自山苦瓜單離植物雌激素及其化學鑑定與生物活性探討

Isolation, Chemical Identification and Biological Characterization of Phytoestrogens from Wild Bitter Gourd (Momordica Charantia)

徐瑨(Chin Hsu)

指導教授 ： 黃青真

國立臺灣大學/生命科學院/生化科技學系/博士(2011年)

https://doi.org/10.6342/NTU.2011.00734

摘要

婦女在停經後因體內雌激素濃度下降，而產生更年期症候群，長期更面臨罹患骨質疏鬆、心血管疾病、代謝症候群之風險增高。許多研究致力於尋找具有雌激素受器選擇性調節劑 (SERM) 功能之植物雌激素，以取代有副作用之賀爾蒙替代療法 (HRT)。我們發現山苦瓜乙酸乙酯萃物之不皂化物區分 (non-saponifiable fraction, NS) 可促進雌激素受器 (Estrogen receptor, ER) 之轉錄活性，暗示山苦瓜可能含有植物雌激素活性成份。本實驗之目的即為探討山苦瓜之植物雌激素效應，並從山苦瓜中純化分離出具有雌激素活性之成份並鑑定其結構，進一步以雌激素依賴型癌細胞，探討其在雌激素相關生理作用中所扮演之可能角色。
先以三週齡剛離乳之母鼠，初探討餵食山苦瓜是否影響雌激素作用之相關生理狀態。將12 隻三週齡剛離乳之母鼠依體重隨機分為 B組 (basal diet，n=4)、E2 組 (2 mg 17β-estradiol/kg diet) 及 BGP 組 (5% 山苦瓜凍乾粉末)。結果發現 E2 組小鼠之陰道開口時間顯著較 B 組提早了 4.5 天 (p<0.01)，而 BGP 組亦提早了 1.8 天 (p = 0.12)。繼以陰道抹片觀察其動情週期的變化，發現 BGP 組小鼠停留於動情期 (Estrus phase) 的時間佔完所有時間之 61%，顯著較 B 組停留於動情期之時間 (41%) 長 (p<0.01)，而 E2 組小鼠則是長期處於動情期 (100%，p<0.01)。
次以卵巢剔除母鼠，餵以高油飼料 (HF 組) 或分別添加 E2、5%山苦瓜凍乾粉末 (BGP 組)、0.2% 不皂化物區分 (NS組) 等飼料15 週或 30 週。結果山苦瓜與 NS 的攝取不會產生如 E2 補充時子宮脹大的效果。餵食實驗飼料三週後進行鈣平衡分析實驗，則觀察到E2 組 (n=5) 糞便鈣排出量較其控制組 (HF 組) 少 (p<0.01)，鈣平衡 (p=0.12) 與鈣保留率 (p=0.07) 則有較高之趨勢。而 BGP 組 (n=5) 亦有糞便鈣與尿鈣排出量較少的現象 (p<0.01)，且 BGP 組與 NS 組之鈣平衡及鈣保留率均提升至與 E2 組沒有顯著差異的效果 (p>0.05)。顯示山苦瓜中成份對缺乏雌激素所引起之骨質疏鬆應有預防保護之功效。乃進一步對其雌激素活性成份進行純化分離如下。
將山苦瓜凍乾粉依序以乙酸乙酯及 95% 酒精萃取，再以開放式管柱層析及製備式 HPLC 分別對乙酸乙酯萃取物之不皂化物區分 (NS) 及酒精萃取物之酸水解產物 (acid-hydrolyzed product, AH) 進行純化分離，並利用ER轉錄活性分析 (transactivation assay) 追蹤活性成份。結果從 NS 中分得葉黃素、loliolide、植醇以及一已知之cucurbitane-type 三萜類化合物5β,19-epoxycucurbita-6,24-dien-3β,23-diol (6)；從 AH 中單離出五個cucurbitane-type 三萜類新化合物，經結構鑑定知其分別為 cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide (1)、 4β,19-epoxycucurbita-6,22(E),24-trien-3β,19-diol (2)、3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3)、19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3β-ol (4) 與 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol (5)。在沒有細胞毒性之濃度處理下，除了化合物 3 與 4，其餘化合物均可活化 ER 轉錄活性。然而相對於輕微活化 ER 的效果，化合物 1、2、3、5與 6 則可以顯著拮抗 E2 所誘發之 ER 轉錄活性。而這些化合物中除了化合物 3 與 4 不具活化 PPARγ 之能力，其他化合物則亦有活化 PPARα與 PPARγ 轉錄活性之效果。
最後以雌激素依賴型乳癌細胞 MCF-7 與子宮內膜癌細胞 Ishikawa 探討山苦瓜區分物及各單離成份之雌激素相關效應。先以 MTT 分析 MCF-7 與 Ishikawa 細胞之增生，結果顯示葉黃素與化合物 2、3、6 可顯著降低 MCF-7之 MTT吸光值。而除了化合物 4 不影響 Ishikawa 之細胞增生，葉黃素及其他五個三萜類化合物均會顯著降低 Ishikawa 之 MTT 吸光值。又在 E2 共同處理下，葉黃素與六個三萜類化合物均可顯著抑制 E2 所誘發之 MCF-7 與 Ishikawa細胞增生，且降低 Ishikawa 細胞內源性 ALP 的活性。而以相同濃度處理非雌激素依賴型癌細胞 MDA-MB-231，則不影響其 MTT 吸光值。
綜之，本研究自酸水解山苦瓜萃物 (AH) 中單離出 5 個新的三萜類化合物，期中 4 個具有植物雌激素活性，另自 NS 中也找到 4 個植物雌激素化合物。這些化合物具有顯著之拮抗雌激素活性。

