WATER-SOLUBLE ELASTIN AS A COSMETIC MATERIAL 21 material because it contains a high amount of hydrophobic amino acids and after its coacer- vation, high-molecular-weight polypeptides are produced. In this study, we focused on the moisturizing and whitening effects of HAPA-elastin. As shown in Figure 4, the application of HAPA-elastin to LabCyte resulted in a high water retention in the epidermis. The water content of the cells increased in a concentration- dependent manner until a concentration of 8.0% was reached, following which, the water content decreased (Figure 4c). These results indicate that, at an optimum HAPA-elastin concentration, moisture can be suffi ciently retained in the skin. Fractions 1 and 2 exhib- ited the same dose-dependent effects as HAPA-elastin. In particular, fraction 1 showed a signifi cant moisturizing effect, producing the highest water content of 77.2% at a 1.0% elastin concentration. Some reports have claimed that oral ingestion of elastin can sup- press water loss from the surface of the skin (3,4). These reports together with our results indicate that HAPA-elastin possesses a moisturizing effect, which is good for maintain- ing a healthy skin. Based on the present study, there is a possibility that fraction 1, owing to its high content of low-molecular-weight peptides, can penetrate the epidermis. This could possibly explain the relatively high water content observed in the LabCyte cells after treatment with frac- tion 1. Generally, low-molecular-weight substances ( 500 Da) can penetrate the skin (23) however, hyaluronic acid has a molecular weight of 400,000 Da, but it has been reported to penetrate both the epidermis and the dermis (24). It was reported that the water con- tent of the skin increased by approximately 10% after 3 weeks of hyaluronic acid inges- tion (25). Similarly, HAPA-elastin may be expected to exhibit moisture-retaining effects. Fraction 2 showed similar moisturizing effects that were comparable with HAPA-elastin, as shown in Figure 4. Since it is diffi cult for high-molecular-weight proteins to penetrate the epidermis, fraction 2 may produce a skin-covering effect and result in water retention, thereby moisturizing the skin. It can be suggested that the low-molecular-weight elastin fragments penetrate the skin to maintain its water content, whereas the high-molecular- weight fragments may remain on the skin and act as a barrier against water evaporation. However, there is a need to further explore this effect. The whitening effect of cosmetics has been widely studied in terms of effective inhibition of melanin production (26). Melanin is the main pigment present in the surface structures of vertebrates and is widely distributed in animals and plants. It is the principal factor re- sponsible for the skin color and pigmentation. The oxidation of tyrosine by enzyme tyrosi- nase, which involves the hydroxylation of tyrosine to L-DOPA and the oxidation of L-DOPA to dopaquinone, is the critical step in melanin biosynthesis. The melanin biosynthesis, which consists of rate-limiting steps, is regulated by tyrosinase at an initial stage (27). Thus, inhibition of tyrosinase activity inhibits the melanin synthesis. HAPA-elastin inhibited the synthesis of melanin (Figure 5). Kojic acid and polyphenols such as fl avonols are well- known tyrosinase inhibitors (28). Furthermore, some studies have reported that several proteins and peptides derived from animals and plants inhibit tyrosinase activity (29,30). This inhibition of tyrosinase activity is mainly caused by hydrophobic and aliphatic amino acids such as Val, Ala, Leu, and Met (29). HAPA-elastin contains a large amount of all the hydrophobic and aliphatic amino acids listed earlier except Met (Table I). HAPA-elastin inhibited the tyrosinase activity by 2.0–11.3% in this study. This can be attributed to the rich hydrophobic and aliphatic amino acid content of HAPA-elastin. However, the HAPA- elastin-mediated tyrosinase inhibition was lower than that mediated by other compounds

