Table of Content

    01 February 2015, Volume 24 Issue 2
    Original articles
    Development and validation of an LC-MS/MS method for quantification of dipalmitoylphosphatidylcholine as a promising biomarker for renal failure in urine
    Dandan Li, Xin Xiong, Qiong Bai, Wenling Yang, Rongsheng Zhao, Aihua Zhang
    2015, 24(2):  73-79.  DOI: 10.5246/jcps.2015.02.008
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    Urinary phospholipids are shown to be sensitive biomarkers for kidney injury. Many works have been reported on qualitative analysis of phospholipids and relative components in human urine while quantitative analysis is rare. We have therefore developed a reliable and convenient quantitative method to evaluate the accuracy and specificity of dipalmitoylphosphatidylcholine (DPPC) as a biomarker for kidney injury diagnosis. Chromatographic separation was achieved using a HILIC Silica Column (2.1 mm×50 mm, 5 µm).Gradient elution was performed with 5 mM ammonium formate (0.1% formic acid) and acetonitrile (0.1% formic acid), at a flow rate of 0.3 mL/min. This method was validated in a linear range of 2200 ng/mL DPPC with inter-day precision of less than 6.5% and accuracy within 111.2% in human urine. Recovery rate, stability, dilution effect and matrix effect also met the requirements for drug evaluation and research issued by FDA. As the first HPLC-MS/MS method for quantitative determination of DPPC, it has been successfully applied to determine levels of DPPC in clinical urine samples and evaluated for potential use in the measurement of DPPC as a biomarker for kidney injury.

    Two alkaloids as α-amylase inhibitors: enzyme kinetics and molecular modeling investigations
    Yi Liang, Fen Pei, Hong Wang, Shizhong Chen
    2015, 24(2):  80-87.  DOI: 10.5246/jcps.2015.02.009
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    In the present study, we studied the inhibitory effects of chelidonine and rutaecarpin on porcine pancreatic α-amylase (PPA) catalyzed hydrolysis using 2-chloro-4-nitrophenyl-4-O-β-D-galactopyranosylmaltoside (Gal-G2-α-CNP). We, for the first time, provided kinetic report and detailed inhibitory effects of both compounds on PPA. Lineweaver-Burk plot revealed that the inhibition was a mixed-noncompetitive type, and only one molecule of inhibitor bound to the enzyme or to the enzyme-substrate complex. Kinetic constants calculated from secondary plots were in millimole range. Dissociation constants of enzyme-inhibitor complex (KEI) were 0.9 mM and 3.5 mM, respectively. Moreover, dissociation constants of enzyme-inhibitor-substrate complex (KESI) were 0.04 mM and 0.31 mM, respectively. These data indicated that the inhibition was more inclined to competitive to Gal-G2-α-CNP hydrolysis. Further molecular docking study manifested that hydrogen bonding formed between acarbose and aspartic acid (Asp300), histidine (His305) and glycine (Gly306), while hydrogen bonding was observed between chelidonine and glutamic acid (Glu233), lysine (Lys200) and His305. In addition, rutaecarpine had only one hydrogen bond with Lys200. Our data indicated that chelidonine and rutaecarpine were two promising drug candidates, and chelidonine possessed stronger inhibitory effect compared with rutaecarpine.

    Simultaneous determination of three main analytes of Murraya exotica by HPLC
    Bingyu Liu, Chen Zhang, Haining Lv, Pengfei Tu, Jianyong Xing, Zhengzhou Han, Yong Jiang
    2015, 24(2):  88-94.  DOI: 10.5246/jcps.2015.02.010
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    Murraya exotica L. is one of the two official source species of traditional Chinese medicine Murrayae Folium et Cacumen. At present, a rapid HPLC analysis method to simultaneously determine two major coumarins, hainanmurpanin (1) and meranzin (2), and one main flavonoid,3,5,6,7,3′,4′,5′-heptamethoxyflavone (3), from the leaves and twigs of M. exotica was established. The analysis was performed on a DIKMA Spursil C18 column (4.6 mm×250 mm, 5 µm) at a flow rate of 1 mL/min, using acetonitrileH2O (5:5, v/v) as mobile phase. The column temperature was 25 ºC, and the detected wavelength was at 320 nm. The three analytes (13) were separated well with good linearity, precision, stability and repeatability. The average recoveries were in the range of 100.52%101.97%, with RSD less than 1.73%. Twenty batches of M. exotica from different habitats were detected, and the sum of the contents of 13 was in the range of 1.557.45 mg/g, respectively. Besides, the contents of these three analytes were also determined for the samples from different medicinal parts (stems, lateral branches, mixture of twigs and leaves) and different harvest times. The results showed that the contents of these three analytes in leaves and twigs are much higher than those in the stems or lateral branches; the plants harvested from June or October contain more active compoundsthan those from the other months. The above results proved that this high performance and simple HPLC assay can be readily utilized as a practical method for the quality control of M. exotica.

