臺灣博碩士論文加值系統

已知洛神花萼萃取物含有許多天然的活性物質，包括：多酚類、花青素、原兒茶酸及類黃酮等，多數具有抗氧化活性功能，本研究推測可能具有抑制酪胺酸酶的活性，可以應用於美白產品。此外，亦有多數的研究指出，某些特殊植物，例如：黑米、稻米、大麥與紅薯等，經酵母菌發酵後，其發酵物具有抑制酪氨酸酶活性及黑色素生成之功能。而本研究則特定選擇乳酸克鲁维酵母菌（Kluyveromyces lactis），進行洛神花萼萃取物發酵，並將發酵後的水萃取物與酒精萃取物，加入B16F10黑色素瘤細胞內培養48 hrs，測試其抑制酪氨酸酶及黑色素生成之效果，探討洛神花萼發酵萃取物是否具有美白的加成功效。
本研究將乾燥的洛神花萼粉末溶於YPD（yeast extraction, peptone and dextrose）培養液中，並進行乳酸克鲁维酵母菌發酵。於發酵24 hrs 後，分別以蒸餾水及95 % 酒精進行萃取，此外也將未發酵的蒸餾水萃取物及未發酵的酒精萃取物進行之比較。首先，以濃度100、200及400 µg/mL的萃取物進行B16F10黑色素瘤細胞之MTT（3-( 4,5-Dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide, 101-298-93-1, MD, Bio, U.S.A）細胞存活率分析，結果顯示：400 µg/mL之濃度會顯著降低細胞存活率，（p < 0.001, vs. 正常組），故往後黑色素含量及酪胺酸酶活性的試驗，皆採用100及200 µg/mL 的濃度進行分析。以促黑激素（α-melanocyte-stimulating hormone, α-MSH）刺激B16F10之黑色素生成，作為處理對照組進行比較，結果顯示：100 µg/mL 及200 µg/mL 的洛神花萼發酵酒精萃取物對於抑制黑色素生成之效果，顯著高於發酵水萃取物與未發酵者 （p < 0.001），其黑色素含量分別降低至52 ± 4 % 及42 ± 7 %（p < 0.001）；酪胺酸酶活性則分別降低至66 ± 8 % 及59 ± 5 %（p < 0.001），顯示具有顯著美白效果。接著以qPCR（Real-time polymerase chain reaction）檢測洛神花萼發酵酒精萃取物，對於抑制黑色素生成相關基因表現量之影響：MITF（Microphthalmia-associated transcription factor）基因表現量分別降低至0.51 ± 0.09及0.35 ± 0.08（p < 0.001）；tyrosinase 基因表現量分別降低至0.78 ± 0.12及0.56 ± 0.17（p < 0.001）；tyrosinase-related protein 1 （TRP-1） 基因表現量分別降低至0.85 ± 0.1及0.73 ± 0.11（p < 0.01）；tyrosinase-related protein 2 （TRP-2）基因表現量分別降低至0.66 ± 0.24及0.55 ± 0.18（p < 0.01）。此外，亦利用DCFDA法檢測洛神花萼發酵酒精萃取物，是否具有抑制B16F10黑色素瘤細胞之ROS（Reactive oxygen species）之功效，結果顯示：細胞內的ROS則分別降低至35.5 ± 5.1 % 及28.9 ± 3.4 %（p < 0.001），證實具有抑制ROS之效果。總言之，本研究發現洛神花萼發酵酒精萃取物具有抑制ROS與黑色素生成以及其相關基因之功能，推測具有應用於美容保健市場之潛力。

Previous studies have suggested that the extract of roselle calyx contains plenty of natural active substances, including polyphenols, anthocyanins, protocatechuic acid, and flavonoids with the antioxidant activity. We considered that it might inhibit the activity of tyrosinase, able to be a kind of skin-whitening product. In addition, there were some reports detecting about ferments for skin whitening, such as black rice, rice, barley, sweet potatoes of yeast fermentation. These ferments have been testified that they were able to reduce tyrosinase activity and melanogenesis in B16F10 melanoma cells. Therefore, in this study, we selected Kluyveromyces lactis (K. lactis) to ferment with roselle calyx and supposed that the ferment extract could be more powerful to inhibit the tyrosinase activity. The ferment was extracted by distilled water or 95 % ethanol, and then treated with B16F10 melanoma cells for 48 hrs, to assess the inhibition of tyrosinase activity and melanin synthesis.
At first, the concentrations of 100, 200 and 400 μg/mL of dried ferment extract were used to test the cell viability by MTT assay. The data showed the cell viability reduced significantly on the 400 μg/mL. It indicated that the appropriated concentrations to test the following experiments were 100 and 200 μg/mL.
To compare the concentrations of 100 and 200 μg/mL with fermentation for 24 hrs, we extracted the material by water firstly. Melanin content decreased to 52 ± 4% and 42 ± 7%, p <0.001; and activity of tyrosinase decreased to 66 ± 8% and 59 ± 5% respectively, ,p <0.001, with a significant skin-whitening effect. Continuously, followed by quantitative polymerase chain reaction (qPCR), we investigated the melanin-related genes. The amount of microphthalmia - associated transcription factor (MITF) gene expression was reduced to 0.51 ± 0.09 and 0.35 ± 0.08, p <0.001; tyrosinase gene expression was decreased to 0.78 ± 0.12 and 0.56 ± 0.17, p <0.001; tyrosinase-related protein 1 (TRP-1) gene expression was reduced to 0.85 ± 0.1 and 0.73 ± 0.11, p <0.01; tyrosinase-related protein 2 ( TRP-2) gene expression was reduced to 0.66 ± 0.24 and 0.55 ± 0.18 respectively, p <0.01. In addition, we also used DCFDA to analyze the antioxidant effect of the ferments to suppress amount of reactive oxygen species (ROS) on the B16F10 melanoma cell. The intracellular ROS reduced to 35.5 ± 5.1% and 28.9 ± 3.4% respectively, p <0.001. Based on the results, it suggested that the ethanol extract of roselle calyx ferment has the good activity to inhibit ROS, melanin synthesis, and melanin-relative gene expressions, which has potential to develop a novel whitening healthy product.

