臺灣博碩士論文加值系統

奈米科技是目前最熱門的技術之一，隨著奈米技術的興起，也為傳統化妝品技術帶來革命性的變化。長久以來，無論局部外用藥物或化妝品，一直努力尋求更有效的皮膚傳輸方式，同時也致力於提高某些不穩定或易氧化成份的穩定性。有些文獻指出奈米技術的開發，如微脂粒技術、奈米乳化技術或奈米膠囊化技術等，可以克服過去活性物質不穩定或油性成份黏膩不易吸收等問題。同時由於活性成份粒子微小化之後，更容易穿透皮膚，使其達到更好的吸收效果。有些研究指出，奈米化後之安定性受奧士瓦熟成（Ostwald Ripening）及凡得瓦爾力（Van der Waals force）影響甚鉅，其可能造成產品絮凝（Flocculation），分層（Creaming）、聚結(Coalescence)等不安定因素，以及奈米乳化高壓過程中可能造成成份分解破壞，皆須考量。本研究利用高壓均質方式將含美白成分之配方奈米乳化為前提，針對製備方法及其性質做一探討，再藉由儀器測試其安定性、安全性及有效弁鄔妗?禲A藉此研究來確認是否達到我們研究的目標。
本研究利用奈米技術所研發的化妝品，主要是將化妝品基劑，如氨基酸、硬脂酸酯、鮫鯊烷、三甘油酯、荷荷葩油、醇類、純水及有效成分如?熊果素(?Arbutin )、神經醯胺( Ceramide)、輔酶Q10、維他命C酯等、以高壓均質方式，壓力控制50~100Mpa，加工次數2~8次處理成為奈米結構，其中壓力大小、乳化劑及油脂選擇、及加工數等，皆會影響其粒徑大小及安定性。
配方篩選後，其粒徑大小約在60~100nm之間，此外亦發現高壓均質的配方，不易孳生細菌，達到物理性防腐的目的，將可使奈米化的附加價值更加提升至不含防腐劑的訴求。再則因為利用高壓製程的關係，研究以HPLC計算回收率，不管有沒有經過高壓均質的產物回收率在98~104%，均在合乎規定的範圍之間。再搭配耐受性測試，目標以-10℃、25℃、45℃三種溫度測試，測其安定性，以目測結果並無分層及嚴重霧化現象，以3000 rpm，離心15分鐘亦無分層現象。藉由膠體粒子穩定測試及電導度，測試其轉相法（PIT法）與高壓均質法之奈米穩定性，比較其差異性，其相圖在溫度與電壓值倒數（1/V）或與電導度值間有明顯差異，轉相法在轉相溫度以上時，會有明顯不安定現象，高壓均質法對溫度則相對安定。在MTT細胞毒性研究中，我們發現傳統製程與奈米製程之產品，在相同濃度時對B16黑色素細胞之毒性相當，顯示並不會因劑型差異，而使毒性增高。微生物驗證其劑型差異是否會明顯提高微生物增生率，結果發現傳統製程較易受微生物污染，奈米劑型有其一定的抗微生物污染性。在經皮吸收實驗過程研究發現，奈米乳化製品中之活性物質穿透人工皮膚細胞之速率遠高於傳統製品。以Lab色度系統測試美白效能方面，雖然測試時間較短，但比起美白精華液略有較好之成效。

Nano-science is the one of the most popular know-how at present. With the nanotechnology rise and development, it brings the revolutionary change for traditional cosmetics technology. No matter the localized medicine for external use and cosmetics has been looking for more effective delivery systems. In the meantime, the stability of cosmetic ingredients was improved. The development of nanotechnology from some searches shows the liposome、nanoemulsion or nanocapsule could overcome the difficulties of the unstability and unabsorbability of active material and oil ingredient sticky . It will approach the best absorb effect after the particle was miniaturized and absorbable easily by skin. Some researches show that Ostwald Ripening and Van der Waals force significantly affected the stability of nanoemulsion. It must be considered that the unstable elements of flocculation、creaming、coalescence, and ingredients could be destroyed and breakdown in the process of nanoemulsion by high pressure. To evaluation preparation method and characteristics of nanoemulsion cosmetics, we prepare some cosmetics containing whitening ingredient using high pressure homogenizer .To confirm the goal of our research, the stability, safety and effectively of nanoemulsion product were evaluated by relative instruments.
