White Ginseng Ethanol Extract (WGEE) Inhibits Tyrosinase and Melanogenesis via MITF Downregulation in B16F10 Cells

White Ginseng Ethanol Extract (WGEE) Inhibits Tyrosinase and Melanogenesis via MITF Downregulation in B16F10 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article White Ginseng Ethanol Extract (WGEE) Inhibits Tyrosinase and Melanogenesis via MITF Downregulation in B16F10 Cells Min Jung Kim, Ye Jin Yang, Ji Woong Heo, Han Nim Choi, Chae Un Lim, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7125633/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hyperpigmentation disorders such as melasma and freckles are common cosmetic concerns that have increased interest in safe, naturally occurring compounds for melanin production control. This study investigated white ginseng ethanol extract (WGEE) from the Geonsam cultivar of Panax ginseng as a potential functional ingredient for skin-whitening applications. WGEE was prepared by extracting white ginseng with 70% ethanol at 40°C for 24 hours. Chemical composition was analyzed using UPLC and colorimetric assays. Antioxidant capacity was evaluated via DPPH and ABTS assays, and tyrosinase inhibition was measured spectrophotometrically. B16F10 melanoma cells were assessed for cytotoxicity, melanin content, and melanogenesis-related proteins using MTT assay and Western blot analysis. Molecular docking evaluated ginsenosides Rg1 and Rb1 binding with tyrosinase. WGEE showed antioxidant activity with DPPH and ABTS radical scavenging of 18.18% and 48.54% at 200 µg/mL. In α-MSH-stimulated B16F10 cells, WGEE at 40 µg/mL reduced melanin production by 33.65% and downregulated MITF, tyrosinase, TRP-1, and TRP-2 expression similar to arbutin. Molecular docking revealed binding energies of -8.1 kcal/mol for Rg1 and − 6.2 kcal/mol for Rb1, suggesting direct tyrosinase inhibition. WGEE demonstrates potential as a natural cosmetic ingredient for skin whitening and pigmentation control. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Drug discovery Biological sciences/Plant sciences Antioxidant B16F10 Cell MITF Tyrosinase White Ginseng Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Alpha-melanocyte stimulating hormone (α-MSH) is a melanocortin peptide hormone derived from proopiomelanocortin (POMC), a proprotein produced in the pituitary gland 1 . When exposed to ultraviolet (UV) radiation, α-MSH binds to the melanocortin 1 receptor (MC1R) found on melanocytes, leading to the activation of adenylyl cyclase and an increase in cyclic adenosine monophosphate (cAMP) levels 2 . This elevation in cAMP activates protein kinase A (PKA), which in turn phosphorylates Cyclic AMP-responsive element-binding protein (CREB), leading to the induction of Microphthalmia-associated transcription factor (MITF) expression, a key regulator of melanogenesis 3 . The MITF protein regulates the production of key enzymes involved in melanin synthesis, such as tyrosinase and tyrosinase-related proteins (TRPs). Melanin exists in two primary forms: eumelanin (black or brown pigment) and pheomelanin (yellow or red pigment). Tyrosine is enzymatically converted into 3,4-dihydroxy-L-phenylalanine (L-DOPA) by the enzyme tyrosinase, subsequently leading to its conversion into dopaquinone 4 . Dopaquinone undergoes additional processing by TRP-1 and TRP-2 enzymes to generate eumelanin. There are multiple strategies available to inhibit melanin production, with primary emphasis on the inhibition of tyrosinase activity. Chemical compounds found in nature, such as hydroquinone and licorice extract, have been shown to impede the enzymatic conversion of tyrosine to L-DOPA, whereas antioxidants like vitamin C have been demonstrated to inhibit the oxidative processes involving tyrosine 5 . Additionally, niacinamide has the capability to inhibit the transfer of melanin to keratinocytes, and chemical bleaching agents are commonly utilized in treatments aimed at skin whitening 6 . Nevertheless, many of the agents mentioned above are closely linked to negative effects such as stinging, contact dermatitis, irritation, high toxicity, and sensitivity. As a result, current studies from cosmetic companies and research institutions have focused on creating new whitening agents that specifically block tyrosinase activity in order to diminish hyperpigmentation, without harming normal, healthy melanocytes. The term Panax, originating from the botanical name of ginseng (Panax ginseng), translates in Greek to panacea 7 . Ginseng has