射干中乳酸脱氢酶抑制剂的筛选分离与质谱分析

本研究采用高效液相色谱-电喷雾质谱联用技术(HPLC-ESI-MS)对射干提取物中异黄酮类化学成分进行分析鉴定,以乳酸脱氢酶作为生物靶分子,运用超滤质谱技术筛选出6种酶抑制剂,分别为鸢尾苷(tectoridin)、鸢尾新苷A(Iristectorin A)、野鸢尾苷(iridin)、鸢尾苷元(tectorigenin)、野鸢尾黄素(irigenin)和次野鸢尾黄素(irisflorentin),并采用半制备型高效液相色谱技术(Semi-prep·HPLC)分离纯化射干提取物中的活性成分。所得单体分离收集液经分析型高效液相色谱法检测,纯度均大于90%。结果证明利用液相色谱-超滤-质谱-半制备型高效液相色谱联用技术可以快速筛选、鉴定、分离射干中的活性物质。此方法对于筛选酶抑制剂有快速和灵敏等优势,具有广泛的应用前景。     

HPLC法测定抗宫炎凝胶剂中金石蚕苷含量的研究

目的:采用高效液相色谱法(HPLC)测定抗宫炎凝胶剂中金石蚕苷的含量,为制定其质量标准提供基础。方法:采用十八烷基硅烷键合胶为填充剂,乙腈-0.5%磷酸(18:82)为流动相;检测波长为332 nm;流速为1.0 ml/min,探讨HPLC测定抗宫炎凝胶剂中金石蚕苷含量的专属性、精密度、稳定性、重现性等。结果:金石蚕苷在0.104~0.468 mg/ml范围内呈良好的线性关系(r=0.9999),平均回收率为99.76%,RSD为1.56%。结论:采用HPLC测定抗宫炎凝胶剂中金石蚕苷含量的专属性、精密度、稳定性、重现性均良好,该方法简便可靠,可用于抗宫炎凝胶剂的质量标准的研究。     

高效液相色谱法测定多抗原肽MAP4原料药中醋酸和三氟醋酸的含量

目的:测定多抗原肽MAP4原料药中醋酸及三氟乙酸的残留量。方法:采用反相高效液相色谱法,采用十八烷基硅烷键合硅胶为填充剂的Kromasil(250mm×4.6mm,10μm),200色谱柱;pH3.0磷酸盐缓冲液及甲醇为流动相,进行梯度洗脱;检测波长:210nm;柱温为25℃;流速:0.8ml/min;进样体积:40μl。结果:该方法中醋酸和三氟乙酸的分离度符合要求;醋酸的检测限0.1μg;三氟乙酸的检测限0.1μg;样品中未检出三氟醋酸。醋酸含量为5.7%。结论:该方法快速,准确,专属性强,精密度高,对多抗原肽MAP4原料药的质量控制有一定的意义。     

灯盏花素粉针剂在不同pH值溶媒中的稳定性观察

目的观察灯盏花素粉针剂在不同pH值溶液中的稳定性。方法在室温(20℃)下,将5%、10%葡萄糖注射液及0.9%氯化钠注射液的pH值调整为3.3、3.6、3.9、4.2后,再分别加入灯盏花素粉针剂,并采用紫外分光光度法在6 h内的不同时间取样测定其含量。结果灯盏花素粉针剂在溶液pH值为3.3时,含量下降很快;pH值为3.6、3.9时,含量下降变缓;pH值为4.2时,含量趋于稳定,无明显变化。结论影响灯盏花素粉针剂稳定性的主要因素是稀释溶媒的pH值。当pH值在3.3~3.6时,灯盏花素易产生浑浊沉淀现象,故建议临床不用葡萄糖注射液作灯盏花素粉针剂的稀释溶媒为好。     

高效液相色谱法检测格列齐特缓释片中格列齐特含量的分析

目的采用高效液相色谱法对格列齐特缓释片中的格列齐特含量进行检测。方法用型号为Hypersil ODS2的250mm×4.6mm液相色谱柱,流动相为甲醇-水(60:40,磷酸调至pH 3.3),填料颗粒直径为5μm,检测波长为228nm,流速为1.0ml/min。结果格列齐特在0.6~6.0μg/ml浓度范围内呈良好的线性关系,平均回收率为99.32%,RSD=1.28%。结论应用高效液相色谱法对格列齐特缓释片中格列齐特含量进行测定,具有操作简便、重现性好,可用于该制剂格列齐特含量测定。     

