基于化学成分敲除与活血化瘀功效定量的川芎质量评价研究

Chuanxiong (the rhizome of Ligusticum chuanxiong Hort.) is a common used Chinese herb for activating blood and dissolving stasis. Its quality cannot be effectively assessed by the chemical marker recorded in Chinese Pharmacopeia (2015 edition). In fact, the pharmaceutical efficacy of Chuanxiong is the synergistic action of multi-effective compounds. The kinds of effective compounds are almost the same among different grades of Chuanxiong herbs, while the contents and ratios are different among the various grades. So, the quality of Chuanxiong can be effectively assessed after the kinds and ratios of these effective compounds (Q-marker) are discovered. Applicants have found out the chemical varieties among Chuanxiong samples in different quality and in different storage periods, and therefore have studied their bioactivities on antiplatelet aggregation in vitro. In this proposal, the pharmaceutical efficacy for activating blood and dissolving stasis is the core, and the integration intensities (pharmacodynamics indexes) of antiplatelet aggregation, anticoagulation and decreasing blood viscosity are used as pharmacodynamics markers for quality assessment of Chuanxiong. The study content includes four parts. .Firstly, Chuanxiong sample is extracted in ethanol and water respectively by reflux. The extraction solutions are mixed and condensed to obtain the total extract. This total extract is separated by preparative HPLC techniques to obtain the chemical samples of extract lacking one chemical compound and the chemical sample of single compound, this procedure is named as Chemical component knock-out. These chemical samples are further investigated on the pharmacological functions and mechanism by rats experiment in vivo. After the principal component analysis (PCA) on the chemical samples and pharmacological data, the kind of effective compounds are found in Chuanxiong. .Secondly, more than 40 Chuanxiong samples are collected and sorted into three grades by macroscopic characters. These samples are analyzed by HPLC-DAD-MS techniques to quantify the level of effective compounds and collect HPLC chromatograms, and further bioassay the intensity of pharmacological effects by rats experiment in vivo. By multiple regression analysis on the contents of effective compounds, HPLC chromatograms, intensity of pharmacological effects, the correlations are found among the macroscopic character, effective compounds and pharmacological effects. The contents and ratios among effective compounds are found among the various grades Chuanxiong. .Thirdly, the chemical analysis methods are developed to quantitatively analyse on these effective compounds in Chuanxiong for quality assessment. .Finally, over 60 Chuanxiong samples are collected from Authentic and Non-authentic Cultivation Areas. The effective compounds and pharmacological effects are quantified by chemical analysis and rats experiment in vivo. The correlations among herbs cultivation area, pharmaceutical efficacy and quality are found by multiple regression analysis. After the certification of this new development model for quality assessment, the new method for effectively assessing the quality of Chuanxiong is developed.

川芎为活血化瘀药，现行《药典》的指标难以有效控制其质量。川芎的功效是多种药效成分协同作用的结果。不同质量川芎药效成分的种类几乎相同，仅含量及比例不同；只要明确了药效成分的种类及其比例（质量标志物），便可有效评价其质量。申请者已发现不同品质和不同贮藏期川芎的化学成分不同，并进行了动物体外抗血小板聚集作用研究。本项目以活血化瘀功效为核心，以抗血小板聚集、抗凝血和降低血液黏度为药效指标，通过对川芎化学成分敲除，大鼠体内实验，研究各化学部位/成分的药理作用及其机制，经主成分分析，明确川芎药效成分的种类。通过测定不同性状川芎的药效成分和药理作用，谱-效关系的多元回归分析，阐明药材性状、药效成分与药理作用的相关性，找出川芎质量差异的药效成分含量及比例。再建立测定这些药效成分的方法。并测定道地与非道地产区川芎药材，分析产地、药效、质量的关系，验证本质量评价模式的实用性，为有效评价川芎质量提供新方法。

川芎等中药是多种成分协同发挥药效作用，目前选择的1种或几种指标成分与功效的相关性不紧密，难以有效评价其质量。《中国药典》以乙醇浸出物和阿魏酸的含量为川芎质量评价指标，特征性不强。虽然藁本内酯是川芎的主要药效成分，但是为挥发性油状物，不稳定，难以作为对照品用于含量测定。为此，本项目以川芎活血化瘀功效为核心，研究药效成分的种类及其对药效的贡献度，建立川芎质量评价的新方法：第一、通过网络药理学和分子对接技术，预测出川芎发挥活血化瘀功效的11种药效成分及其机制。第二、选择抗血小板聚集、抗凝血和抗凝胶作用为活血化瘀功效的药效定量指标，建立了测定这些药效的生物检定方法；并根据各药效的作用机制，确定了综合药效指数的计算方法，这三种药效的权重系数分别为50%、37.5%和12.5%。第三、用制备HPLC技术逐个敲除川芎总提取物中10种主要成分，制备缺乏某种成分的阴性样品；并测定和比较这些成分敲除前、后这三种药效及其综合药效的变化，筛选出6种药效成分。第四、根据传统经验鉴别方法，将37份川芎药材分为三等，分别测定性状（重量、颜色、气味）、化学成分（8种成分的含量和指纹图谱）和药效作用，并进行两者之间和三者之间的相关性分析，选出与药效作用相关性强的4种成分（阿魏酸、丁烯基苯酞、Z-藁本内酯、洋川芎内酯A）。第五、综合前面选出的药效成分及其在川芎中的含量，选择Z-藁本内酯、洋川芎内酯A和阿魏酸为川芎质量评价的指标成分；再经多元回归分析，得到质量指标的计算公式为Y = 0.574X1 + 0.496X2 + 0.466X3。第六、选择欧当归内酯A为内标，建立了HPLC测定这些质量指标成分的一测多评方法。第七、对川芎、茶芎和云芎相近药材进行了性状、化学和药效的测定和比较，发现川芎的质量最优。本项目以川芎功效为核心，筛选对药效作用贡献大的主要成分为化学指标，建立的化学评价方法包含了药效贡献系数，能有效评价川芎的质量，为中药的质量评价提供了一种新型、实用的新模式。