基于CF-PK-PD模式的苗药飞龙掌血止血活性组分及药理作用机制研究

Miao medicine Radix toddaliae asiaticae is a kind of conventional medicinal herbs of Guizhou Miao nationality, and has the effect of dissipating blood stasis & hemostasis, dispelling wind & eliminating dampness, subdhing swelling & detoxicating. Miao people often use Radix toddaliae asiaticae for treatment of bleeding wounds, its hemostatic efficacy is very exact. However, the hemostatic activity-related chemical constituents of Miao medicine Radix toddaliae asiaticae and its corresponding pharmacological mechanisms haven't been reported so far. The application of PK-PD technology for research and development of the drugs is a potential shortcut, but the current PK-PD model is applied to understand the in-vivo dynamic process of the drug, the studied objects are relatively few and limited, some bioactive chemical constituents and the pharmacological effects are still unable to clarify clear for ethnodrugs or traditional Chinese medicines. Herein we first propose CF-PK-PD model to study Miao medicine Radix toddaliae asiaticae. Using fingerprint, PK-PD model etc technologies, the research on hemostatic activity-related chemical constituents, pharmacodynamics, pharmacological mechanisms of Radix toddaliae asiaticae with self-complex constituents and multi-pathway pharmacological effects have been carried out. Through this scientific research, the bioactive sites and constituents in Miao medicine Radix toddaliae asiaticae will be clear, CF-PK-PD model of hemostatic activity-related chemical constituents of Radix toddaliae asiaticae will also be set up, the pharmacological mechanisms of its hemostatic activity-related chemical sites and constituents will be found out. The results of the study will also provide the necessary basis and technical guidance for future work in Radix toddaliae asiaticae drug development.

苗药飞龙掌血为贵州苗族习用药材，具有散瘀止血，祛风除湿，消肿解毒之功效。苗族民众借助飞龙掌血治疗刀伤出血，其止血疗效确切，但是尚未见苗药飞龙掌血的相关止血活性化学成分与药理作用机制的系统研究报道。运用PK-PD技术研究开发药物是一条很有潜力的捷径，然而目前的PK-PD模型研究药物的体内动态过程，研究的对象往往相对少而局限；针对于民族药或者中药而言，一些生物活性相关化学成分和药理作用仍然无法阐明清楚。因此我们首次提出CF-PK-PD模式来研究苗药飞龙掌血，利用指纹图谱、PK-PD模型等技术开展自身成分复杂、多途径药理作用飞龙掌血的止血活性化学成分研究、药效药理作用机制的探讨。通过此次科学研究，我们将明确苗药飞龙掌血活性部位和活性成分，建立苗药飞龙掌血止血活性成分的CF-PK-PD模型，明确苗药飞龙掌血活性部位和活性成分的止血作用机制，也为飞龙掌血相关药品开发提供必要的研究基础和技术指导。

我们开展了飞龙掌血药材的生药学研究, 描述药材性状、组织结构、粉末显微特征。建立了一种Carb/NH2-SPE-GC-MS法测定飞龙掌血药材的农药残留。.以小鼠BT、BA、CT为评价指标, 发现飞龙掌血根皮的冷浸提取物、回流提取物都具有良好止血作用。建议在该药材提取制备中应该浸渍和回流相结合。确定了冷浸提取物的乙酸乙酯部位和正丁醇部位的止血活性比较强。.研究了飞龙掌血根皮冷浸提取物的乙酸乙酯部位和正丁醇部位的化学成分。从乙酸乙酯部位分离得到7个化合物, 鉴定了其中的4个香豆素类成分。获得量达到克级的异茴芹内酯、茴芹内酯、珊瑚菜素均为呋喃香豆素类, 推测其与药材止血活性密切相关。UPLC-ESI-Q-TOF MSE法进一步分析乙酸乙酯部位的天然化合物, 31个成分被鉴定, 4个含量大的化合物被验证。从飞龙掌血根皮的正丁醇部位分离得到25个化合物, 鉴定出14个活性单体，其中香豆素类化合物7个, 有机酸酯类化合物5个, 生物碱类化合物1个, 黄酮类化合物1个。我们的化学工作结果提示呋喃香豆素类化合物可能是飞龙掌血主要止血活性成分类型, 比如: 异茴芹内酯、茴芹内酯。.基于化学工作基础, 将CF-PK-PD与凝血系统、纤溶系统相结合探索止血成分与药理机制。在凝血系统方面, 飞龙掌血不同极性部位活性数据说明乙酸乙酯部位的止血活性最强, 基于LC-MS数据的CF分析揭示lomatin、5-methoxy-8-hydroxy psoralen通过激活内源性凝血途径和纤溶系统, 达到止血凝血的作用。PK-PD分析发现, 异茴芹内酯、茴芹内酯与飞龙掌血的体内止血活性存在关联性, 特别是FIB指标。建立了PK-PD关联模型，计算得到异茴芹内酯、茴芹内酯的Emax、EC50、ke0数值。在纤溶系统方面, 分析评价DD、FDP。乙酸乙酯部位、正丁醇部位显著提升DD和FDP水平, 正丁醇部位对FDP水平影响突出, 而乙酸乙酯部位对DD调控存在明显倾向性。异茴芹内酯、茴芹内酯与DD、FDP的PK-PD关联性分析不显著。我们认为飞龙掌血根皮主要通过凝血系统、纤溶系统达到止血凝血作用, lomatin, 异茴芹内酯、茴芹内酯和5-methoxy-8-hydroxy psoralen作为该药材的重要活性成分主要通过影响凝血系统来实现止血凝血作用。