臺灣博碩士論文加值系統

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。

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.

目錄
內文目錄--------------------------------- I
圖表目錄------------------------- IV
中文摘要------------------------------------------ XIII
英文摘要------------------------------------------ XV
第一章 緒論--------------------------------------- 1
第一節 前言-------------------------------- 1
第二節 研究背景與動機-------------------------------- 3
第三節 研究目的 --------------------------------- 62
第二章 研究材料與方法 --------------------------- 63
第一節 基因轉殖植物之培養條件測試 --------------- 63
第二節 pER8:GUS雌激素活性測試 ---------------- 65
第三節 pER8:GFP抗雌激素活性測試 ----------------- 67
第四節 蘇木之分離與純化 ------------------------- 68
第三章 類雌激素/抗雌激素篩選模式建立 ------------ 75
第一節 pER8:GFP抗雌激素活性篩選模式之建立------- 75
第二節 pER8:GUS雌激素活性篩選模式之建立 ---------- 78
第三節 中草藥之雌激素活性篩選 --------------------- 83


第四章 蘇木之化學結構鑑定 ----------------------- 86
第一節 Brazilein (1) 之結構證明------------------- 86
第二節 Brazilin (2) 之結構證明 ------------------- 92
第三節 Protosappain A (3) 之結構證明-------------- 99
第四節 3,7-Dihydroxy-4H-chromen-4-one (4)之結構證明- 104
第五節 (S)-2,3-dihydro-3,7-dihydroxychromen-4-one (5)
之結構證明----------------------- 110
第六節 3’-Deoxyepisappanol (6) 之結構證明----------- 118
第七節 3’-Deoxysappanol (7) 之結構證明-------------- 127
第八節 3-Dexoysappanone B (8) 之結構證明----------- 134
第九節 4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9)
之結構證明 ---------------------------- 140
第十節 3-Deoxysappanchalcone (10) 之結構證明 -------- 150
第十一節 3’-Deoxysappanone B (11) 之結構證明 ---- 158
第五章 生物活性試驗 --------------------------- 164
第一節 化合物之雌激素與抗雌激素活性結果------- 164
第二節 其他生物活性試驗之實驗方法 ------------ 171
第三節 純化合物之活性結果 --------------- 175



第六章 結論------------------------------ 177
第七章 結果討論 ------------------------- 179
第八章 相關實驗部份--------------------------------- 183
第一節 分離實驗之儀器與材料 ------------------------- 183
第二節 化學藥品與材料------------------------ 185
第三節 各化合物之物理與光譜數據總整理 ---------- 189
第九章 參考文獻------------------------------- 195

























