I. 基因轉殖植物雌激素/抗雌激素活性篩選平台建立 暨 II. 蘇木之化學成分與雌激素/抗雌激素活性之研究

I.Estrogenic/antiestrogenic Bioassay Using Transgenic Plants, pER8:GUS and pER8:GFP II.Studies on the Chemical Constituents and Bioactivities of Caesalpinia sappan L.

賴宛君(Wan-Chun Lai)

指導教授 ： 張芳榮

共同指導教授 ： 吳永昌(Yang-Chang Wu)

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2008年)

摘要

植物雌激素(Phytoestrogen)於近年來成為替代人工合成雌激素，作為女性補充雌激素之主要來源，用以改善女性更年期產生的不適症狀。植物雌激素於化學分類上多屬異黃酮類，這類天然物質廣泛的存在我們日常生活的飲食中，相較於人體中之雌激素(Estrogen)活性強度約為1/102 ~ 1/105。目前的相關研究中，指出這類植物雌激素對於腫瘤的生長具有促進作用，然而另有許多研究指出，植物雌激素亦具有抗雌激素作用，故而對於其生理作用的利弊並無一致的結論，因此相關研究實為迫切重要且值得深入研究的重點。
本研究利用已建立雌激素活性篩選工具之基因轉殖植物pER8:GFP，進一步利用臨床用藥Tamoxifen建立其抗雌激素(anti-estrogen)之篩選模式；並取得另一基因轉殖植物pER8:GUS，應用pER8:GUS以建立更為快速簡便的篩選平台。另一方面，根據中醫藥理論，選取活血化瘀、補腎益氣之婦科相關二十種中草藥，經由此跨動、植物界之篩選模式，篩選出具有或拮抗雌激素活性之天然物，並選取蘇木作為本研究化學成分分離之主題。
蘇木於中醫藥典籍中屬活血化瘀藥，性平，味甘、鹹，功能活血破瘀、消腫止痛；主治經閉痛經、產後瘀阻、胸腹刺痛、外傷腫痛等，與婦科疾病之治療息息相關，且於近年之文獻指出具有抗血管新生之活性。於本研究中，配合新建立之的pER8:GUS雌激素篩選模式，依據活性導引分離法，研究其活性成分。
於蘇木中共分離得到10個已知化合物: brazilein (1)、brazilin (2)、protosappain A (3)、3,7-dihydroxy-4H-chromen-4-one (4)、3’-deoxyepisappanol (6)、3’-deoxysappanol (7)、3-deoxysappanone B (8)、4-(7-hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-
3a-ylmethyl-)-benzene-1,2-diol (9)、3-deoxysappanchalcone (10)、3’-deoxysappanone B (11)以及1個新化合物: (S)-2,3-dihydro-3,7-dihydroxychromen-4-one (5)。
化合物大多屬於Homoisoflavonoids，其中 3、4、8、10、11具雌激素活性，且化合物3、8、11同時具有抗雌激素之作用，並進一步分析化合物之SAR。另外化合物1、2、5、10具細胞毒殺作用，1、3、4、7、10具抗血小板凝集作用。

關鍵字

雌激素 ； 基因轉殖植物 ； pER8:GUS ； pER8:GFP

並列摘要

In recent years, phytoestrogens have substituted synthetic estrogens, as the supplement source of estrogen, used to modify symptoms of postmenopausal women. Most of Phytoestrogens belong to isoflavonoids widely existent in our daily diet. The bioactivity of phytoestrogen is lower than estrogen around 1/102 ~ 1/105. According to some researches, phytoestrogens induced cell proliferation of hormone-dependent carcinoma. In the other articles, phytoestrogens also showed antiestrogenic activity. Therefore, investigations on the physiological functions of phytoestrogens still have no identical conclusion about the advantage and harm. For the reason, the research of phytoestrogens is imperative and very important.
In this study, we applied pER8:GFP, a transgenic plant as a tool of estrogenic activity assay model to screen anti-estrogenic activity combined with the clinical drug Tamoxifen. Further, we used the other transgenic plant pER8:GUS as a detector for estrogenic activity. The new estrogenic assay model showed higher sensitivity and needed shorter time to culture. In addition, we determined twenty Chinese traditional herbs according to traditional Chinese medical theory by pER8:GUS estrogenic activity assay. Then, we got an active extract from Su Mu, Caesalpinia sappan.
Su Mu is a natural, sweet and salty traditional Chinese medicine, which can invigorate circulation, break up blood stasis condition, and promote menstruation including injuries from impact, swollen boils, irregular menses, and pain from blood stasis after birth. It is an important Chinese medicine in gynecology. Bioactivity-guided fractionation of the active extract of Su Mu led to the isolation of eleven compounds including ten known compounds and one new compound.
Ten known compounds are as follows: brazilein (1)、brazilin (2)、protosappain A (3)、3,7-dihydroxy-4H-chromen-4-one (4)、3’-deoxyepisappanol (6)、3’-deoxysappanol (7)、3-deoxysappanone B (8)、4-(7-hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-
3a-ylmethyl-)-benzene-1,2-diol (9)、3-deoxysappanchalcone (10)、3’-deoxysappanone B (11). Moreover, (S)-2,3-dihydro-3,7-dihydroxychromen-4-one (5) is a new compound.
Most pure compounds belong to homoisoflavonoids. Compounds 3、4、8、10 and 11 showed estrogenic activity. Among those five compounds , compounds 3、8 and 11 also proposed antiestrogenic activity. Compounds 1、2、5 and 10 exhibited cytotoxicity to different cancer cell. Furthermore, compounds 1、3、4、7 and 10 inhibited platelet aggregation.

並列關鍵字

phytoestrogen ； transgenic plant ； pER8:GUS ； pER8:GFP

參考文獻

1. J. Zuo, Q.W. Niu, N.H. Chua. An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. The Plant Journal, 2000, 24, 265-273.

2. F.R. Chang, K.I. Hayashi, N.H. Chua, S. Kamio, Z.Y. Huang, H. Nozaki, and Y.C. Wu. The transgenic Arabidopsis plant system, pER8-GFP, as a powerful tool in searching for natural product estrogen-agonists/antagonists. Journal of Naural Products, 2005, 68, 971-973.

3. L. Brand, M. Horler, E. Nuesch, S. Vassalli, P. Barrell, W. Yang, R.A. Jefferson, U. Grossniklaus, M.D. Curtis. A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiology, 2006, 141, 1194-1204.

4. R.J. Ruggiero, F.E. Likis. Estrogen: physiology, pharmacology, and formulations for replacement therapy. Journal of Midwifery & Women's Health, 2002, 47, 130-138.

5. J.M. Hall, J.F. Couse, K.S. Korach. The multifaceted mechanisms of estradiol and estrogen receptor signaling. Journal of Biological Chemistry, 2001, 276, 36869-36872.