JOURNAL OF COSMETIC SCIENCE 366 obtained on prescription for the treatment of melasma and other hyperpigmentary disorders (5–7). For this reason, there is an increasing concern about developing and fi nding new depigmenting agents that are effective and not cytotoxic so they can be used as an alterna- tive to hydroquinone (6). The use of synergistic inhibition would be of interest to increase the inhibition and to reduce the dose needed to produce the desired effect too. Melanin synthesis can be inhibited at different levels of the pathway. This has led to the idea that compounds with different mechanisms of action can be combined to obtain synergistic effects, so we can get a greater reduction of the melanin synthesis. Regarding the background in the combination of some of these agents, the one that has proved to be the most effective is a standard triple combination cream, which contains hydroquinone (which inhibits tyrosinase activity), an exfoliating agent (which stimulates the epidermal turnover and reduces the oxidation caused by hydroquinone), and a corticosteroid to re- duce infl ammation. Clinical trials have been performed with this cream and it has shown a good effectiveness. Arbutin, a derivative compound from hydroquinone, inhibits tyrosinase in a competitive way (as an alternative substrate of tyrosine or L -3,4-dihydroxyphenylalanine [L-DOPA]), without affecting the expression of the enzyme (3,8–11). Kojic acid, which is a fungal metabolite obtained from Aspergillus or Penicillium, also inhibits tyrosinase activity by chelating copper atoms, which are essential for its function, in the active site of the en- zyme. It also has antioxidant properties, preventing the conversion of o-quinone to L -DOPA, which will form melanin at the end (8,10,12–14). Azelaic acid, synthesized by the fungus Pityrosporum ovale, acts as a competitive inhibitor of tyrosinase, besides affect- ing the mitochondrial metabolism and having an antioxidant effect neutralizing free radicals. It also has a certain anti-proliferative and cytotoxic effect for melanocytes by inhibiting enzymes involved in DNA synthesis (2,10,15,16). α-Lipoic acid (or thioctic acid) also has a double action, as azelaic acid and kojic acid. On one hand, it inhibits the expression of microphthalmia-associated transcription factor (MITF), thus inhibiting the expression of the melanogenic enzymes (Tyrosinase, TYRP-1, TYRP-2). On the other hand, it has antioxidant properties in its oxidized form, although it is more antioxidant in its reduced form (dihydropholic acid) (17–20). In this work, some widely used depigmenting cosmetic agents, which are arbutin, kojic acid, azelaic acid, and α-lipoic acid (see Figure 1), will be evaluated individually and in different combinations to look for synergistic effects between them. Mechanisms of action will be studied with a cell-free mushroom tyrosinase assay, although for some of them it has been already suggested (1,8,9). Pair combinations will be done to assess synergistic effects over mushroom tyrosinase and melanin content. For the determination of melanin content, maximum non-cytotoxic concentrations will be obtained for these compounds. METHODS MATERIALS Arbutin, kojic acid, azelaic acid, α-lipoic acid, L -DOPA, synthetic melanin, mushroom tyrosinase, α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium hydroxide (NaOH) was purchased from Panreac (Barcelona, Spain).

COMBINATION OF DEPIGMENTING AGENTS IN VITRO 367 CELL CULTURE Human skin melanocytes (HSM ATCC, ATCC number: CRL-2208) in passes 8 to 11 were cultured with Dermal Cell Basal Medium (ATCC, Primary Cell Solutions, ATCC Number: PCS-200-030) supplemented with insulin (5 mg/ml), ascorbic acid (50 mg/ml), glutamine (6 mM), epinephrin (1 mM), calcium chloride (1.5 mM), peptide growth fac- tor, M8 supplement, penicillin (10 units/ml), and streptomycin (10 units/ml). They were incubated at 37°C and 5% of CO2 and culture medium was changed every 2 to 3 days. CELL VIABILITY ASSAY WST-1 assay (WST-1, Cat. No. 11 644 807 001, Roche, Mannheim, Germany) was per- formed to determine cell viability. HSMs (1–2 × 105 cells/ml) were cultured in 96-well microplates for 24 h. The next day, different concentrations (1:10 dilutions from 1 mg/ml solution or 5 mg/ml for the less cytotoxic ones) for each compound were added and cells were cultured for 72 h. After treatment, the cells were treated with 10 μl of WST-1 and incubated at 37°C for 4 h. Cell viability was determined by measuring the optical density at 450 nm. Cells in culture medium without inhibitors were used as a control. Experi- ments were run in triplicate. MUSHROOM TYROSINASE KINETICS ANALYSIS Kinetic analysis of mushroom tyrosinase inhibition was performed as previously de- scribed with some modifi cations (24). Fifty microliters of mushroom tyrosinase (1000 units), various concentrations of L -DOPA (0.5, 1, 2, and 3 mM) as a substrate and Figure 1. Chemical structure of (A) hydroquinone, (B) arbutin, (C) kojic acid, (D) azelaic acid, and (E) α-lipoic acid.