Structure and identification of immune fragments of acid-degraded oligosaccharides from active Astragalus polysaccharides APS-Ⅱ / 药学学报

We previously reported that active Astragalus polysaccharides APS-Ⅱ generate strong immune activity. Here we establish the optimal method for APS-II acid degradation. After preliminary structural studies and separation and preparation of the degradation products, the oligosaccharide active center with the strongest immune activity was identified by in vitro immune cell culture experiments. The optimum acid degradation conditions for APS-II were determined by a single factor experiment and an orthogonal experiment. Astragalus oligosaccharides prepared under the optimal conditions were subjected to structural analysis by hydrophilic interaction chromatography - electrospray ionization source - high resolution time-of-flight mass spectrometry. The products were separated and oligosaccharide fragments with different degrees of polymerization were isolated by preparative purification chromatography. Finally, fragments of the immunologically active centers were identified by in vitro immune cell cultures from multiple perspectives. The results show that the optimal acid hydrolysis conditions for APS-Ⅱ are hydrolysis temperature 80 ℃, trifluoroacetic acid concentration 1.0 mol·L-1, hydrolysis time 1 h. The degradation conditions have good repeatability. The degradation product is a six-carbon aldehyde glycan structure with the main chain 1→4 connected. The immune activity screening experiment for six oligosaccharide fragments showed that larger molecular weight oligosaccharides have stronger immune-promoting effects. It is speculated that the immunologically active center of Astragalus oligosaccharide is located in the sugar chain of DP9-DP19. The animal welfare and the experimental process in this study follow the requirements of the Animal Ethics Committee of Shanxi University. This result suggests a foundation for the structural characterization and structure-activity relationship research of Astragalus oligosaccharides, and may promote the development of Astragalus oligosaccharide drugs.

Study on antipyretic effect of rhubarb on rats and its antipyretic ingredients / 中国中药杂志

A combination of LC-MS technology and activity evaluation was used to identify the antipyretic ingredients in rhubarb. The rat model of fever was established with dried yeast and then was administered ethanol extract and different polar fractions of rhubarb. Next, the anal temperature of these rats was measured and recorded at 0.5, 1, 2, 3, 4 and 5 h after administration, and the inhibition rate of each part on the rise of body temperature was calculated. The inhibition rate is higher and the antipyretic effect is better. The chemical composition of the effective fraction was analyzed with UPLC-ESI-Orbitrap-MS/MS technology. Compared with the model group, the increase of body temperature of ethanol extract group all reduced at each measurement time especially after 3 h, and the inhibition rate were 38.7%(P<0.05), 78.2%(P<0.01) and 72.4%(P<0.01) at 3 h, 4 h, and 5 h after administration, respectively. Both n-butanol and water fraction showed some antipyretic activity in the early stage, with the inhibition rate of 28.1%(P<0.01) and 24.9%(P<0.05) at 1 h after administration, respectively, while other fractions were not active. Thirty-three and twelve compounds were identified from n-butanol and water fraction by LC-MS/MS analysis, respectively, including ten tannins, fifteen anthraquinone glycosides, four anthrone glycosides, one phenolic glycoside, one naphthaline derivative, one anthraquinone and one sucrose. These results revealed that rhubarb had antipyretic activity on rats, and tannin and anthraquinone glycosides were the main active ingredients inside.

Proteomic analysis and verification of protein expression after upregulation of human CD99 in Hodgkin lymphoma cell line L428 / 中华血液学杂志

Objective@#To investigate the proteins expression difference after upregulation of human CD99 in Hodgkin Lymphoma cell line, L428 cell, and verify the function of differential proteins.@*Methods@#The differential proteins were detected by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis, cluster analysis was done by GOfact.@*Results@#There were 38 proteins screened out, of which 21 proteins were positively associated with CD99, while 17 proteins were negative. Among the 38 proteins, 32 proteins participated in biological process, and 35 proteins were involved in the composition and construction. And 28 proteins participated in multifaceted biological activities including antioxidation, protein binding, catalytic activity, regulation of enzyme, signal transduction, molecular structure, regulation of translation and ion transport.@*Conclusions@#The changes of the differential proteins, correlated with cytoskeleton, cell differentiation, signal pathway and regulating gene expression, are closely relevant to the translation between Hodgkin/Reed-Sternberg and B lymphocyte cell.

