基于植物代谢组学方法的马钱子油炸炮制前后化学差异研究

为对马钱子油炸炮制前后提取物的差异进行定性、定量研究,实验采用超高效液相色谱-四级杆串联飞行时间质谱(UPLC-Q-TOF-MS)采集数据,结合Peak View软件、在线数据库等进行化学成分定性分析,并采用Analyst工作站进行主要化学成分的含量测定,将炮制前后离子数据导入SIMCA-P软件,建立OPLS-DA数学模型,进行差异性次生代谢产物分析。结果显示马钱子的成分类别主要包括生物碱、糖苷、脂肪酸酯和醇类,其中检测到已知成分共29个,未曾报道的化合物达13个;OPLS-DA分析中最显著的化合物是士的宁、麦芽糖、Dattelic acid和油酸;定量分析结果表明油炸炮制后马钱子碱和士的宁的含量有所降低,且士的宁的下降幅度略大于马钱子碱,均能达到《中国药典》2015年版马钱子项下要求。本研究通过多种手段探讨了油炸炮制前后马钱子化学成分变化情况,结果表明炮制方法、减毒效果及化学成分间存在相关性,为马钱子炮制机理解释及药效物质基础阐明提供新的思路和数据参考。     

酶浸提取方法对士的宁产率的影响

目的：研究了纤维素酶在提取生物碱过程中的应用。方法：采用酶浸法和氯仿法两种不同的工艺提取马钱子生物总碱,高效液相色谱法（HPLC）测定了马钱子生物总碱中士的宁的含量。结果：酶浸法提取士的宁和氯仿法提取士的宁的含量分别为1．83％、1．32％;酶浸法和氯仿法提取马钱子生物总碱的产率分别为：2．85％、1．86％。     

药用植物盆架树中的马钱子苷及其衍生物

从药用植物盆架树(Winchia calophylla A．Dc．)的茎皮中分离得到4个环烯醚萜苷类化合物,其结构经现代波谱技术鉴定为马钱子苷、7-O-甲酰基马钱子苷、6 '-O-甲酰基马钱子苷和6',7-O-二甲酰基马钱子苷,其中后3个化合物为新化合物。     

荷包牡丹碱和马钱子碱对小鼠皮层及下丘听神经元声反应潜伏期的影响

频率和强度是声音的两个重要参数,通常以听觉神经元动作电位发放频次编码这两个参数 . 研究表明,声反应潜伏期也可编码声音频率和强度,但尚不清楚潜伏期编码这两个参数究竟发生于哪一级听觉核团 . 如果声音参数由同级中枢编码,则这样的编码方式可能发生改变 . 反之,如果编码方式未发生变化,则意味着声音信息是由低位神经元编码的 . GABA 和甘氨酸 (Gly) 是听中枢普遍分布的抑制性递质 . 通过施加它们的拮抗剂荷包牡丹碱和马钱子碱,观测小鼠皮层和下丘听觉神经元声反应潜伏期的变化 . 结果表明,由反应潜伏期表征声音频率和强度的关系不因 GABA 和 Gly 作用的改变而发生变化,提示频率和强度与反应潜伏期之间的编码关系可能是由低位听神经元编码并向上传递的,而不是在同级中枢 ( 皮层或下丘 ) 完成的 .     

马钱子致大鼠体内毒性的研究*——“装袋”算法和16S rRNA基因测序技术在毒理学研究中的应用

目的 中药马钱子（Strychnos nux-vomica L.,SN）在临床上具有消肿止痛的功效,然而,由于含有生物碱类成分,马钱子具有一定毒性。人们对马钱子毒性所引起的大鼠内源性代谢变化及其对肠道微生物群代谢失调的潜在影响知之甚少,因此,马钱子的毒理学研究对其安全性评价具有重要意义。本研究将代谢组学和16S rRNA基因测序技术相结合来探索马钱子的致毒机制。方法 通过急性、蓄积性和亚急性毒性试验,分别确定马钱子的中毒剂量、毒性强度和毒性靶器官。超高效液相色谱-质谱联用技术用于分析大鼠灌胃马钱子后的血清、肝脏和肾脏样本。利用基于装袋算法的决策树和K最近邻（K nearest neighbor,KNN）模型对组学数据进行分类。从大鼠粪便中提取样本后,使用高通量测序平台对细菌的16s rRNA V3-V4区域进行分析。结果 装袋算法提高了样本分类的准确率。共鉴定出12个生物标志物,这些生物标志物的代谢失调可能是马钱子致体内毒性的原因。拟杆菌、粪厌氧棒菌、颤螺菌、双茎体菌等与肾肝功能的生理指标密切相关,这表明马钱子引起的肝肾损害可能与这些肠道细菌的代谢紊乱有关。结论 本文揭示了马钱子的体内致毒机制,为马钱子临床上的安全合理使用提供了科学依据。     

