治疗糖尿病的中草药活性组分与牛血清白蛋白相互作用的研究
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摘要
糖尿病是一种常见的慢性代谢性疾病,由于其高患病率和死亡率,已逐渐发展成为继癌症、心脑血管疾病之后威胁人类健康的“第三大杀手”。研究开发既能降糖又能治疗并发症的药物,成为目前糖尿病药物开发的方向。我国利用中草药治疗糖尿病的历史悠久,但机理不清,限制了其发展推广。本文选用三七和知母活性组分作为模型药物,在总结前人工作的基础之上,开展了如下具有原始创新性的研究：
(1)将不同极性的组分作用于牛血清白蛋白(BSA),筛选与蛋白质作用的组分,为进一步确定三七中的有效成分提供理论依据。
(2)利用荧光光谱等方法研究了单体皂苷(Rb1,Rg1,Re)及其二元混合物与BSA的相互作用,对中草药二组分混合后相互间的作用机制进行了探索性的研究。
(3)对比研究了超滤法、平衡透析法、凝胶过滤色谱法等研究药物与蛋白结合作用的方法,为进一步探讨药物的作用机制提供参考,并力图寻找筛选中草药中的活性组分的简便、快捷的方法。
论文共分为五章。
第一章：简要介绍了糖尿病的概况及中草药在治疗糖尿病方面的应用,综述了研究药物小分子与蛋白质结合作用的各种方法。
第二章：采用硅胶柱层析对三七的主要活性成分(三七总皂苷)进行了粗分,将不同极性的组分作用于牛血清白蛋白,利用荧光光谱法等方法筛选与蛋白质作用的组分,应用软电离质谱(ESI-MS)及红外光谱并依据相关文献值推测出各组分可能的主要成分及结构。
第三章：应用荧光光谱法在磷酸缓冲体系(pH 7.4)中分别对单组分(人参皂苷Rb1 Rg1, Re)及二元混合物(Rb1-Rg1, Rb1-Re, Rg1-Re)与BSA的相互作用进行研究。这些实验结果能为从分子水平阐释药物单独使用和混合使用的药理机制提供依据,并确定药物组合是否具有拮抗作用。
第四章：采用平衡透析法、超滤法结合高效液相色谱法及凝胶过滤色谱法首次测定芒果苷与BSA的结合率,比较了不同方法的测定结果,证明凝胶色谱在测定蛋白质-药物结合率上存在局限性。
第五章：选用聚苯乙烯树脂作为基质树脂将其进行改性,并将牛血清白蛋白嫁接于材料表面,考察其对药物小分子的吸附能力以及用作色谱柱填料的可能性。
As a very common chronic metabolic disease, diabetes is becoming the third "killer" to the health of mankind along with cancer, cardiovascular and cerebrovascular diseases because of its high prevalence, morbidity and mortality. In our country, there are a lot of Chinese traditional medicine used to cure or alleviate diabetes and its complications. Up to now, it is bewildering the mechnism of these Chinese medicines affecting the disease and there are too much unclear on how and why to use these Chinese medicines.In this paper, we chose Panax notoginseng (Burk.) F. H. Chen as the studied object. The following works were carried out:
1.Total saponins of Panax notoginseng were separated into different polar groups by silica gel column chromatography. The interaction between these substances and BSA were studied using fluorescence and which substance(s) affected the protein be found. It would provide scientific and experimental evidence for confirming the effective constituents of Panax notoginseng.
2.The interactions of the single ingredient (Ginsenoside Rb1, Rg1 and Re) and the multi-ingredients (Rb1 plus Rg1,Rb1 plus Re, Rg1 plus Re) with BSA at physiological pH were investigated by fluorescence spectroscopy. It provides a molecular basis for elucidating the mechanism of each drug acting alone and its combinations and determining whether or not a given drug combination would gain a synergistic effect.
3.Equilibrium dialysis, ultrafiltration combined with high-performance liquid chromatography, and gel-filtration chromatography were used to determine the binding ratio of mangiferin to BSA. The aim of this study was to find simple and rapid methods used for isolating active components of Chinese medicinal plants.
This dissertation consists of five chapters:
Chapter 1:Diabetes and the traditional Chinese medicines used for diabetes therapy were briefly described. The studied methods of drug and protein binding were reviewed.
Chapter 2:Total saponins of Panax notoginseng were separated into four different polar groups by silica gel column chromatography. The interaction between these substances and BSA were studied using fluorescence and which substance(s) affected the protein be found.
Chapter 3:The interactions of the single ingredient (Ginsenoside Rb1,Rg1 and Re) and the multi-ingredients (Rb1 plus Rg1,Rb1 plus Re, Rg1 plus Re) with BSA at physiological pH were investigated by fluorescence spectroscopy. It can provide a molecular basis for elucidating the mechanism of each drug acting alone and its combinations and determining whether or not a given drug combination would gain a synergistic effect.
Chapter 4:Equilibrium dialysis, ultrafiltration combined with high-performance liquid chromatography, and gel-filtration chromatography were used to determine the binding ratio of mangiferin to BSA. Compared the results get from different methods, gel-filtration is unsuitable for the aim.
