臺灣博碩士論文加值系統

近年來的許多研究指出基質金屬蛋白酶(matrix metalloproteinases, MMPs)不論在正常生理功能的維持，或許多疾病的病理機轉上均有重要影響；因為MMPs可以分解膠原蛋白(collagen)，彈力蛋白(elastin)等細胞間質，所以對於皮膚的老化有負面的影響。本實驗以34種中藥的酒精萃取物，對小鼠3T3 纖維母細胞所分泌MMPs活性的影響，經由gelatin-based zymography 進行分析。我們發現人蔘(Panax ginseng)、當歸(Angelica sinensis)、六神花(Spilanthes acmella)、南瓜子(Cucurbita moschata)對MMP-2的活性有明顯抑制作用；而容水沉香樹(Aquilaria malaccensis)、穿心蓮(Androraphis paniculata)對MMP-9活性的抑制有顯著的效果。由於MMP-2可以分解 collagenⅠ與collagen Ⅲ，MMP-9則是分解collagen IV，因此我們認為人蔘(P. ginseng)、當歸(A. sinensis)、六神花(S. acmella)、南瓜子(C. moschata) 、容水沉香樹(A. malaccensis)、穿心蓮(A. paniculata) 的酒精萃取物對於皮膚抗老化方面具有顯著效果，可以應用在抗老保養品的開發。而在細胞存活率方面，經MTT assay顯示牛蒡(Arctium lappa)、容水沉香樹(Aquilaria malaccensis)、血竭(Nelumbo nucifera)、穿心蓮(Andrographis paniculata)、茴香(Aenthum graveolens)、荷葉(Nelumbo nucifera)、金銀花(Lonicera japonica)、仙鶴草(Agrimonia pilosa)、蒺藜(Tribulus ierrestris)、山楂(Crataegus pinnatifida)、銀杏(Ginkgo biloba)、百合(Lilium lancifolium)、柿葉(Diospyros kali)、白花蛇舌草(Hedyotis diffusa) 等14種中草藥的酒精萃取物在處理3T3細胞48小時後，顯示有細胞增生作用。因此，推測這14種中草藥的酒精萃取物具有促進細胞增生的作用，可應用於開發活化細胞的保養化粧品原料。

Previous studies reported that the matrix metalloproteinases (matrix metalloproteinases, MMPs) play an important role both in addition to the maintenance of normal physiological function and the pathogenesis of many diseases. Because MMPs can be broken down collagen, elastin and other extracellular matrix, they may be involved in the process of skin aging. In this study, we selected the alcohol extracts from thirty-four kinds of Chinese herbal medicines, and analyzed MMPs activity secerted from mouse 3T3 fibroblasts by gelatin-based zymography. We found that the alcohol extracts of Panax ginseng、Angelica sinensis、Spilanthes acmella、Cucurbita moschata could inhibit the activity of MMP-2 significantly, and the alcohol extracts of Aquilaria malaccensis、Androraphis paniculata decreased the activity of MMP-9 significantly. Because MMP-2 is able to damage collagens Ⅰ and Ⅲ, and MMP-9 can destory collagen IV, we consider that the alcohol extract of P. ginseng, A. sinensis, S. acmella, C. moschata, A. malaccensis, A. paniculata have intensive anti-aging effects and they can be applied in anti-aging skin care products. On the other hand, we found that alcohol extracts of 14 kinds of Chinese herbal medicines (Arctium lappa, Aquilaria malaccensis, Nelumbo nucifera, Andrographis paniculata, Aenthum graveolens、Nelumbo nucifera, Lonicera japonica, Agrimonia pilosa, Tribulus ierrestris, Crataegus pinnatifida, Ginkgo biloba, Lilium lancifolium, Diospyros kali, Hedyotis diffusa) were tested by MTT assay and the results showed the phenomenon of cell proliferation. These Chinese herbal medicine extracts can be used in cosmetics for cell activation.

中文摘要………………………………………………………………………Ⅰ
英文摘要………………………………………………………………………Ⅳ
誌謝……………………………………………………………………………V
表目錄…………………………………………………………………………Ⅷ
圖目錄…………………………………………………………………………Ⅸ
中英文縮寫表…………………………………………………………………Ⅹ
研究動機與目的…………………………………………………………1
壹、 緒論
前言………………………………………………………………………2
一、皮膚的生理構造……………………………………………………3
二、膠原蛋白……………………………………………………………7
三、基質金屬蛋白脢……………………………………………………8
四、皮膚的老化機制………………………………………………..…11

貳、材料與方法
一、實驗藥品、儀器…………………………………………………15
二、實驗藥材………………………………………………………….17
三、細胞培養…………………………………………………………17


四、實驗方法…………………………………………………………17
1.34種中草藥之酒精萃取液製備……………………………………17
2.細胞存活率測試(MTT assay)……………………………………….18
3.抑制MMPs活性分析……………………………………………….19

