基于中草药的直接凝血酶抑制剂分子模拟研究

从天然产物中筛选与凝血酶靶点具有相互作用的抑制剂,是研究抗凝抗栓类药物的有效方法。本文使用分子对接和分子动力学方法研究了从治疗和预防中风的中草药中筛选出的有效成分与凝血酶的相互作用。结果表明,筛选出的丹参酮I、人参皂苷F1和柯诺辛B三种成分与凝血酶的结合能力最强,可能是具有抗凝抗栓活性的中药有效成分。通过分子动力学方法获取了三种化合物与凝血酶结合的最优构象,三种配体均结合于凝血酶的活性位点,静电力和疏水作用是主要结合作用力。人参皂苷F1与凝血酶形成氢键,其结合能力强于丹参酮I和柯诺辛B。     

超高效液相色谱-轨道阱高分辨质谱法对人参寡糖提取物与蓖麻凝集素120相互作用的研究

利用超高效液相色谱-轨道阱高分辨质谱(UHPLC-Orbitrap-MS)结合链霉亲和素包被的96孔板的方法筛选人参复杂体系中蓖麻凝集素120 (RCA 120)的寡糖结合剂。将RCA 120修饰的96孔板与溶菌酶修饰的96孔板分别与寡糖标准品孵育,对未结合的寡糖利用UHPLC-Orbitrap-MS进行分析,利用峰面积的变化表征寡糖是否与RCA 120发生相互作用。对影响亲和力的因素包括孵育时间、孵育温度和缓冲液等进行了优化。在优化条件下,对生晒参、红参和西洋参的寡糖提取物进行RCA 120结合剂的筛选,从生晒参中筛选出3种二糖化合物和2种三糖化合物,从西洋参中筛选出2种二糖化合物和2种三糖化合物,发现生晒参和西洋参中具有相同单糖残基的寡糖与RCA 120的结合强度均具有显著性差异。但是,从红参中未筛选到RCA 120结合剂,推测这是由红参炮制工艺导致的差异。本方法简单、快速、准确,可高通量地识别和筛选复杂体系中多个糖类组分与蛋白质的相互作用,为糖和蛋白质相互作用的研究提供了新方法。     

人工合成的水蛭素基因在酵母中的表达

本文按水蛭素HV2的氨基酸序列,设计并合成了水蛭素基因,并在酵母α因子表达系统中表达。通过诱变得到的稳定携带水蛭素表达质粒的酵母菌株,在丰富培养基中培养36—48 h后,可在培养液中测得10—20 ATU/ml的分泌的水蛭素表达产物。经过较简单的纯化步骤,得到HPLC纯的水蛭素,其N-端氨基酸序列与天然产物完全相同,并有强烈的抗凝血和抑制凝血酶的活力。从500 ml培养液中可得到约3000 ATU的纯水蛭素,其比活力为6600 ATU/mg。     

红车轴草总异黄酮成分DNA结合剂的超滤质谱筛选

采用离心超滤和液相色谱-质谱联用的方法, 从红车轴草异黄酮提取物中筛选DNA结合剂. 结果表明, 红车轴草中10种异黄酮成分与DNA具有不同的结合能力. 采用液相色谱-串联质谱联用技术对其结构进行了鉴定, DNA结合能力较强的5种化合物分别是德鸢尾素-4'-O-β-D-葡萄糖苷、 德鸢尾素、 黄豆黄葡萄糖苷、 红车轴草素和鹰嘴豆牙素A. 为从中药提取物等复杂体系筛选并鉴定DNA结合剂建立了快速的超滤质谱平台.     

PEG在线定点修饰水蛭素及其修饰位点的理论预测与分析

比较了液相和固相修饰水蛭素的实验结果, 并在实验的基础上对水蛭素以及水蛭素/凝血酶复合物进行分子动力学模拟, 分析了容易被修饰的水蛭素赖氨酸残基的位点. 结果表明, 在液相修饰的时候, 水蛭素在水溶液中可被修饰的赖氨酸残基除Lys47外, 都容易被修饰. 而在固相修饰的时候, 水蛭素仅Lys35和Lys27容易被修饰.     

