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Title: | 苜蓿芽乙酸乙酯萃物中雌激素活性成分之分離純化及其對雌激素依賴型癌細胞和骨骼細胞株之效應 Isolation of estrogenic compounds in the ethyl acetate extract of alfalfa sprouts and effects of the compounds on estrogen-dependent carcinoma cell lines and bone cell lines |
Authors: | Ssu-Ching Wang 王思晴 |
Advisor: | 黃青真(Ching-Jang Huang) |
Keyword: | 苜蓿芽,SERM,植物雌激素,雌激素依賴型癌,骨骼, alfalfa,SERM,phytoestrogen,estrogen-dependent carcinoma,bone, |
Publication Year : | 2008 |
Degree: | 碩士 |
Abstract: | 婦女約至45~55歲,因卵巢逐漸萎縮,體內雌激素與黃體素濃度下降,造成停經及產生更年期症狀,並提高骨質疏鬆症及心血管疾病之風險。然而,荷爾蒙替代療法(HRT)卻可能提高乳癌、子宮內膜癌、心臟疾病等風險,故相關研究人員皆希望能找到理想選擇性雌激素受器調節劑(Selective estrogen receptor modulators, SERM),於不產生副作用下,維持雌激素之保護作用,以取代 HRT。本實驗目的為自苜蓿芽乙酸乙酯萃取物(AEA)中,純化分離出異於coumestrol之植物雌激素,並進一步探討其對於雌激素依賴型癌細胞及骨骼細胞之作用。
將澳洲紫花苜蓿芽 (Medicago Satiya L),以乙酸乙酯萃取,其萃物AEA再以開放式矽膠管柱層析及製備式HPLC進行純化分離,並以ER transactivation assay 追蹤活性成分,最終得6種化合物:甘草素(liquiritigenin)、異甘草素(iso- liquiritigenin)、黑麥草內酯(loliolide)、4-OH-6-alk-Py2one(S,S) (當alk = nona- decyl,為(4S,6S)-4-Hydroxy-6-nonadecyl-tetrahydro-pyran-2-one)、4-OH-6-alk-Py2one(R,S) (當alk = nonadecyl,為(4R,6S)-4-Hydroxy-6-nonadecyl-tetrahydro-pyran-2-one)及香豆雌酚(coumestrol),其中4-OH-6-alk-Py2one(S,S)及4-OH-6-alk-Py2one(R,S)為混合物,其內含新化合物,且AEA單離樣品ERβ之EC50皆小於ERα 。並進行PPAR轉錄活性分析,發現AEA活化PPARα/γ 之大部分活性,可能來自脂肪酸,因六種單離化合物活化 PPARα 及 PPARγ 之活性均不高。 為觀察AEA及其單離樣品對雌激素依賴型癌細胞及骨骼細胞之作用,以 MCF-7及Ishikawa作為雌激素依賴型癌細胞模式,骨骼細胞方面,則以分化之 MC3T3-E1為造骨細胞模式,分化之Raw264.7為破骨細胞模式,自兩方面進行探討。實驗結果顯示,isoliquiritigenin可抑制MCF-7及Ishikawa之細胞增生,且降低Ishikawa內源性ALP活性,coumestrol反而會增加雌激素依賴型癌細胞增生及 ER下游基因表現(ALP活性),顯示各單離樣品中isoliquiritigenin最具抗雌激素依賴型癌發展之能力,但coumestrol為一危險因子。骨骼細胞方面,loliolide 可增加造骨細胞增生、ALP活性及FasL基因表現,減少造骨細胞IL-6基因表現及破骨細胞TRAP活性,其各測試之結果,皆與雌激素相同,因此認為於眾樣品中,loliolide骨質保護作用最為全面。 本研究之結果顯示,苜蓿芽除已知之coumestrol外,尚含有其他五種具雌激素活性之化合物。其於受雌激素作用之細胞株中,分別呈現致效劑(agonist)或拮抗劑(antagonist)之功能。預期其於缺乏雌激素之狀態下,可能具有植物雌激素之功能。 Due to estrogen deficiency, postmenopausal women not only suffer from menopausal syndromes but also have increased risks of osteoporosis, cardiovascular disease and metabolic syndrome. However, hormone replacement therapy (HRT) was found to increase the risk of estrogen-dependent carcinoma and other diseases. Selective estrogen receptor modulator (SERM) is therefore the current research focus as an alternative of HRT that has benefits of estrogen without the side-effects. SERMs are compounds that bind to estrogen receptor and have tissue-specific effects distinct from those of endogenous estrogen. Whereas coumestrol has been attributed to the estrogenic effect of alfalfa sprout, there was evidences that the estrogenic properties of alfalfa sprout ethyl acetate extract (AEA) is distinct from coumestrol. This study, therefore, aims at systematical isolation and identification of estrogenic compounds from AEA. In addition, effects of isolated compounds on estrogen-dependent carcinoma cells (MCF-7 cell and Ishikawa cell) and bone cells (osteoblast model: differentiated MC3T3-E1 cell; osteoclast model: differentiated Raw264.7 cell) were also characterized. AEA was fractionated by silica gel column chromatography and prepared HPLC, and estrogenic activity tracked by an ER transactivation assay. Ultimately, liquiritigenin, isoliquiritigenin, loliolide, 4-OH-6-alk-Py2one(S,S) (when alk = nonadecyl,(4S,6S)-4-Hydroxy-6-nonadecyl-tetrahydro-pyran-2-one) and 4-OH-6-alk- Py2one(R,S) (when alk = nonadecyl, (4R,6S)-4-Hydroxy-6-nonadecyl-tetrahydro- pyran-2-one) as well as coumestrol were isolated and identified. 4-OH-6-alk- Py2one(S,S) and 4-OH-6-alk-Py2one(S,S) are mixtures of compounds with various chain length in the side chain. The compounds with side chain length of 17-19 were novel. AEA and all the active fractions activated ERβ better than ERα. AEA can also activate PPARα and PPARγ, but the activity is mostly attributed to fatty acids . In carcinoma cell model, isoliquiritigenin inhibits cell proliferation and Ishikawa endogenous ALP activity, presents the best inhibitive effect. Coumestrol acts just like estrogen, and may be a risk factor of estrogen-dependent cancer. In bone cell model, loliolide increased osteoblast proliferation, ALP activity, FasL gene expression, and decreases osteoblast IL-6 gene expression and osteoclast TRAP activity. Despite a minimal estrogenic activity, loliolide showed significant antagonistic action on 17β-estradiol in the transactivation assay and inhibitions of the growth of estrogen-dependent carcinoma cell line. In conclusion, five estrogenic compounds other than coumestrol were isolated from AEA and their potential as SERM was evaluated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41222 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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