Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice / 南方医科大学学报

OBJECTIVE@#To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis.@*METHODS@#The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice.@*RESULTS@#We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs.@*CONCLUSION@#The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.

Analysis of chemical constituents of Ginseng-Douchi compound fermentation products based on UPLC-Q-TOF-MS / 中国中药杂志

In this experiment, ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to analyze and identify chemical constituents of Ginseng-Douchi(GD) compound fermentation, and explore the conversion rules of ginsenosides and soybean isoflavones after compound fermentation. Waters Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) was adopted, with 0.1% formic acid aqueous solution(A)-0.1% formic acid acetonitrile solution(B) as mobile phase for gradient elution; electrospray ion source(ESI) was used to collect data in positive and negative ion modes; according to the exact mass number, the secondary spectrum comparison of the database and the existing literature reports, Peakview 2.0/masterview 1.0 software was used to determine the common ion structure formula. Finally, a total of 133 chemical constituents were analyzed and identified from the GD. Ginseng saponins and isoflavone glycosides were significantly converted after fermentation. Among them, peak areas of prototype ginsenosides Rk_3, Rh_1, Rh_2, Rh_3, daidzin, glycitin and genistin decreased significantly; whereas peak areas of se-condary ginsenoside Rb_1, Rb_2, Rk_1, glycitein, genistein and daidzein increased significantly. In this experiment, liquid-mass spectrometry technique was used to investigate the conversion of active ingredients of GD compound fermented products after co-fermentation, so as to provide a scientific basis for elucidating pharmacodynamics material basis and quality control.

Identification and characterization of major constituents in Juglans mandshurica using ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) / 中国天然药物

The present study was designed to identify and characterize the major constituents in Juglans mandshurica Maxim. A simple, efficient and sensitive ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) method was established and validated under positive and negative ion modes. The separation was performed on a Waters ACQUITY UPLC BEH C column (2.1 mm × 100 mm, 1.7 μm) by gradient elution with a mobile phase (Phase A: 0.1% aqueous formic acid solution, Phase B: 0.1% formic acid acetonitrile solution). A total of 165 compounds were rapidly selected by Targeted and Non-Targeted Peak Finding approaches, and then tentatively identifled by comparing with reference substances or inferred through mass spectrometry fragment ion analysis and literature data. These compounds included 68 naphthalenequinones, 20 diarylheptanoids, 29 flavonoids, 20 triterpenes, and 28 phenolic acids. In conclusion, the present study provided an effective approach to identifying components in complex matrices of herbal medicines such as Juglans mandshurica Maxim.

Identification and characterization of major constituents in Juglans mandshurica using ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) / 中国天然药物

The present study was designed to identify and characterize the major constituents in Juglans mandshurica Maxim. A simple, efficient and sensitive ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) method was established and validated under positive and negative ion modes. The separation was performed on a Waters ACQUITY UPLC BEH C column (2.1 mm × 100 mm, 1.7 μm) by gradient elution with a mobile phase (Phase A: 0.1% aqueous formic acid solution, Phase B: 0.1% formic acid acetonitrile solution). A total of 165 compounds were rapidly selected by Targeted and Non-Targeted Peak Finding approaches, and then tentatively identifled by comparing with reference substances or inferred through mass spectrometry fragment ion analysis and literature data. These compounds included 68 naphthalenequinones, 20 diarylheptanoids, 29 flavonoids, 20 triterpenes, and 28 phenolic acids. In conclusion, the present study provided an effective approach to identifying components in complex matrices of herbal medicines such as Juglans mandshurica Maxim.

Dynamic variation of components in exocarp of Juglans mandshurica with browning based on UPLC-Q-TOF/MS / 中国中药杂志

To analyze the dynamic changes in components in exocarp of Juglans mandshurica at different browning periods. Twenty-six batches of exocarp of J. mandshurica samples from thirteen browning periods were assessed by UPLC-Q-TOF-MS/MS. The formula of different compounds were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/masterview1.0 software. Then their structures were determined by analysis of MS/MS fragment or comparison with standard substances and references. The contents of chemical components were changed significantly in different browning periods and twenty five compounds were identified or inferred. Of the 13 naphthoquinone compounds, the contents of 6 compounds with similar parent nucleus as juglone and 3 naphthoquinone glycosides compounds were decreased significantly, and 4 naphthoquinone derivatives such as regiolone were produced; the contents of four flavones and two phenolic acids compounds were decreased significantly; and the contents of 6 diarylheptanoids compounds were increased significantly. UPLC-Q-TOF/MS method can be used to identify and analyze the chemical constituents from exocarp of J. mandshurica rapidly and accurately, and analyze the rules of dynamic changes, to reveal the browning of Chinese medicinal materials and its effects on compositions of fruits and vegetables.

