【作者】 叶海涌；

【导师】 楼宜嘉；

【作者基本信息】 浙江大学 ， 药理学， 2004， 硕士

【摘要】 淫羊藿（Herba Epimedii）为传统补益中药，常用于治疗男女性功能障碍、骨质疏松、更年期综合征、高血压、冠心病等疾病。淫羊藿提取物具有典型的植物雌激素样作用，因此推测其上述主治功效与药材中某种（或某些）具有雌激素样作用的成分有关。淫羊藿苷（Icariin,ICA）被认为是淫羊藿属植物的特征成分，《中国药典》规定其含量作为淫羊藿药材及其制剂的质控标准。体内代谢研究发现，芒柄花黄素（Formononetin）代谢成大豆黄素（Daidzein），鹰嘴豆素A（Biochanin A）代谢成金雀异黄素（Genistein）后雌激素样活性大大增强，推测ICA可能存在与此相同的体内代谢行为。本研究采用现代细胞分子生物学实验方法研究ICA及其拟代谢物淫羊藿素（Icaritin,ICT）和去甲淫羊藿素（Desmethyliearitin,DICT）的雌激素样作用及其构效关系，为阐明中药淫羊藿主治功效的物质基础提供科学依据。 1．ICT和DICT的制备及质谱分析 ICA在纤维素酶催化下水解葡萄糖和鼠李糖，乙酸乙酯萃取得到ICT。质谱结果显示分子离子峰m/z为368，比ICA分子量少308，失去的原子量刚好是一分子葡萄糖和一分子鼠李糖之和，与ICT分子量完全一致。 ICT在BBr₃和CH₂Cl₂的体系中脱去4′甲基，乙酸乙酯萃取反应产物，制备型高效液相纯化得到DICT。质谱结果显示分子离子峰m/z为354，比ICT分子量少14，失去的原子量刚好相当于一个甲基，与DICT分子量完全一致。 2．ICA、ICT、DICT对细胞增殖率的影响 用人乳腺癌MCF-7细胞和T47D细胞体外增殖实验（E-SCREEN法）评价受浙江大学硕士学位论文淫羊箱普及其拟代谢产物的雌激素样作用体外研究试物的雌激素样作用。MCF一7细胞和T47D细胞以适当密度接种24孔板，常规培养ld后换成实验培养液（无内源性雌激素），预培养1^Zd后，换液并加受试物和阳性药雌二醇（Estradiol，几），分别继续培养6d、9d，M竹法检测细胞增殖率。 实验结果显示IcT和DlcT都能显著诱导McF一7细胞增殖，10一mol几达到最大，分别是溶剂组的4.巧、4.34倍（P<0.01，E:为4.61倍），IcA未呈现促MCF一7细胞增殖作用（P>0.05）。ICT和DICT也均能显著诱导T47D细胞增殖，一。一mo比达到最大，分别是溶剂组的1.75、1.36倍护<0.01，E2为2.65倍)，nICT促增殖作用较ICT弱，两者有显著性差异（P<0.05）。提示ICT和DICT有雌激素样作用，而ICA则未体现该作用;DICT促增殖作用较ICT弱，可能与DICT对T47D细胞中ERp比ICT具有更高的选择性有关。3.ICI 182，780（ICI）对MCF-7细胞和T4，细胞增殖的拮抗作用 为验证受试物促MCF一7细胞和T47D细胞的增殖作用是否通过雌激素受体（Estrogen Reeeptors，ERs）介导，E:组和ICA、ICT、DICT各浓度组同时加入终浓度为10’7mo比雌激素受体完全拮抗剂ICI，培养及检测方法同上。实验结果显示EZ、ICT和DICT诱导MCF一7细胞和T47n细胞增值作用被10一mo比Iel完全拮抗，几+ICI、ICA+ICI、ICT+ICI和DICT+ICI组与溶剂对照组的细胞增殖情况无显著性差异（办0.05）。提示ICT和DICT通过ERs介导发挥促McF一7细胞和T47D细胞增殖作用。4.ICA、ICT、DICT对T47D细胞周期的影响 T47D细胞常规培养ld，换实验培养液预培养2d，换液并加10石m。比ICA、ICT和DICT，继续培养3d，收获细胞，固定、碘化丙咤DNA染色，上流式细胞仪检测。结果显示ICT和DICT均能改变T47D细胞周期，使Gl期细胞减少，S期细胞增加，提高细胞增殖指数（P roliferating index，pl=（s+GZM）/（Gl+S+qM）x 100%），Pl分别是溶剂对照的1.45、1.41倍（E2为1.74倍），此作用能被ICI完全拮抗。提示ICT和DICT确实具有促T47D细胞增殖的雌激素样作用。浙江大学硕士学位论文淫羊若昔及其拟代谢产物的雌激素样作用体外研究5.PR、ERa、E邓基因mRNA表达的变化 MCF一7细胞和T47D细胞培养方法同上，用Trizol试剂提取细胞总RNA，M一MuLV酶逆转录得到cDNA，上述cDNA加入PCR反应体系中用几q DNA聚合酶扩增目标片段，1 .5%琼脂糖凝胶电泳，凝胶成像系统观察结果，以p一actin为内参照，定量分析PR、ERa、ERp的mRNA的表达。 实验结果显示，ICT和DICT能明显诱导MCF一7细胞和T47b细胞中PR基因mRNA的表达，ICT组分别是溶剂组的6.19和2.33倍，DICT组分别是溶剂组的3.69和2.1倍（EZ分别为11.77和3.87倍），此作用被ICI完全拮抗。ICA未体现诱导MCF一7细胞PR基因的mRNA表达作用，对T47D细胞稍有诱导作用，为溶剂组的1.34倍。进一步从基因转录水平确认了ICT和DICT具有显著的雌激素样作用。 ICT和DICT对MCF一7细胞和T47D细胞ERa基因mRNA表达影响都相对较小，ICI对MCF一7细胞ERa基因mRNA表达有一定的抑制作用，最低表达只有溶剂组的55%（ICA+ICI）:ICI对T47D细胞ERa基因mRNA影响相对较小，最低表达量为溶剂组的81%（ICT+ICI组）。MCF一7细胞ERp基因mRNA目标片段呈阴性。而ICT和DICT均明显增加T47D细胞ERp mRNA的表达，分别为溶剂组的1 .79、2.27倍（EZ为3.51倍），ICI可抑制这种诱导作用。提示MCF一7细胞ERp在mRNA水平不表达，DICT对T47D细胞ERp mRNA诱导作用较ICT强。6.MCF一7细胞和T47D细胞E助和ERp蛋白的表达更多还原

