柱后活性筛选丹参中直接抗凝血酶有效成分的研究

发布时间：2018-01-21 17:04

本文关键词： 凝血酶丹参酚酸 HPLC-DAD-MS/MS 活性成分筛选等温滴定量热技术(ITC)　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:凝血酶是抗凝防栓药物研究所涉及的关键酶,而现有的直接凝血酶抑制剂具有出血等众多不良反应。丹参为活血化瘀方剂中使用频次前五的药物,现代研究表明其水溶性成分以抗氧化、抗凝血作用为主,因此选择丹参水溶性成分作为待筛选对象。生色底物法可研究药物与凝血酶的直接相互作用,但由于丹参水溶性成分多与生色底物法的产物-对硝基苯胺的最大吸收(280nm)相近,故无法采用传统生色底物法对其进行直接筛选。因此本研究拟建立一种新的快速筛选、测定中药复杂提取物中潜在抗凝血酶物质的方法,对丹参水溶性成分进行HPLC分离-鉴定-柱后活性筛选,并采用等温滴定量热技术对所筛选出的潜在活性成分进一步验证与探究,以期得到具有体外直接抗凝血酶活性的有效成分。方法:1.以HPLC法分离丹参水溶性成分,并对方法的精密度、重复性及稳定性进行考察,从而为后续HPLC柱后馏分的收集做铺垫。采用HPLC-DAD-MS/MS质谱仪对丹参水溶性成分进行鉴别,为后续所筛选出活性成分的指认做参考。2.建立乙酸乙酯半微量萃取-HPLC方法对化学成分抗凝血酶活性进行测定。在生色底物法基础上进行改良,采用控制反应时间、反应温度等因素,对生成的产物-对硝基苯胺进行乙酸乙酯半微量萃取,HPLC法分离分析,并考察不同凝血酶浓度、缓冲溶液pH、孵育时间及测定时间对方法的影响。同时以测定阳性药物阿加曲班在生色底物法改良前后的酶活性抑制趋势来衡量方法的可靠性。3.以建立好的方法对丹参水溶性成分进行HPLC分离-鉴定-柱后活性筛选,并对所筛选出的潜在抗凝血酶活性成分进行活力初步测定。4.以等温滴定量热法(ITC)测定准活性成分与凝血酶的ITC曲线,通过计算总热力学参数来对准活性成分与凝血酶之间的相互作用做进一步研究。结果:1.实验建立了丹参水溶性成分的HPLC测定方法,成功在HPLC上分离了15个色谱峰,结果显示丹参水提物样品溶液室温放置10小时内该溶液各成分色谱峰面积及保留时间变化均无显著性差异,重复进样各色谱峰出峰时间稳定。实验结合样品及对照品溶液的二级质谱信息与相关参考文献比对,确认了其中19种丹参酚酸类成分,为后续柱后馏分接取及活性成分的指认提供了分离基础及实验依据。2.实验成功建立乙酸乙酯半微量萃取-HPLC法用于测定抗凝血酶活性成分,有效排除了样品本身的吸收干扰。经考察,确定体系的凝血酶浓度为5U·mL-1,缓冲溶液的最佳pH为8.3,反应开始后在37℃下孵育5min,以3h内测定为最佳。用该方法测定丹参素钠、丹酚酸A与丹酚酸B对凝血酶的抑制率百分率,分别为3.06%、77.77%、2.35%。3.对丹参水溶性成分柱后接取馏分进行全面筛选,其抑制活性曲线有两个明显抑制峰。经确认为丹酚酸D、异阿魏酸和丹酚酸A所在位置。对各潜在活性成分进行高、中、低浓度抑制活性测定,结果显示,丹酚酸D与异阿魏酸混合后加入体系中时,其凝血酶抑制活性显著提高。后又发现异阿魏酸在一定程度上也可增强丹酚酸A的抑制活性。4.等温滴定量热法测定各潜在活性成分与凝血酶的ITC曲线及热力学参数测定结果显示,阳性药物阿加曲班与凝血酶结合常数最大、丹酚酸A次之,丹酚酸D或异阿魏酸单独加入凝血酶溶液中时,结合常数较低。二者混合后滴定凝血酶溶液时,其结合常数显著提高,大于二者单独滴定时结合常数之和,且其ITC曲线类型有显著变化,证明丹酚酸D与异阿魏酸可能作用在凝血酶的不同靶点。各反应所测得吉布斯自由能△G均小于0,反应均自发发生。各反应焓变△H小于0,熵变△S大于0,初步推断各药物与凝血酶的结合以离子键形式发生。结论:本研究为从复杂的天然药物中寻找抗凝血酶活性成分提供了可靠的筛选方法与检测手段;为其他酶的抑制剂筛选提供了新的思路;为小分子药物与酶等生物大分子的相互作用研究提供了有效方法;同时对于丹参活血的物质基础及作用机制的探讨提供了参考和依据。
[Abstract]:Objective: To study the anti thrombin is a key enzyme of anticoagulant antithrombotic agents involved, and the direct thrombin inhibitor has many existing bleeding adverse reactions. For the use of the frequency of the drug before Salvia miltiorrhiza five Huoxue Huayu Decoction, modern research shows that the water soluble constituents with antioxidant, mainly anti clotting effect, so the choice of water soluble components of Salvia miltiorrhiza as the object to be screened. The direct interaction of drugs with thrombin chromogenic method, but the maximum absorption of water soluble constituents of Salvia miltiorrhiza multi product and chromogenic method of p-nitroaniline (280nm) are similar, so can not use traditional chromogenic method to screen the directly. Therefore, this study intends to establish a quick screening, determination method of latent antithrombin substance in extracts of traditional Chinese medicine complex, water soluble components of Salvia miltiorrhiza by HPLC separation and identification of post column activity screening and using isothermal droplet Quantitative thermal technology of potential active ingredients selected for further validation and inquiry, in order to find effective components with in vitro direct antithrombin activity. Methods: 1. with HPLC method for the separation of water soluble constituents of Salvia miltiorrhiza, and the method's precision, repeatability and stability were investigated, and for the subsequent HPLC column fractions collected pave the way for using HPLC-DAD-MS/MS mass spectrometry of water soluble components of Salvia miltiorrhiza were identified and screened for subsequent active components identified as reference.2. ethyl acetate extraction method of -HPLC semi micro chemical composition for determination of antithrombin activity. Based on the modified chromogenic substrate method, by controlling the reaction time, reaction temperature and other factors. The product of the ethyl acetate extraction of semi trace p-nitroaniline, separation and analysis of HPLC method, and the effects of different concentrations of thrombin, pH buffer, incubation time and determination Effect of time on method. At the same time to determine the