This study aims to establish a high-performance size-exclusion chromatography (HPSEC) method for determining the content of the classical swine fever virus (CSFV) E2 protein and screen the optimal stabilizer to enhance the stability of this protein. The optimal detection conditions were determined by optimizing the composition of the mobile phase, and characteristic chromatographic peaks were identified by SDS-PAGE and Western blotting. The specificity, repeatability, precision, linearity, limit of detection (LOD), and limit of quantitation (LOQ) of the method were assessed. The method established was used to determine the content of CSFV E2 protein antigen and vaccine. Differential scanning fluorimetry (DSF) was employed to screen the buffer system, pH, and salt ion concentrations, and sugar, amino acid, and alcohol stabilizers were further screened. The results showed that using a 200 mmol/L phosphate buffer provided the best column efficiency. An antigen-specific chromatographic peak appeared at the retention time of 18 min, which was identified as the CSFV E2 protein by SDS-PAGE and Western blotting. The method exhibited high specificity for detecting the CSFV E2 protein, with no absorbance peak observed in the blank control. The relative standard deviation (RSD) of the peak area for six repeated injections of the CSFV E2 protein was 0.74%, indicating good repeatability of the method. The RSD for repeated detection of two different concentrations of CSFV E2 protein samples by different operators at different time points was less than 2%, suggesting good intermediate precision of the method. The peak area of the CSFV E2 protein was linearly related to its concentration, with the regression equation showing R² of 1.000. The LOD and LOQ of the method were 14.88 μg/mL and 29.75 μg/mL, respectively. Application of the developed method in the detection of three batches of CSFV E2 protein antigen and three batches of vaccine demonstrated results consistent with those from the bicinchoninic acid (BCA) assay, which meant that the method could accurately determine the content of CSFV E2 protein antigen and vaccine. The DSF method identified 50 mmol/L Tris-HCl at pH 8.0 as the optimal buffer, and the addition of sugar and alcohol stabilizers further improved the stability of the CSFV E2 protein. The HPSEC method established in this study is simple, fast, and exhibits good accuracy and repeatability, enabling precise measurement of the CSFV E2 protein content. It is expected to play a crucial role in the quality control of the CSFV E2 vaccine. Furthermore, the strategy for improving the CSFV E2 protein stability, identified through DSF screening, has significant implications for enhancing the stability of the CSFV E2 vaccine.
Classical Swine Fever Virus/chemistry* ; Chromatography, Gel/methods* ; Animals ; Swine ; Viral Envelope Proteins/immunology*

BACKGROUND/AIMS: Acute exacerbations in chronic obstructive pulmonary disease may be related to air pollution, of which ozone is an important constituent. In this study, we investigated the protein profiles associated with ozone-induced exacerbations in a smoking-induced emphysema model. METHODS: Mice were divided into the following groups: group I, no smoking and no ozone (NS + NO); group II, no smoking and ozone (NS + O); group III, smoking and no ozone (S + NO); and group IV, smoking and ozone (S + O). Bronchoalveolar lavage, the mean linear intercept (MLI) on hematoxylin and eosin staining, nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Western blotting analyses were performed. RESULTS: The MLIs of groups III (S + NO) and IV (S + O) (45 +/- 2 and 44 +/- 3 microm, respectively) were significantly higher than those of groups I (NS + NO) and II (NS + O) (26 +/- 2 and 23 +/- 2 microm, respectively; p < 0.05). Fourteen spots that showed significantly different intensities on image analyses of two-dimensional (2D) protein electrophoresis in group I (NS + NO) were identified by LC-MS/MS. The levels of six proteins were higher in group IV (S + O). The levels of vimentin, lactate dehydrogenase A, and triose phosphate isomerase were decreased by both smoking and ozone treatment in Western blotting and proteomic analyses. In contrast, TBC1 domain family 5 (TBC1D5) and lamin A were increased by both smoking and ozone treatment. CONCLUSIONS: TBC1D5 could be a biomarker of ozone-induced lung injury in emphysema.
Animals ; Biological Markers/metabolism ; Blotting, Western ; Bronchoalveolar Lavage Fluid/chemistry/cytology ; Chromatography, Liquid ; Disease Models, Animal ; Electrophoresis, Gel, Two-Dimensional ; Lung/*metabolism/pathology ; Male ; Mice, Inbred C57BL ; *Ozone ; Proteins/*metabolism ; *Proteomics/methods ; Pulmonary Disease, Chronic Obstructive/etiology/*metabolism/pathology ; Pulmonary Emphysema/etiology/*metabolism/pathology ; Smoking/*adverse effects ; Tandem Mass Spectrometry

