發(fā)布時間：2018-01-21 17:04

  本文關(guān)鍵詞： 凝血酶 丹參酚酸 HPLC-DAD-MS/MS 活性成分篩選 等溫滴定量熱技術(shù)(ITC)　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:凝血酶是抗凝防栓藥物研究所涉及的關(guān)鍵酶,而現(xiàn)有的直接凝血酶抑制劑具有出血等眾多不良反應(yīng)。丹參為活血化瘀方劑中使用頻次前五的藥物,現(xiàn)代研究表明其水溶性成分以抗氧化、抗凝血作用為主,因此選擇丹參水溶性成分作為待篩選對象。生色底物法可研究藥物與凝血酶的直接相互作用,但由于丹參水溶性成分多與生色底物法的產(chǎn)物-對硝基苯胺的最大吸收(280nm)相近,故無法采用傳統(tǒng)生色底物法對其進(jìn)行直接篩選。因此本研究擬建立一種新的快速篩選、測定中藥復(fù)雜提取物中潛在抗凝血酶物質(zhì)的方法,對丹參水溶性成分進(jìn)行HPLC分離-鑒定-柱后活性篩選,并采用等溫滴定量熱技術(shù)對所篩選出的潛在活性成分進(jìn)一步驗證與探究,以期得到具有體外直接抗凝血酶活性的有效成分。方法:1.以HPLC法分離丹參水溶性成分,并對方法的精密度、重復(fù)性及穩(wěn)定性進(jìn)行考察,從而為后續(xù)HPLC柱后餾分的收集做鋪墊。采用HPLC-DAD-MS/MS質(zhì)譜儀對丹參水溶性成分進(jìn)行鑒別,為后續(xù)所篩選出活性成分的指認(rèn)做參考。2.建立乙酸乙酯半微量萃取-HPLC方法對化學(xué)成分抗凝血酶活性進(jìn)行測定。在生色底物法基礎(chǔ)上進(jìn)行改良,采用控制反應(yīng)時間、反應(yīng)溫度等因素,對生成的產(chǎn)物-對硝基苯胺進(jìn)行乙酸乙酯半微量萃取,HPLC法分離分析,并考察不同凝血酶濃度、緩沖溶液pH、孵育時間及測定時間對方法的影響。同時以測定陽性藥物阿加曲班在生色底物法改良前后的酶活性抑制趨勢來衡量方法的可靠性。3.以建立好的方法對丹參水溶性成分進(jìn)行HPLC分離-鑒定-柱后活性篩選,并對所篩選出的潛在抗凝血酶活性成分進(jìn)行活力初步測定。4.以等溫滴定量熱法(ITC)測定準(zhǔn)活性成分與凝血酶的ITC曲線,通過計算總熱力學(xué)參數(shù)來對準(zhǔn)活性成分與凝血酶之間的相互作用做進(jìn)一步研究。結(jié)果:1.實驗建立了丹參水溶性成分的HPLC測定方法,成功在HPLC上分離了15個色譜峰,結(jié)果顯示丹參水提物樣品溶液室溫放置10小時內(nèi)該溶液各成分色譜峰面積及保留時間變化均無顯著性差異,重復(fù)進(jìn)樣各色譜峰出峰時間穩(wěn)定。實驗結(jié)合樣品及對照品溶液的二級質(zhì)譜信息與相關(guān)參考文獻(xiàn)比對,確認(rèn)了其中19種丹參酚酸類成分,為后續(xù)柱后餾分接取及活性成分的指認(rèn)提供了分離基礎(chǔ)及實驗依據(jù)。2.實驗成功建立乙酸乙酯半微量萃取-HPLC法用于測定抗凝血酶活性成分,有效排除了樣品本身的吸收干擾。經(jīng)考察,確定體系的凝血酶濃度為5U·mL-1,緩沖溶液的最佳pH為8.3,反應(yīng)開始后在37℃下孵育5min,以3h內(nèi)測定為最佳。用該方法測定丹參素鈉、丹酚酸A與丹酚酸B對凝血酶的抑制率百分率,分別為3.06%、77.77%、2.35%。3.對丹參水溶性成分柱后接取餾分進(jìn)行全面篩選,其抑制活性曲線有兩個明顯抑制峰。經(jīng)確認(rèn)為丹酚酸D、異阿魏酸和丹酚酸A所在位置。對各潛在活性成分進(jìn)行高、中、低濃度抑制活性測定,結(jié)果顯示,丹酚酸D與異阿魏酸混合后加入體系中時,其凝血酶抑制活性顯著提高。后又發(fā)現(xiàn)異阿魏酸在一定程度上也可增強(qiáng)丹酚酸A的抑制活性。4.等溫滴定量熱法測定各潛在活性成分與凝血酶的ITC曲線及熱力學(xué)參數(shù)測定結(jié)果顯示,陽性藥物阿加曲班與凝血酶結(jié)合常數(shù)最大、丹酚酸A次之,丹酚酸D或異阿魏酸單獨加入凝血酶溶液中時,結(jié)合常數(shù)較低。二者混合后滴定凝血酶溶液時,其結(jié)合常數(shù)顯著提高,大于二者單獨滴定時結(jié)合常數(shù)之和,且其ITC曲線類型有顯著變化,證明丹酚酸D與異阿魏酸可能作用在凝血酶的不同靶點。各反應(yīng)所測得吉布斯自由能△G均小于0,反應(yīng)均自發(fā)發(fā)生。各反應(yīng)焓變△H小于0,熵變△S大于0,初步推斷各藥物與凝血酶的結(jié)合以離子鍵形式發(fā)生。結(jié)論:本研究為從復(fù)雜的天然藥物中尋找抗凝血酶活性成分提供了可靠的篩選方法與檢測手段;為其他酶的抑制劑篩選提供了新的思路;為小分子藥物與酶等生物大分子的相互作用研究提供了有效方法;同時對于丹參活血的物質(zhì)基礎(chǔ)及作用機(jī)制的探討提供了參考和依據(jù)。
[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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284.1

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