【作者】 段煜；

【导师】 吴大诚；

【作者基本信息】 四川大学 ， 材料学， 2006， 博士

【摘要】 黄酮类化合物（flavonoids）广泛分布于绿色植物中，具有抗衰老、抗炎、抗肿瘤、抗病毒、免疫调节等多种生物活性，在保健食品、化妆品和药剂配置等领域有很大的应用前景。然而黄酮类化合物在自然界多以糖苷的形式存在，糖苷极性大，具有一定的亲水性，它与脂肪和油类的相容性很差，难以在脂肪类溶剂中稳定存在，这就限制了黄酮类化合物在这些领域的应用。据研究黄酮类化合物的各种生物活性都是以抗氧化活性为基础的，与抗自由基或抗脂质过氧化有关。特殊的结构赋予它一系列独特的化学性质，如能与多种金属离子发生络合或静电作用，具有还原和捕获自由基的特性以及诸多衍生化反应活性等。黄酮类化合物抑制低密度脂蛋白LDL氧化和抑制类胡萝卜素及不饱和脂肪酸氧化的研究表明，苷元比糖苷具有更优秀的抗氧化活性。这是由于苷元亲脂性强，能嵌入生物膜疏水层的内核中发挥作用。黄酮苷类化合物的多羟基及糖基结构使其具有一定的亲水性，这势必会影响抗氧化活性。黄酮类化合物通过酯化修饰，在分子中引入长烃链可以增加它的脂溶性，从而增加和脂肪的相容性及提高抗氧化性。然而通常的化学修饰反应选择性差，黄酮类化合物的多个羟基都有可能参与酯化，往往结合或屏蔽了主要活性基团，虽然增加了其脂溶性，但降低了本身的抗氧化效果。因此，化学修饰酯化反应通常要经“基团保护-酯化-脱保护基团”三步。酶催化反应具有较强的专一性与选择性，可以选择酯化黄酮类化合物的某位羟基。到目前为止，黄酮苷在酶催化下的酯化已有比较详细的研究，但反应中分子筛添加时间的不同对酯化反应的影响还未见有文献报道。目前引入黄酮苷分子中的最长烃基链也只有16个碳长度，烃链进一步增长可能赋予酯化产物新的性质，比如两亲性。正是基于这些原因，本文以硬脂酸、月桂酸和正己酸为酰基供体，以脂肪酶Novozym 435为催化剂对芦丁进行选择性酯化，合成并提纯出了三种含不同烃基链长度的芦丁脂肪酸酯。用红外光谱和核磁共振波谱对其进行结构鉴定，结果表明该类酯化物的酯化反应位为鼠李糖的C4’’’位羟基。以高效液相色谱监测酯化反应进程，首次研究了分子筛添加时间不同对酯化率的影响。结果显示，添加了分子筛的酯化反应的酯化率和反应速率远大于不添加分子筛的情况。分子筛添加时间不同，酯化率也不同。对于硬脂酸为酰基供体的情况，反应24小时后添加分子筛的酯化反应可以得到最大的酯化转化率46％。以月桂酸为酰基供体的酯化反应，反应11小时后添加分子筛可以得到最大的酯化转化率64.5％。本实验首次得到了芦丁脂肪酸酯的紫外-可见光谱，芦丁脂肪酸酯在256nm和357nm有特征吸收。生物分子的吸收代谢涉及它们和生物膜的相互作用，生物膜的结构形态和生理性质如通透性、流动性等随之发生变化。黄酮类化合物和脂质体双层膜相互作用的研究比较多，很少有和单分子膜相互作用的报道。磷脂单分子膜通常作为生物膜的简化体系，研究黄酮类化合物和磷脂单分子膜的相互作用可以从分子水平上揭示黄酮类化合物的抗氧化机理。但是黄酮类化合物的分子为多苯环排列的平面结构，不能在气/液界面铺展，因此以黄酮类化合物为模型物的单分子膜研究很少。然而通过酯化将长碳链引入黄酮类化合物分子中，这将赋予它两亲性。本文合成的芦丁硬脂酸酯（RS），芦丁月桂酸酯（RL）和芦丁正己酸酯（RC）具有两亲分子的特征，亲水基为带芸香糖和多个酚羟基的芦丁母核，疏水基为多碳烃基链，分子具有两亲性而且水溶性很低，能在空气/水界面铺展为单分子膜，可以作为一种研究单分子膜的新型模型分子。而且芦丁脂肪酸酯单独成膜性质的研究能够为它和生物膜相互作用的研究打下基础。本文用LB膜分析仪研究了RS、RL和RC在空气/水界面的成膜特性，及亚相中的Al³⁺对其成膜性质的影响，为进一步研究它们和磷脂单分子膜的相互作用打下基础。研究结果表明芦丁脂肪酸酯的成膜性与引入的碳链长度、压膜速度和亚相性质有关。RS及RL在空气/水界面都铺展为液态扩张单分子膜，它们在水面的形态为苷元部分保留在空气/水界面，芸香糖的两个糖基紧密排列于水面下，疏水链松散地伸向空气中。RS能铺展成较RL稳定的单分子膜。但相对于庞大的亲水基，两者引入的碳链较短，均不能在水面形成足够稳定的单分子膜。RC在水中有很大的溶解度，不能在水表面形成单分子膜。在Al³⁺的水溶液表面，由于亲水基与Al³⁺络合，使更多的芦丁脂肪酸酯分子能够保留在亚相表面，因此RS、RL在崩溃压时的平均分子面积较亚相为水时大；而RC也能在表面形成液态扩张膜。本文合成的芦丁脂肪酸酯是一类新型两亲分子，根据其在水面Langmuir单分子膜的研究发现，虽然芦丁脂肪酸酯的疏水性很强，能在水面铺展为单分子膜，但它在水中仍有一定的溶解度，尽管非常低。研究芦丁脂肪酸酯在溶液表面的吸附特性，可以作为其铺展单分子膜（解吸过程）的补充和对照。而且至今还未见研究芦丁脂肪酸酯吸附单分子膜的文献。本文在研究芦丁脂肪酸酯的吸附膜过程中发现它在水中的溶解度极低，1000ml水中还不能溶解1mg芦丁脂肪酸酯。如此低的浓度，再加上表面张力仪的测量误差，最终很可能测出的表面张力值和纯水的几乎相等。芦丁脂肪酸酯在碱水溶液中的溶解度比水中大，但芦丁在pH＞8.77的情况下会电离形成钠盐，因此，本文将三种烃基链长的芦丁脂肪酸酯溶解在很稀的NaOH水溶液（pH≈8）中，确保它们既能以一定浓度溶解又不会发生电离成盐。用Wilhelmy吊片法测芦丁脂肪酸酯溶液的表面张力，并用研究吸附膜的常规方法讨论了吸附过程的一些性质。三种芦丁脂肪酸酯碱水溶液的表面张力都随溶液浓度的增加而降低。它们的表面性质和含有的疏水链长度有关，疏水链越长降低碱水溶液表面张力的能力越强，CMC值大。三种芦丁脂肪酸酯NaOH水溶液的dγ/dlgC都为负值，说明芦丁脂肪酸酯发生正吸附。RS、RL和RC溶液的表面饱和吸附量，分别为1.71×10^-11mol/cm²、1.35×10^-11mol/cm²和1.41×10^-11mol/cm²。表面吸附量达到饱和时的浓度分别是3×10^-3mg/mL、1.65×10^-3mg/mL和1.3×10^-3mg/mL，对应于它们表面张力降到最小值时的浓度。它们的等温线都属于Langmuir型等温线。三种芦丁脂肪酸酯溶液的表面吸附在标准状态下能自发进行，但体系吸附能力比较弱。生物膜中含有很多饱和及不饱和的脂肪酸，黄酮类化合物的抗氧化活性部分体现在它和脂肪酸的相互作用方面。由混合单分子膜的π-A曲线可得到单分子膜的崩溃压、平均分子面积、超额自由能、可压缩性等信息，它们不仅反映成膜性质，还能提供成膜分子间的相互作用力等微观信息。硬脂酸通常都作为研究混合单分子膜的一种重要组分。基于这些原因，本文用LB膜分析仪对能在水面铺展成单分子膜的RS及RL与硬脂酸混合单分子膜在不同摩尔配比不同膜压下的相容性、热力学稳定性、膜收缩/膨胀性和压缩性等成膜性质作了研究。研究结果表明，在任何摩尔配比和膜压下两种混合体系中的组分都相容。SA的加入使膜压缩性发生的变化与膜压及SA的摩尔含量都有关。对于RS/SA混合膜体系，随SA摩尔含量增大，膜分子间作用力由吸引力逐渐变为排斥力，组分间的相容性变差。在各种膜压下，X_SA=0.167附近膜收缩效应最大，能形成最稳定的混合膜。