OBJECTIVETo purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies.

METHODCaprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.

RESULTSAntibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 microg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 microg/mL and 1.03 microg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 microg/mL, and the linear working range and LOD were 10.91-11412.29 microg/mL and 3.42 microg/mL, respectively.

CONCLUSIONAntibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.

Antibodies, Monoclonal ; immunology ; Chromatography, Affinity ; Chromatography, Agarose ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Food Contamination ; Fruit ; Insecticides ; immunology ; Organothiophosphorus Compounds ; immunology ; Pesticide Residues ; analysis ; immunology ; Vegetables

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

Two new azaphilone derivatives containing 1,3-dioxolane moiety, penidioxolanes A (1) and B (2), were isolated from marine-derived fungus Penicillium sp. KCB12C078, together with four known compounds (3-6) by chemical investigation. Compounds 1 - 6 were isolated by combination of silica gel, ODS column chromatography and preparative HPLC. Their structures were determined by analysis of spectroscopic data including 1D-, 2D-NMR, and MS techniques. The isolates were evaluated against cancer cell growth inhibition effects and antimicrobial activity.
Chromatography ; Chromatography, High Pressure Liquid ; Fungi ; Penicillium* ; Silica Gel

OBJECTIVETo investigate the chemical constituents from the aerial part of Atractylodes macrocephala.

METHODThe constituents were isolated and purified by repeated column chromatography on silica gel, Sephadex LH-20 and ODS. Their structures were identified by physicochemical properties and spectrum analysis.

RESULTFourteen compounds were isolated and identified as atractylenolide I-III (1-3), 2-[(2E) -3, 7-dimethyl-2, 6-octadienyl]-6-methyl-2, 5-cyclohexadiene-1, 4-dione(4), 2, 6-dimethoxyphenol (5), scopoletin (6), 4-methoxycinnamic acid (7), caffeic acid (8), ferulic acid (9) protocatechuic acid (10), 3-hydroxy-1-(4-hydroxy-3- methoxyphenyl) propan-1-one (11), dictamnoside A (12), syringin (13), D-mannitol (14).

CONCLUSIONAll the compounds were isolated from the aerial part of A. macrocephala for the first time and compounds 4, 5, 7-12, 14 were isolated from this species for the first time.

Atractylodes ; chemistry ; Chromatography, Gel ; Plant Components, Aerial ; chemistry ; Spectrum Analysis

A method for simultaneously identifying antioxidative compounds was developed using time-based LC-MS coupled with DPPH assay regardless of the time consuming process. The methanolic extract of Polygonum aviculare (Polygonaceae) showed significant DPPH radical scavenging activity. Time-based DPPH assay for simultaneous identification of active compounds from the extracts of P. aviculare was used. Major peaks of ethyl acetate fraction of P. aviculare showed high DPPH radical scavenging activity. A simple phenolic compound (1) and six flavonoids (2-7) were isolated from the ethyl acetate fraction of P. aviculare by silica gel and sephadex LH-20 column chromatography. The structures of seven compounds were determined to be protocatechuic acid (1), catechin (2), myricitrin (3), epicatechin-3-O-gallate (4), avicularin (5), quercitrin (6), and juglanin (7) based on the analysis of the 1H-NMR, 13C-NMR and ESI-MS data. All compounds exhibited significant antioxidant activity on DPPH assay and active compounds were well correlated with predicted one.
Catechin ; Chromatography ; Flavonoids ; Methanol ; Phenol ; Polygonum* ; Silica Gel

Two new thiodiketopiperazines (TDKPs), designated graphiumins I (1) and J (2), were isolated from the culture broth of the marine-derived fungus Graphium sp. OPMF00224 by solvent extraction, silica gel column chromatography, and HPLC. Their absolute structures were elucidated by spectroscopic analyses (1D and 2D NMR data, ROESY correlations, and CD data) and chemical methods. They were found to be structurally rare TDKPs with a phenylalanine-derived indolin substructure. Compounds 1 and 2 inhibited yellow pigment production by methicillin-resistant Staphylococcus aureus (MRSA) with IC50 values of 63.5 and 76.5 microg/ml, respectively, without inhibiting its growth, even at 250 microg/ml.
Chromatography ; Chromatography, High Pressure Liquid ; Fungi* ; Inhibitory Concentration 50 ; Methicillin-Resistant Staphylococcus aureus ; Silica Gel

OBJECTIVETo study the chemical constituents in ethyl acetate fraction from the root of Scutelliaria regeliana.

