臺灣龍膽石斑魚(鞍帶石斑魚)為一產量高且高經濟價值之養殖魚類，由於近年來養殖技術之成長，擁有穩定之產量，故本實驗以石斑魚受精卵測試其萃出物之活性，探討其受精卵蛋白經酵素水解後之胜肽分子量分布，及抗氧化等效果，進而研究發展為高附加價值之美容保養品。石斑魚受精卵先經過前處理後，以鳳梨酵素或木瓜酵素水解，多組不同水解時間的受精卵蛋白經過FPLC分離後，經鳳梨酵素及木瓜酵素皆為12小時水解後，可得12500 Da以下的胜肽，其中以木瓜酵素水解12小時的受精卵蛋白具有較高的清除DPPH能力，其IC50為0.17687 mg∕ml，此約為Vit.C 的1/44倍，而鳳梨酵素水解12小時的膠原蛋白，其IC50則只有0.9055 mg∕ml，此約為Vit. C的1/226倍；但就還原力，則以鳳梨酵素水解12小時的受精卵蛋白具有較高的還原能力。使用未經水解且分子量5000Da以下之萃出物來做人體皮膚測試及細胞培養上，發現在人體皮膚測試上，受試者使用一週後，皮膚黑色素斑點便有減少，且保濕程度大幅提升，顯示出龍膽石斑魚受精卵萃出物具有發展為市售保養品之潛力。而在細胞培養基上，在沒有胎牛血清的情況下，含魚卵萃取物的培養基，確實有提升HK2細胞生長速率的效果，可提升細胞生長速率(p=0.062, 有顯著差異)，然而，仍無法完全取代含10%胎牛血清的效果。

Taiwan Epinephelus Lanceolatus is a high economic-valued, high production cultured fish species. In the last decade, the farming technique of Taiwan Epinephelus Lanceolatus has been improved and shown a steady production. Taiwan Epinephelus Lanceolatus is the biggest cultured fish with high amount of proteins. In this study the bioactivity of the zygote extract, the hydrolyzed proteins were assayed the antioxidant activity and the extract without enzymatic hydrolysis and with the molecular weight under 5 kDa was tested and used as cosmetic component.
We use FPLC to measure the hydrolyzed peptide molecule weight and found that hydrolyzed zygote extract showing molecular weight under 12500 Da after enzymatic hydrolysis by papain enzyme and bromelain for 12 hours. And it showed that papain exhibited higher antioxidant activity than bromelain with IC50 of 0.17687 mg∕ml versus 0.9055 mg∕ml. Interestingly, bromelain hydrolyzed peptides showed higher reduction ability than papain hydrolyzed peptides toward reducing Fe3+ (Prussian Blue test ).
The extracts of zygote of Taiwan Epinephelus Lanceolatus without enzymatic hydrolysis and with the molecular weight lower than 5 kDa showed the activity to reduce the melanin spots after one week test. And it also showed the activity to enhance the moisture ability. These results showed that the extract without enzymatic hydrolysis and with molecular weight lower than 5 kDa is a good cosmetic source.

1 Adler-Nissen, J. Enzymic hydrolysis of food proteins. (Elsevier Applied Science Publishers, 1986).
2 Ala-Kokko, L., Pihlajaniemi, T., Myers, J. C., Kivirikko, K. & Savolainen, E. Gene expression of type I, III and IV collagens in hepatic fibrosis induced by dimethylnitrosamine in the rat. Biochem. J 244, 75-79 (1987).
3 Allen, M. et al. Hedgehog signaling regulates sebaceous gland development. The American journal of pathology 163, 2173-2178 (2003).
4 Anderson, D. & Phillips, B. Comparative< i> In Vitro</i> and< i> In Vivo</i> Effects of Antioxidants. Food and Chemical Toxicology 37, 1015-1025 (1999).
5 Bae, I. et al. Biochemical properties of acid-soluble collagens extracted from the skins of underutilised fishes. Food Chemistry 108, 49-54 (2008).
6 Benzie, I. F. & Strain, J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical biochemistry 239, 70-76 (1996).
7 Berlett, B. S. & Stadtman, E. R. Protein oxidation in aging, disease, and oxidative stress. Journal of Biological Chemistry 272, 20313-20316 (1997).
8 Bigg, H. F. & Rowan, A. The inhibition of metalloproteinases as a therapeutic target in rheumatoid arthritis and osteoarthritis. Current opinion in pharmacology 1, 314-320 (2001).
