The evaluation of depigmenting efficacy in the skin for the development of new whitening agents in Korea
Correspondence: Moon Young Heo, College of Pharmacy, Kangwon National University, Chunchon 200-701, Korea. Tel.: + 82 33 250 6914; fax: +82 33 255 7865; e-mail: firstname.lastname@example.org
In this review, the evaluation methods for the screening of depigmenting substrates were investigated. For this purpose, the evaluation method of tyrosinase, a key enzyme of melanin biosynthesis, is most frequently used, but evaluating methods based on the regulation of cellular signal transfer factors or the inhibition of melanosome transfer have also been developed. Evaluation of the depigmenting effect using melanocytes is complex. It has the advantage of being capable of analysing overall effects on melanin biosynthesis at cellular levels. Before the final clinical testing of depigmenting agents, in vitro testing should be conducted to confirm the depigmenting efficacy and safety. Clinical studies for depigmenting agents can be used to investigate the prevention of melanin biosynthesis and to determine whether melanin disappears from skin. Therefore, the most appropriate protocol has to be employed, depending on the mechanism of action of the depigmenting agent.
Dans cette revue, les méthodes d'évaluation pour sélectionner des substances dépigmentantes ont été étudiées. Le procédé d'évaluation de la tyrosinase, enzyme clé de la biosynthèse de la mélanine, est le plus fréquemment utilisé, mais des méthodes de l'évaluation basées sur la régulation des facteurs de transfert de signaux cellulaires, ou sur l'inhibition du transfert des mélanosomes ont également été développées. L'évaluation de l'efficacité de dépigmentation à l'aide de mélanocytes est complexe. Elle a l'avantage d'être capable d'analyser l'ensemble des effets sur la biosynthèse de la mélanine au niveau cellulaire. Avant l'essai clinique final des agents de la dépigmentation, des tests in vitro doivent être menées pour confirmer l'efficacité dépigmentante et la sécurité. Les études cliniques d'agents dépigmentants peuvent être utilisées pour étudier la prévention de la biosynthèse de la mélanine et de déterminer si la mélanine de la peau disparaît. Par conséquent, le protocole le plus approprié doit être utilisé, en fonction du mécanisme d'action de l'agent dépigmentant.
With the improvement of the living standard in the global world, more people have begun to pursue well-being and beauty. In the healthcare field, the market share of products that help people realize well-being and beauty has been expanding at increasing levels. Even in the cosmetics market, many functional products such as those developed for skin moisturizing, skin whitening, wrinkle care, anti-ageing, pimple care, reducing cellulite and improving atopic skin have been launched. Such phenomena have often resulted in misleading, confusing advertisements for medical products. To protect consumers from such misleading promotional activities, the government of the Republic of Korea has enacted the Cosmetics Act and defined the scope of functional cosmetics as follows: cosmetic products for skin whitening effects, cosmetic products for wrinkle care and cosmetic products for protection of skin from UV rays.
As the evaluation of functional cosmetics was introduced according to the Korea Cosmetics Act (2000), many research activities for screening new functional substances have been activated in the Korean cosmetics industry. Specifically, the category of whitening products makes up 22.4% of the local functional cosmetics market in 2011, which indicates that it is one of the most important cosmetics categories in the Republic of Korea . Such phenomenon may be a result of the traditional Korean idea that white skin is the most important factor in beauty.
The ideal depigmenting compounds should have a potent, rapid and selective bleaching effect on hyper-activated melanocytes, carry no short- or long-term side effects, and lead to a permanent removal of undesired pigment, acting on one or more steps of the pigmentation process. In the course of screening such functional substances, various in vitro and/or in vivo studies to determine the efficacy and safety must be conducted.
To find a new depigmenting agent, it is important to understand the process and mechanism of pigmentation. Epidermal and dermal pigmentations, such as melasma, freckling and solar lentigo, are related to an increased number of melanocytes and activity of enzymes involved in melanin production [2, 3]. Melanocytes, the cells that produce melanin, move from the embryonic neural crest to the skin, where they are differentiated to form melanosomes. Melanin synthesized in the melanosomes moves to the dendritic ends of melanocytes. The melanosomes are then transferred into keratinocytes and disappear with the sloughing of keratin.
As a result of the key role played by tyrosinase in melanin biosynthesis, most depigmenting agents act specifically to reduce the function of this enzyme by means of several mechanisms: (i) interference with its transcription and/or glycosylation, (ii) inhibition by different modalities, (iii) reduction in by-products and (iv) post-transcriptional control [4, 5].
Although a depigmenting agent may be highly effective, it must be free of safety issues if it is to be made commercially available. In particular, a depigmenting agent used in the manufacture of functional cosmetics should have good safety characteristics. For example, hydroquinone, retinoic acid and organic mercury compounds have good bleaching effects, but their use in cosmetic products is prohibited in the Republic of Korea because of safety issues . In short, it is necessary to conduct safety studies at early screening stages together with efficacy studies.
