TOPICAL APPLICATION OF SOY MILK EXTRACT 477 duction in human keratinocyte culture maintained at room temperature for two years (unpublished data). These observations indicate that BE is safe and stable as a cosmetic ingredient. In the forearm skin of healthy volunteers, topical application of a gel formula containing 10% BE significantly improved skin elasticity (Ua/Uf) at month 3 (Figure 5). The Ua/Uf of skin treated with the gel formula tended to be higher than that of skin treated with the vehicle formula during the test period, but some parameters of skin viscoelasticity were not affected by topical application. Some parameters measured by cutometer are used in the evaluation of skin elasticity, and, one of them, Ur, is affected by skin thickness (1). Because the results using mouse skin showed no differences among Ur, other elastic parameters (Ua, Ur/Uf, and Ua/Uf), and Ur normalized to skin thickness (Ur*), our animal study employed Ur, the simplest and most typical parameter, as a measure of skin elasticity. On the other hand, the Ur, Uf, and Ua of forearm skin were markedly varied in each human volunteer. Thus, we employed Ua/Uf (Ua normalized by Uf) for evaluation of human skin elasticity. Skin thickness, age, and environmental conditions show considerable differences between animal model and human studies. Mainly due to the activity of Gen released from Gen-Glc during the fermentation of soy milk, topically applied BE increases the amount of cutaneous HA (15), which has specific rheological characteristics, good water-holding capacity, and certain important biological roles. Beta-estradiol (Est) increases the content of HA in murine skin (17), and its activity is more than 1000-fold higher than that of Gen and Dai (11). Also, Est has more than 1000-fold higher potential than Gen and Dai to bind with the beta-estrogen receptor (18), which is abundant in human skin (19). It is hypothesized that Gen in BE 11o 105 lOO -0- Gel formula with 10% BE -0- Vehicle formula -•- No treatment * 95 0 1 2 3 Application period (months) Figure 5. Effect of topical application of gel formula containing Bifidobacterium-fermented soy milk extract (BE) on elasticity of human forearm skin. Values are mean e SE (n = 3). *(p 0.05) indicates a significant difference from no treatment.

478 JOURNAL OF COSMETIC SCIENCE penetrates the epidermal barrier and the cellular membrane of keratinocytes and/or dermal fibroblasts like estrogen, and acts as a mild estrogen agonist to stimulate HA production via binding to the beta-estrogen receptor (11). Thus, it is indicated that the increase of cutaneous HA by BE is associated with not only hydration but also the thickening and recovery of changes in elasticity and viscoelasticity of the mouse skin. BE, containing 15.2 mM L-lactate (15), hydrated mouse skin together with an increase in HA content. Generally, lactate or lactic acid is added to moisturizing formulas at more than 1% (111 mM), indicating that the content of L-lactate (15.2 mM) in BE is not sufficient to express a significant moisturizing effect. Therefore, we speculate that hydration of mouse skin by topical BE is based on a synergic action of BE and L-lactate to increase cutaneous HA. CONCLUSIONS We have found that topical application of BE restores changes in the elasticity and viscoelasticity of mouse skin, increases the HA content, hydrates and thickens mouse skin, and inhibits wrinkle formation in ultraviolet B-irradiated hairless mouse skin (unpublished data). Also, topical application of a gel formula containing 10% BE slows the decrease in elasticity of human skin. In conclusion, this study supports the theory that a quantitative change in cutaneous HA markedly affects several skin features associated with cutaneous aging (2,3). BE is expected to become a new cosmetic ingre- dient with the potential to prevent cutaneous aging through the enhancement of HA production. REFERENCES (10) (1) Y. Takema, Y. Yorimoto, M. Kawai, and G. Imokawa, Age-related changes in the elastic properties and thickness of human facial skin, Br. J. Dermato/., 131,641-648 (1994). (2) R. Tammi, U. M. Agren, A. L. Tuhkanen, and M. Tammi, Hyaluronan metabolism in skin, Prog. HiJtochem. Cytochem., 29, 1-81 (1994). (3) M. O. Longas, Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging, Carbohydr. Res., 159, 127-136 (1987). (4) I. Ghersetich, T. Lotti, G. Campanile, C. Grappone, and G. Dini, Hyaluronic acid in cutaneous intrinsic aging, I,t. J. Dermato/., 33, 119-122 (1994). (5) A. Lundin, B. Berne, and G. Michaelsson, Topical retinoic acid treatment of photoaged skin: Its effects on hyaluronan distribution in epidermis and on hyaluronan and retinoic acid in suction blister fluid, Acta Derre. Ve,ereo/. (Stochh.), 72, 423-427 (1992). (6) A.M. Kligman, D. Dogadkina, and R. M. Lavker, Effects of topical tretinoin on non-sun-exposed protected skin of the elderly,J. Am. Acaa'. Dermato/., 29, 25-33 (1993). (7) T. Tadaki, M. Watanabe, K. Kumasaka, Y. Tanira, T. Karo, H. Tagami, I. Horii, T. Yokoi, Y. Nakayama, and A.M. Kligman, The effect of topical tretinoin on the photodamaged skin of the Japanese, Toho•J. Ex] . RIed., 169, 131-139 (1993). (8) J.J. Anderson, M. S. Anthony, J. M. Cline, S. A. Washburn, and S.C. Garner, Health potential of soy isofiavones for menopausal women, P,b/ic Health N,tr., 2,489-504 (1999). (9) S. Widyarini, N. Spinks, A.J. Husband, and V. E. Reeve, Isofiavonoid compounds from red clover (Trifolium pratense) protect from inflammation and immune suppression induced by UV radiation, Photochem. Photobiol., 74, 465-470 (2001). E. Q. Shyong, Y. Lu, A. Lazinsky, R. N. Saladi, R. G. Phelps, L. M. Austin, M. Lebwohl, and H. Wei, Effects of the isofiavone 4',5,7-trihydroxyisoflavone (genistein) on psoralen 1 plus ultraviolet A ra- diation (PUVA)-induced photodamage, Carci,oge,esis, 23, 317-321 (2002).