Chapter 5:Polystyrene resin used as the matrix resin was modified and grafted with the BSA surface, and its adsorption ability of small molecule drugs as well as the possibility of column packing were studied.
(1)将不同极性的组分作用于牛血清白蛋白(BSA),筛选与蛋白质作用的组分,为进一步确定三七中的有效成分提供理论依据。
(2)利用荧光光谱等方法研究了单体皂苷(Rb1,Rg1,Re)及其二元混合物与BSA的相互作用,对中草药二组分混合后相互间的作用机制进行了探索性的研究。
(3)对比研究了超滤法、平衡透析法、凝胶过滤色谱法等研究药物与蛋白结合作用的方法,为进一步探讨药物的作用机制提供参考,并力图寻找筛选中草药中的活性组分的简便、快捷的方法。
论文共分为五章。
第一章：简要介绍了糖尿病的概况及中草药在治疗糖尿病方面的应用,综述了研究药物小分子与蛋白质结合作用的各种方法。
第二章：采用硅胶柱层析对三七的主要活性成分(三七总皂苷)进行了粗分,将不同极性的组分作用于牛血清白蛋白,利用荧光光谱法等方法筛选与蛋白质作用的组分,应用软电离质谱(ESI-MS)及红外光谱并依据相关文献值推测出各组分可能的主要成分及结构。
第三章：应用荧光光谱法在磷酸缓冲体系(pH 7.4)中分别对单组分(人参皂苷Rb1 Rg1, Re)及二元混合物(Rb1-Rg1, Rb1-Re, Rg1-Re)与BSA的相互作用进行研究。这些实验结果能为从分子水平阐释药物单独使用和混合使用的药理机制提供依据,并确定药物组合是否具有拮抗作用。
第四章：采用平衡透析法、超滤法结合高效液相色谱法及凝胶过滤色谱法首次测定芒果苷与BSA的结合率,比较了不同方法的测定结果,证明凝胶色谱在测定蛋白质-药物结合率上存在局限性。
第五章：选用聚苯乙烯树脂作为基质树脂将其进行改性,并将牛血清白蛋白嫁接于材料表面,考察其对药物小分子的吸附能力以及用作色谱柱填料的可能性。
As a very common chronic metabolic disease, diabetes is becoming the third "killer" to the health of mankind along with cancer, cardiovascular and cerebrovascular diseases because of its high prevalence, morbidity and mortality. In our country, there are a lot of Chinese traditional medicine used to cure or alleviate diabetes and its complications. Up to now, it is bewildering the mechnism of these Chinese medicines affecting the disease and there are too much unclear on how and why to use these Chinese medicines.In this paper, we chose Panax notoginseng (Burk.) F. H. Chen as the studied object. The following works were carried out:
1.Total saponins of Panax notoginseng were separated into different polar groups by silica gel column chromatography. The interaction between these substances and BSA were studied using fluorescence and which substance(s) affected the protein be found. It would provide scientific and experimental evidence for confirming the effective constituents of Panax notoginseng.
2.The interactions of the single ingredient (Ginsenoside Rb1, Rg1 and Re) and the multi-ingredients (Rb1 plus Rg1,Rb1 plus Re, Rg1 plus Re) with BSA at physiological pH were investigated by fluorescence spectroscopy. It provides a molecular basis for elucidating the mechanism of each drug acting alone and its combinations and determining whether or not a given drug combination would gain a synergistic effect.
3.Equilibrium dialysis, ultrafiltration combined with high-performance liquid chromatography, and gel-filtration chromatography were used to determine the binding ratio of mangiferin to BSA. The aim of this study was to find simple and rapid methods used for isolating active components of Chinese medicinal plants.
This dissertation consists of five chapters:
Chapter 1:Diabetes and the traditional Chinese medicines used for diabetes therapy were briefly described. The studied methods of drug and protein binding were reviewed.
Chapter 2:Total saponins of Panax notoginseng were separated into four different polar groups by silica gel column chromatography. The interaction between these substances and BSA were studied using fluorescence and which substance(s) affected the protein be found.
Chapter 3:The interactions of the single ingredient (Ginsenoside Rb1,Rg1 and Re) and the multi-ingredients (Rb1 plus Rg1,Rb1 plus Re, Rg1 plus Re) with BSA at physiological pH were investigated by fluorescence spectroscopy. It can provide a molecular basis for elucidating the mechanism of each drug acting alone and its combinations and determining whether or not a given drug combination would gain a synergistic effect.
Chapter 4:Equilibrium dialysis, ultrafiltration combined with high-performance liquid chromatography, and gel-filtration chromatography were used to determine the binding ratio of mangiferin to BSA. Compared the results get from different methods, gel-filtration is unsuitable for the aim.
Chapter 5:Polystyrene resin used as the matrix resin was modified and grafted with the BSA surface, and its adsorption ability of small molecule drugs as well as the possibility of column packing were studied.
引文
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