Innovatory approach fights pigment disturbances

In order to control this biological phenomenon, it would be interesting to develop a natural depigmentating active ingredient capable of limiting the main signalling pathways involved in skin pigmentation: melanogenesis and transport of melanosomes.

The process of melanogenesis involves various enzymes, which catalyse reactions leading to the formation of melanin pigments.

Among them, tyrosinase is the limiting enzyme in melanogenesis since it catalyses the first two reactions of the melanin synthesis pathway: hydroxylation of tyrosine into dihydroxyphenylalanine (3,4-dopa) and oxidation of dopa into dopaquinone. An increase in melanogenesis results from an increase in tyrosinase activity and melanin synthesis in all cases.

As they are the key molecules of all signalling pathways involved in the human skin pigmentation, tyrosinase activity and melanin synthesis, although classic, nevertheless remain essential targets for all depigmenting agents.

Transport of melanosomes occurs as follows: melanin synthesis takes place within melanosomes which are transported from the peri-nuclear region to the dendritic extremities of melanocytes. On the one hand, this transport occurs along the actin fibres thanks to an anchoring complex composed of Rab27a, melanophilin (or slac2-a) and a molecular motor called myosin Va, which links melanosomes to the actin fibres. (Goud et al., 2002; Passeron et al., 2004, Steingrimsson et al., 2006). On the other hand, motor proteins linked to the microtubules lead to migration of the melanosomes. Kinesin thus permits centrifugal movement, whereas dynein enables centripetal movement of the melanosomes (Ichihashi et al., 2003); (Fig. 1).

Transport linked with the actin fibres enables localised displacement of melanosomes and their accumulation at the dendritic extremities, where they can then be transferred to the neighboring keratinocytes (Marks et al., 2001). If expression of Rab27a is underregulated, recruitment of melanophilin and myosin Va is limited. In the absence of the anchoring complex, melanosomes incapable of binding to actin filaments are not retained in the dendritic extremities of melanocytes and concentrate in the peri-nuclear zone, preventing their transfer to the adjacent keratinocytes (Marks et al., 2001; Seabra et al., 2002; Izumi, et al., 2003).

This biological mechanism could be an interesting target to prevent the accumulation of melanosomes at the dendritic extremities and their transfer to keratinocytes. In this way, we could limit the human skin pigmentation.

Effect of Palmaria palmata extract on melanogenesis

Quantification of anti-tyrosinase activity and melanin synthesis in B16F1 melanocytes.
B16F1 melanocytes were treated with the P. palmata extract at increasing concentrations or with the positive control kojic acid at 0.12 mg/ml in complete DMEM medium and were incubated for 48 hours at 37°C in a humid atmosphere containing 5% CO2. After trypsinisation, cells were counted by tryptan blue inclusion under microscopy and were lysed. After centrifugation, the supernatant was recovered for the assay of tyrosinase activity and the pellet for the melanin assay.

Visualisation of anti-tyrosinase activity in normal human melanocytes.
Normal human melanocytes were cultured on a chamber slide system in complete culture medium. Normal human melanocytes whose melanogenic activity was stimulated by adding IBMX (3-isobutyl- 1-methyl xanthine) were then treated with the P. palmata extract at increasing concentrations during 5 days and incubated at 37°C in a humid atmosphere containing 5% CO2. After fixation and L-DOPA staining, observation was made with an IX 70 microscope (Olympus, Japan) coupled to an image analysis system (Fig. 2).

Effect of extract from Palmaria palmata on the transport of melanosomes

Study by Western Blot of the synthesis of Rab27 and melanophilin by normal human melanocytes.
Normal human melanocytes were cultured in complete culture medium. Normal human melanocytes whose melanogenic activity was stimulated by adding Forskolin were then grown for 48 hours in the presence of the P. palmata extract at increasing concentrations for 48 hours.

At the end of incubation, cell-free extracts were recovered and stored at -80°C. Total proteins were assayed with a BCA assay kit (Sigma, BCA1); equal amounts of each protein extract were resolved using 12% SDS polyacrylamide gel and transblotted onto a membrane. After blocking, the membrane was incubated with a primary antibody anti-Rab27a raised in goats and then with a secondary antibody HRP-conjugated anti-goat IgG or with an anti-melanophilin produced in rabbit and a secondary antibody HRP-conjugated antirabbit IgG.

The visualisation system was peroxidase substrate and chromogen solution. Bands were semi-quantified by densitometry after image analysis with software (Fig. 3).