Investigation of binding between fluoroquinolones and pepsin by fluorescence spectroscopy and molecular simulation.

In this paper, the interactions of pepsin with fluoroquinolones, including norfloxacin (NFX) or ofloxacin (OFX), were investigated using fluorescence spectroscopy. The effects of NFX or OFX on pepsin showed that the molecular conformation of pepsin and the microenvironment of tryptophan residues were changed under mimicked physiological conditions. Static quenching was suggested as a factor. Quenching constants and binding constants were determined and thermodynamic parameters were calculated at three temperatures (25°C, 31°C and 37°C). Molecular interaction distances (binding distance r) were obtained. Binding was enthalpy driven and the process was spontaneous. Synchronous fluorescence, three-dimensional fluorescence spectroscopy and molecular simulation were used for analysis. Interactions were further tested using molecular modelling. Quenching and binding constants of NFX with pepsin were the highest when testing NFX/OFX/fleroxacin/gatifloxacin with pepsin combinations. NFX was the strongest quencher, and affinity of NFX for pepsin was higher than that of OFX/fleroxacin/gatifloxacin.

Off-line two-dimensional liquid chromatography coupled with diode array detection and quadrupole-time of flight mass spectrometry for the biotransformation kinetics of Ginkgo biloba leaves extract by diabetic rat liver microsomes.

Ginkgo biloba leaves extract (GBE), one of the most widely used traditional Chinese medicines worldwide, can be used for the treatment of diabetes mellitus (DM). However, its biotransformation in liver is not fully known under the state of DM. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (q/TOF-MS) was established for the qualification and quantification of the biotransformation of GBE in normal and diabetic rat liver microsomes (RLMs). 6 metabolites were tentatively identified according to the exact molecular weights and the characteristic fragment ions provided by q/TOF-MS data. The results of metabolic stability showed that the metabolic ratio of four target compounds including quercetin, genistein, kaempferol and isorhamnetin in diabetic RLMs were significantly enhanced when comparing with normal RLMs. The results of enzyme kinetics showed that compared with normal RLMs, the Michaelis-Menten constant (Km) value of genistein was obvious increased while its maximal velocity (Vmax) and intrinsic clearance (CLint) values were significantly decreased by diabetic RLMs, and the Vmax and CLint values of kaempferol and isorhamnetin were notably enhanced while their Km values were markedly reduced. For the half-time (t1/2) values of four target compounds and the Km, Vmax and CLint values of quercetin, there were not statistically significant changes between normal and diabetic RLMs. The results suggest that the developed off-line 2D LC-DAD-q/TOF-MS method is an easy and accurate approach for the study of GBE biotransformation in RLMs and may provide the essential data for further pharmacological and clinical studies of GBE.

Predictive metabolic signatures for the occurrence and development of diabetic nephropathy and the intervention of Ginkgo biloba leaves extract based on gas or liquid chromatography with mass spectrometry.

Diabetic nephropathy (DN) is one of the leading causes of death in diabetes mellitus (DM). Early warning and therapy has significant clinical value for DN. This research sought to find biomarkers to predict the occurrence and development of DN and the intervention of Ginkgo biloba leaves extract (GBE) by quantifying fatty acids, amino acids, and nucleosides and nucleobases in rat plasma. Samples were respectively collected at the weekend of 5-10 weeks after diabetic rats induced by streptozotocin were defined. Plasma fasting blood-glucose, kidney index, blood urea nitrogen, creatinine, urine albumin excretion and ultrastructural morphology of kidney were measured or observed. Fatty acids, amino acids and nucleosides and nucleobases in rat plasma were analyzed by gas chromatography or liquid phase chromatography and mass spectrometry, respectively. From the biochemical index and morphological change of kidney, the rats from the 5th to 7th week were in the stage of DM while from the begin of 8th week the rats were suggested in the early stage of DN. The results of quantitative metabolomics showed that 16 differential metabolites were related to the progression of DN, and oleic acid, glutamate and guanosine might be the potential biomarkers of kidney injury. 14 differential metabolites were related to GBE against the progression of DN, while oleic acid and glutamate might be the potential biomarkers of GBE against kidney injury. Those findings potentially promote the understanding of the pathogenic progression of DN and reveal the therapeutic mechanism of GBE against DN.

