In vitro effectiveness of several whitening cosmetic components in human melanocytes

J. Soc. Cosmet. Chem., 42, 361-368 (November/December 1991) In vitro effectiveness of several whitening cosmetic components in human melanocytes KAZUHISA MAEDA and MINORU FUKUDA, Shiseido Research Center, Yokohama, Japan. Received March 20, 1991. Synopsis The inhibitory action of arbutin, kojic acid, and ascorbic acid on tyrosinase activity in human melanocytes was compared. These substances are active whitening cosmeti components. Hydroquinone was used as a positive control. The depigmenting effect of linoleic acid, which has been reported to inhibit melanin synthesis, was compared with those of arbutin, kojic acid, and ascorbic acid. Human melanocytes were cultured with each agent in multiwell plates for three days, and the tyrosinase activity was assayed using L-DOPA as a substrate. In addition, cell viability of three-day cultures was evaluated by the MTT test. Arbutin dose-dependently reduced tyrosinase activity at final concentrations between 0.0! mm and 1.0 mm, at which no change in cell viability was seen. This action was about 1/100 that of hydroquinone, and was stronger than that of kojic acid and ascorbic acid. Linoleic acid did not reduce tyrosinase activity at non-cytotoxic ranges. Furthermore, at concentrations of 0.5 mm, the amount of melanin was reduced significantly by arbutin. These results suggesthat arbutin inhibits melanin production in humans by reducing tyrosinase activity and that the depigmenting action of this agent is stronger than that of kojic acid or ascorbic acid. INTRODUCTION In Japan various whitening beauty cosmetics that contain arbutin (hydroquinone-[3-dglucopyranoside; Figure la), kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone; Figure lb), or ascorbic acid as their principal components are commercially available for clinical use. These cosmetics are considered to be effective for preventing freckles due to ultraviolet exposure. Arbutin, the active component of the crude drug uvae ursi folium described in the Japanese Pharmacopoeia, is a hydroquinone glycoside. Kojic acid is a substancextracted from the fermentation fluid of koji mold. Mishima et al. suggested that this agent inhibits tyrosinase activity by chelating the copper in tyrosinase [EC. 1.14.18.1] (1), the key enzyme for melanin formation. Shono et al. reported that the long-chain fatty acid, linoleic acid, also inhibits melanin production (2). Pigmentation, such as freckles, in the skin is caused by enhanced melanin production 361

362 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS HOCH 0 OH H H I HO H OH 0 CHOH (a) Figure 1. Chemical structures of arbutin (a) and kojic acid (b). or melanocyte proliferation due to such factors as ultraviolet (UV) rays. Pigmentation in the skin is prevented by 1) reducing tyrosinase activity, either by inhibiting the synthesis of tyrosinase, which is an important enzyme for melanin synthesis, or by using an antagonist of the substrate for tyrosinase; 2) decreasing melanocyte functions such as proliferation or decreasing melanin production by using agents that are cytotoxic to melanocytes; 3) reducing dopa to prevent its auto-oxidation; and 4) suppressing inflammatory reactions, such as erythema, that occur following UV irradiation (3). Hydroquinone drugs are widely used by dermatologists to treat abnormal hyperpigmentation such as freckles, senile lentigines, melasma, and other forms of melanin hyperpigmentation. The use of these drugs in bleaching creams is permitted in some countries (4,5), but in Japan, their use as cosmeticomponents is prohibited, as they have been reported to cause irritation and dermatitis under certain conditions (6,7). The selective depigmenting effect of hydroquinone on melanocytes, in association with its chemical structure, has been studied by biochemical methods and electron microscopy (8-12). Its effects are considered to be due to the inhibition of tyrosinase in melanocytes (8,9) and to its cytotoxicity to the melanocytes (10-12). Depigmentation and its mechanism vary among arbutin, kojic acid (1), ascorbic acid (1), and finoleic acid. To compare the depigmenting effects of these agents, we evaluated their effects on tyrosinase activity, cell viability, and melanin synthesis in cultured human melanocytes, using hydroquinone as a positive control. (b) MATERIALS AND METHODS MATERIALS Hydroquinone was obtained from Mitsui Petrochemical Industries (Tokyo, Japan); arbutin was obtained from Nippon Fine Chemical Co. Ltd. (Osaka, Japan); kojic acid was purchased from Sigma Chemical Co. (St. Louis, MO); ascorbic acid and linoleic acid were purchased from Wako Junyaku Co. (Osaka, Japan). MCDB153 medium was purchased from Sanko Junyaku Co. Ltd. (Tokyo, Japan). And MTT assay kit was from Chemicon International, Inc. (Temecula, CA). CULTURE OF HUMAN MELANOCYTES Human melanocytes were obtained from neonatal Caucasian foreskins. Seventh passage

