In Asia, lighter skin is a desirable feature and it is considered a symbol of beauty and femininity. The use of skin brighteners is widely extended by traditional beliefs. In Western countries, skin brightening agents are applied for the treatment of irregular pigmentation such as age spots and the creation of a more even skin tone. Skin brightening products have a worldwide market, and, beyond the treatment of hyperpigmentation disorders, they are powerful candidates for active ingredients in anti-ageing and beauty-enhancing products.
Most current skin brightening agents show numerous adverse effects such as high irritative and sensitising potential, melanocyte cytotoxicity and instability in formulations. Also, their lightening activity appears to be quite limited when it is tested in vivo on human volunteers. Chromabright™ is a novel skin brightener that has been tested for efficacy and safety with satisfactory results. The product presents no side effects, good efficacy in vitro and in vivo and, uniquely, a photoprotective effect.
Abnormalities of human skin pigmentation occur as a result of both genetic and environmental factors. Acute or persistent UVB exposure can result in the formation of hyperpigmented lesions in skin, such as melasma, age spots, freckles and other lentigines.1 There are numerous internal and external stresses that affect human skin pigmentation. Exposure to certain drugs and chemicals as well as the existence of certain disease states can result in hyperpigmentation.2 Skin brightening agents are used to treat these pigmentation disorders and to lighten skin colour. The colour of our skin is mainly determined by the degree and distribution of melanin pigmentation.3 Melanins are pigmented biopolymers that impart skin typology and tan. They are synthesised by the dendritic melanocytes dispersed at the dermo-epidermal junction. Melanin synthesis takes place in membrane-bound organelles termed melanosomes, which contain specific enzymes controlling the production of the pigments.4 Then, the mature organelles migrate towards the extremities of the melanocyte dendrites where they are transferred in skin to keratinocytes and in hair bulbs to the hair shaft, where the final distribution patterns of the pigment are determined.5 The role of melanin is to protect the skin from ultraviolet (UV) damage by absorbing UV sunlight and removing reactive oxygen species.1 The redistribution of melanin to upper layers of the skin following a relatively mild dose of UV is critical to the increased photo-protection of the skin.6 Skin hypopigmentation is normally achieved by inhibiting melanogenesis (melanin synthesis), so the skin is deprived of this photoprotective shield. Tyrosinase is the enzyme that catalyses the rate-limiting step of pigmentation in melanogenesis. Thus, tyrosinase inhibition is the most common approach for the screening of new skin brightening agents.7 Despite the large number of tyrosinase inhibitors in vitro, only a few are able to induce an effective hypopigmenting effect measurable in clinical trials. Additionally, statistically significant demonstrations of comparative clinical efficacy and safety of these products are not always achieved. Most known skin depigmenting chemicals act by destroying the epidermal melanocytes. Many skin lightening agents can cause skin irritation and require months of use before results appear, and some agents are only partly effective.8
Ideal skin brightening agent
Current topical hypopigmenting agents present on the market target melanocytes, the diverse steps in melanogenesis and the melanin transfer to keratinocytes. As it has been previously mentioned, tyrosinase inhibition is the most common approach to determine the in vitro efficacy of potential skin brighteners. The use of mammalianderived tyrosinase should be considered much better than mushroom tyrosinase, because of the different substrate and cofactor requirements, as well as sensitivity to inhibitors. Therefore, it is important to use human tyrosinase for the screening and the evaluation of skin brightening agents.7 Many inhibitors identified in cell-free enzymatic assays are likely to have some toxicity or delivery problems in cell-based assays.4 Furthermore, the extrapolation to the in vivo efficacy is not always satisfactory. Clinical trials performed with human volunteers are critical to determine the efficacy profile of these topical products. A skin brightener with photo-protective properties would also help to protect the skin from the harmful effects of UV radiation when melanin production is diminished. Accordingly, there is a need for novel skin brightening agents with reliable efficacy and safety. New depigmentation products should include the following desirable features: a) inhibition of mammalian tyrosinase; b) lack of toxicologic or mutagenic potential; c) clinical efficacy; d) formulation stability; and e) novelty and patent protection of the agent and/or formulation.1 Chromabright is a highly stable new, patented molecule designed for skin brightening applications that has been tested for efficacy and toxicity. It fulfils all the desirable qualities for a new skin brightener. Its hypopigmenting activity has been proven in in vitro and in vivo studies, and it does not carry any of the side effects that are normally associated with depigmenting agents. The new molecule proved not to act by destroying melanocytes in a cell culture, and it did not show any irritation reaction or sentitisation in the in vivo test with human volunteers (skin sensitisation). The product lacks toxicologic and mutagenic potential, and therefore it is considered a good candidate to be a completely safe skin brightening active for cosmetic applications. In addition, it is a highly stable ingredient in final formulations, and presents a high solubility in oils and detectability at very low concentrations. It was also tested for a photo-protective effect on human epidermal keratinocytes.
Materials and methods
Inhibition of tyrosinase activity
To assess the direct effect of the skin brightener on tyrosinase activity in vitro, both the mushroom tyrosinase inhibition assay and the endogenous human tyrosinase inhibition assay were performed.
