Depigmentation Agents Research Articles

Chalcones as potent tyrosinase inhibitors: the effect of hydroxyl positions and numbers

Chalcones with non-, mono-, di-, tri-, or tetra-substituted hydroxyl groups were examined as new family of tyrosinase inhibitors, demonstrating their potential whitening potency. The inhibition of tyrosinase is one of the major strategies to treat hyperpigmentation. Various limitations are associated with many of these inhibitors, such as high cytotoxicity, poor skin penetration and low stability in formulations. In continuation of our previous study [J. Agric. Food Chem. 51 (2003) 1201], showing that isoliquiritigenin chalcone (ILC) is a potent tyrosinase inhibitor, the present study aims to characterize the chalcone family as new tyrosinase inhibitors, and demonstrate their potential whitening potency. Nine mono-, di-, tri- and tetrahydroxychalcones were tested as inhibitors of tyrosinase mono- and diphenolase activities, showing that the most important factor in their efficacy is the location of the hydroxyl groups on both aromatic rings, with a significant preference to a 4-substituted B ring, rather than a substituted A ring. Neither the number of hydroxyls nor the presence of a catechol moiety on ring B correlated with increasing tyrosinase inhibition potency. 4-Hydroxychalcone (4-HC), ILC and Butein inhibited tyrosinase and shortened the lag period of enzyme monophenolase activity from about 490 min (control) to 30 min (ILC). As pigmentation also results from auto-oxidation, the antioxidant activity of 4-HC, ILC and Butein, were tested. Results showed that chalcones are also potent antioxidants, with Butein the most potent. We may conclude that chalcones are potentially potent new depigmentation agents, with their double effect of reduction and antioxidant activity. A deeper understanding of the relation between their structures to their potency will contribute to designing the optimal agents.

Effects of hydroxystilbene derivatives on tyrosinase activity

Synthesis of melanin starts from the conversion of l-tyrosine to 3,4-dihydroxyphenylalanine ( l-dopa) and then the oxidation of l-dopa yields dopaquinone by tyrosinase. Therefore, tyrosinase inhibitors have been established as important constituents of depigmentation agents. Recently, polyhydroxystilbene compounds, which are trans-resveratrol (3,4 ′,5-trihydroxy- trans-stilbene) analogs, have been demonstrated as potent tyrosinase inhibitors. However, their detailed inhibitory mechanisms are not clearly understood. In the present study, a variety of synthesized hydroxystilbene compounds were tested for their inhibitory effects against murine tyrosinase activity. The inhibitory potencies of the hydroxy- trans-stilbene compounds were remarkably elevated by increasing number of phenolic hydroxy substituents. Methylated hydroxy- trans-stilbene lost the inhibitory activity. Furthermore, hydrogenated hydroxystilbene or hydroxy- cis-stilbene exerted little or no inhibitory effect compared with hydroxy- trans-stilbene on tyrosinase activity. The structure–activity relationships demonstrated in the present study suggest that the phenolic hydroxy groups and trans-olefin structure of the parent stilbene skeleton contribute to the inhibitory potency of hydroxystilbene for tyrosinase activity.

Depigmenting activity and low cytotoxicity of alkoxy benzoates or alkoxy cinnamte in cultured melanocytes.

To obtain effective and safe topical depigmenting agents, we synthesized hydroxybenzoates, alkoxybenzoates, and 3,4,5-trimethoxycinnamate containing a thymol moiety and screened then for high-level inhibitory activity against melanin synthesis in cultured melanocytes. Eight compounds were tested for their depigmenting effect and cytotoxicity using a murine melanocyte cell line. We found that 3,4,5-trialkoxybenzoates and 3,4,5-trimethoxycinnamate, synthesized by conjugating 3,4,5-trialkoxybenzoic acids and 3,4,5-trimethoxycinnmic acid with thymol, showed a potent depigmenting effect and low cytotoxicity. Compound 4h, 5-methyl-2-(methylethyl)phenyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate, showed the most potent depigmenting effect (IC50=10 microM) with low cytotoxicity (IC50=200 microM).