靶向亲和-液相色谱-质谱联用技术快速筛选黄藤总生物碱中乙酰胆碱酯酶抑制剂

采用靶向亲和-液相色谱-质谱联用技术(Target molecule affinity-LC-ESI-MSn)快速筛选黄藤总生物碱中能够抑制乙酰胆碱酯酶活性的成分,共筛选出12种具有潜在抑制乙酰胆碱酯酶的活性成分,并鉴定了6种成分,分别为黄藤素(Palmatine)、小檗碱(Berberine)、药根碱(Jatrorrhizine)、巴马汀红碱(Palmatrubine)、7,8-二氢-8-羟基小檗碱(7,8-Dihydro-8-hydroxyberberine)、Groenlandicine,结合体外酶学实验对这6种化合物进行了活性验证实验.结果表明,黄藤素抑制活性最强,其抑制作用强于阳性对照药盐酸多奈哌齐,说明黄藤素具有开发成抗阿尔茨海默症药物的潜力.本方法简单、快速、准确地从复杂的中药提取物中筛选出具有抑制乙酰胆碱酯酶活性的成分,适用于复杂体系中的高通量筛选.     

毛叶鹰爪花中一个新颖的裂环多氧取代环己烯类化合物

综合运用多种现代色谱学分离方法对毛叶鹰爪花中的化学成分进行了研究,从其枝叶的乙醇提取物中分离得到了3个裂环多氧取代环己烯类化合物,采用多种现代波谱技术确定了这些化合物的化学结构,分别鉴定为artapilosol A(1),microcarpin A(2)和uvarisubol B(3).其中化合物1为一个新化合物,化合物2和3为首次从鹰爪花属植物中分离得到的化合物.化合物1~3的体外细胞毒活性筛选结果表明它们对5种肿瘤细胞株(HL-60,A549,SMMC-7721,MCF-7和SW480)均显示出了较强的体外生长抑制活性,它们的细胞毒活性与抗肿瘤阳性对照药顺铂相当.     

异叶三宝木枝叶中一个新的Stemodane型二萜类化合物

综合运用硅胶柱色谱、反相硅胶柱色谱、Sephadex LH-20凝胶柱色谱以及制备型高效液相色谱等色谱分离技术对大戟科三宝木属植物异叶三宝木Trigonostemon heterophyllus枝叶中的化学成分进行了系统研究,从其枝叶的85%乙醇提取物中分离得到了8个二萜类化合物.采用多种波谱鉴定技术确定了这些化合物的化学结构.其中一个化合物为新的罕见的stemodane型二萜类化合物,七个化合物为首次从三宝木属植物中分离得到的化合物.所有化合物的体外细胞毒活性评价结果表明,它们对五种肿瘤细胞株(HL-60、A549、SMMC-7721、MCF-7和SW480)均显示出了较为显著的体外生长抑制活性,它们的细胞毒活性与抗肿瘤阳性对照药顺铂的活性相当.     

苯并呋喃衍生物的合成和结构表征

天然化合物常表现出各种独特的生物活性,使其成为寻找和筛选各种生物活性物质如医药、农药等的天然宝库.楝酰胺(rocaglamide)(结构式如图式1)是从楝属植物中分离出的具有良好杀虫活性的化合物[1],对疆叶蛾的LC50为0.91μg/mL,与已知的天然杀虫剂苦楝素Azadirachtin(LC50为0.70μg/mL)的活性相当[2-4],这表明楝酰胺有可能成为一种极为重要的天然杀虫剂.     

冬凌草的化学成分研究

采用硅胶柱层析方法从冬凌草的全草乙醇提取物中分离了7个化合物,通过理化性质和波谱分析方法分别鉴定为豆甾醇(Ⅰ),β-谷甾醇(Ⅱ),冬凌草乙素(Ⅲ),lasiodon in(Ⅳ),冬凌草甲素(Ⅴ),5,3,′4′-三羟基-6,7-二甲氧基黄酮(Ⅵ)和胡麻素(Ⅶ).化合物Ⅰ为首次从该属植物中分得,化合物Ⅵ为首次从该植物中分得.     

