Jaborandi extract reveals whitening properties

Skin pigmentation consists of melanin biosynthesis which proceeds through a complex series of enzymatic and chemical reactions in melanocytes. Production of melanin takes place in melanosomes which are transported to the extremity of dendrites to be transferred from melanocytes to nearby keratinocytes. Synthesis of melanin starts from the conversion of amino acid L-tyrosine to L-dopa and then the oxidation of L-dopa to dopaquinone by a specific enzyme, tyrosinase. Several other factors and multiple genes implicated in skin pigmentation regulate the melanogenesis. In this study, murine B16 melanoma cells have been used in order to examine the effect of isopilosine on tyrosinase activity and melanin content. We demonstrated that isopilosine was able to induce a significant decrease of tyrosinase activity. Their results were confirmed using 3D StratiCELL pigmented reconstituted epidermis model allowing interactions between melanocytes and keratinocytes. Following the results obtained with StratiCELL specifically customised TaqMan array focused on genes involved in skin pigmentation, isopilosine can be considered as a new non toxic modulator of the melanogenesis pathway to be included in cosmetic skin whitening formulations.

 Pilocarpus microphyllus (Rutaceae) is widely distributed in the northern region of Brazil, where it is popularly known as jaborandi. Jaborandi leaves contain important imidazole alkaloids, such as pilocarpine, pilosine, pilosinine, pilocarpidine, anhydropilosine and 13-nor-8(11)-dihydropilocarpine, whose pharmacological and physiological properties are still unknown. Isopilosine is another imidazole alkaloid extracted from the leaves of jaborandi. In this study, isopilosine was evaluated for its benefits in skin whitening using 2D and 3D models. Production of melanin is regulated by numerous pathways leading to an increase of microphthalmia-associated transcription factor (MITF) expression. Increased expression of MITF and its activation by phosphorylation stimulate the transcription of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and dopachrome tautomerase (DCT), which are the key enzymes in the production of melanin. Tyrosinase has a role in the oxidation of L-tyrosine to L-3-(3,4-dihydroxyphenyl)- alanine (L-DOPA) and of DOPA to dopaquinone, which is the initial step in melanin synthesis. Dopaquinone spontaneously converts to dopachrome. DCT catalyses the conversion of dopachrome to 5,6-dihydroxyindole-2- carboxylic acid (DHICA). TRP1 catalyses the oxidation of DHICA to indole-5,6-quinone- 2-carboxylic acid. These two closely-related structures, DCT and TRP1, produce unstable quinines that undergo further polymerisation, finally producing melanin.1 Melanogenesis is accomplished in melanosomes, specialised organelles where the production and accumulation of melanin takes place. Melanosomes are transported along melanocytes microtubules and acting network, via the action of motor proteins kinesin and dynein,2,3 to the dendrite tip. Dendricity of melanocytes, mainly regulated by the Rho family proteins4 allows an interaction with a large number of keratinocytes to transfer the melanin produced. The mechanism by which melanocytes transfer melanin is influenced by the interactions of lectins, glycoproteins, E-cadherin, SNAREs, Rab and Rho GTPases.5 In the present study, the effect of isopilosine on tyrosinase activity and melanin content was assessed on murine melanoma B16 cells. The skin-whitening mechanism of isopilosine was assessed by a gene expression analysis on StratiCELL reconstructed human epidermis containing melanocytes. In the 2D model, isopilosine was applied in cell culture medium of B16 murine melanoma cells. Melanin content and tyrosinase activity were measured and compared to arbutin, a well-known depigmentation agent and isobutylmethylxanthine (IBMX), used as pro-pigmentation reference (phosphodiesterase inhibitor). In the 3D model, a dark-tanned epidermis (MEL/001) was used to evaluate the lightening effect of isopilosine as compared to the tyrosinase inhibitor kojic acid and IBMX, using biochemical and morphological procedures. The melanin content was quantified after solvent extraction and by image analysis of Fontana Masson stained histological slides. Using a specifically customised TaqMan array, focused on genes involved in melanogenesis, the effect of isopilosine was also assessed on the expression of more than 90 relevant target genes.

 Results and discussion