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研究生:許純真
研究生(外文):Chun-Chen Hsu
論文名稱:紅麴萃取物對酪胺酸酶及黑色素腫瘤細胞活性之影響
論文名稱(外文):The Inhibitory Effects of Red Mold Rice Extracts on Mushroom Tyrosinase and Melanoma Cell''s Activities
指導教授:李春燕李春燕引用關係王志傑王志傑引用關係
指導教授(外文):Chung-Yann LeeJ J Wang
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:製藥科技研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:113
中文關鍵詞:細胞凋亡黑色素細胞瘤monacolin K酪氨酸酶紅麴
外文關鍵詞:Monascusmonacolin Ktyrosinasemelanomaapoptosis
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酪胺酸酶 (Tyrosinase: EC 1.14.18.1) 為一含銅之多酚氧化酶,於蔬果之褐變或皮膚黑色素的形成中,皆扮演著重要的角色。近年來釵h研究嘗試由天然物或微生物中,尋求可以有效抑制酪胺酸酶之活性物質,並應用於食品、醫藥及化妝品之相關產業製造。因此,本研究首先探討紅麴萃取物對酪胺酸酶活性之抑制作用。研究中以紅麴菌株Monascus purpureus TJ-5醱酵所得之麴米,經不同極性之溶劑萃取後所得產物,以L-dopa當作受質,進一步探討其抗氧化能力和對酪胺酸酶活性抑制效果及阻斷酪胺酸酶生合成作用之生化特性。結果顯示,三種不同溶劑之紅麴萃取物於體外抗氧化作用之研究中,以水層萃取物的抗氧化能力最佳。另外,麴酸在1000 μg/mL濃度下對酪胺酸酶的抑制率為12.23%,而紅麴之水、酒精及乙酸乙酯萃取物則於100 μg/mL濃度下,對酪胺酸酶分別可達78.53%,63.59% 及81.33% 之抑制效果,在紅麴萃取物於不同溫度對酪胺酸酶之抑制能力中,以45℃-55℃間之抑制效果最佳,且三種不同溶劑之萃取物皆具有還原Cu2+之能力,。
本研究並進一步分析、探討紅麴不同溶劑之萃取物對黑色素瘤細胞B16-F0內之酪胺酸酶活性抑制之影響。結果發現紅麴萃取物除水萃取物對黑色素瘤細胞之酪胺酸酶不具抑制效果外,其餘之酒精與乙酸乙酯萃取物,對黑色素瘤細胞之酪胺酸酶皆具有良好之抑制效果,且呈現劑量依賴性關係。另以tetrazolium salt (MTT)、trypan blue exclusion、細胞形態學、DNA 電泳及Western blotting方法來偵測紅麴萃取物對黑色素瘤細胞B16-F0之細胞增生 (proliferation) 及細胞凋亡 (apoptosis) 之影響。結果得知紅麴萃取物中除了水層不具抑制效果外,酒精層和乙酸乙酯萃取物對B16-F0皆具有抑制其增殖的能力,其IC50分別為66.5、63.2 μg/mL。在觀察細胞型態上發現經紅麴萃取物作用之細胞,呈現凋亡之型態特徵,細胞形狀有發泡 (blebbing) 的現象,並產生一些細小顆粒,同時伴隨有凋亡小體 (apoptotic body) 的產生。經電泳分析,亦可見到細胞DNA呈現階梯狀 (laddering) 之小片段 。此外,利用Western blotting觀察,以紅麴萃取物處理30小時後之B16-F0細胞,由其蛋白質表現之變化,發現在Bax、caspase 9及caspase 3之表現上較控制組明顯增加。綜合上述實驗之結果,本研究認為紅麴萃取物具有良好之抗氧化、抑制酪胺酸酶活性及引發黑色素瘤細胞B16-F0細胞凋亡之能力,因此在皮膚美白、護膚產品及皮膚癌的預防深具開發潛能。
Tyrosinase ( EC 1.14.18.1) is a copper-containing enzyme that is widespread in nature. It is responsible for not only melanization in animal but also browning in plants. In recent years, there is an increasing interest in finding natural tyrosinase inhibitors from plants and microorganisms. The tyrosinase inhibitors should have broad applications, especially in medicinal and cosmetics products in relation to hyperpigmentation. This research was focused on investigating the effects of red mold rice extracts on mushroom tyrosinase and melanoma cell for the antioxidant activity and inhibitory activity. Since tyrosinase is a rate-limiting enzyme in the melanogensis process, we first used rice through fermentation on the Monascus purpureus TJ-5, and the fermentated rice was then extracted by using various extraction media. In addition, the antioxidant activity and the inhibitory activity of the extracts on mushroom tyrosinase activity in the presence of L-dopa were determined.The result showed that three extracts all exhibited antioxidant activity and inhibitory activity on the activity of the mushroom tyrosinase. The highest antioxidant activity was found to be in the water extract. The inhibition activity of tyrosinase was found to be 12.23% when the kojic acid was at 1000 μg/mL. The tyrosinase inhibition from water extract, ethanol extract and ethyl acetate extract was found to be 78.53%, 63.59% and 81.33%, respectively when the red mold rice extracts was at 100 μg/mL. The optimum temperature of the inhibition activity of tyrosinase was found to be in the range between 45℃ and 55℃. We also found that the Cu2+ ion exhibited reductive ability on the red mold rice extracts.