Interactions Between Thrombin with Flavonoids from Abelmoschus manihot (L.) Medicus by CZE

A capillary zone electrophoretic method was applied to determine the interactions between flavonoids from Abelmoschus manihot (L.) Medicus and thrombin. Samples containing flavonoids and thrombin at various ratios were incubated at 25 °C and then were separated by CZE with tris-acetate buffer at pH 7.2. Each run could be accomplished within 10 min. In CZE, the peak width broadened due to the affinity interactions between flavonoids and thrombin. Compared with positive and negative control, hirudin interacted with thrombin but heparin had no binding to thrombin, we concluded that the total flavone LXY1 and flavonoids LXY3, LXY4, LXY5 and LXY7 from Abelmoschus manihot (L.) Medicus interacted with thrombin; the aqueous extract LXY2 and flavonoid LXY6 had no binding to thrombin. Both qualification and quantification characterizations of the binding were determined. The experimental results showed that the reported method by capillary zone electrophoresis for the determination of flavonoids and thrombin interactions is powerful, sensitive and fast, requires less amounts of reagents, and further, it can be employed as a reliable alternative to other methods.     

Determination of recombinant hirudin structural deviants by capillary zone electrophoresis augmented with buffer additives

The polypeptide hirudin is a potent and specific thrombin inhibitor used in anticoagulant therapy and naturally occurring in medicinal leech. Using gene-technology methods, recombinant (r) hirudin can be produced on a large scale. Purity evaluation of the synthesized r-hirudin is essential to monitor co-expressed structural deviants and degradation products before therapeutic use. Although the well established RP-HPLC analysis appears to be the method of choice, in the case of r-hirudin baseline separation of the structural deviants is not necessarily achieved. Capillary zone electrophoresis augmented with buffer additives was used as a complementary technique to separate r-hirudin successfully from several similar species, in order to provide characterization information, as well as performing purity control and stability studies.     

Anticoagulatory Substances of Bloodsucking Animals: From Hirudin to Hirudin Mimetics

Bloodsucking animals contain substances in their saliva that specifically interfere with the blood clotting system. These substances are mainly low molecular weight proteins with a molecular mass of between 4 and 50 kDa. Some have become accessible in large quantities with the help of genetic engineering, and as a result their structures and structure—activity relationships have been studied and clinically tested. In the light of what is known about the mode of action of these natural products at the molecular level, new compounds with possible therapeutic effect can be derived. In the last ten years this approach has been tested with the proteinase inhibitor hirudin, obtained from medicinal leeches, and with the thrombin/hirudin complex. The serine protease thrombin plays a central role in the complex pathway of the blood clotting process and its pathophysiological effect finally results in thrombosis. The selectivity of the formation of complexes from hirudin and thrombin lies in the bivalent interaction of the inhibitor with the active site of the enzyme as well as with a substrate recognition site outside of the active site, the so-called fibrin-(ogen) binding site (FBS). The knowledge of this mode of action enabled the synthesis of bifunctional thrombin inhibitors based on hirudin peptides. Hirudin and hirudin mimetics in vivo have been shown to be highly potent anticoagulants for the treatment of arterial and venous thrombosis.     

Interactions between thrombin and natural products of Millettia speciosa Champ. using capillary zone electrophoresis

In Chinese medicine Suberect Spatholobus Stem is used to treat menoxenia, blood deficiency, numb paralyses, and so on. In folk, Millettia speciosa is often used as a substitute for Suberect Spatholobus Stem in some areas but it has not been reported whether M. speciosa is the eligible substitute for Suberect Spatholobus Stem or not till now. In this study, a capillary zone electrophoretic method was applied to determinate the interactions between natural products isolated from M. speciosa Champ. and thrombin for the first time. Both qualitative and quantitative characterizations of the molecule-enzyme binding were determined. Twenty ingredients were isolated from M. speciosa Champ. and the results showed that compared with positive and negative control, the compounds YT-1, YT-2, YT-3, YT-8, YT-9, YT-10, YT-11, YT-12, YT-14, YT-15, YT-16, and YT-20 interacted with thrombin while the other eight had no binding to thrombin. The binding constants of the interaction between compounds and thrombin were calculated by the Scatchard analysis formula. Because M. speciosa contains these compounds which have different levels of anticoagulant activity, it may be the eligible substitute for Suberect Spatholobus Stem.     

Capillary zone electrophoresis for the analysis of peptides synthesized by recombinant DNA technology

Capillary zone electrophoresis is applied to investigate the recombinant insulin-like growth factor and recombinant hirudin. During the production of these peptides in S. cerevisiae, byproducts with small variations in the structure of the polypeptide chain are obtained. The different peptides are separated in a fused silica capillary and detected on-column by ultraviolet absorption or fluorescence. Separation times are 10–40 min. The excellent separation efficiencies obtained indicate that capillary zone electrophoresis is complementary to liquid chromatography in the analysis of these peptides.     