目錄

中文摘要 I
Abstract II
目錄 IV
壹、緒論 1
貳、文獻探討 2
一、洛神花 2
（一） 洛神花之簡介 2
（二） 洛神花之產地 2
（三） 洛神花之型態 2
（四） 洛神花萼之成分及功效 2
二、酵母菌 5
（一） 酵母菌之特性 5
（二） 酵母菌之分類 5
（三） 酵母菌之功能 5
（四） 乳酸克鲁维酵母菌之功能 5
三、發酵 6
（一） 發酵過程 6
（二） 發酵物中可能的功能性成分 6
四、黑色素細胞 8
（一） 黑色素之種類 8
（二） 黑色素形成之機轉 8
（三） 黑色素之形成與轉移 11
（四） 抑制黑色素形成之機轉 12
參、材料與方法 15
一、 實驗細胞株來源及培養基配製 15
二、 DMEM培養基之配製 15
三、 細胞培養與繼代 15
四、 細胞數之計數 15
五、 冷凍細胞 16
六、 解凍細胞 16
七、 YPD 培養基與酵母菌之備製 16
八、 洛神花萼萃取物之備製 16
九、 實驗架構及流程 18
十、 細胞存活率分析 19
十一、細胞內黑色素含量分析 20
十二、細胞內酪胺酸酶活性分析 21
十三、黑色素細胞拍照 22
十四、細胞內ROS檢測 23
十五、基因表現量檢測 qPCR 24
十六、數據統計 26
肆、結果 27
伍、討論 31
參考文獻 34

 
圖目錄

圖 1. 洛神花之外觀型態 4
圖 2. 黑色素生成機轉圖 10
圖 3. 黑色素之形成與轉移 11
圖 4. 熊果素抑制黑色素生成機轉圖 12
圖 5. 本研究之實驗大綱 18
圖 6. 本研究之細胞存活率分析流程圖 19
圖 7. 本研究之細胞內黑色素含量分析流程圖 20
圖 8. 本研究測試細胞內酪胺酸酶活性之試驗流程圖 21
圖 9. 本研究測試黑色素細胞拍照試驗流程圖 22
圖 10. 本研究之ROS實驗流程圖 23
圖 11. 本研究之qPCR 實驗流程圖 25
圖 12. 洛神花萼水萃取物對B16F10黑色素瘤細胞之細胞存活率分析 43
圖 13. 洛神花萼酒精萃取物對B16F10黑色素瘤細胞之細胞存活率分析 44
圖 14. 酒精影響B16F10黑色素瘤細胞之細胞存活率分析 45
圖 15. 洛神花萼萃取物影響B16F10黑色素瘤細胞之黑色素含量分析 46
圖 16. 酒精影響B16F10黑色素瘤細胞之黑色素含量分析 47
圖 17. 洛神花萼發酵酒精萃取物抑制B16F10黑色素瘤細胞之酪胺酸酶活性 48
圖 18. 洛神花萼發酵酒精萃取物對B16F10黑色素瘤細胞之黑色素拍照 49
圖 19. 洛神花萼發酵酒精萃取物抑制B16F10黑色素瘤細胞之ROS生成 50
圖 20. 洛神花萼發酵酒精萃取物抑制人類骨髓幹細胞之ROS生成 52
圖 21. 洛神花萼發酵酒精萃取物抑制mRNA表現量 54
圖 22. 洛神花萼發酵酒精萃取物對抑制B16F10黑色素瘤細胞之作用機制 33


表目錄

表1. 衛生福利部核定之美白成分 13
表2. 基因表現量（qPCR）之引子設計表 25

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