Chemical Profiling and Comparison of Sangju Ganmao Tablet and Its Component Herbs Using Two-Dimensional Liquid Chromatography to Explore Compatibility Mechanism of Herbs.

Sangju Ganmao tablet (SGT), a well-known Chinese patent medicine used to treat cold symptoms, is made from eight herbal medicines. In this study, an off-line hydrophilic interaction × reversed-phase two-dimensional liquid chromatography (HILIC × RP 2D-LC) method was developed to comprehensively separate the chemical constituents of SGT. Through optimization of the experimental conditions, a total of 465 peaks were finally detected in SGT, and the structures of 54 selected compounds were fully identified or tentatively characterized by quadrupole time-of-flight mass spectrometry (qTOF-MS) analysis. The established 2D-LC analysis showed high orthogonality (63.62%) and approximate 11-fold improvement in peak capacity (2399 and 1099, obtained by two calculation methods), in contrast to conventional one-dimensional RPLC separation. The eight component herbs of SGT were also respectively separated by using the 2D-LC system, and we found that a total of 12 peaks detected in SGT were not discovered in any component herbs. These newly generated chemical constituents would benefit better understanding of the compatibility mechanism of the component herbs. The strategy established in this study could be used for systematic chemical comparison of SGT and its component herbs, which contributes to exploration of herbal compatibility mechanism.

Dynamic Characteristics and HIV Infection of Men who have Sex with Men from 2011 to 2017 in Yinchuan, Ningxia, China.

OBJECTIVES: Ningxia Hui Autonomous Region, an important area for ethnic Hui settlement in Northwest China, is a low HIV prevalence region. However, HIV infection rates among men who have sex with men (MSM) in Ningxia have increased to an alarming level, despite scale-up of control measures in recent years. This study aimed to understand the demographical and sexual behavior dynamics of MSM and to explore the factors associated with HIV infection. METHODS: Annual cross-sectional surveys were carried out among MSM during 2011~2017 in Yinchuan, the capital city of Ningxia. Information regarding social demographics, sexual behavior and HIV prevention knowledge was collected. Blood samples were taken for HIV, HCV serological and genetic analysis, and syphilis serological analysis. The dynamic trend was analyzed with trend χ2 test and factors associated with HIV infection were identified by multivariate logistic regression analysis. RESULTS: The study found a decreasing trend for mean age of the MSM population over the study period. MSMs with a college education or higher increased significantly, while the proportions that were in a marriage significantly decreased over the study period. The rate of HIV positive among MSM increased during the study period (p<0.05), however, the rate of recently diagnosed infections decreased from 2012 (p<0.05). Overall, a very high proportion (98%) of MSM had basic knowledge of HIV prevention, however, only approximately 40% of them used condoms consistently during anal sex with male partners. Unprotected anal sex was identified as a risk factor associated with HIV infection, as was syphilis infection. Local residency status and MSM who received intervention and detection services were the factors that decreased HIV infection risk. Sequence analysis identified the HIV-1 CRF55_01B subtype from MSM for the first time in Yinchuan. CONCLUSION: The reduction of recent HIV diagnoses is an encouraging sign of successful HIV control measures in MSM in Ningxia. The finding that a high proportion of MSM had knowledge of HIV prevention but still conducted unprotected sex highlights the need for further control measures to change unsafe sexual practices among MSM.

Simultaneous Determination of 11 Components in Yinzhihuang Preparations and Their Constituent Herbs by High-Performance Liquid Chromatography with Diode Array Detector.

A simple and sensitive liquid chromatography method with diode array detector was established for simultaneous determination of 11 components (geniposidic acid, chlorogenic acid, caffeic acid, geniposide, luteoloside, isochlorogenic acid C, baicalin, luteolin, wogonoside, baicalein and wogonin) in various commercial Yinzhihuang preparations and their herbs by optimizing the extraction, separation and analytical conditions. Eleven components were identified on the basis of their retention times and mass spectra. Chromatographic separation was performed on a C18 analytical column with a gradient elution of acetonitrile and 0.1% formic acid water solution at a flow rate of 1.0 mL/min. The linearity, precision and accuracy of the data obtained were acceptable. The method was used to analyze four Yinzhihuang preparations (powder, capsule, oral liquid and injection) and related herbs (Radix Scutellariae, Flos Lonicerae, Herba Artemisiae Scopariae and Fructus gardeniae). Results suggested that the optimized method could be considered as a good approach to control the quality of Yinzhihuang preparations and their herbs.