Safety studies required for the development of cosmetic products include the acute toxicity, subacute and chronic toxicity, skin and mucous irritation, skin sensitization, carcinogenicity, reproductive toxicity and genotoxicity studies. In addition, they can be classified into in vitro tests, in vivo studies and human patch studies in terms of methodology. In the past, in vivo studies based on animals such as mice, rats or guinea pigs were mainly employed. However, the spread of the animal welfare movement has resulted in a decrease in the number of animal studies. A law stating step-by-step prohibition of animal studies using raw materials for cosmetic products was enacted in Europe . As a result, alternative in vitro methods are actively being developed.
In this review, various methods and mechanisms to demonstrate the efficacy of depigmenting agents are discussed and the most efficient and standardized methods with which to screen the depigmenting agents are investigated. To these ends, various methods that can be used to evaluate efficacy and safety as well as the mechanism of action of the depigmenting agents in connection with the formation, movement and degradation of melanin were identified from published literature.
Efficacy evaluation of depigmenting agents
Methods for the efficacy evaluation of depigmenting agents include in vitro tests, in vivo studies and clinical trials. In vitro tests focus on the screening of depigmenting agents through the investigations of mechanisms for the activation or inhibition of signal transfer factors or enzymes involved in the formation of melanin. A report was recently published on a method of evaluation that employed the mechanism of inhibiting the transfer of melanosomes formed by melanocytes into keratinocytes [8, 9]. For clinical studies, ultraviolet (UV) rays are irradiated to induce pigmentation. And the measurement of whitening effect is conducted for these pigmented areas. The depigmenting agent is applied to individuals with pigmented lesions, and then, measurement of the whitening effect is conducted.
In vitro tests of depigmenting agents
The in vitro test methods used for the evaluation of depigmenting agents can be divided into those acting before, during and after biosynthesis of melanin (Table 1) [4, 10]. For in vitro test methods that act before melanin biosynthesis, the effects of a depigmenting agent can be evaluated according to the expression or activity of factors suppressing MITF, ERK or other melanin biosynthesis signal transfer factors [11-13]. For in vitro test methods that act during melanin biosynthesis, the mechanism of inhibiting enzymes that are directly involved in biosynthesis such as tyrosinase, TRP-1, TRP-2 and dopachrometautomerase or the ROS elimination activity can be evaluated. Melanins produced by melanocytes are transported from melanosomes into keratinocytes and then cause epidermal pigmentation. Therefore, one report described the screening of depigmenting agents by evaluating the inhibition of melanosome movement .
Table 1. Classification of depigmenting agents by a mechanism of interference with melanin synthesis and deposition
|Before melanin synthesis||Tyrosinase transcription||C2-ceramide, Tretinon|
|During melanin synthesis||Tyrosinase inhibition||Hydroquinone, 4-Hydroxy anisole, Arbutin, Kojic acid, Aloesin, Azelaic acid, Ellagic acid, Resveratrol, Oxyresveratrol|
|Product reduction and ROS scavengers||Ascorbic acid, Ascorbyl glucoside|
|Thiotic acid, α-Tocopherol, Hydroxycumarins|
|After melanin synthesis ɑ-linoleic acid||Tyrosinase degradation|| α-Linoleic acid,|
|Inhibition of melanosome transfer||Niacinamide, Soybean/milk extract, Lecithins and Neoglycoproteins|
|Skin turnover acceleration||Lactic acid, Glycolic acid, Retinoic acid, Linoleic acid|
In addition, for in vitro evaluation of depigmenting agents, a method to measure the amount of melanin formed through cell culture is used widely. This method has the advantage of analysing overall effects on the intracellular melanin production system, regardless of the mechanisms of action, so it is widely used immediately prior to in vivo studies.
Regulation of cellular signal transfer
In addition to the evaluation of the whitening effect through direct inhibition of tyrosinase, studies to evaluate depigmenting agents through the regulation of cellular signal transfer factors involved in melanin production have been attempted. It is known that melanin biosynthesis begins when signals induced by UV rays or hormones, such as α-MSH, are transferred into cellular factors to induce expression of tyrosinase.
The stimulating factors such as α-MSH and forskolin, which induce melanin production act to elevate the intracellular concentration of cAMP, activate PKA and phosphorylate CREB for binding with CRE, leading to increased expression of MITF, a transcription factor. MITF regulates the expression of tyrosinase, an enzyme that determines the rate of melanin biosynthesis. The increased expression of tyrosinase later promotes the production of melanin. Therefore, evaluation of MITF expression will allow efficient screening of depigmenting agents.
It is known that MITF is a transcription factor that regulates the proliferation, survival and pigmentation of melanocytes [14, 15]. Further, it has been reported that MITF is an important transcription factor for several of the enzymes involved in melanin production, including tyrosinase, TRP-1 and TRP-2 [16-18]. Moreover, it is well-known that α-MSH induces the expression of MITF [17-19].
In addition to the MITF expression pathway, it has been reported that melanin biosynthesis is regulated through the ERK pathway for MITF degradation , and lysophosphatidic acid, EGCG and hinokitiol affect the signal transfer pathways to regulate the expression of MITF and inhibit melanin biosynthesis [11, 12]. Even though these materials have no or very weak inhibition of tyrosinase activity, they show a whitening effect. Although it is complicated and difficult to evaluate the depigmenting effect by measuring the degree of MITF expression, this method has been highlighted because it is useful in screening depigmenting agents without direct inhibition of tyrosinase activity.
