198 JOURNAL OF COSMETIC SCIENCE benzenediol (Fig. I) was by far the most active compound within this compound class. It exhibited an ICso of 0.50 µM, thus reducing tyrosinase activity approximately 22 times more effectively than Kojic acid with an ICso= 11.05 µM under identical test conditions. Because of its excellent tyrosinase inhibitory activity, further detailed in vitro studies in melanocyte cultures and on 3D skin models as well as first human in vivo studies were performed with 4- ( l-phenylethyl)l,3-benzenediol. In the cellular lightening assay on B16V mouse melanoma cells, a pronounced efficacy of 4-( 1-phenylethyl) 1,3-benzenediol was found. It was by far the most potent inhibitor of melanin synthesis with an IC50 of 2.1 µM, whereas the ICso of 8-arbutin and Kojic acid was 67 µM and 440 µM, respectively, under identical test conditions. This means that 4-(1-phenylethyl)l,3-benzenediol was approx. 200 times stronger than Kojic acid in the cellular assay. In the pigmented 3D skin model, 4-(1-phenylethyl)l,3-benzenediol reduced the melanin content of pigmented 3D epidermis models at least IO times more efficiently than Kojic acid within a period of 19 days (Fig. 2). To confirm that 4-( 1-phenylethyl) 1,3-benzenediol sufficiently penetrates into skin and is effective also under human in vivo conditions a clinical study was performed with Asian subjects during a duration period of 4 weeks (Fig. 3). Product GS05048SL-A and -B (0.5 % 4-( 1-phenylethyl)1,3-benzenediol each) efficiently lighten the natural tone of Asian skin in vivo after 2x daily treatment for 28 days. 0.5 % 4-(1-phenylethyl)l,3-benzenediol is more efficient than 1.0 % Kojic acid. A more pronounced effect is achieved when 4-( 1-phenylethyl) 1,3-benzenediol is directly incorporated into an aqueous gel formulation with low oil phase content (GS05048SL-A). Pre-solubilising 4-(1- phenylethyl)l,3-benzenediol in a formulation with high oil phase content (GS05048SL-B) was less effective. Despite the considerable concentration gradient between the applied formulation and the skin itself, 4-(1- phenylethyl)l,3-benzenediol is obviously only poorly released from the oil phase because of it high lipophilicity Conclusion Systematic screening of plant extracts, isolated natural products and nature-derived synthetic derivatives thereof for potential skin lightening activity showed, that 4-(1-phenylethyl)l,3-benzenediol, a dihydroxylated diphenylmetbane derivative, possesses potent tyrosinase inhibitory activity. Further investigations on the lightening activity in a cellular lightening assay (Bl6V mouse melanoma cells) and on pigmented 3D skin models (MelanoDerm.™ skin type N). delivered the unequivocal proof in vitro, that 4-(1-phenylethyl)l,3-benzenediol is one of the most potent lightening agents ever reported. First studies on Asian skin confirmed that it also shows good lightening activity in a human in vivo situation. Figure 2: Lightening activity of 4-(1-phenylethyl) 1,3-benzenediol on pi � ented 3D epidermis models (MelanoDerm , skin type N) photos were taken after 19 days of daily treatment with Kojic acid and 4-(1-phenylethyl) 1,3-benzenediol (Bio377), respectively. Reduction oflMlanln ReducllonL.....---..,__.,;;;.._.,._.__oiliiiii__, oflMlanln Figure 3: In vivo efficacy of 4-(1-phenylethyl) 1,3- benzenediol on non-irradiated skin (mean chromameter readings to baseline after 28 days) 4 cosmetic formulations were studied: GS05048SL-A (0,5% 4-(1-Phenylethyl)l,3- benzenediol) GS05048SL-B (5% of a 10% solution of 4- (1-Phenylethyl)l,3-benzenediol in neutral oil) GS05048SL-C (1 % Kojic acid) GS05048SL-D (placebo formulation). 1.1•----------- .... ... • .... lr 11 2.11 ,: , .. i· .... ..• •UI - ... u, 'lM

2005 ANNUAL SCIENTIFIC MEETING 199 IMPROVING EFFICACY OF SKIN LIGHTENING PRODUCTS BY USING MULTIPLE PIGMENTATION INHIBITION MECHANISIMS Di Qu, Ph.D., Jesse C. Leverett and John V. Scimeca R&D - Skincare & Cosmetics, Access Business Group, Ada, MI Summary: The efficacy of skin lightening products containing multiple pigmentation inhibition mechanisms was examined via in-vitro and in-vivo studies. The results were compared with that of formulas containing only a single inhibition mechanism. The multiple mechanism products showed higher reduction in total melanin than the single mechanism ones in the in-vitro efficacy studies where cultures ofreconstructmed human epidermis were