historically been utilized for the prevention and treatment of a variety of illnesses, and contemporary studies have confirmed its broad spectrum of pharmacological benefits. Ginseng is categorized based on its processing technique, where white ginseng pertains to dried, unaltered ginseng, while red ginseng goes through a steaming and drying procedure 8 . Though there have been many studies investigating the pharmacological advantages of ginseng, there is a scarcity of research on white ginseng. Recent interest in skin whitening has led to ongoing research into the use of natural and synthetic substances for treating skin conditions like pigmentation. In this study, we employed an ethanol extract of white ginseng to examine its bioactive compound content, antioxidant properties, and effects on cell whitening in B16F10 cells. Moreover, our goal is to showcase the potential of processed white ginseng, which is a type of Panax ginseng cultivated in Korea, as a valuable ingredient in functional cosmetics for skin brightening, as well as its application in food products. Results The concentration of polyphenols and flavonoids in ethanol extracts of white ginseng The concentration of 200 µg/ml was used to quantify the total polyphenol and flavonoid content of WGEEs. The WGEE contained a total polyphenol content of 33.11 ± 0.43 mg/g at the specified concentration, and a total flavonoid content of 33.33 ± 0.41 mg/g as indicated in Table 1 . Table 1 Total phenolic and flavonoids contents of WGEE Items Concentration WGEE (2mg/mL) Total polyphenol (GAE mg/g) 1) 33.11 ± 0.43 Total flavonoid (QE mg/g) 2) 33.33 ± 0.41 1 Total polyphenol content is expressed as gallic acid equivalents (GAE). 2 Total flavonoid content is expressed as quercetin equivalent (QE). Antioxidant Activity of WGEE The radical scavenging activity of WGEE was observed at concentrations ranging from 40 to 300 µg/mL, with scavenging rates increasing proportionally. Vit. C demonstrated a scavenging activity of 91.1%, significantly higher by 65.3% compared to the 300 µg/mL concentration of WGEE at 100 µg/mL (Fig. 1 A). The ABTS radical scavenging activity of WGEE was measured at various concentrations (10 µg/mL to 300 µg/mL), showing scavenging rates ranging from 5.3–60.9%. Vit. C demonstrated a scavenging activity of 94.0%, exceeding 33.1% the activity observed in the 300 µg/mL concentration of WGEE at 100 µg/mL as shown in Fig. 1 B. Inhibitory Effects of WGEE on Melanogenesis and Tyrosinase Activity The study assessed the anti-melanogenic properties of WGEE by analyzing melanin content and tyrosinase activity in B16F10 melanoma cells. Cytotoxicity assessment revealed a notable reduction in cell viability at concentrations equal to or greater than 60 µg/mL (p < 0.01), leading to the adoption of non-cytotoxic concentrations of 20 and 40 µg/mL for subsequent experiments (Fig. 2 A). The results presented in Fig. 2 B indicate that treatment with 100 nM α-MSH led to a significant increase in melanin production, reaching 127.97% compared to the control group (p < 0.001), thus confirming its melanogenic properties. Treatment with 20 µg/mL of WGEE resulted in 104.88% melanin content, indicating no significant alteration. In contrast, treatment with 40 µg/mL of WGEE led to a significant suppression of melanin synthesis to 94.31%, in comparison to both the α-MSH group (p < 0.001) and control (p < 0.001). The findings demonstrate that the inhibitory effect of WGEE on melanin synthesis is dependent on the dose. Additionally, experiments conducted to assess tyrosinase inhibition in vitro showed that WGEE reduced L-tyrosinase activity in a dose-dependent manner ranging from 20 to 200 µg/mL (Fig. 2 C). The inhibition rates ranged from 9.80 ± 1.1% to 23.00 ± 1.7%, with Vit. C at a concentration of 200 µg/mL used as a positive control exhibiting 66.05 ± 2.3% inhibition (p < 0.001). Suppressive Effects of WGEE on MITF and Tyrosinase Expression To examine the impact of WGEE on melanogenesis, levels of key melanogenesis-related proteins like tyrosinase, TRP-1, and MITF were measured through western blot analysis in α-MSH-stimulated B16F10 melanoma cells as shown in Fig. 3 A. In comparison to the control group treated with α-MSH alone, administration of WGEE led to a notable reduction in the expression levels of tyrosinase, TRP-1, and MITF. Significantly, the group administered with 40 µg/mL white ginseng extract showed a reduction of approximately 19.05% in tyrosinase expression (p < 0.05, 40% reduction