reliability of.3. with positive drug, argatroban inhibition trend to measure method in enzyme activity before and after chromogenic method improved to establish a good method of water soluble components of Salvia miltiorrhiza HPLC separation - Identification - post activity screening, and the potential components of antithrombin activity were screened out preliminary determination of.4. viability by isothermal titration calorimetry (ITC) determination of ITC curve quasi active ingredient and thrombin, by calculating the total thermodynamic parameters on the interaction between the active component and thrombin to do further study. Results: the experiment to establish a method for determination of 1. water soluble components of Salvia miltiorrhiza HPLC, HPLC on the success of separation the 15 peaks, results showed that the water extract from Salvia miltiorrhiza sample solution at room temperature for 10 hours the solution of each component chromatographic peak area and retention time were no significant difference, repeated sampling Every peak peak time. Experiments with samples and two control MS information associated with the references on solution, confirmed 19 salvianolic acids which, for the subsequent post column fraction access and active ingredients identified provides the basis and experimental basis for.2. experiment successfully established semi micro ethyl acetate -HPLC extraction method for determination of antithrombin activity components, effectively eliminate the interference absorption sample itself. After investigation, to determine the concentration of thrombin system for 5U and mL-1, the best pH buffer solution is 8.3, after the reaction starts at 37 DEG C and incubated for 5min with 3H in the determination of the best. Determination of sodium Danshensu by this method, salvianolic acid A and salvianolic acid B on thrombin inhibition rate was respectively 3.06%, 77.77%, 2.35%.3. of water soluble components of Salvia miltiorrhiza column access fractions of comprehensive screening, the inhibition curve has two obvious peak suppression. Identified salvianolic acid D, isoferulic acid and salvianolic acid A. The location of the high potential of active components, low concentration of inhibitory activity determination showed that salvianolic acid D and isoferulic acid added after mixing system, the thrombin inhibitory activity increased significantly. After it was found that the results of ITC curves and thermodynamic parameters of ferulic acid in a certain extent can also determine the potential active components and enhanced thrombin inhibitory activity.4. isothermal titration calorimetry of salvianolic acid A showed positive drug argatroban and thrombin binding constants, salvianolic acid A and salvianolic acid D or ISO ferulic acid adding thrombin solution, the binding constant is low. The two mixed solution of thrombin titration, the binding constant increased significantly, more than two separate titrations of binding constants and the ITC curve type has changed significantly, that of salvianolic acid D and ISO o Ferulic acid may play a role in different target thrombin. Measured the reaction Gibbs free energy G is less than 0, the reactions were spontaneous. The reaction enthalpy change of H is less than 0, the entropy change of delta S is greater than 0, combined with the preliminary inference drug and thrombin by ionic bond form. Conclusion: This study provides reliable screening methods and detection methods for active components from natural drugs antithrombin complex; inhibitors for other enzymes provides a new way of screening; provides an effective method for the study of interaction of small molecule drugs and enzymes and other biological macromolecules; at the same time to provide reference and basis for discussion on material basis and action the mechanism of Danshen Huoxue.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284.1

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