叁、結果……………………………………………………………….22
肆、討論……………………………………………………………….25
一、人蔘(Panax ginseng)……………………………………………….25
二、容水沉香樹(Aquilaria malaccensis)………………………………26
三、穿心蓮(Androraphis paniculata)…………………………………26
四、南瓜子(Cucurbita moschata)………………………………………27
五、當歸(Angelica sinensis)與六神花(Spilanthes acmella)……………27
伍、結論……………………………………………………………….28
陸、參考文獻…………………………………………………………..29







表目錄
表一、人體組織中存在的膠原蛋白種類……………………………35
表二、MMPs和細胞外基質…………………………………………36
表三、34種中草藥的資料與使用部位………………………………37
表四、細胞培養基之組成……………………………………………43

表五、separating gel………………………………………………….44
表六、stacking gel…………………………………………………….45
表七、1X tank buffer………………………………………………….46
表八、2X sample buffer……………………………………………….47
表九、reaction buffer………………………………………………….48
表十、fixation buffer…………………………………………………..49
表十一、staining buffer………………………………………………..50
表十二、destaining buffer……………………………………………..51
表十三、中草藥萃取物對3T3細胞之細胞存活率(a)……………….52
表十四、中草藥萃取物對3T3細胞之細胞存活率(b)……………….53











圖目錄
圖一、中草藥萃取物對老鼠3T3細胞之細胞存活率(a)……………54
圖二、中草藥萃取物對老鼠3T3細胞之細胞存活率(b)……………55
圖三、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..56
圖四、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..57
圖五、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..58
圖六、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..59圖七、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..60圖八、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..61圖九、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..62
圖十、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析..63圖十一、不同中草藥酒精萃取物(濃度1mg/ml )對MMPs的活性分析…………………………………………………………………64
圖十二、不同濃度之中草藥酒精萃取物(濃度0.5-2mg/ml)對MMPs的活性分析……………………………………………………………65
圖十三、人蔘酒精萃取物對MMPs的活性分析……………………66
圖十四、容水沉香樹酒精萃取物對MMPs的活性分析……………67
圖十五、穿心蓮酒精萃取物對MMPs的活性分析………………...68
圖十六、當歸酒精萃取物對MMPs的活性分析…………………...69
圖十七、六神花酒精萃取物對MMPs的活性分析……………….70