膜压越小SA对混合膜的收缩或膨胀效果越显著。对于RUSA混合单分子膜，膜分子间作用力始终为排斥力，膜呈膨胀状态，混合体系不稳定。RS的碳链长，并且具有和SA相同的疏水链碳数，因此RS/SA较RL/SA混合膜体系的相容性和稳定性好。脂质的氧化通常是自由基进攻含不饱和键的磷脂，因此研究黄酮类化合物与含不饱和键磷脂的相互作用是非常有意义的。基于这些原因，本文选择L-α二油酸磷脂酰胆碱（DOPC）单分子膜为生物膜的简化体系，用LB膜分析仪讨论了它和RS及RL混合单分子膜的组分相容性、热力学稳定性和膜压缩性，并得到了膜分子间相互作用的相关信息。这些信息对从分子水平上解释芦丁脂肪酸酯对磷脂膜的抗氧化机理是非常有价值的。不同温度下磷脂的存在状态不同，这必然导致混合单分子膜某些性质发生改变，同时会影响分子间相互作用。因此本文还讨论了温度对DOPC/芦丁脂肪酸酯混合单分子膜的成膜性质。任何温度和任何混合比例下的DOPC/RS和DOPC/RL混合单分子膜中的两组分是完全互容的。温度越高组分相容性越好。在各种温度下，RS对DOPC单分子膜的收缩效果随表面压降低而更加显著。RS对DOPC单分子膜的收缩作用是由于两种分子形状匹配，可以以相互咬合的方式排列。DOPC与RL的相容性比较差，这是由于RL的烃链短，在某些条件下两组分的分子结构不匹配。但升温或降低RL含量都能改善两组分的相容性。DOPC/RS混合体系在三个实验温度的某些条件下都是热力学稳定的，而DOPC/RL体系只有在37℃时才在0.2＜X_RL＜0.5范围内处于热力学稳定状态。温度对DOPC/RS和DOPC/RL混合单分子膜的C_s没有影响。任何温度下，混合单分子膜中芦丁脂肪酸酯的摩尔含量越大膜越容易被压缩，且C_s随膜压的增大而减小。芦丁是一类重要的黄酮类化合物，通过酶催化酯化引入长烃基链得到的芦丁脂肪酸酯具有较强的脂溶性，由此推测它在脂类中的抗氧化性将增强。然而至今国内外还未见有关黄酮糖苷脂肪酸酯的抗氧化性研究。本文以反映脂质氧化修饰程度的硫代巴比妥酸反应物质（TBARS）为指标，研究了含不同长度烃链的RS、RL和RC对Fe²⁺介导的卵磷脂氧化修饰的影响。结果表明芦丁脂肪酸酯的抗氧化活性优于芦丁。而且RC具有最好的抗氧化活性，能使氧化体系中卵磷脂的MDA含量在四小时内都保持非常低。黄酮类化合物在人体内主要通过体液吸收代谢，而RS、RL及RC在水中的溶解度非常小，这将影响它们的抗氧化效果。本文以芦丁月桂酸酯（RL）为代表，自制了RL-聚乙二醇固体分散体。结果表明以PEG4000为载体，采用甲醇溶解法制备RL为固体分散体，可以显著提高RL的溶解度，并起到缓释RL的效果。RL在固体分散体中的分散程度随着PEG4000含量的增加而增大。在固体分散体的制备中，RL与载体PEG4000无化学键生成。制备过程不改变RL的分子结构。更多还原

【Abstract】 Flavonoids are widely distributed in different parts of miscellaneous plants and exhibit a wide range of biological activities, such as anticancer, anti-inflammatory, anti-virus, anticoagulant, anti-atherosclerosis, and inhibitory effects on low-density lipoprotein （LDL） oxidation. Due to their biological properties, the use of flavonoids in food, cosmetic and pharmaceutical preparations is currently the subject of great interest. Unfortunately, most of naturally occurring flavonoids （mainly flavonoid glycosides） show a low solubility and stability in the lipophilic media and ineffectiveness in stabilizing fats and oils. These properties limit the development of nutrition food and commodity containing flavonoids. Most of biological activities of flavonoids may arise from their resistance to oxidation including scavenging free radicals, chelating transition metal ions, and protecting the lipid from peroxidization. Aglucone show more excellent anti-oxidation compared with glucosides because the former exhibit strong lipotropy and partition preferentially into the hydrophobic core of the bio-membrane. These results are confirmed by protecting LDL and the complex of carotenoids and unsaturated fatty acids from oxidation by flavonoids. Generally speaking, the hydrophilic property of flavonoid glucosides, which dues to polyhydroxyl and saccharide groups, will compress their anti-oxidative activity. Esterification of the hydroxyl groups in molecules by fatty acids is a possible way to improve the hydrophilic property of flavonoid glucosides. In the classical esterification catalyzed by chemical catalyst, most of hydroxyl groups may be acylated, which leads to a mixture of products with various degree of esterification. Once the active hydroxyls are acylated, the biological activity of flavonoids