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研究生:朱志維
研究生(外文):Chih-Wei Chu
論文名稱:利用麴酸抑制黑色素細胞生成與釋放黑色素研究進而建立一套美白機制
論文名稱(外文):To establish a skin whitening system : Inhibition of melanin synthesis and release from melanoma cells treated with kojic acid
指導教授:楊重熙
指導教授(外文):Chung-Shi Yang
學位類別:碩士
校院名稱:國立暨南國際大學
系所名稱:應用化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:62
中文關鍵詞: 麴酸 美白 黑色素癌細胞
外文關鍵詞:kojic acidmelanoma cell
相關次數:
  • 被引用被引用:1
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  • 下載下載:425
  • 收藏至我的研究室書目清單書目收藏:2
麴酸( kojic acid )為已知抑制黑色素生成相當有效的藥物,因 kojic acid 會與
tyrosinase 中心的銅離子結合而抑制 tyrosinase 的活性。 forskolin 能刺激老鼠
的黑色素癌細胞(B16-F10)生成大量的黑色素,forskolin 是一種adenylate
cyclase(AC)的活化劑,在加入 forskolin 的黑色素癌細胞中,可以觀察的到黑
色素明顯的累積。
在實驗中分成兩個部份,第一部份是給予forskolin (100 μM)刺激黑色素生
成,並且同時加入kojic acid 去抑制, 而在kojic acid 10 mM 時可以看到明顯
的抑制黑色素生成效果,約可以抑制79 % 的黑色素生成。
第二個部份是沒有加入forskolin 刺激生成黑色素,而是直接加入kojic acid
抑制生成黑色素,結果發現放置三天後取出細胞,將細胞打破取出胞液後放置
於96 孔盤中,置入ELISA Readers 在波長405nm 下來探測細胞內黑色素生成
量,並且測試細胞內tyrosinase 的活性,當kojic acid 為10mM 時可抑制約68 %
黑色素生成,接下來希望能夠得知kojic acid 在細胞內抑制的有效濃度為何,
所以將細胞打破,並利用HPLC 偵測kojic acid 在細胞內的濃度,但卻偵測不
到,原因有可能是kojic acid 與tyrosinase 作用的關係或是細胞內濃度低於偵測
極限,而在未來的實驗中希望能找到方法去偵測到kojic acid 在細胞內的濃度,
最後加入L-ascorbic acid 觀察對於抑制黑色素生成的效果,但由於L-ascorbic
acid 很容易降解,所以在與細胞培養三天後,對於抑制黑色素生成的效果不明
顯。
往後利用這一系列的數據並且與in tube 實驗配合作為依據進而建立一套檢
驗美白藥品能力的比對機制。
Kojic acid is known to be able to suppress the generation of melanin effectively,
because kojic acid can bind with the copper ion located in the active site of
tyrosinas which is a key enzyme responsible for melanin synthesis. Forskolin can
induce the melanin production in melanoma cells via the activation of adenylate
cyclase to increase intracellular cyclic AMP concentration.
We first investigated the effect of kojic acid treatment on the forskolin (100
μM)-induced melanin production in melanoma cells (B16-F10). When 10mM of
kojic acid was employed, the generation of melanin can be effectively suppressed.
This amount of kojic acid can control up to about 79% of the synthesis of melanin.
We then studied the effect of kojic acid treatment on the endogenous melanin
production in the melanoma cells. The results indicated that 10mM of Kojic Acid
can control about 68% of the synthesis of melanin.
Next, in order to findout the intracellular concentration of kojic acid following
the extracellular treatment, the cells were lysed and the concentration of kojic acid
was analyzed by HPLC. Unfortunately, we were not able to observe detectable
amount of kojic acid in the cells. The reason might be that the intracellular
concentration of kojic acid is too low probably due to its strong binding with
tyrosnase.
Finally, L-ascorbic Acid was added to observe its efficiency in suppressing
melanin. L-ascorbic Acid can be oxidized easily; thus, there appeared to be no
apparent result after three days of cultivation. These series of data along with in
tube experiment can be an effective comparison mechanism to test the efficiency of
5
whitening medicine in the future.
