Genetic Variation Affects the Anti-Melanogenic Efficacy of Platycodon grandiflorus Flowers

: This study investigated the utilization of by-products from Platycodon grandiflorus and the role of genetic diversity in their anti-melanogenic properties, focusing on the purple-flower (PF) and white-flower (WF) varieties. Our results show that the WF variety exhibited significantly higher anti-melanogenic activity, attributed to higher concentrations of key saponins such as platycodin D3 and platycodin D. These saponins demonstrated strong tyrosinase inhibitory effects as confirmed by molecular docking analysis. Further, the WF variety showed increased expression of genes involved in saponin biosynthesis, highlighting the role of genetic diversity in determining phytochemical composition and pharmacological efficacy. The superior anti-melanogenic activity of WF suggests its potential as a valuable ingredient in the cosmetic industry for skin-whitening products. Our findings emphasize the importance of utilizing by-products and selecting specific genotypes to enhance the quality and efficacy of plant-derived products. Future research should explore the full spectrum of bioactive compounds in P. grandiflorus , investigate sustainable extraction methods, and conduct clinical trials to validate the safety and effectiveness of these compounds in cosmetic and therapeutic applications.


Introduction
Platycodon grandiflorus, commonly known as balloon flower, is a single species belonging to the family Campanulaceae and is extensively utilized for ornamental, edible, and medicinal purposes in East Asia [1].Traditionally, the roots of P. grandiflorus have been employed for medicinal applications, particularly in the management of respiratory diseases including bronchitis, tonsillitis, asthma, and pneumonia [1][2][3].Although considerable attention has been directed towards elucidating the pharmacological attributes of its root, recent investigations have begun to explore the utilization of the aerial parts of P. grandiflorus to repurpose by-products generated during harvesting [4,5].For example, extracts derived from the aerial parts of P. grandiflorus have demonstrated notable antioxidant, whitening, and anti-inflammatory properties [6,7].Furthermore, flower extracts exhibit skincare benefits, including anti-inflammatory properties and promotion of skin wound healing [8].These properties indicate the potential of balloon flower by-products as valuable biomaterials for applications in the food, cosmetic, and medical industries.
In Korea, P. grandiflorus is found in two prevalent varieties: those with purple flowers (PFs) and those with white flowers (WFs), each distinguished by unique phytochemical compositions, notably anthocyanins [9].Additionally, distinctive triterpene saponins, including platycoside E (PE), platycodin D3 (PD3), and platycodin D (PD), have been identified as critical factors for discriminating between the varieties of P. grandiflorus [10].Notably, PD has a broad spectrum of pharmaceutical activities, including anti-inflammatory, anti-melanogenic, antiviral, anti-obesity, anti-coagulant, and anti-tumor properties [4,11].These observations suggest that variations in the content and composition of phytochemicals, depending on the variety, may lead to differences in pharmacological properties.
Appl.Sci.2024, 14, 6867 2 of 8 Consequently, the selection of genotypes assumes paramount importance for facilitating the potential utilization of specific genotypes across various industries, such as cosmetics and pharmaceuticals.However, variations in bioactivities between PFs and WFs are poorly known.
In this study, we compared and analyzed the differences in anti-melanogenic activity between two common varieties of P. grandiflorus to highlight the value of flowers as functional ingredients rather than merely as agricultural by-products.Based on HPLC and molecular docking analyses, we demonstrated that the variation in the anti-melanogenic activities of P. grandiflorus varieties is derived from different levels of several triterpene saponins including PD3 and PD.Our findings suggest that understanding genetic diversity is crucial for ensuring the quality control of medicinal plant-derived products.

Preparation of Plant Extracts
Flowers of P. grandiflorus, encompassing both WFs and PFs, were cultivated and harvested from the research farms at Chungbuk National University, South Korea, and immediately frozen in liquid nitrogen.The extracts from P. grandiflorus flowers were prepared using 70% ethanol and evaporated using a rotary vacuum evaporator.Subsequently, the crude extracts were suspended in water and sequentially partitioned with equal volumes of hexane, ethyl acetate (EtOAc), and n-butanol (water-saturated BuOH).The remaining aqueous extract was designated as the aqueous fraction.Each fraction was then further evaporated using a vacuum evaporator.