Control of the cultivation process of antithrombin III and its characterization by capillary electrophoresis

The production by baby hamster kidney cells of recombinant antithrombin III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotachophoresis using mixtures of continuous spacers. The results were compared with those obtained by capillary zone electrophoresis (CZE). The downstream process, which incorporated anion-exchange and heparin affinity chromatography, was monitored by CZE under acidic conditions and voltage ramping. The purified product was characterized by its isoelectric point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectric focusing using a pH range of 4–6 and various mobilization procedures. The molecular mass of AT III was investigated by capillary gel electrophoresis (CGE), applying removable dextran gels. Both parameters could be determined within 30 min using only one coated capillary. The results showed an excellent correspondence with those achieved with conventional slab gels. The affinity complex between AT III and thrombin could also be detected by CGE and the heparin dependence of the affinity reaction could be investigated.     

Evaluation of affinity interaction between small molecules and platelets by open tubular affinity capillary electrochromatography

In this paper, an open tubular affinity capillary electrochromatography (OT‐ACEC) was developed by physical adsorption of rabbit platelets on the inner surface of capillary. The interactions between small molecules include adenosine diphosphate (ADP) (positive control), protocatechuic acid (negative control) and seven natural products (salvianolic acid B, salvianic acid A sodium, hydroxysafflor yellow A, ferulic acid, chlorogenic acid, sinapic acid, caffeic acid) and platelets were evaluated by their retention factors and binding constants obtained based on peak‐shift assay. Then, the activities of anti‐platelet aggregation induced by thrombin (THR), ADP and arachidonic acid (AA) for those small molecules (except ADP) were evaluated by turbidimetric method. The results indicate that: (i) ADP, a platelet aggregation inducer, had strong interaction with platelet, while protocatechuic acid that had no inhibition on platelet aggregation behaved no specific interaction; (ii) there was a positive correlation between the anti‐platelet aggregation activities of small molecules and their interactions with platelet, generally those compounds with higher binding constants with platelet exhibited higher activities. Therefore, the OT‐ACEC method developed in the present study can be a potential method to evaluate affinity interactions between small molecules and platelets, so as to predict the biological activities such as anti‐platelet aggregation for the small molecules.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

Mapping of stereoselective recognition sites on human serum transferrin by capillary electrophoresis and molecular modelling

Stereoselective recognition of chiral compounds can be used for mapping of surface interaction sites on proteins. Iron-free human serum transferrin is a suitable chiral selector in capillary electrophoresis used in native form in solution. Separation of optical isomers of tryptophan-methylester, tryptophan-ethylester and tryptophan-butylester and various drugs were studied in capillary zone electrophoresis applying a distinct transferrin zone prior to sample injection. Changes in the electrophoretic patterns (i.e., in the migration properties) of the molecules reflected the possible interactions with the protein. The tryptophan derivatives and eight drugs possessed stereoselective interactions, seven drugs showed interactions without appreciable chiral separation, and the others did not present any direct complexation with the protein molecules. Molecular modelling was performed to characterize the binding areas at the iron binding site of iron-free transferrin. The docking of tryptophan derivatives on transferrin showed that the R-enantiomers possess a stronger complexation with transferrin, whereas the S-enantiomers are bound by weaker interactions, which is in excellent agreement with the capillary electrophoresis results, where the R-enantiomers were always retarded stronger by transferrin. A ranking of drugs by the lipo score parameter of the docking shows an accordance with the stereoselective interactions by the protein.     

Detection of recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of recombinant hirudin (r-hirudin), a specific and reproducible analysis method is required. Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules. Recently, CE combined with immunoassay has emerged as a new analytical technique. CE-based immunoassay (CEIA) is a sensitive and specific method combining laser-induced fluorescence (LIF) and immunoassay. Therefore, in this study, we specifically investigated fluorescence labeling and determination of r-hirudin by CEIA with a LIF detector using labeled r-hirudin and polyclonal antibody. r-Hirudin was labeled with fluorescein isothiocyanate (FITC). FITC-labeled r-hirudin was purified using high-performance liquid chromatography (HPLC). The method is based on preincubation of r-hirudin and antibody for 20 min, followed by CE analysis using an uncoated capillary. Free and bound r-hirudin were separated within 5 min using CE with high reproducibility. This study demonstrated that the CEIA method could be applied to quantitative analysis of r-hirudin in biological fluids.