Serum metabonomics in mice infected with mycoplasma pneumoniae by UPLC-Q-TOF-MS / 中国中药杂志

Ultra high performance liquid chromatography coupled with tandem quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS) was applied to metabonomics study in BALB/c mice infected with mycoplasma pneumoniae(MP) to analyze the changes in serum endogenous metabolites, identify potential biomarkers associated with mycoplasma pneumoniae pneumonia(MPP), analyze the metabolic pathway and explore the pathogenic mechanism of MPP. The BALB/c mice were inoculated with MP by repeated intranasal infectious routes to establish MPP models, and the results of the lung tissue biopsy, IgM and mycoplasma nucleic acid content determination showed that the models of MP in BALB/c mice were successfully established. UPLC-Q-TOF-MS was used to analyze the serum metabolic profiling of BALB/c mice infected with MP, and then principal component analysis(PCA) was combined with orthogonal partial least squares discriminant analysis(OPLS-DA) for data processing. The results showed that there were significant differences in serum metabolic profile between the MP infected mice and the normal mice. Forty-seven potential biomarkers such as ornithine, cortisol, vitamin A and tryptophan were screened out by database searching and MS information matching. These potential biomarkers related to 17 metabolic pathways including retinol metabolism, arginine and proline metabolism, steroid hormone synthesis and so on. The metabonomic research method for serum of mice infected with mycoplasma pneumoniae based on UPLC-Q-TOF-MS was established in this study. The metabolic changes of endogenous small molecules in mice infected with MP were reflected in the overall level, laying the foundation for the selection and evaluation of MPP drugs.

Serum pharmacochemistry of Qinbai Qingfei concentrated pellets based on UPLC-Q-TOF-MS / 中国中药杂志

To analyze the main components of Qinbai Qingfei concentrated pellets in rat serum with UPLC-Q-TOF-MS technology and serum pharmacochemistry theory. After gavage administration with Qinbai Qingfei concentrated pellets, blood was collected from hepatic portal vein. ACQUITY UPLC BEH C₁₈(2.1 mm×100 mm, 1.7 μm) was used, with 0.1% formic acid agueous solution(A)-0.1%formic acid and acetonitrile(B) as the mobile phase for gradient elution. The flow rate was 0.3 mL•min⁻¹, the column temperature was maintained at 35 ℃. Through the comparative analysis fingerprints of Qinbai Qingfei concentrated pellets, drug containing-serum and blank serum, and with the help Peakview and Metabolitepilot software, components in serum were defined. A total of 28 compounds were identified, including 18 prototypes and 10 metabolites. As a result, UPLC-Q-TOF-MS technology and serum pharmacochemistry theory were applied to comprehensively expound Qinbai Qingfei concentrated pellets'constituents migrating to rat serum, and provide scientific basis for further studies for in vivo metabolic process and effective material base.

Dynamic variation of major effective components in fresh rejuvenated fruit of Juglans mandshurica based on UPLC-Q-TOF-MS / 中国中药杂志

The changes in effective components of Juglans mandshurica at different harvest periods were analyzed by UPLC-Q-TOF-MS/MS. Eighteen batch samples of J. mandshurica from six harvest periods were assessed by multivariate statistical analysis with Markerview software. The formula of different compounds were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/Masterview1.0 software. Then their structure were determined by analysis of MS/MS fragment or comparison with standard substances and references. Naphthoquinone are the major markers in samples of Juglans mandshurica from different harvest periods. Thirty-eight of naphthalenequinones were identified or inferred in J. mandshurica and contents decline gradually. UPLC-Q-TOF-MS method which develops a new strategy can identify and analyze chemical constituents from J. mandshurica rapidly and accurately, main chemical constituents can be used for quality evaluation and efficacy material research. The dynamic changes in the metabolite accumulation of J. mandshurica the basic data for harvesting medicinal plants at different times.

Analysis on major chemical compounds in exocarp of Juglans mandshurica based on UPLC-Q-TOF/MS / 中草药

Objective: To identify the major chemical compounds in 78 batches of the exocarp of Juglans mandshurica from different origins by ultra performance liquid chromatography coupled with time-of-fight mass spectrometry (UFLC-Q-TOF/MS) and determine the major active chemical components. Methods: The separation was performed on Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), with a mobile phase using water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) for gradient elution; Q-TOF/MS and electrospray ion (ESI) source were applied for the analysis under the positive ion mode; One thousand ions were extracted through Markerview 1.2.1 software from 78 batches. And common ions (compounds) were selected according to the following principles: One ion can be detected in all samples, and the relative strength is greater than the e4. Then the formula of common ions were determined by accurate mass and isotopic abundance ratio from target screening function of Peakview 2.0/masterview1.0 software. Their structures were determined by analysis of MS/MS fragment or comparison with standard substances and references. Results: Thirty-one major chemical compounds including eleven naphthalene quinones, three diarylheptanoids, three flavonoids, eight triterpenes, and six other compounds were identified or inferred in the exocarp of J. mandshurica. Conclusion: UPLC-Q-TOF/MS method which develops a new strategy can identify the main chemical constituents from the exocarp of J. mandshurica rapidly and accurately, main chemical constituents can be used for the quality evaluation and efficacy material research.

Identfication of new naphthalenones from Juglans mandshurica and evalution of their anticancer activities / 中国天然药物

Two new naphthalenone compounds were isolated from green walnut husks of Juglans mandshurica and their structures were identified as 4-butoxybutoxy-5,8-dihydroxy-3,4-dihydro-2H-naphthalen-1-one (1), 4-ethoxyethoxy-5,8-dihydroxy-3,4-dihydro-2H-naphthalen-1-one (2). Compounds 1 and 2 were named as Juglanstetralone A (1) and Juglanstetralone B (2). Compound 1 showed more significant anti-tumor activity than 2 against gastric cancer BGC-823 cells, wih the IC50 of 125.89 (g(mL(-1).