Cell culture: The B16 melanoma cell line or another equivalent cell line (1 × 106) is inoculated into 60-mm dishes containing DMEM with 10% foetal bovine serum (FBS), 100 nM TPA, 1 nM cholera toxin, 50 μg mL−1 streptomycin and 50 μg mL−1 penicillin or other equivalent media and is then incubated at 5% CO2 and 37 °C.
Determination of concentration: The concentration is determined after a crystal violet assay  for the toxicity of a sample. Cell culture is conducted with specific concentrations of samples, media are cautiously removed from wells, and cells are washed with phosphate-buffered saline (PBS). Then, 500 μL of crystal violet solution (0.1% crystal violet per 10 mL ethanol, 90 mL D.W. added) is added to each of 24 wells. Incubation is conducted at room temperature for 10 min, and washing is performed four times with distilled water in a manner that does not cause the removal of cells. Then, 1000 μL of 95% ethanol is added to dissolve pigments, and absorbance is measured at 590 nm.
Sample treatment: When more than approximately 80% of the well bottom is occupied by cells, media are exchanged with those without FBS and incubation is conducted for 24 h. After the removal of media, new media with 1 μM α-MSH and sample without FBS are added and incubation is conducted at 5% CO2 and 37 °C for 48 h. Cells are washed two times with PBS and are then suspended in buffer solution.
Measurement of the amount of MITF expressed: The suspended cells are subjected to protein determination, after which, 10 μg of protein per sample is separated by SDS–polyacrylamide gel electrophoresis. The separated proteins are adsorbed into nitrocellulose membranes or PVDF sheets, and dried milk is used for blocking of the membrane at room temperature for 1 h. MITF-specific antibodies as the primary antibody are added and incubated at a temperature below 4 °C for 16 h with shaking. After washing, a secondary antibody with bound enzyme is added to induce binding and incubated for 2 h. Film is then exposed to luminescence generated by an enzyme reaction, and a densitometer is used to assay the target protein. Actin antibody is treated according to the aforementioned procedures. The actin assay value is used to correct for the amount of MITF present [11, 12].
Statistics: Control group and α-MSH-treated group are used as positive controls. The degree of the decrease in α-MSH-induced MITF expression by sample is measured. This procedure is repeated more than three times. When statistical analysis of results shows the significant decrease, the sample is considered to have the depigmenting effect.
Tyrosinase inhibition assay
Tyrosinase is an enzyme that is engaged in the most important rate-determining step in melanin biosynthesis. Melanogenesis is initiated by tyrosine that is metabolized into DOPA and then dopaquinone by tyrosinase. This first reaction is the rate-determining step in melanogenesis.
Tyrosinase has various forms, including mettyrosinase, oxytyrosinase and deoxytyrosinase. Oxytyrosinase is an activated form, whereas deoxytyrosinase is an inactivated form [18, 22, 23].
The function of tyrosinase is closely related to disease. In animal studies, disorder in melanin biosynthesis has been found to lead to condition of abnormal melanin coloration such as vitiligo or hyper-pigmentation. In severe cases, skin cancer may result from such disorders . Therefore, studies on tyrosinase inhibitors are highly important for the development of new drugs for the treatment of abnormal melanin coloration as well as functional cosmetics intended to produce a depigmenting effect.
The method used to evaluate the degree of tyrosinase inhibition is widely used in the screening of depigmenting agents. As it is easy to use and can be applied to evaluate many samples at the same time, it is considered to be the most appropriate method for early screening of candidate substances. This method, which is based on an enzyme–substrate reaction, is to induce the reaction of tyrosine and tyrosinase for a specific period and to measure the amount of products after the reaction.
Mushroom tyrosinase , melanoma tyrosinase  or human melanocytes tyrosinase  can be used. In general, mushroom tyrosinase is widely used because it is easy to obtain. DOPA can be the end product for measurement, but it is not widely employed because of the need for the use of a radioactive isotope. HPLC , another method that can be used to measure DOPA, is not used frequently because it requires a long period of time to analyse many samples. The most appropriate method used to measure dopachrome, one of the reaction products, is to perform spectrometry at 475 nm  or 490 nm .
In general, the test method of tyrosinase inhibition assay is performed as follows . The sample is dissolved in ethanol or other appropriate solvent and diluted to an appropriate concentration range for the inhibition of tyrosinase. These dilutions are used as test solutions (at least five concentrations). Then, 220 μL of 0.1 M phosphate buffer (pH 6.5), 20 μL of test solution and 20 μL of mushroom tyrosinase (1500–2000 U mL−1) (or human tyrosinase) are added into a test tube in order. Forty microlitres of 1.5 mM tyrosine solution is then added into the tube, and the reaction is conducted at 37 °C for 10 to 15 min. The absorbance is then measured at 490 nm with an ELISA reader. For a blank solution, 0.1 M phosphate buffer (pH 6.5) is added, instead of test solution. The concentration of test solution showing 50% inhibition activity (IC50) is calculated using an appropriate program. Depending on the conditions, this test can be scaled up or down as necessary. Arbutin or ethyl ascorbyl ether may also be used as a positive control.
a: absorbance of blank solution after reaction.
b: absorbance of test solution after reaction.
a′ and b′: absorbance when tested with the addition of buffer instead of tyrosinase.