Intradermal concentration of hydroquinone after application of hydroquinone ointments is higher than its cytotoxic concentration.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild topical side effects including irritation. In this paper, the cytotoxicity of HQ was assessed in vitro using rat skin fibroblasts as the concentration with 50% survival after 24 h exposure to be 16.5 microM. The intradermal concentrations at 2 h after application of the HQ ointments was also estimated to be 358 mM and 51.7 mM in stratum corneum and viable tissue (viable epidermis+dermis), respectively, by an in vitro rat skin permeation study with rat full-thickness abdominal skin and Franz-type diffusion cells. It was demonstrated that the intradermal concentration of HQ was much higher than that eliciting cytotoxicity, suggesting that the topical side effects after application of HQ ointment were due to the cytotoxicity of HQ.

Whitening Effect of Cosmetics Containing Magnesium L-Ascorbyl-2-Phosphate(VC-PMG, Vitamin C Derivatives) Assessed by Colorimeter

Backgrounds : An inhibitory effect of magnesium L-ascorbyl-2-phosphate (VC-PMG, a stable derivative of ascorbic acid) on melanogenesis has been described. Furthermore, glabridin in licorice is known to have inhibitory effects on melanogenesis and widely used for raw materials for depigmenting agents. Objective : The purposes of this study are to provide objective data by measuring the visual clinical effects of VC-PMG with the colorimeter and to promote the development of de-pigmenting agents. Methods : 20 volunteers joined the study. With an artificial UVB irradiation, eight tanned areas were made on the inner side of the forearm. During two months, each tanned area was treated with five whitening cosmetics with 3% VC-PMG and increasing concentration of licorice from 0% to 3%. Darkness degree of each area was measured weekly by the colorimeter and the visual assessment. Results : For all cosmetics, whitening effect was measured by colorimeter and visual as-sessment. The cosmetic containing VC-PMG 3% and licorice 1% had more whitening effect than any other cosmetics of different concentrations. Moreover, VC-PMG 3% alone also had whitening effect in some volunteers. Conclusion : VC-PMG was clinically found to have whitening effect.

Pharmaceutical and Clinical Assessment of Hydroquinone Ointment Prepared by Extemporaneous Nonsterile Compounding.

Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in Japan by imitating skin lightening creams commercially available in the U.S.A. and European Union. In our hospital, HQ ointments consisting of 5 or 10% HQ, 1.6% L(+)-ascorbic acid (AsA), 0.5% (w/w) Na2SO3, 10% (v/w) glycerin and hydrophilic ointment have been prepared. However, various problems have been observed including chromatic aberration of HQ ointments, relatively large variability of efficacy, and undesirable side effects although they were mild. Herein, the pharmaceutical and clinical properties of the HQ ointments were evaluated. HQ ointments were highly effective for treatment of various types of skin pigmentation. Chromatic aberration occurred during 3 months of storage, but this could be suppressed by storage at 4 degrees C. Chromatic aberration was independent of prescribed HQ content, and was not explained by alterations of HQ or p-benzoquinone (p-BQ) contents. Unexpectedly, removal of both antioxidants resulted in suppression of chromatic aberration, but an increase in p-BQ content. Acidification by removal of Na2SO3 only was further effective for the suppression of chromatic aberration, but with a decrease of p-BQ content except in the initial period. Chromatic aberration was due to water soluble material and insoluble material both formed by co-existence of HQ and p-BQ at a molecular ratio of 5:3 to 1:1. 1H-NMR analysis elucidated that the water soluble material was not HQ or p-BQ, and the insoluble material was a complex of HQ and p-BQ with non-covalent binding.

Isolation and Synthesis of Cosmetic Substances from African Dietary Leaves, Celosia argentea L. for Skin Depigmentation.

Six compounds were isolated from the leaves of Celosia argentea L.. Tyrosinase inhibitory and superoxide scavenging activity of the compounds was examined so as to obtain a skin depigmentation agent for cosmetic application. Eugenyl O-β-D-glucopyranoside (citrusin C, 1) showed strong tyrosinase inhibitory activity. Two-step inhibitory effects of successive tyrosine and DOPA oxidation for tyrosinase were stronger compared to arbutin used commercially at present (47.20% inhibition of tyrosine oxidation and 87.93%, DOPA oxidation, compared to 63.00% for tyrosine and 7.70% for DOPA oxidation in the case of arbutin). Moreover, we succeed in providing compound 1 abundantly in the synthetic study which used acetobromo-α-D-glucose and Sn(OTf)2.

