364 JOURNAL OF COSMETIC SCIENCE Figure 2. Cytotoxic effect of whitening agent on Melanoderrn. A, C: cross sections. B, D: top view. A, B: vehicle control. C, D: whitening agent. All subjects applied both anhydrous and water-base products containing kojic acid and a retail product containing hydroquinone to their forearms twice daily. Chromameter (Minolta CR200) measurements of skin darkness (L*) were taken at each of the test sites before product application and again 24 hours later. RESULTS AND DISCUSSION Pt variety of whitening agents at different concentrations was tested on melanocytes for an initial screening. After the initial screening, ingredients were selected for further analysis. One percent ascorbic acid, 1% magnesium ascorbyl phosphate, 3% lactic acid, 1% kojic acid, and a plant extract were selected and tested on Melanoderm in a non-ionic aqueous base (Figure 1). After the treatment with whitening agents, Melanoderm was treated with L-DOPA as described in Materials and Methods and examined under the microscope. The intensity of melanocyte staining in Melanoderm depends on the tyros- inase-inhibitory activity of the treated material. Lactic acid (Figure 1C) and kojic acid (Figure 1D) treated samples showed a dramatic decrease in tyrosinase activity as com- pared to other whitening agents. The MTT assay and dendritic and healthy melanocytes in Melanoderm suggested that these whitening agents were not cytotoxic. However, a whitening agent (Figure 2C,D) showed necrosis of melanocytes and keratinocytes, in- dicating a cytotoxic effect on Melanoderm. To determine the relative activity of these whitening agents, melanin was extracted from the treated Melanoderm for quantifica- tion. A quantitative analysis (Figure 3) showed 48%, 46%, and 33% inhibition by kojic acid, lactic acid, and MAP, respectively. The plant extract did not show any inhibitory activity. Kojic acid was found to be the most effective however, its activity was lost after a month when it was held at room temperature in the aqueous non-ionic base (Figure 4). To confirm our results and check the stability of kojic acid, we developed a new HPLC

SKIN-WHITENING PRODUCTS 365 Figure 3. Effect of whitening agents on tyrosinase inhibition in Melanoderm. Tissues were treated with whitening agents. Melanin was extracted from the tissues after L-DOPA treatment.