Fourteen compounds were isolated from Dalbergia odoriferae and purified by repeated column chromatography on silica and sephadex LH-20 gel and structurally identified by spectral analysis. These compounds were identified as 4, 9-dimethoxy-3-hydroxypterocarpan (1), medicarpin (2), 2', 4', 5-trihydroxy-7-methoxyisoflavone (3), 2', 3', 7-trihydroxy-4'-methoxyisoflavan (4), formononetin (5), 3, 8-dihydroxy-9-methoxypterocarpan (6), koparin (7), 3-hydroxy-9-methoxypterocarp-6a-ene (8), 2'-hydroxyformononetin (9), stevenin (10), 2', 7-dihydroxy-4', 5'-dimethoxyisoflavone (11), lyoniresinol (12), 2, 4-dihydroxy-5-methoxy-benzophenone (13) and neokhriol A (14). Compounds 1, 3, 4, 6, 8, 12 and 14 were isolated from this plant for the first time. Antibacterial activity assay showed that compound 4 had inhibitory effect on Ralstonia solanacearum.
Anisoles ; chemistry ; isolation & purification ; pharmacology ; Anti-Bacterial Agents ; chemistry ; isolation & purification ; pharmacology ; Benzophenones ; chemistry ; isolation & purification ; pharmacology ; Chromatography ; methods ; Dalbergia ; chemistry ; Dextrans ; Gels ; Isoflavones ; chemistry ; isolation & purification ; pharmacology ; Microbial Sensitivity Tests ; Naphthalenes ; chemistry ; isolation & purification ; pharmacology ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Pterocarpans ; chemistry ; isolation & purification ; pharmacology ; Ralstonia solanacearum ; drug effects ; growth & development ; Silica Gel

The chemical constituents of 95% ethanol extract of Melastoma dodecandrum were isolated and purified by chromatography on silica gel, Sephadex LH-20, and HPLC, to obtain thirteen compounds eventually. On the basis of their physico-chemical properties and spectroscopic data, these compounds were identified as quercetin (1), quercetin-3-O-β-D-glucopyranoside (2), quercetin-3-O-(6"-O-p-coumaroyl) -β-D-glucopyranoside (3), kaempferol (4), kaempferol-3-O-β-D-glucopyranoside (5), kaempferol-3-O- [2",6"-di-O-(E)-coumaroyl]-β-D-glucopyra-noside (6), luteolin (7), luteolin-7-O-(6"-p-coumaroyl) -β-D-glucopyranoside (8), apigenin (9), apigenin-7-(6"-acetyl-glucopyranoside) (10) , naringenin (11), isovitexin (12), and epicatechin-[8,7-e] -4β-(4-hydroxyphenyl)-3,4-dyhydroxyl-2(3H)-pyranone (13). Eight compounds(3,5,6,8-11 and 13) were obtained from M. dodecandrum for the first time.
Apigenin ; analysis ; Chromatography ; methods ; Chromatography, High Pressure Liquid ; Dextrans ; Flavanones ; analysis ; Flavonoids ; analysis ; chemistry ; Glycosides ; analysis ; chemistry ; Kaempferols ; analysis ; Luteolin ; analysis ; Magnoliopsida ; chemistry ; Plants, Medicinal ; chemistry ; Quercetin ; analysis ; Silica Gel

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.
Chemical Fractionation/methods ; Chromatography, Liquid/methods/veterinary ; Cryptosporidium parvum/*chemistry/growth & development/metabolism ; Electrophoresis, Polyacrylamide Gel/methods/veterinary ; Gene Expression Profiling/*methods/veterinary ; Proteome/analysis ; Proteomics/*methods ; Protozoan Proteins/*analysis ; Sporozoites/chemistry/metabolism ; Tandem Mass Spectrometry/methods/veterinary

The column chromatography on silica gel, Sephadex LH-20 and semi-preparative HPLC were used to separate and purify the compounds from the EtOAc extract of medium and MeOH extract of cell cultures of Morus alba. Eight compounds were isolated. Based on physico-chemical properties and spectroscopic data, their structures were identified as isobavachalcone (1), genistein (2), norartocarpetin (3), albanin A (4), guangsangon E (5), mulberrofuran F (6), chalcomoracin (7), kuwanon J (8). Compounds 3-6 were isolated from the cell cultures of M. alba for the first time.
Benzofurans ; isolation & purification ; Cell Culture Techniques ; methods ; Chalcones ; isolation & purification ; Chromatography, Gel ; methods ; Chromatography, High Pressure Liquid ; Dextrans ; Genistein ; isolation & purification ; Morus ; chemistry ; cytology ; Plant Leaves ; chemistry ; cytology ; Plants, Medicinal ; chemistry ; cytology ; Silica Gel ; Terpenes ; isolation & purification

OBJECTIVETo study the chemical constituents of aerial parts of Ammopiptanthus mongolicus.

METHODIsolation and purification were carried out on silica gel, Sephadex LH-20 and HPLC column chromatography. The structures of the compounds were identified by physico-chemical properties and spectral analysis.