聚合物纳米微球分离纯化放线菌素D的研究（英文）

本实验考察从链霉菌发酵产生的放线菌素D粗提物中分离制备高纯度放线菌素D的工艺,以聚合物纳米微球为固相载体,对聚合物纳米微球型号、洗脱条件进行优化。最终确立放线菌素D的分离纯化工艺为:采用PS40-300(以聚苯乙烯/二乙烯基苯聚合物为基质,粒径40μm,孔径300)纳米微球作为层析填料;以链霉菌发酵产生的放线菌素D粗提物为上样样品,用适量95%乙醇(V/V)充分溶解,上样量为0.5 g/100 m L,控制洗脱速度为5.0 m L/min,以65%乙醇水(V/V)洗脱,收集纯度95%以上的洗脱液,真空浓缩得到高纯度的放线菌素D。结果表明该分离纯化工艺,纯度与收率都较高,流程简单、可行,为该产品产业化开发奠定基础。     

刺葡萄籽中多酚的超声波提取及提取物中齐墩果酸的含量分析

本文研究了超声波法提取刺葡萄籽中多酚类物质的方法,考察了提取剂、提取时间、料液比等因素对提取率的影响。结果表明,用超声波法进行多酚类物质提取的最佳工艺条件为：以70％的丙酮水溶液为提取剂,料液比为1：10,室温下超声波提取两次,每次30min。采用Folin—Ciocaheau方法测定粗提物中多酚的含量,得出刺葡萄籽多酚粗提物得率为4．95％,纯度为49．89％。本文还对多酚粗提物中活性成分齐墩果酸进行了进一步的分离、鉴定与检测。     

辣椒总碱主要组分改进的HPLC测定法

本文采用HPLC法对辣椒总碱样品的主要组分进行了定量测定。色谱条件为：ODS C18柱,柱温：25℃,流动相：甲醇-乙腈-水(65：25：10),流速：1mL／min,检测波长：280nm,进样量为2μL。分析过程中,辣椒总碱的组分与其他杂质达到基线分离,能够准确测出辣椒碱及二氢辣椒碱的含量。粗提物辣椒总碱中辣椒碱及二氢辣椒碱的含量分别为2．36％和2．89％,方法的平均加样回收率分别为99．22％和99．06％。结果表明,本实验提供的测定方法较以往的文献所提供的辣椒总碱的检测方法更快速、简便、准确。     

毒药猛剂善起沉疴——马钱子

正相信大家对古代宫斗剧中"毒酒夺命"的场景并不陌生。其中,马钱子和鹤顶红、钩吻并列为宫廷三大毒药。因为服用后腹中剧痛,致头足相接,状如牵机,马钱子也被人叫作"牵机毒"。马钱子是史上皇上赐毒的首选,历史上著名的南唐后主李煜就是被宋太宗用马钱子毒死的。     

核壳型离子印迹聚合物的制备与应用

离子印迹聚合物(IIPs)是利用分子印迹技术对目标离子进行印迹、聚合进而得到对模板离子有选择性吸附的聚合物。核壳型离子印迹聚合物作为一种新型吸附材料被应用于样品的处理和实际检测中。本文对核壳型离子印迹聚合物的核壳类型、印迹聚合物的制备方法及实际检测应用等最新研究进展进行综述,为核壳型离子印迹聚合物的研究与应用提供参考。     

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

A selective, simple and efficient method-ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C(18) column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48-496.4 ng/ml for strychnine and 2.64-528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.     

Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.     