LC-MS/MS method for the simultaneous quantification of 11 compounds of Ginkgo biloba extract in lysates of mesangial cell cultured by high glucose.

The mesangial cell (MC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN), but the compounds interacting with cell are still unknown, which may be potential bioactive components. Based on this, the determination of GBE in MC lysates was proposed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in this study. The MC was cultured with normal or high glucose with GBE for 4, 8, 12, 16, 24 and 48h. The harvested cell was extracted with 40% acetic acid in water and further analyzed by LC-MS/MS. All the validation data including linearity, intra-day and inter-day precision, limit of detection and quantification, matrix effect, and stability were within the required limits. The validated method was successfully applied to quantify 11 compounds of GBE in cell lysates. The results showed that high glucose prolonged the peak time of all observed 11 compounds and peak concentrations of bilobalide, ginkgolide C, ginkgolide B, quercetin, luteolin, kaempferol, isorhamnetin and genkwanin in cell lysates, which hinted that these compounds may be the potential bioactive components of GBE with preventive effect against DN.

Screening for Potential Bioactive Components in Ginkgo biloba Extract by the Rat Renal Tubular Epithelial Cell Extraction and LC-MS/MS.

Rat renal tubular epithelial cell (RTEC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN). The compounds in GBE binding with cell membrane or entering into cell are still unknown, which may be potential bioactive components. In this paper, a powerful method for screening and analyzing the potential bioactive components from GBE was developed using cell extraction coupled with high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). 8 prototype compounds and 5 metabolites were obtained, among which 6 prototype compounds and 1 metabolite were identified or tentatively characterized as rutin, bilobalide, ginkgolide B, ginkgolide C, genkwanin, apigenin and diosmetin by comparing their retention times and MS spectra with those of authentic standards or literature data. The 6 prototype compounds were further quantitatively analyzed using electrospray ionization in negative mode multiple reaction monitoring (MRM). The results showed that high glucose changed the Tmax, MRT(0-t), Cmax and AUC(0-t) of all observed compounds and decreased the t1/2 of genkwanin and apigenin, significantly. The overall findings indicate that 8 prototype compounds may be the potential bioactive components of GBE with preventive effect against DN and the method of RTEC extraction coupled with LC-MS/MS technology screening method we developed is a feasible, rapid, and useful tool for screening and analyzing potential bioactive components.

Target cell extraction coupled with LC-MS/MS analysis for screening potential bioactive components in Ginkgo biloba extract with preventive effect against diabetic nephropathy.

A rapid and useful approach for screening potential bioactive components in Ginkgo biloba extract (GBE) with preventive effect against diabetic nephropathy (DN) was developed using mesangial cells extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Mesangial cells were first divided into two groups according to their treatments with high glucose or high glucose plus GBE. After incubation for 4, 8, 12, 16, 24 and 48 h, the cells were harvested and extracted with 40% acetic acid in water before LC-MS/MS analysis. Then, 19 compounds and five metabolites were found to selectively combine with mesangial cells. Notably, compounds including quercetin and rutin were identified or tentatively characterized according to the results of retention time and MS spectra, which is highly consistent with our previous reports that quercetin and rutin are potent protective agents against glomerulosclerosis in DN. Therefore, all these results indicate that target cell extraction coupled with LC-MS/MS analysis can be successfully applied for predicting the bioactive components in GBE with preventive effect against DN.

Two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to study the excretion and metabolic interaction of edaravone and taurine in rats.

In this study, two independent and complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the determination of edaravone or taurine in rat urine, feces and bile after intravenous administration, using 3-methyl-l-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Edaravone was separated on an Agilent Eclipse Plus C18 column (100×2.1 mm, 3.5 µm) using methanol and water (containing 5 mM ammonium formate and 0.02% formic acid) as mobile phase, while taurine was performed on a Waters Atlantis HILIC Silica column (150×2.1 mm, 3 µm) using acetonitrile and water (containing 5mM ammonium formate and 0.2% formic acid) as mobile phase. The mass analysis was performed in a Triple Quadrupole mass spectrometer via multiple reaction monitoring (MRM) with negative ionization mode. The optimized mass transition ion pairs (m/z) for quantification were 173.1→92.2 and 187.2→106.0 for edaravone and its IS, 124.1→80.0 and 172.0→80.0 for taurine and its IS, respectively. The validated methods have been successfully applied to the excretion and metabolism interaction study of edaravone and taurine in rats after independent intravenous administration and co-administration with a single dose. The results demonstrated that there were no significant alternations on the metabolism and cumulative excretion rate of edaravone and taurine, implying that the proposed combination therapy was pharmacologically viable.