IN VITRO EFFECT OF WHITENING COSMETICS 363 melanocytes were cultured for about two weeks in MCDB153 medium containing 0.13 mm CaCI 2, 10 ng/ml epidermal growth factor (EGF), 5 g/ml insulin, 0.5 g/ml hydrocortisone, 1 ng/ml recombinant basic fibroblast growth factor (rbfgf), 10 ng/ml phorbol 12-myristate, 13-acetate (PMA) and 0.2% v/v of bovine pituitary extract. After replacement of the medium with MCDB 153 without rbfgf and PMA, the cells were cultured for a further two days. The cultured melanocytes were placed in 96-well plates at a density of 12,500 cells/cm 2. Two plates, one for the measurement of tyrosinase activity and the other for the measurement of cell viability, were cultured for one day. Hydroquinone and linoleic acid at final concentrations of 0.001, 0.005, 0.01, and 0.05 mm, and arbutin, kojic acid, and ascorbic acid at final concentrations of 0.01, 0.05, 0.10, 0.50, 1.0, and 5.0 mm, were added to triplicate wells and cultured at 37øC for three days. CELL VIABILITY ASSAY The cell viability in the presence of each agent was evaluated by the MTT test (13). After culture, phosphate buffer (ph 7.4) containing 5 mg/ml MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrasolium bromide) was added to each well. The plate was incubated at 37øC for 4 hours, after which isopropanol containing 0.04 N HC1 was added. Viable cells formed dark blue formazan by cleaving MTT with mitochondria. After 30 minutes, absorbance was measured at 570 nm using 655 nm as a reference. To eliminate agent interference in the measurement, wells containing each test agent alone were incubated and reacted with MTT. Cell survival was calculated from the absorbance. TYROSINASE ACTIVITY ASSAY The cells were washed with PBS and lysed with 45 1 of 1% Triton-X/PBS. After vibration, 5 1 of 10 mm L-DOPA was added to the wells. After incubation of the plates at 37øC for 60 min, absorbance was measured at 475 nm in a Model-3550 ELISA Reader (Bio-Rad Lab., Richmond, CA). The absorbance values were compared with a standard curve obtained with mushroom tyrosinase (Sigma Chemical Co., St. Louis, MO); the standard curve was linear within the range of experimental values. The coefficient of correlation was determined as 0.999. MELANIN ASSAY Melanin content was determined according to the method described by Oikawa (14), which we modified. Briefly, human melanocytes (40,000 cells/well) cultured in 12-well plates in 1.0 ml of medium were cultured with arbutin (0.5 mm), kojic acid (0.5 mm), and ascorbic acid (0.5 mm) at 37øC for three days. The culture solution was aspirated, and 1.0 ml of 2.5% trypsin solution was added to each well. The cells were detached, placed in an Eppendorf tube, and centrifuged at 1,000 rpm in a Hitachi CR 15 refrigerated centrifuge to obtain cell pellets. The pellets were mixed with 5% trichloroacetic acid, agitated well, and centrifuged at 10,000 rpm to deposit melanin. The melanin sediment was washed with PBS and mixed with 1N NaOH for dissolution, and the absorbance at 475 nm was measured. The amount of melanin per cell was calculated

364 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS from the absorbance. A standard curve was obtained with synthetic melanin (Sigma Chemical Co., St. Louis, MO). STATISTICAL ANALYSIS The mean value and standard deviation at each concentration of each agent were calculated. Student's t-test was used to compare the means (-+SD) of the determinations. RESULTS VIABLE CELLS PER WELL Figure 2 shows human melanocyte cell viability after culture for three days with hydroquinone, linoleic acid, arbutin, kojic acid, or ascorbic acid. Hydroquinone caused no significant changes in cell viability at final concentrations between 0.00! mm and 0.0! mm, but markedly reduced it at 0.05 mm. Linoleic acid reduced cell viability to about 70% at final concentrations between 0.00! mm and 0.005 mm and reduced viability to less than 20% at concentrations of 0.01 mm and 0.05 mm. Arbutin did not reduce cell viability at final concentrations between 0.0! mm and 1.0 mm, but reduced it to about 74% at 5.0 mm. Kojic acid did not reduce cell viability at final concentrations between 0.0! mm and 1.0 mm, but reduced it to 3000 2000 o none --- hydroquinone -- linoleic acid -e-arbutin kojic acid [] ascorbic acid looo ß.....al ß,,.an..I ß. ß nl=..i ß ß. o.ool 0.010 0.100 1.000 10.000 rnm Figure 2. Effects of hydroquinone, arbutin, kojic acid, ascorbic acid, and linoleic acid on cell survival per well in human melanocytes. Cultures of 12,500 cells/cm 2 were incubated with these agents for three days. Results are expressed as cell survival number per well. Bars represent of standardeviation from the mean.

