200 JOURNAL OF COSMETIC SCIENCE about 20% whereas the multiple mechanism formulas achieved about 35%. Figure 2 shows the images of surface melanin on the 3-D skin models where (A) is the control, (B) from a single mechanism formula, and (C) from a multiple mechanism formula. We see that much less melanin is shown on (C) than on (B) which indicates the formula with multiple mechanisms is more efficacious than that of a single one. The morphology of the melanocytes is shown in Figure 3. The melanocytes treated with the control (A) were full of melanosomes. Much less melanosomes and melanocytes were seen in the product treated samples. The shape of the melanocytes became thinner, and the color of their dendrites was more transluscent. The multiple-mechanism sample (C) had clearly less melanin and melanocytes than the single-mechanism one (B) indicating a better skin lightening efficacy. The in-vitro results were then confirmed through a couple of clinical studies. As shown in Figures 4- 6, the multiple mechanism formula out performed the single mechanism formula in a 12-week study in the U.S., and a 4-week study in Japan. Figure 7 shows the before and after photos comparing the overall skin conditions, such as mottled pigmentation, skin tone and clarity, of a panelist's face. Figure 4. Figure 5. IIMW � in u S.-Da1SlanTone 0% ,...., _____ -y-. -5% Figure 6 2....... 8-w.k 12-week II Single Mec:hanam ■ Multiple Mecnam..,. lnvlvo Elllcacy Study ,n J;apen {4 --■) Mottled P,grnenta!JOn Skin Tone II Sinfle Mechanism ■ Mulltple Mechaniaml 2-Wlillk IISuigla� Figure 7. Sample pictures from a 12-week in-vivo study in the U.S. 8-weak 12-week ...... MechaniarTa Discussion: It was noticed from the in vitro study that single ingredient performed better than a complete formula. We believe the phenomenon was caused by the interactions among various ingredients as we had seen certain ingredients promoted pigmentation in the in-vitro study and we might have used some ingredients in a formula for other functions without knowing their counteractive effects in pigment inhibition. Conclusion: Formulas with multiple pigment inhibition mechanisms show better efficacy than single mechanism ones in our in-vitro and in-vivo studies. The combined action is clearly better than a single action. This approach provides a meaningful way to increasing performance of skin lightening products in the OTC-equivalent category. Reference I. Petit, L., et al. International Journal of Cosmetic Science, 54( 4 ), p 169-181. 2003 2. Giuseppe Prata, Melanins and Melanogenesis. Academic Press. 1992. 3. Sigma-Aldrich Quality Control Test Procedure, Enzymatic Assay of Tyrosinase (EC.1.14.18.1) 4. Bessou-Touya, S., et al. J. Invest. Dermatol., 111 (6), p 1103-1108. 1998 5. Minwalla, L., et al. Pigment Cell Res., 14(3), pl85-194. 2001

2005 ANNUAL SCIENTIFIC MEETING 201 BOTANICALS FROM TR ADITIONAL CHINESE MEDICINE (TCM) AND THEIR POTENTIAL AS NATURAL ANTI-IRRITANTS IN COSMETIC APPLICATIONS Gabriele Vielhaber, Ph.D., Imke Meyer, Holger Joppe, Helge Franke and Martina Herrmann, Ph.D. Symrise GmbH & Co. KG, Muhlenfeldstr. 1, D-37603, Holzminden, Germany Introduction Many plants are known that are traditionally used for their anti-inflammatory activity. In most cases, the evidence for the anti-inflammatory activity is derived from animal models or immunocompetent cells but efficacy proof on human skin cells is scarce. In skin, interleukin-I (IL-la) is a key mediators in the inflammatory process. Increased cutaneous levels of IL-la are found in conditions with a weakened epidermal barrier such as aged skin, dry skin or sensitive skin as well as in photodamaged skin [1-4]. However, specific IL-la inhibitors are rare. We have evaluated the IL-la inhibitory efficacy of plant extracts from traditional chinese medicine (TCM) on human keratinocytes. Since reactive oxygen species are a major trigger in the inflammatory cascade, we have also investigated whether the radical scavenging activity of the extracts significantly contributes to their anti-irritant activity. Materials and Methods IL- I a Assay: HaCaT keratinocytes were seeded into 96well microplates and grown to 90-100 % confluency. The medium was exchanged against solutions containing the test compounds. After 1 h, IL-Ia biosynthesis was stimulated by addition of 0.4 µM A23187 (Sigma, Taufkirchen, Germany). After a further 6 h, the cells were lysed with 1 % Triton X 100 in PBS and the IL-Ia content was measured with the Human IL-la ELISA Kit from Perbio Science (Bonn, Germany). Oexamethasone was used as a reference (1 µM = 43% IL-la inhibition). LOH Assay: Lactate dehydrogenase (LOH) activity in the supernatant was determined by use of the Cytotoxicity Detection Kit from Roche (Mannheim, Germany). ABTS Assay: Samples were incubated for 10 min at 30°C with the 2,2'-Azino}!is-(3-ethylbenzo!hiazoline-6- §.Ulfonate (ABTS) radical cation which was prepared by reaction of ABTS with potassium persulfate. The degree of decolorization corresponds to the radical scavenging activity and was determined spectrophotometrically at 734 nm . Results and Discussion For measurement of IL-la the human keratinocyte cell line HaCaT was used since these cells were easier to handle than primary keratinocytes. To assure that the enhanced levels of IL- I a were not due to cytotoxicity, a LDH assay with the supernatant was performed in parallel to the anti-irritant assay. Japanese honeysuckle flowers (Lonicera japonica), kudzu roots (Pueraria lobata) and Japanese pagoda tree flowers (Sophorajaponica) were found to possess considerable anti-irritant activity. At 1 %, the extract preparations containing 3% of dry extract (trade name: Extrapone®) inhibited IL-la by 40 % (Honeysuckle) and 29 % {Pueraria, Sophora), respectively. Additionally, 0.1 % of the extracts also exhibited potent radical scavenging capacity in the cell-free ABTS assay with a maximum antioxidant capacity of 25 % (Honeysuckle), 81 % (Pueraria) and 22 % {Sophora), respectively. To elucidate the respective active principles of the TCM extracts, phytochemical characterization was performed by HPLC-OAD-MS analysis. As major UV-detectable components chlorogenic acid (0.08%), puerarin (0.17%), and rutin {0.10%), respectively were identified. However, the anti-irritant and radical scavenging activity of the single compounds and the extracts did not correlate. Only in the case of Sophora the radical scavenging activity could be fully ascribed to its major component rutin.