盐肤木治疗冠心病活性三萜的分离筛选及其作用机制研究

The balance of cholesterol influx, esterification and release is necessary to avoid lipid overload within macrophages, then inducing the formation of foam cells, and ultimately leading to atheroma development. Therefore, targeting these three pathways and suppressing the formation of foam cells have attracted more and more attention in research for therapies for coronary heart disease (CHD). The conclusive efficacy of Shuguantong Syrup (its raw materials were roots of Rhus chinensis) to treat CHD was confirmed by clinical trials. However, its therapeutic basis has not been well clarified. In our previous research, the triterpenoids-rich fraction derived from extract of R. Chinensis was proved to meliorate CHD of the zebrafish significantly. The phytochemical study of this fraction yielded altogether eleven compounds. Among them, five are dammarane triterpenoids, including semialactone, hydroxydammanrenone Ⅱ and three analogues with novel structures. The purified triterpenoid semialactone exhibited better therapeutic effect on zebrafish model than the fraction. Moreover, both semialactone and hydroxydammanrenone Ⅱwere reported to demonstrate ACAT inhibitory activities, considered to be promising lead compounds for the treatment of CHD. It is rather remarkable that semialactone, which shows good activities both in-vivo and in-vitro, possesses a hemiketal formed between the primary alcohol at CH3-19 and keto group at C-3. Compounds with similar structures are quite rare in nature, with only 66 natural-derived compounds and just 4 from R. chinensis reported till now. Globally profiling and effectively matching novel natural product analogues using diagnostic product ion network based on LC-MS of the targeted fraction characterized more than 40 semialactone analogues with a hemiketal in A ring, suggesting that the fraction was rich in novel compounds. This study will continue to isolate triterpenoids induced by LC-MS detection, and then to evaluate activities of single compounds using high throughput screening based on zebrafish model and THP-1 derived foam cells. This study will also explore the mechanism of active compounds on the formation of foam cells, and ultimately elucidate the therapeutic basis of R. Chinensis.

调节巨噬细胞脂质平衡，抑制泡沫细胞形成是冠心病治疗的研究热点。以盐肤木为原药材的国药准字中成药舒冠通糖浆，临床治疗“气滞血瘀”型冠心病疗效确切，然药效物质基础不明。课题组前期发现盐肤木三萜部位明显改善斑马鱼冠心病，从中分离11个化合物，包括2个抑制泡沫细胞形成的达玛烷型三萜（hydroxydammanrenone Ⅱ和semialactone）及3个结构相似的新化合物。药理实验表明semialactone改善斑马鱼冠心病作用强于来源部位。体内外均显示良好活性的semialactone具独特半缩酮基团，该类成分盐肤木仅报道4种，LC-MS分析表明该部位含40余种semialactone类似物，提示大量结构新颖的活性成分有待研究。本项目运用质谱引导纯化系统等现代分离技术，对该部位进行化学成分研究，采用斑马鱼高通筛选结合泡沫细胞模型评价单体活性，从抑制泡沫细胞形成角度初步阐明盐肤木药效物质基础。

本项目旨在研究盐肤木根治疗冠心病的药效物质基础。首先，采用质谱导向分离技术，对盐肤木根预防斑马鱼心衰的活性部位进行化学成分研究，得到20个化合物（达玛烷型三萜13个、齐墩果烷型三萜3个、线型三萜1个、长链脂肪酸3个）。 其中11个达玛烷型三萜为新化合物，且10个新三萜具稀有17位α侧链。其次，基于维拉帕米诱导的斑马鱼心衰模型，以及普纳替尼诱导的斑马鱼血栓模型，对足量化合物进行活性筛选，其中9个达玛烷型、3个齐墩果烷型以及1个线型三萜化合物均在其安全浓度下展现了显著的预防斑马鱼心衰作用，具体表现在减小心衰斑马鱼心脏扩大以及静脉瘀血面积，增加心衰斑马鱼的心输出量、血流量以及心率。同时，3个达玛烷型以及2个齐墩果烷型三萜能显著减少斑马鱼血栓形成。由于所检测三萜在预防心衰作用上表现尤为良好，我们认为很有必要对此进行深入挖掘。我们选取了其中活性较好的且结构具代表性的达玛烷型[rhuslactone和(Z)-rhuslactic acid]和齐墩果烷型（oleanonic acid）三萜，采用异丙肾上腺素诱导的斑马鱼心衰模型以及具心脏特异性荧光的转基因cmlc2:EGFP斑马鱼模型，进行进一步预防心衰作用评价，所检测三萜均能显著改善心包瘀血；(Z)-rhuslactic acid、oleanonic acid能改善心输出量；oleanonic acid能减轻心肌细胞凋亡。.本研究工作在一定程度上证明盐肤木根中所含的三萜类成分为其改善心血管疾病的药效物质基础之一，为继续深入研究该闽产中成药的物质基础及作用机制打下坚实基础。