j. Cosmet. Sci., 50, 171-184 (May/June 1999) Skin-softening effect of acetylhyaluronate on stratum corneum T. OKA, M. UEMURA, N. UENO, and T. YANAKI, Shiseido Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa, 223-8553Japan. Accepted for publication April 30, 1999. Presented in part at the Third Scientific Conj•rence of the Asian Societies of Cosmetic Scientists, Taipei, Taiwan, May 24, 1997. Synopsis Sodium hyaluronate (HA), which is made from safe biological sources, has a very high moisturizing effect. To endow HA with precious functions, we synthesized varieties of HA derivatives and evaluated their usefulness for cosmetic products. After numerous investigations for seeking useful HA derivatives, we discovered a novel HA derivative, sodium acetylhyaluronate (AcHA), which has superb moisturizing and excellent skin-softening effects on stratum corneum. To investigate the usefulness of AcHA in vivo, the efficacy of AcHA on skin physiology was evaluated. It was found that AcHA raised water content in stratum corneum more markedly than did HA. Apparently, AcHA could enhance the intrinsic water-holding capacity of the stratum corneum. The results suggested that there was an interaction between AcHA and stratum comeurn and that this could induce the strong skin-softening effect. This agreed with the fact that AcHA is an amphiphilic polymer having an effect of lowering surface tension. The results also suggested that AcHA adheres to human skin, enhances the water-holding capacity of the stratum comeurn, and shows a strong skin-softening effect. Upon the use of AcHA in a cosmetic formulation, it was observed that a lotion containing 0.2 w/w% AcHA could improve dry skin condition. INTRODUCTION It has long been recognized that epidermis is the main obstruction to free movement of molecules across skin and that stratum corneum is the principal barrier within epider- mis. It is well known that the stratum corneum is a remarkably tough and resilient membrane consisting of approximately 20 stacked layers of flattened and dead epidermal cells (1-5). The lipid composition of human stratum corneum displays striking regional variations that could reflect differences in stratum corneum thickness, turnover, des- quamation, and/or permeability, and the stratum corneum lipids play an important role in the retention of water in skin (6,7). Normal human stratum corneum contains 10 - 20 w/w% water, which maintains essentially healthy and fresh skin. Water also influ- ences the flexibility of the stratum corneum (8-11). However, the stratum corneum always has a tendency to lose its normal water content due to aging, surroundings, physical constitution, and other factors, and the reduction of water content in stratum 171

172 JOURNAL OF COSMETIC SCIENCE corneum results in dry skin and the formation of wrinkles. Therefore, it is useful to apply a humectant to skin to maintain the normal water content (12,13). As a humectant, sodium hyaluronate (HA) is a biopolymer derived from cartilage, vitreous humor, and synovial fluid, and shows useful physical characteristics such as high viscosity, viscoelasticity, high thread-forming ability, and high biocompatibility (14). For example, HA is used as a thickener in aqueous solvent systems, as a coating enforcement for liposomes, as an embedded base for organisms, capsule bases, and so on. On the other hand, many kinds of HA derivatives were synthesized and evaluated for useful functions. Abatangelo et •/. reported that sulfated hyaluronic acid derivatives showed no cytotoxic effects on mouse fibroblasts and that the introduction of sulfate groups along the hyaluronic acid chain made the macromolecules resistant to enzymatic digestion (15). In our study, to endow HA with precious functions, we synthesized a variety of HA derivatives and evaluated their usefulness for cosmetic products. After numerous investigations, we eventually discovered a novel HA derivative, sodium acetylhyaluronate (AcHA), which increases HA's moisturizing effect and has a very high skin-softening effect on stratum comeurn. In this study, we report the synthesis of AcHA that could show excellent moisturizing and skin-softening effects. We also report an i, vivo treatment effect of AcHA, which was used as an active ingredient in a lotion for dry skin condition. EXPERIMENTAL MATERIALS Chemicals used in this study were cosmetic grade or reagent grade without further purification. One 6-week-old male Hartley guinea pig (Japan SLC, Shizuoka, Japan) was housed individually in a wire mesh cage (260 x 380 x 180 mm, Japan Clea, Tokyo) and kept under standard laboratory animal conditions, which were maintained at a tem- perature of 21øC to 25øC with 40% to 70% relative humidity (RH). The room air was ventilated l0 to 15 times per hour automatically and a 12 hr/12 hr light-dark cycle (lighting 07:00-19:00) was imposed. The animal was fed •a•/ib•t•m with a commercial diet (RC-4, Oriental Yeast Industry, Tokyo) and sterilized water through an automatic water supply system. The animal was deeply anesthetized with pentobarbital sodium and the skin was dissected out. Animal care and experiments were performed in accor- dance with the guidelines of the National Institute of Health. The stratum corneum was removed from the dorsal skin by the heat-trypsinization method described by Chris- tophers and Kligman (16,17). The sheets of the stratum corneum were stored in a vacuum desiccator over silica gel. The water used in this study was purified by an ion-exchanged water purification system Type G-10B (Organ0, Tokyo, Japan). SYNTHESIS OF AcHA In a 300-ml conical glass flask, a mixture (100 ml) of acetic acid and acetic anhydride (1:4 - 4:1 by volume) was placed, and 6 g of fine powder of Biohyalo-12 © (sodium hyaluronate molecular weight: approx. 1,200 kilodaltons Shiseido, Tokyo) was gradu- ally added with stirring. Then, 3 - 5 ml of concentrated sulfuric acid as a catalyst was