臺灣博碩士論文加值系統

茶葉中所富含多酚已被證實具有抗氧化、抑菌、抗發炎、抑制黑色素細胞生成等活性，對人體皮膚保健有很大幫助。本研究以大葉種臺茶8號、18號及21號茶菁製成紅茶與綠茶為原料，比較小葉種臺茶12號，探討不同茶樣多酚成分、抗氧化活性變化及抑制酪胺酸酶活性之效果，並探討不同茶葉對黑色素瘤細胞 (B16-10)產生黑色素的抑制作用，以MTT來評估細胞存活率，包含角質細胞 (HaCaT)對UVB輻射的保護能力，及對表皮癌細胞(A-431)的抗增殖作用。研究結果發現大葉種綠茶抗氧化活性、還原能力與抑制皮膚癌細胞株 (A-431)增殖活性中，均較相同茶種所製成之紅茶為佳，顯示這些效果與其所含有的高含量總酚、兒茶素有關，其中以大葉種臺茶21號綠茶最佳。由細胞週期分析結果證明，大葉種臺茶21號綠茶萃取物會使人類表皮癌細胞(A-431)受到抑制，在G0/G1 phase的訊息傳遞路徑受損，細胞被阻斷局限停滯，使S phase與G2-M phase細胞量增多，無法正常的進行細胞分裂，而走向細胞凋亡。但在抑制酪胺酸酶活性、抑制黑色素瘤細胞生成黑色素能力與在角質細胞的抗UVB效果均以紅茶較綠茶佳，顯示紅茶的皮膚保健活性，除兒茶素外，來自如茶黃質與茶紅質等高聚合多酚化合物的調控作用也有很大的影響。本研究結果建議不易作成「紅玉」與「紅韻」的臺茶18號與21號之冬茶，可製成綠茶應用於抗氧化和抗皮膚癌，或製成紅茶作為美白和抗UVB輻射用途，如此可將大葉種冬茶應用於皮膚保健用途提高冬產值。

關鍵詞：抗增殖、抗輻射、多酚、皮膚癌、茶

Teas contained abundant polyphenolic compounds. Previous researches have demonstrated that polyphenolic compounds physiologically play critical roles as antioxidant, antibacterial, anti-inflammatory, inhibiting melanin production and in many functions for skin health. Tea extracts can be very beneficial for human skin care. Effects of extracts from various Taiwan teas on skin care and skin cancer antiproliferation effects were investigated in this study. Green tea and black tea samples were processed from 4 Taiwan Tea varieties (TTES No. 8, 12 , 18, 21), and those tea extracts were analyzed to determine polyphenols composition, antioxidant activity, tyrosinase inhibitory activity, and melanin inhibition of melanoma cells (B16-10). Cell viability of keratinocytes (HaCaT) was assessed by MTT assay to evaluate protection against UVB radiation, and anti-proliferative effect on human epidermoid carcinoma cells (A-431).
Results indicated that green tea from large-leafed variety (TTES No. 8, 18, 21) exhibited higher levels of antioxidant activity, reducing power and antiproliferation activity on skin cancer cell line (A-431), better than those of the same varities of tea extracts obtained from black tea products. Our results showed that these effects related with high levels of total phenolics and catechins. Among all tested samples, large-leafed variety TTES No.21 green tea exhibited the highest inhibition activities. Cell cycle analysis demonstrated that large-leafed TTES No.21 green tea extract can suppress human epidermoid carcinoma cells (A-431). The signaling pathways of G0/G1 phase was malfunction and further blocked S phase and G2-M phase leading to cell numbers increased in S phase and G2-M phase and not able to go through normal cell division. Eventually those cells went through apoptosis. However, for the inhibition of tyrosinase activity, inhibitions of melanoma cells to produce melanin ability and anti-UVB effect on keratinocytes were significantly higher in black tea compared with those of green tea. For skin care activity of black tea, not only catechins, theaflavins and thearubigins, and other active polymerized polyphenol compounds also might be contributed function. These findings suggested that TTES No. 18 and 21 are not easily to be processed to famous tea products like "Ruby" and "Hong Yun" as popular summer tea, however, winter tea can still be utilized to produce green tea products for antioxidant and anti-skin cancer, and black tea can serve as cosmetic whitening and anti-UVB radiation skin care product. Therefore, large-leafed variety winter tea products have potential for developing novel skin care products and further adding agricultural value for winter tea products.

Keywords: Anti-proliferative, radioprotective, polyphenols, skin cancer, tea.

