## 3 Results
### Effect of Solvent Fractions from R. rugosa Crude Extract on Cell Viability
rugosa_ crude extract on extra/intracellular melanin content and tyrosinase activity. (**A**) Extracellular melanin content; (**B**) Intracellular melanin content; (**C**) Intracellular tyrosinase activity. \({}^{\alpha-n}\) 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. B: unstimulated untreated blank group; C: \(\alpha\)-MSH-stimulated untreated control group; KA: kojic acid (positive control). Solvent Fractions from R. rugosa Inhibit Melanogenesis via Supressed Activation of the PKA/CREB Pathway  
To elucidate the anti-melanogenesis mechanism of H\({}_{2}\)O, \(n\)-BuOH, 85% aq. MeOH, and \(n\)-hexane solvent fractions from _Rosa rugosa_, their effect on the PKA/CREB signaling pathway and its downstream melanogenesis-related proteins (tyrosinase, MITF, TRP-1, and TRP-2) were investigated using Western blot analysis. As shown in Figure 6, the protein expression levels of PKA, p-PKA, CREB, and p-CREB were markedly up-regulated in the \(\alpha\)-MSH-stimulated B16F10 cells compared to the unstimulated blank group. However, expression of these proteins was significantly suppressed following treatment with 20 ug/mL solvent fractions from _R. rugosa_, which indicates that fractions might possess the ability to inhibit the phosphorylation of PKA and subsequent activation of CREB to suppress melanin production. Furthermore, the protein expression levels of tyrosinase, MITF, TRP-1, and TRP-2 were also detected to determine whether the effect on PKA/CREB activation was translated into suppressed production of melanogenesis-related proteins. Consistent with the mRNA expression results, the H\({}_{2}\)O (Figure 7A), \(n\)-BuOH (Figure 7B), 85% aq. MeOH (Figure 7C), and \(n\)-hexane (Figure 7D) solvent fractions from _R. rugosa_ dose-dependently decreased the protein levels of tyrosinase, MITF, TRP-1, and TRP-2. The above results reveal that solvent fractions from _R. rugosa_ could attenuate overproduction of melanin by inhibiting its synthesis via suppression of the PKA/CREB pathway. Figure 5: Effect of solvent fractions from _R. rugosa_ crude extract on mRNA expression levels of tyrosinase, MITF, TRP-1, and TRP-2. The mRNA levels are given as a relative percentage of the stimulated untreated group. The mRNA expression was quantified using RT-qPCR and normalized against \(\beta\)-actin. a-e 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. B: unstimulated untreated blank group; C: \(\alpha\)-MSH-stimulated untreated control group; KA: kojic acid (positive control). Figure 6: Effect of solvent fractions from _R. rugosa_ crude extract on the protein expression of PKA/CREB-signaling-pathway-related proteins. **(A)** Effect of H\({}_{2}\)O, _n_-BuOH, 85% aq. MeOH, and _n_-hexane solvent fractions on protein expression of PKA, p-PKA, CREB, and p-CREB at a concentration of 20 \(\upmu\)g/mL in B16F10 cells; **(B)** Quantification of protein expression levels with densitometric analysis of Western blot bands *-8 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. B: unstimulated untreated blank group; C: \(\alpha\)-MSH-stimulated untreated control group; KA: kojic acid (positive control).