臺灣博碩士論文加值系統

紫外線 (Ultraviolet irradiation) 對於皮膚所引起的傷害會造成黑色素 (Melanin) 的產生形成色素沉著 (Pigmentation)，嚴重可能造黑色素瘤 (Melanoma) 的誘發；長期的紫外線照射還會導致自由基 (Free radical) 生成造成皮膚老化現象。在台灣每年有可觀的蓮霧 (Syzygium samaragense) 產量，果農為維持蓮霧品質會進行蔬果，將過多的蓮霧花 (Wax apple flowers) 摘下丟棄。若能開發出蓮霧花的新功效，即能將廢棄之蓮霧花再利用，更能提升蓮霧果樹在台灣的經濟地位。有研究指出蓮霧本身具抗氧化能力，本研究室也證實，蓮霧花本身亦具有強抗氧化力。再者針對促進膠原蛋白增生及抗老化的研究，是目前保建開發關注的主流。因此本研究採用蓮霧蔬果時捨棄的花，利用乙醇萃取獲得 AEW (Alcohol extract of wax apple flowers)，評估美白功能性以及紫外線減少膠原蛋白 (Collagen) 的影響。本研究首先進行體外試管酪胺酸酶抑制率測定 (In tube tyrosinase inhibition assay)，再以小鼠黑色素瘤細胞 (Murine melanoma B16F10, B16 cell) 模式確定 AEW 對於 B16 cell 之存活率 (MTT viability assay)。接著測定黑色素含量分析 (Melanin assay)、細胞內酪胺酸酶活性分析 (Cellular tyrosinase activity assay) 以及 MITF analysis (Microphthalmia-associated transcription factor analysis)。另外以人類纖維母細胞 (Human foreskin fibroblast cell, Hs68 cell) 模式，以 MTT assay 確立 AEW 對細胞生長之影響。另將 Hs68 cell 以 UVA 照射 (20 J/cm2) 進行西方墨點法 (Western blot analysis) 及明膠蛋白酵素電泳法 (Gelatin Zymography)，觀察 type І procollagen、MMP-1 蛋白表現量及 MMP-2 之活性。並檢測 MDA、NFκB (Nuclear factor κB) 和 TIMP-1 (Tissue inhibitor of metalloproteinases 1) 含量。實驗結果顯示5 μg/mL AEW 有良好的酪胺酸酶抑制率且對於 B16 cell 無細胞毒性。經300 nM α-MSH 刺激後顯著增加2.8倍之黑色素含量。當 AEW 提高至 50 μg/mL 時可顯著降低 69.4% 黑色素，且細胞內酪胺酸酶抑制活性可達10.9%。結果中亦發現 50 μg/mL AEW 對於 Hs68 cell 並無細胞毒性。將 Hs68 cell 經 20 J/cm2 UVA 照射後與控制組相比可顯著降低 85.0% type І procollagen 蛋白質含量，且促進 1.2 倍之 MMP-1 蛋白及 2.7 倍 MMP-2 活性，並產生 1.3 倍之 MDA。將經 UVA 照射的 Hs68 cell 加入 50 μg/mL AEW 可顯著地增加 7.3 倍 type І procollagen 含量，並抑制 MMP-1 (91.9%) 和 MMP-2 (91.2%)，同時可降低 27.5% MDA 產量。因此由以上結果可知，AEW 的添加可有效降低黑色素的含量可能是經由細胞內對於酪胺酸酶活性的抑制；對於 UVA 的傷害可有效保護 type І procollagen 不被降解可能是透過抑制 MMP-1 的表現，降低 MMP-2 的活性和 MDA 的產生所致。我們研究中得知，AEW 能有效降低黑色素的生成，對於經 UVA 所造成的傷害，AEW 可能經抑制 MDA (降低活性氧生成) 進而使 MMP-1 和 MMP-2 降低，最終防止膠原蛋白被分解。