    Docetaxel-loaded mixed micelles composed of Solutol HS15 and Pluronic F127 or folate-conjugated F127: preparation, optimization and in vitro comparative characterization
    Wenting Wu, Zhiyu Guan
    2015, 24(2):  95-103.  DOI: 10.5246/jcps.2015.02.011
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    In this study, two kinds of docetaxel (DTX)-loaded mixed micelles, composed of Solutol HS15 (HS15)/Pluronic F127 (F127) or folate-conjugated F127, (SF-DTX and FSF-DTX), were prepared by the thin-film hydration method and evaluated in vitro. Both SF-DTX and FSF-DTX were spherical with diameter close to 23 nm. They had high encapsulating efficiency (99.05% and 90.28% for SF-DTX and FSF-DTX, respectively) and sustained-release property. SF and FSF were able to enhance the cellular accumulation of DTX in KBv cells and reduce ATP content in A-549 cells. They also were able to reverse multidrug resistance (MDR). In vitro cytotoxicity and cellular accumulation of DTX suggested an active targeting of FSF-DTX. It could be concluded from the results that the novel F127/HS15 system could serve as a potential nanocarrier with the ability of overcoming MDR, and folate-conjugated F127/HS15 might achieve active targeting at the same time.

    Preparation, optimization and in vitro evaluation of core-shell paclitaxel-loaded nanoparticles composed of MPEG-PLA and PLA
    Yu Sun, Xinming Zhang, Lin Tang
    2015, 24(2):  104-110.  DOI: 10.5246/jcps.2015.02.012
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    Nanoparticles with typical core-shell structure were prepared with a blend of methoxypoly(ethylene glycol)-poly(lactide)copolymer (MPEG-PLA) and poly (lactic acid) (PLA) along with paclitaxel by the O/W solvent evaporation method. An orthogonalexperiment L9(3)3 was applied to get the best preparation conditions. The core-shell paclitaxel-loaded MPEG-PLA/PLA nanoparticleswith the highest drug loading efficiency were obtained when amount of MPEG-PLA, time of ultrasonication and volume of deionized water were 300 mg, 10 min and 30 mL, respectively. The release behavior of paclitaxel from the optimal MPEG-PLA/PLA nanoparticles showed that 22% of paclitaxel was released in 14 d. When incubating with human nasopharyngeal carcinoma cells expressing LMP1, these optimal nanoparticles showed a little lower tumor growth compared with free paclitaxel.

    Cytotoxicity effects of Rhizoma Coptidis on L929 murine fibroblast cells
    Manman Gu, Yanfei He, Chunyang Han, Qi Zhou, Tengfei Liu, Yanfei Huang, Cuiyan Liu
    2015, 24(2):  111-120.  DOI: 10.5246/jcps.2015.02.013
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    Rhizoma Coptidis (RC), a widely used traditional Chinese medicine, is commonly believed to be non-toxic. However, little is known about its cytotoxicity and relevant mechanisms at cellular and genetic levels. The present study aimed to explore the cytotoxicity of RC and its possible mechanisms related to cell cycle arrest, DNA damage and reactive oxygen species (ROS) level in L929 murine fibroblast cells. The cells were cultured in vitro and treated with different RC concentrations for 24 h. Cell viability was determined by CCK-8 method, morphological changes were observed with an inverted microscope, cell cycle and ROS level were examined by flow cytometry, and DNA damages were detected by comet assay. Our results showed that cell viability was significantly decreased in a dose-dependent manner when the RC concentration was higher than 1 mg/mL. A RC concentration above 1 mg/mL altered the morphology of L929 cells. Both cells at G2/M phase and the ROS level increased in the 2 mg/mL group. Each DNA damage indicator score increased in the groups with the RC concentration of above 0.05 mg/mL. Taken together, our studysuggested that RC at a high dosage exhibited cytotoxicity on L929 cells, which was likely to be the consequences of cell cycle arrest, DNA damage and accumulation ofintracellular ROS.