in TRP-1 (p < 0.001), 76.47% TRP-2 (p < 0.001) and 33.33% reduction in MITF expression (p < 0.001) (Fig. 3 B) compared to the group that received only α-MSH. These results suggest a potent inhibitory effect of white ginseng extract on melanin biosynthesis machinery. Determination of Ginsenosides Rg1 and Rb1 in WGEE and Evaluation of Their Bioactive Potential UPLC analysis enabled the quantitative determination of the ginsenosides (Rg1 and Rb1) (Fig. 4 A). Ginsenosides Rg1 and Rb1 were identified in a 1000 ppm WGEE at concentrations of 12.0252 ppm and 37.5906 ppm, respectively, indicating the presence of ginsenoside components in the extract. While other minor compounds were also observed on the chromatogram, Rg1 and Rb1 constituted the major peaks (Fig. 4 B). In vitro and in silico insights into tyrosinase inhibitors with Ginsenoside The protein is shown as cartoon ribbons, with red and blue colors representing different subunits, and the ligands (Rg1, Rb1) are displayed as stick models. You can see interaction types like hydrogen bonding, Van der Waals forces, and hydrophobic interactions with Discovery Studio Visualizer. We used molecular docking simulations to predict how tyrosinase (PDB ID: 3NM8) interacts with ginsenosides Rg1 and Rb1, which are key active compounds found in Panax ginseng. Both the ligands had strong binding affinities and interacted well within the active site of tyrosinase. Ginsenoside Rg1 was placed inside the enzyme's hydrophobic pocket, creating several regular hydrogen bonds and Van der Waals interactions with nearby residues (Fig. 5 A). Important amino acids that participate in the binding of Rg1 are histidine A204, glutamate A208, glycine A216, and methionine A261, which are vital for an enzymatic function. The 2D diagram in Fig. 5 B showed that the ligand is stabilized by interactions like pi-sigma and alkyl bonding with PRO A253 and GLY A209 in the active site, helping keep its shape stable. The binding of ginsenoside Rb1 with tyrosinase is 3D diagram in Fig. 5 C. In Fig. 5 D, ARG 75, TRP 269, TYR 72, THR 271, THR 272, PRO 273, PHE 262, ASN 249, TYR 250, GLN 242, ASN 278, TRP 238, MET 277, GLU 274, VAL 276, ASP 275, PRO 67, MET 266, ARG 70, GLU 71, TRP 68, LEU 74, ALA 64, HIS 69, LEU 66, LEU 282 and PHE 65 were identified as important interacting residues connected through hydrogen bonds, hydrophobic interactions, and amide-π stacking. Discussion This study demonstrates that WGEE exhibits significant anti-melanogenic properties, positioning it as a promising natural ingredient for hyperpigmentation treatment. WGEE effectively reduced melanin production in α-MSH-stimulated B16F10 cells and downregulated key melanogenesis proteins including MITF, tyrosinase, TRP-1, and TRP-2. The antioxidant capacity and molecular docking results suggest that WGEE's skin-whitening effects involve both indirect antioxidant mechanisms and direct tyrosinase inhibition. Polyphenols and flavonoids are important antioxidant compounds present in plants, serving as participants in redox reactions and demonstrating diverse physiological functions 9 . Various phenolic compounds, including salicylic acid 10 , p-benzoxybenzoic acid, gentisic acid, vanillic acid, and ascorbic acid, have been identified in ginseng for their noteworthy antioxidant properties 11 . Moreover, research suggests that the overall flavonoid content may differ depending on the extraction technique 12 and different organs (leaves, stems, and roots) utilized 13 . For example, Chung 11 and colleagues identified variations in phenolic compounds and their levels based on the specific parts of the ginseng plant, the geographical location of its habitat, and the methods used for cultivation. Therefore, it is apparent that both polyphenol and flavonoid levels can vary not only depending on the extraction technique employed, even for identical samples, but also based on the geographical location of cultivation. The stability of the DPPH radical is affected by factors such as light exposure, temperature variations, and pH levels, making it a commonly utilized compound in assays for antioxidant activity 14 , 15 . This technique is based on the transformation of the purple DPPH radical into a yellow hue as a result of its interaction with antioxidants 16 . The ABTS assay is a commonly utilized indirect method recognized for its resistance to pH variations, especially in the evaluation of food and natural water-soluble phenolic substances 16 . ABTS displays durability in the