基于中药模拟炮制和细胞膜色谱法的新疆准噶尔乌头生物碱水解机制及产物毒效活性研究

Aconitum soongaricum is one of the poisonous grass to seriously impair grass quality in xinjiang in recent years.The research makes clear its rich standing stock and high alkali content in Xinjiang. At the same time，it can be reduced toxic reaction by processed with Heating boiled method，it has the effect in analgesia，anti-inflmmatories and immunizing，and it has the unique advantage to cure common rheumatism in Xinjiang，it is worthy to research deeply and become poison to treasure.But we need the intensive study on its hydrolysis processing， its regular pattern of time history in hydrolysis product， its toxic effect activity of hydrolysis products of various alkaloids，and in what level of processing the poisonous attenuation of term of validity can be reached. Therefore，the topic firstly aimed at its alkaloid extracting，separation and evaluation，and then it adopts processing methods of Simulation processing methods of Chinese medicine to process the monomer and composition obtained，observes the change law of hydrolysis with heating time; adopts CMC and HPLC-MS technique to screen and separate activitived compents from their hydrolysis products, proves thd changes of simulation processing in truly processing at the same time, and illuminates hydrolysis mechanism. Accordingly we can make clear the toxic effective material basis in processed products of aconitum soongaricum on the basis of investigating of aconitum soongaricum，the in vitro cytotoxicity-test and cardiotoxicity，anti-inflammatory activity in vitro，the anticancer effect and its mechanism, determine the toxic effect material base; Analysis of correlation between various components and the toxic effect, the foundation of optimizing the processing technology of aconitum soongaricum can be moved forward. Processing mechanism can be clarified from molecular level.Lay the foundation for establish the quality standard.

准噶尔乌头是危害新疆草场的毒害草，前期研究表明其蕴藏量丰富、生物碱含量高，与川、草乌有较近亲缘关系，可通过炮制降低毒性，炮制后仍具镇痛、抗炎、调节免疫的药理作用，在治疗新疆多发的风湿类疾病方面有独特优势，值得深入研究，变毒为宝，但其生物碱专属性成分、炮制水解途径、水解产物毒效活性仍未知，难以建立法定质量标准，使其利用受限。本课题在前期研究基础上，提取、分离准噶尔乌头中生物碱单体，采用中药模拟炮制方法，按已确定的药材炮制方法--加热水煮法，炮制所得总生物碱和单体，考察水解变化途径，并在实际饮片炮制中验证模拟炮制变化，阐明炮制水解机制；其次，采用细胞膜色谱与HPLC/MS联用技术筛选和分离水解产物活性成分,同时考察并验证其生物碱和水解产物的体内外毒性、抗类风湿和抗肿瘤活性及机制；最后，采用统计方法分析成分与毒效活性之间的相关性，确定毒效物质基础，为确定其炮制质控指标、临床安全有效应用奠定基础。

新疆准噶尔乌头（Aconitum soongaricum Stapf.）是危害新疆草场的毒害草，在治疗新疆多发的风湿类疾病方面有独特优势，值得深入研究，变毒为宝，但其生物碱专属性成分、炮制水解途径、水解产物毒效活性仍未知，难以建立法定质量标准，使其利用受限。.本课题在前期研究基础上，采用溶剂提取分离方法，从准噶尔乌头中分离并鉴定5种生物碱单体：乌头碱、准噶尔乌头碱、16,17-二氢-12β,16β-环氧欧乌头碱、12-表-欧乌碱、脱氧乌头碱。在此基础上，首先采用HPLC法以准噶尔乌头对准噶尔乌头中4种化学成分进行含量测定，并将这4种成分进行模拟炮制；其次采用中药模拟炮制方法，结合HPLC和HPLC-MS测定结果，经过不同时间的模拟炮制后，准噶尔乌头4种化学成分中乌头碱以及脱氧乌头碱水解为毒性较小的苯甲酰乌头原碱；准噶尔乌头碱与12-表-欧乌头碱经过不同时间的模拟炮制后未发生水解反应。.其次，采用细胞膜色谱与HPLC/MS联用技术筛选和分离水解产物活性成分,首先采用高表达表皮生长因子受体细胞膜色谱（EGFR/CMC）法筛选出乌头碱、12-表-欧乌碱、准噶尔乌头碱、水解产物苯甲酰乌头原碱为作用于EGFR的有效成分；其次采用体外细胞生物实验研究筛选成分的抗类风湿活性，表明乌头碱，准噶尔乌头碱，苯甲酰乌头原碱对人类风湿成纤维样滑膜细胞（HFLS-RA）有增殖抑制作用，在体外均有一定的抗类风湿活性，可抑制HFLS-RA细胞的增殖作用。采用CCK-8检测准噶尔乌头主要生物碱及水解产物对A549细胞、PANC-1细胞、SKOV-3细胞增殖抑制作用，结果表明新疆准噶尔乌头生物碱对人肺癌细胞A549，人胰腺癌细胞PANC-1，人卵巢癌细胞SKOV-3具有抑制作用，其中准噶尔乌头碱对SKOV-3细胞抑制作用较强。采用裸鼠卵巢癌动物模型的构建，准噶尔乌头碱、苯甲酰乌头碱干预后对成瘤裸鼠瘤块增长的抑制率，结果表明准噶尔乌头碱高剂量组可显著抑制瘤块的生长。.综上，明晰了准噶尔乌头炮制减毒机制和抗炎抗肿瘤活性成分，但抗炎体内活性和抗肿瘤作用机制仍需进一步深入研究。