數種植物雌激素食材萃物對 PPAR、脂肪細胞生成及雌激素活性特質鑑定

Abstract

婦女於停經後因雌激素分泌不足，而產生停經症候群症狀並會增加罹患代謝症候群的風險，進而有可能引發第二型糖尿病或心血管疾病。目前已知 PPAR (peroxisome proliferators activated receptor) α/γ 雙效活化物是治療代謝症候群重要標的，且 PPARγ 的活化會影響脂肪細胞的分化與脂質生合成。本研究即是檢驗已知有植物雌激素活性的山藥與苜蓿芽乙酸乙酯萃取物與六種山藥雌激素純化物，19-6-3-1、19-8-3-2-1、26-3、26-5-2、28-1 與 61-1-6 活化 PPARα 與 PPARγ 的能力並了解其對是否會影響前脂肪細胞 3T3-L1 的增生與脂質生合成。另一方面，又篩選鑑定新的五種花草藥：菊花、茉莉、蓮花、桂花及月季花 80% 甲醇(aq) 萃取物的植物雌激素效應。 將山藥與苜蓿芽凍乾粉末以乙酸乙酯萃取，萃取物回溶於絕對酒精，處理經共轉染 Gal4-rPPARα/γ chimeric receptor 與 (UAS)4-ALP (alkaline phosphatase) reporter 之 CHO-K1 細胞，分析報導基因 ALP 之活性，測試山藥與苜蓿芽乙酸乙酯萃取物與山藥雌激素純化物的 PPAR 活性。3T3-L1 的增生效應是將細胞以樣品處理 24 與48 小時後，以 MTT 染色法評估細胞增生，比較與 vehicle 組之差異。3T3-L1 分化實驗則是以樣品於各種分化期處理不同時間來了解山藥、苜蓿芽與苦瓜乙酸乙酯萃取物處理的時間點對於分化的影響，分別是將萃取物於分化全期、分化前期、分化後期及已分化期處理 3T3-L1 細胞，分化完成後測定其三酸甘油酯含量作為分化程度之指標。 山藥與苜蓿芽乙酸乙酯萃取物於 PPARα 之最大相對活化百分比分別約可達正對照組 10 μM WY 14643 之 40% 與 50%，於 PPARγ 則分別約為 0.1 μM BRL 49653 之 25% 與20%。兩種萃取物皆以活化 PPARα 的效果較佳；山藥雌激素純化物 19-6-3-1 與 26-3 活化 PPARα 與 PPARγ 的能力則皆為 12% 左右。而在 3T3-L1 增生方面，山藥乙酸乙酯萃取物於 pre-confluent (起始細胞數 2500/well) 及 confluent (起始細胞數 5000/well) 時期皆促進細胞增生。而苜蓿芽乙酸乙酯萃取物則於 pre-confluent 時期促進細胞增生，但於 confluent 則降低 MTT 染色值。山藥乙酸乙酯萃取物於 3T3-L1 分化全期、後期與已分化期皆顯著增加三酸甘油酯堆積。苦瓜乙酸乙酯萃取物處理可增加三酸甘油酯堆積，且有劑量效應。但苜蓿芽乙酸乙酯萃取物於各時期添加皆不影響細胞三酸甘油酯堆積。總括來看，山藥與苜蓿芽乙酸乙酯萃取物及山藥純化物 19-6-3-1 與 26-3 可活化 PPARα 與 PPARγ，為 (或含) PPARα/γ 雙效活化物，且山藥乙酸乙酯萃取物會促進前脂肪細胞 3T3-L1 的增生、分化與脂質累積能力，而苜蓿芽乙酸乙酯萃取物則在 PPARα/γ 活化的同時卻不易促進脂肪組織增生。 五種花草藥萃取物先以 CHO-K1 細胞共轉染 Gal4-hER (estrogen receptor) α/β chimeric receptor 與 (UAS)4-ALP reporter 測試活化 ERα 與 ERβ 的能力。再進行人類乳癌細胞株 MCF-7 與子宮內膜癌細胞株 Ishikawa 增生試驗 (MTT 試驗)，以了解萃取物對雌激素依賴型癌細胞的活性。另亦測試樣品對 Ishikawa 細胞中 ER 標的基因 ALP (alkaline phosphatase) 活性之影響，以評估具 ER 活性之 花草藥萃取物樣品於含 ER 細胞中，透過 ER 調控基因表現之狀況。 菊花、茉莉、蓮花、桂花與月季花萃取物皆是以活化 ERβ 的活性較高，Ishikawa ER 標的基因 ALP 的活性大致與 ER 轉染活性相符，以菊花萃取物的活性最高。於 MCF-7 增生試驗中，五種花草藥萃取物皆會使 MTT 染色值上升，但以茉莉萃取物的效應為最低。Ishikawa 子宮內膜癌細胞增生試驗中，桂花萃取物可以抑制細胞的增生，其他萃取物皆會促使細胞 MTT 染色值增加，但仍是以茉莉萃取物的效應最低。縱觀 MCF-7 與 Ishikawa 細胞以 MTT 染色值為指標之增生試驗結果，以茉莉萃取物的結果最為理想，因其於 50 μg/mL 濃度下 ERβ 的活性約可達最大相對活化百分比的 19% (與 Ishikawa ALP 試驗結果相似)，但是於此濃度下，子宮內膜癌細胞株 Ishikawa 並不會被促進增生，乳癌細胞株 MCF-7 則只有少量的增生 (<15%)，故可提供植物雌激素活性，但不易刺激癌細胞增生。 由本論文結果可得知具有植物雌激素活性的山藥與苜蓿芽乙酸乙酯萃取物有活化 PPARα 與 γ 的能力，但前者促進前脂肪細胞 3T3-L1 的增生並分化，但後者則否。並篩選出五種新的具植物雌激素活性之花草藥，其中茉莉 80% 甲醇(aq) 萃取物不致引發乳癌及子宮內膜癌細胞之顯著生長，具有開發的潛力。