The nanoemulsion product, having basis ingredients such as amino acid、stearate ester、squalane、triglycerides、jojoba oil、alcohols、aqua and other effective materials such as ?arbutin、ceramide、Q10、vitamin C ester, is manufactured by optimum condition (50~100Mpa and 2~8 times)using high pressure by high homogenizer. Pressure、emulsifying agent、oil type and times of procedure will influence the particle size and stability.
The bacteria are not multiplied in the nanoemulsion product when the particle size of oil droplet falls in between 60nm and 100nm. It has additional value of preservative function. To evaluate the recovery of the ingredient content of cosmetics obtained through high-pressure homogenizer, we use HPLC to determine the active material content. It is found that the recovery rate of active material is 98~104%.
The stabilities of nanoemulsion products are carried out by three different temperatures （-10℃、25℃、45℃）and the result is satisfied （without stratification and nebulization）. Colloidal particle stability and conductance tests were used to compare the differences of nanoemulsion products obtained through PIT or high-pressure methods. PIT method has obvious unstable state when the phase inverse temperature is arrived but the product of high-pressure method is stable for temperature. Both of conventional emulsion and nanoemulsion products also show similar toxicity to B16 melanin cells using MTT assay. It is also found that conventional emulsion product is polluted by microorganism easier than nanoemulsion product. In percutaneous absorption experiment, we found that the quantity of active material of nanoemulsion product is higher than that of convention emulsion product crossing artificial skin within the same period. LAB chrominance instrument was used to assess the whitening efficiency. As a result of short term, we found that the effect of nanoemulsion product is better than that of convention whitening essence fluid.

中文摘要----------------------------------------------------------------------------I
英文摘要--------------------------------------------------------------------------III
誌謝---------------------------------------------------------------------------------V
內文目錄--------------------------------------------------------------------------VI
表目錄-----------------------------------------------------------------------------XI
圖目錄-----------------------------------------------------------------------------XIII

壹、 緒論---------------------------------------------------------------------------------1
1-1 前言------------------------------------------------------------------------------ 1
1-2 研究背景與動機----------------------------------------------------------------2
1-3 研究目的-------------------------------------------------------------------------4
1-4 研究流程-------------------------------------------------------------------------5
貳、文獻回顧----------------------------------------------------------------------------7
2-1 皮膚生理現象與吸收---------------------------------------------------------7
2-1-1 皮膚基本構造----------------------------------------------------------------7
2-1-1-1 表皮層----------------------------------------------------------------------8
2-1-1-1-1 角質形成細胞 (Keratinocytes)--------------------------------------8
2-1-1-1-2 樹枝狀細胞（Dendritic cells）-------------------------------------8
2-1-1-2 真皮層---------------------------------------------------------------------9
2-1-1-3 皮下組織-------------------------------------------------------------------9
2-1-2 皮膚的顏色及老化--------------------------------------------------------10
2-1-2-1 皮膚的顏色---------------------------------------------------------------10
2-1-2-2 皮膚的老化---------------------------------------------------------------12
2-1-3 皮膚表皮吸收---------------------------------------------------------------12
2-2 乳化 (Emulsifications)-------------------------------------------------------13
2-3 奈米化粧品技術--------------------------------------------------------------17
2-3-1 奈米粉體色料---------------------------------------------------------------18
2-3-2 奈米膠囊（Nanocapsule）-----------------------------------------------18
2-3-3 微乳化（Microemulsion）-----------------------------------------------19
2-3-4 微脂粒（Liposome）-----------------------------------------------------19
2-3-5 奈米乳化（Nanoemulsion）---------------------------------------------21
