenylproplonylglycine and xanthurenic acid. These results are helpful to elucidate the metabolism and disposition of celastrol in vivo,and its mechanism of action.

Role of Metabolic Activation in Elemicin-Induced Cellular Toxicity.

Elemicin, an alkenylbenzene constituent of natural oils of several plant species, is widely distributed in food, dietary supplements, and medicinal plants. 1'-Hydroxylation is known to cause metabolic activation of alkenylbenzenes leading to their potential toxicity. The aim of this study was to explore the relationship between elemicin metabolism and its toxicity through comparing the metabolic maps between elemicin and 1'-hydroxyelemicin. Elemicin was transformed into a reactive metabolite of 1'-hydroxyelemicin, which was subsequently conjugated with cysteine (Cys) and N-acetylcysteine (NAC). Administration of NAC could significantly ameliorate the elemicin- and 1'-hydroxyelemicin-induced cytotoxicity of HepG2 cells, while depletion of Cys with diethyl maleate (DEM) increased cytotoxicity. Recombinant human CYP screening and CYP inhibition experiments revealed that multiple CYPs, notably CYP1A1, CYP1A2, and CYP3A4, were responsible for the metabolic activation of elemicin. This study revealed that metabolic activation plays a critical role in elemicin cytotoxicity.

Metabolic Activation of Myristicin and Its Role in Cellular Toxicity.

Myristicin is widely distributed in spices and medicinal plants. The aim of this study was to explore the role of metabolic activation of myristicin in its potential toxicity through a metabolomic approach. The myristicin- N-acetylcysteine adduct was identified by comparing the metabolic maps of myristicin and 1'-hydroxymyristicin. The supplement of N-acetylcysteine could protect against the cytotoxicity of myristicin and 1'-hydroxymyristicin in primary mouse hepatocytes. When the depletion of intracellular N-acetylcysteine was pretreated with diethyl maleate in hepatocytes, the cytotoxicity induced by myristicin and 1'-hydroxymyristicin was deteriorated. It suggested that the N-acetylcysteine adduct resulting from myristicin bioactivation was closely associated with myristicin toxicity. Screening of human recombinant cytochrome P450s (CYPs) and treatment with CYP inhibitors revealed that CYP1A1 was mainly involved in the formation of 1'-hydroxymyristicin. Collectively, this study provided a global view of myristicin metabolism and identified the N-acetylcysteine adduct resulting from myristicin bioactivation, which could be used for understanding the mechanism of myristicin toxicity.

Optimization of extraction and analytical protocol for mass spectrometry-based metabolomics analysis of hepatotoxicity.

Drug-induced liver injury is a clinically leading side-effect of drugs. In the present study, a liquid chromatography mass spectrometry-based metabolomics protocol was optimized for extraction and analysis of endogenous metabolites from liver tissue during hepatotoxicity. Various extraction solutions, resuspension solutions, extraction folds and dissolution methods for the supernatant were compared using the number of extracted total ions, relative response and relative extraction efficiency of targeted metabolites from liver tissue. The polar and nonpolar endogenous metabolites associated with liver injury were analyzed by hydrophilic interaction chromatography and reversed-phase liquid chromatography with UPLC-QTOFMS. The results indicated that extraction with 10-fold 50% acetonitrile in water and the supernatant diluted (1:1) with 100% acetonitrile rather than resuspension was the optimal extraction protocol. Subsequently, the optimized method was able to examine the change in metabolites in mouse liver tissue resulting from treatment with a toxic natural product, toosendanin. Taken together, the optimized extraction and analytical protocol provides high reliability and reproducibility for polar and nonpolar metabolites in liver tissue and may be suitable for metabolomics analysis of liver injury induced by drugs or chemicals.

PPARα Mediates the Hepatoprotective Effects of Nutmeg.

Nutmeg is a Traditional Chinese Medicine used to treat gastrointestinal diseases. Some reports have indicated that nutmeg has hepatoprotective activity. In this study, a thioacetamide (TAA)-induced acute liver injury model in mice was used to explore the mechanism of the protective effects of nutmeg extract (NME), including its major bioactive component myrislignan. The results indicated that NME could effectively protect TAA-induced liver damage as assessed by recovery of increased serumtransaminases, decrease in hepatic oxidative stress, and lower hepatic inflammation. Metabolomics analysis further revealed that treatment with NME led to the recovery of a series of lipids including lysophosphatidylcholines that were decreased and a lowering of acylcarnitines that were increased in mouse plasma and liver after TAA exposure. Gene expression analysis demonstrated that the hepatoprotective effect of NME was achieved by modulation of the peroxisome proliferator-activated receptor alpha (PPARα) as well as the decrease in oxidative stress. NME could not protect from TAA-induced liver injury in Ppara-null mice, suggesting that its protective effect was dependent on PPARα. Myrislignan, a representative neolignan in nutmeg, showed potent protective activity against TAA-induced liver toxicity. These data demonstrate that nutmeg alleviates TAA-induced liver injury through the modulation of PPARα and that the lignan compounds in nutmeg such as myrislignan partly contributed to this action.