从葛根探讨基于分子聚集态的中药药效物质基础

Previous work has shown that it is a common physical phenomenon the Traditional Chinese Medicine(TCM) form aggregates in the aqueous solution, rather hazy as to what their mechanism. Exceptional bioavailability properties of TCM were hypothesized to stem from their ability to form the congeries of different molecules held together by chemical forces, we shall perhaps explain the efficient substance of multi-constituents cooperate with each other which are from the gastrointestinal tract uptake pathway into systemic circulation..This project we take Pueraria lobata (Willd.) Ohwi (PUE) as an example and study the molecular morphology composed of aggregation was related in the inhibiting angiotensin converting enzyme (ACE) based on angiotensinⅡand its ATn receptor (AngⅡ-ATR) signal pathway. Using dynamic light scattering (DLS) and transmission electron microscopy (TEM), formation of drug aggregates was demonstrated. By means of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the active components of the aggregates in PUE will be identified. Rabbits in the vertebrobasilar arterial insufficiency(VBI) were intragastrically administrated with the same doses of water extract with differing size in diameter. To investigate the therapeutic effect on cerebral blood flow velocity, detected the level of plasma AngⅡ, and the expressions of AT1R and AT2R in the vertebrobasilar arterial tissue were measured by western blotting and reverse transcription-polymerase chain reaction（RT-PCR）methods. The relationship between the content of efficient components contained in aggregation, the character of pharmacokinetics, and therapeutic activities in the vivo level will be study so as to find key chemical substances..By using molecule dynamics (MD), we can obtain trajectories of the molecular system and investigate the thermodynamics and kinetics of the system, include, the property of a weak interaction between the molecules, the molecules aggregate/water interface , the structure of multi-components aggregation -based starch prepared from PUE so that we will probe deeply into the mechanisms of resistance ACE. Single active molecule and active components of the aggregates culture rabbit's basilar arterial smooth muscle cell (BASMCs), the expressions of mRNA and protein of ACE are tested by western blotting and RT-PCR methods. These results provide evidence of interaction between drug and target..The project purpose is to reveal the integrity and specificity of TCM that play a role of therapy with "molecular cluster". Similar to the concept of nanoparticles, the aggregation morphology is expected to become the new perspectives to study pharmacodynamic material basis of TCM.

课题前期工作表明，60种单味药和24种复方水煎液，分子聚集体普遍存在，机制研究有待深入。若把聚集体作为药效的基本作用单位，将有可能解释中药多组分协同作用的物质基础。本项目以葛根为例，从AngⅡ-ATR通路，探讨聚集体的形成对药效的影响。DLS法考察聚集体在胃肠环境的动态变化；UPLC-MS/MS鉴定聚集分子结构；椎基底动脉供血不足模型兔灌胃给予同剂量不同聚集态葛根水煎液，检测脑血流速度，血浆AngⅡ；脑椎基底动脉组织AT1、AT2受体基因和蛋白表达的变化。依不同聚集体化学成分绝对量差异、药代动力学特征与药理活性的相关性，发现葛根聚集体中关键效应分子（群）。分子动力学方法计算机模拟多组分效应分子聚集体系；以此体外干预兔BASMCs考察靶酶ACE的变化，探讨聚集效应机制；揭示中药多组分以"分子团簇"发挥药效的整体性和特殊性。类似于纳米概念，分子聚集态有望成为研究中药独特药效物质基础的新视点。

中药是复杂的多组分体系，水煎液是临床常用的口服剂型，由于形成化学键或氢键和范德华力，分子之间存在着强烈的相互作用，彼此结合在一起，形成“分子聚集体”，分子聚集体的大小随浓度变化而变化，与浓度成正比。.本项目以葛根为例，如四种不同浓度的葛根水煎液（0.019 g/mL、0.038 g/mL、0.075 g/mL、0.30 g/mL），其浓度大小的顺序为G-1 <G-2< G-3< G-4；聚集分子粒径范围G-1 10.14μm~28.10μm、G-2 10.80μm~30.10μm，G-3 22.42μm~31.96μm，G-4 36.20μm~ 50.49μm即聚集体粒径（直径）大小顺序为G-1 <G-2< G-3< G-4。水煎液中分子聚集体的形成，影响中药的药效和生物利用度；等剂量的G-2和G-3葛根水煎液VBI兔灌胃的入血成分的种类和数量较多；从AngⅡ-ATR通路，对VBI兔血浆AngⅡ含量、椎基底动脉组织AT1R mRNA、AT2R mRNA表达的影响较大，药效好；而G-1和G-4药效较差。聚集分子粒径太小，由于药效成分达血药浓度的峰值太小；或聚合分子太大，不利于体内吸收；因此，粒径适中聚集体更容易吸收入血产生药效。.分子动力学方法计算机模拟多组分效应分子聚集体系，以此体外干预兔BASMCs考察靶酶ACE的变化。模拟多分子聚集体对靶标的作用优于单分子，葛根水煎液中形成以直链淀粉为基质的多组分稳定体系，显示出多组分以“分子团簇”发挥药效的整体性和特殊性。.类似于纳米概念，分子聚集态有望成为研究中药独特药效物质基础的新视点。