Characterization of PPAR, Adipogenesis and Estrogenic Activities of Some Phytoestrogenic Food Materials

• Main Authors: Yung-Ju Chen, 陳永如
• Other Authors: 黃青真
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/84380639238261130752

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 95 === 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.