目錄
謝誌
中文摘要
英文摘要
目錄
圖次
第一章 緒論
1.1 前言---------------------------------------------------------------------------1
1.2 皮膚的構造------------------------------------------------------------------2
1.2.1 皮膚的層次--------------------------------------------------------2
1.2.2 皮膚的功能--------------------------------------------------------3
1.3 黑色素細胞(Melanocyte)-------------------------------------------------6
1.4 酪胺酸酶(Tyrosinase)-----------------------------------------------------7
1.5 黑色素(Melanin)的生成--------------------------------------------------8
1.6 影響黑色素生成的機制-------------------------------------------------10
1.7 麴酸(kojic acid)與抗氧化劑(Ascorbic acid)--------------------------11
7
第二章 實驗材料與方法
2.1 實驗材料--------------------------------------------------------------------14
2.1.1 實驗細胞株-------------------------------------------------------14
2.1.2 實驗藥品----------------------------------------------------------14
2.1.3 實驗儀器設備----------------------------------------------------15
2.2 實驗方法-------------------------------------------------------------------16
2.2.1 細胞株培養-------------------------------------------------------16
2.2.2 利用Forskolin 刺激細胞內黑色素生成試驗---------------17
2.2.2.1 細胞內酪胺酸酶(Tyrosinase)活性試驗---------17
2.2.2.2 測量細胞內黑色素含量----------------------------18
2.2.3 同時加入Forskolin 與麴酸測定黑色素生成與酪胺酸活酶
活性---------------------------------------------------------------19
2.2.4 利用麴酸抑制黑色素生成與酪胺酸酶活性試驗---------19
2.2.5 利用高效率液相層析儀觀察細胞外與細胞內藥品濃度-20
2.2.5.1 高效率液相層析儀沖提液的配置----------------20
2.2.5.2 高效率液相層析儀分離條件----------------------20
2.2.6 利用抗氧化劑( L-Ascorbic acid)抑制黑色素生成試驗--21
2.2.7 同時加入麴酸與抗氧化劑抑制黑色素生成試驗----------21
8
第三章 實驗結果
3.1 利用Forskolin 刺激黑色素細胞之試驗結果-------------------------23
3.1.1 加入Forskolin 後黑色素細胞生成黑色素之影響--------23
3.1.2 同時加入Forskolin 與麴酸對於黑色素生成之影響-----23
3.2 在細胞中加入麴酸抑制黑色素生成與酪胺酸酶活性試驗-------26
3.2.1 不同濃度下麴酸抑制酪胺酸酶活性之結果----------------26
3.2.2 不同濃度下麴酸抑制生成黑色素之結果-------------------26
3.2.3 利用HPLC 測試麴酸在細胞內的濃度之結果------------29
3.2.4 利用HPLC 測試麴酸在細胞外濃度之結果---------------29
3.2.5 同時加入Forskolin 與麴酸並利用HPLC 測試麴酸在細
胞內濃度之結果------------------------------------------------32
3.2.6 加入麴酸與不同濃度酪胺酸酶並利用HPLC 測量麴酸濃
度之變化----------------------------------------------------------34
3.3 在細胞中加入L-ascorbic acid 抑制黑色素生成試驗---------------35
3.3.1 不同濃度下抗氧化劑抑制酪胺酸酶活性之結果----------35
3.3.2 不同濃度下抗氧化劑抑制黑色素生成之結果-------------35
3.3.3 利用HPLC 測試抗氧化劑在細胞內的濃度之結果-------38
3.3.4 利用HPLC 測試抗氧化劑在細胞外的濃度之結果-------38
9
3.4 在細胞中同時加入麴酸與L-ascorbic acid 抑制黑色素生成試驗--41
3.4.1 同時加入麴酸與抗氧化劑對於酪胺酸酶活性之影響----41
3.4.2 同時加入麴酸與抗氧化劑對於黑色素生成之影響-------41
第四章 結果與討論--------------------------------------------------------------44
第五章 未來展望-----------------------------------------------------------------46
參考文獻------------------------------------------------------------------------------47
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