    Pathogens and possible opinion for antibiotic treatment in adult patients with 1-14 fecal WBC/HPF of acute non-bloody diarrhea
    Fengqin Hou, Xinting Sun, Guiqiang Wang
    2015, 24(2):  121-127.  DOI: 10.5246/jcps.2015.02.014
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    The pathogens and opinion for antibiotic treatment in adult patients with 1–14 fecal white blood cells per high power field (WBC/HPF) of acute non-bloody diarrhea remain obscure. The study attempts to clarify it. Stool specimens of adult patients with acute non-bloody diarrhea and stool examination showing 1–14 fecal WBC/HPF were collected for bacterial culture and viral detection. Patients included in this study were 196 cases with mean age of (37.9±17.4) years and 42.3% was women. The bacterial and viral detection rates were 63 (32.1%) and 21 (10.7%), respectively. Of the isolated pathogens, campylobacteria was present in 14 (22.0%) samples and was the most common bacteria and calicivirus was found in 10 (47.6%) samples and was the most common virus. Based on single pathogens, 46 cases were caused by invasive pathogens, 26 cases were caused by non-invasive pathogens. The body temperature was significantly higher in feverish patients caused by invasive pathogens than those caused by non-invasive pathogens ((38.4±0.7) ºC vs (37.7±0.4) ºC, P = 0.002). The probability of diarrhea caused by invasive pathogens was higher in patients with T>38.4 ºC than those with T≤38.4 ºC (RR = 1.5). When T>38.4 ºC is used as the threshold for antibiotic treatment, the misuse rate of antibiotics would decrease from 26.9% to 3.8% (P = 0.021). So T>38.4 ºC may be used as a possible reference value for antibiotic treatment in adult patients with 1–14 fecal WBC/HPF of acute non-bloody diarrhea.

    Case report
    Hypersensitive syndrome reaction of antiepileptic drug: two case reports and literature review
    Xiangji Dang, Haisheng Jiao, Faqin Wang, Fei Yang, Hui Li
    2015, 24(2):  128-132.  DOI: 10.5246/jcps.2015.02.015
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    Hypersensitivity syndrome reaction of antiepileptic drug (AED) can induce serious cutaneous, hematological and hepaticevents. In severe cases, fulminant hepatic failure may necessitate liver transplantation, and most patients die due to the liver failure. Severe adverse cutaneous reactions, including Steven-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN) and hypersensitivity syndrome, are rare but life-threatening. Its morality rate is as high as 5%–50%. Accurate early diagnosis and timely treatment may contribute to decreased morality rate. In this paper, we reported cases of hypersensitive syndrome reaction to carbamazepine (CBZ) or phenobarbital (PB) in two patients with epilepsy. Clarification of the therapeutic process and the early manifestation of epilepsy may be helpful to improve the epilepsy therapy while avoiding the potential severe adverse cutaneous reactions of AED. The two reported cases highlighted that the therapeutic process of CBZ and PB might lead to the fatal allergic reaction, which was mainly caused by the absence of epoxide-hydroxylase and the defect of hepatocytes.

    Short communication
    Synthesis of impurity B of Flumazenil
    Li Pan, Xi Wen, Yu Sha, Wenting Zhu, Maosheng Cheng
    2015, 24(2):  133-136.  DOI: 10.5246/jcps.2015.02.016
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    We report herein the synthesis of impurity B of Flumazenil via demethylation, benzyl protection, cyclization and debenzylation from 7-methoxyl-3,4-dihydro-4-methyl-2H-1,4-benzo-diazepine-2,5(1H)-dione. The structure of impurity B of Flumazenil was confirmed by 1H NMR, 13C NMR and HRMS, and the overall yield was 29.3%. The finding will be helpful for optimizing the synthetic process and quality control of Flumazenil.