Proteomic analysis and verification of protein expression after upregulation of human CD99 in Hodgkin lymphoma cell line L428 / 中华血液学杂志

Objective: To investigate the proteins expression difference after upregulation of human CD99 in Hodgkin Lymphoma cell line, L428 cell, and verify the function of differential proteins. Methods: The differential proteins were detected by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis, cluster analysis was done by GOfact. Results: There were 38 proteins screened out, of which 21 proteins were positively associated with CD99, while 17 proteins were negative. Among the 38 proteins, 32 proteins participated in biological process, and 35 proteins were involved in the composition and construction. And 28 proteins participated in multifaceted biological activities including antioxidation, protein binding, catalytic activity, regulation of enzyme, signal transduction, molecular structure, regulation of translation and ion transport. Conclusions: The changes of the differential proteins, correlated with cytoskeleton, cell differentiation, signal pathway and regulating gene expression, are closely relevant to the translation between Hodgkin/Reed-Sternberg and B lymphocyte cell.

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

Background Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. Methods The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Results Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. Conclusion We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.(AU)

Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata

Abstract Background Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. Methods The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Results Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. Conclusion We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.

Structure and Property of Ladder Electrode Linear Ion Trap Mass Analyzer / 分析化学

Theoretical study and experimental test of a new ion trap mass analyzer-ladder electrode linear ion trap (LeLIT) were reported. The LeLIT was composed of two pairs of ladder electrodes and one pair of end-electrodes. By optimizing the geometric structure of ladder electrodes, we optimized the electric field distribution for optimum mass analysis. Since the distribution of electric field can be controlled by the adjustment of LeLIT structure, LeLIT have better performance than rectilinear ion trap theoretically. Due to its simpler structure than hyperbolic electrode LIT, LeLIT can be easily built. In this work, the height, width and field ratio were adjusted for optimizing the property of LeLIT, and a mass resolution of 10150 was obtained when the mass scan speed was 225 Da / s for a LeLIT with X0 ×Y0 = 9 mm×5 mm dimension. The preliminary experimental result showed that the LeLIT has proper tandem mass spectrometric performance as any conventional ion trap mass analyzer.

Diagnostic effect of metabonomics determination method on acute paraquat poisoning / 中华危重病急救医学

Objective To screen the potential biomarkers in plasma of rats with acute paraquat (PQ) poisoning using gas chromatography-mass spectrometry (GC-MS) based metabonomics technology,and to provide concrete evidence for early diagnosis.Methods Eight Sprague-Dawley (SD) rats were randomly divided into PQ poisoning group (intragastricly administrated with PQ solution 100 mg/kg) and control group (intragastricly administrated with the same volume of normal saline) according to the random number table,with 4 rats in each group.The general situation of rats was observed at 2,24 and 48 hours after administration.The blood of eye sockets was collected,the endogenous small molecule metabolites in plasma were determined with GC-MS method,and metabolic profile analysis and random forest analysis were performed to filter the potential biomarkers.Results ① The rats in PQ poisoning group gradually appeared lack movement,tachypnea,abdominal seizure and other symptoms of poisoning.In control group,the vital signs were stable.② The metabolites in plasma of rat were analyzed with GC-MS analysis,and the diagrammatic figure was plot as combined with principal component analysis (PCA) and partial least squares-discriminated analysis (PLS-DA) model,which showed that the distribution of plasma metabolism in PQ poisoning group was more diffuse but in the control group was more intensive,indicating that the metabolic patterns in two groups were different.From 2 hours after PQ administration,the metabolic trajectory in PQ poisoning group was significantly deflected compared with that of the control group,which was similar to control group until 48 hours,indicating that the metabolites in plasma of rat showed obvious difference in the early period.Five kinds of potential biomarkers with large weights were selected by random forest method which were serine,L-asparagine,hexadecanoic acid,octadecanoic acid,and arachidonic acid,the retention time was 15.259,24.345,33.334,37.695,and 40.254 minutes,respectively.The levels of serine,L-asparagine,arachidonic acid in PQ poisoning group were significantly higher than those of the control group,peaked at 48,48 and 24 hours,respectively (40.884-5.38 vs.28.85±2.32,6.61±1.31 vs.0.76±0.65,14.21±4.28 vs.4.42±1.19,all P ＜ 0.01),and the levels of hexadecanoic acid and octadecanoic acid were significantly lowered,reached tough at 48 hours (39.09 ± 10.23 vs.83.99 ± 20.49,44.03 ± 3.60 vs.140.76 ± 73.91,P ＜ 0.05 and P ＜ 0.01).The changes in these biomarkers were related to the toxicity of PQ,indicating that PQ could interfere the energy and lipid metabolism in rats.Conclusion Combine with the metabonomics analysis,screened plasma serine,L-asparagine,arachidonic acid content in PQ poisoning rats increased significantly,and hexadecanoic acid and octadecanoic acid content decreased significantly,which can preliminary diagnose acute PQ poisoning with animal general performance.