absence of phenolic compounds 17 . Nevertheless, it displays a pronounced reactivity towards hydrogen donors, such as phenolic compounds, resulting in the formation of colorless ABTS 16 . Although WGEE demonstrated moderate melanin production inhibitory activity relative to vitamin C, its gradual inhibitory effect suggests the presence of bioactive phytochemicals, including protocatechuic acid and ginsenosides, which may synergistically modulate melanogenesis through antioxidant-mediated and melanin synthesis-suppressive mechanisms. Collectively, these findings suggest that WGEE successfully hinders melanin production by reducing both melanin synthesis and tyrosinase activity. The expression level of MITF, a crucial regulator of tyrosinase and TRP-1, was significantly decreased, indicating that white ginseng extract may downregulate melanogenesis by suppressing transcription. The inhibition of MITF signifies upstream control, as MITF triggers the transcription of melanogenic enzymes such as tyrosinase and TRP-1 18 . Considering the confirmed role of MITF in pigmentation, it is probable that its decreased expression is responsible for the observed reduction in downstream enzymes, consequently impacting melanin synthesis. To summarize, the ethanol extract of white ginseng demonstrates inhibitory effects on melanin production by regulating MITF and its downstream genes, indicating its possible use as a botanical skin-lightening agent. Ginsenosides are major bioactive components of ginseng, known to possess diverse physiological effects, including antioxidant 19 , anti-inflammatory 20 , and immunomodulatory activities 12 . These findings are consistent with previous studies that report the whitening effects of ginsenosides such as Rg1 and Rb1 via suppression of MITF and melanogenic enzymes 21 . Rg1 and Rb1 exhibit whitening, antioxidant, and skin-protective effects, suggesting their potential application as functional cosmetic ingredients. The findings of this study suggest that white ginseng possess sufficient bioactivity to be utilized as a functional material. The results of the molecular docking analysis showed that Rg1 exhibited a stronger binding affinity (− 8.1 kcal/mol) compared to Rb1 (− 6.2 kcal/mol) at the active site of tyrosinase (PDB ID: 3NM8). This suggests Rb1 has better antioxidants and anti-melanogenic properties than Rg1 in living organisms. Additionally, the way both compounds interact with histidine and glutamate residues close to the copper center of tyrosinase might prevent the oxidation of phenolic substrates like L-DOPA through a chelating mechanism or steric hindrance. These results show that ginsenosides, especially Rb1, could work as natural tyrosinase inhibitors and show potential as functional ingredients in cosmetics or pharmaceuticals for treating hyperpigmentation and oxidative stress-related disorders. Further investigations are warranted to elucidate the molecular mechanisms underlying WGEE's melanogenesis inhibitory effects and to evaluate its safety profile as a natural alternative to synthetic whitening agents. In vivo studies are essential to validate these findings and assess clinical efficacy in human subjects. Future research should also optimize extraction methodologies and evaluate ginsenoside stability to establish standardized protocols for cosmetic applications. Conclusion As summarized in Fig. 6 , WGEE inhibits melanogenesis in B16F10 cells by blocking tyrosinase activity, reducing MITF expression downregulating TRP1/TRP2 expression, and enhancing antioxidant activity, making it a promising candidate for natural skin-whitening applications. Material and methods Crude extract methods White ginseng was extracted with 70% ethanol at 40°C for 24 hours. A mixture of white ginseng and white ginseng fine roots, blended in a weight ratio of 6:4 (w/w), was subjected to two rounds of extraction using consistent conditions. The final extract was filtered and concentrated at reduced pressure, resulting in the production of the WGEE. Analysis of total flavonoid and total polyphenol concentration To measure the total polyphenol content, white ginseng extract (2 mg/mL) was mixed with 500 µL of 2N Folin’s phenol reagent and allowed to stand at room temperature for 3 min. Next, 400 microliters of a 7.5% sodium carbonate solution were added. Subsequently, the solution was transferred to a 96-well plate and allowed incubate in the absence of light at an ambient temperature for a duration of 1 hour. After