維他命C又稱抗壞血酸(L-Ascorbic acid)，因具有生理的活性效果，因此在化妝品上廣為應用，主要有三種功能。首先，維他命C是一種良好的抗氧化劑，能有效保護皮膚細胞免於自由基的侵害。另一方面，維他命C能促進膠原蛋白生成，使得組織與組織之間能重新被修復。第三，維他命C被視為抑制黑色素的重要成分之一，它能減少黑色素生成。但是維他命C的缺點是經不起熱度及氧化容易被分解，因此開發其穩定衍生物以便保存使用。
維他命C醣苷(2-O-α-D-glucopyranosyl-L-ascorbic acid)為維他命C的醣化衍生物之一，不容易氧化，對環境穩定性佳，安全性高，因此用於取代維他命C。而熊果素(hydroquinone-O-beta-D-glucopyranoside)是一種由熊果葉抽取出來的成分，為藥用美白成分對苯二酚的醣化衍生物，其美白機制與對苯二酚類似但是熊果素對皮膚的刺激性較弱，衛生署對於此兩種成分個別有限制使用的濃度，因此本研究在此規範濃度之下，希望能在安全評估之下，以不同階段的美白機制，結合維他命C醣苷和熊果素兩種美白成分，確定結合後之美白成效，並利用HPLC分析維他命C醣苷和熊果素水解的程度。
在本研究中，我們以B16F0黑色素瘤細胞為測試平台，實驗結果說明在未調整pH值，16mM的維他命C醣苷培養液中，投藥12小時後，黑色素含量逐漸減少，酪胺酸酶相關活性(Phenolase activity)也隨著維他命C醣苷濃度增加而遞減。另外，在HPLC分析實驗結果顯示澱粉水解酵素(Amyloglucosidase)也能將維他命C醣苷分解出維他命C。調整維他命C醣苷培養液的pH值之後，發現抑制黑色素含量的效果變差。因此在同樣16mM維他命C醣苷，投藥12小時的環境下，結合不同濃度的熊果素和澱粉水解酵素，改善其抑制黑色素效果。以細胞實驗結果發現，在16mM維他命C醣苷、2mM熊果素和5 units/ml澱粉水解酵素培養液中，發現黑色素含量減少了48.8%，因此本研究結果，能提供結合不同美白機制的美白複合成分作為初步之參考。

1. Meeting Beauty & Wellness Needs through Cosmeceuticals. 2007, Datamonitor Corporation.
2. Darr, D., et al., Topical vitamin C protects porcine skin from ultraviolet radiation-induced damage. Br J Dermatol, 1992. 127(3): p. 247-253.
3. Pinnell, S.R., Regulation of collagen biosynthesis by ascorbic acid: a review. Yale J Biol Med, 1985. 58(6): p. 553-559.
4. Shindo, Y., et al., Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin. J Invest Dermatol, 1994. 102(1): p. 122-124.
5. 曹汝智, 中西醫結合皮膚美容學, ed. 皮膚醫學美容專業系列叢書. 2000: 學苑出版社. p.3-6.
6. Segre, J.A., Epidermal barrier formation and recovery in skin disorders. J Clin Invest, 2006. 116(5): p. 1150-1158.
7. Boissy, R.E., Melanosome transfer to and translocation in the keratinocyte. Exp Dermatol, 2003. 12 Suppl 2: p. 5-12.
8. Thong, H.Y., et al., The patterns of melanosome distribution in keratinocytes of human skin as one determining factor of skin colour. Br J Dermatol, 2003. 149(3): p. 498-505.
9. Slominski, A., et al., Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev, 2004. 84(4): p. 1155-1228.
10. Sulaimon, S.S. and B.E. Kitchell, The biology of melanocytes. Vet Dermatol, 2003. 14(2): p. 57-65.
11. Parvez, S., et al., Survey and mechanism of skin depigmenting and lightening agents. Phytother Res, 2006. 20(11): p. 921-934.
12. Yamamoto, I., et al., L-ascorbic acid alpha-glucoside formed by regioselective transglucosylation with rat intestinal and rice seed alpha-glucosidases: its improved stability and structure determination. Chem Pharm Bull (Tokyo), 1990. 38(11): p. 3020-3023.
13. Gokmen, V., et al., Enzymatically validated liquid chromatographic method for the determination of ascorbic and dehydroascorbic acids in fruit and vegetables. J Chromatogr A, 2000. 881(1-2): p. 309-316.
14. Austria, R., A. Semenzato, and A. Bettero, Stability of vitamin C derivatives in solution and topical formulations. J Pharm Biomed Anal, 1997. 15(6): p. 795-801.