Ultraviolet (UV) irradiation may induce skin injury, and pigmentation via melanin production, and it can induce melanoma seriously. UV irradiation may cause skin aging and free radicals formation. In Taiwan, there are a lot of harvests in wax apple (Syzygium samaragense) every year. In order to keep the wax apple quality, it is necessary to controlling the amount of wax apple flowers by getting rid of excess flowers. Hence, studying and creating the new special effect with recycling these excess flowers will be helpful to raise the economic value of wax apple. Studies indicate that wax apple has potent effect on anti-oxidation, and our previous results also reveal the anti-oxidative ability in wax apple flowers. Additionally, investigating on collage increment and anti-aging is the main concerned choice in development of health care, including cosmetic field. Therefore, the present study evaluates the effects of recycling excess wax apple flowers on whitening and the UV-induced collagen degeneration function. Firstly, the study observed the tyrosinase inhibition of wax apple flowers after ethanol extract (AEW) in tube experiment. We examined MTT viability assay by using murine melanoma B16F10 cells (B16 cells). Consequently, cellular tyrosinase activity, melanin content assay, and microphthalmia-associated transcription factor analysis (MITF analysis) were also monitored. In addition, we used human foreskin fibroblast cells (Hs68 cells) to not only obtain the MTT viability of AEW, but also determined type І procollagen, MMP-1 and MMP-2 activity by western blot analysis and gelatin zymography methods with or without 20 J/cm2 UVA irradiation in Hs68 cells. We continuously tried to explore the undergoing mechanism, and then examined MDA, nuclear factor κB (NFκB) and tissue inhibitor of metalloproteinases 1 (TIMP-1) expression in Hs68 cells experiments. The present results exhibit that 5 μg/mL AEW display the better tyrosinase inhibitory rate and no toxicity for B16 cell, and that can increase melanin content 2.8 times after 300 nm α-MSH treatment. AEW 50 μg/mL treatment, almost no toxicity in Hs68 cells model, can apparently reduce 69.4% melanin content and attain to 10.9% for cellular tyrosinase inhibitory rate. Type І procollagen protein content clearly decreases 85.0% than controls after 20 J/cm2 UVA irradiation exposure, but enhances MMP-1 expression to 1.2 times, MMP-2 activity to 2.7 times, and MDA to 1.3 times than controls. However, 50 μg/mL AEW treatment in Hs68cells after 20 J/cm2 UVA irradiation exposure can obviously increases type І procollagen content to 7.3 times, but suppresses 91.9% of MMP-1, 91.2% of MMP-2 and 27.5% of MDA. According to our present results, we demonstrate that AEW can significantly decreases melanin content, and may have effective on inhibiting cellular tyrosinase activity. Treatment with AEW may prevent type І procollagen from UVA- induced damage, and probably diminish the MMP-1 expression, MMP-2 activity, and MDA formation. Hence, we speculate that extracts of wax apple flowers can reduce melanin produce effectively, and against UVA-induced collagen degeneration through alleviating MDA production (reduce ROS formation), MMP-1 and MMP-2 displays.

中文摘要 Ⅰ
英文摘要 Ⅳ
誌謝 Ⅵ
目錄 Ⅷ
表目錄 Ⅸ
圖目錄 Ⅹ

第壹章　前言

第一節　皮膚結構 1
第二節　紫外線介紹 4
第三節　黑色素之生合成路徑 6
第四節　紫外線對皮膚之傷害 8
第五節　蓮霧簡介 12
第六節　蓮霧花簡介 16
第七節　研究目的 17

第貳章、材料與方法

第一節　實驗材料 20
第二節　蓮霧花酒精萃取 25
第三節　蛋白質含量測定 26
第四節　細胞培養 26
第五節　細胞存活率分析 28
第六節　黑色素含量測定 29
第七節　UVA 照射處理 29
第八節　西方墨點法 29
第九節　明膠蛋白酵素電泳法 31
第十節　脂質過氧化測定 32
第十一節　細胞內酪胺酸酶抑制率 32
第十二節　MITF, TIMP-1 and NFκB analysis 32
第十三節　統計分析 33

第叄章　結果

第一節　AEW 於 B16 細胞模式中美白之相關評估 34
第二節　AEW 於 Hs68 細胞模式中之膠原蛋白及相關代謝探討 36

第肆章　討論

第一節　美白評估 39
第二節　抗紫外線 A 傷害之影響及其相關路徑 41

第伍章　結論

第一節　美白評估 43
第二節　膠原蛋白及其相關路徑 44

第陸章　參考文獻 45

附錄表圖 51

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