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研究生:孫慧慈
研究生(外文):Hui-Tzu Sun
論文名稱:蜂王漿美白功效成份分析與研究
論文名稱(外文):Study on the Bioactive Components Identified from Royal Jelly and Their Whitening Effects
指導教授:彭及忠
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:79
中文關鍵詞:蜂王漿蜜蜂美白黑色素瘤細胞酪胺酸酶
外文關鍵詞:Royal jellyhoney beewhiteningmelanoma cellstyrosinase
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  • 被引用被引用:1
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蜂王漿是由年輕蜜蜂工蜂的下咽頭腺及大顎腺所分泌的黏稠狀乳白色液
體,為蜂王一生主要的食物來源,目前已被證實的藥理活性包含:抗氧化、抗菌、
抑制血管新生達到抗癌效果、能舒緩血管達到降血壓的功能還有降膽固醇等功
效,因此蜂王漿被視為養生藥品,在功能性食品及商業醫療產品上廣泛的被使
用,近年更發現蜂王漿具有美白淡斑的能力,因此現今各大藥妝品牌都紛紛添加
蜂王漿來替他們的保養產品提高其附加價值。為了釐清蜂王漿那一成分促使其具
有美白的效果,本研究利用甲醇來萃取蜂王漿,再將甲醇萃取物經管柱層析純化
分離,從中分離出10 個分離樣品,樣品進一步進行體外酪胺酸酶抑制活性測
試,發現第6 個樣品的活性最佳,藉用NMR 與GC-MS 分析鑑定,確定讓蜂王
漿具有抑制酪胺酸酶活性的成份主要為: trans-10-hydroxy-2-decenoic
acid(10-HDA)。10-HDA 是蜂王漿脂肪酸中的主要成份,為蜂王漿特有的化合物,
已有文獻指出其具有抗菌、抗癌、增進膠原蛋白增生等藥理活性,但對於美白方
面之研究則少有這方面的探討,為深入瞭解10-HDA 美白功效的機制,我們利用
B16F10 黑色素瘤細胞來探討10-HDA 對於細胞內酪胺酸酶及黑色素含量的變
化,和西方墨點法觀察培養後細胞中與黑色素生成相關蛋白質的變化量,由結果
得知10-HDA 可以減少酪胺酸酶及黑色素含量並且抑制黑色素生成相關蛋白質:
Microphthalmia-associated transcription factor (MITF)、tyrosinase、TRP-1(tyrosinase
relates protein 1)與TRP-2(dopachrome tautomerase)的表現量,因此確定10-HDA
是促使蜂王漿具有美白能力的活性成份。

Royal jelly (RJ) is produced by the hypopharingeal and mandibular glands of the young worker honey bees . It is the major food source of the queen honey bee . RJ has been confirmed to showed many functional properties such us anti-oxidant,anti-bacterial, anti-angiogenesis, anti-tumor, vasodilative, hypertension and anti-hypercholesterolemic effects .It has been widely used in healthy foods and
commercial medical products in many countries. Recently, some researches have found that RJ has depigmenting effect, therefore nowadays many cosmetic products have added RJ to improve the value of those products. However, until now the
ingredient of the RJ that have whitening effect still unknown . We extracted the whitening component from royal jelly by methanol , and then separated it by column
chromatography. To make sure which component have whitening effect , samples tested in in vitro tyrosinase inhibitory activity assay, and then identified is as
trans-10-hydroxy-2-decenoic acid (10-HDA) by MMR and GC-MS. 10-HDA, a major fatty acid ingredient of royal jelly, is a special active compound which exists only in royal jelly in the nature. It has many pharmacological activities, such as anti-bacterial, anti-cancer and collagen production promoting activity, but less studies about 10-HDA whitening effect. To study the mechanism of the 10-HDA whitening effect we used B16F10 melanoma cells co-culture with 10-HDA to observe the changes inintracellular tyrosinase and melanin content , and Western blot assay observed the
variation of the cells of melanin production-related proteins, by the results we found 10-HDA not only can reduced tyrosinase and melanin content , also inhibit the
expression of melanogenesis enzyme group such us Microphthalmia-associated transcription factor (MITF), tyrosinase, TRP-1(tyrosinase relates protein 1), and
TRP-2(dopachrome tautomerase). Therefore, we infer the 10-HDA is the active component of royal jelly has whitening ability.

