雄性禿(Androgenetic alopecia，AGA)為最常見的脫髮形式，約有90%的落髮原因來自基因或荷爾蒙作用。雄性禿的特性是由頭皮上的毛髮轉變成細短的絨毛，使毛囊微小化且一直重複出現在毛髮週期的生長期，進而縮短生長期的階段。許多研究顯示雄性禿的主要原因是禿髮部位毛囊裡二氫睪固酮(dihydrotesterone，DHT)含量太高所引起，5α還原酶(5α-reductase)的主要功能是催化睪固酮(testosterone，T)轉換成更具活性的二氫睪固酮，因此5α-reductase的濃度太高也會導致二氫睪固酮的產量增加。
5α-reductase又分兩種類型：第一型(TypeⅠ)及第二型(TypeⅡ)，此次實驗主要是利用本實驗室建立的細胞平台，於2006及2008年建立的5αR1及5αR2穩定細胞株，細胞測試是用來評估萃取物對細胞的毒性且快速篩選出潛在抑制5α-reductase的能力。本研究利用四種不同萃取方式(99.5%乙醇微波萃取、99.5%乙醇回流裝置萃取、99.5%乙醇冷泡萃取及乙酸乙酯冷泡萃取)萃取天然植物(紅花三葉草)並以高效能液相層析儀(High-performance liquid chromatography，HPLC)針對紅花三葉草萃取物中的異黃酮(Daidzein、Genistein、formononetin 及 BiochaninA)含量分別進行定量。經HPLC分析後篩選出有效含量最高者進行細胞測試，接著更進一步選擇7週大的母鼠C57BL/6Jmice進行動物試驗。實驗結果顯示：紅花三葉草以99.5%乙醇微波萃取含量最高(formononetin：2.510%，BiochaninA：2.009%)，在細胞實驗方面，同時具有5αR1及5αR2抑制效果，5αR1的IC50濃度為0.464mg/ml；5αR2的IC50濃度為0.524mg/ml；動物實驗方面，不僅讓屬於休止期毛髮階段的老鼠再生毛髮，皮膚組織切片也有明顯皮層厚度、毛囊大小及毛囊數量增深變大的現象發生。 Androgenetic alopecia (AGA) is the most common form of hair loss, about 90% of male baldness comes from genetics and hormones influences. AGA is characterized by vellus transformation of scalp hairs, corresponding to hair follicle miniaturization during repeated hair cycles with shortened anagen phase. Many studies have reported that the main reason causing male bald is the high activity of 5α-reductase, which can catalyze testosterone (T) to the more bioactive metabolite dihydrotesterone (DHT). Therefore, the unusual higher activity of 5α-reductase will lead to increased DHT production. There are two isoforms of the enzyme (type Ⅰ and type Ⅱ), and both are capable of producing DHT from testosterone.
In this thesis, we applied previously established 5α-reductase enzyme conversion assay and human embryonic kidney (HEK) 293 cell lines stably expressing either type I or type II 5α-reductase to quickly screen potential inhibitors of 5α-reductase from red clover and evaluate their cytotoxicity. We used four different ways (microwave, reflux, soak ethanol, soak ethyl acetate) to extract isoflavones (genistein, daidzein, biochanin A, formononetin) from red clover and applied HPLC for quantification of their contents. The extracted fractions containing the highest levels of these isoflavones were further subjected to cell culture model test. Fractions showed high inhibitory activity toward 5α-reductase and low cytotoxicity to HEK293 cells were further tested in seven-week-old female C57BL/6J mice. The results showed that the microwave extraction method could extract the highest amounts of isoflavones (formononetin：2.510%，BiochaninA：2.009%), and the extracts could effectively inhibit both type I (IC50, 0.464 mg/ml) and type II 5α-reductase (IC50, 0.524 mg/ml). The animal test results demonstrated the effectiveness of red clover extracts in promoting hair re-growth. Further examination of the biopsy samples from the red clover extracts-treated mouse showed enhanced thickness of skin and proliferative hair follicles with significantly enlarged sizes. The screening system described here is a convenient and reliable model for evaluating potential inhibitions of 5α-reductase, and the extracts of red clover might represent good lead materials to develop 5α-reductase inhibitors for preventing AGA in the future.