2-3-5-1 轉相法 (PIT)------------------------------------------------------------22
2-3-5-2 高壓均質法--------------------------------------------------------------25
參、實驗---------------------------------------------------------------------------30
3-1 原料、儀器與試劑---------------------------------------------------------30
3-1-1 化粧品原料----------------------------------------------------------------30
3-1-2 其他試藥藥品與試劑----------------------------------------------------31
3-1-3 儀器設備-------------------------------------------------------------------32
3-2 操作條件測試---------------------------------------------------------------33
3-3 配方開發---------------------------------------------------------------------34
3-3-1 高壓均質法---------------------------------------------------------------34
3-3-2 轉相法---------------------------------------------------------------------38
3-4 粒徑測試--------------------------------------------------------------------40
3-4-1 DLS動態光散射分析儀------------------------------------------------40
3-4-2 雷射繞射粒度分佈分析儀（Laser Diffraction Scattering）-----42
3-5 安定性評估----------------------------------------------------------------43
3-5-1 膠體粒子穩定測試系統-----------------------------------------------44
3-5-2 電導度計測試-----------------------------------------------------------46
3-5-3 離心及耐受性試驗-----------------------------------------------------47
3-6 安全性評估-----------------------------------------------------------------48
3-6-1 MTT細胞測試------------------------------------------------------------49
3-6-2 一次刺激貼布測試-----------------------------------------------------52
3-6-3 微生物測試--------------------------------------------------------------54
3-7 有效性測試----------------------------------------------------------------55
3-7-1 有效成份含量測試------------------------------------------------------56
3-7-2 經皮吸收測試------------------------------------------------------------59
3-7-3 人體感官及美白效能測試--------------------------------------------63
肆、結果與討論----------------------------------------------------------------66
4-1 原料準備與物性測試----------------------------------------------------66
4-2 操作條件評估-------------------------------------------------------------66
4-3 配方開發實驗-------------------------------------------------------------66
4-4 粒徑測試-------------------------------------------------------------------70
4-5 離心及耐受性測試-------------------------------------------------------74
4-6 電導度測試----------------------------------------------------------------75
4-7 膠體粒子穩定測系統----------------------------------------------------77
4-8 MTT細胞測試-------------------------------------------------------------79
4-9 一次刺激之貼膚測試---------------------------------------------------84
4-10 微生物滋生測試-------------------------------------------------------86
4-11 有效成份含量測定----------------------------------------------------90
4-12 經皮吸收測試----------------------------------------------------------94
4-13 人體感官測試及美白效能測試------------------------------------100
伍、總結---------------------------------------------------------------------103
陸、參考文獻---------------------------------------------------------------108

1.郭俊賢、殷正華. 奈米生技化妝品專利地圖及分析. 行政院國家科學委員會科技資料中心. Vol 7,Jun 2004:1-2,53-57
2.http://www.bccresearch.com/
3.Ruysschaert, T. Germain, M. da Silva Gomes, J.F.P. Fournier, D. Sukhorukov, G.B. Meier, W. Winterhalter, M. Liposome-Based Nanocapsules. IEEE Xplore. 2004 Mar; 3(1): 49-55.
4.林書毅. 以w/o為乳劑系統製備奈米膠囊藥物載體之研究. 國立台灣大學藥學研究所 碩士論文. 90年系統編號90NTU01551002
5.Fang JY, Hong CT, Chiu WT, Wang YY. Effect of liposomes and niosomes on skin permeation of enoxacin. Int J Pharm. 2001 May; 219(1-2): 61-72.
6.Tharwat Tadros,Lorna Kessell. Stabilizing Nanodispersions in personal Care and cosmetic Applications. Cosmetics&toiletries, 119(8), Aug 2004:41-46
7.Somasundaran P, Chakraborty S, Qiang Q, Deo P, Wang J, Zhang R. Surfactants, polymers and their nanoparticles for personal care applications. J Cosmet Sci. 2004; 55 Suppl: S1-17.
8.Alvarez-Roman R, Barre G, Guy RH, Fessi H. Biodegradable polymer nanocapsules containing a sunscreen agent: preparation and photoprotection. Eur J Pharm Biopharm. 2001 Sep;52(2):191-5
9.C. Solans , P. Izquierdo, J. Nolla, N. Azemar, M.J. Garcia-Celma. Nano-emulsions. Current Opinion in Colloid & Interface Science. 2005 Oct; 10:102–110
10.Sonneville-Aubrun O, Simonnet JT, L''Alloret F. Nanoemulsions: a new vehicle for skincare products. Adv Colloid Interface Sci. 2004 May; 108-109:145-9.
