# Genetic Variation Affects the Anti-Melanogenic Efficacy of _Platycodon grandiflora_ Flowers
## 3 Results and Discussion
### Variation in Saponin Content and Biosynthesis between Two Varieties  
The intricate relationship between the content and composition of phytochemicals in medicinal plants and their diverse biological activities has been a focus of considerable scientific research [27]. Particularly, genetic diversity greatly influences phytochemical biosynthesis and content, resulting in distinct metabolic profiles and variations in biological activity across different plant genotypes [28; 29]. In addition, the fractionation of crude extracts using butanol is a frequently employed method to enrich saponins from plant materials [30; 31]. Based on the analysis of total saponin content, we found that WF_BU contained a higher amount of saponins (230.83 \(\pm\) 9.07 \(\upmu\)g/mg) than PF_BU (205.14 \(\pm\) 2.16 \(\upmu\)g/mg) (Figure S1). Therefore, we hypothesized that the differences in anti-melanogenic activity between PF_BU and WF_BU (Figure 1) are attributed to variations in saponin content and composition. To investigate this hypothesis, we quantified five major saponins from _P. grandiflora_ (PE, PD3, PD, PcA, and PgD) in WF_BU and PF_BU. As shown in Figure 2A,  
Figure 1: Comparison of anti-melanogenic activities among different genotypes of _Platyodon grandiflora_. (**A**) Inhibitory effects of crude extracts and their solvent fractions on IBMX-induced melanin production in B16F10 melanoma cells. (**B**) Cell viability of B16F10 cells after incubation with crude extracts and their solvent fraction for 48 h. (**C**) IC50 values for the inhibition of IBMX-induced melanin production by BuOH fractions. (**D**) Inhibitory effects of BuOH fractions on intracellular tyrosinase activity in IBMX-stimulated B16F10 cells. (**E**) Effects of BuOH fractions on the expression levels of melanogenesis-related genes in IBMX-stimulated B16F10 cells. The transcription level of each gene in the samples is expressed relative to that of the mock control (Mock). (**F**) Inhibitory effects of BuOH fractions on mushroom tyrosinase activity under cell-free conditions. Data are presented as mean \(\pm\) SE of three independent experiments. Statistical significance is indicated by **\(p\)** < 0.05. Different letters denote significant differences as determined by [PERSON]’s multiple range test (\(p\) < 0.05). HX, hexane fraction; Et, EtOAc fraction; BU, BuOH fraction; AQ, Aqueous fraction; PF_BU, BuOH fraction of purple-flower extract; WF_BU, BuOH fraction of white-flower extract; YYR, tyrosinase; TRPM, tyrosinase-related protein 1; TRP2, tyrosinase-related protein 2.  
WF_BU exhibited higher quantities of PD3, PD, PcA, and PgD compared to PF_BU, suggesting that genetic diversity affects saponin content in _P. grandiflora_. Comprehensive metabolomic and transcriptomic analyses in diverse plant species have elucidated the correlation between specific components and key genes involved in saponin biosynthesis [32; 33; 34]. These studies indicate that saponin accumulation is regulated by a complex transcriptional network. Consistent with these findings, our qRT-PCR analysis revealed a significant, approximately two-fold increase in the expression levels of squalene synthase, squalene epoxidase, and \(\beta\)-ayrin synthase in WFs compared to PFs (Figure 2B), suggesting that the biosynthesis of saponin is affected by genetic diversity. The conversion of PE and PD3 into PD is catalyzed by specific glycosyltransferases (GTs) [35]. Notably, PF exhibited higher levels of PE compared to WFs, whereas WFs showed elevated levels of PD3 and PD relative to PF (Figure 2A). These findings suggest that genetic variation influences the expression levels or activity of specific GTs. PD exhibited TYR inhibitory activity (81.67% inhibitory effect at 3 mg/mL of PD) and inhibited melanin production in \(\alpha\)-MSH-stimulated B16F10 melanocytes [7], indicating that the variations in major saponins, particularly PD, contributed to differences in the anti-melanogenic activity of _P. grandiflora_.