DOPA auto-oxidation inhibition assay
This assay is used to measure the DOPA oxidation inhibited by tyrosinase. This method is similar to the aforementioned tyrosinase inhibition assay, except it includes the use of DOPA as a substrate. A description of this method follows . The sample is dissolved in ethanol or another appropriate solvent, which is diluted with an appropriate buffer, such as 0.1 M phosphate buffer (pH 7.0), to reach an appropriate range of concentration for the inhibition of DOPA oxidation. These dilutions are used as test solutions (at least five concentrations). Then, 850 μL of 0.1 M phosphate buffer (pH 7.0), 50 μL of test solution and 50 μL of mushroom tyrosinase (1500–2000 U mL−1) (or human tyrosinase) are added into a test tube in this order, and the reaction is conducted at 37 °C for 6 min. Fifty microlitres of 0.06 mM L-DOPA (L-3,4-dihydroxyphenylalanine) solution is added to this solution, and the reaction is conducted at 37 °C for 1 min. The absorbance is then measured at 475 nm with a microplate reader. For a blank solution, 0.1 M phosphate buffer (pH 7.0) is added, instead of test solution. The concentration of test solution showing 50% inhibition activity (IC50) is then calculated using an appropriate program. Depending on the conditions, this test can be scaled up or down as necessary. Arbutin or ethyl ascorbyl ether may be used as a positive control.
Dopachrometautomerase inhibition assay
In melanin biosynthesis, tyrosine is converted into dopaquinone by tyrosinase. In the presence of cystein, dopaquinone produces cysteinyldopa, which finally leads to the production of pheomelanin . However, in the absence of cystein, dopaquinone transforms to the non-enzymatic generating dopachrome. Dopachrome undergoes either spontaneous decarboxylation to form 5,6-dihydroxyindole (DHI) or tautomerization to form 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The formation of DHICA from a dopachrome is controlled by dopachrometautomerase. This dopachrometautomerase is another major enzyme correlating with the melanogenesis pathway . Dopachrometautomerase has a subcellular distribution within the melanocytes, which is similar to that of tyrosinase. It catalyses the transformation of dopachrome into DHICA. This means that dopachrometautomerase acts as a ‘dopachrome conversion’ factor . The production of DHICA induced by dopachrometautomerase eventually leads to the brown DHICA-eumlanin, which is followed by further oxidation and polymerization.
Dopachrome is produced by mixing cold DOPA (0.5 mg L-DOPA per mL in 0.05 M sodium phosphate, pH 6.8) with silver oxide (6 mg Ag2O mg−1 DOPA) for 3 min. After filtering through a 0.22-μm millipore filter, the supernatant is treated with Chelex 100 (Bio-Rad Laboratories, Richmond, CA, U.S.A.) to remove all traces of sliver. The dopachrome is prepared immediately before use owing to its instability.
The assay reagents consisted of 125 μL of dopachrometautomerase (0.9 mg total protein), 250 μl of the dopachrome solution, 525 μL of a 0.05 M sodium phosphate buffer, pH 6.8, and 100 μL of either MeOH or 1 mM of the test sample dissolved in MeOH. The dopachrometautomerase activity is determined by measuring the increase in absorbance at 308 nm, indicating the enzyme-catalysed formation of DHICA from dopachrome.
The sample is dissolved in ethanol or other appropriate solvent, which is diluted with 0.05 M phosphate buffer (pH 6.8). These dilutions are used as test solutions (at least five concentrations). A volume of 525 μL of 0.05 M phosphate buffer (pH 6.8), 100 μL of test solution and 125 μL of dopachrometautomerase is added into a test tube in this order, and reaction is conducted at 37 °C for 10 min. Then, the absorbance is measured at 308 nm with an ELISA reader. For a blank solution, 0.05 M phosphate buffer (pH 6.8) is added, instead of test solution. The concentration of test solution showing 50% of inhibition activity (IC50) is calculated with an appropriate program.
Antioxidants can have depigmenting effects by interacting with o-quinones, thus avoiding the oxidative polymerization of melanin intermediates, or with the copper at the active site. Moreover, antioxidants, by scavenging ROS generated in the skin following UV exposure, can inhibit possible second messengers that are able to stimulate epidermal melanogenesis either directly or indirectly [34, 35].
Ascorbic acid interferes with the different steps of melanization, by interacting with copper ions at the tyrosinase active site  and reducing dopaquinone and by blocking DHICA oxidation . The antioxidant properties of a-tocopherol, which interferes with lipid peroxidation of melanocyte membranes and increases the intracellular glutathione content, could explain its depigmenting effect [38, 39]. 6-Hydroxy-3,4-dihydrocumarins have been recently reported to have an anti-melanogenic activity in cultured normal human melanocytes at non-cytotoxic concentration without interfering with tyrosinase activity . The acceleration in the biosynthesis of glutathione within melanocytes, leading to the inhibition of tyrosinase transfer to premelanosomes, could be the mechanism of action . In general, the degree of DPPH free radical removal or the degree of lipid peroxidation inhibition is measured for anti-oxidation assay.
Free radical scavenging activity: A volume of 0.1 mL of sample is added to 1.0 mL of 100 μM DPPH-ethanol solution, and reaction is conducted at 37 °C for 30 min. Then, the absorbance is measured at 520 nm .
