Mortality and teratogenic effect of drugs on zebrafish embryos were observed daily between 48 to 144 hpf by using inverted microscope (Nikon Eclipse TS 100 Shinagawa, Japan). Effect of Chalcone Derivatives on Cellular Melanin Content Melanin is the final product of a complete melanin biosynthesis pathway. A potent depigmenting agent is usually described with respect of decrement in melanin formation. The depigmenting effect of both FLA and FLB on Rabbit Polyclonal to NSF -MSH-induced B16/F10 cells is usually described as in Physique 2. Induced melanogenesis by -MSH results in significant elevation of cellular melanin production. In order to study the decrement production of cellular melanin content, induced B16/F10 cells were treated Mitomycin C with FLA and FLB by 4-fold serial dilution of compounds (25, 6.25 and 1.56 M). Results demonstrated high extent of depigmenting effect of FLA (0.84 0.06 g melanin/ g protein) and FLB (0.38 0.03 g melanin/ g protein) at highest concentration of 25 M. In the absence of compounds, -MSH-induced B16/F10 cells produced 3.75 0.05 g melanin/ g protein. It was also found that both compounds showed inhibition towards cellular melanin production in dose-dependent manner. Interestingly, Mitomycin C cellular melanin content of B16/F10 cells were reduced albeit at the lowest concentration of 1 1.56 M. Open in a separate window Physique 2 Effect of chalcone derivatives on melanin production of -MSH-induced B16/F10 cells. Cellular melanin content after treated with (A) FLA and (B) FLB. Both compounds were tested at different concentrations (25, 6.25 and 1.56 M) and incubated Mitomycin C for 72 h. Arbutin (50 M) was used as positive control. All values are the mean S.E.M. of three impartial experiments. ** 0.01 and *** 0.01 were considered significantly different to -MSH-induced control group. 2.3. Inhibitory Effect Mitomycin C of Chalcone Derivatives on Cellular Tyrosinase Activity Tyrosinase plays a major role in the initial biosynthesis reaction of melanogenesis. Melanogenic inhibition targeting on tyrosinase has been a major interest in developing new depigmenting agent. The melanogenic effect of chalcone derivatives was evaluated on cellular tyrosinase activity of -MSH-induced B16/F10 cells (Physique 3). In the study, enzyme activity was measured by using l-DOPA as enzyme substrate while cells were treated in 4-fold serial dilution of compounds dosages descending from 25 to 1 1.56 M. The induced tyrosinase activity of cells was observed to decline tremendously with the treatment of FLA (1.23 0.04 U/g protein) and FLB (0.96 0.02 U/g protein) at highest concentration of 25 M. In the absence of compounds, -MSH-induced B16/F10 cells exhibited 8.64 0.15 U/g protein of tyrosinase activity. It was also exhibited that enzyme inhibitory activity of FLA and FLB exhibited a dose-dependent manner in which significant inhibition was evident up to 6.25 M. Open in a separate window Mitomycin C Physique 3 Effect of chalcone derivatives on cellular tyrosinase activity of -MSH-induced B16/F10 cells. Cellular tyrosinase activity after treated with (A) FLA and (B) FLB. Both compounds were tested at different concentrations (25, 6.25 and 1.56 M) and incubated for 72 h. Arbutin (50 M) was used as positive control. All values are the mean S.E.M. of three impartial experiments. A value of *** 0.001, was considered significantly different to -MSH-induced control group. 2.4. Down-Regulation Effect of Chalcone Derivatives on Tyr, Trp-1, Trp-2 and Mitf Genes Expression in B16/F10 Cells as well as genes expression (Physique 4). In the present study, B16/F10 cells were exposed to various concentrations of chalcone derivatives (1.56, 6.25 and 25 M). Induced expression of genes after treatment with FLA and FLB. At highest concentration of 25 M, both FLA and FLB showed suppression on gene with 0.20 0.01 and 0.15 0.04-fold expression respectively. Suppression around the grasp regulator of gene was also found significant on both FLA and FLB at highest concentration of 25 M with 0.41 0.07 and 0.32 0.03-fold expression, respectively. The other melanogenic-related genes of and and mRNA levels. Data are expressed as the mean SEM of three individual experiments. A value of ** 0.01 and *** 0.001 were considered significantly different to -MSH-induced control group. 2.5. Zebrafish Toxicity Assessment 3-isobutyl-1-methylxanthine (IBMX), a chemical compound was used in the experiment.