The fruit of mountain pepper （Litsea cubeba） is hailed by the aborigines as "the black pearl in the forest", and was called the " Makao " in the Atayal language. It was reveled that The essential oil extracted from L. cubeba fruit with good antibacterial effect and It was suitable for developing into a natural preservative for food and cosmetics. The main ingredient of the fruit essential oil of L. cubeba （LCEO） could used as a synthetic raw material for vitamins A, E and K. Vitamin K was used for the prevent the loss of collagen and vitamin E could be used as an antioxidant. LCEO Can be developed into a skin care product without the need for additional spices. In view of the above points, the LCEO has great potential in cosmetic applications. At present, LCEO were extracted from L. cubeba fruits from two different regions in Taiwan and Northern Thailand by water distillation. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT assay） was used to estimate survival rate on mouse B16-F10 melanoma cells and CHO-K1 breast cells after the treatment of LCEO. It was shown less toxic effect on the CHO-K1 cells than the B16-F10 cells. After that, the DPPH test was also shown that LCEO has good antioxidant activity. In the whitening test, the result showed that LCEO can reduce the production of melanin by B16-F10 cell. By Western blot analysis, It was evidenced that the high dosage of LCEO reduced the Melanocyte Inducing Transcription Factor （MITF） content. It indicated that Litsea cubeba essential oil can inhibit the transcription of MITF, reduce the transcription of melanin-producing enzymes, and achieve the whitening effect. After that, the principle of high-pressure injection to fill LCEO into the patch, and Aedes albopictus was used for repellent effect. The result was shown that the LCEO can repellent mosquitoes.
目錄
中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VI
表目錄 VIII
壹、緒論 1
一、研究動機 1
二、研究背景 2
貳、文獻回顧 5
一、山胡椒的功能 5
二、山胡椒精油之成分分析 6
三、山胡椒樹之無性繁殖方法 6
四、人體皮膚黑色素形成的機制 9
五、山胡椒水萃物的功能分析 14
參、研究架構 18
肆、材料與方法 19
一、山胡椒果實精油萃取及成分分析 19
二、動物細胞活性檢測 21
三、山胡椒水萃物之抗氧化美白功效分析 25
四、山胡椒水萃物驅蚊功效測試 30
伍、試驗結果與討論 31
一、台灣及泰北山胡椒果實精油萃取及成分分析 31
二、動物細胞活性檢測 37
三、山胡椒水萃物之抗氧化美白功效分析 40
四、山胡椒水萃物驅蚊功效測試 51
陸、結論 52
柒、參考文獻 53
附錄一、名詞解釋 58


圖目錄
圖 1. 山胡椒樹 2
圖 2. 台灣山胡椒花 2
圖 3. 泰北山胡椒果實 3
圖 4. 乾燥的山胡椒果實 3
圖 5. 山胡椒組織培養種苗繁殖流程圖 8
圖 6. 人體皮膚層結構圖 9
圖 7. 黑色素細胞在表皮和毛髮中的位置 10
圖 8. 黑色素細胞生成成熟黑色素的四階段 11
圖 9. 真黑色素和褐黑素的合成途徑 13
圖 10. 參與黑色素生成調控的主要信號傳遞路徑 14
圖 11. 本論文研究實驗之架構圖 18
圖 12. 精油萃取裝置 19
圖 13. 經過台盼藍染色後B16-F10活細胞呈色情形 23
圖 14. 自製試驗箱成品 30
圖 15. 台灣山胡椒精油GC-MS圖譜 32
圖 16. 泰北山胡椒精油GC-MS圖譜 34
圖 17. 泰北及台灣山胡椒精油GC-MS分析化合物成分含量百分比 35
圖 18. 台灣山胡椒精油及泰北山胡椒精油對B16-F10細胞存活率之影響 37
圖 19. 台灣山胡椒精油及泰北山胡椒精油對CHO-K1細胞存活率之影響 38
圖 20. 台灣山胡椒精油及泰北山胡椒精油自由基清除率 40
圖 21. 台灣山胡椒精油及泰北山胡椒精油對B16-F10黑色素含量之影響 42
圖 22. 台灣山胡椒精油對B16-F10所產生黑色素之影響 43
圖 23. 泰北山胡椒精油對B16-F10所產生黑色素之影響 44
圖 24. BSA檢量線 45
圖 25. 台灣山胡椒精油及泰北山胡椒精油對B16-F10合成TRP-1、TRP-2、MITF之影響 46
圖 26. 台灣山胡椒精油及泰北山胡椒精油對B16-F10合成TRP-1之影響西方墨點法量化圖 47
圖 27. 台灣山胡椒精油及泰北山胡椒精油對B16-F10合成TRP-2之影響西方墨點法量化圖 48
圖 28. 台灣山胡椒精油及泰北山胡椒精油對B16-F10合成MITF之影響西方墨點法量化圖 49
圖 29. 山胡椒精油驅蚊效果之影響 51


表目錄
表 1. 衛福部於2016年公告核准使用的13種美白成分 16
表 2. 8 %分離膠用量配置表 28
表 3. 5 %集集膠用量配置表 28
表 4. 台灣山胡椒精油GC-MS圖譜峰值時間、KI值、化合物成分含量百分比 31
表 5. 泰北山胡椒精油GC-MS圖譜峰值時間、KI值、化合物成分含量百分比 33


柯季宏。（2004）。植物抽出成分之抗病媒蚊活性（Vol. 37）。中華林學季刊，353-365。
李淑真。（2020）。桃園區農業專訊（Vol. 113），9-11。
徐健。（2010）。山蒼子種子繁育和組織培養技術初步研究。（碩士），西南大學，重慶市，中國。
羅英妃。（2016）。種苗科技專訓 No.93。
張伊芸。（2016）。燈光及植物精油對重要吸血雙翅目昆蟲之推-拉防治策略探討：以蚊科及臺灣鋏蠓為例。（碩士），國立台灣大學，台北市，台灣。
Abdulla-Kamil, A. （2014）. Seroprevalence of Herpes Simplex Virus Type 2 （ HSV-2 ） in Pregnant Women and its Relation to Some Blood Cells and IL-2 in Kirkuk , Iraq. Middle East Journal of Internal Medicine, 7（3）, 19-27. Doi:10.5742/MEIM.2014.92551
Adams, Robert. （2005）. Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Carol Stream, 16, 65-120.
