## 3 Results
### Effect of Solvent Fractions from R. rugosa Crude Extract on Cell Viability  
Apart from the effect of _R. rugosa_ crude extract, we also evaluated the anti-melanogenesis effect of H\({}_{2}\)O, _n_-BuOH, 85% aq. MeOH, and _n_-hexane solvent fractions, separated from the crude extract by solvent fractionation, in \(\alpha\)-MSH-stimulated B16F10 cells. The results in Figure 3 show that all four fractions were not toxic to the cells at the concentrations ranging from 5 to 20 \(\upmu\)g/mL; hence, 5, 10, and 20 \(\upmu\)g/mL doses were used for the subsequent experiments. Figure 2: Effect of _R. rugosa_ crude extract on protein and mRNA expression levels of the melanogenesis- and PKA/CREB-signaling-pathway-related genes. **(A)** Protein expression bands of PKA, P-PKA, CREB, and P-CREB; **(B)** Protein expression bands of tyrosinase, MITE, TRP-1, and TRP-2; **(C)** Quantification of protein expression levels with densitometric analysis of Western blot bands; **(D)** The mRNA levels of tyrosinase, MITE, TRP-1, and TRP-2 given as relative percentages of mRNA levels in the stimulated untreated group. All mRNA levels were normalized against \(\beta\)-actin levels. The mRNA expression was measured using RT-qPCR \({}^{\text{a-e}}\) Bars with different letters in the same treatment group are significantly different while the same letters mean no significance (\(p<0.05\)) according to a one-way ANOVA test followed by the [PERSON]’s multiple range test. B: unstimulated untreated blank group; C: \(\alpha\)-MSH-stimulated untreated control group; KA: kojic acid (positive control). Effect of Solvent Fractions of R. rugosa Extract on Active Tyrosine Levels and Extracellular and Intracellular Melanin Content  
After treatment for 24 h at the concentrations of 5, 10, and 20 \(\upmu\)g/mL with solvent fractions from _R. rugosa_ extract, the extra/intracellular melanin contents were determined. As shown in Figure 4A,B, the four solvent fractions significantly inhibited both extracellular and intracellular melanin content of \(\alpha\)-MSH-stimulated cells in a dose-dependent manner (\(p<0.05\)). The tyrosinase activity in \(\alpha\)-MSH-stimulated cells was also measured, following treatment with solvent fractions from _R. rugosa_ extract. The result showed that the tyrosinase activity in the untreated \(\alpha\)-MSH-stimulated control group was highest and that in the unstimulated untreated blank group was the lowest (Figure 4C). It is worth noting that the tyrosinase activity decreased significantly in the H\({}_{2}\)O, \(n\)-BuOH, 85% aq. MeOH, and \(n\)-hexane solvent fractions groups, in which the inhibition effect was most effective at 20 \(\upmu\)g/mL. Therefore, the above results indicate that solvent fractions of _R. rugosa_ extract contained potentially effective ingredients that could inhibit the synthesis of melanin. Figure 3: Effect of H\({}_{2}\)O, \(n\)-BuOH, 85% aq. MeOH, and \(n\)-hexane solvent fractions on cell viability. \({}^{\mathrm{a-d}}\) Bars with different letters in the same treatment group are significantly different (\(p<0.05\)) while the same letters indicate no significance according to a one-way ANOVA test followed by the [PERSON]’s multiple range test. Effect of Solvent Fractions from R. rugosa Crude Extract on mRNA Expression Levels of Melanogenesis-Related Genes  
To verify that the decrease in melanin and tyrosinase levels was related to the down-regulation of melanogenesis-related genes, the expression levels of tyrosinase, MITF, TRP-1, and TRP-2 mRNA were determined using RT-qPCR. As demonstrated in Figure 5, the expression levels of the above genes in \(\alpha\)-MSH-stimulated cells were notably higher than those of unstimulated cells (\(p<0.05\)). However, the mRNA expression levels of tyrosinase, MITF, TRP-1, and TRP-2 were all down-regulated after the cells were treated with solvent fractions from _R. rugosa_ crude extract, where the concentration of 20 ug/mL showed the most effective inhibition. Figure 4: Effect of solvent fractions from _R.