(3.235.25.169) 您好!臺灣時間:2021/04/17 20:38
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:林姵妏
研究生(外文):Pei-Wen Lin
論文名稱:甲醇層的穿山龍乙醇萃取物在抑制黑色素生成之研究
論文名稱(外文):Methanol Partition Fraction of Ethanol Extract of Discorea nipponica Makino Inhibits Melanogenesis
指導教授:張德生
指導教授(外文):Te-Sheng Cheng
口試委員:吳聲祺江建民丁秀玉張德生
口試委員(外文):Sheng-Chi WuChien-Min ChiangHsiou-Yu DingTe-Sheng Cheng
口試日期:2014-06-26
學位類別:碩士
校院名稱:國立臺南大學
系所名稱:生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:78
中文關鍵詞:抑制黑色素
外文關鍵詞:melanogenesis
相關次數:
  • 被引用被引用:0
  • 點閱點閱:290
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
本研究將二十種中草藥萃取物利用B16F1小鼠黑色素瘤細胞(B16F1細胞)平台篩選到具有美白活性的萃取物- 穿山龍萃取物(Dioscorea nipponica Makino;DNM)具有抑制黑色素生成之效用。以體外試驗之平台- B16F1細胞及活體平台-斑馬魚胚胎與小黑鼠來評估穿山龍萃取物抑制黑色素生成的作用及機轉。本研究也進一步探討中草藥穿山龍萃取物具有抗氧化之效用。
在B16F1細胞平台上,加入不同劑量的DNM處理B16F1細胞,細胞再經IBMX誘導黑色素生成,由實驗結果顯示DNM有效的抑制黑色素生成。以不同DNM濃度分析B16F1細胞內外的酪胺酸酶之活性,結果發現細胞內外酪胺酸酶之活性皆無抑制,因此再利用酵素圖譜法(Zymography)與西方墨點法(Western blot)檢測細胞內酪胺酸酶的活性及蛋白質的表現,結果顯示DNM也並無影響酪胺酸酶的活性與蛋白量。另一方面,DNM透過DPPH、 ABTS+清除自由基試驗及B16F1細胞內ROS的含量來評估其抗氧化能力。實驗結果中,DNM在清除DPPH及ABTS+自由基試驗皆具有抗氧化能力。在細胞內ROS的含量測定上,DNM明顯著地降低細胞內ROS的含量。此外在活體平台-斑馬魚試驗下,DNM有效地降低斑馬魚幼蟲的皮膚黑色素生成,且顯示出劑量依賴的效應。最後在另一個活體平台-小黑鼠皮膚美白試驗下,在小黑鼠背部塗抹2 % DNM的凝膠製劑,使用2週後略有增加肌膚美白的程度。綜合以上實驗結果證實DNM具有降低黑色素生成及抗氧化之效用。
本研究證實DNM在小鼠黑色素瘤細胞、斑馬魚活體具有抑制黑色素生成之能力,且降低了細胞內的ROS含量,因此DNM在皮膚美白、抗氧化方面可做為高潛力之有效皮膚抗老與美白成分,DNM能用於化妝品產業。

Melanogenesis inhibition by the methanol layer of Dioscorea nipponica Makino extract (DNM) was investigated both in vitro in cultivated murine B16 melanoma cells and in vivo in zebrafish and mice. In B16 cells, results showed that DNM dose-dependently inhibited melanogenesis under nontoxic concentrations. Despite the potent inhibition of melanogenesis, results from evaluating intracellular tyrosinase activity and tyrosinase zomographic staining showed that DNM did not reduce intracellular tyrosinase activity and the active protein amount of intracellular tyrosinase in B16 cells.However, DNM showed strong antioxidant activities against cell-free DPPH and ABTS+ free radical and intracellular Reactive oxygen species (ROS).
These results reveal that DNM inhibits melanogenesis of B16 cells through antioxidant activity but not through inhibiting tyrosinase activity. The melanogenesis inhibitory activity of DNM was also evaluated in vivo in zebrafish and mice. In zebrafish, melanogenesis of larvae skin was significantly and dose-dependently inhibited by DNM treatment. In mice, application of a 2 % gel preparation of DNM applied to mouse skin slightly increased the degree of skin whitening after 2 weeks of treatment. Based on our findings reported here, DNM is a good candidate for use as a skin-whitening agent for treating skin hyperpigmentation disorders.

