摘要
异槲皮苷(isoquercitrin, IQ)是广泛存在于药用植物、食品饮料、蔬菜水果中的天然黄酮类化合物,具有多种重要的生理活性及多方面优良的药理活性。研究发现某些金属离子与天然药物配合后,形成了具有较强的清除超氧阴离子自由基能力的配合物,提高了药物的抗氧化性,同时也降低了天然药物的毒副作用。
本文以异槲皮苷为研究对象,运用分析化学和生物化学手段,对异槲皮苷及其金属配合物的光谱特征和抗氧化特性及异槲皮苷的药代动力学进行了研究。
全文主要分以下五部分：
第一章本章简要综述了活性氧自由基和抗氧化作用。活性氧自由基是生物体内与氧代谢有关的含氧自由基,它有极高的反应活性,参与了许多生命过程,在生命活动中起着十分重要的作用。研究表明人体摄入外源抗氧化剂可清除体内多余的自由基,并对多种与自由基有关的疾病有治疗和改善的作用。异槲皮苷作为一种重要的天然黄酮类化合物,因其具有多方面生物活性而广泛应用于食品和医药领域。文献表明异槲皮苷具有清除自由基作用,但其金属配合物的抗氧化作用则鲜见报道。
第二章异槲皮苷及其金属配合物与牛血清白蛋白作用的光谱特征研究。运用紫外光谱法、荧光光谱法研究异槲皮苷与牛血清白蛋白(BSA)相互作用,机理及BSA和异槲皮苷-BSA结合物的紫外吸收光谱,实验测定了在26℃和37℃时异槲皮苷与BSA结合的猝灭常数(2.65×1012L·mol-1·s-1,2.57×1012L·mol-1·s-1)、结合常数(1.56×103L·mol-1,1.47×103L·mol-1)、热力学参数平均值(△H=4.16kJ·mol-1,△S=74.56J·K-1,△G=-18.54kJ·mol-1)、作用位点数n(0.73和0.71)、给体(BSA)与受体(IQ)间距离r(3.73 nm)。本文还研究了BSA和异槲皮苷-BSA结合物的紫外吸收光谱,发现吸收峰发生了蓝移,提示有新的复合物生成。同时确定了铁锌离子与异槲皮苷的配合比(1：1型结合；2：1型结合),并研究了铁锌离子-BSA及离子-异槲皮苷-BSA的光谱特征。结果提示异槲皮苷与BSA主要是凭借疏水作用力结合,以静态猝灭方式使BSA强度减弱,且异槲皮苷的部分片断能够插入BSA分子内部进而影响其猝灭过程。金属离子介入影响了异槲皮苷与BSA的结合。
第三章研究了异槲皮苷及其金属配合物体内、体外的抗氧化能力。体外实验测定了异槲皮苷及其金属配合物清除超氧阴离子自由基、羟自由基和亚硝酸阴离子的能力。实验表明异槲皮苷铁或锌配合物清除体外自由基的能力比异槲皮苷强。体内试验测定了小鼠肝脏和肾脏异槲皮苷及其金属配合物对氯化镉诱导小鼠产生氧化损伤的抑制作用。实验表明镉引起小鼠肝或肾组织超氧化物歧化酶和过氧化氢酶活性下降及丙二醛、一氧化氮、蛋白质羰基、蛋白质与DNA交联率水平的增高,而异槲皮苷及其金属配合物却能改善氧化状态,表明异槲皮苷及其金属配合物具有抗氧化能力。
第四章建立了动物体内IQ水平的高效液相色谱-紫外检测分析法,并对异槲皮苷药代动力学进行研究。该方法线性范围为0.021-0.126μg,回归方程：Y=5924.01+1.11234×106C,相关系数r=0.9991；样品中最低定量限为15.8 ng/ml；日内和日间RSD<5.0%。方法是准确、灵敏、可靠的。小鼠尾静脉注射异槲皮苷后,异槲皮苷能迅速的分布在小鼠血液中,30min吸收水平达到峰值,在240min后仍然能检测到IQ。然而,异槲皮苷灌胃后,小鼠肝脏中未检测到其单体。
第五章对以上实验进行了总结,并对异槲皮苷及其金属配合物的研究进行了展望。
总之,本文的实验结果表明异槲皮苷可与蛋白(如BSA)结合形成复合物,且金属离子介入能影响异槲皮苷与BSA的结合,引起光谱特征的变化,将直接或间接地影响蛋白质在体内的生理作用；异槲皮苷及其金属配合物对外来毒物引起的氧化损伤具有抑制作用；异槲皮苷能迅速进入血液并维持较长时间。这些结果提示异槲皮苷可通过与蛋白质结合及抗氧化作用在临床治疗中发挥作用,且由于异槲皮苷金属配合物能影响异槲皮苷和蛋白结合的光谱特性及抗氧化性,可为改造异槲皮苷分子、寻找新的药物分子提供有价值的信息。
Isoquercitrin (IQ, Quercetin-3-glucose) is one of the important flavonoids, widely presented in medicinal plants, food, beverages, fruits and vegetables. It exhibits a variety of important physiological activity and pharmacological activity. The recent studies found that the complexes between natural medicines and certain metal ions possessed a strong ability to remove superoxide anion radical, not only increasing antioxidant property of drugs, but also reducing the side effects of natural medicines.
