Biochemical and molecular study of members of euphorbiaceae family for tyrosinase inhibitory effect

Abstract

The synthesis of the visible pigment melanin by melanocytes is the basis of human pigmentary called melanogenesis. During this process, the expression of tyrosinase and tyrosinase-related proteins is up-regulated. The rate-limiting step in this pathway is controlled by the enzyme tyrosinase. A variety of physiological factors including sunlight can stimulate differentiation of melanocytes to increase pigmentation. Overproduction of melanin leads to the development of melasma, which can be treated by hydroquinone compound presented in some skin care products, but with some side effects e.g. an allergic reaction or skin burning. Previously, the extracts from plant in Euphorbiaceae family have been claimed to treat melasma. Therefore, we screened the extracts from two euphorbiaceous members found mainly in Thailand namely Mallotus spodocarpus and Excoecaria bicolor for their anti-tyrosinase propertiy using cell-free mushroom tyrosinase and melanocyte cell-based assays. The results demonstrated that the crude extract from E. bicolor was more effective in mushroom tyrosinase inhibitory assay than that of the extract from M. spodocarpus in a does-dependent manner (0.125 mg/ml-2 mg/ml). Using melanocyte cell culture system and by means of MTT cytotoxic assay, the result was found that the extract from M. spodncarpus possessed a very potent cytotoxic effect, compare to that of the extract from E. bicolor, which had an approximately 13% cytotoxicity at the concentration of 0.125 mg/ml. However, the result was shown by means of RT-PCR assay that both extracts reduced RNA expression level of tyrosinase and its transcription factor, MITF, in a does-dependent manner (10 µg/ml and 100 µg/ml). Therefore, the extract from E. bicolor was selected for further examination on the signaling cascades leading to MlTF- mediated tyrosinase expression by means of western blot analysis. The results demonstrated that the E. bicolor extract increased protein expression level of phosphorylated form of ERK, a key regulator for MITF degradation, in a does-dependent manner (10 µg/ml and 100 µg/ml). In summary, the findings indicated that the extract from E. bicolor possesed anti-tyrosinase property and its mechanism of action was involved ERK- mediated MITF protein degradation, as well as and a down-regulation of the MITF RNA. Therefore, E. bicolor extract could be cosmetically useful for targeting melasma.