11.Kieche Meleson, Sara Graves, Thomas G. Mason. Formation of Concentrated Nanoemulsions by Extreme Shear. Soft Materials. 2004; 2(2-3): 109 - 123
12.M. Porrasa, C. Solansb, C. Gonz´aleza, A. Mart´ıneza, A. Guinarta, J.M. Guti´erreza. Studies of formation of W/O Nano-emulsions. Colloids and Surfaces A: Physicochem. Eng. Aspects. 2004; 249: 115–118
13.Huailiang Wu, Chandrasekharan Ramachandran,Noman D. Weiner, Blake J Roessler,Topical transpost of Hydrophilic compounds using water-in-oil nanoemulsion,international Journal of Pharmaceutics, 220,2001:63-75.
14.T.Tadros,E.Vandekerckhove,A.Vandamme,B.Levecke and K.Booten, A Hydrophobically modified inulin surfactant for preparing and stabilizing nanoemulsions, cosmetics&toiletries, 120(2),Feb 2005: 45–50
15.P Izquierdo,J.Feng,J.Esquena,Th.F.Tadros,Joseph C.Dederen,M. J. Garca,N.Azemar,C.Solans.The influence of surfactant mixing ratio on nano-emulsion formation by the PIT method.Journal of colloid and interface SCIENCE 285(2005)388-394
16.Kang, Ki-Choon; Pyo, Hyeong-Bae; Lee, Jong-Dae; Jeong, Noh-Hee. Properties of nanoemulsions containing lecithin in cosmetics. Journal of Industrial and Engineering Chemistry 10(4), 2004: 564-568.
17.Forgiarini, A.; Esquena, J.; Gonzalez, C.; Solans, C. Formation and stability of nano-emulsions in mixed nonionic surfactant systems. Progress in Colloid & Polymer Science (2001), 118, 184-189.
18.Graves, S.; Meleson, K.; Wilking, J.; Lin, M. Y.; Mason, T. G. Structure of concentrated nanoemulsions. Journal of Chemical Physics (2005), 122(13), 134703/1-134703/6.
19.P.Izquierdo,J.Esquena,Th.F.Tadros,C. Dederen,M.J.Garcia-Celma, N.Azemar,et al.Formation and stability of nano-emulsions prepared using the phase inversion temperature method,Langmuir 18(1), 2002:26-30
20.KR2004105169 Preparation method of stable nano-emulsion using arbutin and lecithin and cosmetic formulation containing the same
21.JP 2004359587 Method for producing stable emulsified nanoparticle using arbutin and cosmetic composition containing emulsified nanoparticle
22.H. Aro , G.Dahms, Kemira Pigments Oy. Compatibility of micro- fine titanium dioxide with organic UV filters. Cosmetics and Toiletries Manufacture Worldwide. 2004; p115-118.
23.F. Menzel, T. Reinert, J. Vogt, T. Butz. Investigations of percutaneous uptake of ultrafine TiO2 particles at the high-energy ion nanoprobe LIPSION. Nucl Instrum Methods Phys Res B. 2004 June; (219-220): 82-86.
24.L. Petit; G. E. Piérard. Skin-lightening products revisited. Int. J Cosmetic Sci. 2003 Aug; 25(4): pp. 169-181.
25. P. S piclin, M.Gasˇperlin, V. Kmetec. Stability of ascorbyl palmitate in topical microemulsions. International Journal of Pharmaceutics 222,2001:271–279
26. 劉瑋, 張懷亮.皮膚科學與化妝品幼警?? 化學工業出版社. 2005; 1-16.
27. Duval C, Smit NP, Kolb AM, Regnier M, Pavel S, Schmidt R. Keratinocytes control the pheo/eumelanin ratio in cultured normal human melanocytes. Pigment Cell Res. 2002 Dec; 15(6): 440-6.
28. C Nicolas, D Schmitt. Langerhans'' cells: methods of identification. Pathol Biol (Paris). 1984 Mar; 32(3): 199-208.
29. J M Austyn. Dendritic cells. Curr Opin Hematol . 1998 Jan; 5(1): 3-15.
30. I.Moll, M. Roessler, J. M. Brandner, A. C. Eispert, P. Houdek and R. Moll. Human Merkel cells aspects of cell biology, distribution and functions. Eur. J. Cell Biol. 2005; 84(2–3): 259–71.
