發(fā)布時間：2018-05-27 17:34

  本文選題：中藥藥效物質(zhì) + 化學(xué)生物學(xué)　； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：中藥藥效物質(zhì)基礎(chǔ)研究是中藥作用機(jī)理探討、創(chuàng)新藥物研制和建立與藥效相關(guān)的中藥質(zhì)量控制標(biāo)準(zhǔn)的基礎(chǔ),是中藥現(xiàn)代化研究中的重中之重。而中藥藥效物質(zhì)發(fā)現(xiàn)是中藥藥效物質(zhì)基礎(chǔ)研究的核心內(nèi)容,建立中藥藥效物質(zhì)的快速篩選方法對中藥現(xiàn)代化研究具有重要的推動作用。本論文聚焦于中藥藥效物質(zhì)發(fā)現(xiàn)方法的技術(shù)性問題,著重研究基于化學(xué)生物學(xué)方法結(jié)合高分辨質(zhì)譜技術(shù)的中藥藥效物質(zhì)快速篩選新方法,其主要研究內(nèi)容如下:1.建立HPLC-ESI-Q-TOF-MS/MS方法對養(yǎng)心氏片的化學(xué)成分進(jìn)行了系統(tǒng)性分析,共鑒定了其中127個化合物,對其結(jié)構(gòu)進(jìn)行了分類和質(zhì)譜裂解規(guī)律的解析。采用DPPH-LC-MS分析技術(shù)對養(yǎng)心氏片中的抗氧化物質(zhì)基礎(chǔ)作了進(jìn)一步的研究;從中快速篩選到34個潛在的抗氧化成分。其中丹酚酸A和丹酚酸B為最強(qiáng)的抗氧化物質(zhì),其DPPH自由基清除活性高于VC。通過對大鼠口服養(yǎng)心氏片的入血成分進(jìn)行分析,發(fā)現(xiàn)丹酚酸A和丹酚酸B可以入血,可能是養(yǎng)心氏片發(fā)揮體內(nèi)抗氧化作用的重要藥效物質(zhì)。2.創(chuàng)新性地提出了一種血腦屏障人造膜平行性透過試驗與DPPH探針識別相結(jié)合的PAMPA-BBB-DPPH方法,并將其應(yīng)用于中藥中可透過血腦屏障的抗氧化物質(zhì)篩選。發(fā)現(xiàn)知母乙酸乙酯提取物為活性中藥提取物。通過DPPH-HPLC結(jié)合高分辨質(zhì)譜技術(shù),鑒定出知母提取物中的可透過血腦屏障的抗氧化物質(zhì)為2,6,4'-Trihydroxy-4-methoxybenzophenone。體外研究表明,該物質(zhì)具有中等強(qiáng)度的抗氧化活性,其DPPH自由基清除能力略弱于VC等抗氧化劑,但是其血腦屏障人造膜透過能力很強(qiáng),具有深入開發(fā)為腦保護(hù)藥物的價值。3.將親和超濾LC-MS方法與分子對接技術(shù)相結(jié)合,首次應(yīng)用于中藥注射劑與人血清白蛋白相互作用的研究。發(fā)現(xiàn)丹紅注射液中的丹酚酸類成分有很強(qiáng)的人.血清白蛋白結(jié)合作用,且與聯(lián)用藥阿司匹林的主要代謝產(chǎn)物水楊酸存在競爭作用。通過競爭性結(jié)合試驗、分子對接、結(jié)合位點(diǎn)探針替代試驗等方法解釋了其競爭機(jī)制。研究內(nèi)容為丹紅注射液與人血清白蛋白的相互作用研究奠定了基礎(chǔ),同時也為中藥注射劑與人血清白蛋白的相互作用研究提供了新的思路。4.創(chuàng)新性地提出一種基于固定化融合靶點(diǎn)親和色譜的親和選擇方法,將其應(yīng)用于PPARy配體的篩選。通過基因工程技術(shù)將靶點(diǎn)受體與親和標(biāo)簽融合,可以實現(xiàn)靶點(diǎn)蛋白的快速制備、定向固定及純化,從而應(yīng)用于天然產(chǎn)物中相應(yīng)配體的篩選。相比于之前報道的親和選擇篩選方法,可低成本地獲得大量靶點(diǎn)蛋白,實現(xiàn)快速、定向的靶點(diǎn)固定,并能提高篩選通量。應(yīng)用該方法發(fā)現(xiàn)野菊花中的異綠原酸A具有較強(qiáng)的PPARγ結(jié)合能力。該研究為親和選擇方法的建立提供了參考。
[Abstract]:The basic research on the substance of traditional Chinese medicine (TCM) is the most important part in the study of TCM modernization. It is the basis of innovating the development of Chinese medicine and establishing the quality control standard of Chinese medicine related to its efficacy. The discovery of effective substances of traditional Chinese medicine is the core of the basic research on the substance of traditional Chinese medicine and the establishment of a rapid screening method of Chinese medicine pharmacodynamic substances has an important role in promoting the modernization of traditional Chinese medicine. In this paper, we focus on the technical problems of the discovery methods of Chinese traditional medicine pharmacodynamic substances, and focus on the study of a new method for rapid screening of Chinese traditional medicine pharmacodynamic substances based on chemical biological method and high resolution mass spectrometry. The main research contents are as follows: 1. A HPLC-ESI-Q-TOF-MS/MS method was established for the systematic analysis of the chemical constituents of Yangxin tablets. 