浅析高效液相色谱在药物分析中的应用

当前,随着药品检测的发展,高效液相色谱技术是常用的定性和定量分析的技术,我们可以使用此技术同时检测多种防腐剂,不仅省时省力,还具有精确科学的优势。但同时,我们也发现,在使用高效相色谱进行药物分析检测时,难点在于确定高效液相色谱的流动相的配置比例、要素组成、流速以及检测时的波长;高效液相色谱在确定方案之后,就需要进行样品的前处理、样品的检测、检测方法的精确性和稳定性。本文就主要分析高效液相色谱技术的发展现状,对高效液相色谱检测存在的问题进行分析,并提出了一些合理化的建议,希望有所帮助。     

盐酸司来吉兰中右旋异构体含量的HPLC分析

采用高效液相色谱法分离和检测盐酸司来吉兰的光学异构体。对影响分离和检测的色谱柱、流动相、检测波长和样品的配制方法等进行了筛选,并对建立的方法进行了线性、精密度、回收率、最小检出量和溶液的稳定性研究。结果表明在v(环已烷)∶v(异丙醇)为99.8∶0.2,流速1 mL.min-1,柱温10~30℃条件下,盐酸司来吉兰的光学异构体得到了基线分离,所建立的自身对照方法能有效的测定出盐酸司来吉兰中右旋异构体的含量。     

高效液相色谱法测定山楂叶中熊果酸含量的研究

本文建立了一种可靠性高、重现性好的高效液相色谱(HPLC)测定山楂叶中熊果酸含量的方法,在测定中采用富集和固相萃取组合纯化工艺去除干扰物质。高效液相色谱测定条件为Hypersil(ODS)色谱柱,流动相为甲醇:0.2%磷酸二氢钠(90∶10,V/V),检测波长210nm,流速0.8mL/min。熊果酸浓度在100~800μg/mL与峰面积存在良好线性关系(r2=0.9992),该方法准确可靠,日内稳定性标准偏差在0.6%~1.5%,日间稳定性标准偏差在0.7%~2.6%。为不同产地山楂叶中熊果酸含量建立有效的分析方法。     

高效液相色谱法制备Ⅰ型胶原蛋白及其性质研究

从猪皮中分离纯化I型胶原蛋白,并对其部分性质进行研究。采用粗提法,高效液相色谱半制备法进行分离纯化,用分析型高效液相色谱检测纯度,同时对其理化性质进行鉴定,并对所提取的胶原进行全身急性毒性试验及皮肤致敏试验。高效液相色谱测定结果显示所得样品为一单峰,理化性质测定结果符合I型胶原蛋白特征;通过整体水平的安全性评价,表明该方法提取的I型胶原蛋白具有较大的安全性和可靠性。因此,用高效液相色谱可制得高纯度且具有良好生物安全性的I型胶原蛋白。     

超临界流体萃取——高效液相色谱法测定百合中秋水仙碱

分别用超临界二氧化碳流体和有机溶剂萃取百合中的秋水仙碱,然后用高效液相色谱法直接测定萃取物中秋水仙碱的含量,从而测得百合中秋水仙碱的含量。超临界流体萃取的条件是：用乙醇作提携剂,萃取压力为18MPa,萃取温度为40℃,高效液相色谱测定条件为：ODS柱,甲醇：磷酸二氢钾溶液作流动相,检测波长为220nm,此法快速,简便,准确,可应用于秋水仙碱原料,制剂及其它植物中秋水仙碱含量的测定。     

Amperometric detection of insulin at renewable sol-gel derived carbon ceramic electrode modified with nickel powder and potassium octacyanomolybdate(IV)

A renewable three-dimensional chemically modified carbon ceramic electrode (CCE) containing nickel powder and K4[Mo(CN)8] was constructed by sol-gel technique. The electrochemical properties and stability of modified electrode was evaluated by cyclic voltammetry in pH range 4-10. The redox couple of [Mo(CN)8] (4-/3-) was shown both as a solute in electrolyte solution and as a component of a carbon based conducting composite electrode. The apparent electron transfer rate constant (ks) and transfer coefficient (alpha) were determined by cyclic voltammetry and they were about 17.1 and 0.57 s(-1), respectively. The catalytic activity of the modified CCE toward insulin oxidation was investigated at pH range of 3-8 by cyclic votammetry. The modified electrode showed excellent electrocatalytic activity toward insulin electroxidation at physiological pH value. The modified electrode was used for insulin detection chronoamperometrically at pH 7. Under optimized condition in amperometry method, the concentration calibration range, detection limit and sensitivity were 0.5-500 nM, 0.45 nM and 6140 nA/microM, respectively. Flow injection amperometric determination of insulin at pH 7.4, at this modified electrode yielded a calibration curve with the following characteristics, linear dynamic range 100-500 pM; sensitivity 8.1 nA/nM and detection limit 40 pM (based on S/N = 3). The inherent stability at wide pH range, high sensitivity, low detection limit, low cost and ease of preparation are of advantageous of this insulin sensor. This sensor indicates great promise for monitory insulin in chromatographic effluents.     