IN VITRO EFFECT OF WHITENING COSMETICS 365 about 54% at 5.0 mm. Ascorbic acid did not markedly reduce cell viability at final concentrations between 0.01 mm and 0.5 mm, but reduced it to about 78% at 1.0 mm and to about 35% at 5.0 mm. TYROSINASE ACTIVITY PER WELL Figure 3 shows human melanocyte tyrosinase activity after culture for three days with hydroquinone, linoleic acid, arbutin, kojic acid, or ascorbic acid. The activity was calculated within concentration ranges that did not markedly reduce cell viability. Hydroquinone dose-dependently reduced tyrosinase activity per well at final concentrations between 0.00! mm and 0.0! mm. Linoleic acid did not reduce tyrosinase activity at final concentrations between 0.001 mm and 0.005 mm. Arbutin dose-dependently reduced tyrosinase activity at final concentrations between 0.01 mm and 1.0 mm. Kojic acid and ascorbic acid showed similar dose-inhibitory curves; they did not markedly reduce tyrosinase activity at final concentrations between 0.01 mm and 0.50 mm, but rapidly reduced it at higher concentrations. CELLULAR MELANIN CONTENT Cell pellets were obtained after human melanocytes were cultured for three days with - 3 o none * hydroquinone x linolei½ -e-arbutin kojic acid ascorbic acid _ 2 acid z o 0... I. ß.....I... ill. ß 0.001 0.010 0.100 1.000 rnm Figure 3. Effects of hydroquinone, arbutin, kojic acid, ascorbic acid, and linoleic acid on tyrosinase activity per well in human melanocytes. Cultures of 12,500 cells/era 2 were incubated with these agents for three days. Results are expressed as units of mushroom tyrosinase per well. Bars represent of standard deviation from the mean.

366 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 0.5 mm of arbutin, 0.5 mm of kojic acid, or 0.5 mm of ascorbic acid. The intensity of the melanin color in cells cultured with arbutin was lighter than in the untreated cells. However, the melanin color in cells cultured with kojic acid or ascorbic acid was similar to that of the untreated cells. Table I compares the effects of each agent on melanin production. The amount of melanin was obtained from the absorbance at 475 nm after dissolution of intracellular melanin with 1N NaOH. Melanin production was significantly reduced by 0.5 mm of arbutin, but not by 0.5 mm of kojic acid or 0.5 mm of ascorbic acid. DISCUSSION It has been reported that cultured neonatal human melanocytes are an excellent source for the study of pigment cell biology (15,16), even though there may be fundamental biochemical differences, such as proliferation and melanin production in adult versus neonatal melanocytes (17,18). In this study, we used neonatal human melanocytes for in vitro assay of some depigmenting agents. We compared the inhibitory effects of these agents on melanin synthesis, using cell viability, tyrosinase activity, and the amount of melanin as an index. Hydroquinone dose-dependently reduced tyrosinase activity per well at concentrations which no change in cell viability was seen. The specifi cytotoxicity of hydroquinone melanoma has previously been investigated (11, 12). An explanation of this cytotoxicity may be that hydroquinone acts as a substrate for tyrosinase, thereby being converted into toxic semiquinone radicals (9). The degradation of melanosomes observed in the presence of topically applied hydroquinone in guinea pig skin melanocytes has been described (10). It is possible that reduction of tyrosinase activity occurs by melanosomal specific cytotoxicity. Furthermore, under certain conditions, hydroquinone is a better substrate for tyrosinase than tyrosine itself (8). This suggests that the depigmentation induced by this agent is strongly associated with its inhibition of tyrosinase activity apart from cytotoxicity. Whether these two mechanisms are responsible for the reduction of tyrosinase caused by hydroquinone remains unknown. Linoleic acid (0.001 mm and 0.005 mm) did not reduce tyrosinase activity in the wells. However, this agent might have marked cytotoxic effects at higher concentrations, which it reduced cell viability to less than 20%. Therefore, linoleic acid seems to have reduced tyrosinase activity per well at higher concentrations by decreasing the number of viable cells. The cytotoxic action of linoleic acid has also been reported in the presence of the culture medium containing EGF (19). We studied the in vivo effect of linoleic acid Table Effects of Arbutin, Kojic Acid, and Ascorbic Acid on Melanin Content in Human Melanocytes Melanin per cell Agent Concentration (ng/cell) Control 0.794 + 0.032 Arbutin 0.5 mm 0.592 + 0.012' Kojic acid 0.5 mm 0.768 + 0.162 Ascorbic acid 0.5 mm 0.746 -+ 0.004 Cultures of 40,000 cells/well were incubated with these agents for three days. Results are expressed as ng of melanin per cell and represent mean -+ SD. * P < 0.001, vs control. I