incubation, the absorbance at 750 nm was quantified using a microplate reader, and the total polyphenol content was calculated using a standard curve. To determine the total flavonoid content, 100 µL of the same extract was mixed in a 96-well plate with 860 µL of 80% ethanol, 20 µL of 10% aluminum chloride, and 20 µL of 1M potassium acetate. Following a 40-minute incubation period at room temperature, the absorbance at 415 nm was recorded to determine the total flavonoid content through the utilization of a standard curve. DPPH and ABTS assays were used to evaluate radical scavenging activity To conduct the DPPH assay, a solution of DPPH was created through the dilution of the DPPH reagent in pure methanol to ensure an absorbance reading of 1.00 ± 0.10 at a wavelength of 517 nm. Samples were additionally diluted with 100% methanol to different concentrations. A 100 µL portion of the DPPH solution was combined with 100 microliters of the sample, and the resulting mixture underwent incubation at 37°C in the absence of light for a duration of 30 minutes. Following this, the absorbance was recorded at 517 nm utilizing a microplate spectrophotometer. ABTS + radicals were produced through the combination of 7.4 mM ABTS and 2.6 mM potassium persulfate in distilled water at a 1:1 ratio. The mixture underwent a reaction period of 16–24 hours in darkness. The ABTS + solution was subsequently diluted with distilled water until reaching an absorbance of 0.7 ± 0.02 at 734 nm. In a 96-well plate, 100 microliters of the diluted ABTS + solution were mixed with 100 microliters of the sample, and the absorbance was promptly measured at 734 nanometers using a microplate spectrometer. Cell Culture and Cytotoxicity Assay B16F10 cells sourced from the Korean Cell Line Bank were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Waltham, MA, USA) and 0.2% NaHCO 3 (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated at a temperature of 37°C in a controlled environment with 5% carbon dioxide. A cytotoxicity assay was conducted using MTT to evaluate cell viability. Cells were distributed into 96-well plates at a concentration of 5 × 10 3 cells per well and left to incubate for 24 hours. Following this, the culture medium was exchanged with a fresh medium that contained different concentrations of white ginseng extract ranging from 10 to 300 µg/mL. Subsequently, the cells were exposed to 10 µL of 1.0 mg/mL MTT solution and allowed to incubate at 37°C for a duration of 2 hours. After incubation, the MTT solution was aspirated, and the cells were rinsed with D-PBS. The resulting formazan crystals were dissolved in 100 µL of DMSO for 30 minutes, and the absorbance was measured at 570 nm using a Synergy H1 reader. Intracellular Melanin Content Measurement Intracellular melanin content was measured using a modified Hosoi method 22 . B16F10 cells were seeded at a density of 1 × 10 5 cells/well in 60 mm plates and incubated at 37°C for 24 hours. Subsequently, cells were treated with α-MSH and 20 and 40 µg/mL α-MSH (100nM) targeting polypeptides for 48 hours at 37°C. The culture medium supernatant was then collected, and extracellular melanin content was measured at 490 nm. For each group, 2 × 10 6 cells were lysed using radio immunoprecipitation assay (RIPA) buffer. The cellular pellets, acquired through centrifugation, were rinsed with alcohol before being dissolved in a 1 N NaOH solution containing 10% DMSO at a temperature of 90°C for a duration of 1 hour, followed by the measurement of absorbance at 490 nm. Tyrosinase inhibition assay conducted in vitro The activity of tyrosinase inhibition was assessed through a modified methodology based on the approach outlined in the study by Tomita et al. (1990) 23 , 24 . In this experimental procedure, 100 microliters of a sample solution at a concentration of 1 milligram per milliliter was combined with 3.2 milliliters of a 0.1 molar sodium phosphate buffer at a pH of 6.5. Subsequently, 50 microliters of mushroom tyrosinase with an activity of 2000 units per milliliter, obtained from Sigma-Aldrich in the United States, was added to the mixture. After mixing, 100 µL of 1.5 mM L-tyrosine (Sigma-Aldrich, USA) was added. The solution was allowed to incubate at a temperature of 37°C for a duration of 15 minutes, followed by measurement of absorbance at a wavelength of 475nm using a UV/Vis spectrophotometer (OPTIZEN POP, Mecasys, Korea). For the control group, a 0.1 M sodium phosphate buffer with a pH of 6.5 was employed instead