摘要.......................................................................................................................................i
Abstract................................................................................................................................ ii
誌 謝 .................................................................................................................................iii
目 錄 .................................................................................................................................iv
圖 目 錄 ..........................................................................................................................vi
縮寫對照表.......................................................................................................................vii
第1章 緒論..................................................................................................................1
1.1 研究背景...........................................................................................................1
1.2 研究目的...........................................................................................................1
1.3 研究架構...........................................................................................................2
第2章 文獻回顧..........................................................................................................3
2.1 黑色素的生成機轉.............................................................................................3
2.1.1 皮膚的結構與功能....................................................................................3
2.1.2 黑色素生成................................................................................................6
2.2 抑制黑色素生成之調控機轉...........................................................................14
2.3 穿山龍(Dioscorea nipponica Makino)背景介紹..............................................16
2.3.1 穿山龍(Dioscorea nipponica Makino).....................................................16
2.3.2 穿山龍的相關文獻研究及專利..............................................................17
第3章 材料與方法...................................................................................................18
3.1 材料.......................................................................................................................18
3.1.1 藥品................................................................................................................19
3.1.2 抗體................................................................................................................19
3.1.3 溶液配製........................................................................................................20
3.1.4 實驗儀器........................................................................................................23
3.1.5 中草藥萃取物來源........................................................................................23
3.1.6 細胞株來源 ..................................................................................................24
3.1.7 活體系統平台 ..............................................................................................24
3.2 方法.........................................................................................................................26
3.2.1 細胞繼代培養 ............................................................................................26
3.2.2 細胞冷凍保存..............................................................................................26
3.2.3 細胞計數......................................................................................................27
3.2.4 細胞毒性測試(MTT法) ..............................................................................28
3.2.5 B16-F1 細胞內黑色素含量測定................................................................28
3.2.6 B16-F1 細胞內酪胺酸酶(Cellular tyrosinase)活性試驗...........................28
3.2.7 B16-F1 細胞外酪胺酸酶(Cell-free tyrosinase)活性試驗..........................29
3.2.8 酪胺酸酶活性試驗之酵素圖譜法(Zymography) ......................................30
3.2.9 酪胺酸酶之西方墨點法(Western Blot).………………………..………...31
3.2.10 清除 DPPH自由基能力之測定..................................................................33
3.2.11 清除 ABTS自由基測定分析方法..............................................................34
3.2.12 細胞內的ROS含量測定..............................................................................34
3.2.13 斑馬魚胚胎致死率試驗.……………………………………………….…35
3.2.14 斑馬魚胚胎顯微鏡攝影拍攝之皮膚美白篩選分析……………….….…35
3.2.15 小鼠之皮膚美白分析………………………………………………….….36
3.2.16 蛋白質含量試驗…………………………………………………………..36
3.2.17 數據統計分析……………………………………………………………..37
第4章 結果……………………………………………………………….………….38
4.1 DNM 對小鼠B16F1細胞毒性測試……………………………..………..38
4.2 DNM 對小鼠B16F1細胞黑色素生成之抑制測試………………..……..39
4.3 DNM 對酪胺酸酶(Tyrosinase)的活性影響 ……………….………........40
4.4 DNM 對B16F1細胞內酪胺酸酶活性及蛋白含量分析………..………..42
4.5 DNM 對於DPPH自由基的清除能力…………………………..………...44
4.6 DNM 對於ABTS+自由基的清除能力…………………………..……….45
4.7 DNM 對於細胞內的ROS含量之測定………………………..…………..46
4.8 DNM 對斑馬魚胚胎之毒性測試…………………………………………48
4.9 DNM 對斑馬魚胚胎之黑色素生成抑制測試……………..……………..49
4.10 DNM 對小黑鼠活體之皮膚美白分析…………………….….…………..50
第5章 結論與未來展望 ................................................................................................51
5.1 結論...................................................................................................................51
5.2 未來展望...........................................................................................................51
參考文獻………......................................................................................................52
附 錄 ………..………………………………………………………………………56
附錄一 個人履歷…………………………………………………………………56
附錄二 論文著述 Publication paper……………………………………………..57

Abdel-Malek, Z., Swope, V. B., Suzuki, I., Akcali, C., Harriger, M. D., Boyce, S. T., . . . Hearing, V. J. (1995). Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proceedings of the National Academy of Sciences, 92(5), 1789-1793.
Alhaidari, Z., Olivry, T., & Ortonne, J. (1999). Melanocytogenesis and melanogenesis: genetic regulation and comparative clinical diseases. Veterinary Dermatology (United Kingdom).