Chemical constituents in the essential oil of the endemic plant Cotula cinerea (Del.) from the southwest of Algeria

Objective: To extract and identify the main constituents of the essential oil of Cotula cinerea (Del.) (Asteraceae family) from southwest of Algeria. Methods: The essential oils obtained by hydrodistillation, from the aerial parts of the endemic plant Cotula cinerea which was collected in the region of Sahara from southwest of Algeria, were analyzed by gas chromatography-mass spectrometry. Results: A total of 33 compounds were identified representing 98.66% of the oil. The main compounds were (E)-citral (24.01%), limonene epoxide cis- (18.26%), thymol methyl ether (15.04%), carvacrol (15.03%), trans-carveol (13.79%), carvone (3.06%) and trans-piperitol (2.54%). Conclusions: The main constituents in essential oil of the aerial part of the plant from southwest of Algeria were different from that collected from southeast of Algeria or in Morocco.

Gas chromatography mass spectrometry analysis and in vitro antibacterial activity of essential oil from Trigonella foenum-graecum

Objective: To evaluate the antibacterial activity of essential oil from Trigonella foenum-graecum seeds powder, and identify the compounds from the extracted oil. Methods: The seeds powder of Trigonella foenum-graecum was subjected to Clevenger extractor. Seven strains of bacteria were used to test antibacterial activity of the extract. The activity against bacteria was tested by disk diffusion method using Whatman No. 1 filter paper. Gas chromatography mass spectrometry analysis was performed with an Agilent7890/5975B-gas chromatography/mass selective detector. Results: The hydrodistillation of seeds powder yielded 0.285% (v/w) of oil. Disk diffusion of the oil showed bactericidal activity against both Gram negative and Gram positive bacteria of tasted strains. The inhibition zone ranged from (8 ± 0) mm to (15.0 ± 0.7) mm depending on microbial strains. Gas chromatography mass spectrometry analysis showed 14 different compounds. The total compounds represented 80.96% of the oil. Conclusions: The antibacterial activity is due to the effects of different biological active compounds present in the extract. Identification of the compounds may help to develop new effective antimicrobial agent(s). Further researches on purification, characterization and toxicology of the active compounds are needed.

Gas chromatography mass spectrometry analysis and in vitro antibacterial activity of essential oil from Trigonella foenum-graecum

Objective: To evaluate the antibacterial activity of essential oil from Trigonella foenum-graecum seeds powder, and identify the compounds from the extracted oil. Methods: The seeds powder of Trigonella foenum-graecum was subjected to Clevenger extractor. Seven strains of bacteria were used to test antibacterial activity of the extract. The activity against bacteria was tested by disk diffusion method using Whatman No. 1 filter paper. Gas chromatography mass spectrometry analysis was performed with an Agilent7890/5975B-gas chromatography/mass selective detector. Results: The hydrodistillation of seeds powder yielded 0.285%(v/w) of oil. Disk diffu-sion of the oil showed bactericidal activity against both Gram negative and Gram positive bacteria of tasted strains. The inhibition zone ranged from (8 ± 0) mm to (15.0 ± 0.7) mm depending on microbial strains. Gas chromatography mass spectrometry analysis showed 14 different compounds. The total compounds represented 80.96%of the oil. Conclusions: The antibacterial activity is due to the effects of different biological active compounds present in the extract. Identification of the compounds may help to develop new effective antimicrobial agent(s). Further researches on purification, characterization and toxicology of the active compounds are needed.

Adulticidal activity of essential oil of Lantana camara leaves against mosquitoes.