used. In- vivo studies confirmed the advantage of such an approach. More than 30% reduction in mottled pigmentation and 50% reduction in dark skin tone were achieved after a 12-week use period. This study shows that multiple inhibition mechanism improves the skin lightening efficacy of a product. Introduction: Skin lightening products take a large market share in Asian countries. Consumers continue to demand for more efficacious products which often marketed under an OTC-equivalent product category, such as quasi-drug cosmetics in Japan, special use cosmetics in China, and functional cosmetics in Korea. The levels and functions of ingredients under this category are usually regulated. To increase product efficacy, utilization of multiple pigmentation inhibition mechanisms provides a way [I]. Melanogcnesis of the skin involves multiple pathways and skin lightening can be achieved through many mechanisms other than tyrosinase inhibition [2]. In this study, we formulated products with ingredients exhibiting various pigmentation inhibition properties such as tyrosinase inhibition, rnelanosome transfer inhibition, a-MSH inhibition, eumelanin-pheomelenin ratio alteration, and existing melanin removal. In-vitro and in-vivo studies were conducted to confirm the efficacy. Material and Methods: Tyrosinase activity assay [3] was used to screen melanin inhibition activity of various ingredients. In-vitro efficacy was tested using a 3-D model of reconstructured human epidermis with melanocytes (Melanoderm from MatTek). The samples of skin model were incubated with various ingredients or products three times a week for two weeks. Total melanin, surface coloration, cell liability, and microscopic images were examined after that [ 4 ]. In-vivo studies were conducted via clinical tests in an independent testing lab on the face of 60 female Asian subjects. Skin lightening parameters such as skin clarity, skin tone, and mottled pigmentation were clinically graded based on a IO point scale. Color pictures were taken at the time of evaluation. Results: Various ingredients were screened for pigment inhibition activity. Some significant results are listed in Table I where tyrosinase inhibition activity (column 3) was compared with total melanin and surface coloration results (columns 4 & 5). Notice the trend in column 3 do not translate into column 4 indicating some ingredients worked under mechanisms other than tyrosinase inhibition. For instance, while citrus unshiu extract showed a much lower tyrosinase inhibition than bearberry extract and the botanical blend, their levels ofinhibition in melanin production in 3-D skin model were comparable. For wheat germ extract, it had a 10% inhibition in tyrosinase activity. However, it inhibited about 20% melanin production in 3-D skin model. Its coloration score was also very low indicating not much eumelanin was produced on the surface of the skin model. These results supported literature and vendor data, as citrus unshiu extract is known to inhibit melanin production in addition to tyrosinase inhibition. Wheat germ extract is believed to switch the melanin production from eumelanin (a dark/brown melanin) to pheomelanin (a light/yellow melanin) while the content of lectins in the material inhibits transfer ofmelanosomes from the melanocytes to the keratinocytes [5]. Comparative efficacy data were obtained when incorporating ingredients with various pigment inhibition mechanisms into some cosmetic formulas. Their efficacy of melanin inhibition was measured against the formulas with single inhibition mechanism. In both systems a 3% magnesium ascorbyl phosphate was used to establish an OTC-equivalent status. Figure I shows the total melanin content of the 3-D skin models after treated with various products for two weeks. It is seen that the single mechanism formulas had an average reduction in total melanin of Table 1 Cone. T� I�(%) Canlral (C&Aft medium) - - Mg-Ascabyl Phosphala 3% - Na-AsccrbylPholphale 3% - 8-b9r,yEldraf;I 2% 99.7 Bolanteal llend4 2% 91.0 BdalicalExtraC12 01%&0lids 703 Rasptiarry Ellhel 2% 393 Otnm Ulllliu EllllaCI 2% 24.2 'M.-GermEldra:I 2% 11.4 T01111Malril1 Coloration (%Conlrol) (1-5 scale) 1000 3 530 2 49.0 2 &20 2 690 3 450 2 87 0 4 62.0 1 81.0 1 Figure 1. Comparison of In-Vitro Elllcacr Control Toner Lotion Cream 0 20 40 60 80 Total Melanin (of% Control) 100 Cl Multiple mechanisms ■ Single mechanism