【Abstract】 Epimedii Herba is an important traditional Chinese herbal medicine widely used as a tonic and aphrodisiac in China, Japan and Korea, and has been proven to possess efficacy on sexual function disorder, menopause syndrome, osteoporosis and cardiovascular diseases et al. Recent studies have indicated that the extracts or drug preparations of Epimedii Herba possess classic phytoestrogen-like activities, therefore, we speculate that the in vivo pharmacological activity of Epimedii Herba is related to the estrogen-like activities of its component(s). Icariin (ICA) is considered to be the major active component of Epimedii Herba, therefore, the content of ICA is employed as the standard for quality control in Epimedii herba and its drug preparations. In vivo metabolism studies have shown that the estrogenic activities of metabolite usually markedly increase, such as biochanin A metabolized to genistein, formononetin to daidzein. It is most likely that the metabolism process of ICA in vivo is similar to that described above. To determine the material foundation of pharmacological efficacy in Epimedii herba and its drug preparations, and to provide scientific data for traditional Chinese herbal medicine entering international market, the present paper studied the estrogenic activity and its structure-relationship of ICA and its possible metabolites icaritin (ICT) and desmethylicaritin (DICT) through cell biology and molecular biology techniques.1. Synthesis and identification by MS of ICT and DICTICT was derived from ICA by hydrolysis using a removing-glycoside specific cellulase, the target product was extracted with ethyl acetate. El mass spectrum displayed a molecular ion peak at m/z 368 corresponding to that of icaritin. The lost atomic mass units are exactly equal to that of total molecular weight of a rhamnosyl and a glucosyl. DICT was derived from ICT demethylation by boron tribromide, the target product was extracted and dried as above described. El mass spectrum displayed a molecular ion peak at m/z 354 corresponding to that of DICT. The lost atomic mass units are exactly equal to that of total molecular weight of a methyl.2. The effects on cell proliferation of ICA ICT and DICTThe estrogenic activities were assessed by MCF-7 cells and T47D cells proliferation assay in vitro (E-SCREEN). MCF-7 cells and T47D cells were seeded into 24-well plate at appropriate density, and after 1 day the medium was changed to experimental medium (endogenous estrogen-free), cultured for 1 or 2 day(s) for MCF-7 cells and T47D cells, respectively, The medium was changed to fresh estrogen-free medium and the cells were exposed to test chemicals and positive control estradiol (?), and cultured for another 6 or 9 days for MCF-7 and T47D cells, respectively. Cell proliferation was assessed by MTT.ICT and DICT significantly enhanced the proliferation of MCF-7 cells in a concentration-dependent manner from 10to 10mol/L, and the maximal stimulation was observed at 10mol/L. Compared with control, relative preliferation (RP) for ICT and DICT were calculated as 4.15, 4.34 fold respectively (p<0.01, E2as 4.61 fold). Whereas, ICA did not present stimulation of cell growth. Also, ICT and DICT significantly enhanced the proliferation of T47D cells, and the maximal stimulation was observed at 10mol/L. Compared with control, RP for ICT and DICT were calculated asl.78, 1.36 fold respectively (p<0.01, E2 as 2.65 fold), and there were significantly different between ICT and DICT (p<0.01). These results that ICT and DICT possess estrogen-like activities, however, icariin appears to have no estrogenicity in vitro; the significantly different of stimulation on T47D cells betweenICT and DICT is probably relevant to the selectivity to ERp of DICT better than ICT.3. Inhibition of ICI 182, 780 on cell proliferationIn order to ensure that the cell proliferation of MCF-7 cells and T47D cells induced by test compounds were mediated by estrogen receptors, cells were simultaneously exposed to 10~7mol/L ICI in the presence of test chemicals, a更多还原