野扇花属植物中具有促雌激素生成作用生物碱的发现与作用机制研究

The deficiency of estrogen was the main cause of many diseases, such as the hypofunction of ovary, climacteric syndrome, and osteoporosis. Estrogen replacement therapy had to need long term use and was prone to induce endometrial carcinoma and breast cancer. Promoting estragen biosynthesis in local tissue was the new therapy for diseases caused by the deficiency of estrogen.It was first discovered that characteristic constituents, alkaloids, in Sarcococca hookeriana var. digyna could significantly increase estradiol concentration in KGN cells. The activtiy of two seperated compounds was stronger than those of orther compounds from literatures. Thus, it was suggested that alkaloids in Sarcococca hookeriana var. digyna may be the source finding promoting estragen biosynthesis drug. Thus, on the base of our previous works, in order to establish a library of diversiform structure compounds, the plants of Sarcococca will be systematic seperated and high-content compounds will be derived througy sytheses. The works of screening bioactivity compounds will be carried out with three cell models and the better activity compounds will be reassay with a ovariectomized rat model. The mechanisms on increasing estradiol concentration will be discussed at enzyme and gene levels. the results, that this project will be accomplished to obtain, will provide theoretical basis for dicovering drug regulating ovarianfunction and treating premature ovarian failure,infertility and aged male osteoporosis caused by lacking estrogen.

雌激素缺乏是引起卵巢功能低下、更年期综合症以及骨质疏松等多种疾病的首要因素。外源性雌激素替代疗法不仅需长期用药，且易诱发子宫内膜癌和乳腺癌等疾病。促进局部组织内源性雌激素分泌是治疗由雌激素缺乏引起相关疾病的新途径。本课题组首次发现野扇花属植物中的特征性成分——甾体类生物碱能显著提高人卵巢颗粒细胞模型中雌二醇(E2)水平，且其中两个化合物活性高于文献报道的其他类型化合物，因此有望从该类生物碱中筛选出具有促进体内雌激素分泌的先导化合物。本项目拟进行，1）系统分离野扇花属植物中的甾体类生物碱，对高含量化合物进行结构改造，合成系列衍生物，建立结构丰富的化合物库；2）选择3种细胞进行活性筛选，同时采用切除雌性大鼠卵巢动物模型对活性显著化合物进行活性再评价；3）从酶及基因水平研究高活性化合物促E2生成的作用机理。该研究旨在寻找有促内源性雌激素生成作用的活性物质，为新型雌激素水平调节药物的开发奠定基础。

以研究发现调控雌激素天然产物、阐明作用机制与构效关系为目的，本课题开展了7种植物的化学研究，评价了它们对雌激素合成的作用及机制，并探讨了构效关系。取得如下进展： (1)分离和合成化合物111个，结构以孕甾烷类生物碱为主，外延到柠檬苦素衍生物、联苯环辛烯型木脂素、螺二烯酮型木脂素、联苯四氢呋喃型木脂素、螺环化合物、齐墩果烷型皂苷、香豆素、黄酮等天然产物结构，新化合物12个，所有化合物均从相应植物中首次得到，为这些植物的开发提供研究基础。 (2) 以KGN细胞模型筛选出促雌激素合成活性化合物6个，其中生物碱3个、柠檬苦素类2个、香豆素1个。EC50分别为69.5±5.56、84.9±5.03、82.2±9.61、165.4±3.61、162.9±28.6、187.87±1.13μM。(3) 甾体生物碱促雌激素合成构效关系研究发现3位羟基对活性影响较大，如果羟基发生酯化和醚化，其活性基本丧失，如果氧化成羰基，活性进一步加强。为该类化合物构效关系研究提供方向。(4)采用实时定量 RT-PCR和western blotting、芳香化酶活性检测技术阐明3个物碱促雌激素合成的机制是直接作用于芳香化酶，而非上调芳香化酶表达而产生作用。上述结果为阐明雌激素调控机制，针对芳香化酶进行药物设计提供了重要的活性化合物及理论基础。