Ali, S.A., & Naaz, I. （2015）. Current Challenges in Understanding the Story of Skin Pigmentation — Bridging the Morpho-Anatomical and Functional Aspects of Mammalian Melanocytes. Muscle Cell and Tissue, Doi: 10.5772/60714
Giovanni, B., Roman, P., Rianasoambolanoro, R., Franks, K.N., Angelo, C., Marcello, N., & Filippo, M. （2020）. Insecticidal and Mosquito Repellent Efficacy of the Essential Oils from Stem Bark and Wood of Hazomalania Voyronii. Journal of Ethnopharmacology, 248（112333）. Doi: 10.1016/j.jep.2019.112333
Boruah, N., Borah, S., Sarkar, P., & Sharma, H. （2019）. Chemical Analysis of Litsea Cubeba Essential Oil Harvested from Arunachal Pradesh, India and Estimation of Mosquito Repellent Activity with Reference to Network Analysis. Asian Journal of Pharmaceutical and Clinical Research, 241-244. Doi:10.22159/ajpcr.2019.v12i5.32672
Chung, B.Y., Choi S.R., Moon I.J., Park C.W., Kim, Y.H., & Chang, S.E. （2016）. The Glutathione Derivative, GSH Monoethyl Ester, May Effectively Whiten Skin but GSH Does Not. International Journal of Molecular Sciences, 17（5）, 629. Doi:10.3390/ijms17050629
Cichorek, M., Wachulska, M., & Skoniecka, A. （2013）. Heterogeneity of Neural Crest-Derived Melanocytes. Open Life Sciences, 8（4）, 315 – 330. Doi:10.2478/s11535-013-0141-1
D'Mello, S., Finlay, G.J., Baguley, B.C., & Askarian-Amiri, M. （2016）. Signaling Pathways in Melanogenesis. International Journal of Molecular Sciences, 17（7）, 1144. Doi:10.3390/ijms17071144
Egawa, G., & Kabashima, K. （2017）. Barrier Dysfunction in the Skin Allergy. Allergology International, 67（1）, 3-11. Doi: 10.1016/j.alit.2017.10.002
Emílio-Silva, M.T., Rodrigues, V.P., Bueno, G., Ohara, R., Martins, M.G., Horta-Júnior, J.A.C., Branco, L.G.S., Rocha, L.R.M., & Hiruma-Lima, C.A. （2020）. Hypothermic Effect of Acute Citral Treatment during LPS-induced Systemic Inflammation in Obese Mice: Reduction of Serum TNF-α and Leptin Levels. Biomolecules, 10（10）, 1454. Doi: 10.3390/biom10101454
Hartman, M.L., & Malgorzata, C. （2015）. MITF in Melanoma: Mechanisms Behind its Expression and Activity. Cellular and Molecular Life Sciences , （72）7, 1249-1260. Doi:10.1007/s00018-014-1791-0
Hirobe, T. （1995）. Structure and Function of Melanocytes: Microscopic Morphology and Cell Biology of Mouse Melanocytes in the Epidermis and Hair Follicle. Histol Histopathol, 10（1）, 223-37.
