308 JOURNAL OF COSMETIC SCIENCE (A) (B) Figure 1. (A) Optical photoinicrograph ofchitosan immobilized protease (B) Scanning electron micrographs of the surface of chitosan immobilized protease. Thermal Stability of Chitosan Immobilized Enzyme in a cosmetic formulation The water-soluble chitosan immobilized protease stability provided 80% retention of the activity at 40D after 2 months in cosmetic products(Fig. 2). And when stored in 4D the stabilitics of immobilized enzymes were more increased than high temperature. However, free protease in a cosmetic formulation was severely inactivatcd in a few days. Skin color measurement When daily applied to the skin, cream formulation containing 1% chitosan immobilized enzyme, was more effective in exfoliating stratum comeurn of forearm than that containing 5% hydroxy acid(Fig. 3). 120% lOO% 80% 60% 40% 20% 0% -- -- 10 20 30 40 50 60 Days Figure 2. Thermal stability of chitosan immobilized and free proteases in cosmetic formulation when stored at 4 ( : immobilized, : free) and 40 ( : immobilized, : free). • 15 • lO t• 5 0 A B C O E sam p'• Figure 3. The effect of immobilized protease on skin coloration index. A:gel base, B:1% free protease, C:1% chitosan immobilized protease, D:1% papain, E:5% AHA CONCLUSIONS A new protease produced by Bacillus' sp. HB-5 was immobilized on various carriers by different methods of immobilization. A new procedure for entrapping enzyme into chitosan was developed and investigated the properties of immobilized enzymes. The stability of immobilized enzyme were prepared by physical adsorption on chitin, ionic binding onto DEAE-cellulose, covalent binding on CNBr-Agarose, and entrapment in alginate had the highest activities. The chitosan immobilized protease had the higher resistance to high temperature than free ones. The chitosan immobilized protease stability provided 80% retention of the activity at 40 after 2 months in cosmetic products. Cream formulation containing 1% chitosan immobilized protease, exhibited 20% increase on the skin coloration index. REFERENCES 1. Takuji M. et al. Jpn. J. Soc. Cosmet. Chem., 27, 276 (1993). 2. Watanebe, T. et al. J. Solid-Phase Blochem., 3, 161 (1978).

2002 ANNUAL SCIENTIFIC SEMINAR 309 Persea Gratissima (AVOCADO) STEROLS DECREASE UVB INOUCEO PROINFLAMMATORY MEDIATORS Laurie B. Joseph, Ph.D. and Kathy Koukouras Croda, Inc., North American Technical Center, Edison, NJ 08837 •lntroduction It is well known in the literature that the exposure of skin to low levels of UVB radiation can cause the production of proinflammatory chemical mediators, which can cause premature aging. Changes in the production of these mediators, cytokines and prostanoids, in the skin can decrease or increase the potentially damaging effects of UVB irradiation. The ability to modulate the production of these inflammatory mediators through consumer use of substances such as Avocado derived sterols can, in turn, decrease the potentially damaging effects of UVB irradiation. In this series of experiments we investigated the effects of phytosterols (up to 80% sterol) on the production of pro- inflammatory mediators IL-lct, PGE2, and IL-8 caused by low dose UVB exposure (1). IL-lct is a known mediator of fever, inflammation, and an activator of T lymphocytes. It is a nonspecific cytokine produced by epithelial cells, activated macrophages and dead cells. Prostaglandin E 2 is produced when cell membrane phospholipids are perturbed. PGE2 is a nonspecific mediator of inflammation. IL-8 is a member of the Chemokine Family of Interleukins (a chemical mediator which causes cell migration into the site of inflammation), and is produced by various cell types including epithelial cells. IL-8 can recruit acute as well as chronic inflammatory cells (neutrophils and lymphocytes, respectively), and has been implicated in psoriasis (2,3). Materials and Methods A thin film of lotion containing Persea Gratissima (Avocado) Sterols, or lotion base was applied to EpiDerm TM (4) 24H prior to UVB exposure (400joules/m2), and remained on the tissue for a maximum of 48H. At 12, 24, and 48H post UVB irradiation, cells were sacrificed to determine cell viability (4), cytokine (5,6) and prostaglandin production (7). Results Pre-treatment of EpiDerm with either lotion was not cytotoxic (Figure 1). By 12 and 24 H post-UVB exposure there was an across the board decrease in the cell viability of 5% to 95%, as compared to the matched controls. After 48 H UVB exposure, both lotions protected the cells as compared to the EpiDerm exposed to UVB alone. The production of PGE2 was suppressed for both lotions at 12H (Figure 2). At 48H, there was no significant difference in the production of PGE2. At 12H post UVB exposure there was a complete suppression of IL-lct (Figure 3). By 48H post UVB exposure, there was a difference (p=0.1) in the IL-1 ct levels in EpiDerm TM pre-treated with lotion containing phytosterols vs. those treated with either the lotion base or UVB alone. IL-8 production was not completely suppressed 12H after UVB exposure (Figure 4). Twenty-four and 48H after exposure, the Avocado containing lotion had a profound effect on IL-8 production. Interestingly, 48H after UVB exposure, IL-8 production by untreated unexposed control tissue and tissue treated with phytosterol containing lotion plus UVB was not significantly different. From these experiments, we can conclude: 1. 2. 3. 4. Conclusions Avocado sterols are neither cytotoxic nor phototoxic. Neither lotion had an effect on reducing the PGE2 production at 24 or 48 H post UVB exposure. There was no significant difference in PGE2 production among the 3 tested treatments. Application of Avocado lotion prior to UVB exposure decreased IL-1 ct production at 48 hours to 468 pg/ml as compared to either lotion or UVB alone (1242 pg/ml and 1464 pg/ml, respectively). Finally, 48 hours post UVB exposure, the tissue treated with Avocado lotion prior to UVB exposure decreased IL-8 production to the level of the 48 H untreated matched control.