Furthermore, we also investigated the red mold rice extracts in mouse melanoma B16-F0 cell on tyrosinase activeness suppression. Results showed that red mold rice extracts did not have the suppression effect in the water extract. However, the red mold rice in the ethanol and ethyl acetate extracts did show very good suppression effect and the suppression effect was found to be dose dependent. In addition, We further determined the proliferation rate and apoptosis of red mold rice extracts in mouse melanoma B16-F0 cell by using tetrazolium salt (MTT), tyrpan blue exclusion, DNA fragment, morphological change and western blotting assays. It showed that the red mold rice in both ethanol and ethyl acetate extracts significantly inhibited the melanoma cell’s proliferation rate and their IC50 values were found to be 66.5 and 63.2 μg/mL, respectivly . Morphological changes were observed, cell revealed evident characteristics of apoptosis including cell blebbing and formation of apoptotic bodies. DNA displayed a characteristic inter-nucleosomal ladder of DNA fragment. Furthermore, in cell treated with red mold rice extracts for 30 hours, the level of Bax, caspase-9 and caspase 3 increased, comparing to the control group. Based on the results, we could make sure the red mold rice on all the extracts has highest antioxidant activity.
However, the ethanol and ethyl acetate extracts showed better inhibitory activity on mushroom tyrosinase and induction of apoptosis in melanoma cells. Therefore, we expect the red mold rice extracts possess great potential on the development of the skin-whitening and melanoma-cytotoxic agents.
目錄………………………………………………………………… Ι
縮寫表……………………………………………………………… Ⅲ
圖次………………………………………………………………… Ⅴ
表次………………………………………………………………… Ⅶ
中文摘要…………………………………………………………… Ⅷ
英文摘要………………………………………………………….. Ⅹ
第一章 緒言………………………………………………………. 1
第二章 文獻回顧………………………………………………… 3
第一節 黑色素細胞與黑色素的形成…………………………..3
第二節 黑色素瘤 (Melanoma)………………………………….8
第三節 細胞凋亡 (Apoptosis) ………...................8
第四節 紅麴之簡介………………………………………….. 15
第五節 lovastatin (monacolin K) 抗腫瘤細胞之相關研究.26
第六節 研究動機與目的…………………………………......31
第三章 材料與方法…………………………………………......33
第一節 菌株之培養 …………………………………………...33
第二節 實驗儀器..……………………………………………..33
第三節 藥品……………………………………………………..35
第四節 固態培養方法與條件………………………………....37
第五節 紅麴萃取樣品之製備及Total phenol和Monacolin K
之成份分析……………………………..............39
第六節 紅麴萃取物體外抗氧化作用之研究…………....... 42
第七節 紅麴萃取物體外抑制酪胺酸酶活性之研究..........45
第八節 紅麴萃取物體外抑制Melanoma B16-F0之研究……...46
第四章 結果與討論………………………………………………..57
第一節 紅麴萃取樣品之製備及Total phenol和Monacolin K 之
成份分析..................................... 57
第二節 紅麴萃取物體外抗氧化作用之研究………..........64
第三節 紅麴萃取物體外抑制酪胺酸酶活性之研究..........67
第四節 紅麴萃取物體外抑制Melanoma B16-F0之研究……...74
第五章 結論………………………………………………………..93
第六章 參考文獻…………………………………………………..95
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