Vitiligo. Pathogenesis and treatment.

Vitiligo is an acquired skin disorder caused by the disappearance of pigment cells from the epidermis that gives rise to well defined white patches which are often symmetrically distributed. The lack of melanin pigment makes the lesional skin more sensitive to sunburn. Vitiligo can be cosmetically disfiguring and it is a stigmatizing condition, leading to serious psychologic problems in daily life. It occurs worldwide in about 0.5% of the population and it occurs as frequently in males as it does in females. The cause is unknown, but might involve genetic factors, autoimmunity, neurologic factors, toxic metabolites, and lack of melanocyte growth factors. Since a causative (gene) treatment is not (yet) available, current modalities are directed towards stopping progression and to achieving repigmentation in order to repair the morphology and functional deficiencies of the depigmented skin areas. Many treatments have been used for some time; however; there are some new developments: narrowband ultraviolet (UV) B (311 nm) therapy, the combination of corticosteroid cream + UVA therapy, and the transplantation of autologous pigment cells in various modalities. In widespread vitiligo, residual pigment can be removed by depigmentation agents. Sunscreens, camouflage products, and good guidance may help the patient cope better with the disease.

Occupational Leukoderma in Workers Engaged in 4‐(p‐Hydroxyphenyl)‐2‐Butanone Manufacturing

Occupational Leukoderma in Workers Engaged in 4‐(p‐Hydroxyphenyl)‐2‐Butanone Manufacturing: Yoshiharu Fukuda, et al. Department of Public Health, Kumamoto University School of Medicine—Occupational leukoderma was found to be caused by exposure to depigmentation agents. This is the first report associated with the depigmenting activity of 4‐(p‐Hydroxyphenyl)‐2‐butanone and its crude products. The purpose of this paper is to present three cases of occupational leukoderma in the upper extremities of workers engaged in the manufacturing of 4‐(p‐Hydroxyphenyl)‐2‐butanone. Two workers had symptoms of dermatitis in the same areas before depigmentation. An epidemiological study, their clinical courses and results of phototesting suggest that these cases of leukoderma were due to exposure to 4‐(p‐ hydroxyphenyl)‐2‐butanone and its crude products.

Topical skin depigmentation agents

Novel topical dermatologic or cosme-ceutical agents are needed to treat cutaneous hyperpigmentation, since most currently available products typically contain hydroquinone (HQ), which has significant disadvantages. Although most studies have found HQ therapies to be efficacious, this compound is highly toxic and mutagenic to mammalian cells. Skin pigmentation results from the enzymatic conversion of tyrosine into melanin within the melanosomes of melanocytes, followed by transfer of the melanosomes into the keratinocytes of the epidermis. In order to identify novel inhibitors of skin pigmentation based on inhibition of tyrosinase, the key enzyme responsible for initiating melanogenesis, agent screening methods have been developed using both melanocyte cell cultures and cell-free enzymatic assays. An example of an HQ analog that demonstrates the desired activity in these assays is methyl gentisate (MG), which was discovered as an inhibitor of de novo melanogenesis in cultured melanocytes, with significantly reduced cytotoxicity and lack of mutagenic potential compared to HQ. A strategy is summarized for discovering new compounds capable of inhibiting melanogenesis, such as MG, and developing them into dermatologic or cosmeceutical products. In addition, consideration is given to the major differences in product registration requirements in various global markets for topical skin-light-ening agents.