Determination of catalase-like activity in plants based on the amperometric monitoring of hydrogen peroxide consumption using a carbon paste electrode modified with ruthenium(IV) Oxide

A carbon paste electrode containing ruthenium(IV) oxide as a modifier was tested as an effective hydrogen peroxide amperometric sensor in bulk measurements (hydrodynamic amperometry). Factors that influence its overall analytical perform ance, such as pH and the applied potential, were examined. The RuO2-modified electrode displayed high sensitivity towards hydrogen peroxide, with detection limits as low as 0.02 mm at pH 7.4 and 0.007 mM at pH 9.0. The method was applied for monitoring the decomposition of hydrogen peroxide (by catalase) in phosphate buffer of pH 7.4. The relative response of the electrode towards ascorbic acid was assessed and it was found that the selectivity of the RuO2-modified electrode towards hydrogen peroxide over ascorbic acid could be significantly improved by electro-polymerizing m-phenylenediamine on its surface prior to measurements. The RuO2-modified electrode was used for the kinetic (fixed time) determination of catalase activity in the range of 4-40 U/mL (detection limit 1.2 U/mL). The method was applied to the determination of catalase-like activity in various plant materials (recov-ery ranged from 93 to 101%, detection limit 480 U/100 g).     

A central role for gamma-glutamyl hydrolases in plant folate homeostasis

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.     

HPLC法测定灵芝孢子粉中三萜含量

为准确测定灵芝孢子粉中三萜的含量,运用高效液相建立适合孢子粉的分析测定方法。通过对前处理条件的优化,确定40%乙醇为孢子粉中等极性三萜酸类的最佳提取溶剂,浓缩倍数是子实体提取条件的50倍。通过色谱柱和洗脱条件的优化,建立了包括灵芝酸I、灵芝烯酸C、灵芝酸C2等13种标准品测定方法,方法学考察显示该分析方法精密度、重复性、稳定性的RSD值均小于5%,可以用于灵芝孢子粉中三萜类成分的定量检测。通过5组样品的分析发现,灵芝酸C6、灵芝酸G、灵芝酸A、灵芝酸D、灵芝酸F是灵芝孢子粉中的主要三萜类成分,其中灵芝酸A含量最高,平均占样品三萜总量的比例达19.71%;三萜类成分的溶出量与是否破壁没有相关性。三萜类成分在灵芝孢子粉和灵芝孢子油产品中的含量非常低,孢子粉的三萜含量为14.24-99.70μg/g,仅为子实体的1/100,灵芝孢子油中三萜含量也均低于50μg/g,因此三萜类成分不适合作为灵芝孢子粉及其相关产品的定量检测指标。     

生物传感器法测定麦芽汁中赖氨酸含量的研究

本研究利用固定化赖氨酸氧化酶和生物传感器,建立了麦芽汁中赖氨酸含量的测定方法。研究了该方法的线性范围、检出限、最适pH值范围、稳定性及专一性。以大生产麦芽汁为样品,与其它检测方法进行了比较。结果表明,生物传感器法测定赖氨酸含量操作简单而准确,加标回收率为97．41％～103．23％,RSD为2．23％;线性范围为2～100 mg/100 mL,检出限为2 mg/100 mL;稳定性及专一性强,不受其他氨基酸的干扰,能够用于氨基酸种类复杂的麦汁赖氨酸的检测。本方法与氨基酸分析仪、DBL、HPLC等方法相比较,测定结果无显著差异（P＞0．05）。     

Comparison of pretreatment,preservation and determination methods for foliar pH of plant samples

