Analysis of nephroloxic and carcinogenic aristolochic acids in Aristolochia plants by capillary electrophoresis with electrochemical detection at a carbon fiber microdisk electrode

Aristolochic acids (AAs) are the main bioactive ingredients in the most of Aristolochia plants, which are used to make dietary supplements, slimming pills and Traditional Chinese Medicines (TCMs). Excessive ingestion of AAs can lead to serious nephropathy. Therefore, quantitative analysis and quality control for the plants containing AAs is of great importance. In this paper, capillary electrophoresis (CE) with electrochemical detection (ED) at a 33 mu m carbon fiber microdisk electrode (CFE) has been applied to detect AA-I and AA-II in Aristolochia plants. Under the optimum conditions: detection potential at 1.20 V, 2.0 x 10(-2) mol L-1 phosphate buffer solution (PBS) (pH 10.0), injection time 25 s at a height of 17 cm and separation voltage at 12.5 kV, the AA-I and AA-II were baseline separated within 5 min. Low detection limits for AA-I and AA-II were 4.0 x 10(-8) mol L-1 and 1.0 x 10(-7) mol L-1, respectively. Wide linear ranges were from 4.0 x 10(-8) mol L-1 to 1.9 x 10(-5) mol L-1 and 1.0 X 10(-7) mol L-1 to 5.0 x 10(-5) mol L-1 for AA-I and AA-II, respectively. The proposed method has been successfully applied to analyze AAs contents in plant extracts. The results indicated that the contents of AAs in each part of Aristolochia debilis Sieb. Et Zucc.

Characterization of triterpenoidic saponin mixture in crude extracts from leaves of Acanthopanax senticosus Harms by saponin structural correlation and mass spectrometry

Eighteen triterpenoidic saponins in crude extracts from leaves of Acanthopanax senticous Harms have been investigated by electrospray ionization multi-stage tandem mass spectrometry and high-resolution mass spectrometry. In ESI-MS spectra, predominant [M + Na](+) ions in the positive ion mode have been observed for molecular mass information. Meanwhile, specific structural correlations between these ions are firstly found. The 18 peaks (ions) can be classified into three groups (group D, E, and F with mass increase) with each group including six peaks. There is a mass difference of 132 Da between group D and E for each corresponding peak in turn (for example peak 1 to peak 7), indicating one more pentose residue was attached to saponins in group E than those corresponding in group D. The mass difference of 146 Da between group E and F implies one more deoxy-hexose attached to saponins in group F than those corresponding in group E. The structural correlations of the corresponding ions are confirmed by tandem mass spectrometry and high-re solution mass spectrometry. These structural features can not only facilitate the rapid characterization of the native known saponins in crude plant extracts, thus avoiding tedious derivation and separation of saponins, but also help find novel compounds of the same type in a specific medicinal plant.

Synergistic extraction of rare earths by mixture of bis(2,4,4-trimethylpentyl)phosphinic acid and Sec-nonylphenoxy acetic acid

Synergistic extraction of trivalent rare earths (RE=Sc, Y, La, Gd, Yb) from hydrochloride medium using mixture of bis(2,4,4-trimethylpentyl)phosphinic acid (HL, Cyanex272) and Sec-nonylphenoxy acetic acid (HA, CA-100) in n-heptane has been studied. The synergistic enhancement coefficients were observed for La (1.30), Gd (1.97), Y (3.59), Yb (8.21) and Sc (14.41). The results indicated yttrium was extracted into n-heptane as YH(5)A(4)L(4) mixed species instead of Y(HL2)(3), Y(OH)(2)A(HA)(3) which were extracted by Cyanex272 and CA-100, respectively. A cation exchange mechanism was proposed and further clarified by IR spectra. The equilibrium constants, formation constants and thermodynamic functions such as Delta G, Delta H and Delta S were determined. The Cyanex272 + CA-100 system not only enhanced the extraction efficiency of RE but also improved the selectivities significantly. The mutual separation factors of these ions suggested the mixture system would be of practical value in extraction and separation of rare earths.