Development of an in vitro Primary Screen for Skin Depigmentation and Antimelanoma Agents

An in vitro cell culture assay was developed to identify inhibitors of melanogenesis and agents which produce cytostatic or cytotoxic effects specifically in melanocytes. A total of 50 compounds related to tyrosine, dihydroxyphenylalanine, and hydroquinone (HQ) were tested in vitro in order to determine their effects upon a murine melanocyte cell line, Mel-Ab, that produces copious amounts of melanin in culture. The agents that demonstrated an inhibition of growth or pigment production by 50% (IC50) at < 100 micrograms/ml were considered active. The cytotoxicity of melanocyte-active compounds were also tested in vitro on a control nonmelanocyte cell line (HT 1080), using a simple crystal violet staining method to quantitate adherent cell number after treatment. The cell culture assay was validated with known potent melanocyte cytotoxic agents, including HQ and 4-S-cysteaminylphenol (4-S-CAP). Although most cytotoxic chemicals were nonspecific in this primary screen (i.e. killing both Mel-Ab and HT-1080 cells), several of the compounds tested exhibited high melanocyte-specific cytotoxicity, similar to HQ and 4-S-CAP. Potentially these compounds may be useful as either antimelanoma or skin depigmentation agents. All of the compounds identified as active in this primary screen were cytotoxic or cytostatic to melanocytes, except for the methyl ester of gentisic acid, which uniquely inhibited the de novo synthesis of melanin without cytotoxicity.

Occupational Vitiligo Due to Unsuspected Presence of Phenolic Antioxidant Byproducts in Commercial Bulk Rubber

We investigated the occurrence of cutaneous depigmentation (vitiligo) among employees of a company that manufactured hydraulic pumps. The interiors of these pumps were injection-molded with rubber. We identified a small but significant cluster of vitiligo cases among a group of employees who frequently handled the rubber used in this injection molding process. Although none of the additives specified in the rubber formulations was a phenolic or catecholic derivative, known to be potential causes of chemically induced vitiligo, gas chromatographic analysis identified a para-substituted phenol (2,4-di-tert-butylphenol, DTBP) in solid samples of the most frequently used rubber. Surface wipe analysis confirmed that workers could be exposed to DTBP from simple handling of the rubber. We subsequently established that the solid bulk rubber used as the base in these stock rubber formulations contained both DTBP and smaller quantities of p-tert-butylphenol. Both had formed as unsuspected byproducts during chemical synthesis of two antioxidants added to the solid bulk rubber by a major rubber supplier. We conclude that the unsuspected presence of potential chemical depigmenting agents in solid bulk rubber, from which industrial rubber products are formulated, may contribute to the occurrence of occupational vitiligo, and that a simple review of ingredients in rubber formulations is inadequate to detect their presence.

An Evaluation of the Effectiveness of Azelaic Acid As a Depigmenting and Chemotherapeutic Agent

In the past five years, it has been reported that certain dicarboxylic acids (C8-C13) and azelaic acid (C9) (AZA), in particular, have a remarkable effect in the management of lentigo maligna, human malignant melanoma, and certain disorders of hyperpigmentation. Preclinical trials, therefore, were undertaken in order to evaluate the effectiveness of AZA as a depigmenting agent and as a chemotherapeutic agent. Twenty-seven uniformly black pigmented guinea pigs were given topical applications of various concentrations (3, 5, 10, 15, and 20%) of AZA preparations for 8 weeks, and their effects on the melanocytes of epilated skin of the backs and the nonepilated ears of guinea pigs were compared to the effects of well-known depigmenting agents. Whereas 4-isopropylcatechol, monobenzylether of hydroquinone, monoethylether of hydroquinone, hydroquinone, and 4-hydroxyanisole were found to be selectively cytotoxic to melanocytes in black-skinned guinea pigs, AZA has little or no visually recognizable effect on melanocytes in these animals. The therapeutic effect of local s.c. injections of various concentrations of AZA preparations on the development of s.c. implanted B-16 melanoma tumor was evaluated in 96 C57BL/6J mice. In addition, 31 BDF1 mice, implanted i.p. with B-16 melanoma tumor, were used to assess the effect of 100-500 mg/kg concentrations of AZA administered i.p. In both studies, AZA revealed no significant tumoristatic or tumoricidal effect on the size, color, and growth of melanoma. The effect of AZA was also evaluated on S-91A (melanotic or pigmented) and S-91B (amelanotic) human melanoma cells in culture. Low concentrations (10(-5) and 10(-3) M) of AZA had no inhibitory effect on the growth of these cells. Only at higher concentrations (greater than 10(-3) M) was a cytotoxic effect on cell viability observed. These observations indicate AZA is not selectively cytotoxic to normal and proliferative melanocytes and has no apparent inhibitory effect on the formative process of melanin pigmentation.