摘要 ……………………………………………………………………….……. i
Abstract …………………………………………………………………….…… ii
誌謝 ……………………………………………………………………..……… iv
目錄 ……………………………………………………………………..……… v
表目錄 ……………………………………………………………………..…… viii
圖目錄 …………………………………………………………………..……… ix
第一章緒論……………………………………………………………..……… 1
第二章文獻回顧………………………………………………………..……… 2
2.1 人體皮膚組成與黑色素生成機制…………………………………..……… 2
2.1.1 人體皮膚……………………………………………………………..….… 2
2.2 黑色素……………………………………………………………………..… 3
2.2.1 黑色素細胞…………………………………………………………….….. 3
2.2.2 黑色素生成途徑……………………………………………………...…… 3
2.3 酪胺酸酶……………………………………………………...…….……..… 6
2.4 美白機轉與美白成份的使用及限制…………………………………..…… 6
2.4.1 美白成份作用機制……………………………………………….….……. 6
2.4.2 美白成份介紹…………………………………………………………...…. 7
2.5 蜂王漿……………………………………………………………………..… 10
2.5.1 蜂王漿之簡介…………………………………………………………..…. 10
2.5.2 蜂王漿生理與藥理活性……………………………………………......… 12
2.5.3 蜂王漿已知化合物……………………………………………….…....…. 13
2.6 研究目的……………………………………………………...…….…......… 18
2.7 專利檢索……………………………………………………...…….……..… 18
2.7.1 細胞存活率試驗方法之相關專利………………………………......…… 19
2.7.2 ELISA 方法之相關專利……………………………………………......… 20
2.7.3 美白產品與成份之相關專利……………………………….…..…….….. 21
2.7.4 美白試驗之相關專利……………………………….…..………….....….. 22
2.7.5 葵烯酸(10-HDA)之相關專利……………………………….…..…….….. 22
第三章實驗方法與材料……………………………….…..………….....…….. 23
3.1 實驗架構……………………………………………………...…….……..… 23
3.2 蜂王漿之美白活性成份分離與純化…………………………..…….…..… 24
3.2.1 實驗使用之儀器與藥品……………………………….…..………….…... 24
3.2.2 蜂王漿之美白活性成分之萃取與分離……………………………....…... 24
3.3 高壓液相層析儀(HPLC)分析……………………………….......……....…. 25
3.3.1 實驗使用之儀器與藥品……………………………….…..……...….….... 25
3.3.2 分析定量10-HDA 含量……………………………….…..…….....…...... 25
3.4 蘑菇酪胺酸酶抑制活性之分析……………………………….…..…….…. 25
3.5 B16F10 黑色素瘤細胞細胞株之培養…………………………….….…....... 26
3.5.1 細胞株來源…………………………………………………………....….. 26
3.5.2 細胞株培養…………………………………………………………....….. 26
3.6 B16F10 黑色素瘤細胞存活率之分析………………………………....….. 26
3.7 蛋白質含量之測定………………………………………………….…........ 27
3.8 B16F10 黑色素瘤細胞內酪胺酸酶活性之分析……………………............. 27
3.9 B16F10 黑色素瘤細胞內黑色素含量之分析………………………….....… 28
3.10 西方墨點法分析……………………………………………….….…......... 28
3.10.1 蛋白質電泳分析(SDS-PAGE) ……………………………….….…....... 28
3.10.2 西方墨點法測試探討10-HDA 美白機制……………………………... 29
第四章結果……………………………………………………………..……… 31
4.1 蜂王漿萃取物分離純化……………………………………………….....… 31
4.1.1 蜂王漿萃取物分離純化…………………………………………...…...… 31
4.1.2 以蘑菇酪胺酸酶活性抑制測試進行美白篩選平台………….……........ 31
4.2 利用B16F10 黑色素瘤細胞探討10-HDA 美白機制………….……....... 32
4.2.1 細胞存活率之分析………………………………..……….……............... 32
4.2.2 B16-F10 黑色素瘤細胞內黑色素含量之分析………………………...… 32
4.2.3 B16-F10 黑色素瘤細胞內酪胺酸酶含量之分析……………………...… 33
4.2.4 10-HDA 蘑菇酪胺酸酶活性抑制測試………………………………..…. 33
4.2.5 利用西方墨點法測試探討10-HDA 美白機制…………………….....… 34
第五章討論……………………………………………………………..…...… 35
第六章結論及未來展望………………………………..……….…….............. 37
6.1 結論……………………………………………………………..………...... 37
6.2 未來展望……………………………………………………………..…...… 37
參考文獻……………………………………………………………..……...…... 38
英文論文大綱……………………………………………………………..…….. 55
個人簡歷………………….…………….……….……………...……........…….. 74

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