196 JO URN AL OF COSMETIC SCIENCE A simple lotion formula was preserved with I% PFI 056 and challenge tested using S. aureus, B. subtilis, E.coli, P aeriginosa, C. albicans, and A. niger [Figure 5.]. Plates were counted at 24 hours, 48 hours, and 21 days. [Figure 6.] The results clearly demonstrate the potential PF1056 as a viable part of a cosmetic preservative system. 100 Figure 1 50 25 12.5 6.25 3.13 1 56 0 76 0.39 mwmt •-Bacill;.. - -Escherichia - S�lmon ua I 1 -Shigella -Vibrio -Gandida . -- - --- Figure3 Figure 5 Test Formulation Water 91.2 % Yeast Extract 2.8 % Propylene Glycol 5.0% PF1056 1.0 % 100% BO"k 60% 40% 20% Organism Gram+ Gram- Yeast Mold Figure2 6 9 12 15 18 21 24 Figure4 Figure6 Challenge Test 24Hour 48Hour 21 Day 10 IO 10 10 10 10 10 10 10 8 X 10 1 IO IO

2005 ANNUAL SCIENTIFIC MEETING 4-(1-PHENYLETHYL) 1,3-BENZENEDIOL: A NEW HIGHLY POTENT LIGHTENING AGENT Gerhard Schmaus, Ph.D., Gabriele Vielhaber, Ph.D., Karin Jacobs and Helge Franke Symrise GmbH & Co. KG, Muhlenfeldstr. I, D-37603, Holzminden, Germany Introduction 197 There is an increasing worldwide demand for skin lightening active ingredients. Whereas a pale skin is the beauty ideal � Asian countries, Caucasian skin types from Europe and the US aim to treat pigment spots. Current skin lightening actives such as Kojic acid, Arbutin or Ascorbic acid derivatives have several disadvantages regarding their safety or stability. Since nature is without any doubt an inexhaustible source of inspiration for new actives for cosmetics, we recently focused within a continuously running natural product research program on different wood extracts and pure isolates, obtained thereof. The goal was to evaluate their tyrosinase inhibitory activity and to identify a new skin lightening agent, lacking those negative side effects described above. Materials and Methods Origin of test material: Phenolic compounds with different substitution patterns were either isolated from plant extracts by preparative liquid chormatography or they were synthesized. Mushroom Tyrosinase Assay: Tyrosinase reactions were performed in a 96well rnicroplate with 66.7 mM phosphate buffer (pH 6.8) containing 50 u/ml mushroom tyrosinase (EC.1.14.18.l, Fluka Chemie GmbH, Buchs, Switzerland) and test compounds. After pre-incubating at 37°C for IO min, L-3,4-dihydroxyphenylalanine (L-DOPA) was added to the mixture and the formed dopachrome was measured photometrically at 475 nm. The inhibitory effect of the test samples was expressed relative to the control and IC50 (50 % inhibitory concentration) values were calculated. Kojic acid was used as a reference. In vitro Toning Assay with B16V Mouse Melanoma Cells: B16V cells were seeded into 96well microplates. After adhesion (24 h) the medium was replaced by freshly prepared solutions of the test compounds at non cytotoxic concentrations (solvent: cell culture medium containing IO nM cx.-Melanocyte .Stimulating Hormone). After incubation for another 96 h, melanin was extracted with NaOH and the absorption at 400 nm was measured. In vitro Toning Assay with Pigmented 3D Epidermis Models: Freshly prepared solutions of the test compounds (solvent: PBS) were applied at non cytotoxic concentrations on the top of MelanoDerm™ MEL-300-B models (skin type IV). The test compounds were reapplied daily. After overall incubation time of 7 and 19 days, respectively, the melanin was extracted with soluene and the absorption at 400 run was measured. In Vivo Lightening Efficacy was studied in a human model on Asian skin. Test products and placebo formulation were applied 2 times daily. Subjects were requested to avoid any UV exposition during the test period. Measurement of the lightening efficacy was done by chromametry and visual assessment at t = 0 and after 28 days. Results and Discussion Within our natural products screening the tyrosinase inhibiting activity of ,,Scotch Pine" (Pinus sylvestris) heart wood extracts and pure isolates thereof attracted our specific interest. From this extract we isolated stilbene derivatives like Pinosylvin-3-O-methylether and Pinosylvin. Since we observed that such stilbene derivatives are fairly unstable under light condition, more stable partially hydrogenated derivatives like Dihydropinosylvin-3-O- methylether and Dihydropinosylvin, both also occurring in trace amouts in pine heart wood, as well as a couple of nature-derived substances with very similar structure like 4-benzyl-1,3-benzenediol, 2-( 1- phenylethyl) 1,3-benzenediol and 4-(1-phenylethyl) 1,3-benzenediol were synthesizd. The inhibitory activity of all compounds was measured with the same mushroom tyrosinase assay and their IC50 values relative to Kojic acid were calculated. Several compounds like Pinosylvin-3-O- methylether, Dihydropinosylvin-3-O-methylether, 4-benzyl-1,3- benzenediol and 2-(1-phenylethyl)I,3-benzenediol showed only weak to moderate tyrosinase inhibitory activity. 4-( 1-phenylethyl) I ,3- Figure 1: Structural formula of 4-( 1-phenylethyl)1,3-benzenediol