麻杏石甘汤胶体颗粒细胞活性分析

利用 Nile Red 荧光染料标记汤剂中的胶体颗粒，通过荧光显微镜和流式细胞 仪检测颗粒与不同细胞的作用，显示麻杏石甘汤中的胶体颗粒能结合在不同细胞 上，结合能力由荧光强度表示：MRC5>HepG2>Caco2>MDCK。其结合能力可能 与细胞活性如增殖有关。
将离心和醋酸纤维素膜滤后的麻杏石甘汤在不同细胞（MRC5, HepG2, L02, NR8383, MDCK）中评估其增殖活性，结果表明 5000 g 离心样品对细胞的增殖 活性优于其他组，其在 NR8383 细胞中增殖率达 169%。5000 g 离心再经 0.22 μm 和 0.45 μm 醋酸纤维素膜微滤后，样品毒性增加且增殖活性下降，表明胶体颗粒 可能是麻杏石甘汤的生物活性组分。
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upon cells from different mammal species or tissues in a dose-dependent manner. Colloidal particles from the decoction, labeled by Nile red, bound to and
In Ma-xing-shi-gan decoction, the total amino acids, free amino acids, protein and reducing sugar were significantly lower than the sum of those from individual herbs. The reduction in the chemicals and darkening in colour indicate the occurrence of Maillard reaction during the boiling process of Ma-xing-shi-gan decoction.
Ma-xing-shi-gan decoction improved the neutral red uptake by macrophage (NR8383 cell line) in vitro. The decoction conducted different proliferative activity
Tyndall phenomenon was observed in Ma-xing-shi-gan decoction. The morphological properties of colloidal particles were studied by phase contrast microscope, atomic force microscope (AFM) and scanning electron microscopy (SEM). The colloidal particles were spherical with an uneven size distribution at a range of 100 nm to 1000 nm in diameter. This is consistent with the measurement performed with laser light scattering.
麻杏石甘汤在保护、修复两种作用模式下均显示出体外抗 H1N1 流感病毒活 性，且其活性优于利巴韦林。缺方或各个单方煎煮得到的汤药会降低抗病毒活性， 表明药材煎煮过程其化学组分发生的相互作用有利于复方抗病毒活性的形成。
麻杏石甘汤能提高巨噬细胞（NR8383）对中性红的吞噬能力，对不同细胞 的增殖活性具有组织和细胞特异性，且有一定的剂量依赖效应。
丁达尔现象表明，麻杏石甘汤汤剂为典型的胶体体系，相差显微镜下可见明 显的胶体颗粒聚集体。通过原子力显微镜和扫描电镜，发现麻杏石甘汤中存在的 胶体颗粒呈球形，易发生聚团。激光粒度仪检测发现麻杏石甘汤胶粒的粒径分布 在 100 nm ~ 1000 nm 之间。离心和醋酸纤维素膜滤可分离胶体颗粒，5000 g 离心 后胶粒平均粒径明显高于 7000 g、15000 g 离心及 0.22 μm 和 0.45 μm 醋酸纤维素 膜微滤。
综上所述，麻杏石甘汤不仅具有抗病毒活性和细胞增殖活性，而且可以增强 肺泡巨噬细胞吞噬能力。麻杏石甘汤煎煮过程中各组分相互作用形成了胶体颗 粒，该颗粒很可能对麻杏石甘汤生物活性有显著影响。
关键词：麻杏石甘汤，胶体颗粒，抗流感病毒，增殖活性，美拉德反应
I of colloid particles in Ma-xing-shi-gan decoction
In summary, the Ma-xing-shi-gan decoction possesses potent anti-influenza virus and cell proliferation activities with a preference to pulmonary macrophages. The components in Ma-xing-shi-gan decoction interact with each other during the boiling process, which assists the formation of nano-scale colloidal particles. The particles have notable impacts on the bioactivities of Ma-xing-shi-gan decoction.
Abstract
Ma-xing-shi-gan decoction is composed of E. sinica (9 g), radix Glycyrrhizae (9 g), Armeniacae Semen (9 g) and gypsum (36 g). It is a famous Chinese herbal tonic widely used as a treatment for respiratory inflammations, i.g. asthma, acute pneumonia. However, its effective compositions and mechanism remains unclear.
enriched in different cells in vitro with a preference to the cell types, revealed by fluorescence microscopy and flow cytometry. This preference was indicated by the fluorescence intensity and numbers of Neil red labeled cells, in a sequence of MRC5>HepG2>Caco2>MDCK. The binding of the colloidal particles provide a possibility of affecting the cellular life-cycle, i.g. proliferation.
The cell proliferation activities of fractions of the decoction, separated by centrifuge or microfiltration were evaluated with cells in vitro, i.e. MRC5, HepG2, L02, NR8383, MDCK. The fraction of 5000 g centrifuge conducted the better cell proliferation activities than other fractions, while achieved the most potent proliferation rate of 169% on NR8383 cells. The 0.22 μm and 0.45 μm membrane filtration increased its cyto-toxicity when decreased its proliferation activity, which indicates the presence of colloidal particles may be dominant to the bioactivities of Ma-xing-shi-gan decoction.
Keywords: Ma-xing-shi-gan decoction, colloidal particles, anti-influenza virus activities, proliferation activity, Maillard reaction
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目录
第一章 引言 ...............................................................................................................1 1.1 中药研究现状..................................................................................................1 1.1.1 中药简介................................................................................................1 1.1.2 中药炮制................................................................................................1 1.1.3 中药配伍与拆方研究............................................................................1 1.1.4 中药发展的机遇和挑战........................................................................2 1.2 麻杏石甘汤研究现状 .....................................................................................2 1.2.1 麻杏石甘汤简介....................................................................................2 1.2.2 麻杏石甘汤化学成分研究....................................................................4 1.2.2.1 麻黄化学成分研究......................................................................4 1.2.2.2 杏仁化学成分研究......................................................................4 1.2.2.3 石膏化学成分研究......................................................................4 1.2.2.4 甘草化学成分研究......................................................................5 1.2.3 麻杏石甘汤药效活性研究....................................................................5 1.3 美拉德反应简介 .............................................................................................7 1.3.1 美拉德反应机理....................................................................................7 1.3.2 美拉德反应研究现状............................................................................8 1.3.3 美拉德反应与中药炮制........................................................................9 1.4 中药汤剂的胶体体系 .....................................................................................9 1.4.1 中药汤剂中的胶体................................................................................9 1.4.2 中药配伍、炮制和胶体颗粒..............................................................10 1.5 流感病毒简介 ............................................................................................... 11 1.5.1 流感病毒简介...................................................................................... 11 1.5.2 流感病毒分类和生物学特性.............................................................. 11 1.5.3 流感病毒颗粒的结构简介..................................................................12 1.5.4 流感病毒的增殖..................................................................................13 1.5.5 中药抗病毒研究机制..........................................................................14 1.6 本论文的意义和工作内容 ...........................................................................15 1.6.1 课题意义..............................................................................................15 1.6.2 课题实验设计......................................................................................15