will be reduced or even lost. However, the enzymatic catalysis is an alternative because enzymes are very regioselective. More attention has recently been paid to enzymatic esterification of flavonoid glucosides. By now a lot of literatures have reported the effect of water content, acyl donor structure, flavonoid backbone, enzyme origin and solvent nature on the results of the acylation. However, the effect of addition time of molecular sieves on the conversion in esterification has not yet been reported in some detail. The longest alkyl chain of acyl donor that has been reported by now is hexadecyl. The esterification by acyl donors with longer alkyl chains will bring rutin esters some new properties, such as amphipathy. Thus, in our experiments, rutin was acylated with a series aliphatic acid in esterification reaction catalyzed by immobilized Candida antarctica lipase B （Novozym 435） in tert-arnyl alcohol. The lipophilic rutin stearate was synthesized by this method. The structure of turin esters were characterized by spectra methods of ¹H-NMR and ¹³C-NMR, FT-IR and UV-Vis. The results suggested that the regioselectivity of the lipase-catalyzed esterification of rutin was specific at the C4’" position of the rhamnose moiety. It was found that the addition Of molecular sieves increased both of the reaction rate and the yield obviously. For the esterification which stearic acid was the acyl donor, the final conversion for the case to add molecular sieves at 24 h after the beginning of reaction was the highest, i. e. about 46%. And the highest conversion, i. e. about 64.5%, for the esterification which lauric acid was acyl donor was obtained with adding molecular sieves at 11 h. The UV-Vis spectrums of rutin esters, what have not been reported until now in other literatures, were very similar to that of rutin. The main peaks were 256 and 357 nm in the UV-Vis spectrum for rutin esters.The absorption and metabolism of pharmaceuticals involve the interaction of pharmaceuticals with bio-membrane, which leads to the changes of structure and physio-properties of bio-membrane, such as permeation, fluidity. Lipid monolayer is usually chosen as simple model of bio-membrane, so the investigation on interaction of flavonoids with lipid monolayer may be a simple as well as effective way to reveal the antioxidative activity mechanism of flavonoids. However, few papers have reported the interaction of flavonoids with lipid monolayer, more with the lipid bilayers. This probably because flavonoids can not spread at air/water interface, as other conjugated aromatic molecules. However, amphiphilic flavonoids esters which are obtained by esterification could spread at air/water interface to form monolayer. The properties of monolayers formation of amphiphilic flavonoid esters at the air/water is the precondition for investigating the interaction of flavonoids with lipid monolayer. However, little attention has by now been paid to this subject. In our experiments, we explored the monolayers formation of