绿藻多糖CH1-1的可控降解及其寡糖的制备研究

目的 研究绿藻多糖CH1-1的可控降解及其寡糖的制备。方法 采用不同浓度的三氟乙酸（TFA）或盐酸（HCl）对绿藻多糖CH1-1进行水解,通过薄层色谱和凝胶渗透色谱法研究了水解过程中不同的反应温度和反应时间对多糖降解产物的影响;通过控制酸浓度、反应温度和反应时间,通过Bio-Gel P4凝胶色谱柱分离得到寡糖产物;采用质谱法对得到的寡糖产物进行分析。结果 采用0.1 mol?L-1 和0.05 mol?L-1 TFA或HCl,随着反应温度的升高和反应时间的延长,多糖的分子量降低,其中0.1 mol?L-1 TFA、80 ℃及0.1 mol?L-1 HCl、80 ℃水解条件对多糖的降解作用较好;最终选取0.1 mol?L-1 HCl、80 ℃、3 h的水解条件对多糖进行降解制备寡糖,并采用Bio-Gel P4凝胶色谱柱对寡糖混合物进行分离纯化,电喷雾质谱分析表明,这些寡糖组分为聚合度为1～8的硫酸阿拉伯糖。结论 建立了绿藻多糖CH1-1可控降解方法,制备得到新颖的海洋硫酸寡糖。     

Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways

Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.     

海洋硫酸鼠李寡糖的制备研究

目的 探究海洋硫酸鼠李寡糖的制备方法并对所得寡糖的特征进行了研究。方法 采用三氟乙酸(TFA)和盐酸(HCl)对海洋硫酸鼠李多糖进行降解,通过薄层色谱法和凝胶渗透色谱法对降解条件进行分析,确定寡糖的制备方法;制备得到一系列硫酸鼠李寡糖,采用质谱法对其特征进行研究。结果 0.1 mol.L-1 TFA 80℃及0.1 mol.L-1 HCl 80℃水解条件对多糖的降解作用较好,且通过Bio-Gel P4凝胶色谱柱对寡糖能进行有效分离;最终选取0.1 mol.L-1 HCl 80℃10 h的水解条件对硫酸鼠李多糖进行降解,采用Bio-Gel P4凝胶色谱柱对寡糖混合物进行分离纯化,负离子模式下的电喷雾质谱分析表明,这些寡糖为聚合度为1–11的硫酸化鼠李寡糖。结论 首次建立了海洋新型硫酸鼠李寡糖的制备方法,这些硫酸鼠李寡糖为新型海洋特征寡糖。     

Development and validation of a HPLC–ES-MS/MS method for the determination of glucosamine in human synovial fluid

A new HPLC method for the determination of glucosamine (2-amino-2-deoxy-d-glucose) in human synovial fluid was developed and validated. Synovial fluid samples were analyzed after a simple protein precipitation step with trichloroacetic acid using a polymer-based amino column with a mobile phase composed of 10 mM ammonium acetate (pH 7.5)–acetonitrile (20:80, v/v) at 0.3 mL/min flow rate. d-[1-¹³C]glucosamine was used as internal standard. Selective detection was performed by tandem mass spectrometry with electrospray source, operating in positive ionization mode and in multiple reaction monitoring acquisition (m/z 180 → 72 and 181 → 73 for glucosamine and internal standard, respectively). The limit of quantification (injected volume = 3 μL) was 0.02 ng, corresponding to 10 ng/mL in synovial fluid. Calibration curves obtained using matrix-matched calibration standards and internal standard at 600 ng/mL were linear up to 2000 ng/mL. Precision values (%R.S.D.) were ≤14% in the entire analytical range. Accuracy (%bias) ranged from −11% to 10%. The recoveries measured at three concentration levels (50, 800, and 1500 ng/mL) were higher than 89%. The method was successfully applied to measure endogenous glucosamine levels in synovial fluid samples collected from patients with knee osteoarthritis and glucosamine levels after oral administration of glucosamine sulfate (DONA^®) at the dose of 1500 mg/day for 14 consecutive days (steady-state).     

Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita

Aim of the study

Artemisia vestita is a common traditional Tibetan medicinal plant which has been used widely in China for treating various inflammatory diseases. Since little is known about its active components, the purpose of this study was to isolate and identify the immunosuppressive compounds from Artemisia vestita.

Materials and methods

A bioassay-guided isolation was performed with picryl chloride-induced contact hypersensitivity in mice. MTT assay and Flow cytometric analysis were used for determining Con A-induced lymphocyte proliferation and CD25 expression in T cells, respectively.

Results

The ethanol extract of the Artemisia vestita was found to possess significant inhibitory activity against the picryl chloride-induced contact hypersensitivity in mice. Then 4 fractions were isolated by macroporous adsorption resin and one of these fractions (AV3), which showed the highest activity in in vivo test, was further subjected to column chromatography. Nine known flavones were isolated and identified as pectolinarigenin (1), jaceosidin (2), cirsilineol (3), cirsimaritin (4), hispidulin (5), quercetin (6), 6-methoxytricin (7), acacetin (8), and apigenin (9). The structures of the 9 flavones were elucidated by spectral techniques. All the compounds were evaluated for their inhibitory activity on the proliferation and activation of T cells in vitro. Among the 9 flavones, cirsilineol (3), 6-methoxytricin (7) and apigenin (9) significantly inhibited T cell proliferation and activation in the bioassays.

Conclusion

The result suggests that cirsilineol, 6-methoxytricin and apigenin are the major active components in Artemisia vestita.