Background & objectives: Development of insect resistance to synthetic pesticides, high operational cost and environmental pollution have created the need for developing alternative approaches to control vector-borne diseases. In the present study we have investigated the insecticidal activity of essential oil isolated from the leaves of Lantana camara against mosquito vectors. Methods: Essential oil was isolated from the leaves of L. camara using hydro-distillation method. Bioassay test was carried out by WHO method for determination of adulticidal activity against mosquitoes. Different compounds were identified by gas chromatography-mass spectrometry analysis. Results: LD50 values of the oil were 0.06, 0.05, 0.05, 0.05 and 0.06 mg/cm2 while LD90 values were 0.10, 0.10, 0.09, 0.09 and 0.10 mg/cm2 against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluvialitis and An. stephensi respectively. KDT50 of the oil were 20, 18, 15, 12, and 14 min and KDT90 values were 35, 28 25, 18, 23 min against Ae. aegypti, Cx. quinquefasciatus, An. culicifacies, An. fluviatilis and An. stephensi, respectively on 0.208 mg/cm2 impregnated paper. Studies on persistence of essential oil of L. camara on impregnated paper revealed that it has more adulticidal activity for longer period at low storage temperature. Gas chromatographic-mass spectrometric analysis of essential oil showed 45 peaks. Caryophyllene (16.37%), eucalyptol (10.75%), α-humelene (8.22%) and germacrene (7.41%) were present in major amounts and contributed 42.75 per cent of the total constituents. Interpretation &conclusion: Essential oil from the leaves of L. camara possesses adulticidal activity against different mosquito species that could be utilized for development of oil-based insecticide as supplementary to synthetic insecticides.

A proteomic study on the insulin resistant HepG_2 cell treated by Ecdysterone / 中国药理学通报

Aim To screen the express-altered proteins before or after effect of Ecdysterone on HepG_2 cell model of insulin resistance by the strategy of comparative proteomics, which may approach new proves exploring the target of sensitizer.Methods HepG_2 cells were incubated with 5×10~(-7) mol·L~(-1) insulin for 16 h, and glucose consumption was determined. After treatment, the insulin resistant cells were incubated with 10~(-5) mol·L~(-1) Ecdysterone for 24 h.Then glucose consumption contents were determined. The proteins of two groups before and after treatment with Ecdysterone were extracted by lysis buffers. The express-altered proteins were screened by 2-DE technique.Some of them were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Results 53 express-altered protein spots of insulin resistant HepG_2 cells before and after treated by Ecdysterone were screened by 2-DE technique,in which 35 ones were up-regulated and the others down-regulated, 6 spots of which were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Conclusion The target of Ecdysterone as a sensitizer involves many proteins and kinases which correlate insulin resistant. These results lay a foundation for further studies on the function of these target proteins.

Preliminary comparative proteomics study of cervical carcinoma tissues with different sensitivity to concurrent chemoradiotherapy / 中华放射肿瘤学杂志

Objective To investigate the proteomics differences between the high-sensitivity(HS) and the low-sensitivity(LS)groups of cervical carcinoma treated by concurrent chemoradiotherapy,and to confirm the sensitivity associated proteins in intermediate stage and advanced cervical carcinoma.Methods Fresh carcinoma tissues were collected from 10 untreated cervical carcinoma patients.According to the response to concurrent chemoradiotherapy,the tissues were classified into HS group and LS group.In the first part of our experiment,protein separation was performed using two-dimensional gel electrophoresis(2-DE)with Amersham 18 am linear pH 3-10 immobilized pH gradient(IPG)strips.The images of the gels were analyzed by PD-quest 7.0 software to find the differentially expressed protein-spots in each group.Then the differentially expressed protein-spots were incised from the gels and digested by trypsin.The peptide mass flngerprintings(PMF)was acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).The proteins were identified by data searched in the Mascot-database.Two differentially expressed proteins were assayed by western blot and immunohistochemical methods.Results Most of the gels were clear and successfully analyzed by PD-quest 7.0 software.Most of the protein-spots concentrated on the area of 20-100 KDa(Mw)and pH4-8.The average number of the protein-spots was 781±74 in HS group and 766±52 in LS group.The match rate was 87.6%between the two groups.Eight proteins highly in HS group but lowly expressed in LS group included hemoglobin subunit beta,caspase-14 precursor,calmodulin-like,S100-A9 protein(MRP-14),galectin-7,HSKERC4,keratin 19 and actin.Ten proteins highly in LS group but lowly expression in HS group included anti HBs antibody light-chain Fab,laminB1,WARS protein,flavin reductase,glutamate dehydrogenase 1,nuclear matrix protein 238,retinal dehydrogenase 1,AFl 65172,subunit of replicative DNA polymerase and HSP70.The higher expression of HSP70 in LS group and galectin7 in HS groups were further confirmed by western blot and immunohistochemical method.Conclusions The 2-DE gels images are successfully acquired from high-sensitivity group and low-sensitivity group of intermediate stage and advanced cervical carcinoma tissues treated by concurrent chemoradiotherapy.Some differentially expressed proteins between the two groups can be further confirmed by western blot and immunohistochemical method.