Fingerprint analysis and multi-ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection.

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C18 column (50 × 2.1 mm id, 1.8 µm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9-104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi-ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.

Quantitative and qualitative analysis of common peaks in chemical fingerprint of Yuanhu Zhitong tablet by HPLC-DAD-MS/MS.

A quality control (QC) strategy for quantitative and qualitative analysis of "common peaks" in chemical fingerprint was proposed to analyze Yuanhu Zhitong tablet (YZT), using high performance liquid chromatography with diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS). The chromatographic separation was achieved on an Agilent Eclipse plus C18 column with a gradient elution using a mixture of 0.4‰ ammonium acetate aqueous (pH 6.0 adjusted with glacial acetic acid) and acetonitrile. In chemical fingerprint, 40 peaks were assigned as the "common peaks". For quantification of "common peaks", the detection wavelength was set at 254 nm, 270 nm, 280 nm and 345 nm, respectively. The method was validated and good results were obtained to simultaneously determine 10 analytes (protopine, jatrorrhizine, coptisine, palmatine, berberine, xanthotoxin, bergapten, tetrahydropalmatine, imperatorin and isoimperatorin). For qualification of "common peaks", 33 compounds including 10 quantitative analytes were identified or tentatively characterized using LC-MS/MS. These results demonstrated that the present approach may be a powerful and useful tool to tackle the complex quality issue of YZT.

Development and application of a LC-MS/MS assay for the simultaneous quantification of edaravone and taurine in beagle plasma.

An LC-MS/MS method was developed and validated for the simultaneous quantification of edaravone and taurine in beagle plasma. The plasma sample was deproteinized using acetonitrile containing formic acid. Chromatographic separations were achieved on an Agilent Zorbax SB-Aq (100 × 2.1 mm, 3.5 µm) column, with a gradient of water (containing 0.03% formic acid) and methanol as the mobile phase at a flow rate of 0.3 mL/min. The analyte detection was carried out in multiple reaction monitoring mode and the optimized precursor-to-product transitions of m/z [M+H](+) 175.1 → 133.0 (edaravone), m/z [M+H](+) 189.1 → 147.0 (3-methyl-1-p-tolyl-5-pyrazolone, internal standard, IS), m/z [M-H](-) 124.1→80.0 (taurine), and m/z [M-H](-) 172.0 → 80.0 (sulfanilic acid, IS) were employed to quantify edaravone, taurine, and their corresponding ISs, respectively. The LOD and the lower LOQ were 0.01 and 0.05 µg/mL for edaravone and 0.66 and 2 µg/mL for taurine, respectively. The calibration curves of these two analytes demonstrated good linearity (r ＞ 0.99). All the validation data including the specificity, precision, recovery, and stability conformed to the acceptable requirements. This validated method has successfully been applied in the pharmacokinetic study of edaravone and taurine mixture in beagle dogs.

Multidimensional information-based HPLC technologies to evaluate traditional chinese medicine.

Traditional Chinese medicines (TCMs) are usually complex mixtures and contain hundreds of chemically different constituents, which make the quality control (QC) of crude drugs and their medical preparations extremely difficult. In the past years, with the rapid development of modern instrumental analysis and computer-aided data processing techniques, great progress has been made in the research of quality standards and the development of QC techniques. Among them, the use of the high-performance liquid chromatography (HPLC) technique is one of the best approaches because of its high separation efficiency. However, one-way separation, single detection methods or data processing cannot meet the needs of the QC of TCMs. Multidimensional information-based HPLC technologies such as two-dimensional HPLC, HPLC coupled with several different detection methods and HPLC fingerprint combined with multicomponent quantification have solved this problem with their comprehensive analysis; these methods have gradually been accepted by more researchers for further in-depth study. The present work provides an overview of the development of QC for TCMs based on HPLC technologies with modern hyphenated techniques, multiseparation methods and some common data processing methods in fingerprint spectra over the last six years.