Preparation and characterization of poly(ethyl acrylate)/bentonite nanocomposites by in situ emulsion polymerization

Transparent poly(ethyl acrylate) (PEA)/bentonite nanocomposites containing intercalated-exfoliated combinatory structures of clay were synthesized by in situ emulsion polymerizations in aqueous dispersions containing bentonite. The samples for characterization were prepared through direct-forming films of the resulting emulsions without coagulation and separation. An examination with X-ray diffraction and transmission electron microscopy showed that intercalated and exfoliated structures of clay coexisted in the PEA/bentonite nanocomposites. The measurements of mechanical properties showed that PEA properties were greatly improved, with the tensile strength and modulus increasing from 0.65 and 0.24 to 11.16 and 88.41 MPa, respectively. Dynamic mechanical analysis revealed a very marked improvement of the storage modulus above the glass-transition temperature. In addition, because of the uniform dispersion of silicate layers in the PEA matrix, the barrier properties of the materials were dramatically improved. The permeability coefficient of water vapor decreased from 30.8 x 10(-6) to 8.3 x 10(-6) g cm/cm(2)s cmHg. (C) 2002 Wiley Periodicals, Inc.

Rare earth separation with 1-hexyl-4-ethyloctyl isopropylphosphonic acid extractant

A new extractant 1-hexyl-4-ethyloctyl isopropylphosphonic acid (HHEOIPP or HA) in heptane was employed to extract rare earths from hydrochloric acid medium. The dependence of extraction distribution ratio on equilibrium aqueous pH and the concentration of extractant were investigated. On the basis of slope analysis,it was proposed that two different kinds of extracted species were formed. For rare earth elements (La similar to Ho) the extracted species was LnA(3)(HA)(3) and for heavy rare earth elements (Er similar to Lu) the species was LnClA(2)(HA)(3). The steric hindrance plays an important role in forming the species. The extraction constants and separation factors of the adjacent rare earths were calculated too. Compared with HDEHP and HEH/EHP, HHEOIPP is a valuable extractant with high separation selectivity. The "tetrad effect" between K-ex and atomic number was observed.

Simultaneous determination of 2-aminothiazole, 2-aminobenzothiazole and 2-mercaptobenzothiazole by capillary electrophoresis with end-column amperometric detection

Capillary electrophoresis with amperometric detection is evaluated for the simultaneous determination of 2-aminothiazole (A), 2-amino-benzothiazole (AB), 2-mercaptobenzothiazole (AM). The cyclic voltammogram, hydrodynamic voltammogram, effect of pH, concentration of buffer and separation voltage on the separation and the detection were studied. The conditions were optimized as follows: 50 mM phosphate buffet; pH 6.0, 2s at 17.5 kV sample injection, separation at 17.5 kV, 1.2 V as detection potential. The method provided low detection limit as 0.5 mu M, 0.05 mu M and 0.01 mu M, wide linear range 2-200 mu M, 10-200 mu M and 0.025-100 mu M for A, AB, and AM, respectively. The variations in peak current and migration time for 15 consecutive injections of a standard containing 5 mu M each compound were 3.7, 2.1, and 3.9%, and 1.2, 0.8, and 1.2%, for A, AB and AM, respectively. This method was employed to analyze river water.

Separation of zinc(II) from europium(III) by using hollow fiber membrane

Extraction and separation of Eu3+ and Zn2+ in sulfuric acid solution was investigated by hollow fiber membrane with cyanex 302 (bis (2,4,4-trimethylpentyl) monothiophosphinic acid) in counter-currently circulating operation. Reaction mechanism of membrane extraction and effect of extractant concentration and H+ concentration in aqueous phase on the mass transfer coefficient were discussed. It can be concluded that Zn2+ can be extracted completely from Eu3+ sulfate solution according to the kinetics competing difference. In one extractor process, extraction percentage of Zn2+ was not completely and Eu3+ was not extracted. Extraction percentage of Zn2+ reached 94.92%, but Eu3+ only reached 8.59% after 100 minutes extraction in two series connectors and that of Zn2+ and Eu3+ reached 99.9% and 6.53% respectively after 40 minutes extraction in three series connectors.