RS, RL and RC at the air/water interface, changing the compression rate. We also report the influences of Al³⁺ in subphase toπ-A isotherms of RS and RL monolayer. Although the introduction of alkyl chains makes the rutin, which does not spread at air/water interfaces, amphiphilic, the properties of monolayers depend on the alkyl chain length, cmpression rate and the subphase. Independent of subphase, the isotherms of RS and RL monolayers with liquid-phase are observed. Near to the collapse of monolayers, the aglucons of rutin esters lie on the surface with a tightly packing of glucose and rhamnose, which are oriented perpendicularly to the surface and point into the water phase, and alkyl chains point into air loosely. Both RS and RL have not enough hydrophobicity to form monolayers which are stable enough to maintain the surface pressure for a long period. RL with shorter alkyl chain shows a more pronounced tendency of dissolution. The molecular area of the RS and RL monolayers decreased as the compression rate was reduced, however, theπ_coll increased. RC was so soluble that it was not capable of forming monolayer at air/water interface. When RS, RL and RC were spread on aqueous aluminum solution, the values of a_coll andπ_coll of monolayers are larger compared to spread at air/water interface, and RC could spread to form monolayer with liquid-phase.Although amphipathic rutin esters could spread at water/air interface, they were soluble in water a little. The surface properties of rutin esters adsorbed at the water/air interface from bulk phase could be the supplement and comparison for that of spread monolayer of rutin esters. However, the adsorption of rutin esters at water/air from bulk phase has not been reported by now. In our experiments, the surface properties of rutin esters adsorbed at the surface of diluted alkali solution. The reason why choose diluted alkali solution was that the solubility of rutin esters in water was so low that 1 mg rutin esters could not dissolve in 1000 ml water, while rutin esters could dissolve in alkali solution. But rutin would ionize and form sodium salt in the alkali solution if pH＞8.77, thus we chose diluted alkali solution, of which the value of pH was about 8, as experiment solution. The measurement of surface pressure of rutin esters in diluted alkali solution was performed using a KSV instrument of interfacial tension with a Wilhelmy type microbalance using a platinum plate. The suface properties of rutin esters adsorbed at the surface were investigated. The surface pressure of RS, RL and RC decreased with the increasing of their concentration. Surface activity was more effective and the value of CMC was higher with longer alkyl chain of rutin esters. The values of d_γ/dlgC of all rutin esters were negative, which indicated the positive adsorption. Respectively, the maximum of surface excess of RS, RL and RC were 1.71×10^-11 mol/cm²、1.35×10^-11 mol/cm² and 1.41×10^-11 mol/cm² and the concentration of which were 3×10^-3 mg/mL、1.65×10^-3 mg/mL