127 of them were identified and their structures were classified and analyzed by mass spectrometry. The basis of antioxidant substances in Yangxin tablets was further studied by DPPH-LC-MS analysis, and 34 potential antioxidants were screened from them. Salvianolic acid A and Salvianolic acid B were the strongest antioxidants and the scavenging activity of DPPH radical was higher than that of VC. By analyzing the components of Yangxin tablet in rats, it was found that Salvianolic acid A and Salvianolic acid B could enter the blood, which may be an important antioxidation substance of Yangxin tablet. A novel PAMPA-BBB-DPPH method combining parallel permeability test of blood brain barrier (BBB) with DPPH probe identification was proposed and applied to the screening of antioxidants in Chinese medicine. It was found that the ethyl acetate extract of Anemarrhena emarginosa was active Chinese medicine extract. By means of DPPH-HPLC and high resolution mass spectrometry, the antioxidants in the extracts of Anemarrhena emarginosa through the blood-brain barrier were identified to be 2x6O4- Trihydroxy-4-methoxybenzophenone. In vitro studies showed that the compound had moderate antioxidant activity, and its scavenging ability of DPPH free radical was slightly weaker than that of VC, but its blood-brain barrier artificial membrane was highly permeable, and had the value of further development as a brain protection drug. The affinity ultrafiltration (LC-MS) method was combined with molecular docking technique to study the interaction between traditional Chinese medicine injection and human serum albumin (HSA) for the first time. It was found that Salvianolic acid in Danhong injection had strong human. Serum albumin binds and competes with salicylic acid, the main metabolite of aspirin. The competitive mechanism was explained by competitive binding test, molecular docking and site probe substitution test. The study laid a foundation for the study of the interaction between Danhong injection and human serum albumin, and also provided a new idea for the study of the interaction between traditional Chinese medicine injection and human serum albumin. A novel affinity selection method based on immobilized fusion target affinity chromatography was proposed and applied to the screening of PPARy ligands. Through the fusion of target receptor and affinity label by genetic engineering technology, the target protein can be quickly prepared, immobilized and purified, and applied to the screening of corresponding ligands in natural products. Compared with the previously reported affinity selection method, a large number of target proteins can be obtained at low cost, which can achieve fast, targeted target fixation and improve screening flux. By using this method, it was found that Isochrysanthemum Lv Yuan A had strong binding ability to PPAR 緯. This study provides a reference for the establishment of affinity selection method.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R284

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本文編號：1943156