Inhibition of lipid peroxidation: Add test sample 0.1 mL and ethyl linoleate 10 μM to an incubation medium(4.89 mL) containing 2% sodium dodecyl sulfate, 1 μM ferrous chloride and 0.5 mM hydrogen peroxide. Keep the incubation medium at 55 °C for 16 h. Transferred each reaction mixture (0.3 mL) into test tube, which was followed by the addition of 4% BHT (50 μL) in order to prevent further oxidation. Add 1 mL of 0.67% TBA to the reaction mixture. Vortex the sample and incubate at 95 °C for 30 min. After cooling, add 4 mL of a 15% methanolic butanol solution and stir the solution. Centrifuge the reaction mixture for 10 min (1,000 × g) and measure the absorbance of the supernatant at 532 nm. The extent of lipid peroxidation is determined by measuring the quantity of the TBA reactive substance (TBARS). 1,1,3,3-Tetraethoxypropane is used as the standard, and the lipid peroxide concentration is expressed as the amount of malondialdehyde and is shown as the percentage inhibition of MDA formation .
Evaluation of depigmenting effect using melanocytes
The method used for the evaluation of the depigmenting effect using melanocytes is a screening system that simulates condition similar to intracellular conditions compared with in vitro tests of enzyme activity or anti-oxidation and has the advantage of being able to analyse overall effects on melanin biosynthesis by melanocytes. However, as this method is complex and time-consuming, it is believed that this method is appropriate for the confirmatory evaluation of samples that are preliminary screened with an enzyme activity assay.
Evaluation of the depigmenting effect using melanocytes can be performed by measuring the intracellular tyrosinase activity or the amount of intracellular melanin produced. Various methods are used depending on the type of cell line, culture conditions and methods of evaluation. The following table summarizes the melanocytes and culture conditions that are frequently used (Table 2).
Table 2. Types of melanocytes, properties and media for cell culture
|B16 murine melanoma cell line||Cell line derived from C57BL/6 mouse, widely used for screening of depigmenting agents||EMEM media, supplemented with Penicillin 100 000 U L−1, Streptomycin sulphate 100 mg L−1, 10% foetal bovine serum (FBS)|
|Melan-a murine cell line||Cell line derived from C57BL/6 mouse, suitable for tests because they have having most characteristics of normal mouse melanocytes but immortalized.||RPMI-1640, pH 7.0–7.1, supplemented with Penicillin 100 000 U L−1, Streptomycin sulphate 100 mg L−1, Glutamine 2 mM, TPA 200 nM, 10% FBS|
|HM3KO human melanoma cell line||Human melanoma cell line isolated from melanoma spread into peritoneum, active in pigment production||EMEM supplemented with Penicillin 100 U mL−1, Streptomycin sulphate 100 g mL−1, 10% FBS|
|Normal Melanocytes derived from human or animal||Melanoma cell line obtained from normal tissues of human or animal origin, having the advantage of being closest to normal skin tissues, but expensive because of difficulties to obtain lots of cells and to culture||MCDB-153 media, supplemented with Bovine Pituitary Extract 15 g mL−1, hFGF 1 ng mL−1, PMA 10 ng mL−1, Insulin 5 g ml−1, Hydrocortisone 0.5 g mL−1, Gentamycin 50 g mL−1, Amphotericin 50 ng mL−1|
Culturing of melanocytes only enables investigators to evaluate the effects on the amount of melanin production in melanocytes, whereas it does not consider the effects on extracellular signal transfer. Therefore, a melanocyte–keratinocyte coculture and a cultured three-dimensional skin method have been developed. However, such methods are not widely used because they are expensive and require conditions that are difficult to achieve.
Murine melanoma cells (B-16 F1) or other similar cells are inoculated into a 6-well plate containing DMEM with FBS or other equivalent media (1 × 105 cells/well), and incubation is conducted at 5% CO2 and 37 °C until more than approximately 80% of the well bottom is occupied by cells. The media are then replaced with new media containing appropriate dilutions of test sample, and incubation is conducted at 5% CO2 and 37 °C for a specified period of time. An appropriate range of concentration of the test sample is determined through toxicity tests using a crystal violet assay. The media are removed, cells are washed with PBS, and trypsin is applied to recover cells. The number of cells is counted with a haematocytometer. Centrifugation is then conducted at 4,000–16,000 × g for 10 min, and the supernatant is removed to obtain the pellet. The pellet is dried at 60 °C and dissolved with 100 μL of 1 M sodium hydroxide solution or appropriate cell lysis buffer to obtain intracellular melanin in a water bath at 60 °C. The absorbance is measured at 490 nm with a microplate reader, and the amount of melanin is then calculated as specific cell numbers or specific protein content. Normalization is performed with the result from a blank test .
Melanosome Transfer Inhibition Test
Melanins synthesized in melanosomes of melanocytes move to the dendritic ends of melanocytes. Then, the dendritic ends containing melanosomes are transferred into keratinocytes, and keratins are fallen off and disappeared.