of the sample, and a Vitamin C (ascorbic acid) solution served as the positive control. Western blot B16F10 cells were washed with PBS and then dissolved in 100 µL lysis buffer for 20 min with ice. After centrifugation at 12,000 rpm for 15min, the protein suspension was obtained by collecting the liquid supernatant. Then, 20µg proteins were loaded into 8% SDS-PAGE gel before being transferred to 0.45µm PVDF membranes. The membranes were blocked with 5% Bovine Serum Albumin (BSA) buffer for 2h, then washed with tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) three times, and incubated with MITF (sc-515925), TYR (sc-20035), TRP-1 (sc-166857), TRP-2 (sc-74439), and GADPH with TBST. After the reaction with the second antibody, an Chemiluminescence Imager detection SH-Cute 523 system was used to visualize the proteins (Shenhua Science Technology, Hangzhou city, Chinese). Densitometric analysis of the bands were performed using ImageJ (Version 1.54). Western blot results represented at least three independent experiments. UPLC analysis conditions An analysis of WGEE for both qualitative and quantitative purposes was conducted utilizing a Thermo Dionex Ultimate 3000 system. The separation process utilized a Pronto SIL C18 column with dimensions of 150 × 4.6 mm, particle size of 5 µm, and model number 120-5-C18 SH, manufactured by Bischoff Chromatography in Leonberg, Germany. The samples were dissolved in a solution of 70% methanol to achieve a concentration of 1000 ppm before being passed through a 0.45 µm filter. Ginsenoside Rg1 and Ginsenoside Rb1 purchased from ChemFaces (Cat. No. CFN99967 and CFN99964, respectively), located in Wuhan, Hubei, China, were employed as reference compounds ( Supplementary data ). Molecular Docking Analysis Molecular docking was conducted to investigate the binding interactions between ginsenosides (Rg1 and Rb1) and tyrosinase (PDB ID: 3NM8) 25 . The 3D structure of tyrosinase was obtained from the Protein Data Bank, and the structure of ginsenoside Rg1 was retrieved from the PubChem database. Ligand structures were converted from SDF to PDB format using Open Babel (v3.1.1) and energy-minimized with the MMFF94 force field. Protein preparation involved removal of water molecules, addition of hydrogen atoms, and assignment of Kollman charges using AutoDock Tools. Docking simulations for Rg1 were performed using AutoDock Vina (v1.1.2), with the grid box centered on the active site and the exhaustiveness set to 8. Due to the large molecular size and high conformational flexibility of Rb1, docking using open-source tools like AutoDock or PyRx was not feasible. Therefore, BIOVIA Discovery Studio, a commercially licensed software, was used for conformer generation and docking analysis of Rb1. Ligand-protein complexes were visualized in 3D using PyMOL and Discovery Studio Visualizer, and 2D interaction maps were generated to examine hydrogen bonding, hydrophobic interactions, and key amino acid residues involved in binding. Statistical analysis GraphPad Prism version 8.0 was used for all statistical analyses (GraphPad Software, SanDiego, CA, USA). The researchers utilized one-way factorial analysis of variance to assess the variation. The findings are presented as the mean value along with the standard error of the mean or standard deviation. A p-value lower than 0.05 was deemed statistically significant for all tests. Declarations Data availability All data generated or analysed during this study are included in this published article (and its supplementary information files). Funding This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. RS-2022-NR075631), the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Science and ICT (No. RS-2024-00403488), and the Korea Institute of Oriental Medicine grant number KSN2412012. Author contributions M.J.K., Y.J.Y., J.W.H., H.N.C., C.U.L., S.H.J., H.H.K., G.S.K., Y.H.K., J-H.Y. and K.I.P. searched and collected the literature, summarized the contents, and described the articles. M.J.K., Y.J.Y., J.W.H. and H.H.K. organized the tables and created the figures. S.H.J., G.S.K., J.H.Y., Y.H.K. and K.I.P. provided valuable suggestions during manuscript preparation and critically revised the manuscript accordingly. G.S.K., J-H.Y., and K.I.P. conceptualized and wrote the manuscript. All authors have read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Consent for publication All authors have approved the manuscript for publication. Competing interests The authors declare no competing interests. References Slominski, A., Tobin, D. J., Shibahara, S. & Wortsman, J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol. Rev. 84 , 1155–1228. 