15. Kameyama, K., et al., Inhibitory effect of magnesium L-ascorbyl- 2-phosphate (VC-PMG) on melanogenesis in vitro and in vivo. J Am Acad Dermatol, 1996. 34(1): p. 29-33.
16. Lee, S.B., et al., Antioxidative effects of glycosyl-ascorbic acids synthesized by maltogenic amylase to reduce lipid oxidation and volatiles production in cooked chicken meat. Biosci Biotechnol Biochem, 2004. 68(1): p. 36-43.
17. Huh, C.H., et al., A randomized, double-blind, placebo-controlled trial of vitamin C iontophoresis in melasma. Dermatology, 2003. 206(4): p. 316-320.
18. Ebihara, M., et al.,Iontophoresis promotes percutaneous absorption of L-ascorbic acid in rat skin. J Dermatol Sci, 2003. 32(3): p. 217-222.
19. Spiclin, P., M. Gasperlin, and V. Kmetec, Stability of ascorbyl palmitate in topical microemulsions. Int J Pharm, 2001. 222(2): p. 271-279.
20. Takebayashi, J., et al., Characterization of the radical-scavenging reaction of 2-O-substituted ascorbic acid derivatives, AA-2G, AA-2P, and AA-2S: a kinetic and stoichiometric study. Biol Pharm Bull, 2006. 29(4): p. 766-771.
21. Ros, J.R., J.N. Rodriguez-Lopez, and F. Garcia-Canovas, Effect of L-ascorbic acid on the monophenolase activity of tyrosinase. Biochem J, 1993. 295 ( Pt 1): p. 309-312.
22. Garcia-Molina, F., et al., Opposite effects of peroxidase in the initial stages of tyrosinase-catalysed melanin biosynthesis. Int J Biochem Cell Biol, 2005. 37(6): p. 1179-1196.
23. Claus, H. and H. Decker, Bacterial tyrosinases.Syst Appl Microbiol, 2006. 29(1): p. 3-14.
24. Sugimoto, K., et al., Syntheses of arbutin-alpha-glycosides and a comparison of their inhibitory effects with those of alpha-arbutin and arbutin on human tyrosinase. Chem Pharm Bull (Tokyo), 2003. 51(7): p. 798-801.
25. Shimogaki H, T.Y., Tmai H, Masuda M, In vitro and in vivo evaluation of ellagic acid on melanogenesis inhibiton. Int J Cosmet Sci., 2000. 22: p. 291-303.
26. Canovas, F.G., et al., The role of pH in the melanin biosynthesis pathway. J Biol Chem, 1982. 257(15): p. 8738-8744.
27. Murakami, K., et al., Comparison of ascorbic acid and ascorbic acid 2-O-alpha-glucoside on the cytotoxicity and bioavailability to low density cultures of fibroblasts. Biochem Pharmacol, 1992. 44(11): p. 2191-2197.
28. Kumano, Y., et al., In vitro and in vivo prolonged biological activities of novel vitamin C derivative, 2-O-alpha-D-glucopyran osyl-L-ascorbic acid (AA-2G), in cosmetic fields. J Nutr Sci Vitaminol (Tokyo), 1998. 44(3): p. 345-359.
29. Kumano, Y., et al., Enhancing effect of 2-O-alpha-D-glucopyrano syl-L-ascorbic acid, a stable ascorbic acid derivative, on collagen synthesis. Biol Pharm Bull, 1998. 21(7): p. 662-666.
30. Yamamoto, I., et al., Synthesis and characterization of a series of novel monoacylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha- D-glucopyranosyl-L-ascorbic acids, as skin antioxidants. J Med Chem, 2002. 45(2): p. 462-468.
31. Miyai, E., et al., Ascorbic acid 2-O-alpha-glucoside-induced redox modulation in human keratinocyte cell line, SCC: mechanisms of photoprotective effect against ultraviolet light B. Biol Pharm Bull, 1997. 20(6): p. 632-636.
32. Jurkovic, P., et al., Skin protection against ultraviolet induced free radicals with ascorbyl palmitate in microemulsions. Eur J Pharm Biopharm, 2003. 56(1): p. 59-66.
33. Yamamoto, I., et al., Collagen synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-O-alpha-D- glucopyranosyl-L-ascorbic acid. J Nutr, 1992. 122(4): p. 871-877.
34. Muto, N., K. Terasawa, and I. Yamamoto, Evaluation of ascorbic acid 2-O-alpha-glucoside as vitamin C source: mode of intestinal hydrolysis and absorption following oral administration. Int J Vitam Nutr Res, 1992. 62(4): p. 318-323.
35. Sauer, J., et al., Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta, 2000. 1543(2): p. 275-293.