and 1.3×10^-3 mg/mL. The adsorption isotherm of the rutin esters were all Langmuir adsorption. At standard state, they all could adsorb at surface spontaneously.Stearic acid is usually mixed with other amphiphilic molecules to study the two-component mixed monolayer. In the present paper, the characteristic such as miscibility, thermodynamic stability and compressibility of RS/SA and RL/SA mixed monolayers have been investigated by Langmuir film balance to yield quantitative information on the nature of the molecular interaction. These are the precondition for investigating the interaction of flavonoids with lipid monolayer. The collapse pressures of the mixed monolayers increase gradually with increasing proportion of stearic acid and are between that of the pure components, which mean RS/SA and RL/SA monolayers are miscible throughout the mixture composition range. However, with the increasing of X_SA, the attraction between molecules of the two components in RS/SA mixed monolayers turns to be repulsion and the miscibility of two components turns to be bad. At X_SA=0.14-0.16 the prominent negative deviations from ideal molecular areas and the most negative values of△G_ex are observed with an exception for the case of 25 mN/m, where the maximum deviations and the most negative excess free energies are observed at X_SA=0.25. The deviations from ideal molecular areas and the excess free energies of RL/SA are all positive at any mole fraction of stearic acid. The miscibility and stability of RS/SA mixed monolayers are better than those of RL/SA mixed monoalyers. This probably because RS has longer alkyl chains and favors the array of hydrophobic groups of two components of mixture. The compressibility of the mixed monolayers seems to be sensitive to both the amount of stearic acid component and the surface pressure. The considerable fluctuations of comressibility of RS/SA and RL/SA mixed monolayers with the various X_SA were observed and the greatest compressibility were both obtained atπ=5 mN/m.Monolayers of dioleoylphosphatidylcholine （DOPC） are used as model membranes to study their molecular interaction with RS and RL. The characteristic such as miscibility, thermodynamic stability and compressibility of DOPC/RS and DOPC/RL mixed monolayers have been investigated by Langmuir film balance to yield quantitative information on the nature of the molecular interaction, which can provide valuable insights to reveal the antioxidative activity mechanism of flavonoids. The effects of temperature of the subphase on the DOPC-rutin esters interactions are investigated. Pure RS, RL and DOPC were all capable of forming monolayers with liquid-phase at the air/water interface. The two components of DOPC/RS and DOPC/RL monolayers were miscible throughout the mixture composition range. The miscibility was more excellent at higher temperature. At different temperatures, the condensing effect of RS on the DOPC monolayer, which represented the attractive forces between DOPC and RS, is more significant at