圖表目錄
圖:
圖1-2-1、17-β-Estradiol (E2) ---------------------------- 3
圖1-2-2、Estrogen receptor α、β組成之比較 ---------- 4
圖1-2-3、雌激素接受體活化作用機轉 ---------------- 6
圖1-2-4、Estrogen之致癌相關機轉 ---------------------- 7
圖1-2-5、芳香環轉化酶阻斷劑(aromatase inhibitors)作用機轉 8
圖1-2-6、Tamoxifen 治療hormone-responsive 癌症之藥理作用機
轉-- 9
圖1-2-7、Tamoxifen 對於人體各器官之生理作用 ------- 10
圖1-2-8、Phytoestrogens 化學結構分類示意圖 ---------- 13
圖1-2-9、Phytoestrogens 各類化學結構之代表性化合物---14
圖1-2-10、??-Glucuronidase之作用 -------------------- 16
圖1-2-11、組成pER8:GUS 植物之載體------------------- 17
圖1-2-12、蘇木植物外部型態圖------------------------- 19
圖1-2-13、蘇木植物生長外部型態圖--------------------- 20
圖1-2-14、蘇木藥用部位之外觀性狀--------------------- 20
圖2-4-1、分離流程1---------------------------------- 68
圖2-4-2、分離流程2---------------------------------- 70
圖2-4-3、分離流程3---------------------------------- 71
圖3-1-1、Tamoxifen對不同濃度estrogen之抑制反應 ------ 75
圖3-1-2、Tamoxifen對E2之抑制曲線圖 ------------------ 76
圖3-2-1、pER8:GUS種子於1/2MS、MS培養基生長狀況 ------ 78
圖3-2-2、植物於1/2MS、MS培養基第1、3、6天植物乾重 --- 79
圖3-2-3、pER8:GUS測試，對E2雌激素活性敏感度約在1.25~0.63
nM ------80
圖3-2-4、植物盆栽圖。植株至開花約1個月，生長週期約2個月- 81
圖3-2-5、各植株子代pER:GUS-0808、pER:GUS-0810¬、pER:GUS-
0813雌激素活性測試結果 ---------------------------- 82
圖3-3-1、E2 與 大豆萃取物之雌激素活性結果 ------------ 84
圖3-3-2、中草藥萃取物雌激素活性測試結果 ---------------- 85
圖4-1-1、Brazilein (1) 之ESI-MS 圖譜-------------------- 89
圖4-1-2、Brazilein (1) 之IR光譜----------------------- 89
圖4-1-3、Brazilein (1) 之UV光譜 ----------------------- 90
圖4-1-4、Brazilein (1) 之1H-NMR光譜--------------------- 90
圖4-1-5、Brazilein (1) 之13C-NMR光譜------------------- 91
圖4-1-6、Brazilein 生合成途徑 ------------------- 88
圖4-2-1、Brazilin (2) 之ESI-MS圖譜--------------------- 96
圖4-2-2、Brazilin (2) 之IR光譜----------------------- 96
圖4-2-3、Brazilin (2) 之UV光譜----------------- 97
圖4-2-4、Brazilin (2) 之1H-NMR光譜---------------------- 97
圖4-2-5、Brazilin (2) 之13C-NMR光譜------------------- 98
圖4-2-6、Brazilein (1)與brazilin (2) 1H-NMR圖譜比較 --98
圖4-3-1、Protosappain A (3)之ESI-MS 圖譜-------------- 101
圖4-3-2、Protosappain A (3) 之IR光譜------------------- 101
圖4-3-3、Protosappain A (3) 之UV光譜-------------- 102
圖4-3-4、Protosappain A (3)之1H-NMR光譜------------ 102
圖4-3-5、Protosappain A (3)之13C-NMR光譜----------- 103
圖4-4-1、3,7-Dihydroxy-4H-chromen-4-one (4) 之ESI-MS 圖譜 ---- 106
圖4-4-2、3,7-Dihydroxy-4H-chromen-4-one (4) 之IR光譜-- 106
圖4-4-3、3,7-Dihydroxy-4H-chromen-4-one (4) 之UV光譜 - 107
圖4-4-4、3,7-Dihydroxy-4H-chromen-4-one (4) 之1H-NMR光譜 -- 107
圖4-4-5、3,7-Dihydroxy-4H-chromen-4-one (4) 之13C-NMR光譜 --- 108
圖4-4-6、3,7-Dihydroxy-4H-chromen-4-one (4) 之HMQC光譜 ------ 108
圖4-4-7、3,7-Dihydroxy-4H-chromen-4-one (4) 之HMBC光譜 ----- 109
圖4-5-1、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
ESI-MS 圖譜 ----------------------------------- 113
圖4-5-2、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
IR光譜 ----------------------------------- 113
圖4-5-3、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
UV光譜 ---------------------------------- 114
圖4-5-4、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
1H-NMR光譜 ----------------------------------- 114
圖4-5-5、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
13C-NMR光譜 -------------------------------- 115
圖4-5-6、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
DEPT光譜 ------------------------------------- 115
圖4-5-7、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
HMQC光譜------------------------------------- 116
圖4-5-8、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
HMBC光譜------------------------------------- 116
圖4-5-9、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
COSY光譜 --------------------------------- 117
圖4-5-10、(S)-2,3-Dihydro-3,7-dihydroxychromen-4-one (5) 之
NOESY光譜 -------------------------------- 117
圖4-6-1、3’-Deoxyepisappanol (6)之ESI-MS 圖譜 -------- 121
圖4-6-2、3’-Deoxyepisappanol (6) 之IR光譜 ------ 121
圖4-6-3、3’-Deoxyepisappanol (6) 之UV光譜 ----------- 122
圖4-6-4、3’-Deoxyepisappanol (6) 之1H-NMR光譜 -------- 122
圖4-6-5、3’-Deoxyepisappanol (6) 之13C-NMR光譜-------- 123
圖4-6-6、3’-Deoxyepisappanol (6) 之DEPT光譜----------- 123
圖4-6-7、3’-Deoxyepisappanol (6) 之HMQC光譜----------- 124
圖4-6-8、3’-Deoxyepisappanol (6) 之HMBC光譜---------- 124
圖4-6-9、3’-Deoxyepisappanol (6) 之COSY光譜----------- 125
圖4-6-10、3’-Deoxyepisappanol (6) 之NOESY光譜 -------- 125
圖4-6-11、3’-Deoxyepisappanol (6) 之CD光譜 ----------- 126
圖4-7-1、3’-Deoxysappanol (7) 之ESI-MS 圖譜----------- 131
圖4-7-2、3’-Deoxysappanol (7) 之IR光譜------------ 131

圖4-7-3、3’-Deoxysappanol (7) 之UV光譜------------ 132
圖4-7-4、3’-Deoxysappanol (7) 之1H-NMR光譜------- 132
圖4-7-5、3’-Deoxysappanol (7) 之13C-NMR光譜-------- 133
圖4-7-6、3’-Deoxysappanol (7) 之CD光譜 ----------- 133
圖4-7-7、化合物6、7 1H-NMR圖譜與13C-NMR圖譜之比較 --- 128
圖4-8-1、3-Dexoysappanone B (8) 之ESI-MS 圖譜--------- 137
圖4-8-2、3-Dexoysappanone B (8) 之IR光譜--------------- 137
圖4-8-3、3-Dexoysappanone B (8) 之UV光譜--------------- 138
圖4-8-4、3-Dexoysappanone B (8)之1H-NMR光譜------------ 138
圖4-8-5、3-Dexoysappanone B (8)之13C-NMR光譜----------- 139
圖4-9-1、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol(9)之ESI-MS圖譜-144

