Ⅰ.跨界活性篩選系統及Ⅱ.黃豆、葛根異黃酮與萃取物活性最適化之研究

Ⅰ.Cross Kingdom’s Assay & Ⅱ.The Study in Isoflavonoids and Optimazation of Bioactive Extracts from Glycine max and Pueraria lobata

戴佳惠(Jia-Hui Dai)

指導教授 ： 張芳榮

共同指導教授 ： 吳永昌(Yang-Chang Wu)

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2006年)

https://doi.org/10.6832/KMU.2006.00075

摘要

雌激素(Estrogen)是維持女性正常生理機能之重要激素，但含量過高則易引起乳癌及其他激素相關性癌症之危險。越來越多的研究指出，植物性雌激素(Phytoestrogen)之活性雖約為雌激素1/100~1/1000，但其卻具有抗雌激素之效用，且對乳癌，骨質疏鬆症，心血管疾病，甚至精神方面失調等均具有預防效果，並能減緩女性更年期障礙。
目前已知植物性雌激素大都屬於異黃酮類化合物，但自天然界尋求新的植物性雌激素，仍屬相當熱門且重要的課題。
本研究即利用pER8-GFP阿拉伯芥轉殖株，建立其篩選模式，找出最適合此系統穩定表現之培養環境。並從中醫藥典籍中選定多種與婦科相關如月經不調、痛經、經閉或活血化瘀等症狀的中藥材及民間常用之中藥材，利用此系統篩選出多種具有植物性雌激素活性之天然物。本實驗並利用螢光表現程度之不同，建立螢光表現比色方法，共分為11級分。研究中並選定黃豆與葛根作為雌激素活性研究之天然物，黃豆富含異黃酮類等化學物質，被認為有助於捕捉自由基，減輕婦女更年期症狀，及預防癌症之功效；葛根根據文獻記載，具有治療高血壓、心絞痛、心腦血管疾病、防止腦血栓、降血壓，及延緩衰老等藥理作用。將兩者乙醇萃取物以特定比例混合後，配合分離純化及化學結構鑑定，以Biological及Chemical profiles之方法，找尋最適當之活性萃取物及其主要雌激素活性化合物。從黃豆分離出主要雌激素活性化合物，結構經由光譜分析及化學方法確認証實依序為genistein﹙1﹚、daidzein﹙2﹚、genistin﹙3﹚、 daidzin﹙4﹚、glycitin﹙5﹚，葛根主要雌激素活性化合物，結構經由光譜分析及化學方法確認証實依序為genistein﹙1﹚、daidzein﹙2﹚、daidzin﹙4﹚、puerarin﹙6﹚。
在合理的設計下，利用轉基因植物建立快速、簡單的生物活性測試方法，在未來，可以將其應用於自天然物中發現新的雌激素或抗雌激素物質。

關鍵字

植物雌激素 ； 異黃酮 ； 阿拉伯芥 ； 活性篩選

並列摘要

Estrogen is the important hormone to maintain the physiological function of female. However, high amount of estrogen may cause risks of breast cancer and other cancers. Comparing their estrogenic effect, phytoestrogen exhibits 1/100~ 1/1000 fold effect less than estrogen, and provides antiestrogen effect. Phytoestrogen was reported to prevent breast cancer, osteoporosis, cardiovascular diseases, neurodegenerative disorders, and relieve of menopausal ailments. The isoflavones are the major constituents of phytoestrogen, but to find out the new phytoestrogen compounds from natural sources is still the popular topic.
Recently, pER8-GFP, transgenic Arabidopsis plant system, was established as the screening model for discovering natural phytoestrogen products. We further finds the suitable cultural environment for the fluorescent expression of transgenic Arabidopsis expression, which induced by estrogenic substances. Various Chinese medicines with functions to irregularity of menstruation, dysmenorrheal, and simulating the circulation of blood were chosen and screened to find out their estrogenic activity. Based on the variation of fluorescence expression levels, the fluorescent intensity was divided into eleven degrees, which can be applied to be as a standard scale in experiments. The extracts of Glycine max and Pueraria lobata exhibited significant activity to the assay. Glycine max contains various isoflavones and other phytocomponents, which contribute to scavenge free radicals, reduce menopausal symptoms, and possible prevent the cancer. In addition, the isoflavones from extract of Pueraria lobata are important ingredients for medicine and healthy food with curative effect in lowering hypolipidemia, blood pressure, and blood sugar, treating angina pectoris and cardiovascular diseases, preventing of cerebral thrombus, and postponing aging. In this investigation, the ethanol/H2O extracts of both original materials mixed in ratios in associating with the modern techs in isolation, purification, and structural elucidation will be studied using the concepts of chemical and biological profiles. The major phytoestrogens from Glycine max were genistein (1), daidzein (2), genistin (3), daidzin (4), and glycitin (5) as well as the major phytoestrogens from Pueraia lobata were genistein (1), daidzein (2), daidzin (4), puerarin (6).
A rapid and simple bioassay by using transgenic Arabidopsis thaliana has been established under a rational design, and it can be performed easily and reliably in future to discover the new estrogens/ antiestrogens from natural sources.

並列關鍵字

phytoestrogen ； isoflavone ； Arabidopsis ； bioactivity screening

參考文獻

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