【作者】 肖建波；

【导师】 任凤莲； 杨春生；

【作者基本信息】 中南大学 ， 应用化学， 2009， 博士

【摘要】 近年来,中药活性成分与血清蛋白质的相互作用已经引起研究者的广泛关注。中药活性小分子在人体血液中与血浆载体蛋白的相互作用直接影响药物的分布、排泄、代谢、活性和毒副作用。中药活性成分与血液载体蛋白的相互作用研究可以为营养学和临床医学提供重要的信息,对了解药物在体内的代谢过程,阐明药物的作用机制,研究药代动力学以及药物的毒性,解释蛋白质结构和功能的关系和开发新药等都有重要的意义。本论文主要研究多酚类化合物和牛血清白蛋白之间的相互作用,选择常见的有代表性的黄酮类化合物和白藜芦醇,研究中药小分子与生物大分子相互作用的结构-结合力关系、理论模型及其应用。1.多光谱法研究葛根素与牛血清白蛋白的相互作用采用荧光光谱,红外光谱,共振散射光谱和同步荧光光谱考察了葛根素与牛血清白蛋白的相互作用。在20℃和30℃时,葛根素和牛血清白蛋白之间的表观结合常数（K_a）分别是1.13×10⁴ L/mol（20℃）和1.54×10⁴L/mol（30℃）,结合位点数（n）是0.95±0.02。实验结果显示,葛根素可以通过插入到牛血清白蛋白分子内部,通过形成葛根素-牛血清白蛋白的配合物来猝灭其内源荧光。随着葛根素浓度的增加,牛血清白蛋白的共振散射强度减弱,在溶液中呈现聚集的形态。静态猝灭和无辐射能量转移都是导致荧光猝灭的主要原因。正的熵变和焓变表明葛根素和牛血清白蛋白之间主要是疏水作用力。吉布斯自由能变是负值说明这个过程是自发的。2.黄酮醇B环羟基对其与血清白蛋白作用的影响黄酮醇B环多羟基对电子共振和转移起重要作用,黄酮醇B环多羟基也是其清除自由基能力的决定因素。然而黄酮醇B环羟基对其与血清蛋白作用的影响还不清楚。高良姜素,山奈酚,槲皮素和杨梅素具有相同的A环和C环结构,仅B环上的羟基数目不同,通过测定它们与牛血清白蛋白的结合常数和结合位点发现具有相同A环和C环结构的黄酮醇,其B环羟基的增加将增强其与血清白蛋白的作用,B环上每增一个羟基可以提高结合常数1个数量级。同时研究了黄酮醇的疏水常数(lgK_ow)和在C₁₈柱上的色谱保留指数（K′）与结合常数（lgK_a）的关系。亲脂性越高的黄酮醇,其与牛血清白蛋白的结合力越弱;黄酮醇的色谱保留指数（K′）随B环羟基的增加而减小。研究结果还发现黄酮醇的抗氧化性越强则与牛血清白蛋白的结合力越大。3.黄酮羟基的糖苷化对其与血清蛋白作用的影响天然的生物类黄酮多是以糖苷形式存在于植物中,在人体内被酶水解成黄酮苷元才能发挥其药效作用。黄酮苷元比黄酮苷更易被人体吸收。然而黄酮羟基的糖苷化对其与血清蛋白作用的影响还不清楚。论文研究了四种黄酮苷元及其常见的黄酮苷与牛血清白蛋白的结合常数与结合位点数,结果表明黄酮母环上的羟基被糖苷取代后将削弱其与血清白蛋白的作用,导致其与牛血清白蛋白的结合常数降低1-3个数量级。槲皮素与牛血清白蛋白的结合常数是槲皮苷与牛血清白蛋白的结合常数的5000多倍,而染料木素与牛血清白蛋白的结合常数仅为染料木苷与牛血清白蛋白的结合常数的6倍。4.应用药物与牛血清白蛋白相互作用研究表没食子儿茶酚没食子酸酯（EGCG）增强石杉碱甲对乙酰胆碱酯酶的抑制效果课题组研究发现EGCG可以在体内增强石杉碱甲对乙酰胆碱酯酶的抑制效果,降低石杉碱甲的使用量而具有同样的效果,然而相关机理还不清楚。论文通过研究EGCG和石杉碱甲与牛血清白蛋白的相互作用来解释增强机理。荧光光谱显示EGCG与牛血清白蛋白之间有很强的结合力,石杉碱甲与牛血清白蛋白之间几乎没有作用力,但在EGCG存在下,石杉碱甲可以接近牛血清白蛋白的氨基酸残基并与牛血清白蛋白发生作用。同步荧光研究显示,EGCG与牛血清白蛋白形成配合物后对酪氨酸残基和色氨酸残基的结构和微环境都有影响;在EGCG存在下,石杉碱甲接近牛血清白蛋白酪氨酸残基,石杉碱甲的加入增强了EGCG和牛血清白蛋白酪氨酸残基的结合力。酪氨酸残基的结构和微环境受到明显的改变,而石杉碱甲远离色氨酸残基,对色氨酸残基没有影响。红外光谱显示EGCG比石杉碱甲更能影响牛血清白蛋白的二级结构。因此,可能的机理是EGCG首先和血清白蛋白的酪氨酸残基通过氢键形成稳定的配合物,然后石杉碱甲通过氢键与EGCG-血清白蛋白配合物中的EGCG发生作用,并通过EGCG远距离传递电子给血清白蛋白酪氨酸残基,增加了EGCG和血清白蛋白酪氨酸残基的结合力。与EGCG相比,GCG与牛血清白蛋白的结合力降低了100倍。EGCG与GCG具有相似的结构,差异仅在2位上空间结构,说明手性异构体影响其与BSA的相互作用。5.基于生物大分子荧光猝灭的二项式分布模型用于分析药物小分子与生物大分子的相互作用——以白藜芦醇和牛血清白蛋白为例药物小分子和血清白蛋白存在高结合力和低结合力的结合位点,通常通过Stern-Volmer方程和双对数曲线方程及其修改形式获得的结合常数仅反映具有高结合力的结合位点。与具有高结合力的位点相比,低结合力的位点实际上并没有被药物分子占据。因此药物实际占据的位点数和结合位点数是不同的,许多模型没有考虑到这一点。本文在此基础上,以白藜芦醇和牛血清白蛋白为例,建立了一个基于生物大分子荧光猝灭的二项式分布模型用于研究药物小分子和生物大分子的相互作用,并用同步荧光和红外光谱深入了解白藜芦醇和牛血清白蛋白的相互作用。白藜芦醇与牛血清白蛋白的主要作用方式是范德华力和氢键作用力,△G是负值说明该过程是自发的。同步荧光光谱表明白藜芦醇与牛血清白蛋白色氨酸残基和酪氨酸残基很接近,影响了色氨酸残基和酪氨酸残基的微环境。白藜芦醇更能影响酪氨酸残基,不仅猝灭酪氨酸残基的荧光强度而且引起酪氨酸残基的最大发射波长蓝移。在牛血清白蛋白的红外光谱中表征其二级结构中酰胺Ⅰ的特征峰(1620-1700 cm^-1)几乎消失,酰胺Ⅱ谱带从1541.60 cm^-1移动为1544.10 cm^-1,这表明牛血清白蛋白的二级结构已发生改变。研究还发现白藜芦醇和牛血清白蛋白结合的临界距离（R₀）几乎不随温度变化而变化,且与荧光测量参数的取值无关,如激发光栅狭缝宽度,发射光栅狭缝宽度和灯电压等。更多还原

【Abstract】 The interaction between bio-active compounds from Chinese medicine and proteins has attracted great interests among researchers,which can provide important information for their nutritional and medical effects on human health. The work in this thesis mainly concerns about the interaction between polyphenols and BSA.The flavonoids and resveratrol were selected to study the structure-affinity relationship,theory model and application about the interaction between small molecules and biomacromolecules.1.Analysis of interaction between puerarin and BSA by multispectroscopic methodThe interaction between puerarin and BSA is investigated by means of fluorescence spectroscopy,resonance light-scattering spectroscopy,infrared