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研究生: 黃育亭
Yu-Ting Huang
論文名稱: 山苦瓜萃取物抑制黑色素生成及抗光老化效應的評估
Evaulation of anti-melanogenic and anti-photoaging activity of wild bitter melon extracts
指導教授: 蔡帛蓉
Tsai, Po-Jung
學位類別: 碩士
Master
系所名稱: 人類發展與家庭學系
Department of Human Development and Family Studies
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 153
中文關鍵詞: 山苦瓜紫外線MMP-1黑色素生成
英文關鍵詞: wild bitter melon, UV irradiation, MMP expression, melanogensis
論文種類: 學術論文
相關次數: 點閱:1033下載:48
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  • 黑色素可保護皮膚免於紫外線傷害,但是過多的黑色素生成會造成黑色素沉澱形成色素斑塊。黑色素生合成主要受到酪胺酸酶(tyrosinase)調控。荷爾蒙、壓力和紫外線照射...等因素會促進黑色素生成。光老化(photoaging)為長期暴露在紫外線下造成皮膚提早老化的現象。紫外線誘導reactive oxygen species (ROS)的產生會提高皮膚中的氧化壓力、增加matrix metalloproteinases (MMP-1, collagenases)表現而降低皮膚中type I procollagen含量,亦促進cyclooxygenase (COX), PGE2和 nitric oxide (NO)等生成誘導發炎反應,導致皮膚乾燥形成皺紋。
    苦瓜(bitter melon)或山苦瓜(wild bitter melon)的果實不僅當成蔬菜食用,也被認為具有抗菌、抗病毒、抗腫瘤、抗胃潰瘍、抗瘧、抗發炎及抗氧化等活性。在東南亞,苦瓜的葉子被用來煮汁治療皮膚病。
    本研究以in vitro細胞模式評估台灣山苦瓜萃取物對於黑色素生成以及光老化的影響,再以in vivo人體試驗評估山苦瓜萃取物是否具有皮膚保健功效。
    研究結果顯示,山苦瓜葉子甲醇萃取物具有良好清除自由基(DPPH, NO, superoxide, and hydroxyl radical)的能力。於in vitro細胞實驗模式中,山苦瓜葉子甲醇萃取物與果實乙酸乙酯萃取物可顯著抑制α-MSH刺激之B16-F10 melanoma細胞的黑色素生成與tyrosinase活性。另觀察UVB誘導人類角質細胞株(HaCaT)的ROS, MMP-1, and COX-2表現,山苦瓜果實萃取物(乙酸乙酯、乙醇、正己烷萃取物)、葉子(甲醇萃取物)或藤(甲醇萃取物)皆可以抑制UVB誘導HaCaT細胞的MMP-1與COX-2 mRNA表現。花蓮1號山苦瓜葉子甲醇萃取物與果實乙酸乙酯萃取物可以抑制UVB誘導HaCaT cells產生的ROS生成和MMP-1與COX-2 之mRNA與蛋白質表現。進一步探討其抑制作用機轉,發現UVB會顯著促進HaCaT cells ERK與JNK的磷酸化,而花蓮1號山苦瓜葉子甲醇萃取物與果實乙酸乙酯萃取物則能抑制此ERK磷酸化情形,故推測山苦瓜萃物經由抑制ROS生成、ERK的磷酸化而降低MMP-1表現。綜合細胞實驗結果,推測山苦瓜萃物應具有抑制黑色素生成與預防光老化的潛力。
    另外招募29名受試者進行人體試驗,結果發現山苦瓜萃取物不會產生皮膚過敏或不適等症狀,具良好的外用安全性。而且塗抹含山苦瓜萃物乳霜於第8週、第12週均顯著改善斑點與細紋,與細胞實驗結果相符,故本研究已初步證實山苦瓜萃取物的皮膚保健功效。

    Tyrosinase is a key enzyme in melanogenesis. Melanin is essential for protecting human skin against radiation, but the accumulation of abnormal melanin induces pigmentation disorders. UV radiation is responsible as a causative factor for various skin lesions including photoaging and photocarcinogenesis. Photoaging is characterized by severe wrinkling and pigmentary changes. In addition, UV induced MMP-1and then caused collagen breakdown and induced inflammatory responses.
    Bitter melon (Momordica charantia) extracts exerted hypoglycemic, anti-diabetic, antiviral, antitumor, antiulcer, anti-leukemic, antibacterial, anti-mutagenic, anti-inflammatory, and antioxidant properties. In Asia, the infusion of stems and leaves of bitter melon has been proposed as an agent for skin care.
    In this study, we investigated the anti-melanogenic and anti-photoaging properties of wild bitter melons (WBM; Momordica charantia Linn. var. abbreviata Ser.). Results showed that methanolic (MeOH) extracts of WBM leaves showed significant DPPH free radical, superoxide, nitric oxide and hydroxyl radical scavenging activity. Ethyl acetate (EA) and ethanolic (EtOH) extracts of fruits, MeOH extracts of leaves and viny juice of WBM significantly inhibited mushroom tyrosinase activity. EA fruit extracts and MeOH leaf extracts of WBM significantly suppressed tyrosinase activity and decreased melanin levels in α-MSH-stimulated B16-F10 melanocytes. EA fruit extract and MeOH leaf extract of WBM, Hualien-1 (HL-1), significantly reduced UVB-induced intracellular ROS production and inhibited mRNA and protein levels of COX-2 and MMP-1. Besides, both extract significantly inhibited UVB-enhanced phosphorylation of ERK and JNK. Our findings suggested that both HL-1 extracts inhibited UVB-induced MMP-1 expression, which may be through reduced ROS production and ERK phosphorylation.
    Twenty-nine subjects were enrolled in a randomized, double-blind, placebo-controlled, split-face comparative study. HL-1 extracts was found to be topical safe and well tolerated. A gradual and time-related improvement in pigmentary abnormality and wrinkle was shown under the treatment with HL-1 extracts. HL-1 extracts significantly reduced spot and wrinkle after 8-wk and 12-wk treatment. Our study herein demonstrated the skin care potential of wild bitter melon.