關鍵字

山苦瓜 ； 雌激素受器 ； 植物雌激素 ； 轉錄活性分析 ； 卵巢剔除鼠 ； 雌激素依賴型癌細胞

並列摘要

Postmenopausal women suffer not only from the menopausal syndromes but also higher risks of osteoporosis, cardiovascular diseases and metabolic syndrome. Some phytoestrogens have been characterized as selected estrogen receptor modulator (SERM) and regarded as one of the substitute for hormone-replacement therapy which was shown to have adverse events. The Cucurbitaceae plant Momordica charantia (MC, bitter gourd) is a common tropical vegetable and has been used in traditional medicine. We have found the the non-saponifiable fraction (NS) of a wild bitter gourd (BG) extract activated transcription via estrogen receptors (ER). Studies in this thesis thus aimed at identifying and characterizing phytoestrogenic compounds in BG.
To assess the in vivo estrogen-related response of BG, 3 groups (n=4/group) of weanling C57BL/6J female mice were respectively fed a basal diet (B), basal diet supplemented with E2 (2 mg 17β-estradiol/kg diet) or BGP (5% bitter gourd powder). The vaginal opening of mice treated with E2 and BGP were respectively, 4.5 days (p<0.01) and 1.8 days (p=0.12) earlier than those of the B group. Results of vaginal smear examination indicated that the E2 group persisted on the estrus stage while BGP group showed a prolonged estrus stage (61%) that was longer than B group (41%)(p<0.01).
To assess more in vivo estrogen-related response, ovariectomized mice fed a high fat diet (HF group) were supplemented with E2 (E2 group), 5% bitter gourd powder (BGP group) or 0.2% non-saponifiable fraction from ethyl acetate extract of BG (NS group) for 15 or 30 weeks. The uterus weight was significantly higher in the E2 group (p<0.05) but not in the BGP and the NS group. In the calcium balance analysis after 3 weeks feeding, the fecal Ca excretion was less (p<0.01), and the Ca balance (p=0.12) and Ca retention (p=0.07) were higher in the E2 group. The fecal and urinary Ca excretion of the BGP group were also less (p<0.01) than the control group. The Ca balance and Ca retention of BGP and NS groups were also improved and comparable to those of the E2 group (p>0.05). These results implied that BG may be beneficial for estrogen deficiency.
To elucidate the phytoestrogenic compounds in BG, a cell-based ER transactivation assay were used to track the active compounds. Lyophilized powders of BG fruits were exhaustively extracted with ethyl acetate (EA) and 95% ethanol (EtOH), sequentially. The non-saponifiable fraction (NS) of the EA extract as well as the acid-hydrolyzed EtOH extract (AH) was fractionated by repeated column chromatography and isolates were further purified by preparative HPLC or RP-HPLC. Lutein, loliolide, phytol, and one known cucurbitane-type triterpenoid 5β,19-epoxycucurbita-6,24-dien-3β,23-diol (6) were identified from NS. Five new cucurbitane-type triterpenoids, cucurbita-6,22(E),24-trien-3β-ol-19,5β-olide (1), 4β,19-epoxycucurbita-6,22(E),24-trien-3β,19-diol (2), 3β-hydroxycucurbita-5(10),6,22(E),24-tetraen-19-al (3), 19-dimethoxycucurbita-5(10),6,22(E),24-tetraen-3β-ol (4), together with 19-nor-cucurbita-5(10),6,8,22(E),24-pentaen-3β-ol (5) were isolated from AH. Except for compound 3 and 4, the remaining compounds showed weak agonistic activity via ERα and β in the non-cytotoxic concentration range. Compounds 1, 2, 3, 5 and 6 showed significant antagonist activity on ERs in the transactivation assay.
These ER active extract/compounds were then assessed for their effect on the proliferation of estrogen-dependent human breast cancer cell MCF-7 and endometrial-derived Ishikawa cells by MTT assay in the absence or presence of 1 nM E2. Lutein, compound 2, 3 and 6 significantly reduced the MTT values of MCF-7. Except for compound 4, lutein and the remaining 5 cucurbitane-type triterpenoids significantly reduced the MTT values of Ishikawa cells. All the 6 cucurbitane-type triterpenoids decreased the ER target gene ALP activity of Ishikawa cells. Besides, lutein and all the 6 cucurbitane-type triterpenoids antagonized the proliferation effect of 1 nM E2 in both of these estrogen-dependent cancer cell lines. These inhibition effects were not observed in the estrogen-independent MDA-MB-231 cells within a similar concentration range.
In conclusion, five new cucurbitane-type triterpenoids were isolated and identified from the AH of Momordica charantia, and four of them showed estrogenic/antiestrogenic activities. Besides, another four known compounds were isolated and identified from the NS as phytoestrogens of MC. These compounds showed significant antagonistic activity toward E2. These results provide basic evidence that Momordica charantia might be a source of beneficial phytoestrogens isolated and identified.

並列關鍵字

Momordica charantia ； estrogen receptor ； phytoestrogen ； transactivation assay ； ovariectomized mice ； estrogen-dependent carcinoma

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