lower surface pressures. This condensing effect could be ascribed to the match of molecular shapes of RS and DOPC on the monolayers packing. The less miscibility of RL and DOPC may be dues to its shorter alkyl chain. At some mole fraction of RS and surface pressure, DOPC and RL molecular shapes may mismatch, and the cohesion between RL and RL, DOPC and DOPC is greater than RL and DOPC. But high temperature and low X_RL both could improve the miscibility of RL and DOPC. The values of△G_ex of RS/DOPC monolayers are all negative at any X_RS and surface pressures, with the exception for the case ofπ= 30mN/m, X_RS=0.75, where△G_ex is positive. In the range of 0.33＜X_RS＜0.47 for all surface pressure studied, the mixture exhibits the greatest thermodynamic stability compared with the pure component monolayers. The two components of DOPC/RL monolayers mix spontaneously only for X_RL＞0.5 andπ＜25 mN/m. At any temperatures studied, the compressibility of DOPC/RS monolayers increased with the increasing of X_RS and the decreasing of the surface pressure. This phenomenon was also observed for DOPC/RL monolayers.Rutin is famous and typical one of flavonoids. Esterification of the hydroxyl groups in molecules by aliphatic acid is a possible way to improve the hydrophilic property of rutin. We expect that rutin esters will inset into bio-membrane with alkyl chains locating at the hydrophobic core. The hydrophobic interaction between the alkyl chains steadies the location of rutin esters in the bio-membrane. Furthermore, the anti-oxidation groups keep retentive during the esterification of rutin. Thus, more prominent antioxidative activity may be expected for rutin esters. However, the investigations about the antioxidation of rutin esters have been reported by now. In our experiments, using the content of thiobabituric acid reactive substance （TBARS） as index, the inhibitory effects of rutin and rutin esters on oxidative modification of lecithin induced by Fe₂₊ were compared. The results showed that, rutin and rutin esters could inhibit the oxidative modification and the role of rutin esters was better than that of rutin; rutin caporate, which has moderate solubility in water, was the best in three kinds of rutin esters.Rutin is adsorbed and metabolized via body fluid in vivo, while its three aliphatic esters, i. e. stearic ester, lauric ester and caproic ester, are weak soluble in water. In our experiments, rutin laurie ester was chosen as sample to prepare solid dispersions for increasing its water solubility. PEG4000 was taken as a carrier, and solid dispersions of rutin laurie ester with different ratio were prepared by methanol solution method. The solubility of the solid dispersion was significantly increased, compared with the rutin laurie eater, and its solubility increased with the increase of content of PEG4000. IR spectrophotometer and UV spectrophotometer were used to investigate the solid dispersion. It is clear that there was no chemical reaction between rutin laurie ester and PEG4000.更多还原