三种广西特色珍珠菜属植物中抗肿瘤三萜皂苷及其构效关系和作用机制研究

Malignant tumor is a great threat to human health and life. Lysimachia clethroides Duby, one of the species of genus Lysimachia (Primulaceae), has been used for the treatment of throat ache, edema, and menoschesis, etc. In vivo and in clinical studies have demonstrated that it has significant antitumor activity, but there is still lack of research on its effective constituents. In our previous research, 73 compounds, including 22 new ones had been isolated and elucidated, and 13β,28-epoxyoleananes were believed to be its effective constituents on the basis of antitumor activity test. Among the 73 compounds, 14 were triterpenoid saponins, which could be classified into four types, and two types were isolated for the first time from this genus. To obtain more active triterpenoid saponins to validate and enrich the structure-activity relationship （SAR）, and to verify the types of saponins in genus Lysimachia based on phytochemical taxonomy, we will investigate systematically the triterpenoid saponins in the active fractions of three species of Lysimachia genus plants in Guangxi, following a bioassay-guided approach. The isolated triterpenoid saponins will be tested next for their antitumor activities, together with the previous obtained triterpenoid saponins, the SAR will be constructed. Mechanism of 13β,28-epoxyoleananes will be studied through ROS-mediated endoplasmic reticulum stress, ang in vivo activity will be evaluated by SCID mice transplanted tumor model. The purpose of this project is to obtain at least 40 triterpenoid saponins, so as to discover 1 or 2 leading compounds for further antitumor research.

报春花科珍珠菜属植物珍珠菜在临床和体内均证实具有显著的抗肿瘤活性，尚缺乏物质基础的系统研究。申请者前期从珍珠菜中分离鉴定73个化合物，22个新化合物，活性测试证实其抗肿瘤物质基础为三萜皂苷；发现14个三萜皂苷，其中两种皂苷类型为珍珠菜属首次发现，且不同结构类型的皂苷抗肿瘤活性不同。为了获得更多活性三萜皂苷，验证和充实构效关系，同时从植物化学分类学上探究该属两种新皂苷类型是否普遍存在，本项目拟采用活性跟踪的手段，对同属广西特色植物灵香草、临桂香草和阳朔过路黄活性部位的三萜皂苷进行系统研究；对分离得到的化合物进行体外抗肿瘤活性评价，结合珍珠菜中三萜皂苷，构建构效关系。拟通过ROS介导的内质网应激途径开展作用机制研究，并采用SCID小鼠移植肿瘤模型进行体内药效学评价。期望获得三萜皂苷40?50个，并从中优选出1?2个体内外均具有显著抗肿瘤活性，具有进一步新药研究价值的先导化合物。

珍珠菜属植物珍珠菜在临床和体内均证实具有显著的抗肿瘤活性，尚缺乏物质基础的系统研究。申请者前期从珍珠菜中分离鉴定了73个化合物，证实三萜皂苷为其发挥抗肿瘤活性的主要物质基础，且不同结构类型的皂苷抗肿瘤活性不同。为了获得更多活性三萜皂苷，验证和充实构效关系，同时从植物化学分类学上丰富该属植物中三萜皂苷的结构类型，本项目对三种广西特色珍珠菜属植物灵香草、星宿菜和临时救的三萜皂苷富集部位进行了系统的化学成分研究。从以上三种植物中共分离鉴定了40个齐墩果烷型三萜皂苷，新化合物有20个，主要结构类型为13β,28-环氧-齐墩果烷型和△12-齐墩果烯型，还包含较为少见的28,30-内酯-齐墩果烷型。对所有三萜皂苷进行了体外抗肿瘤活性评价，发现了11个活性较好的化合物，这些活性化合物均属于苷元为cyclamiretin A或protoprimulagenin A的13β,28-环氧-齐墩果烷型三萜皂苷，初步的构效关系分析证实苷元母核的C-12、C-21、C-22、C-28、C-29等位置引入羟基会使活性丧失。完成了△12-齐墩果烯型到12-羰基齐墩果烷型三萜皂苷的结构转化。通过Annexin V/PI共染色、Hoechst 33258荧光染色、ROS测定实验等方法初步探讨了其中一个活性三萜皂苷的凋亡诱导机制；考察了部分三萜皂苷对Ras/Raf信号通路传导的影响，得出了和体外活性测试类似的构效关系。对其中一个活性显著、含量丰富的三萜皂苷进行了体内抗肿瘤活性评价，证实该化合物高剂量组（5mg/kg）对人肝癌细胞HepG2裸小鼠异种移植瘤生长具有明显的抑制作用，并显示较好的安全性。以上研究结果丰富了珍珠菜属植物三萜皂苷的类型，为扩大三种广西特色植物的产业开发或临床应用提供化学和药理学基础。