In this paper, IQ as the research object, spectrum characteristic, antioxidative property and pharmacokinetics of IQ and its complexes with iron and zinc were studied using spectrophotometry. Full-text mainly consists of the following five parts, summarized as follows:
Chapter I:Reactive oxygen free radicals such as superoxide anion (02-) and hydroxyl radical (·OH) have high reactivity, they participate in many life process and play an important role in life events. Studies have shown that the exogenous antioxidants intaked into human body may clear excess of free radicals in vivo, treat and improve a variety of free radical-related diseases. IQ is an important natural flavonoid compounds with multiple biological activities and widely used in food and medicines. It is suggested that IQ had potent free radical scavenging effect. However, few studies on the scavenging capacities of free oxygen radical and the anti-oxidant activities of IQ and its metal complexes against oxidation induced by xenobiotics in mice were involved.
Chapter II:IQ and its complexes with metal were characterized by UV-Vis absorption spectrometry. The results indicated that the combination reaction of them was a single static quenching process. In aqueous solution, IQ combined strongly with BSA at 26℃and 37℃, respectively.The quenching constant (2.65×1012 L·mol-1·s-1,2.57×1012 L·mol-1·s-1), binding constants (1.56×103 L·mol-1,1.47×103 L·mol-1), the average thermodynamic parameters (△H=4.16 kJ·mol-1,△S=74.56 J·K-1,△G=-18.54 kJ·mol-1), the binding sites number (n=0.73,n=0.71) were measured. The binding distance (3.73nm) between IQ and BSA were also obtained. This paper also studied UV absorption spectrum of BSA and IQ-BSA. The result showed that absorption peak wavelength blue-shift occurred, suggesting that there might be a new generation of composites to influence the conformation of BSA. On the other hand, the match ratio of Fe3+or Zn2+with IQ was found as 1:1 or 2:1, the spectrum characteristic of the interaction between the Fe3+or Zn2+-BSA and ion-IQ-BSA were also studied. These experimental results showed that hydrophobic interaction played an important role in the binding of IQ with BSA and static gravitation was the major force of the combination. The fragment of IQ was able to insert into the BSA molecule to affect its quenching process. Moreover, it suggested that the interventions of metal ions affected the combination of IQ and BSA.
Chapter III:The free oxygen radical (ROS) scavenging activities of IQ and its metal complexes (IQ-Fe3+ and IQ-Zn2+) and their preventive effects against ROS formation in liver and kidney of mice induced by cadmium (Cd) were investigated. The results showed IQ and its complexes had positive scavenging abilities in vitro for superoxide anion, hydroxyl radical and nitrite, and the capacity of ROS scavenging of IQ-Fe3+ or IQ-Zn2+ was stronger than that of IQ. CdCl2 (2.5 mg/kg b.w., i.p.) significantly inhibited the activities of superoxide dismutase and catalase and increased the levels of malondialdehyde, nitric oxide, protein carbonyl and the coefficients of DNA-protein crosslinks in mouse liver or/and kidney. Treatment with IQ and its complexes (5 and 50μmol/L, i.p.) prior to Cd2+ intoxication significantly improved antioxidant markers. The observations suggested that IQ and its complexes had the significant role in protecting animals from Cd2+-induced toxicity.
Chapter IV:The metabolites of IQ and its pharmacokinetics in mice mouse plasma and livers were determined by high-performance liquid chromatography (HPLC) with UV detection. The recursive equation was shown in the following way:Y=5924.01+1.11234×106C, r=0.9991; the standard curve was linear in the range from 0.021μg to 0.126. The limit of quantitation (LOQ) of IQ in the samples was 15.8 ng/ml. The intra-day and inter-day RSDs were less than 5.0%. The developed method was specific, accurate and sensitive. IQ could quickly spread in mouse plasma after tail vein injection and the concentration of IQ in mouse plasma achieved the peakvalue at 30 min, and could be still detected in plasma after 240 min. However, the monomer of IQ was not detected in mouse livers after intragastric administration.
Chapter V:The experiments above are summarized, and the further researches about isoquercitrin and its metal complexes were predicted.
In conclusion, the experimental results showed IQ could be coupled with protein such as BSA to form complex compounds. The interventions of metal ions affected the combination of IQ and BSA and change the spectrum characteristic of IQ, in turn directly or indirectly affect the body's physiological functions. Moreover, Cd2+ significantly induced oxidative damage of lipids and proteins in livers and kidneys of mice while IQ and its complexes could inhibit such damage effects, eventually protecting lives and kidneys of body. In addition, IQ readily distributed the blood and could be stay in the blood for a long time. This provides important experimental evidences for elucidating the binding mechanism of IQ and BSA and effects of Fe3+ and Zn2+ on the interaction and further for looking for new drug molecules.
引文
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