RESULTNine compounds were isolated and identified as (+)-maackiain (1), brevifolin (2), 7-hydroxy-4'-methoxy isoflavanone (3), daidzein 4',7-diglucoside (4), genistein 4', 7-di-O-beta-D-glucoside (5), isolupalbigenin (6), ononin (7), beta-sitosterol (8), beta-daucosterol (9).

CONCLUSIONCompounds 2, 4 - 6 were obtained from the genus Ammopiptanthus for the first time.

Chromatography, Agarose ; methods ; Chromatography, High Pressure Liquid ; methods ; Fabaceae ; chemistry ; Glucosides ; chemistry ; isolation & purification ; Isoflavones ; chemistry ; isolation & purification ; Plant Extracts ; chemistry ; isolation & purification ; Plant Leaves ; chemistry ; Pterocarpans ; chemistry ; isolation & purification ; Silica Gel ; Sitosterols ; chemistry ; isolation & purification ; Taxoids ; chemistry ; isolation & purification

The paper is to report the establishment of a method for the determination of multi-residue organochlorine and pyrethroid pesticides in traditional Chinese medicines (TCMs). Fifty-six pesticides were extracted by high-speed homogenization, and then purified through gel permeation chromatography (GPC) and solid phase extraction (SPE) cartridges. The residues were simultaneously identified and quantified by GC-ECD equipped with dual tower, dual column and two micro-ECD detectors. The analytical performance was demonstrated by the analysis of 3 TCMs samples' extracts, spiked at three concentration levels for each pesticide. In general, the recoveries ranging from 70% to 110%, with relative standard deviations (RSDs) better than 15%, were obtained. The limit of detection (LOD) for most of the targeted pesticides tested was below 0.01 mg kg(-1). The method had good extraction efficiency, purification effect and good reproducibility, which could be applied to the determination of organochlorine and pyrethroid pesticide residues in the routine analysis of TCMs.
Chromatography, Gas ; methods ; Chromatography, Gel ; Drug Contamination ; Drugs, Chinese Herbal ; chemistry ; Hydrocarbons, Chlorinated ; analysis ; Limit of Detection ; Pesticide Residues ; analysis ; Pyrethrins ; analysis ; Reproducibility of Results ; Solid Phase Extraction

OBJECTIVETo investigate the alkaloids from Senecio scandens.

METHODCompounds were isolated with silica gel and Sephadex LH-20 column chromatography and their structures were determined by spectral analysis and chemical evidence. The hepatic cytotoxicity of isolated compounds was tested by MTT method in vitro.

RESULTSix alkaloids were obtained and identified as adonifoline (1), 7-angeloylturneforcidine (2), hordenine (3), 1, 3, 6, 6-tetramethyl-5, 6, 7, 8-tetrahydro-isoquinolin-8-one (4), 4-(pyrrolidin-2-one) -phenyl acetic acid (5), (4-pyrrolidinophenyl) acetic acid (6).

CONCLUSIONCompound 6 is a new natural product, compounds 3, 4 were obtained from the genus Senecio for the first time, compounds 2, 5 were obtained from this plant for the first time. Compound 1 showed significant growth inhibitory effect against hepatocyte at 100 micromol x L(-1).

Acetic Acid ; chemistry ; Alkaloids ; chemistry ; isolation & purification ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Dextrans ; chemistry ; Hepatocytes ; Lactones ; isolation & purification ; metabolism ; pharmacology ; Mice ; Mice, Inbred ICR ; Pyrrolizidine Alkaloids ; isolation & purification ; metabolism ; pharmacology ; Senecio ; chemistry ; Silica Gel ; Tyramine ; analogs & derivatives ; isolation & purification ; pharmacology

OBJECTIVETo study the chemical constituents of the whole plant of Rhododendron concinnum.

METHODThe compounds were isolated and purified by chromatography on silica gel and polyamide. Their structures were identified on the basis of spectroscopic data (MS,1H-NMR and 13C-NMR) and chemical evidence.

RESULTFive dihydroflavones were isolated and identified as (2R)-farrerol-7-O-glucopyranoside (1), (2R,3R)-(-)-dihydroquercetin-3-O-beta-D-xylopyranoside(2), (2S,3S)-(-)-dihydroquercetin-3-O-beta-D-glucopyranoside(3), eriodictyol-7-O-beta-D-glucopyranoside (4) , (2R, 3R)-(+)-dihydroquercetin (5).

CONCLUSIONExcept compound 5, others were firstly isolated from the genus Rhododendron.

Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Chromatography, High Pressure Liquid ; methods ; Chromones ; chemistry ; Humans ; Inhibitory Concentration 50 ; Magnetic Resonance Spectroscopy ; Quercetin ; analogs & derivatives ; analysis ; pharmacology ; Rhododendron ; chemistry ; Silica Gel ; Silicon Dioxide ; analysis ; pharmacology