Bolognia, J. L., & Pawelek, J. M. (1988). Biology of hypopigmentation. Journal of the American Academy of Dermatology, 19(2), 217-255.
Brash, D. E., Rudolph, J. A., Simon, J. A., Lin, A., McKenna, G. J., Baden, H. P., . . . Ponten, J. (1991). A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proceedings of the National Academy of Sciences, 88(22), 10124-10128.
Briganti, S., Camera, E., & Picardo, M. (2003). Chemical and instrumental approaches to treat hyperpigmentation. Pigment cell research/sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 16(2), 101-110.
Chang, T.-S. (2009). An updated review of tyrosinase inhibitors. International journal of molecular sciences, 10(6), 2440-2475.
Chang, T.-S., & Lin, J.-J. (2010). Inhibitory effect of danazol on melanogenesis in mouse B16 melanoma cells. Archives of pharmacal research, 33(12), 1959-1965.
Chang, T.-S., Lin, M.-Y., & Lin, H.-J. (2010). Identifying 8-hydroxynaringenin as a suicide substrate of mushroom tyrosinase. Journal of cosmetic science, 61(3), 205.
Choi, T., Kim, J., Ko, D., Kim, C., Hwang, J., Ahn, S., . . . Yoon, T. (2007). Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds. Pigment cell research/sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society, 20(2), 120-127.
Choi, T. Y., Kim, J. H., Ko, D. H., Kim, C. H., Hwang, J. S., Ahn, S., . . . Yoon, T. J. (2007). Zebrafish as a new model for phenotype‐based screening of melanogenic regulatory compounds. Pigment Cell Research, 20(2), 120-127.
Costin, G.-E., & Hearing, V. J. (2007). Human skin pigmentation: melanocytes modulate skin color in response to stress. The FASEB Journal, 21(4), 976-994.
del Marmol, V., & Beermann, F. (1996). Tyrosinase and related proteins in mammalian pigmentation. FEBS letters, 381(3), 165-168.
Dong, Y., Wang, H., Cao, J., Ren, J., Fan, R., He, X., . . . Dong, C. (2011). Nitric oxide enhances melanogenesis of alpaca skin melanocytes in vitro by activating the MITF phosphorylation. Molecular and cellular biochemistry, 352(1-2), 255-260.
Fu, T., Huang, Y., Ji, Y., Li, B., Liu, Z., Qi, W., & Zou, W. (2005). Pharmaceutical composition containing steroidal saponins, the preparation method and use thereof: Google Patents.
Gillbro, J., & Olsson, M. (2011). The melanogenesis and mechanisms of skin‐lightening agents–existing and new approaches. International journal of cosmetic science, 33(3), 210-221.
Halaban, R., Langdon, R., Birchall, N., Cuono, C., Baird, A., Scott, G., . . . McGuire, J. (1988). Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. The Journal of cell biology, 107(4), 1611-1619.
Hamid, M. A., Sarmidi, M. R., & Park, C. S. (2012). Mangosteen leaf extract increases melanogenesis in B16F1 melanoma cells by stimulating tyrosinase activity in vitro and by up-regulating tyrosinase gene expression. International journal of molecular medicine, 29(2), 209.
Ho, M.-L., Hsieh, Y.-S., Chen, J.-Y., Chen, K.-S., Chen, J.-J., Kuo, W.-H., . . . Chen, P.-N. (2011). Antimetastatic potentials of Dioscorea nipponica on melanoma in vitro and in vivo. Evidence-Based Complementary and Alternative Medicine, 2011.
Ito, S., & Wakamatsu, K. (2008). Chemistry of Mixed Melanogenesis—Pivotal Roles of Dopaquinone†. Photochem Photobiol, 84(3), 582-592.
Jimbow, K. (1995). Current update and trends in melanin pigmentation and melanin biology. The Keio journal of medicine, 44(1), 9.
Kalt, W., Forney, C. F., Martin, A., & Prior, R. L. (1999). Antioxidant capacity, vitamin C, phenolics, and anthocyanins after fresh storage of small fruits. Journal of agricultural and food chemistry, 47(11), 4638-4644.
Kim, Y.-J., & Uyama, H. (2005). Tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future. Cellular and Molecular Life Sciences CMLS, 62(15), 1707-1723.