spectroscopy,and synchronous fluorescence spectra.The binding constants（K_a） between puerarin and BSA are 1.13×10⁴ L/mol（20℃）,and 1.54×10⁴ L/mol （30℃）,and the number of binding site（n） is 0.95±0.02.The experimental results shows that puerarin can be inserted into BSA and quenches the inner fluorescence by forming the puerarin-BSA complex.The addition of increasing puerarin to BSA solution leads to the gradual enhancement in RLS intensity, exhibiting the formation of the aggregate in solution.It is found that both static quenching and non-radiation energy transfer are the main reasons for the fluorescence quenching.The positive entropy change and enthalpy change indicate that the interaction of puerarin and BSA is driven mainly by hydrophobic interaction.The process of binding is a spontaneous process in which Gibbs free energy change is negative.2.Effect of hydroxyl groups in the ring B of flavonols on binding with BSAThe hydroxyl group in ring B of flavonols is a significant structural feature as free radical scavengers and antioxidants.In this paper,four flavonols （galangin,kaempferol,quercetin,and myricetin） with different hydroxyl groups in ring B are studied for their affinities for BSA by quenching the protein intrinsic fluorescence.From the spectra obtained,the quenching constants,the binding constants,and the number of binding sites are calculated.The hydroxyl groups in ring B of flavonols significantly affect the binding process;in general, the binding affinity increases with increasing hydroxyl groups in the ring B. The binding constants（K_a） are determined as:myricetin（4.54×10⁸ L/mol）＞quercetin（3.65×10⁷ L/mol）＞kaempferol（2.57×10⁶ L/mol）＞galangin（6.43×10⁵ L/mol）.Addition of another hydroxyl group on the ring B can enhance the affinity for BSA by one order of magnitude.The hydrophobicity of these four flavonols is assessed by the partition coefficient values(K_ow) and the chromatographic retention factors（K’） of these four flavonols are determined by HPLC.K’ and lgK_ow are inversely proportional to lgK_a for flavonols with increasing hydroxyl groups on the ring B.These results showed that the hydrogen bond force plays an important role in binding flavonols to BSA. These results are also in agreement with the generally accepted structure -dependent free radical scavenger and antioxidant abilities of flavonols.3.Effect of glycosylation of flavonids on binding with serum albuminThe dietary sources of flavonoids are almost always glycosides,which in most cases are hydrolyzed to aglycones to produce effects.Four flavonoid aglycones（baicalein,quercetin,daidzein,and genistein） and their glycosides （baicalin,quercitrin,daidzin,and genistin） are studied for their affinities for BSA.The glycosylation of flavonoids affects the binding process;in general, the glycosylation decreases the binding affinity.Glycosidation could lower the affinity for BSA by one to three orders of magnitude depending on the conjugation site.For quercetin and quercitrin,the binding constants for BSA are 3.65×10⁷ and 6.47×10³ L/mol,respectively.For baicalein and baicalin,the binding constants are 4.54×10⁸ and 1.63×10⁶ L/mol,respectively.This result partly supports that the flavonoid aglycones are easier absorbed than flavonoid glycosides.The E_1/2 values of flavonoids are