Hushcha-Yekatsiaryna, B. I., Oton-Gonzalez, L., Mauro, G. D., Martini, F., Tognon, M., Mattei, M. D. （2021）. MicroRNAs in the Regulation of Melanogenesis. International Journal of Molecular Sciences, （22） 11, 6104. Doi:10.3390/ijms22116104
Vieira, A.J., Beserra, F.P., Souza, M.C., Totti, B.M., Rozza, A.L. （2018）. Limonene: Aroma of innovation in health and disease. Chemico-Biological Interactions, （1）283, 97-106. Doi: 10.1016/j.cbi.2018.02.007
Thielmann, J., & Muranyi, P. （2019）. Review on the Chemical Composition of Litsea Cubeba Essential Oils and the Bioactivity of its Major Constituents Citral and Limonene. Journal of Essential Oil Research, （31）5, 361-378. DOI: 10.1080/10412905.2019.1611671
Kamle, M., Mahato, D.K., Lee, K.E., Bajpai, V.K., Gajurel, P.R., Gu, K.S., & Kumar, P. （2019）. Ethnopharmacological Properties and Medicinal Uses of Litsea cubeba. Plants, 8（6）, 150. Doi: 10.3390/plants8060150
Kremer, J.L., Melo, G.P., Marinello, P.C., Bordini, H.P., Rossaneis, A.C., Sábio, L.R., Cecchini, R., Cecchini, A.L., Verri, W.A., & Luiz, R.C. （2019）. Citral Prevents UVB-induced Skin Carcinogenesis in Hairless Mice. Journal of Photochemistry and Photobiology B: Biology, 198, 111565. Doi: 10.1016/j.jphotobiol.2019.111565
Kumar, P., Nagarajan, A., & Uchil, P.D. （2018）. Analysis of Cell Viability by the MTT Assay. Cold Spring Harbor Protocols, 2018（6）. Doi: 10.1101/pdb.prot095505
Levy, C., Khaled, M., & Fisher, D.E. （2006）. MITF: Master Regulator of Melanocyte Development and Melanoma Oncogene. Trends in Molecular Medicine, 12（9）, 406-414. Doi: 10.1016/j.molmed.2006.07.008
Li, W.R., Shi, Q.S., Liang, Q., Xie, X.B., Huang, X.M., & Chen, Y.B. （2014）. Antibacterial Activity and Kinetics of Litsea Cubeba Oil on Escherichia Coli. PLoS One, 9（11）, e110983. Doi: 10.1371/journal.pone.0110983
Lin, J., & Fisher, D. （2007）. Melanocyte Biology and Skin Pigmentation. Nature, 445（7130）, 843–850. Doi:10.1038/nature05660
Mitsunaga, T., & Yamauchi, K. （2015）. Effect of Quercetin Derivatives on Melanogenesis Stimulation of Melanoma Cells. Journal of Wood Science, 61（4）, 351–363. Doi:10.1007/s10086-015-1476-9
Noosidum, A., Chareonviriyaphap, T., & Chandrapatya, A. （2014）. Synergistic Repellent and Irritant Effect of Combined Essential Oils on Aedes Aegypti （L.） Mosquitoes. Journal of Vector Ecology, 39（2）, 298-305. Doi: 10.1111/jvec.12104. PMID: 25424258
Nigjeh, S.E., Yeap, S.K., Nordin, N., Kamalideghan, B., Ky, H., & Rosli, R. （2018）. Citral Induced Apoptosis in MDA-MB-231 Spheroid Cells. BMC Complementary and Alternative Medicine, 18（1）, 56. Doi: 10.1186/s12906-018-2115-y
Riedhammer, C., Halbritter, D., Weissert, R. （2014）. Peripheral Blood Mononuclear Cells: Isolation, Freezing, Thawing, and Culture. Multiple Sclerosis, 1304, 53-61. Doi:10.1007/7651_2014_99
Shirai, Y., Kamimura, K., Seki, T., & Morohashi, M. （2001）. L-lactic Acid as a Mosquito （Diptera: Culicidae） Repellent on Human and Mouse Skin. Journal of Medical Entomology, 38（1）, 51-54. Doi: 10.1603/0022-2585-38.1.51
Si, L., Chen, Y., Han, X., Zhan, Z., Tian, S., Cui, Q., & Wang, Y. （2012）. Chemical Composition of Essential Oils of Litsea Cubeba Harvested from its Distribution Areas in China. Molecules, 17（6）, 7057-7066. Doi: 10.3390/molecules17067057
Wang, J., Oussama Khelissa, S.O., Chihib, N.E., Dumas, E., & Gharsallaoui, A. （2019）. Effect of Drying and Interfacial Membrane Composition on the Antimicrobial Activity of Emulsified Citral. Food Chemistry, 298,125079. Doi: 10.1016/j.foodchem.2019.125079
Zhao, M., Hu, J., Ni, H., Jiang, Z., & Wang, L. （2019）. Research Progress in Melanogenesis Signaling Pathway. Chinese Journal of Aeronautics, 35（9）, 1633-1642. Doi: 10.13345/j.cjb.190084
Zhang, X.T., Wei, K.J., Chen, Y., Shi, Z., Liu, L., Li, J., Zhang, G., & Ji, Wei. （2018）. Molecular Cloning and Expression Analysis of tyr and tyrp1 Genes in Normal and Albino Yellow Catfish Tachysurus Fulvidraco. Journal of fish biology, 92（4）, 979-998. Doi:10.1111/jfb.13556
Zhou, Q., Zheng, Dongfang., Zhao, Yong., Wang, T., & Yang, Yafeng. （2020）. Muhammad Ashraf Active Constituents of Litsea Cubeba. Thermal Science, 24（3A）, 1745-1752. Doi: 10.2298/TSCI190526047Z