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1、板蓝根汤剂纳米颗粒的分离与表征中文摘要板蓝根是十字花科植物菘蓝的干燥根，具有清热解毒，凉血利咽的功效。在治疗流行性病毒时有较强的抗菌、抗病、解毒的作用，是用于治疗病毒感染时的常用临床中草药。但目前其抗菌抗病毒的作用物质基础还不明确，同时由于其以汤剂的用药方式增加了其研究的复杂性。其复杂性在于板蓝根药材化学成分多，汤剂的给药方式往往是多种有效物质相互协同或拮抗，同时，汤剂的胶体学性质也在一定程度上影响着其药效的发挥。本课题围绕着前期科学假说“中药有效成分群是通过自组装成特殊的纳米构造来实现药物在体内的递送、释放和发挥药效”开展，运用胶体化学和生物化学的研究方法，对板蓝根汤剂中纳米颗粒的存在形态及
2、其基本性质等方面进行研究。板蓝根汤剂经过离心除杂后发现其具有明显的丁达尔现象，这说明板蓝根汤剂存在着胶体体系；通过AFM、SEM等手段观察板蓝根汤剂胶体颗粒的形态特征，发现板蓝根汤剂胶体颗粒呈大小不等的球形和椭球形，并且颗粒不稳定会相互聚集成小丘状或粘连成串；通过超滤的方法去除掉板蓝根汤剂中的胶体颗粒后发现其抗病毒活性大大降低。利用凝胶色谱对板蓝根汤剂中的纳米颗粒进行分离纯化。板蓝根汤剂5000g离心10min后上样，色谱条件为：Sephacryl S-1000 柱(1.0 cm100 cm)，缓冲液为0.02 mol L-1 磷酸盐缓冲液(pH5.0)，流速为0.35mL min-1，上样量
3、为2mL，检测器为马尔文动态光散射仪。对Sephacryl S-1000凝胶色谱柱分离得到的纳米颗粒进行表征。纳米颗粒大小主要分布在10100 nm之间，Zeta电位为-9.01.0 mV的范围内；对其稳定性进行研究表明：pH、温度对其稳定性有一定的程度的影响，时间对其稳定性影响很小；对其进行组成成分的分析，其中干物质、总糖、总氨基酸分别占总上样量的48%、97.6%、1%，其颗粒中总糖以多糖的形式存在；另外，表告依春、靛蓝、靛玉红在纳米颗粒中的含量较少。 对分离得到的板蓝根汤剂纳米颗粒进行MDCK细胞抗流感病毒活性实验，发现其具有一定的抗病毒活性，修复率达到39%。同时，颗粒与抗病毒药物利巴
4、韦林结合后作用于受流感病毒侵染的MDCK细胞，能明显增强抗病毒活性。关键字：板蓝根汤剂，纳米颗粒，粒径，Zeta电位，抗病毒Isolation and Characterization of Nanoparticles from Radix Isatidis DecoctionAbstract Radix isatidis, the dried root of isatis which is a kind of cruciferous plant, is proved to be effective in clearing heat, detoxification, cooling blood
5、 and relieving sore throat. With strong effects in antibacterial, disease-resistant and detoxification in the treatment of pandemic virus, Radix isatidis is generally considered to be a common clinical herbal medicine when treating viral infections, however, its antibacterial and antiviral material
6、basis is not clear. Besides, the traditional decoction when using radix isatidis increases the complexity in research, that is, the active ingredients of radix isatidis include multiple chemical composition and the effect of medical broth is the results of mutual synergistic or antagonistic effects
7、of these active ingredients. Moreover, the colloidal nature of the decoction also has an impact upon efficacy of play. In this issue, we are trying to utilize chemically colloidal and biochemical methods to investigate the presence and basic properties of the nanoparticle form in radix decoction on
8、the basis of former hypothesis, which is “the active ingredient group of TCM(Traditional Chinese Medicine) is self-assembled into a special nano-structure to achieve the delivery of the drug in the body, release, and play a pharmacodynamics”.After centrifugation to remove impurities,It have been fou
9、nd that obvious Tyndall phenomenon Exist in the Radix Isatidis Decoction,This Indicate that there is a colloidal system;Morphological characteristics of colloidal particles in Radix Isatidis Decoction observed by AFM,SEM and other means,And found that colloidal particles with size ranging from spher
10、ical and ellipsoidal,The colloidal particles is unstable and always mutual gathered into a mound-like or adhesion clusters;Get rid of colloidal particles by ultrafiltration was found that its antiviral activity have been greatly reduced. The nanoparticles from Radix Isatidis Decoction were isolated
11、and purified by gel chromatography.After 5000g centrifugation for 10 minutes pretreatment,the sample on the sample.Chromatographic conditions:SephacrylS-1000 column (1.0 cm x 100 cm),The buffer was phosphate buffer,and a concentration of 0.02 mol L-1 (pH5.0,The flow rate was 0.35mL min-1,Sample volu
12、me was 2mL,Detector was Malvern dynamic light scatting(DLS). Characterization of nanoparticles obtained on the Sephacryl S-1000 gel column chromatography.The nanoparticle size distribution between 10 to 100nm,Zeta potential was within the scope of -9.01.0 mV;Through studies have shown that pH and te
13、mperature have a certain degree of influence on its stability while the temperature does not;The composition of nanoparticles isolated by Sephacryl S-1000 gel chromatography were investigated,and the dry matter,total sugar and the total amino acids accounted for 48%, 97.6%,1% respectively.The majori
14、ty of the particles of sugar is polysaccharide;In addition,Epigoitrin,indigo,indirubin are rarely present in the nanoparticle. MDCK cells anti-influenza virus activity experiments showed that the nanoparticles isolated by Sephacryl S-1000 gel chromatography has a certain anti-viral activity,The repa
15、ir rate is 40%.At the same time,Combined ribavirin can significantly enhance the anti-viral activity in MDCK cells.Keywords: Radix Isatidis Decoction, nanoparticles, particle size, Zeta potential,Anti-virus 第一章 引言11.1板蓝根研究进展11.1.1 板蓝根简介11.1.2 板蓝根的现代药理作用研究11.1.3 板蓝根主要有效成分的研究41.2 中药汤剂中纳米颗粒的研究61.2.1 中药
16、汤剂中的胶体体系与有效活性成分61.2.2 中药与纳米化技术71.2.4 纳米技术在现代中药研究中的应用101.2.4 中药纳米颗粒的研究方法111.3本研究的目的与意义11第二章 材料与方法142.1 实验材料与设备142.1.1 主要仪器设备142.1.2 材料与试剂152.2 实验方法162.2.1 板蓝根汤剂的准备162.2.2 板蓝根汤剂中胶体颗粒的存在及其显微镜观察172.2.3 板蓝根汤剂中纳米颗粒的分离纯化182.2.4板蓝根汤剂中纳米颗粒抗流感病毒的测定192.2.5 板蓝根汤剂中纳米颗粒的表征24第三章 结果与讨论303.1 板蓝根汤剂中胶体颗粒的存在及其显微形貌观察303.1.1 板蓝根汤剂的丁达尔现象303.1.2 相差显微镜观察板蓝根汤剂中的胶体颗粒303.1.
