Studies On The Screening Of Melanogenisis Inhibitors And Their Percutaneous Formulations

Shengjie Bian (Specialty: Pharmaceutics) Advisors: Prof. Junmin Zheng, Prof. Dae-Duk Kim and Prof. Chi-Ho LeeHyperpigmentation including melasma, freckles, and senile lentigines is caused by the abnormal production of melanin, a pigment in the human skin as a major defense mechanism against ultraviolet light. Melanin is produced in melanocytes mediated by several enzymes, among which tyrosinase is essential in catalyzing the oxidation of tyrosine into dopa and subsequently to dopaquinone. Thus, it was suggested that hindering melanogenesis either by the inhibition of the tyrosinase activity or suppression of the expression and synthesis of the enzyme itself could be the solution. Several melanogenisis inhibitors, such as gentisic acid (GA), arbutin (AR), L-ascorbic acid (VC), hydroquinone (HO) and melanoston (MN) were found to be effective in the therapy of skin pigmentary disorders. However, the effect of formulations was seldom considered and the maintenance of drug concentration in the target site in the skin was ignored in the former practice. A lot of studies indicated that appropriate strategy of formulations can improve the effect of therapy for a lot of drugs. Therefore, the selection of proper formulations for topical delivery of melanogenisis inhibitors to the target site in the skin could be useful and necessary in improving the effect of therapy and the reduction of side effect.The main objectives of this research was to develop effective topical delivery systems for the therapy of skin hyperpigmentation and to investigate the mechanism of melanogenisis inhibitory effect with these five drugs.In biological studies, the effect of melanogenisis inhibitors on the melanin release of B16 melanoma cells was investigated and the toxicity of each drug was also studied by B16 cell culture. AR, MN, VC and HQ showed to be effective in the determination of inhibitory effect on the melanin release of B16 melanoma cells. The sequence of the inhibition is HQ>MN>AR. The result of GA was not accessible because GA can react with the culture medium. And VC didn't show obvious dose-dependent inhibition. In the viability study of B16 melanoma cells by MTT assay, HQ is very toxic to the cells with the IC50 of 3.167 ug/ml. GA and AR are very mild melanogenisis inhibitors with high cell viability. The sequence of toxicity to B16 cells is HQ>MN>VC>AR>GA. The inhibitory effect of the five drugs on mushroom tyrosinase was studied. The results of inhibition of mushroom tyrosinase indicated that gentisic acid, arbutin, L-ascorbic acid and hydroquinone are tyrosinase inhibitor with the sequence of inhibition:HQ>GA>AR>VC. While MN did not show inhibitory effect on mushroom tyrosinase. The Line-weaver Burk Plot also showed that AR and GA are competitive inhibitors of mushroom tyrosinase. These results showed that AR and GA are mild and safe while effective tyrosinase inhibitors and can be applied in the therapy of hyperpigmentation disorders. MN is also very effective melanogenisis inhibitors, but it takes effect not by inhibition of tyrosinase. The safty of hydroquinone should be well considered in application.The effect of liposomes with edge-activator on the skin permeation and skin deposition of arbutin (AR) was investigated compared with arbutin aqueous solution. The abutin liposomes was prepared by extrusion method. The particle size distribution of liposomes was measured by laser light scattering and the entrapment efficiency was determined by sephadex gel chromatography. The skin permeation and skin deposition of AR in various skin layers were also investigated. The results showed the particle size distribution of liposomes was 91.7 nm (0.203% variability). The entrapment efficiency decreased with the increase of surfactant, also decreased with the increase of drug concentration in the formulations. While the entrapped drug amount in per mg lecithin reached the highest amount with the 5% AR liposomes. In skin permeation studies, the permeation rate of 5% AR in liposomes was (8.91 +1.33 ug/ml), close to the...