Postmenopausal women suffer not only from peri-menopausal syndromes but also from increased risks of metabolic syndrome, type 2 diabetes, and cardiovascular diseases. A few of phytoestrogens, such as isoflavones, have been shown to activate PPAR (peroxisome proliferators activated reportor) α/γ which is speculated as one of the mechanism of its beneficial effects in improving metabolic syndrome. This study first aimed at testing the phytoestrogenic yam and alfalfa EAEs (ethyl acetate extracts) and six yam estrogenic purified compounds—19-6-3-1, 19-8-3-2-1, 26-3, 26-5-2, 28-1 and 61-1-6—for their PPAR activity and effects on proliferation and differentiation of 3T3-L1 adipocyte, as PPARγ is known for regulating adipocyte differentiation. A second aim is to characterize five phytoestrogenic flower herb extracts, Chrysanthemum morifolium, Jasminum sambac, Nelumbo nucifera, Osmanthus fragran, and Rosa chinensis, were tested for estrogenic effects. Lyophilized powder of yam and alfalfa were extracted by ethyl acetate and their EAEs were obtained. To test PPAR activity, CHO-K1 cells were co-transfected with vectors carrying Gal4-rPPARα/γ chimeric receptor and (UAS)4-ALP (alkaline phosphatase) reporter, and treated with yam and alfalfa EAEs and yam estrogenic active compounds. The maximal % of induction relative to 10 μM WY 14643 on PPARα were 40% (yam EAE) and 50% (alfalfa EAE), and to 0.1 μM BRL 49653 on PPARγ were 25% (yam EAE) and 20% (alfalfa EAE). Yam estrogenic purified compound 19-6-3-1 and 26-3 can also activated both PPARα and PPARγ. Proliferation of 3T3-L1 was monitored by the MTT assay after 24 or 48 hr treatment at 2 different starting cell density, to examine the effect before and after confluence. Yam EAE increased 3T3-L1 cell growth at all doses tested. On the other hand, alfalfa EAE suppresses 3T3-L1 proliferation at confluent stage but enhances at pre-confluent stage. To test EAE samples on differentiation, differentiating 3T3-L1 cells were treated with yam, alfalfa, and 2381 bitter gourd EAEs at full stage, early stage only, late stage only or post-differentiation stage only. Cellular TG, protein and DNA contents were measured 8 days post differentiation. Yam EAE treatment throughout full stages of differentiation, at late stage, or at post-differentiation, all stimulates adpogenesis as indicated by TG/DNA. 2381 bitter gourd EAE can only stimulates adpogenesis when treated throughout full stages of differentiation. However, alfalfa EAE did not change these parameters measured. Taking together, both yam and alfalfa EAEs and yam estrogenic purified compounds 19-6-3-1 and 26-3 activated both PPARα and PPARγ, but only yam significantly increased 3T3-L1 proliferation and differentiation. To test ER (estrogen receptor) activity of the five flower herbs, CHO-K1 cells are co-transfected with vectors carrying Gal4-hERα/β chimeric receptor and (UAS)4-ALP reporter, and treated with 80% methanol extracts of the five flower herbs. All five flower herb extracts exhibited higher activity on ERβ than on ER α. The five extracts were then tested for their effect on the proliferation on human breast cancer cell line MCF-7 and endometrial cancer cell line Ishikawa using the MTT assay. Except for Jasminum sambac extract, all the remaining 4 flower herb extracts increased MTT values to various extent, support the data on ER activation. Jasminum sambac extract did not significantly increased MTT value of Ichikawa cells and increase MTT value of MCF-7 cells to a low extent. To test the sample extract on the ER target gene expression, Ishikawa cells treated with sample extracts were assayed for ALP activity. Except for OF, all the remaining four extracts increased ALP activity of Ishikawa cells indicating ER active compounds in these extract can also act on natural ER. Among the five samples, Jasminum sambac extract showed significant estrogenic activity in both of the transactivation assay and the Ishikawa cell ALP assay, but did not increase the proliferation of Ishikawa cells and enhanced MCF-7 cell growth on to a limited extent. In conclusion, phytoestrogenic yam and alflalfa EAEs are able to activate both PPARα and PPARγ, but only yam EAEs increased 3T3-L1 proliferation and differentiation. Five flower herbs were demonstrated to contain phytoestrogenic compounds. Among these, Jasminum sambac extract might have highest potential to be developed as a dietary supplement for post-menopausal women.