SKIN WHITENING
spectrophotometric method at 405 nm. The following results (Fig. 3) show that
the lightening effect of mango is not impacted by an adsorption specific to the melanocytes.
Tissue engineering: an intermediate approach to in vivo study Developed as a research resource and often used for safety and efficacy studies in cosmetics, reconstructed skin models offer proprieties close to human skin. Epidermic reconstruction is based on the basal layer (collagen IV, laminin, etc.) and then, the corneous layer is created by the differentiation. It is a real technology established on the ability of living cells to gather and create a three-dimensional tissue that resembles the in vivo context. Studies carried out on the healing mechanism have shown the keratinocytes’ leading role, particularly in the differentiation process. This phenomenon is not really visible in monolayers when those cells are treated separately. For instance, keratinocytes normally need various additives in culture media to maintain their appearance, morphology and growth rate in monolayer models (in vitro). The main advantage of reconstructed
epidermis is that exchanges of growth factors, particularly between the various cells types, enable the replacement of those additives essential to keep them alive. Besides, without dermic fibroblasts, the growth speed of keratinocytes is modified and they grow up faster. Thus, with this skin model, understanding of dermoepidermal interactions has been proved and the potential of the fruit active on the melanin synthesis is more realistic thanks to the presence of normal human keratinocytes (NHK), essential to the melanin release in the different cellular layers.
Lightening effect on reconstructed tanned epidermis To validate the in vitro efficacy of the tested products, an ex vivo evaluation of the depigmenting potential has been carried out on Reconstructed Human Tanned Epidermis (RHTE – Fig. 4), provided by the
Composition
Melanocyte Nucleus
Tyrosinase expression
Tyrosinase activity
+CU2+
L-Tyrosine O2
L-DOPA O2
Dopaquinone
Eumelanin TRP1
TRP2
Pheomelanin Cystein
Glutathione Melanin
Focus on a melanosome
Figure 1: Melanogenesis.
company, SkinEthic. The experimental approach of melanin content during the whole studied period is comparatively identical to the monolayer test. That is to say, epidermises have been tested with and without the samples in non-cytotoxic conditions.
Study protocol Epidermises are reconstructed with cell culture at the air-liquid interface of human keratinocytes and melanocytes (phototype VI – Figs. 5 and 6) for 10 days on a polycarbonate filter with the following proportions: 10 keratinocytes for 1 melanocyte, in an adapted growth medium. Before treatment of each epidermis,
a stabilisation stage is necessary for 48 hours at 37˚C. Epidermises are then transferred into a 6-well plate with 1 mL
100 75 50 25 0
Control
of culture medium and tested products. Indeed, kojic acid and Melan’oWhite (now referred to as ‘the lightening fruit active’) are formulated at 4% to be topically applied and are tested in the medium at 1% in solution (corresponding to 250 µM of kojic acid). Then, they are compared to the placebo formula (without the fruit active). Treatment is renewed over three
consecutive days and cells are kept in the medium growth for 48 extra hours. At the end of the experiment (after 6 days of treatment on epidermises), melanin is extracted and cell viability is tested. Epidermises are torn off and melanin extraction is carried out with a Soluene solution and heated at 100˚C for 45 minutes. Absorbance is measured on the supernatants obtained by
*Significant: (Student t-test; p<0.05)
–91.3%
–93.2%*
–91.6%*
Kojic acid (250 µM)
0.025% Melan’oWhite Figure 2: Significant inhibition of intracellular melanin content after 72 hours.
Table 1: Composition, shape and appearance of various melanosomes. Melanosomes
Premelanosomes Eumelanosomes
Non-melanised organelle with no particular activity
Highly polymerised with low sulphur content. Correctly protect against UV rays
Phaemelanosomes Low polymerised with high sulphur content. Slightly protective against UV rays
58 PERSONAL CARE March 2012
0.05%
Shape Round shape
Black-brown egg-shaped pigments and larger than phaemelanosomes
Brown-red or yellow pigments with round shape
Appearance
Melanin content (%)
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