【摘要】：目的：國(guó)醫(yī)大師鄧鐵濤教授臨床運(yùn)用加味補(bǔ)中益氣湯治療重癥肌無(wú)力(MG)幾十年,其治療效果十分顯著。本研究以加味補(bǔ)中益氣湯方為例,采用數(shù)據(jù)挖掘、確定靶標(biāo)、建立數(shù)據(jù)庫(kù)和虛擬篩選的現(xiàn)代計(jì)算機(jī)方法,研究加味補(bǔ)中益氣湯對(duì)MG的治療作用。并在君臣佐使中,找到主要針對(duì)乙酰膽堿酯酶(AChE)靶標(biāo)的草藥；對(duì)于主要的抗AChE的草藥,發(fā)現(xiàn)抑制AChE化合物的主要骨架結(jié)構(gòu)；以及發(fā)現(xiàn)新的AChE抑制劑；從傳統(tǒng)中藥復(fù)方得到啟發(fā),快速找到活性化合物的新方法。方法：1.研究MG發(fā)病機(jī)制,確定AChE作為研究靶標(biāo),從蛋白數(shù)據(jù)庫(kù)中獲得AChE的晶體蛋白結(jié)構(gòu)。2.通過(guò)數(shù)據(jù)挖掘建立中藥復(fù)方君臣佐使的化學(xué)成分?jǐn)?shù)據(jù)庫(kù)和已知的AChE抑制劑的數(shù)據(jù)庫(kù)。3.本研究采用了利平斯基五規(guī)則,進(jìn)行初步的化合物的類(lèi)藥性篩選。4.采用分子二維(2D)、三維(3D)相似度疊合技術(shù)和Glide分子對(duì)接技術(shù),對(duì)中藥復(fù)方化學(xué)成分進(jìn)行基于AChE抑制劑配體的虛擬篩選。5.骨架分析,得到潛在的抗AChE活性化合物新骨架,從商業(yè)數(shù)據(jù)庫(kù)中購(gòu)買(mǎi)相同的骨架的化合物。6.酶的活性實(shí)驗(yàn),以donepezil為陽(yáng)性對(duì)照品,測(cè)定24個(gè)化合物的IC50值,通過(guò)Ellman酶分析實(shí)驗(yàn),找到了5個(gè)活性化合物。7用分子動(dòng)力學(xué)模擬的方法進(jìn)行驗(yàn)證,驗(yàn)證基于分子對(duì)接虛擬篩選預(yù)測(cè)活性化合物的可靠性。結(jié)果：通過(guò)對(duì)MG發(fā)病機(jī)制的研究,預(yù)測(cè)AChE該病的主要研究靶標(biāo)。研究發(fā)現(xiàn)加味補(bǔ)中益氣湯中的抗AChE活性成分主要是黃酮類(lèi)的化合物,同時(shí),還發(fā)現(xiàn)了一類(lèi)新的抗AChE的化合物骨架,通過(guò)實(shí)驗(yàn)驗(yàn)證,找到了5個(gè)新的AChE抑制劑X4, X10, X11, X19和X21,IC50分別為6.5±0.23μM,11.4±2.72μM,7.1±1.34μM,10.9±0.48μM,2.3±0.15μm。分子動(dòng)力學(xué)模擬表明,預(yù)測(cè)的結(jié)合自由能△G值與新的AChE抑制劑的生物活性的數(shù)據(jù)是一致的,呈線性關(guān)系。說(shuō)明通過(guò)分子對(duì)接篩選活性化合物的技術(shù)的可靠性。這項(xiàng)研究表明,新的活性化合物的發(fā)現(xiàn)可以從中藥材的活性成分骨架中得到啟發(fā)。結(jié)論：本研究的結(jié)論如下：(1)加味補(bǔ)中益氣湯對(duì)重癥肌無(wú)力具有治療作用,其中君藥和使藥發(fā)揮了主要作用,臣藥和佐藥對(duì)重癥肌無(wú)力的治療可能具有不同的功能。(2)主要的活性成分是黃酮衍生物類(lèi)AChE抑制劑。(3)本文發(fā)現(xiàn)了5個(gè)新骨架的AChE抑制劑。(4)本文提供新的研究方法,從傳統(tǒng)的中醫(yī)藥經(jīng)典處方,結(jié)合數(shù)據(jù)挖掘方法研究中藥復(fù)方活性成分,找到活性化合物。
[Abstract]:Objective: professor Deng Tietao, a master of Chinese medicine, has been using Jiawei Buzhong Yiqi decoction in the treatment of myasthenia gravis (MG) for decades, and its therapeutic effect is very remarkable. In this study, taking Jiawei Buzhong Yiqi decoction as an example, the therapeutic effect of Jiawei Buzhong Yiqi decoction on MG was studied by using data mining, determining target, establishing database and virtual screening. Among the envoys, the herbs targeting acetylcholinesterase (AChE) were found, the main skeleton structures of AChE compounds were found for the main anti-AChE herbs, and the new AChE inhibitors were found. Inspired by traditional Chinese medicine compound, a new method of finding active compounds quickly. Methods: 1. To study the pathogenesis of MG and determine AChE as the research target, the crystal protein structure of AChE was obtained from the protein database. 2. The chemical composition database and known AChE inhibitor database of traditional Chinese medicine compound Junchen Zoran were established by data mining. 3. In this study, the Lipinski five rules were used to screen the properties of the compounds. 4. Two dimensional (2D), three dimensional (3D) similarity superposition technique and Glide molecular docking technique were used to screen the chemical components of traditional Chinese medicine based on AChE inhibitor ligands. 5. Skeleton analysis, a new skeleton of potential anti-AChE active compounds was obtained, and the same skeleton compounds were purchased from commercial databases. 6. The IC50 values of 24 compounds were determined by donepezil as positive reference, and 5 active compounds were found by Ellman enzyme analysis. 7 the results were verified by molecular dynamics simulation. The reliability of predicting active compounds based on virtual screening of molecular docking was verified. Results: through the study of the pathogenesis of MG, the main research targets of AChE were predicted. It was found that the anti-AChE active components in Jiawei Buzhong Yiqi decoction were mainly flavonoids. At the same time, a new kind of anti-AChE compound skeleton was also found. Five new AChE inhibitors X4, X10, X11 were found by experiments. The IC _ (50) of X19 and X21 are 6.5 鹵0.23 渭 M, 11.4 鹵2.72 渭 M, 7.1 鹵1.34 渭 M, 10.9 鹵0.48 渭 M and 2.3 鹵0.15 渭 M, respectively. Molecular dynamics simulation shows that the predicted binding free energy G value is consistent with the data of the biological activity of the new AChE inhibitor, and there is a linear relationship between the predicted binding free energy G value and the biological activity of the new binding free energy G. It shows the reliability of the technology of screening active compounds through molecular docking. This study shows that the discovery of new active compounds can be inspired by the skeleton of active components of Chinese medicinal materials. Conclusion: the conclusions of this study are as follows: (1) Jiawei Buzhong Yiqi decoction has therapeutic effect on myasthenia gravis, in which monarch medicine and make medicine play a major role. Subjects and adjuvants may have different functions in the treatment of myasthenia gravis. (2) the main active components are flavonoids derivative AChE inhibitors. (3) five new skeleton AChE inhibitors have been found in this paper. (4) in this paper, five new skeleton AChE inhibitors have been found. (4) the main active components are flavonoids derivative AChE inhibitors. To provide new research methods, From the traditional classical prescription of traditional Chinese medicine, combined with data mining method, the active components of traditional Chinese medicine compound were studied, and the active compounds were found.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R249;R277.7

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