31. 黃宜純. 美容皮膚生理學. 知音出版社
32. Ito S, Wakamatsu K, Ozeki H. Chemical analysis of melanins and its application to the study of the regulation of melanogenesis. Pigment Cell Res. 2000; 13 Suppl 8:103-9.
33. Eisenhofer G, Tian H, Holmes C, Matsunaga J, Roffler-Tarlov S, Hearing VJ. Tyrosinase : a developmentally specific major determinant of peripheral dopamine. FASEB J. 2003 Jul; 17(10): 1248-55.
34. Simonot D., McColl J., Thome D. Tyrosinase Inhibitors: Activity of a rumex extract in combination with kojic acid and arbutin. Cosmetics & Toiletries Magazine. 2002 Mar; 117 (3): 52-56.
35. Masse M.-O.; Duvallet V.; Borremans M.; Goeyens L. Identification and quantitative analysis of kojic acid and arbutin in skin-whitening cosmetics. Int J Cosmet Sci. 2001 Aug; 23(4): 219-232.
36. K Maeda and M Fukuda. Arbutin: mechanism of its depigmenting action in human melanocyte culture. J Pharmacol Exp Ther. 1996 Feb; 276(2): 765-9.
37. Chakraborty AK, Funasaka Y, Komoto M, Ichihashi M，Effect of arbutin on melanogenic proteins in human melanocytes. Pigment Cell Res. 1998 Aug; 11(4): 206-12.
38. Morisaki K, Ozaki S. Design of novel hybrid vitamin C derivatives: thermal stability and biological activity. Chem Pharm Bull (Tokyo). 1996 Sep; 44(9): 1647-55.
39. Wang CC and Wu SM. Simultaneous determination of L-ascorbic acid, ascorbic acid-2-phosphate magnesium salt, and ascorbic acid-6- almitate in commercial cosmetics by micellar electrokinetic capillary electrophoresis. 2006 Jan. Available online.
40. Desmond J. Tobin. Biochemistry of human skin－our brain on the outside. Chem. Soc. Rev., 2006; 35: 52–67
41. Hans Schaefer, Thomas E. Redelmeier Skin Barrier Principles of Percutaneous Absorption. 1996. (ISBN 3-8055-6326-4)
42. Elias PM, Cooper ER, Korc A, Brown BE. Percutaneous transport in relation to stratum corneum structure and lipid composition. J Invest Dermatol. 1981 Apr; 76(4): 297-301.
43. 蔡義宏. 乳化原理，應用界面化學. 1979; 2（3）：13-19.
44. 吳適. 乳化劑在食品工業上之應用. 食品工業. 1979; 11（8）：28-32.
45. 張炳揚. 食品體系中乳化安定性之控制. 食品工業.2001; 33（6）：42-48.
46. 陳崇賢. 乳液概論. 界面科學雜誌. 1996; 19：1-12.
47. 王金源. 乳化技術. 應用界面化學. 1979; 2（3）：21-26
48. Yuan Fangli, Huang Shulan and Li Jinlin. Preparation of zinc oxide nano-particles coated with aluminum. J Mater Sci Lett. 2001 Aug; 20(16): 1549 - 1551.
49. Berkland C, Kim K, Pack DW. PLG Microsphere Size Controls Drug Release Rate Through Several Competing Factors. Pharm Res. 2003 Jul; 20(7): 1055-62.
50. Xu XL, Zhang ZC, Ge XW, Zhang MW. Emulsifier for microemulsion polymerization. Colloid & Polymer Science. 1998 July; 276(6): 534-538.
51. Lin TL , Hu Y, Lee TT. The effect of alcohols on the size of water-in-oil microemulsion droplets. 1997 Dec; 105(1): 268–271.
52. Li P, Ghosh A, Wagner RF, Krill S, Joshi YM, Serajuddin AT. Effect of combined use of nonionic surfactant on formation of oil-in-water microemulsions. Int J Pharm. 2005 Jan; 288(1): 27-34.
53. Gareth D. Rees, Brian H. Robinson. Microemulsions and organogels: Properties and novel applications. Advanced Materials. 2004 Oct; 5(9): 608–619.
54.Wissing SA, Muller RH. Solid Lipid Nanoparticles as Carrier for Sunscreens: In Vitro Release and In Vivo Skin Penetration, J Control Release. 2002 Jun 17; 81(3): 225 - 33.
