# Genetic Variation Affects the Anti-Melanogenic Efficacy of _Platycodon grandiflora_ Flowers
## 3 Results and Discussion  
### Comparative Evaluation of the Anti-Melanogenic Properties of P. grandiflora Flower Extracts  
In the cosmetic industry, achieving effective skin whitening while ensuring stability and safety poses a significant challenge. Traditional whitening agents such as arbutin and kojic acid have demonstrated promise but are hindered by stability issues [15]. Consequently, there is a rising interest in discovering natural compounds derived from plants with whitening properties that could potentially address these challenges [16; 17]. As described above, _P. grandiflora_ extracts have skincare benefits [7; 18; 19]. However, the anti-melanogenic effects of _P. grandiflora_ flowers require further investigation. Therefore, the anti-melanogenic properties and their variation between PF and WF were investigated using B16F10 murine melanoma cells. Although neither of the crude extracts (100 \(\mathrm{\SIUnitSymbolMicro m}\)/mL) inhibited IBMX-induced melanin production in B16F10 cells, the EOAc fraction at 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL showed the highest anti-melanogenic activity, with PFs and WFs exhibiting 65.5% and 62.5% inhibition, respectively, compared to the mock control (Figure 1A). However, no significant difference was observed between the two EOAc fractions. In contrast, the BuOH fraction at 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL displayed distinct activities, with WF_BU (BuOH fraction of WF) exhibiting 22.9% inhibition, showing significantly better anti-melanogenic activity compared to PF_BU (BuOH fraction of PF), which did not exhibit any anti-melanogenic activity. This distinction was further emphasized when comparing the IC50 values (PF_BU: IC50 = 427.97 \(\pm\) 20.26 \(\mathrm{\SIUnitSymbolMicro g}\)/mL; WF_BU: IC50 = 241.58 \(\pm\) 8.82 \(\mathrm{\SIUnitSymbolMicro g}\)/mL; Figure 1C). Additionally, MTT assays indicated that the anti-melanogenic activity of WF_BU was not accompanied by cytotoxic effects in B16F10 cells (Figure 1B). Consistent with this finding, WFs of various plants have strong anti-melanogenic activities compared to flowers of other colors [20; 21; 22] because of active phytochemicals such as terpenoids and lipid derivatives, distinct from pigment substances [23; 24].  
Tyrosinase is a crucial copper-containing enzyme that initiates pigment synthesis by utilizing its substrate amino acid tyrosine or L-3,4-dihydroxyphenylalanine (L-DOPA). Furthermore, the activity of tyrosinase (TYR) directly influences cellular pigmentation [25]. As shown in Figure 1D, WF_BU reduced intracellular TYR activity in IBMX-stimulated cells, indicating that the anti-melanogenic activities of WF_BU resulted from the reduction in TYR activity. The change in intracellular TYR concentration is consistent with changes in enzyme activity [26]. To assess the effect of WF_BU on _TYR_ expression, the expression levels of melanocyte-specific genes such as _TYR_, TYR-related protein-1 (_TRPI_), and -2 (_TRP2_) were determined using qRT-PCR. WF_BU had no effect on the IBMX-induced increases in melanocyte-specific gene expression (Figure 1E). In addition, the residual TYR activity was 64.2% of the control with 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL of WF_BU under cell-free conditions, suggesting that the anti-melanogenic activity of WF_BU was mediated by inhibition of the L-DOPA oxidation activity of tyrosinase rather than by inhibiting transcription (Figure 1F). This result is similar to the anti-melanogenic activity of _P. grandiflora_ root extract [7].  
Collectively, these findings suggest that WF_BU has the potential as an effective inhibitor of tyrosinase, indicating the possibility of developing WF_BU as a skin-whitening agent.