Although transfer of melanosomes is greatly affected by hormones or UV rays, the most important factor is the genetic one. The activation of protease-activated receptor 2 (PAR-2), a seven trans-membrane G-protein-coupled receptor, which is expressed in keratinocytes and not in melanocytes, was found to activate keratinocyte phagocytosis, enhancing the pigment transfer . Inhibition of PAR-2 cleavage by serine protease inhibitors, such as RWJ-50353, completely avoids the UVB-induced pigmentation of epidermis analogues [46, 47]. This melanosome transfer inhibition test is to measure the amount of melanin in keratinocyte after coculture of melanocyte and keratinocyte, to determine whether the sample is able to inhibit the movement of melanin.
Coculture of melanocyte and keratinocyte: Incubate foreskins in 0.25% trypsin for 2 h at 37 °C. Vortex the skin gently for 30 s to separate the dermis from the epidermal cell suspension. Remove the dermis and then pellet the epidermal cells by centrifuge and resuspend in either melanocyte or keratinocyte growth medium for seeding in 25-cm2 tissue culture flasks. Maintain melanocyte in M154 basal medium supplemented with 4% heat-inactivated FBS, 1% antibiotic/antimycotic solution, 1 μg mL−1 transferrin, 1 μg mL−1 vitamin E, 5 μg mL−1 insulin, 0.6 ng mL−1 hFGF, 10−8 M α-MSH and 10−9 M ET-1. Maintain keratinocyte in M154 basal medium supplemented with human keratinocyte growth supplements and 1% antibiotic/antimycotic solution. Stain subsequently the established cultures of melanocytes with the succinimidyl ester of carboxy fluorescein diacetate (CFDA) at 2 μmol L−1 in Hanks balanced salt solution for 30 min. Add these melanocytes to keratinocytes at a ratio of 1 : 2 and coculture in normal melanocyte growth medium/normal keratinocyte growth media 1 : 2.
Sample Treatment: Maintain coculture in the presence or absence of sample for 6 days. Add the sample to the media of the cocultures every 12 h. On the 7th day, wash cells in 5 mL of PBS containing 0.4% heat-inactivated FBS and 0.2% sodium azide. Prefix the cells in 4% paraformaldehyde for 30 min at 4 °C followed by permeabiliztion in fluorescence-activated cell sorting permeabilizing solution for 10 min at room temperature, and wash twice in PFA. Incubate the cells for 45 min at room temperature with the primary antibody diluted in 10% normal human serum/PFA (PFAN) at 1 : 300. Rinse cells three times with PFA and incubate for 30 min at 37 °C with goat anti-mouse IgG conjugated to phycoerythrin (PE) at 1 : 20 dilution in PFAN. Wash cells twice and post-fix in 1% paraformaldehyde.
Detection of melanosome transfer: Analysis cells by flow cytometry. CFDA and PE is both excited with the 488 nm line of an argon ion laser. Detect fluorescence emission for CFDA and PE selectively by collection with 525 nm and 575 nm band pass filters, respectively. Melanocytes are positive for CFDA only. Keratinocytes are positive for PE. Assessment of melanosome transfer to keratinocyte as determined by recording the expression of CFDA within PE-positive cells .
Clinical test for skin depigmenting agents
Melasma is a dysfunction of pigmentary system, resulting in an irregular brown or grayish-brown facial hyper-melanosis. Although it can occur in both sexes and any skin type, it is more commonly seen in women  and those with darker complexions – Fitzpatrick's skin types IV to VI – especially those living in areas of intense UV radiation, such as Hispanics/Latinos, Asians and African Americans [49-51]. The condition usually develops slowly and symmetrically and can last for many years, with worsening in the summer and improvement during the winter.
The common contributing factors include genetic predisposition , pregnancy  endocrine dysfunction or hormone treatments [53, 54], use of oral contraceptives  and exposure to UV light [50, 56]. In addition, cosmetics and drugs containing phototoxic agents (e.g. anti-seizure medications) have also been linked to melasma .
Clinical studies performed to evaluate the depigmenting effect may be assessed to investigate the effect on the inhibition of melanin pigmentation or the effect on the improvement of pigmented conditions. The subject is human beings pigmented artificially by UV irradiation or had site of hyper-pigmentation, such as freckles, lentigo or blotched skin which may be investigated.
Changes of skin tone can be visually measured by experts or instrumentally measured with the use of equipment such as a Chromameter. The visual evaluation method employs the scoring system to evaluate the degree of whitening. Therefore, it is important to develop well-designed protocols, such as an objective scoring method and the use of qualified evaluators, to establish the reliability of results. The instrumental evaluation method has the advantage of quantitative and objective measurement. However, the results may depend on the size and condition of target sites and the experience and skill of technicians. Therefore, it is desirable to use these two methods in parallel and to make conclusions from the results of both methods.
Melanosomes move to the outside of skin with keratinocytes and are eventually sloughed off with other skin. The capacity of chemical peeling products (e.g. α-hydroxy acids) to disperse melanin pigment and/or accelerate epidermal turnover can result in skin lightening [57, 58]. Clinical tests of the epidermal turnover rate can be used for the evaluation of whitening agents.
Efficacy evaluation on induced pigmentation
Selection of subjects
Healthy male or female adults, aged 18 to 60 years, are normally selected as subjects for human clinical studies. In principle, those with Fitzpatrick types I-III are preferable, but those with types III and IV can also be included. Exposure to UVB must be kept to a minimum. Those whose health status or skin conditions may affect the results and women who are pregnant or lactating must be excluded. In addition, those who have ever experienced adverse reactions to sunlight, skin disorders or abnormal skin symptoms such as erythema and black spots, which may affect the interpretation of results, must be excluded.