Digestion of L-DOPA (tyrosinase substrate) in the presence of the test items and measure of absorbance variations at 475 nm (wavelength of melanin absorption) was used as a validated assay for measuring the potential inhibitory activity of compounds on the tyrosinase activity. The results obtained in all experiments were normalised regarding the absorption of a control experiment (untreated). Kojic acid was used as positive control.
Depigmenting effect on human melanocyte cultures
To assess the depigmenting effect of the tested product, plated human melanocytes were incubated for 5 or 9 days and fresh medium containing 0.1 mM Chromabright or 0.1 mM kojic acid was added daily. The melanin content was visualised directly by image recording through a microscope using a 40x lens. The lightening efficacy was blindly assigned by counting the cells showing melanin staining and the total number of cells. The results obtained in all experiments were normalised regarding the depigmenting efficacy of a control experiment (untreated). Kojic acid was used as positive control (n.d. = not determined).
Photoprotection test on human epidermal keratinocytes
The in vitro HEKa NRU photo-protection test is based on the determination of the protective effect of a chemical when tested the presence of a cytotoxic dose of simulated solar light. The photo-protective effect is expressed as an increase of the uptake of the vital dye Neutral Red (NR), when measured 24 hours after treatment of human epidermal keratinocytes with the test chemical and irradiation. NR is a weak cationic dye that readily penetrates cell membranes by nondiffusion, accumulating intracellularly in lysosomes. Alterations of the cell surface of the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought by the action of solar light result in a decreased uptake and binding of NR. Cell viability is determined by Neutral Red Uptake, measuring the optical density of the NR extract at 540 nm in a spectrophotometer. The responses obtained in presence of 47 μg/mL and 150 μg/mL of the test product were compared to irradiated (CTR+UV) and non-irradiated (CTR-UV) control cells (non-treated cells).
In vivo test: skin brightening effect on a panel of human volunteers
20 healthy Asian female volunteers aged 18 to 46, of all skin types, were selected for this study. The volunteers applied a cream containing 0.1% Chromabright on one side of the face twice daily for 2 months, and a placebo cream on the other side. Measurements were taken before application, and after 30 and 60 days of treatment. The brightening effect was instrumentally evaluated by means of a Chromameter CR-300. The parameters L*, a* and b* were measured (mean values from 5 successive measurements); where L* is luminance which represents relative brightness, from L*=0 (total darkness) to L*=100 (absolute white), a* is the red/green colour axis and b* is the yellowblue colour axis. The best description of a brightening effect is given by combining the L* and b* parameters, in the Individual Typological Angle ITA°, which is obtained according to the formula:4 ITA° = Arctg [(L*-50)/b*]•(180/p) The brighter the skin, the higher are the L* and ITA° parameters.
Results and discussion
Inhibition of tyrosinase activity
1 mM Chromabright performed successfully in both tyrosinase inhibition activity assays. The product was able to inhibit mushroom tyrosinase activity by 37% (Fig. 1) and human endogenous tyrosinase activity by 43% (Fig. 2).
Depigmenting effect on human melanocyte cultures
The new molecule showed a better depigmenting effect than kojic acid after 5 days of incubation on human melanocyte cultures (45.87% vs 30.24% respectively). After 9 days of incubation, the new molecule achieved a 61.2% depigmenting effect. The product was able to induce a significant depigmenting effect on melanocytes compared to untreated cells as can be seen in the microscopy images obtained (Figs. 3 and 4).
Photoprotection test on human epidermal keratinocytes
Irradiated cells treated with the new molecule showed an increased viability respect to irradiated non-treated cells. 47 μg/mL of the product induced a 190.4% increase in cell viability with respect to irradiated control cells, and at 150 μg/mL cell viability increased by 254.88% (Figs. 5 and 6). In vivo test: skin brightening effect on a panel of human volunteers After 30 days of treatment, a significant increase (p<0.01) in the Luminance L* (1.1%) and in the ITA° (7.7%) was observed for the cream containing the new molecule. The placebo cream presented no significant effects (Fig. 4). After 60 days of treatment, the brightening effect induced by the cream containing the new molecule increased as compared with the results at 30 days, with values of DL* = 1.7% and DITA° = 13.7%. This effect was significantly superior (p<0.001) to the effect observed for the placebo cream (Fig. 7). At the end of the study, no adverse effects were reported by the volunteers.
Conclusions
Generally, skin lightening agents have toxicity-related problems and are only partly effective. New skin brighteners should be developed with guaranteed safety and efficacy. A novel skin brightening agent has been designed to be and effective and safe active ingredient. Chromabright showed no cytotoxic effects on melanocytes, keratinocytes or fibroblasts, neither mutagenicity nor any irritation or sensitisation reaction. Its efficacy was tested in cell-free enzymatic assays as well as in cell-based assays. It proved to inhibit both mushroom and endogenous human tyrosinase in vitro, and exhibited a significant depigmenting effect on human melanocytes. The product also proved to have a significant photo-protective effect on human epidermal keratinocyte cell cultures, and can therefore prevent the skin-damaging effects of ultraviolet radiation. Low concentrations of Chromabright have demonstrated a significant skinbrightening effect when tested on a panel of human volunteers. In conclusion, Chromabright can be classified as a safe skin brightening agent due to its impeccable safety profile and contrasted in vitro and in vivo efficacy.
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