JOURNAL OF COSMETIC SCIENCE 22 such as a collagen (31). This gentle effect can be benefi cial in the development of useful biomaterials without any side effects. Surprisingly, a signifi cant difference in tyrosinase in- hibition was observed between fractions 1 and 2 (Figure 5). The average molecular weights of HAPA-elastin, fraction 1, and fraction 2, determined using gel fi ltration chromatogra- phy (Figure 3), were 4,700, 3,900, and 10,000 Da, respectively. The molecular weights were used to calculate the molarities of each sample. The molarities for a 10 mg/ml solution of HAPA-elastin, fraction 1, and fraction 2 were 2.1, 2.6, and 1.0 mM, respectively. Al- though the molarities of HAPA-elastin and fraction 1 at a concentration of 10 mg/ml were almost similar, HAPA-elastin showed relatively higher tyrosinase inhibition than fraction 1 did. Interestingly, fraction 2 showed relatively higher tyrosinase inhibition than HAPA- elastin and fraction 1 did, despite the low molarity of fraction 2 compared with either of them. It suggested that tyrosinase inhibition activity of elastin and elastin derivatives was not simply proportional to their molecular weight. It can be presumed that the observed difference in tyrosinase inhibition was due to the different sequences of amino acids in the various peptides and not merely due to the presence of different amino acids. It has been reported that no amino acid can, by itself, completely inhibit the activity of tyrosinase (32). Melanocytes have binding sites for the elastin-derived Val-Gly-Val-Ala-Pro-Gly (VGVAPG) hexapeptide. This hexapeptide is involved in bioactivities such as cell migration and prolif- eration (33). This may be the mechanism by which HAPA-elastin inhibited tyrosinase ac- tivity, which may subsequently promote skin cell turnover and result in whitening effects. In conclusion, HAPA-elastin is useful not only as a functional food but also as a cosmetic material, because it maintains the water content of the skin and inhibits tyrosinase activity. The fi ndings from this study may contribute to the development of better cosmetics with milder and more moisturizing effects on the skin. ACKNOWLEDGMENT We thank E&C HealthCare Ltd. and ECC Co., Ltd. for the fi nancial support to conduct this study. REFERENCES (1) C. M. Kielty, M. J. Sherratt, and C. A. Shuttleworth, Elastic fi bres, J. Cell Sci., 115, 2817–2828 (2002). (2) M. Hasegawa, T. Kawasaki, M. Saito, H. Ito, K. Okamoto, C. Arai, Y. Kashiwakura, and S. Yoshimura, Determination of aortic medial tissue elastin and collagen in stroke-prone spontaneously hypertensive rats, Nihon Ronen Igakkai Zasshi, 18(5), 336–341 (1981). (3) R. Yamaji, Development of Functional Materials for Diseases and Beauty in Women (CMC Publishing Co., Ltd. Tokyo, 2014), pp. 266–273. (4) T. Hayakawa, M. Sato, A. Saiga-Egusa, Y. Takahata, F. Morimatsu, and Y. Nomura, Effect of porcine arte- rial elastin peptide to the moisture content of mice skin, Nihon Chikusan Gakkaiho, 80(2), 215–222 (2009). (5) S. G. Harvey, J. R. Gibson, and C. A. Burke, L-cysteine, glycine and dl-threonine in the treatment of hypostatic leg ulceration: A placebo-controlled study, Pharmatherapeutica, 4(4), 227–230 (1985). (6) H. Ohara, S. Ichikawa, H. Matsumoto, M. Akiyama, N. Fujimoto, T. Kobayashi, and S. Tajima, Collagen- derived dipeptide, proline-hydroxyproline, stimulates cell proliferation and hyaluronic acid synthesis in cultured human dermal fi broblasts, J. Dermatol., 37(4), 330–338 (2010). (7) H. W. Spier and G. Pascher, Analytical and functional physiology of the skin surface, Hautarzt, 7(2), 55–60 (1956). (8) H. Sage and W. R. Gray, Studies on the evolution of elastin—I. Phylogenetic distribution, Comp. Bio- chem. Physiol. B, 64(4), 313–327 (1979).