Number of subjects
The number of subjects should be selected more than 20 valid data points to allow statistical comparison. If a control group is used, the double-blind method, in principle, has to be employed.
The upper back or upper arm, which is rarely exposed to UV rays, is normally selected as an irradiation site.
A solar simulator equipped with a xenon arc lamp having that as a continuous emission spectrum similar to sunlight and not showing specific peaks or another equivalent light source is used to irradiate UV rays to selected sites. Wavelengths below 290 nm must be removed using an appropriate filter. The light source should maintain a consistent intensity of radiation during the test period. UV rays of 2–3 MEDs are uniformly irradiated, or after the irradiation of UV rays of 1 MED, the intensity can be adjusted depending on the level of the induced pigmentation.
When the intention is to investigate the prevention effect, a sample is applied immediately after UV irradiation. If the purpose is to improve the induced pigmentation condition, the sample is applied once or twice a day for 4–8 weeks from 2 to 3 days after UV irradiation.
Evaluation of depigmenting effect
The site for evaluation has to experience no air flow, and constant temperature and humidity should be maintained. After taking a rest for at least 30 min at the site, subjects are evaluated. The degree of the depigmenting effect on test sites is visually examined by experts to make scores and/or the degree of change in skin colour can be measured with appropriate equipment, such as Chromameter (Minolta, Osaka, Japan), DemaSpectrometer (Cortex Technology, Hadsund, Denmark) or Mexameter (Courage-Khazaka Electronic, Koln, Germany) .
Efficacy evaluation in hyper-melanosis
Selection of subjects
Those with pigmentation, such as freckles, lentigo or blotched skin, are selected as subjects. Refer to criteria described in 3.1.
Number of subjects
The number of subjects should be selected more than 20 valid data points to allow statistical comparison. If a control group is used, the double-blind method, in principle, has to be employed.
Sites showing hyper-pigmentation, such as eye rims or upper arms, are selected. If a piece of equipment is employed for evaluation, the sites of irradiation must be selected while considering the minimum pigmentation area that can be measured by the equipment.
The sample is applied once or twice a day for 4–8 weeks.
Evaluation of depigmenting effect
Refer to those described in 3.1 .
Efficacy evaluation on stratum corneum turnover rate by dansyl chloride test
Healthy male or female volunteers aged 18 to 60 are selected as subjects for clinical studies. Women who are pregnant or lactating are excluded. In general, the subjects have to be in good health and cannot have skin disorders or abnormal skin symptoms such as erythema and black spots, which may affect the interpretation of results. Four sites below both forearms are selected for the application of sample. Sample application is conducted in a randomized manner (upper and lower sides of both forearms). Dansyl chloride (5-methyl-amino-1-naphthalene-sulphonyl chloride) is mixed with petrolatum to make a 5% mixture. Prior to sample application, 5% dansyl chloride is applied to selected sites for 15 h using closed patch. The selected sites are strongly rubbed with the use of a finger coat. A fluorescent irradiator is used to measure the degree of fluorescent pigmentation. After 21-day application of the sample, the degree of disappearance of fluorescent pigments is measured .
Safety evaluation of depigmenting agents
One of the important things to consider in the development of depigmenting agents is safety. For medicinal products, because they are intended to treat diseases, they can be useful if their benefits or efficacies outweigh their side effects. However, in the field of cosmetics, all products are used for the purpose of beauty. Therefore, products of which safety is not established cannot be used, even if they have excellent efficacy. Accordingly, the safety issue has to be addressed from a very early stage in the development of new cosmetic substances.
Although different countries have different scopes of safety evaluation applicable to cosmetics depending on the nature of the substance, the following studies have to be conducted: acute toxicity test, skin irritation and sensitization test, eye irritation test, phototoxicity and sensitization test and a human patch test. In addition, testing of repeated dose toxicity, reproductive toxicity, mutagenicity/genotoxicity or carcinogenicity test may be required, if necessary.
These toxicity tests are conducted in the manner of in vivo studies or clinical trials. Therefore, considerable costs and time are required to perform complete toxicity studies. It is desirable to adopt a good design strategy from the screening stage to identify which toxicity studies should be conducted to ensure efficient and effective evaluation of depigmenting agents. To select the most appropriate and relevant toxicity studies, it is important to understand the natures and properties of the substances. For common chemical substances, expected toxicities can be determined from various published reports on the toxicities of other similar substances. As lots of toxicity data may be exempted for natural substances that are considered to be safe because they have been used traditionally, screening of depigmenting agents to be used in the manufacturing of cosmetics products is active.
Recently, toxicity studies using animals have been restricted by laws, especially in European countries. More specially, a law stating step-by-step prohibition of animal studies using raw materials and finished cosmetic products was enacted in Europe in 2003. As a result, it has become difficult to conduct toxicity studies of cosmetics using animals. To overcome such difficulty, alternative toxicity test methods that do not include animals' experimentation must be developed.
