## 4 Discussion
To elucidate the potential anti-melanogenesis mechanism of _R. rugosa_ further in \(\alpha\)-MSH-induced B16F10 cells, we analyzed the upstream melanogenesis-activating cascade: PKA/CREB signaling. As mentioned previously, the PKA/CREB signaling pathway plays an important role in melanogenesis. At the cellular level, increased cAMP activates PKA; subsequently, activated PKA facilitates the activation of CREB via phosphorylation. Phosphorylated CREB then stimulates the expression of its transcription targets MITF, tyrosinase, TRP-1, and TRP-2, which finally induce the melanocyte to secrete a large amount of melanin and initiate skin pigmentation [37; 38]. Research revealed that pinstrophin could increase melanin content, tyrosinase activity, and melanogenesis-related markers by upregulating the PKA/CREB signaling pathways [39]. A natural citrus flavanone, 5-demethylnolietin, was also confirmed to promote melanogenesis through the cAMP/CREB pathway [40]. Conversely, a long noncoding RNA, UCA1, was found to decrease the levels of melanogenesis-related gene expression by inhibiting the cAMP/PKA pathway [41]. The present results found that the protein expression levels of PKA, P-PKA, CREB, and P-CREB were notably upregulated after the cells were stimulated with \(\alpha\)-MSH. However, the expression levels of the above proteins were downregulated after treating the \(\alpha\)-MSH-stimulated B16F10 cells with _R. rugosa_ crude extract and its solvent fractions at a concentration of 20 \(\mu\)g/mL. Among all tested fractions, the \(n\)-BuOH fraction showed the most consistent results to inhibit melanogenesis. In some tests other fractions showed higher inhibitory effect than \(n\)-BuOH but failed to exhibit similar effects in all areas, resulting in \(n\)-BuOH being the most effective to inhibit both melanin and tyrosinase content, PKA/CREB signaling, MITF, and TRPs. Nevertheless, these results indicate that _R. rugosa_ crude extract and its solvent fractions may contain potential therapeutic ingredients against abnormal pigmentation diseases. Although kojic acid treatment as a positive control indicated that the effect of the samples tested in the current study was not fully comparable to that of kojic acid, the present samples were mixture of compounds that may or may not possess the anti-melanogenic properties. Therefore, future studies to elucidate the active ingredients of _R. rugosa_ are expected to yield more effective compounds compared to fractions. Considering the side effects of continuous use of kojic acid and other tyrosinase inhibitors [27], finding novel natural bioactive agents will pave the way for safer treatment of hyperpigmentation. In this regard, the current results showed that _R. rugosa_ is a potential source for novel anti-melanogenic compounds to produce more effective results than current tyrosinase inhibitors such as kojic acid. To find the potential components that play an effective role in anti-melanogenesis activity in _R. rugosa_ crude extract and its solvent fractions, the total polyphenol content was determined. The results showed that the total polyphenol content of _R. rugosa_ crude extract and its H\({}_{2}\)O, \(n\)-BuOH, 85% aq. MeOH, and \(n\)-Hexane solvent fractions were 1383.9 \(\pm\) 44.5, 2004.7 \(\pm\) 43.4, 7270.3 \(\pm\) 54.5, 2064.1 \(\pm\) 34.8, and 1091.1 \(\pm\) 26.2 mg GAE/100 g extract, respectively, in which the \(n\)-BuOH fraction possessed the highest total polyphenol content. Many studies have verified that plant polyphenols exert various kinds of positive effects on human health, such as antioxidant, anti-inflammation, anti-aging, anti-cancer, and antiallergenic activities, etc. [42; 43]. A previous study detected a total of 531 polyphenol metabolites in rose hips with UPLC-MS/MS. Those results showed that rose hip extract contained phenolic acids (220), flavonoids (219), phlorotannins (50), lignans, and coumarins [44]. It has also been reported that polyphenols can ameliorate skin aging [45] and suppress tyrosinase activity [46].