基于草乌减毒效应的诃子化学成分及缓和药性特征的相关研究

Fructus chebulae is one of the most commonly used drugs in Mongolia medicine and it is called King of medicine. With the reducing toxic effect, the position of Fructus chebulae in Mongolia medicine is similar to the Glycyrrhiza in Chinese medicine. Most of the previous detoxification studies on processing Aconitum kusnezoffii Reichb. with Fructus chebulae decoction were focused on the changes of detoxification effects and chemical components of Aconitum kusnezoffii Reichb., while the researches on how to play the detoxification effects of Fructus chebulae were not enough. This restricted understanding the processing laws of Mongolia medicine and the chemical components changes of Fructus chebulae.In this study, we suggest that the alleviate drug property character of Fructus chebulae is the basis of detoxification effect on Aconitum kusnezoffii Reichb., and there are relationships between toxic effect of alkaloid in Aconitum kusnezoffii Reichb. on myocardial cells and chemical components changes of Fructus chebulae. The detoxification effect of processing Aconitum kusnezoffii Reichb. with Fructus chebulae decoction is taken as the point of penetration. On the levels of cells pulsing effects and apoptosis, the toxic damages on myocardial cells by alkaloid in Aconitum kusnezoffii Reichb. which processed by different techniques is described.Using biochemical methods to observe the impact of myrobalan rat myocardial enzymes.Using biochemical methods to observe the impact of myrobalan rat myocardial enzymes.To analyze the Fructus chebulae soaks solutions that processed using different parameters with fingerprints andIR、 LC-MS methods then processing the data by complex system cluster method. By coupling the chemical components fingerprints of Fructus chebulae with the multiple correlation analysis of toxic effect of alkaloid in Aconitum kusnezoffii Reichb. on myocardial cells, to explore chemical fingerprint characters of Fructus chebulae relate to the detoxification effect of Aconitum kusnezoffii Reichb.. To clarify the alleviate drug property characters and detoxification mechanism of Fructus chebulae, to provide the scientific proofs of drug property theory of Mongolia medicine

诃子是蒙医常用的一味药，被称为"众药之王"。在蒙医药中的地位类似中医药的甘草，有较好的解毒作用，诃子汤炮制草乌减毒的研究以往多集中在对草乌减毒效应及草乌的化学成分变化，对诃子如何发挥解毒作用的研究不足，制约了对蒙药炮制规律及诃子药性特征的理解。我们提出"诃子缓和药性特点是对草乌炮制减毒的药性依据，制草乌总生物碱对心肌细胞的毒性效应与诃子的成分变化有相关性"的假说，拟以诃子汤炮制草乌减毒效应为切入点，从细胞搏动效应、细胞凋亡等多层次描述不同条件所得的制草乌总生物碱对心肌细胞的毒损效应，采用复杂系统聚类分析结果，并结合IR、LC/MS等技术对所得不同条件的诃子浸泡液进行分析，通过偶联不同条件下诃子化学成分指纹图谱与制草乌总生物碱心肌毒性效应的多元相关性分析，寻找与草乌减毒效应相关的诃子化学指纹谱特征，运用生物化学方法观察诃子对心肌酶的作用，从而阐明诃子的缓和药性特点及解毒作用机理。

诃子是蒙医常用的一味药，被称为“众药之王”。在蒙医药中的地位类似中医药的甘草，有较好的解毒作用，诃子汤炮制草乌减毒的研究以往多集中在对草乌减毒效应及草乌的化学成分变化，对诃子如何发挥解毒作用的研究不足，制约了对蒙药炮制规律及诃子药性特征的理解。我们提出“诃子缓和药性特点是对草乌炮制减毒的药性依据，制草乌总生物碱对心肌细胞的毒性效应与诃子的成分变化有相关性”的假说，以诃子汤炮制草乌减毒效应为切入点，以原代培养的乳大鼠心肌细胞为对象研究生草乌、诃子汤制草乌总生物碱对心肌细胞的毒性；以细胞存活率（MTT法）、细胞乳酸脱氢酶（LDH)漏出率等指标反映细胞毒性变化；采用Hoechst33528荧光染色法和Annexin V/PI双染色法研究心肌细胞凋亡；并用流式细胞仪检测心肌细胞[Ca2+]i变化，研究诃子汤制草乌对心肌细胞毒性损伤及减毒机制。研究结果不同浓度的生草乌总生物碱和诃子汤制草乌总生物碱对心肌细胞的影响程度不同，诃子汤制草乌总生物碱通过减轻心肌细胞膜通透性及低细胞凋亡率，从而降低心肌细胞毒性。. 采用傅里叶变换红外光谱技术（FTIR）对诃子原药材及提取物、草乌诃子不同炮制时间、不同比例样品进行分析。发现水提法最有利于诃子中有效活性成分的溶出，诃子的用量对草乌的毒性降低有明显的作用，随着炮制时间的增长，诃子对草乌中的毒性成分的中和反应越充分，减毒效果明显。.采用高效液相色谱技术（HPLC）分别从不同炮制时间、不同比例的角度研究诃子炮制草乌前后诃子中化学成分的含量变化，针对课题组前期实验筛选出的代表性成分没食子酸和鞣花酸的含量进行追踪测定。结果显示，没食子酸和鞣花酸的含量与炮制时间具有正相关性，且与诃子用量成依赖关系。. 采用LC-MS技术对诃子单煎液、草乌单煎液及不同比例草乌诃子炮制样品进行分析，去掉草乌谱峰干扰后，针对样品中诃子相关化学成分进行分析，分别鉴定出没食子酸、安石榴苷、诃子次酸、Tellimagrandin I（2，3-di-O-galloyl-4，6-（S）-HHDP- a/b- D-Glc）、柯里拉京、诃黎勒酸、阿魏酸、鞣花酸8个化合物。本课题全方位、多角度地阐释了诃子的缓和药性特点及解毒作用机理。