At an early stage of the screening of new depigmenting agents, an in vitro cytotoxicity test or HAP-CAM test can be used to estimate toxicity. However, toxicity studies required for commercialization have to be conducted according to validated methods recommended by OECD or other internationally recognized organizations (Table 3).
Table 3. Current state of method validations for the alternatives to animal experiment
|Acute toxicity|| |
Fixed dose method
|Delete Acute oral toxicity, Lethal Dose(LD50)||Up-and-down procedure|
|Skin irritation and corrosive|| |
Human Skin Model Test
Acute Dermal Toxicity-fixed Dose Procedure(draft)
in vitro Membrane Barrier Test Method for Skin Corrosive (draft)
Episkin™ skinCorro-sive test
Epiderm™ skin orro-sive test
Corrositex® assay for skin corrosivity
|Epiderm™, Episkin™ and TER Assays|
|Skin sensitization||Local Lymph Node Assay(LLNA)||Local Lymph Node Assay(LLNA)||–|
|Eye irritation||–||–||EpiOcular™ OCL-200|
|Phototoxicitya||3T3 NRU||3T3 NRU||3T3 NRU|
Out of various skin mechanisms, the hyper-pigmentation mechanism (the mechanism affecting melanin biosynthesis) has been actively researched . Melanin is synthesized from tyrosine. However, there are various biosynthesis pathways, depending on the enzymes present and the presence or absence of materials with an -SH group. Further, the end product may be eumelanin or pheomelanin . In addition, various factors are involved in melanin biosynthesis, including (i) hormones and cytokines, (ii) enzymes, (iii) metal ions and (iv) interferon, prostaglandin and histamine [63, 64]. Accordingly, the effects of such various factors have to be investigated during the screening of new depigmenting agents. However, it is not efficient or cost-effective to investigate all of the factors mentioned previously. It is desirable to take the following step-by-step approaches when demonstrating the safety and efficacy of new depigmenting agents.
The first step is to screen candidate substances by employing in vitro test methods that are able to test all possible substances within a short period of time. Although various factors are involved in melanin biosynthesis, tyrosinase is the most important of the enzymes. Therefore, testing of the effect on tyrosinase activity has been adopted widely to demonstrate the whitening effect of new candidate substances (Table 1). In addition, to screen substances that are not related to tyrosinase activity, anti-oxidation activity or the effect on MITF or other factors regulating the melanin biosynthesis can be additionally investigated.
The second step is to investigate the safety and efficacy of such screened candidate substances at cellular levels. Substances proven to be highly effective in the first step described previously may show cytotoxicity in cell culture or may not have the intended function in cells because they cannot penetrate the cell membrane. Therefore, this step is considered essential to prove the efficacy and safety of new depigmenting agents. In addition, coculture of melanocytes and keratinocytes can be used to develop a new depigmenting agent that inhibits the movement of melanosomes. In this step, the cytotoxicity test, which is the basic test for the evaluation of toxicity, should be performed in parallel with the evaluation of efficacy.
The third step is to conduct various preliminary tests prior to clinical studies. In vivo studies are performed to demonstrate the depigmenting effect. Brown guinea pigs with induced hyper-pigmentation can be used to investigate the depigmenting effect and anticipate the appropriate doses for human subjects. However, limitation in the use of animal studies has spread across the world, leading to the prohibition of studies of cosmetics or their raw materials based on animal models. They are not acceptable to European sensibilities (and legal restrictions) any more. To overcome this situation, a method using three-dimensional artificial skin may be an alternative. In the third step, tests have to be conducted to determine the structures and specifications of depigmenting agents. As this step is the final one for commercialization, it is important that the specifications be established during this step. In particular, for natural substances, it is essential to isolate them and determine their structural characteristics for adequate quality control in the commercialization process. Further, the safety has to be established for new synthetic substances or newly isolated substances. Necessary toxicity studies must be conducted, and safety evaluation based on results from such studies should be performed prior to clinical studies. For toxicity studies, alternative methods have to be actively introduced, instead of animal studies.
The final step is to demonstrate the depigmenting effect in clinical studies and investigate any potential adverse reactions. Clinical tests can include protocols to investigate the prevention of melanin biosynthesis and determine whether melanin disappears. Therefore, the most appropriate protocol has to be employed, depending on the mechanism of action of the depigmenting agent. As the protocol for irradiation with UV rays and for measuring the inhibition of melanin biosynthesis has the advantages of the relative ease of recruitment of subjects and regulation of melanin biosynthesis, it is considered to be preferable to the protocol necessary when testing on subjects with hyper-pigmentation conditions such as freckles. For depigmenting agents to be used in clinical studies, the safety issue is one of the utmost importance. It is an essential requisite to perform clinical studies after the establishment of safety through investigation of published report or toxicity studies.
As described previously, there are various methods that can be utilized to evaluate the efficacies of depigmenting agents. Therefore, it is important that the most efficient and appropriate methods be adopted to demonstrate the efficacy of a new depigmenting agent [31, 65-71]. In addition, when developing a new whitening product containing a combination of several depigmenting agents, it is desirable to select depigmenting agents with different mechanisms of action rather than those with the same mechanism. Such combination products may decrease adverse reactions and show synergistic effects.
This study was partially supported to publish by Kangwon National University (2011) and Institute of Pharmaceutical Sciences, Kangwon National University.