55. Muller RH, Mader K, Gohla S. Solid Lipid Nanoparticles (SLN) for Controlled Drug Delivery – a Review of the State of the Art. Eur J Pharm Biopharm. 2000 Jul; 50(1): 161 - 77.
56. Orlando David Henrique dos Santos, Juliana Violi Miotto, Jacqueline Moreira de Morais, Pedro Alves da Rocha‐Filho, Wanderley Pereira de Oliveira. Attainment of Emulsions with Liquid Crystal from Marigold Oil Using the Required HLB Method. J Dispersion Science and Technology. 2005; 26(2): 243 - 249
57. M.S.E1-Aasser, E.D.sudol. Miniemulsions：overview of research and applications. JCT. Res. 2004; 1(1):21-31.
58. 王培義. 化妝品—原理·配方·生産工藝(第二版). 化學工業出版社.，2006; p244-246.
59. P. W. Voorhees. The theory of Ostwald ripening. J Statistical Physics. 1985 Jan; 38(1-2): 231–252.
60. Yves De Smet, Luc Deriemaeker, Robert Finsy. A Simple Computer Simulation of Ostwald Ripening. American Chemical Society. 1997 Apr; 13 (26): 6884 –6888.
61. www.kruss.de.
62. Jung GD, Yang JY, Song ES, Park JW. Stimulation of melanogenesis by glycyrrhizin in B16 melanoma cells. Exp Mol Med. 2001 Sep; 33(3): 131-135.
63. Seiberg M.; Paine C.; Sharlow E.; Andrade-Gordon P.; Costanzo M.; Eisinger M.; Shapiro S.S. Inhibition of Melanosome Transfer Results in Skin Lightening 1. J Invest Dermatol. 2000 Aug; 115(2):162-167(6).
64.Ogiso T, Niinaka N, Iwaki M. Mechanism for Enhancement Effect of Lipid Disperse System on Percutaneous Absorption. J Pharm Sci. 1996 Jan; 85(1): 57-64.
65. Choe YB, Jang SJ, Jo SJ, Ahn KJ, Youn JI. The difference between the constitutive and facultative skin color does not reflect skin phototype in Asian skin. Skin Res Technol. 2006 Feb; 12(1): 68-72.
66.Chen HB,Wong T,Chang XL,Yang YJ,Yang XL. Preparation of ibuprofen microemulsion and its transdermal absorption.Chin PharmJ. 2004 Jan;39(1):43-45.
67.Li L,Su J,Li Z,Zhou H,Song WW. A study on evaluating the effectiveness of lightening cosmetics.J Environ Occup Med. 2003 Feb,20(1):28-30.
68.行政院衛生署,常用藥物化粧品檢驗方法專輯(六),藥物食品檢驗局,2001年4月:202-203.
69.張麗卿. 化粧品檢驗實務.匯華圖書出版有限公司.1998 初版:109-111.
70.www.cmp-micro.com
71.K.H. Diec, A.Eitrich, T.Schmidt, T.Sokolowski, J.Schreiber.PIT Microemulsions with Low Surfactant content. Cosmetics&Toiletries, July 2001，116(7)：61-66
72.Klaus Raith, Hany Farwanah,Siegfried Wartewig,Reinhard H.H. Neubert.Progress in the analysis of Stratum corneum ceramides.Eur.J.Lipld Sci.Technol.106, 2004:561-571
73. Sottofattori, E., Anzaldi, M., Balbi, A., Tonello, G.Simultaneous HPLC determination of multiple components in a comercial cosmetic cream. Pharm. Biomed.Anal. 18,1998: 213–217.
74. Nakajima, M.et al. Arbutin decreases the pigmentation of Cultured human melanocytes through mechanisms other than induction of tyrosinase activity. Pigment Cell Res. 11 (1), 1998: 12-17.
75. Maeda, K. & Fukuda, M. Arbutin: mechanism of its depigmentating action in human melanocyte culture. J. Pharmacol. Exp. Ther. 276 (2), 1996: 765-9.
76. S,Piacentea, Ely E. S. Camargob, A.Zampellia, J.S. Graciosoc,A.R. Souza Britoc, C. Pizzaa,W.Vilegasd. Flavonoids and Arbutin from Turnera diffusa. Z. Naturforsch. 57c, 2002: 983-985