目 錄
頁次
口試委員審定書 I
誌謝 II
中文摘要 III
英文摘要 .IV
目錄 VI
圖目錄 X
表目錄 XII
附錄目錄 XIII
第一章 緒論 1
1.1 前言 1
1.2 茶葉的種類與加工方法 2
1.2.1 引言 2
1.2.2 茶樹品種 2
1.2.2 茶葉分類 4
1.2.3 茶葉加工 5
1.3茶葉中的化學物質與保健活性 7
1.3.1 茶葉的化學成分 7
1.3.2 茶葉的多酚類物質 9
1.3.3 茶多酚的保健活性 17
1.4 皮膚的構造與保健 19
1.4.1 皮膚構造與功能 19
1.4.2 皮膚受損與老化 24
1.4.3 紫外線對皮膚的傷害 25
1.5自由基與癌症 28
1.5.1 自由基、活性氧與氧化傷害 28
1.5.2 氧化壓力與癌症 29
1.5.3 皮膚癌細胞 32
1.5.3.1 人類鱗狀上皮癌(表皮癌)細胞 32
1.5.3.2 細胞週期 33
1.5.3.3 細胞凋亡 35
1.6 茶多酚的抗癌作用與機制 38
1.7 研究動機 40
1.8 研究目的 40

第二章 研究材料與方法 41
2.1 實驗架構 41
2.1.1 實驗架構流程圖 41
2.1.2 樣品縮寫表 42
2.2 實驗樣品 43
2.2.1 樣品來源 43
2.2.2 樣品製備 45
2.3 實驗細胞株 45
2.4 實驗試藥與試劑 46
2.4.1 溶劑 46
2.4.2 試藥 46
2.4.3 細胞實驗各種溶液及培養基配置 47
2.5 儀器設備 48
2.5.1 樣品前處理相關儀器設備 48
2.5.2 化學分析相關儀器設備 48
2.4.3 細胞培養相關儀器設備 49
2.5.4 其他儀器設備 49
2.6 實驗方法 49
2.6.1 成份特性分析測定 49
2.6.1.1 茶湯品質之茶湯顏色與明亮度之測定 49
2.6.1.2 茶黃質含量測定 49
2.6.1.3 茶紅質含量測定 50
2.6.1.4 吸收光譜分析分析 50
2.6.1.5 總酚含量測定 51
2.6.1.6 總類黃酮含量測定 51
2.6.1.7 縮合型單寧含量測定 51
2.6.1.8 兒茶素與酚酸含量之HPLC高效能液相層析分析 52
2.6.2 抗氧化能力測定 53
2.6.2.1 DPPH自由基清除能力測定 53
2.6.2.2 總抗氧化能力測定 54
2.6.2.3 還原能力測定 54
2.6.2.4 一氧化氮NO自由基抑制能力測定 54
2.6.3 細胞培養 55
2.6.3.1 細胞繼代培養 55
2.6.3.2 細胞解凍 55
2.6.3.3 細胞冷凍保存 56
2.6.3.4 細胞濃度計數 56
2.6.4 阻斷黑色素之美白活性試驗 57
2.6.4.1 抑制酪胺酸酶活性 57
2.6.4.2 抑制黑色素細胞生成黑色素活性試驗 58
2.6.5 細胞活性測定 59
2.6.5.1 細胞存活率評估 59
2.6.5.1.1 Trypan blue染色法 59
2.6.5.1.2 MTT分析法 59
2.6.5.2 細胞損傷分析 60
2.6.5.2.1 乳酸去氫酶LDH活性測定 60
2.6.5.3 黑色素瘤細胞(B16-F10)之毒性試驗 61
2.6.5.4 黑色素瘤細胞(B16-F10)對UVB劑量之存活率試驗 61
2.6.5.5 角質細胞(HaCa T)之毒性試驗 61
2.6.5.6 角質細胞(HaCa T)對UVB劑量的損傷試驗 61
2.6.5.7 角質細胞(HaCa T)抗UVB之存活率試驗 62
2.6.5.8 人類表皮癌細胞(A-431)的抗增殖活性 62
2.6.5.9 細胞週期分析 62
2.7 統計分析 63
第三章 結果與討論 64
3.1 成分分析 64
3.1.1茶湯品質之顏色與明亮度 64
3.1.2茶黃質 65
3.1.3茶紅質 65
3.1.4吸收光譜分析 66
3.1.5總多酚類化合物 71
3.1.6總類黃酮 71
3.1.7縮合單寧 71
3.1.8兒茶素類化合物與酚酸含量分析 73
3.2 抗氧化活性分析 79
3.2.1 DPPH自由基清除能力 79
3.2.2 總抗氧化能力 79
3.2.3 還原能力 79
3.2.4 一氧化氮(NO)自由基清除能力 80
3.3美白活性試驗 82
3.3.1 抑制酪胺酸酶活性 82
3.3.2 不同茶葉萃取物對小鼠黑色素瘤細胞(B16-F10)之細胞毒性試驗 82
3.3.3 不同UVB劑量對小鼠黑色素瘤細胞(B16-F10)之細胞毒性試驗 83
3.3.3 抑制小鼠黑色素瘤細胞(B16-F10)生成黑色素能力 83
3.4細胞存活率試驗 86
3.4.1 不同茶葉萃取物對人類角質細胞(HaCaT)之細胞毒性試驗 86
3.4.2 不同 UVB 劑量對人類角質細胞(HaCaT)之細胞毒性試驗 86
3.4.3 不同 UVB 劑量對人類角質細胞(HaCaT)之損傷試驗 86
3.4.4 人類角質細胞(HaCaT)抗UVB之防護能力試驗 89
3.4.5 不同茶葉萃取物對人類表皮癌細胞(A-431)的抗增殖活性試驗 90
3.5 細胞週期分析 92
第四章 結論 94
參考文獻 95

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