Alkaloids from the leaves of Strychnos wallichiana

The leaves of Strychnos wallichiana Steud. ex. DC. from Bangladesh contain icajine and novacine as their major alkaloids. Smaller amounts of strychnine, brucine, pseudostrychnine, pseudobrucine, N-methyl-sec.-pseudo-β-colubrine, 14-hydroxyicajine, strychnine N-oxide, and brucine N-oxide are also present. The new bases 14 hydroxynovacine and icajine N-oxide have been isolated.     

Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

PREPARATION OF GLUTATHIONE IMPRINTED POLYMER

Glutathione imprinted polymer was prepared using 1-vinyl imidazole and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, in dimethyl sulfoxide. The adsorption selectivity of glutathione-imprinted polymer was tested by reduced glutathione, oxidized glutathione, and L-Gly-Leu-Tyr in 30% phosphate buffer (0.01 M, pH 5.0)–70% acetonitrile and binding affinity values were compared. Reusability of molecularly imprinted polymer particles was also investigated. Molecularly imprinted polymer particles were found to be stable and to maintain glutathione adsorption capacity at 95% when washed with methanol–acetic acid (10%) after seven usages. Functional monomer 1-vinyl imidazole and cross linker ethylene glycol dimethacrylate-based glutathione imprinted polymer could be used as solid phase extraction material for recognition of glutathione in biological samples.     

Development of lipid A-imprinted polymer hydrogels that selectively recognize lipopolysaccharides

To remove lipopolysaccharide (LPS) from pure water, we developed polymer hydrogels that selectively recognize LPS. A molecular imprinting technique was used to prepare the polymer hydrogels. We prepared the polymer hydrogels with LPS-binding sites by using acryloyllysine and acryloylphenylalanine as functional monomers and used lipid A as a template because it is the biologically active part of LPS and contains two phosphate groups. Co-existence of n-octane during the polymerization process was highly effective in promoting the formation of LPS-accessible sites on the surface of the hydrogels. Both an electrostatic and a hydrophobic interaction between the lipid A portion of LPS and the recognition site of the imprinted hydrogel are necessary for LPS recognition. The adsorption isotherm of LPS to the lipid A-imprinted hydrogels was Langmuir-type; the saturated adsorption capacity and the adsorption constant, calculated by applying an equation for Langmuir-type adsorption isotherms, were 1.0×10(-11)mol/cm(2) and 2.5×10(5)M(-1), respectively. The imprinted hydrogels selectively recognized toxic LPS in a competition experiment in which two other kinds of LPS with similar chemical structures to that of the LPS of E. coli (toxic LPS) were adsorbed to the lipid A-imprinted hydrogels.     

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.     

Molecularly imprinted polymer of 5-methyluridine for solid-phase extraction of pyrimidine nucleoside cancer markers in urine

Normal and modified urinary nucleosides represent potential biomarkers for cancer diagnosis. To selectively extract modified nucleosides, we developed a molecularly imprinted polymer (MIP) of 5-methyluridine as selective material for molecularly imprinted solid-phase extraction (MISPE). The MIPs were obtained from vinyl-phenylboronate ester derivative of the template, acrylamide and pentaerythritol triacrylate co-polymer, and were tested in batch and cartridge experiments with aqueous samples. Our results indicated that the imprinted polymer was selective for pyrimidine nucleosides with a K(d) and a B(max) of 46 microM and 18 micromol/g, respectively. Finally, a MISPE of the most common pyrimidine nucleoside cancer markers in urine sample was realized.     

Quantification and confocal imaging of protein specific molecularly imprinted polymers

We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.     

Removal of the fermentation by-product succinyl L-tyrosine from the beta-lactamase inhibitor clavulanic acid using a molecularly imprinted polymer

Clavulanic acid is a beta-lactamase inhibitor used in therapeutic combinations with the penicillin-type antibiotics. During the fermentation leading to clavulanic acid, a succinyl L-tyrosine by-product is unavoidably formed. Occasionally, the amount of this by-product is found to be as high as 2% of the product even after standard purification operations. To further remove this impurity, we prepared a highly specific adsorbent for succinyl L-tyrosine with the molecular imprinting technique. This was performed by simultaneously using vinylbenzyl trimethylammonium chloride and methacrylic acid as the functional monomers. The imprinted polymer selectively bound succinyl L-tyrosine, and could be successfully used to remove this impurity at concentrations of less than 2% in the presence of clavulanic acid.