## 3 Results  
### Anti-Melanogenic Effects of FBG in B16F10 Cells  
The cytotoxicity effects of FBG, BG, and WG in B16F10 cells were measured by MTT assay. As shown in Figure 1A, FBG and WG showed lower toxicity at concentrations below 400 \(\upmu\)g/mL than BG. Therefore, a 100 \(\upmu\)g/mL concentration was selected for further experiments for FBG, BG, and WG.  
We investigated the tyrosinase activity of FBG, BG, and WG on mushroom-tyrosine and IBMX-stimulated B16F10 cells. The oxidation of L-DOPA was measured with mushroom-tyrosine at 0.5, 1, 1.5, 2, and 2.5 mg/mL of FBG, BG, and WG, respectively. As shown in Figure 1B, FBG inhibited mushroom tyrosinase activity better than BG and WG in a concentration-dependent manner. At a 2.5 mg/mL concentration, FBG inhibited mushroom tyrosinase by 60.10%, whereas BG and WG inhibited the enzyme by 54.93% and 49.97%, respectively.  
Isobutylmethylxanthine (IBMX) can induce melanogenesis by increasing cellular cAMP levels. As shown in Figure 1C, tyrosinase activity in IBMX-stimulated B16F10 cells was significantly increased by 4.3-fold (431.4%) compared with unstimulated cells. The tyrosinase activity of FBG, BG, and WG at a concentration of 100 \(\upmu\)g/mL was reduced by 215.7%, 186.8%, and 102.5%, respectively, compared with IBMX-stimulated cells. Arbutin (100 \(\upmu\)M) significantly reduced cellular tyrosinase activity by 230.5%.  
Furthermore, their effects on melanin production were evaluated in B16F10 cells. The B16F10 cells were co-treated with 100 \(\upmu\)M of IBMX and FBG, BG, or WG for 72 h. The results showed that the extracellular (Figure 1D) and intracellular (Figure 1E) melanin content of  
Figure 1: Anti-melanogenic effects of FBG, BG, and WG in B16F10 cells. **(A)** Cytotoxicity effect of FBG, BG, and WG in B16F10 cells for 72 h. The cells were treated with each sample at the indicated concentrations (50, 100, 200, and 400 \(\upmu\)g/mL) for 72 h. The cytotoxicity effect was then determined with MTT assays. **(B)** Inhibition of mushroom tyrosinase was determined in response to the indicated concentration of FBG, BG, and WG (0.5–2.5 mg/mL). **(C)** Intracellular tyrosinase activity in IBMX-stimulated B16F10 cells was measured in response to the concentration of 100 \(\upmu\)g/mL of FBG, BG, WG, or arbutin (100 \(\upmu\)M) for 72 h. The IBMX-stimulated B16F10 cells were treated with a concentration of 100 \(\upmu\)g/mL of FBG, BG, or WG, or arbutin (100 \(\upmu\)M) for 72 h, and **(D)** the extracellular and **(E)** the intracellular melanin contents were measured at 475 and 405 nm, respectively. The graph shows mean \(\pm\) SD values of three replicates. *** \(p<0.001\), as compared with the control group; * \(p<0.05\), **\(p<0.01\)**, as compared with the IBM- induced group.  
IBMX-stimulated cells increased by approximately 2.1 and 3.7-fold, respectively, compared with untreated cells (216% and 372%). In contrast, FBG at 100 \(\upmu\)g/mL significantly decreased extracellular and intracellular melanin content by 110.5% and 129.5%, respectively, whereas 100 \(\upmu\)g/mL of BG decreased extracellular and intracellular melanin content by 135.2% and 160.3%, respectively. Moreover, WG decreased extracellular and intracellular melanin content by 168.9% and 182.6%, respectively. In addition, arbutin at 100 \(\upmu\)M reduced extracellular and intracellular melanin production by 105.5% and 130.11%, respectively. These results suggest that FBG at 100 \(\upmu\)g/mL more strongly inhibited melanin production compared with BG and WG.  
In order to investigate the molecular mechanisms of FBG, BG, and WG in melanogenesis, we examined the changes in the expression of tyrosinase, TYRP-1, TYRP-2, and MITF. As shown in Figure 2, the mRNA expression of tyrosinase, TYRP-1, TYRP-2, and MITF was increased by IBMX stimulation. FBG, BG, and WG suppressed the mRNA expression of tyrosinase, TYRP-1, and MITF, whereas TYRP-2 was unregulated. However, FBG better downregulated the expressions of the tyrosinase, TYRP-1, and MITF genes compared with BG and WG.