Genetic variants of phosphodiesterase 4D gene are associated with an enhanced risk for ischemic stroke in young Chinese population.

BACKGROUND: Previous studies have shown that the phosphodiesterase 4D (PDE4D) gene is a susceptibility gene for ischemic stroke (IS) primarily in elder populations. However, few studies have reported the role of the PDE4D gene polymorphisms in a young cohort. AIMS: To investigate the association between the PDE4D gene polymorphisms and young-onset IS in Chinese population. MATERIALS AND METHODS: A total of 186 young patients (18-45 years) with IS and 232 matched control subjects were recruited. Two SNPs (rs918592 and rs2910829) in PDE4D gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio and 95% confidence intervals (95% CI) were calculated to test the association between the genetic factors and IS. RESULTS: The rs918592A/A genotype frequency and A allele frequency, rs2910829 CT/TT genotype frequency and T allele frequency of young IS group were significantly higher than those of the control group ( P < 0.05). Besides, the frequency of Hap (A-T) was remarkably higher in the young patients than that in the controls (OR =4.047, 95% CI: 3.521-4.652). Hap (A-C) and Hap (G-C) were associated with decreased risk of IS (OR =0.640, 95% CI: 0.452-0.906; OR =0.675, 95% CI: 0.466-0.978, respectively). CONCLUSIONS: Our findings suggest that the rs918592 and rs2910829 polymorphisms and haplotypes of PDE4D gene are significantly associated with IS in Chinese young population.

A new strategy for quality control and qualitative analysis of Yinhuang preparations by HPLC-DAD-MS/MS.

A combinative method using fingerprint analysis (FA) and multi-ingredients quantification (MIQ) was developed and validated for the quality control of Yinhuang (YH) preparations including granule, capsule, and lozenge by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). Common peaks with or without standard references in FA were confirmed or identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The chromatographic separations were achieved on a Sepax GP-C(18) column (250 mm × 4.6 mm i.d., 5 µm) with a gradient elution using a mixture of 0.1 % formic acid methanol solution and 0.1 % formic acid water solution. In quantitative analysis, nine bioactive constituents (chlorogenic acid, caffeic acid, luteoloside, baicalin, luteolin, wogonoside, baicalein, wogonin, and oroxylin A) were simultaneously determined. The detection wavelength was set at 275 nm, 320 nm, and 350 nm according to the absorption properties of the nine quantified compounds. The linearity, recovery, intraday and interday precision, accuracy, limit of detection (LOD) and quantification (LOQ), repeatability and stability were all tested and good results were obtained. In the FA, 320 nm was selected. The correlation coefficients of similarity were determined on the basis of the relative retention time (RRT) and relative peak area (RPA) of 20 common peaks in chromatographic fingerprints. Results indicated that both the RRT and RPA of 20 common peaks shared a close similarity. From the 20 common peaks, 18 compounds, including the nine quantified compounds, were identified or tentatively characterized by comparing their retention times, UV spectra, and MS spectra with those of standard compounds or literature data. The study not only presents a powerful and reliable analytical tool for the quality control of YH preparations, but also provides the chemical evidence for revealing the material basis of their therapeutic effects.

Development of a novel method combining HPLC fingerprint and multi-ingredients quantitative analysis for quality evaluation of traditional Chinese medicine preparation.

A novel method combining high performance liquid chromatography (HPLC) fingerprint and simultaneous quantitative analysis of multiple active components was developed and validated for quality evaluation of one type of traditional Chinese medicine preparations: Shuang-huang-lian (SHL) oral liquid formulation. For fingerprint analysis, 45 peaks were selected as the common peaks to evaluate the similarities among several different SHL oral liquid preparations collected from manufacturers. Additionally, simultaneous quantification of eleven markers, including chlorogenic acid, caffeic acid, rutin, forsythiaside, scutellarin, baicalin, forsythin, luteoloside, apigenin, baicalein and wogonin, was performed. Statistical analysis of the obtained data demonstrated that our method has achieved desired linearity, precision and accuracy. Finally, concentrations of these eleven markers in SHL oral liquid prepared by different manufacturers in China were determined. These results demonstrated that the combination of HPLC chromatographic fingerprint and simultaneous quantification of multi-ingredients offers an efficient and reliable approach for quality evaluation of SHL oral liquid preparations.

Protective effects of rutin on rat glomerular mesangial cells cultured in high glucose conditions.

