178 JOURNAL OF COSMETIC SCIENCE 1.2 0.8 0.6 0.4 0.:2 0.0 0 i i i i i i i i i i i . 50 1 oo Time / min Figure 4. Time course of the changes in the dynamic elastic modulus of guinea pig stratum corneum treated with a combination of various humectants. The y-axis shows the ratio of the dynamic elastic modulus, E'•/E'•_o, where E'• is the dynamic elastic modulus of stratum corneum treated with a test solution at the specific time after the application, and E't_ o is that of non-treated stratum corneum. The stratum corneum was treated with 2 pl of water or AcHA solution. The x marks represent the modulus of the stratum corneum treated with water. The open circle (¸), 0.2% HA the closed upside-down triangle (•'), 5% glycerol the open square (•]), 0.1% HA with 5% glycerol the closed triangle ('), 0.1% AcHA with 5% glycerol the closed circle (0), 0.2% AcHA. the AcHA-treated skin shows a remarkable improvement because skin grooves and skin ridges can be clearly observed as compared with the other test pieces. DISCUSSION To evaluate the skin-softening effect of AcHA, the dynamic elastic modulus of stratum corneum was measured. The method reported by Takahashi eta/. can evaluate quanti- tatively the skin-softening effect in vitro (19). They reported a clear change in the dynamic elastic modulus of the stratum corneum after the application of 10% aqueous glycerol solution. Although stratum corneum was plasticized to the same extent as pure water, the plasticization of stratum corneum treated with the glycerol solution lasted for a longer time than that obtained with water. This effect is due to its low evaporation rate. Upon the use of AcHA with the DS of hydroxyl groups from 2.6 to 3.8, we found that a value between 3.0 and 3.5 maintained the highest skin-softening effect for a long period of time. The important point to notice is that AcHA shows singly an excellent skin-softening effect. The skin-softening effect of AcHA was significantly more effective than that of glycerol. It can be believed that this strong skin-softening effect can be attributed to its amphiphilic properties following the introduction of acetyl groups to

EFFECT OF AcHA ON STRATUM CORNEUM 179 o o o • ! ! ! I i i i I I I i i i 0 50 100 150 Time / min Figure 5. Effect of 0.2% AcHA on human skin surface conductance. The closed circle (0) and the closed triangle (&) represent the conductance of the skin treated with AcHA and HA, respectively. Each data point represents the mean of five trials. The bars indicate the standard deviation. **p 0.01 *p 0.05 statistical significance (Student t-test). HA. Since the DS value of AcHA reflects its hydrophobicity/hydrophilicity balance, there may be found a relationship between that the skin-softening effect and the affinity of AcHA to stratum corneum. Therefore, as the results suggested, the subsequent experiments were carried out using an effective AcHA of which the DS was 3.5. More noteworthy is that the concomitant use of glycerol with AcHA or HA enhanced the skin-softening effect that was already observed with AcHA or HA alone. Ozawa et al. reported that HA could produce the synergic effect of skin softening when HA was used with a small amount of glycerol (13). Moreover, the synergic effect of skin softening of AcHA is more highly effective than that of HA. It is conceivable that AcHA or HA may form an aqueous membrane, which is similar to an oil seal, on the surface of stratum corneum and may significantly enhance the intrinsic skin-softening effect of glycerol. To estimate the moisturizing effect of AcHA in vivo, the skin surface conductance that reflects the water-holding capacity was measured. The water-holding capacity of the human skin that was increased by AcHA was significantly greater than that by HA even at 120 min after the application (Figure 5). Since the treatment with AcHA on stratum corneum enhances its water-holding capacity, there is a possible interaction between AcHA and stratum corneum. This enhancement of water-holding capacity with AcHA suggests a possible mechanism for its excellent skin-softening effect. Freijlarsson et al. showed that a significant amount of an amphiphilic polymer was adsorbed by a hydro- phobic methylated silica surface (24). Because (a) the surface of skin shows hydrophobic properties due to sebum and (b) AcHA appears apparently more hydrophobic than HA,