Determination of Anti-Melanogenic Properties and Cytotoxicity
B16F10 melanoma cells (KCLB No. 80008, Korea Cell Line Bank, Seoul, Republic of Korea) stimulated with 3-isobutyl-1-methylxanthine (IBMX) were treated with various concentrations of the samples.After a 48-h incubation in a CO 2 incubator, melanin production and cell viability in IBMX-stimulated B16F10 cells were assessed as previously described [12].Control refers to untreated cells, while mock refers to cells treated with DMSO and IBMX.To investigate the direct inhibitory effect on tyrosinase activity, a cell-free tyrosinase inhibition assay was conducted using a Tyrosinase Inhibition Screening Kit (BioVision, Milpitas, CA, USA) according to the manufacturer's instructions.

Expression Analysis Using Quantitative Real-Time PCR (qRT-PCR)
Total RNA from B16F10 cells and P. grandiflorus flowers was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the FavorPrep Plant Total RNA Purification Mini Kit (Favogen, PingTung, Taiwan), respectively.After synthesizing cDNA, the expression levels of each gene were determined using qRT-PCR with SYBR Green.GAPDH (for B16F10 cells) and actin (for flowers) were used for the normalization of gene expression, and the primer pairs used for qRT-PCR are listed in Supplemental Table S1.

Saponin Analysis Using HPLC
To quantify saponin compounds in the BuOH fractions, HPLC analysis was conducted using an Agilent 1260 Infinity II LC System coupled with a diode array detector.Saponin compounds were separated on a Poroshell 102 EC-C18 column (4.6 × 150 mm, 4 µm) at 30 • C with a flow rate of 1 mL/min.UV absorption was detected at 205 nm, and 20 µL of each sample was injected into the HPLC system.The mobile phase consisted of distilled water (A) and acetonitrile (B) containing 0.05% phosphoric acid.The concentration was calculated by substituting the peak areas of the samples into the calibration curve of the standards.

Molecular Docking
Molecular docking was conducted using tyrosinase (PDB code: 3NQ1) and the following compounds: PE (CHEBI ID: 70449), PD (ChemSpider ID: 142975), PD3 (ChemSpider ID: 57620425), platyconic acid A (PcA) (CHEBI ID: 70435), and polygalacin D (PgD) (Chem-Spider ID: 58805187).The docking calculations were performed using AutoDock4 with the Lamarckian Genetic Algorithm [13], following the methodology as previously described [14].For the blind docking, a grid box was set to encompass the entire protein structure, with docking parameters configured for 100 runs and 2,500,000 energy evaluations per cycle.Based on the blind docking outcomes, specific grid maps were generated for the defined docking.The parameters for the defined docking were adjusted to 250 runs and 2,500,000 energy evaluations for each iteration.The lowest binding energies and predicted inhibition constants were extracted from the docking log files.

Statistical Analysis
The data were represented as the mean ± SE of three independent biological experiments.The significant differences between each group were obtained using Duncan's multiple range test (p < 0.05) and Student's t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Comparative Evaluation of the Anti-Melanogenic Properties of P. grandiflorus 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. grandiflorus extracts have skincare benefits [7,18,19].However, the antimelanogenic effects of P. grandiflorus 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 µg/mL) inhibited IBMX-induced melanin production in B16F10 cells, the EtOAc fraction at 100 µg/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 EtOAc fractions.In contrast, the BuOH fraction at 100 µg/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± 20.26 µg/mL; WF_BU: IC50 = 241.58± 8.82 µg/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 (TRP1), 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 µg/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. grandiflorus 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.
melanocyte-specific gene expression (Figure 1E).In addition, the residual TYR activity was 64.2% of the control with 200 μg/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. grandiflorus 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.