METHODThe compounds were isolated by silica gel column chromatography and HPLC, and their structures were elucidated by means of spectral analyses.

RESULT23 compounds were isolated and identified.

CONCLUSIONCompound 1 is new, named as scutellariae flavonol, and the others were isolated from S. regeliana for the first time.

Chromatography, Gel ; Chromatography, High Pressure Liquid ; Plant Extracts ; chemistry ; isolation & purification ; Scutellaria ; chemistry

Ginseng damping-off, caused by the fungal pathogens Rhizoctonia solani and Pythium sp., is a critical disease in ginseng seedling. In a continuing effort to find microorganisms with the potential of acting as a biocontrol agent against Rhizoctonia damping-off, we found that a Streptomyces sp. A501 showed significant antifungal activity against Rhizoctonia solani. In field experiment to test the efficacy of Streptomyces sp. A501 in controlling ginseng damping-off, the incidence of damping-off disease was meaningfully reduced when ginseng seeds were soaked in the culture broth of Streptomyces sp. A501 before sowing. To perform characterization of the antifungal compound, we isolated it from the culture broth of strain A501 through Diaion HP-20 and silica gel column chromatographies and preparative high-performance liquid chromatography. The structure of the antifungal compound was assigned as fungichromin by spectroscopic methods, mainly nuclear magnetic resonance and electrospray ionization-mass analysis.
Chromatography ; Chromatography, Liquid ; Incidence ; Magnetic Resonance Spectroscopy ; Panax* ; Pythium ; Rhizoctonia ; Seedlings ; Silica Gel ; Streptomyces*

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45degrees C and the highest activity was exhibited in 35 to 45degrees C. The enzyme lost their activities almost completely (95~100%) at 80 degrees C or above and as well as bellow 25degrees C.
Acrylamide ; Aspergillus ; Aspergillus oryzae ; Benzyl Alcohols ; Cellulase ; Cellulose ; Chromatography, DEAE-Cellulose ; Chromatography, Gel ; Chromatography, Ion Exchange ; DEAE-Cellulose ; Electrophoresis, Polyacrylamide Gel ; Glucosides ; Hydrogen-Ion Concentration ; Molecular Weight ; Oryza

Enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid (CNDE) is the key step in chemoenzymatic synthesis of pregabalin. We purified an intracellular carboxyl esterase from Morganella morganii ZJB-09203, which exhibited high enantioselectivity and activity towards CNDE. The carboxyl esterase was purified to electrophoretic homogeneity by ammonium sulfate fraction precipitation, Phenyl Sepharose 6 FF hydrophobic interaction chromatography, anion exchange with DEAE Sephadex A-50 and Bio-Scale CHT column. The purified enzyme was a monomer with molecular mass of 68 kDa determined by SDS-PAGE and gel chromatography. Substrate specificity of the enzyme towards p-nitrophenyl esters suggested that the purified enzyme was an esterase. The optimal reaction pH for CNDE hydrolysis was 9.0, and optimal temperature was 45 degrees C. The esterase was stable between pH 7.0 and 9.0, and at 40 degrees C. The enzyme activity was enhanced by Ca2+, Cu2+ and Mn2+, whereas strongly inhibited by Co2+, Fe3+, Ni2+ and EDTA. Meanwhile, we investigated the kinetic parameters of the esterase towards p-nitrophenyl esters and effect of CNDE concentration on conversion. The present study reported the esterase capable of stereospecific hydrolysis of CNDE for the first time. Our research will provide foundations for industrial production of Pregabalin using the new biocatalyst.
Chromatography, Gel ; Electrophoresis, Polyacrylamide Gel ; Esterases ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Morganella morganii ; enzymology ; Substrate Specificity ; Temperature