Kwon, C.-S., Sohn, H. Y., Kim, S. H., Kim, J. H., Son, K. H., Lee, J. S., . . . Kim, J.-S. (2003). Anti-obesity effect of Dioscorea nipponica Makino with lipase-inhibitory activity in rodents. Bioscience, biotechnology, and biochemistry, 67(7), 1451-1456.
Lajis, A. F. B., Hamid, M., & Ariff, A. B. (2012). Depigmenting effect of kojic acid esters in hyperpigmented B16F1 melanoma cells. BioMed Research International, 2012.
Luo, D. (2008). Identification of structure and antioxidant activity of a fraction of polysaccharide purified from Dioscorea nipponica Makino. Carbohydrate Polymers, 71(4), 544-549.
Maddodi, N., Jayanthy, A., & Setaluri, V. (2012). Shining light on skin pigmentation: the darker and the brighter side of effects of UV radiation. [Review]. Photochem Photobiol, 88(5), 1075-1082.
Masaki, H. (2010). Role of antioxidants in the skin: anti-aging effects. Journal of dermatological science, 58(2), 85-90.
Nakagawa, M., Kawai, K., & Kawai, K. (1995). Contact allergy to kojic acid in skin care products. Contact dermatitis, 32(1), 9-13.
Packer, L., Witt, E. H., & Tritschler, H. J. (1995). Alpha-lipoic acid as a biological antioxidant. Free Radical Biology and Medicine, 19(2), 227-250.
Parvez, S., Kang, M., Chung, H. S., Cho, C., Hong, M. C., Shin, M. K., & Bae, H. (2006). Survey and mechanism of skin depigmenting and lightening agents. Phytotherapy Research, 20(11), 921-934.
Passi, S., & Nazzaro-Porro, M. (1981). Molecular basis of substrate and inhibitory specificity of tyrosinase: phenolic compounds. British Journal of Dermatology, 104(6), 659-665.
Różanowska, M., Sarna, T., Land, E. J., & Truscott, T. G. (1999). Free radical scavenging properties of melanin: interaction of eu-and pheo-melanin models with reducing and oxidising radicals. Free Radical Biology and Medicine, 26(5), 518-525.
Riley, P. (1997). Melanin. The international journal of biochemistry & cell biology, 29(11), 1235-1239.
Schallreutera, K. U., Wazira, U., Kotharia, S., Gibbonsa, N. C., Moore, J., & Wood, J. M. (2004). Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes. Biochemical and biophysical research communications, 322, 88-92.
Schauer, E., Trautinger, F., Köck, A., Schwarz, A., Bhardwaj, R., Simon, M., . . . Luger, T. (1994). Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes. Journal of Clinical Investigation, 93(5), 2258.
Seeram, N. P., Adams, L. S., Henning, S. M., Niu, Y., Zhang, Y., Nair, M. G., & Heber, D. (2005). In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. The Journal of nutritional biochemistry, 16(6), 360-367.
Shimogaki, H., Tanaka, Y., Tamai, H., & Masuda, M. (2000). In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibition. International journal of cosmetic science, 22(4), 291-304.
Slominski, A., Tobin, D. J., Shibahara, S., & Wortsman, J. (2004). Melanin pigmentation in mammalian skin and its hormonal regulation. Physiological reviews, 84(4), 1155-1228.
Tai, S. S.-K., Lin, C.-G., Wu, M.-H., & Chang, T.-S. (2009). Evaluation of depigmenting activity by 8-hydroxydaidzein in mouse B16 melanoma cells and human volunteers. International journal of molecular sciences, 10(10), 4257-4266.
Wakamatsu, K., Graham, A., Cook, D., & Thody, A. J. (1997). Characterisation of ACTH Peptides in Human Skin and Their Activation of the Melanocortin‐1 Receptor. Pigment cell research, 10(5), 288-297.
Yuan, Y., Zhang, L., Dai, Y., & Yu, J. (2007). Physicochemical properties of starch obtained from Dioscorea nipponica Makino comparison with other tuber starches. Journal of Food Engineering, 82(4), 436-442.
許德發, 鄭智交, 楊濟華, 李仰川, 溫慧萍, & 許照紅. (2002). 化妝品概論, 華格那企業有限公司.
郭秀怡. (2012). 覆盆莓酮抑制黑色素生成之研究. 碩士, 國立臺南大學, 台南市.
趙時玉. (2013). 同異黃酮抑制黑色素生成之研究. 碩士,國立台南大學,台南市
嚴嘉蕙. (2014). 化妝品概論: 新文京開發出版股份有限公司.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