The protective effect of rutin on the glomerulosclerosis of diabetic nephropathy (DN) in rat mesangial cells was investigated. The cultured mesangial cells were divided into eight groups: normal, solvent control, high glucose, low dose of rutin, moderate dose of rutin, high dose of rutin, captopril and Ginkgo biloba extract. The cell cycles, type IV collagen and laminin in cytoplasm, TGF-ß1 mRNA of mesangial cells, Smad 2/3 and Smad 7, and the activities of four antioxidant indexes including T-SOD, MDA, CAT and GSH-Px were measured by flow cytometry, radioimmunoassay, RT-PCR, western blotting and visible spectrophotometry, respectively. Compared with the high glucose group, rutin decreased the cell percentages of the G0/G1 phase and inhibited the expression of Smad 2/3, laminin and type IV collagen, and TGF-ß1 mRNA level, significantly. The antioxidant capacity, the cell percentages of S phase and Smad 7 expression were significantly increased by rutin. These results suggest that rutin is a potent protective agent against glomerulosclerosis in DN.

[Genetic characteristics of entervirus 71 isolated in Ningxia Hui Autonomous region in 2009].

OBJECTIVE: To analyze the genetic characteristics of EV71 strains isolated from HFMD cases in Ningxia Hui Autonomous Region in 2009. METHODS: In 2009, totally 385 specimens from 344 HFMD cases were collected from Ningxia. Enterovirus isolation was performed in RD cell line from all the specimens. EV71 isolates were identified by specific RT-PCR from the positive cultures, and sequences of complete EV71 VP1 encoding region were determined for farther analyses. RESULTS: Totally from 126 EV strains isolated in this study, 58 EV71 strains (46%) were identified. And complete VP1 sequences of 46 EV71 strains were determined, and genetic analyses were performed. It was showed that the nucleotide identity of 46 Ningxia strains with the representatives of A and B genotypes were 81.7%-82.8% and 83.1%-85.2%, and the amino acid identity were 93.9%-95.9% and 96. 2%-97.9% respectively. The nucleotide identity of NingXia EV71 isolates with representatives of subgenotype C1, C2, C3, C4a, C4b, and C5 were 88.3%-90.6% (97.9%-99.6%), 88.3%-90.1% (97.9%-99.3%), 87.8%-89.0% (97.6%-98.9%), 94.2%-98.9% (97.9%-100%), 91.8%-94.1% (98.6%-99.6%), and 86.7%-89.1% (97.9%-98.9%). Phylogenetic tree analysis revealed that 46 stains were clustered with reference stains of subgenotype C4 and the Ningxia EV71 isolates were belonged to subgenotype C4a. CONCLUSION: EV71 of subgenotype C4a had spread widely in Ningxia in 2009, which was absolutely predominant type in Ningxia in 2009 and also as the predominant type in China mainland since 2005.

[Synthesis and absorption spectra properties of Au-Ag alloy nanoparticles using gallic acid as reductant].

Gold-silver alloy nanoparticles with a homogeneous size distribution and good stability were synthesized in aqueous solution by one-step reduction with gallic acid as reductant in the absence of other stabilizers for the first time. The absorption spectra of as prepared gold-silver alloy nanoparticles under different reaction temperatures and with various gold-silver mole ratios were studied. The absorption spectra exhibit only one single peak with the maximum wavelength located between the absorption peaks of Au nanoparticles and Ag nanoparticles, however, the absorption spectrum of the physical mixtures of Au nanoparticles and Ag nanoparticles exhibits two absorption peaks coming from their corresponding monometallic metal nanoparticles, suggesting primarily the formation of the alloy nanoparticles. The maximal absorption peaks of alloy nanoparticles showed gradually a blue-shift with the rising of reaction temperature companying with the increase in absorbance and the narrowing of peak shape. The maximum absorption wavelengths of alloy nanoparticles showed a red-shift with increasing Au/Ag molar ratio in a linear fashion, further indicating the formation of alloy nanoparticles. HRTEM images showed that the as-prepared alloy nanoparticles with Ag/Au molar ratio of 1:1 are close to sphere in shape, homogeneous in size distribution and without capping phenomenon. EDX analysis suggested the present of Au and Ag in individual nanoparticles, and the Au/Ag mole ratio value of a sample with a nominal element composition was found to be close to the nominal value.