基于毒-效跟踪评价导向的千金子炮制减毒的物质基础及作用机理研究

On the basis of the cognition of unique properties of Semen Euphorbiae and the practice of manufacturing pulveratum method for resolving and reducing toxicity, this project will clarify the material basis and mechanism of action of attenuated Semen Euphorbiae pulveratum after processed, by using modern technologies of separation, analysis and evaluation to biological effect, taking the toxicity combined with medicine effect as indexes. We hypothesized that: Semen Euphorbiae pulveratum has less toxicity and alleviated toxicity compared with the unprocessed one, that were achieved for that the content of fatty oil were reduced or the toxic ingredients-the long chain structure of ester part of diterpenoids were changed or metabolized into low-level toxic/non-toxic ingredients.The main contents researched in this project are as follows: ①to study the correlation between toxicity and effect of unprocessed Semen Euphorbiae by using different biological effect evaluations，and to analyze and compare the toxic ingredient of unprocessed and processed Semen Euphorbiae by modern technologies of separation qualitatively and quantitatively, to prepare the toxic ingredients, and to define its mechanism of having a reduced toxicity after processed by toxic and pharmacodynamic evaluations; ②to clarify the toxicity and efficacy change of unprocessed and processed Semen Euphorbiae by ADME methods at different contents and time, and elaborated by Caco-2 cells, in situ single pass intestinal perfusion model, bowel microsomal metabolic experiment;③to explore the material basis that the process of cream is a procedure of efficiency-enhancing and toxicity-reducing on the whole animal ,cellular and molecular level by some techniques such as: morphological examination, the determination of enzymes and inflammatory factors of mucous membrane, and the comparative proteomics of intestinal tissue, to provide?scientific support and theoretical basis for ensuring its clinical safety and effectiveness and promoting the sustainable development for TCM industry.

本项目基于对千金子独特药性理论的认识和中医制霜以解毒降毒、缓和药性的实践，以毒性和药效结合指标为主线，用现代分离分析技术及生物效应评价方法研究其制霜后毒性降低的物质基础及作用机理。提出：千金子制霜减毒存效作用可能是通过降低脂肪油含量，以及化学转化和体内代谢改变其毒-效物质二萜醇酯的长链酯结构生成低(无)毒的效应物质而实现的。研究内容有：①应用不同生物效应评价指标，以毒-效的同步相关性分离制备千金子的毒-效成分，对其炮制前后的毒-效成分进行定性、定量比较分析，以毒性和药效评价指标确认千金子制霜减毒及缓和药性的科学性。②采用ADME系统从Caco-2细胞系、在体单向肠灌流系统、肠微粒体体外代谢法等方面研究千金子炮制前后量-时-效-毒之间的关系。③采用肠组织的差异蛋白组学等技术，从整体动物、细胞、分子水平研究千金子制霜减毒的作用机理，为临床安全有效应用和中药产业可持续发展提供科学支撑和理论依据。

本项目以毒性和药效评价指标为导向，研究比较了千金子制霜前后的药理作用和化学成分含量变化，确定千金子石油醚部位为其主要毒-效部位，用UPLC-Q-TOF-MS等现代分析手段进行了化学成分的分离鉴定，以毒性和药效评价指标确认了千金子制霜减毒及缓和药性的科学性。研究了千金子制霜前后提取物对SD雄性大鼠消化间期胃肠道生物电活动的影响，结果表明生品和霜品提取物均使大鼠胃肠道生物电峰电位数目增多，幅度增大，生品作用强于霜品，说明千金子能提高胃肠道平滑肌的敏感性，促进肠蠕动，制霜后对胃肠道刺激作用减弱。采用ADME系统从Caco-2细胞系、在体单向肠灌流系统、肠微粒体体外代谢法等方面研究千金子制霜前后量-时-效-毒之间的关系，结果表明，千金子中主要的二萜醇酯类成分千金子素L1、L2、L3可能为P-gp的底物，通过阻断P-gp对底物的外排作用，使药物在肠道的跨膜转运增加，生物利用度增加，并且能被肠微粒体CYP450酶所代谢分解。研究了IL-1，IL-2、IL-3、IL-6、IL-8、IgA、TNF-α等细胞因子和免疫因子在炎症过程中的作用，结果表明，霜品对促炎因子的抑制作用高于生品，对肠道粘膜炎症和损伤减弱。研究了千金子制霜前后对正常大鼠小肠消化酶（LDH、MDH和乳糖酶）和胃肠激素（VIP、SP）的调节作用，结果表明，制霜后可提高小肠消化酶活性，升高VIP 和SP 水平，对肠道毒性降低。用iTRAQ技术的定量蛋白质组学研究了千金子制霜前后作用于肠道的差异蛋白，结果表明，千金子作用于肠道组织鉴定的差异蛋白，主要由细胞部分构成，表达部位多位于细胞内及细胞器。生品与霜品提取物共同调控的有G蛋白信号通路、Eph/Ephrin信号通路，霜品提取物调控不同的通路主要有细胞骨架、糖酵解与糖异生等。对所有差异蛋白进行生物信息学分析，分析筛选出Ang-4、STAT1并进行了Western blot验证，推测Ang-4和STAT1可能是千金子作用于机体的靶点蛋白之一。本研究从整体动物、细胞、分子水平研究千金子制霜减毒的作用机理，为千金子临床使用的安全性评价提供参考依据，并对千金子的合理用药起到一定的指导作用。