## 4 Discussion  
_R. rugosa_ is a widely utilized plant appearing in daily diets, cosmetics, and textile products due to its medical properties. Studies have shown that it owes these medical properties to various kinds of bioactive secondary metabolites, such as phenolic acids, flavonoids, and ascorbic acid [24]. The current results suggest that _R. rugosa_ crude extract and its solvent fractions (H\({}_{2}\)O, _n_-BuOH, 85% aq. MeOH, and _n_-hexane) inhibited \(\alpha\)-MSH-stimulated melanogenesis in B16F10 murine melanoma cells by suppressing the activation of the PKA/CREB signaling pathway. Numerous studies have verified that tyrosinase is the rate-limiting enzyme for melanin synthesis, converting tyrosine to DOPA, which is an important intermediate during melanin synthesis [25]. Excess secretion of tyrosinase causes hyperpigmentation in the skin and leads to commercial problems in food industries because this enzyme attends to the unwanted color changes of vegetables, fruits, crustaceans, and fungi [26]. Therefore, searching for and developing tyrosinase inhibitors have drawn the attention of researchers in the fields of medicine, food, and cosmetics. It is widely accepted that inhibiting tyrosinase is the main action mechanism of chemicals that are used for their whitening properties, such as kojic acid, licorice flavonoid, \(\alpha\)-arbutin, and other similar substances. However, reports also show that these agents also exhibit side effects such as cytotoxicity, skin irritation, and itchiness [27]. Many extracts or components from natural plants have shown a tyrosinase-inhibitory effect. A previous study reported that _Euryale ferva_ seed extract showed inhibitory effects on both in vitro tyrosinase activity and cellular tyrosinase activity as well as melanin secretion [28]. _Ishige okamurae_ extracts, _Pueraria lobata_ stem extracts, and _Camelia sinensis_ water extracts were also reported to possess anti-tyrosinase activities [29; 30; 31]. Consistent with these studies, our results indicate that both extract and solvent fractionate from _R. rugosa_ significantly blocked melanin production by inhibiting \(\alpha\)-MSH-induced cellular tyrosinase activity in B16F10 cells. Based on the effect of _R. rugosa_ extracts on extra/intra-cellular melanin content and active tyrosinase levels, the transcription and translation of melanogenesis-related genes, viz., tyrosinase, MITF, TRP-1, and TRP-2, were explored with RT-qPCR and Western blot. MITF is mainly responsible for regulating the expression of tyrosinase, TRP-1, and TRP-2, and its overexpression not only causes an increase in the expression of these proteins but also influences other melanocytic differentiation markers [32]. Therefore, MITF is usually looked at as a diagnostic marker for melanogenesis [33]. Expectedly, the present study found that _R. rugosa_ crude extract and its H\({}_{2}\)O, _n_-BuOH, 85% aq. MeOH, and _n_-hexane fractions could effectively suppress the expression levels of MITF, tyrosinase, TRP-1, and TRP-2 in dose-dependent manners. A previous study revealed that the _R. rugosa_ flower contains aqueous polyphenols in which quercetin glycoside was the main component [34]. Meanwhile, it has been reported that quercetin could inhibit UVA-induced melanogenesis through the Nrf signaling pathway in B16F10 cells [35]. _R. rugosa_ is also rich in polysaccharides that showed hypoglycemic and immunomodulatory activities [36]. For these reasons, _R. rugosa_ is a potential material to be researched and developed for anti-melanogenesis cosmetic products. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & **Crude** & \multirow{2}{*}{**H\({}_{2}\)O fr.**} & \multirow{2}{*}{_n_-BuOH fr.**} & **85\% aq.** & \multirow{2}{*}{_n_-Hexane fr.**} \\  & **Extract** & & & & **MeOH fr.** & \\ \hline Total & & & & & \\ polyphenol content [1] & & & & & \\ \hline \hline \end{tabular} \({}^{1}\)Unit: mg GAE/100 g extract.  
\end{table} 
Table 1: Total polyphenol content of crude extract of _R. rugosa_ and its solvent fractions.