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 ± 9.07 μg/mg) than PF_BU

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 ± 9.07 µg/mg) than PF_BU (205.14 ± 2.16 µg/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. grandiflorus (PE, PD3, PD, PcA, and PgD) in WF_BU and PF_BU.As shown in Figure 2A, Appl.Sci.2024, 14, 6867 5 of 8 WF_BU exhibited higher quantities of PD3, PD, PcA, and PgD compared to PF_BU, suggesting that genetic diversity affects saponin content in P. grandiflorus.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 β-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 α-MSH-stimulated B16F10 melanocytes [7], indicating that the variations in major saponins, particularly PD, contributed to differences in the anti-melanogenic activity of P. grandiflorus.
(205.14 ± 2.16 μg/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. grandiflorus (PE, PD3, PD, PcA, and PgD) in WF_BU and PF_BU.As shown in Figure 2A, WF_BU exhibited higher quantities of PD3, PD, PcA, and PgD compared to PF_BU, suggesting that genetic diversity affects saponin content in P. grandiflorus.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 β-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 α-MSH-stimulated B16F10 melanocytes [7], indicating that the variations in major saponins, particularly PD, contributed to differences in the anti-melanogenic activity of P. grandiflorus.

Molecular Docking Analysis of P. grandiflorus Saponins as Tyrosinase Inhibitors
The molecular docking approach is a pivotal tool in analyzing the interactions between target proteins and small molecules, greatly contributing to the discovery of high-quality enzyme inhibitors that have progressed to clinical trials [36].To elucidate the TYR inhibitory mechanisms of P. grandiflorus, we analyzed the interaction sites and patterns between major saponins and TYR using the structure-based molecular docking approach.Initially, we employed blind molecular docking to identify the binding sites of selected saponins on TYR.Subsequently, using a grid centered around the TYR residues identified in the blind docking, defined molecular docking was performed to predict the binding energies of PE, PD3, PD, PcA, and PgD to Bacillus megaterium TYR (TyrBm).Our findings revealed that PD3 demonstrated the highest binding affinity to tyrosinase among the tested saponins, with a binding energy of −9.39 ± 0.11 kcal/mol, followed by PD (−8.76 ± 2.22 kcal/mol), PE (−4.90 ± 1.85 kcal/mol), PcA (−5.86 ± 0.01 kcal/mol), and PgD (−6.30 ± 0.84 kcal/mol) (Table 1).In TyrBm, Val 218 functions as a gatekeeper residue, modulating the accessibility of the active site entrance.Substitution of this Val with Phe results in decreased TYR activity [37], suggesting that Val 218 plays a regulatory role in substrate binding of TyrBm.In P. grandiflorus, all tested saponins, except for PE, are engaged in hydrophobic interactions with the Val 218 residue (Table 1), indicating that the TYR inhibitory activity of P. grandiflorus saponins is likely mediated by inhibiting substrate binding.

Conclusions
This study highlights the potential of utilizing by-products from P. grandiflorus and underscores the important role of genetic diversity in enhancing their anti-melanogenic properties.The WF variety (WF_BU; IC50 = 241.58± 8.82 µg/mL) demonstrated superior antimelanogenic activity compared to the PF variety (PF_BU; IC50 = 427.97± 20.26 µg/mL), primarily from higher levels of saponins such as PD3 and PD.These findings were supported by molecular docking analysis, which confirmed the strong TYR inhibitory effects of these saponins.Additionally, the study revealed increased expression of genes involved in saponin biosynthesis in the WF variety, further illustrating the impact of genetic diversity on phytochemical composition and pharmacological efficacy.These results suggest that by-products of P. grandiflorus may serve as valuable ingredients in the cosmetic industry, particularly for skin-whitening products.The importance of selecting specific genotypes to optimize the quality and efficacy of plant-derived products is evident.Future studies should focus on exploring the full range of bioactive compounds in P. grandiflorus and conducting clinical trials to ensure the safety and effectiveness of these compounds in cosmetic and therapeutic applications.

Figure 1 .
Figure 1.Comparison of anti-melanogenic activities among different genotypes of Platycodon grandiflorus.(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 IBMXinduced 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 ± SE of three independent experiments.Statistical significance is indicated by ** p < 0.05.Different letters denote significant differences as determined by Duncan'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 whiteflower extract; TYR, tyrosinase; TRP1, tyrosinase-related protein 1; TRP2, tyrosinase-related protein 2.

Figure 1 .
Figure 1.Comparison of anti-melanogenic activities among different genotypes of Platycodon grandiflorus.(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 ± SE of three independent experiments.Statistical significance is indicated by ** p < 0.05.Different letters denote significant differences as determined by Duncan'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; TYR, tyrosinase; TRP1, tyrosinase-related protein 1; TRP2, tyrosinase-related protein 2.