End-column amperometric detection of aesculin and aesculetin by capillary electrophoresis

Determination of aesculin (AL) and aesculetin (AT) by capillary electrophoresis with end-column amperometric detection using a 33 mu m microdisk carbon fiber electrode is described. The HDVs, the effect of pH, buffer concentration, injection voltage, injection time and separation voltage on the peak current response (i(p)) of the analytes and the number of theoretical plates (N) were studied. The method has high sensitivity and good reproducibility. Under the optimum condition - 10 mM, pH 9.00 phosphate buffer, 4 s at 9 kV injection, separation at 15 kV and +1.0 V as the detection potential - low detection limits (S/N = 3) of 0.06 and 0.3 mu M were obtained for AL and AT, respectively. The calibration curve was linear over three orders of magnitude. The relative standard deviations (n = 15) of peak current and migration time were 3.9% and 4.6%, and 0.96% and 0.75% for 15 consecutive injections of 5 mu M AL and AT, respectively. The use of this method for the separation and detection of the two compounds present in the traditional Chinese medicine and human urine samples is also reported. (C) 1999 Elsevier Science B.V. All rights reserved.

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240 000 to 460 000/m, and the relative standard deviation for t(0) and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated. (C) 2000 Elsevier Science B.V. All rights reserved.

Tm:Y_2SiO_5晶体的生长和光谱性质研究

采用中频感应提拉法生长了高质量的Tm:Y2SiO5(Tm:YSO)晶体,测定了晶体的晶格常数和分凝系数.运用劳厄照相法确定了单斜晶系Tm:YSO晶体的三个偏振轴〈010〉,D1和D2,在室温下测量了三个偏振轴方向的吸收光谱、荧光光谱和荧光寿命,计算了晶体吸收峰的吸收线宽和吸收截面.研究发现,相对于其他两个偏振轴方向,D1方向在790 nm处出现较强的吸收峰,同时在2μm附近出现了一定强度的发射峰,D1方向的吸收截面较大,荧光寿命较长.Tm:YSO晶体适用于AlGaAs二极管抽运固体激光器,在2μm波段固体激光器的应用上将有很大的发展潜力.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin. was purified by DEAF A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg, The fibrinopeptides released, identified by HPLC consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it. indicating it is venom serine protease. (C) 2000 Elsevier Science Ltd. All rights reserved.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded a-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin. (C) 2004 Elsevier Ltd. All rights reserved.

CHARACTERIZATION OF OHS1, AN ARGININE/LYSINE AMIDASE FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

In this paper, we present the results of purification and characterization of an arginine/lysine amidase from the venom of Ophiophagus hannah (OhS1). It was purified by Sephadex G-75 gel filtration and ion-exchange chromatography on DEAE-Sepharose CL-6B. It is a protein of about 43,000, consisting of a single polypeptide chain. It is a minor component in the venom. The purified enzyme was capable of hydrolysing several tripeptidyl-p-nitroanilide substrates having either arginine or lysine as the C-terminal residue. We studied the kinetic parameters of OhS1 on six these chromogenic substrates. OhS1 did not clot fibrinogen. Electrophoresis of fibrinogen degraded with OhS1 revealed the disappearance of the alpha- and beta-chains and the appearance of lower mel. wt fragments. OhS1 had no hemorrhagic activity. It did not hydrolyse casein, nor did it act on blood coagulation factor X, prothrombin and plasminogen. The activity of OhS1 was completely inhibited by NPGB, PMSF, DFP, benzamidine and soybean trypsin inhibitor, suggesting it is a serine protease. Metal chelator (EDTA) had no effect on it.