IN VITRO EFFECT OF WHITENING COSMETICS 367 on mouse hair follicle melanocytes. Topically applied 2% linoleic acid markedly suppressed hair growth, but the depigmenting effect was less significant (data not shown). These findings suggest that linoleic acid inhibits melanin production by its cytotoxic action. Arbutin dose-dependently reduced tyrosinase activity per well at concentrations below 1.0 mm. The amount of melanin was reduced to 75% by arbutin. The effect of arbutin was about 1/100 that of hydroquinone. Arbutin can also act as a good substrate for tyrosinase, similar to hydroquinone; the effectiveness of these drugs as depigmenting agents may be related to their ability to act as substrates for tyrosinase. The depigmenting effect of a milky lotion containing 3% arbutin was tested on some forty individual suffering UV irradiation on the inner side of the upper arm. The lotion was applied three times daily. After seven days, skin pigmentation was significantly inhibited compared with placebo lotion (20). Kojic acid markedly inactivated isolated tyrosinase by chelation (1). In cultured human melanocytes, tyrosinase activity per well was slightly reduced at the concentration range between 0.1 mm and 0.5 mm but was rapidly dose-dependently reduced at higher concentrations. At lower concentrations, a dose-dependent decrease was not observed. These findings suggesthat the inhibitory effect of kojic acid on tyrosinase activity in the cell culture system is smaller than that of arbutin at concentrations that do not affect cell viability, even though marked inactivation was observed in isolated tyrosinase. Nair et al., using Yucatan minipigs as an assay in vivo (21), reported that kojic acid topically applied for 12 weeks resulted in no activity. On the contrary, Mishima et al. reported that topically applied kojic acid prevented artificial pigmentation in humans by irradiation with UV (22). The discrepancy between these results may be ascribed to the difference of species or the method of in vivo testing. Further investigations are needed to clarify this. In ascorbic acid-treated cells, tyrosinase activity per well was slightly reduced at final concentrations between 0.05 mm and 0.50 mm but rapidly dose-dependently reduced at higher concentrations. Ascorbic acid was oxidized rapidly in the aqueous phase, with loss of activity in time and very limited transcellular potency owing to its being hydrophilic. Some ascorbic acid derivatives were considered stable and transcutaneous with regard to antipigmenting function (23,24). Magnesium ascorbic acid phosphate, which is a stable ascorbic acid derivative, prevented erythema and postinflammatory hyperpigmentation following UV irradiation in humans (23). Lipophilic ascorbic acid derivatives also prevent freckles and melanin spots on the skin (24). Ascorbic acid is a potent antioxidant; in addition to its anti-enzymatic properties (23,26), it may prevent melanin synthesis by suppressing inflammation (25) and by inhibiting the autooxidation of dopa and dopaquinone. These result suggest that, to clarify those effects not ascribed to cytotoxicity, assays for both tyrosinase activity and cell viability in human melanocyte cultures are necessary to evaluate the depigmenting action. As whitening cosmetics are usually used daily, if the depigmenting effect is caused by cytotoxicity, irreversible hypopigmentation will occur somewhat in the skin or hair. In this assay system, arbutin inhibits melanin production by reducing tyrosinase activity, not by non-specificytotoxicity, and the depigmenting action of arbutin is stronger than that of kojic acid or ascorbic acid. However, the end exposure site for whitening cosmetics will usually be intact skin. We need to consider both in vitro and in vivo effects.

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