植物叶片pH值的测定及样品预处理和保存方法的比较研究 植物叶片pH值是叶片能量运转、新陈代谢、养分平衡等生理活动的重要调节因素。但目前尚缺乏一套合适的植物叶片样品的保存和测试方法，能同时满足叶片样品的长期保存和接近鲜叶片pH值测定的要求。本文通过探索植物叶样品的预处理和保存方式以及叶:水混合比例对叶片pH测定值的影响，提出一种能够长期保存植物叶片，同时对叶片pH值影响较小的测定方法，并且建立不同处理方法间的转换关系。本研究采集多种植物的叶片样品，并分别进行短期冷藏、冷冻和烘干处理保存，以刚采摘的新鲜绿色叶片作为对照，研究叶片样品不同保存方式对其pH值的影响。对烘干绿色叶片按叶片和水1:8的体积比和1:10的质量比分别进行pH值测定，对冷冻叶片：水按质量比为1:10和1:15的比例分别进行测定，分析不同加水比例对所测叶片样品pH值的影响。结果表明，短期冷藏和冷冻处理对植物叶片pH值的测量结果没有显著影响，但是烘干处理一般会使测定值偏高。因此，在长时间的野外采样工作中，通过便携式冷藏箱对植物样品进行冷冻保存，是保持叶片样品pH值稳定、即更接近新鲜叶pH测量值的较好的样品保存方式。对于长时间野外采样，冷冻预处理是植物叶样品保存的最佳选择；而冷藏预处理是短时间保存的最佳选择。不同的叶片：水比例对pH测量值存在显著影响：样品稀释比例 越高，氢离子浓度越低，测量到的叶片pH值越大。因此，为了建立现有不同植物叶pH值测量方法之间的联系，本文为叶片样品的不同前处理和测量方式提供了转换关系。本研究结果将有助于植物叶片pH值的研究，从而提高对这一功能性状的认识。     

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0444-0) contains supplementary material, which is available to authorized users.     

High-performance liquid chromatographic method for the determination of pioglitazone in human plasma using ultraviolet detection and its application to a pharmacokinetic study

An analytical method based on high-performance liquid chromatography (HPLC) with ultraviolet detection (269 nm) was developed for the determination of pioglitazone in human plasma. Rosiglitazone was used as an internal standard. Chromatographic separation was achieved with a reversed-phase Apollo C18 column and a mobile phase of methanol-acetonitrile-mixed phosphate buffer (pH 2.6; 10mM) (40:12:48, v/v/v) with a flow rate of 1.2 ml/min. The calibration curve was linear over the range of 50-2000 ng/ml (r(2)>0.9987) and the lower limit of quantification was 50 ng/ml. The method was validated with excellent sensitivity, accuracy, precision, recovery and stability. The assay has been applied successfully to a pharmacokinetic study with human volunteers.     

海南黄灯笼椒与不同品种辣椒的辣椒素含量测定

目的:建立HPLC法测定海南黄灯笼椒等辣椒的辣椒素含量。方法:采用Diamonsil C18柱(4.6 mm×250mm,5μm),流动相为甲醇-水(体积比70∶30),流速为1.0 mL/min,检测波长为230 nm,柱温:30℃,外标法定量。结果:辣椒素的线性关系良好,线性范围2.0μg/mL～20.0μg/mL,r=0.9990;精密度、稳定性实验的RSD均低于2%;平均回收率100.8%(n=9),RSD=1.87%。对海南黄灯笼椒等7个品种辣椒的辣椒素含量进行测定,海南黄灯笼椒的辣椒素含量最高。结论:该方法简便快捷,可用于辣椒中辣椒素含量的测定;海南黄灯笼椒辣椒素含量最高,这些信息可为海南黄灯笼辣椒的研究开发提供参考。     

Use of methylumbeliferyl-derivative substrates for lipase activity characterization

Lipases and esterases have been recognized as very useful biocatalysts because of their wide-ranging versatility in industrial applications, their stability, low cost, and non-requirement for added cofactors. The physical properties of lipidic substrates, typically water insoluble, have determined a great difficulty in studying lipolytic enzymes. A method for fast and simple detection of lipolytic activity, based on the use of 4-methylumbelliferone (MUF)-derivative substrates was developed. The system has been used for the detection of lipase activity either from microbial colonies, cell culture suspensions, or from proteins separated on SDS-polyacrylamide or isoelectric focusing gels. The use of MUF-derivative substrates has also been extended to the quantitative determination of lipolytic activity from a variety of assays including optimum pH and temperature determination, growth dependency, kinetics or stability studies, or residual activity quantification after treatment with potential inhibitors. The method has shown to be a useful tool for the characterization of a variety of lipases from microbial origin, including those cloned in heterologous hosts.