9 Bornstein, P. The biosynthesis of collagen. Annual review of biochemistry 43, 567-603 (1974).
10 Chai, H.-J. et al. Effects of sizes and conformations of fish-scale collagen peptides on facial skin qualities and transdermal penetration efficiency. Journal of BioMed Research 2010 (2010).
11 Cheng, F.-Y., Hsu, F.-W., Chang, H.-S., Lin, L.-C. & Sakata, R. Effect of different acids on the extraction of pepsin-solubilised collagen containing melanin from silky fowl feet. Food chemistry 113, 563-567 (2009).
12 Dong, S. et al. Antioxidant and biochemical properties of protein hydrolysates prepared from Silver carp (< i> Hypophthalmichthys molitrix</i>). Food Chemistry 107, 1485-1493 (2008).
13 Ebrahimzadeh, M. A., Nabavi, S. M., Nabavi, S. F., Bahramian, F. & Bekhradnia, A. R. Antioxidant and free radical scavenging activity of H. officinalis L. var. angustifolius, V. odorata, B. hyrcana and C. speciosum. Pak J Pharm Sci 23, 29-34 (2010).
14 Ebrahimzadeh, M. A., Pourmorad, F. & Bekhradnia, A. R. Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. African Journal of Biotechnology 7 (2008).
15 Eyre, D. R., Weis, M. A. & Moskowitz, R. W. Cartilage expression of a type II collagen mutation in an inherited form of osteoarthritis associated with a mild chondrodysplasia. Journal of Clinical Investigation 87, 357 (1991).
16 Gill, I., López-Fandiño, R., Jorba, X. & Vulfson, E. N. Biologically active peptides and enzymatic approaches to their production. Enzyme and Microbial Technology 18, 162-183 (1996).
17 Harman, D. The free radical theory of aging. Antioxidants and Redox Signaling 5, 557-561 (2003).
18 Hazane, F. et al. Ageing effects on the expression of cell defence genes after UVA irradiation in human male cutaneous fibroblasts using cDNA arrays. Journal of Photochemistry and Photobiology B: Biology 79, 171-190 (2005).
19 Houck, K. A. et al. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Molecular Endocrinology 5, 1806-1814 (1991).
20 Hwang, K.-A., Yi, B.-R. & Choi, K.-C. Molecular mechanisms and in vivo mouse models of skin aging associated with dermal matrix alterations. Laboratory animal research 27, 1-8 (2011).
21 Jenkins, R. R. Free radical chemistry. Sports Medicine 5, 156-170 (1988).
22 Kishimoto, J., Burgeson, R. E. & Morgan, B. A. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes & development 14, 1181-1185 (2000).
23 Kongsri, S., Janpradit, K., Buapa, K., Techawongstien, S. & Chanthai, S. Nanocrystalline hydroxyapatite from fish scale waste: Preparation, characterization and application for selenium adsorption in aqueous solution. Chemical Engineering Journal 215, 522-532 (2013).
24 Kuivaniemi, H., Tromp, G. & Prockop, D. J. Mutations in fibrillar collagens (types I, II, III, and XI), fibril‐associated collagen (type IX), and network‐forming collagen (type X) cause a spectrum of diseases of bone, cartilage, and blood vessels. Human mutation 9, 300-315 (1997).
25 Kuo, S.-R. & Shao, K.-T. Species composition of fish in the coastal zones of the Tsengwen Estuary, with descriptions of five new records from Taiwan. ZOOLOGICAL STUDIES-TAIPEI- 38, 391-404 (1999).
26 Lahmann, C., Young, A. R., Wittern, K. P. & Bergemann, J. Induction of mRNA for Matrix Metalloproteinase 1 and Tissue Inhibitor of Metalloproteinases 1 in Human Skin in vivo by Solar Simulated Radiation¶. Photochemistry and photobiology 73, 657-663 (2001).
27 Nabavi, S., Ebrahimzadeh, M., Nabavi, S. & Bahramian, F. In vitro antioxidant activity of Phytolacca americana berries. Pharmacologyonline 1, 81-88 (2009).
28 Nagai, T., Araki, Y. & Suzuki, N. Collagen of the skin of ocellate puffer fish (< i> Takifugu rubripes</i>). Food chemistry 78, 173-177 (2002).
29 Nishimoto, M., Sakamoto, R., Mizuta, S. & Yoshinaka, R. Identification and characterization of molecular species of collagen in ordinary muscle and skin of the Japanese flounder< i> Paralichthys olivaceus</i>. Food chemistry 90, 151-156 (2005).