1. 楊博智，(2002)，Andrographolide抑制人類單核球細胞 (THP-1) 誘發第九型基質金屬酵素活化之機轉探討，台北醫學大學醫學研究所碩士論文。
2. 劉珶、張懷亮，皮膚科學與化妝品功效評價，(2005)。初版，中國大陸：化學工業出版社，277-299。
3. 駱郁婷，(2009)，人參皂苷Rb1對老鼠肝臟星狀HSC-T6細胞活化與纖維化之影響，台北醫學大學保健營養學系碩士論文。
4. 賴宜甄，(2006)，藻類多醣體應用於化妝品之抗老化分析，嘉南藥理科技大學化粧品科技研究所碩士論文。
5. Aslam, M. N., Lansky, E. P., Varani, J. (2006), Pomegranate as a cosmeceutical source: pomegranate fractions promote proliferation and procollagen synthesis and inhibit matrix metalloproteinase-1 production in human skin cells, Journal Ethnopharmacol, 103, 311-318.
6. Brew, K., Dinakarpandian, D., Nagase, H. (2000), Tissue inhibitors of metalloproteinases: evolution, structure and function, Archives of Biochemistry and Biophysics, 1477, 267-283.
7. Cury, P. R., Dearaujo, V. C., Canavez, F., Furuse, C., Leite, K. R., Dearaujo, N. S. (2007), The effect of epidermal growth factor on matrix metalloproteinases and tissue inhibitors of metalloproteinase gene expression in cultured human gingival fibroblasts, Archires of Oral Biology, 52, 585-590.
8. Cory, A. H., Owen, T. C., Barltrop, J. A., (1991), Use of anaqueoussoluble tetrazolium/formazan assay for cell growth assays in culture. Cancer communications, 3, 207-212.
9. Desrivieres, S., Lu, H., Soria, C., Legrand, Y., Menashi, S. (1993), Activation of the 92 KDa Type IV collagenase by tissue kallikrein, Cell Physiology, 157, 587-593.
10. El-Domyati, M., Attia, S., Saleh, F., Brown, D., Birk, D. E., Gasparro, F., Ahmad, H., Uitto, J. (2002), Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin, Experimental Dermatology, 11, 398-405.
11. Fortino, V., Maioli, E., Torricelli, C., Davis, P., Valacchi, G. (2007), Cutaneous MMPs are differently modulated by environmental stressors in old and young mice, Toxicology, 173, 73-79.
12. Gelse, K., Poschl, E., Aigner, T. (2003), Collagens--structure, function, and biosynthesis, Advanced Drug Delivery Reviews, 55, 1531-1546.
13. Gary, J. F., Sewon, K., James, V., Zsuzsanna, B. C., Yinsheng, W., Subhash, D., John, J. (2002), Mechanisms of Photoaging and Chronological Skin Aging, Archives of Dermatology,138, 1462-1470.
14. Howard, E. W., Bullen, E. C., Banda, M. J. (1991), Preferential inhibition of 72-kDa and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2, Journal of Biological Chemistry, 266,13070-13075.
15. Hornebeck, W. (2003), Down-regulation of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival, Pathological Biolology, 51, 569-573.
16. Rabe, J. H., Mamelak, A. J., McElgunn, P. J. S., Morison, W. L., Sauder, D. N. (2006), Photoaging: Mechanisms and repair, Journal of American Academy of Dermatology. 55, 1-19.
17. Kupai, K., Szucs, G., Cseh, S., Hajdu, I., Csonka, C., Csont, T., Ferdinandy, P. (2010), Matrix metalloproteinase activity assays: Importance of zymography, Journal of Pharmacological andToxicological Methods, 10, 1016.
18. Karin, W., Nicole S., Petra B., Georges R., Adrian W., Christopher L., Regina G., (2004), β-carotene inhibits UVA-induced matrix metalloprotease 1 and 10 expression in keratinocytes by a singlet oxygen-dependent mechanism Free Radical Biology and Medicine, 37, 654 – 670.
19. Longo, G. W., Chow, L. T., Fan, Y. S., Werb, Z. (2002),Matrixmetalloproteinase 2 and 9 work in concert to produce aortic Aneurysms, Clinical Investigation, 110, 625-632.
20. Lee, C. W., Choi, H. J., Kim, H. S., Kim, D. H., Chang, I. S., Moon, H. T., Lee, S. Y., Oh, W. K., Woo, E. R. (2008), Biflavonoids isolated fromSelaginella tamariscina regulate the expression of matrixmetalloproteinase in human skin fibroblasts, Bioorganic and Medicinal Chemistry, 16, 732-738.
21. Masamitsu, I., Hideya, A., Masaki, Y., Yoko, N., Mary, M. (2009), Photoaging of the skin, Anti-Aging Medicine, 6, 46-59.
22. Madlener, M., Parks, W. C., Werner, S. (1998), Matrixmetalloproteinases (MMPs) and their physiological inhibitors (TIMPs)
are differentially expressed during excisional skin wound repair, Experimental Cell Research, 242, 201-210.
23. Mimura, Y., Ihn, H., Jinnin, M., Asano, Y., Yaman,e K., Tamaki, K. (2006), Epidermal growth factor affects the synthesis and degradation of type I collagen in cultured human dermal fibroblasts, Matrix. Biology, 25, 202-212.
24. Moon, H. I., Lee, J., Chung, J. H. (2006), The effect of erythrodiol-3-acetate on the expressions of matrix metalloproteinase-1 and type-1 procollagen caused by ultraviolet irradiated cultured primary old aged human skin fibroblasts, Phytomedicine, 13, 707-711.
25. Mosmann, T. (1983), Rapid colorimetric assay for cellular growth and
survival application to proliferation and cytotoxicity assay, Immunological
Methods, 65, 55-63.
26. Nakahara, H., Howard, L., Sato, H., Seiki, M.,Yeh, Y., Chen, T. W. (1997), Transmembrane / cytoplasmic domain-mediated membrane type
1-matrix metalloprotease docking to invadopodia is required for cell
Invasion, Biochemistry and cell Biology, 94, 7959-7964.
27. Nagase, H., Sasaki, T., Yadomae, T., Chiba, N., Adachi, Y. (1999), Activation mechanisms of matrix metalloproteinases, Biochemistry, 378, 151-159.
28. Offord, E. A., Gautier, J. C., Avanyi, O., Scaletta, C., Runge, F., Kramer, k., Applegate, L. A. (2002), Photoprotective potential of lycopene, beta-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblast, Free Radical Biology and Medicine 32,1293-1303.
29. Palosaari, H., Pennington, C. J., Larmas, M., Edwards, D. R., Tjaderhane, L., Salo, T.(2003), Expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in mature human odontoblasts and pulp tissue, European Journal of Oral. Sciences, 111, 117-27.
30. Sternlicht, M. D., Werb, Z. (2001), How matrix metalloproteinases regulate cell behavior, Annual Review of Cell and Developmental Biology, 17, 463-516.
31. Uitto, V. J., Overall, C. M., McCulloch, C. (2003), Proteolytic host cell enzymes in gingival crevice fluid, Periodontology, 31, 77-104.
32. Vayalil, P. K., Mittal, A., Hara, Y., Elmets, C. A., Katiyar, S. K. (2004), Green tea polyphenols prevent ultraviolet light-induced oxidative damage and matrix metalloproteinases expression in mouse skin, Investigative Dermatology, 122, 1480-1487.
33. Zhao, W., Berry, M. N., Daemen, M. J. (1999), Photoprotective effect of blacktea extracts agains UVB-induced photoxicity in skin,International Journal of Cosmetics Science, 70, 637-644.