几种艾纳香属植物中佛司可林类二萜化合物的靶向分离及环磷腺苷酶激活活性研究

Forskolin(FSK), a diterpenoid rarely in plants, was highlighted for its adenylate cyclase activation activity. For the first time,we find that FSK type diterpenoids are abundant in Blumea aromatic, which is a Chinese herb medicines. The literature survey suggests that Blumea fistulosa, a herb belong to the Genous Blumea also have forskolin type compounds. Under the regulation and the guidance of our UPLC-QTOF-ESIMS library database, in combination with active tracking method, target compounds of Forskolin type deterpenoids would be isolated from blumea plants, such as B. aromatic, B.fistulosa and so on by means of solvent extraction, column chromatography and preparative HPLC etc. The structures of the isolated compounds will be elucidated on the bases of the modern spectral methods,such as NMR etc, and combination with CD and X-ray single crystal diffraction technology.Followed,a series derivatives of Forsklin will be obtained by structure modification or semisynthesis. The screening of Adenosine cyclophosphate activation of enzyme activity of these compounds will be performed through a competitive protein binding assay with adenylate cyclase (Ac) activity as an index to evaluate structure-activity relationships. This study will provide leading compounds for innovative drug discovery. On the other hand, this project will provide new Chinese medicinal herb sources for FSK type diterpenoids.Third,this study would provide compounds database for pharmacological efficacy mechanism study for B.aromatic,as well as those related medicinal herbs of this genus.

佛司可林二萜化合物因具有腺苷酸环化酶激活活性而成为研究热点。但是该类化合物在植物中十分罕见。我们首次发现艾纳香属植物山风中含有大量具有佛司可林骨架的二萜化合物。活性筛选和文献调查表明同属植物节节红、见霜黄也可能含有此类成分。我们拟在前期已建UPLC-QTOF-ESIMS数据库的指导下，结合活性追踪，选择山风、节节红、见霜黄等4-5种艾纳香属植物，经柱层析及制备HPLC法等，靶向分离纯化这类目标化合物单体，并运用NMR等现代波谱技术结合CD及单晶衍射等对其结构加以鉴定。然后通过结构修饰、改造，得到这类化合物的系列衍生物，并经竞争性蛋白结合分析法，以腺苷酸环化酶(Ac)活性为指标，考察各化合物对环磷腺苷酶激活活性，探讨这类化合物的构效关系，为创新药物的研发提供先导结构。该研究将为佛司可林类活性物质的大量获取提供新的植物资源，同时也为阐明山风等药用植物的活性物质基础提供依据。

据根山风中分离得几个的FSK类化合物的超高效液相色谱-四极杆飞行时间串联质谱（UHPLC-QTOF-MS/MS）分析，结合文献及Chemspider 等数据库信息，建立靶向分离鉴定的方法。对采集到的12种艾纳香属植物：节节红、见霜黄、长圆艾纳香、艾纳香、走马风、山风、尖苞艾纳香、裂苞艾纳香、滇桂艾纳香、高艾纳香、柔毛艾纳香、千头艾纳香通过建立的基于UHPLC-QTOF-MS/MS的FSK类化合物进行靶向检测，寻找具有FSK类化合物艾纳香属植物，根据筛选结果，大量采集了山风、节节红、走马风展开了靶向追踪与分离工作。.获得化合物75个，通过现代谱学技术，特别是1D及2D核磁共振技术，结合必要的单晶衍射对化合物的结构进行鉴定，获得新化合物13个，其中11个FSK类化合物。通过大鼠模型，以心肌细胞中cAMP的含量为指标，筛选得到具有酰胺酸环化酶激活活性的FSK类化合物4个，3个活性较好。通过MTT法，以抑制肺癌细胞（A549）及乳腺癌细胞（BT20）生长活性为指标，筛选得到具有抗肿瘤活性化合物2个，MTT法经EV-71手足口病病毒活性测试获得抗口蹄疫病毒化合物6个，一个含量较高，发现其IC50为4.6，活性强，具有开发价值。一个黄酮类成分，可能是山风主要活性成分之一，并申请地方标准一项《山风中馥芳香艾纳素的测定 高效液相色谱法》，为山风药材的质量控制建立新的测试方法。