    摘要 ................................................ i Abstract ...........................................ii 目錄 .............................................. iii 圖目錄 ......................................... vi 表目錄 .............................................. viii 第一章 緒論 ................................................ 1 一、研究動機 ............................................... 1 二、研究目的 .............................................. 2 第二章 文獻探討 ............................................. 3 第一節 皮膚結構 .................................... 3 一、表皮(epidermis) ................................. 4 二、真皮(dermis) ......................................... 4 第二節人類皮膚色素沉積(human skin pigmentation)..............6 一、黑色素(melanin) ................................. 6 二、黑色素生成(melanogenesis) ................... 6 三、黑色素沉積的治療 ................................... 11 第三節 皮膚老化 ...................................12 一、光老化(photoaging)..........................14 二、光老化的治療 .........................................21 第四節 營養與皮膚老化.................................................23 第五節 實驗材料-山苦瓜 ....................................25 第三章 材料與方法 ..........................27 第一節 細胞實驗(in vitro study) ...................27 一、實驗架構 ..............................27 二、實驗試劑 ......................................28 三、實驗設備 ............................................35 四、實驗方法 ............................36 (一) 樣品製備 ......................36 (二) 抗氧化能力評估 ..............................38 (三) 細胞培養...................................41 (四) 細胞存活率分析(MTT assay)..................42 (五) 抑制黑色素生成能力評估 ...........................43 (六) 抗光老化能力評估 ...............................45 (七) 山苦瓜成份分析-高效液相層析儀(HPLC) .......50 五、統計分析 ........................................50 第二節 人體試驗(in vivo study)..............51 一、實驗架構 .............................................51 二、材料與方法 .............................52 (一) 乳霜製備與安定性評估 ...............................52 (二) 招募受試者 ...................................55 (三) 評估方式與項目 ..................................56 三、統計分析 .....................................57 第四章 結果 .................................58 第一節 山苦瓜萃取物抗氧化能力評估 .........................58 一、山苦瓜萃取物清除DPPH之能力 ...............................58 二、山苦瓜萃取物清除NO之能力 ................................60 三、山苦瓜萃取物清除superoxide之能力 .....................62 四、山苦瓜萃取物清除OH-之能力 .............................64 五、山苦瓜萃取物之細胞保護功效 ..............................67 第二節 細胞實驗 .........................................69 一、樣品對細胞存活的影響 ............................69 二、抗黑色素生成與抗老化能力評估 ............................69 (一) 蘑菇酪胺酸酶(mushroom tyrosinase)活性抑制測定 ...........69 (二) 黑色素瘤細胞(B16-F0)之黑色素含量測定 ..................71 (三) B16-F10 cells之酪胺酸酶活性與黑色素含量測定 ............73 三、評估山苦瓜萃取物的抗光老化能力 ........................77 (一) 急性模式(高UV劑量、短暴露時間)-抗光老化能力評估 .....77 (二) 模擬皮膚慢性(低UV劑量、長暴露時間)光老化-抗光老化能力評估 .....80 (三) UVB誘導ROS生成 ..........................86 (四) 山苦瓜對於UVB-induced ERK and JNK磷酸化之影響 ........88 四、 山苦瓜的成份分析 ...........................91 第二節 人體試驗 ....................................94 一、乳霜製備與安定性試驗 .................................94 (一) 乳霜的小量製備 ........................................94 (二) 乳霜安定性試驗 ....................................96 (三) 大量乳霜製備與安定性試驗 .................................99 二、人體試驗進行 .....................................99 第五章 討論 ......................................... 108 第一節 細胞實驗 .................................... 108 一、山苦瓜萃取物的抗氧化能力與多酚(polyphenol)組成 ........... 109 二、山苦瓜萃取物抑制黑色素生成功效評估 ..................... 110 三、山苦瓜萃取物的光保護效應............................. 115 第二節 人體試驗 ................................... 121 第三節 結論 ........................................ 123 第六章 參考文獻 ....................................... 124 附錄一 招募文宣 ........................................... 134 附錄二 受試者同意書 ................................... 135 附錄三 Baseline Demodraphics ....................... 141 附錄四 Adverse Reaction Report Form ................ 142 附錄五 Initial Evalation Form (baseline) ......... 144 附錄六 Initial Evalation Form (4 weeks) ..... 145 附錄七 Initial Evalation Form (8 weeks) ........... 146 附錄八 Initial Evalation Form (12 weeks) ............ 147 附錄九 Subject’s Static Global Assessment .......... 148 附錄十 Subject Questionnaire ..................... 149 附錄十一 乳霜微生物檢驗報告 .......................... 151 附錄十二 人體委員會審核通過證明函 .................... 153

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