山藥具雌激素活性成份之單離與鑑定研究

Abstract

婦女停經後因卵巢逐漸萎縮，分泌雌激素功能劇降，而產生更年期症狀；長期更使骨質疏鬆且心血管疾病危險率提高。山藥（Dioscorea spp.）是薯蕷科（Dioscoreaceae）、薯蕷屬（Dioscorea）多年生蔓性根莖類植物，某些品系山藥（如D. villosa）製劑已在北美地區供為停經後婦女之膳食補充品，但功效不明。有研究顯示，山藥能改善停經後婦女性激素之理狀況，暗示山藥可能含植物雌激素活性成份。本實驗主要目的為分離鑑定山藥中植物雌激素活性成份。首先建立一分子模式（transactivation assay），用以篩選具雌激素活性之樣品，其次分離純化山藥中具雌激素活性之成分，進一步以雌激素依賴型細胞與雌激素受器結合試驗，評估其在雌激素作用上扮演的角色以供開發為保健食品之基礎。 首先分別建構GAL4-ERα LBD及GAL4-ERβ LBD chimeric receptor質體。將其與(UAS)4-AP 報導基因質體共同短暫轉染入CHO-K1細胞，加入樣品48小時後測定AP活性，代表測定樣品雌激素活性。17β-estradiol與三種已知為植物雌激素之異黃酮皆隨劑量增加而顯著促進AP活性，其中genistein、daidzein與genistin對於ERα之EC50分別為299 nM、875 nM與726 nM；於ERβ之EC50分別為3.4nM、52.4 nM與13.9 nM，可看出異黃酮對ERβ有較好的活性。因此，我們所建立之轉錄活性分析，可用於SERM（Selective Estrogen Receptor Modulator）之篩選。篩選不同食材發現，將山藥以不同溶劑萃取，結果顯示乙酸乙酯萃物可活化hERα與hERβ，甲醇萃物無活化效應。以此系統分析七種不同品系之山藥，其EA萃物對ERα之活性約為1 nM E2之13.72 % ~ 27.33 %之間，對ERβ約為1 nM E2之14.48 % ~ 30.59 %之間。 台農二號山藥經矽膠管柱層析與製備式HPLC分離純化，經NMR及mass鑑定結構，找出山藥中能活化ER之成份包括兩個新化合物：Hydro-Q9 chromene與γ-tocopherol-9，以及四個已知化合物RRR-α-tocopherol、coenzyme Q9、cycloartenol、及1-feruloyl glycerol。進一步進行雌激素受器結合試驗，顯示六種化合物皆會與hERβ結合。另以雌激素依賴型細胞株MCF-7與Ishikawa，證明此六種化合物可作用於天然存在細胞中之ER傳訊途徑。於MCF-7細胞增生實驗中，除了RRR-α-tocopherol (23μM) 顯著增加MCF-7細胞生長外，其餘五種化合物皆對細胞增生無影響。在Ishikawa細胞測定內因性AP活性實驗中，當濃度為20μg/mL，包括RRR-α-tocopherol (2.8 倍)、Coenzyme Q9 (3.6倍)及1-feruloyl glycerol (3.0倍) 等三種化合物可顯著增加AP活性，約達1nM E2活化效果之50%。本研究鑑定出山藥具有雌激素活性之成份，也發現一些新類型的植物雌激素活性化合物，可供發展為停經後婦女保健食品之基礎。

Yam (Dioscorea spp.) is a common food in tropical area and has been shown to improve the status of sex hormone in postmenopausal women. However, phytoestrogenic activity and active compound have never been reported. In this study, estrogenic activity of yam was examined and active compounds were isolated and identified based on the ligand-dependent transcriptional activation through estrogen receptors. Ethyl acetate extracts of various species/varieties of yam were found to activate estrogen receptors α and β to various extents. The extract of Dioscorea alata cv. Tainung No.2 tuber was fractionated by repeated silica gel column chromatography. The active compounds were isolated and purified by preparative HPLC. Based on NMR and mass spectrometry, two new compounds, hydro-Q9 chromene and γ-tocopherol-9, together with four known compounds, RRR-α-tocopherol, coenzyme Q9, cycloartenol and 1-feruloylglycerol were isolated from the TNG2 EA extract. Except for cycloartenol, all of the remaining five compounds showed estrogenic activities. In MCF-7 proliferation assay, only a-tocopherol (23.2μM) induces MCF-7 cell growth. The remaining five compounds did not significantly affect the proliferation of MCF-7 cells. In Ishikawa uterine cancer cell lines, agonist activity was determined by quantitating alkaline phosphatase induction. When the concentration is 20μg/mL, RRR-a-tocopherol (2.8-fold), Coenzyme Q9 (3.6-fold) and 1-feruloylglycerol (3.0-fold) display agonist activity of approximately 50% relative to 17β-estradiol. These results provide basic evidence for the beneficial effect of yam for menopausal women.