30 Olsen, D. et al. Recombinant collagen and gelatin for drug delivery. Advanced Drug Delivery Reviews 55, 1547-1567 (2003).
31 Park, H.-J., Zhang, N. & Park, D. K. Topical application of< i> Polygonum multiflorum</i> extract induces hair growth of resting hair follicles through upregulating Shh and β-catenin expression in C57BL/6 mice. Journal of ethnopharmacology 135, 369-375 (2011).
32 Pillai, S., Oresajo, C. & Hayward, J. Ultraviolet radiation and skin aging: roles of reactive oxygen species, inflammation and protease activation, and strategies for prevention of inflammation‐induced matrix degradation–a review. International journal of cosmetic science 27, 17-34 (2005).
33 Qusti, S. Y., Abo-khatwa, A. N. & Lahwa, M. B. Screening of antioxidant activity and phenolic content of selected food items cited in the Holly Quran. EJBS 2, 40-51 (2010).
34 Rittié, L. & Fisher, G. J. UV-light-induced signal cascades and skin aging. Ageing research reviews 1, 705-720 (2002).
35 Saiga, A., Tanabe, S. & Nishimura, T. Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. Journal of Agricultural and Food Chemistry 51, 3661-3667 (2003).
36 Sarmadi, B. H. & Ismail, A. Antioxidative peptides from food proteins: a review. Peptides 31, 1949-1956 (2010).
37 Sato, N., Leopold, P. L. & Crystal, R. G. Effect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia. Journal of the National Cancer Institute 93, 1858-1864 (2001).
38 Scharffetter–Kochanek, K. et al. Photoaging of the skin from phenotype to mechanisms. Experimental Gerontology 35, 307-316 (2000).
39 Sell, D. R. Ageing promotes the increase of early glycation Amadori product as assessed by< i> ϵ</i>-< i> N</i>-(2-furoylmethyl)-l-lysine (furosine) levels in rodent skin collagen: The relationship to dietary restriction and glycoxidation. Mechanisms of ageing and development 95, 81-99 (1997).
40 Shimada, K., Fujikawa, K., Yahara, K. & Nakamura, T. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of agricultural and food chemistry 40, 945-948 (1992).
41 St-Jacques, B. et al. Sonic hedgehog signaling is essential for hair development. Current biology 8, 1058-1069 (1998).
42 Sun, J., He, H. & Xie, B. J. Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. Journal of Agricultural and Food Chemistry 52, 6646-6652 (2004).
43 Uitto, J. Connective tissue biochemistry of the aging dermis. Age-related alterations in collagen and elastin. Dermatologic clinics 4, 433-446 (1986).
44 Wang, L. et al. Isolation and characterisation of collagens from the skin, scale and bone of deep-sea redfish (< i> Sebastes mentella</i>). Food Chemistry 108, 616-623 (2008).
45 West, M. D., Pereira-Smith, O. M. & Smith, J. R. Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Experimental cell research 184, 138-147 (1989).
46 Wu, H., Pan, B. S., Chang, C.-l. & Shiau, C. Low-molecular-weight peptides as related to antioxidant properties of chicken essence. Journal of Food and Drug Analysis 13, 176 (2005).
47 Wu, H.-C., Chen, H.-M. & Shiau, C.-Y. Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (< i> Scomber austriasicus</i>). Food research international 36, 949-957 (2003).
48 Yamaguchi, T., Takamura, H., Matoba, T. & Terao, J. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1, 1-diphenyl-2-picrylhydrazyl. Bioscience, Biotechnology, and Biochemistry 62, 1201-1204 (1998).

中文文獻
1 Chiang, Y.-H. & 江盈萱. 大目鮪魚卵蛋白質對人類皮膚纖維母細胞生長之影響. (2011).
2 Jhang, S.-P. & 張詩萍. 不同孵化時間點帶石斑魚受精卵萃取物對皮膚纖維母細胞之增生作用. (2010).
3 王建龍.，阿根廷魷及赤魷眼窩組織酵素水解物之抗氧化活性.，國立臺灣海洋 大學食品科學研究所碩士論文 (2002)
4 王綉雲，在人類上皮癌細胞A431 細胞中PHGPx 對EGF 所誘導c-Jun 表現的影響 (2008)
5 周玲, 翁文明, 李金亮 & 賴秋明. 鞍带石斑鱼胚胎發育及仔魚形態發育, 餌 料轉變的觀察研究. 中國農學通報 26, 293-302 (2010).
6 洪雅萍.，膠原蛋白產品的功效.，科學發展.，380期.，30-35