inversely proportional to the binding constants binding to BSA.Higher binding affinities with BSA are associated with higher anti-oxidant and free radical scavenger activities for this class of compounds.4.Investigate the mechanism of enhancement effect of EGCG on huperzine A inhibiting AChE activity by binding with BSA The mechanism of enhanced effect of（-）-epigallocatechin-3-gallate （EGCG） on huperzine A（Hup A） inhibiting acetylcholinesterase（AChE） was investigated by studying the binding process with BSA using multi-spectroscopic techniques.Hup A is a potent and reversible inhibitor of AChE and available currently in the market for Alzheimer’s disease.EGCG is the main polyphenol in green tea and the most widely studied polyphenol for disease prevention.EGCG can enhance the inhibitory effect of Hup A on AChE and also can greatly prolong the inhibitory time.The fluorescence quenching and infrared spectra data indicate that there is a strong binding force between EGCG and BSA and Hup A hardly interacts with the main transport protein in blood.The binding constant（K_a） between EGCG and BSA was 2.96×10⁷ L/mol（37℃）.In the presence of EGCG,Hup A can affect BSA.The synchronous fluorescence data shows that EGCG is close to Trp and Tyr residues and affects their microenvironment.After bound with EGCG,Hup A can affect BSA and Hup A remarkably affected the microenvironment of Tyr residues,but Hup A didn’t affect the microenvironment of Trp residues. According to the above results,here showed the suggested mechanism of enhanced effect of EGCG on Hup A inhibiting AChE.At first,the EGCG-BSA complex forms.Hup A is suggested to bind to EGCG-BSA complex and locates in close proximity to the Tyr residues.The binding Hup A to EGCG also increases the affinity of EGCG for BSA through electron transfer.The enhanced transporting of Hup A in blood might be a cause of the enhanced effect of EGCG on Hup A inhibiting AChE.The binding constant（K_a） between GCG and BSA was 2.98×10⁵ L/mol（R=0.9930） and the number of binding site（n） was 1.22.The affinity of GCG to BSA is 100-times lower than that of EGCG for BSA.GCG and EGCG have the same structure;only the bond of C-2 is a epimer.5.A new model based on binomial distribution to analysis the interaction between drug and protein.A new model based on binomial distribution by means of fluorescence quenching was developed to gain insights into the interaction of small molecules with protein.Trans-resveratrol and BSA were used to test.The binding number maximum of Trans-resveratrol was determined to be 8.86 at 293.15 K,23.42 at 303.15 K and 33.94 at 313.15 K and the binding mechanism analyzed in detail.The apparent binding constants（Ka） between Trans-resveratrol and BSA were 5.02×10⁴（293.15 K）,8.89×10⁴（303.15 K） and 1.60×10⁵ L/mol（313.15 K）,and the binding distances（r） between Trans-resveratrol and BSA were 2.44,3.01,and 3.38 nm at 293.15,303.15,and 313.15 K,respectively.The negative entropy change and enthalpy change indicated that the interaction of Trans-resveratrol and BSA was driven mainly by van der Waals interactions and hydrogen bonds.The process of binding was a spontaneous process in which Gibbs free energy change was negative.The interaction of Trans-resveratrol and BSA was confirmed by synchronous fluorescence and FTIR spectra.更多还原