10.1152/physrev.00044.2003 (2004). Costin, G. E. & Hearing, V. J. 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The antioxidant activities of Korean Red Ginseng (Panax ginseng) and ginsenosides: A systemic review through in vivo and clinical trials. J. ginseng Res. 45 , 41–47. 10.1016/j.jgr.2020.09.006 (2021). Im, D. S. Pro-Resolving Effect of Ginsenosides as an Anti-Inflammatory Mechanism of Panax ginseng. Biomolecules 10 10.3390/biom10030444 (2020). Lee, H. R., Jung, J. M., Seo, J. Y., Chang, S. E. & Song, Y. Anti-melanogenic property of ginsenoside Rf from Panax ginseng via inhibition of CREB/MITF pathway in melanocytes and ex vivo human skin. J. ginseng Res. 45 , 555–564. 10.1016/j.jgr.2020.11.003 (2021). Hosoi, J., Abe, E., Suda, T. & Kuroki, T. Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res. 45 , 1474–1478 (1985). Fatiha, B. et al. Phenolic composition, in vitro antioxidant effects and tyrosinase inhibitory activity of three Algerian Mentha species: M. spicata (L.), M. pulegium (L.) and M. rotundifolia (L.) Huds (Lamiaceae). Industrial Crops and Products 74, 722–730, (2015). https://doi.org/10.1016/j.indcrop.2015.04.038 TOMITA, K. et al. A new screening method for melanin biosynthesis inhibitors using Streptomyces bikiniensis. 43 , 1601–1605 (1990). Zhang, L. et al. Biotransformation of ginsenoside Rb(1) and Rd to four rare ginsenosides and evaluation of their anti-melanogenic effects. J. Nat. Med. 77 , 939–952. 10.1007/s11418-023-01719-5 (2023). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.pdf Supplementarydata2.Westenblotimage.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7125633","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":492375697,"identity":"3637ceb1-8d5b-4a35-8f57-cbe865202c96","order_by":0,"name":"Min Jung Kim","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"Jung","lastName":"Kim","suffix":""},{"id":492375700,"identity":"3331c041-42ba-4daf-ae4b-7d5202070cc1","order_by":1,"name":"Ye Jin Yang","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"Jin","lastName":"Yang","suffix":""},{"id":492375701,"identity":"e1f583cc-b7fa-4507-a366-19e557ff246d","order_by":2,"name":"Ji Woong Heo","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Woong","lastName":"Heo","suffix":""},{"id":492375703,"identity":"da688d1a-1c5a-4016-b93d-dabfc45d56f3","order_by":3,"name":"Han Nim Choi","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"Nim","lastName":"Choi","suffix":""},{"id":492375705,"identity":"f306cb69-5554-45c9-ad5e-50fb2e429824","order_by":4,"name":"Chae Un Lim","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Chae","middleName":"Un","lastName":"Lim","suffix":""},{"id":492375707,"identity":"6722dbc0-0959-40bc-9968-f7f5047fbd1e","order_by":5,"name":"Se Hyo Jeong","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Se","middleName":"Hyo","lastName":"Jeong","suffix":""},{"id":492375708,"identity":"dabb5d8a-b57a-4413-8bf8-1acb37c3ec53","order_by":6,"name":"Hun Hwan Kim","email":"","orcid":"","institution":"Korea Institute of Toxicology (KIT)","correspondingAuthor":false,"prefix":"","firstName":"Hun","middleName":"Hwan","lastName":"Kim","suffix":""},{"id":492375710,"identity":"78087e8b-7939-468d-b52b-fb4011e957ca","order_by":7,"name":"Gon Sup Kim","email":"","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":false,"prefix":"","firstName":"Gon","middleName":"Sup","lastName":"Kim","suffix":""},{"id":492375712,"identity":"fbc40d99-e596-497c-ab95-1f853a071d72","order_by":8,"name":"Young Hun Kim","email":"","orcid":"","institution":"Chosun University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Hun","lastName":"Kim","suffix":""},{"id":492375713,"identity":"9a32e130-57c7-4a06-bd3e-1858f0509840","order_by":9,"name":"Ju-Hye Yang","email":"","orcid":"","institution":"Korea Institute of Oriental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ju-Hye","middleName":"","lastName":"Yang","suffix":""},{"id":492375714,"identity":"2cbc6988-5023-471b-aa41-84403334de3b","order_by":10,"name":"Kwang Il Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACCWYQecAGxk8gWksaKVrA5IHDJGiRbOc9+LnizHm77RIJjB9+MKTlE9QizcyXLHnmxu3knTMSmCV7GHIsGwhpkWPmMZBs+HA72eBGAoM0A0OFAUFbgFqMfzZ8OAfSwvybKC3SzDxmkg03DtgBtbABbckhrEWymS/NsuFMcoLBmYdtlj0GaYS1SJw/e/hmwzE7e4PjyYdv/KhIJqyFgYEHTCY2MDA2MDAQowGmxZ4otaNgFIyCUTAyAQDLZzlAf4Wc+QAAAABJRU5ErkJggg==","orcid":"","institution":"Gyeongsang