圖4-9-2、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9)之IR光譜------ 144

圖4-9-3、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9) 之UV光譜-------- 145

圖4-9-4、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9)之1H-NMR光譜- 145

圖4-9-5、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol(9)之13C-NMR光譜--- 146

圖4-9-6、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9)之DEPT光譜------- 146


圖4-9-7、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9)之HMQC光譜----- 147

圖4-9-8、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9)之HMBC光譜----- 147

圖4-9-9、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9) 之COSY光譜----- 148

圖4-9-10、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9) 之NOESY光譜--- 148

圖4-9-11、4-(7-Hydroxy-2,2-dimethyl-9bH-1,3,5-trioxa-cyclopenta[a]naphthalene-3a-ylmethyl-)-benzene-1,2-diol (9) 之CD光譜--------- 149
圖4-10-1、3-Deoxysappanchalcone (10) 之ESI-MS 圖譜---153
圖4-10-2、3-Deoxysappanchalcone (10) 之IR光譜--------- 153
圖4-10-3、3-Deoxysappanchalcone (10) 之UV光譜------- 154

圖4-10-4、3-Deoxysappanchalcone (10) 之1H-NMR光譜----- 154
圖4-10-5、3-Deoxysappanchalcone (10) 之13C-NMR光譜--- 155
圖4-10-6、3-Deoxysappanchalcone (10) 之DEPT圖譜-------- 155
圖4-10-7、3-Deoxysappanchalcone (10) 之HMQC圖譜-------- 156
圖4-10-8、3-Deoxysappanchalcone (10) 之HMBC圖譜------- 156
圖4-10-9、3-Deoxysappanchalcone (10) 之COSY圖譜-------- 156
圖4-10-10、3-Deoxysappanchalcone (10) 之NOESY圖譜------ 156
圖4-11-1、3’-Deoxysappanone B (11) 之ESI-MS 圖譜------ 161

圖4-11-3、3’-Deoxysappanone B (11) 之IR光譜-------- 161
圖4-11-2、3’-Deoxysappanone B (11) 之UV光譜-------- 162
圖4-11-5、3’-Deoxysappanone B (11) 之1H-NMR光譜 ---- 162
圖4-11-4、3’-Deoxysappanone B (11) 之13C-NMR光譜------ 163
圖4-11-6、3’-Deoxysappanone B (11) 之DEPT光譜 ------- 163
圖5-1-I、化合物CS-1、CS-2、CS-5、CS-6、CS-7 於pER8:GUS
雌激素活性測試結果---------------------------------- 166
圖5-1-II、化合物CS-3、CS-4於pER8:GUS雌激素活性測試結果 167
圖5-1-III、化合物CS-8、CS-10、CS-11於pER8:GUS雌激素活性
測試結果-------------------------------------------- 168
圖5-1-2、化合物於pER8:GUS 抗雌激素活性結果 ------ 169

表:
表1-2-1、荷爾蒙相關癌症治療之用藥 ------------------- 11
表1-2-2、蘇木屬之化合物及其基原統整 ----------------- 25
表2-4-1、本研究分離之化合物統整表 ------------------ 72
表3-2-1、比較植物於1/2MS、MS培養基之生長速率 ---------- 79
表3-3-1、中草藥萃取物之雌激素活性結果 ----------- 84
表4-1、1H-NMR and 13C-NMR (400 MHz, DMSO-d6) spectral data of 1---- 88
表4-2-1、Brazilein (1)、brazilin (2) 1H-NMR之比較。--- 94
表4-2-2、1H-NMR and 13C-NMR (200 MHz, Acetone -d6) spectral data of 2 -- 95
表4-3、1H-NMR and 13C-NMR (400 MHz, Acetone -d6) spectral data of 3 --- 100
表4-4、1H-NMR and 13C-NMR (400 MHz, Methanol-d4) spectral data of 4 --- 105
表4-5-1、化合物5與類似化合物之旋光度比較 ----------- 112
表4-5-2、1H-NMR and 13C-NMR (400 MHz, Methanol-d4) spectral data of 5 - 112
表4-6、1H-NMR and 13C-NMR (400 MHz, Methanol –d4) spectral data of 6 -- 120
表4-7、1H-NMR and 13C-NMR(400 MHz, Methanol –d4) spectral data of 7 -- 130
表4-8、1H-NMR and 13C-NMR (400 MHz, Methanol–d4) spectral data of 8 -- 136
表4-9、1H-NMR and 13C-NMR (400 MHz, Pyridine –d5) spectral data of 9 -- 143
表4-10、1H-NMR and 13C-NMR (400 MHz, Methanol –d4) spectral data of 10 ----------------------------------- 152


表4-11、1H-NMR and 13C-NMR (400 MHz, Methanol –d4) spectral data of 11-------------------------------- 160
表5-1-1、化合物之雌激素活性結果-------------------- 165
表5-3-1、化合物之細胞毒殺活性IC50值------------------- 175
表5-3-2、化合物之抗血小板凝集試驗IC50值------------- 176

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