National University","correspondingAuthor":true,"prefix":"","firstName":"Kwang","middleName":"Il","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2025-07-15 03:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7125633/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7125633/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87990576,"identity":"6ff7fc76-0ae2-4927-86e6-710c1f973fa9","added_by":"auto","created_at":"2025-07-31 08:23:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60211,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of Antioxidants by DPPH Radical Scavenging Activity and ABTS Radical Scavenging Activity.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) DPPH radical scavenging activities of the WGEE in various concentrations. (\u003cstrong\u003eB\u003c/strong\u003e) Hydrogen peroxide scavenging activities of WGEE in various concentrations. Data are Mean± Standard Deviations (SD) (n = 3) and are expressed as a percentage of the untreated control. Statistical significance is expressed as *p \u0026lt;0.05, ***p \u0026lt;0.001. Vit.C: 100 μg/mL ascorbic acid (positive control).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/cb3866b8452e639f9b70cdd9.png"},{"id":87990225,"identity":"607dc750-880d-4fd0-9cc5-e8c025fc610d","added_by":"auto","created_at":"2025-07-31 08:15:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59445,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ethanol extract of WGEE on the Inhibition of melanin production.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Cells treated with WGEE at different concentrations for 1 day was assayed using MTT cytotoxicity. Data are means ± SEM and is expressed as a percentage. Statistical significance is expressed as **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. untreated control group; (\u003cstrong\u003eB\u003c/strong\u003e) The melanin contents were measured in B16F10 cells. Statistical significance is expressed as **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. α-MSH-stimulated group; #p \u0026lt; 0.05 vs. control group. (\u003cstrong\u003eC\u003c/strong\u003e) Tyrosinase inhibitory activities of WGEE. Data are means ± SD (n = 3) and are expressed as a percentage of the untreated control. Statistical significance is expressed as ***p \u0026lt;0.001. Vit.C : 200 μg/mL ascorbic acid (positive control).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/420e26cbd730c1833034a432.png"},{"id":87991444,"identity":"4193c3f7-5b90-47b3-b17b-8ed767664b54","added_by":"auto","created_at":"2025-07-31 08:31:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54862,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis with antibodies against MITF, tyrosinase, TRP1, and TRP2.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Western blot assays were performed to examine melanin synthesis related proteins, MITF, TYR, TRP-1 and TRP-2 in B16F10 cells with WGEE treatment for 48 h. Cropped images shown in each panel were derived from different parts of the same gel or from different gels. Each blot band was obtained from gels run in parallel under identical conditions. (\u003cstrong\u003eB\u003c/strong\u003e) Bar graphs of MITF tyrosinase, TRP1, and TRP2 band densities relative to total form band density. (GADPH was used for normalization) “+” means treat, “-” means no treat. Statistical significance is expressed as *p \u0026lt; 0.05, ***p \u0026lt; 0.001 vs. α-MSH-stimulated group; \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/91cec6921279b62ac5478898.png"},{"id":87991443,"identity":"d8c4df58-2faa-43f3-a4d4-70ab7a513c6e","added_by":"auto","created_at":"2025-07-31 08:31:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35782,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative chromatograms of ginsenosides Rg1 and Rb1 detected at 203 nm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) UPLC chromatogram of ginsenoside (Rg1, Rb1) standard chemical (100 ppm), (\u003cstrong\u003eB\u003c/strong\u003e) UHPLC chromatogram of ginsenoside (Rg1, Rb1) in white ginseng ethanol extrats (1000 ppm).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/2bc215abb4214bed5243d150.png"},{"id":87990233,"identity":"8d80ca4f-4d3e-4e9b-85cb-202a094ebce7","added_by":"auto","created_at":"2025-07-31 08:15:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":584388,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking studies of compounds WGEE\u003cem\u003e \u003c/em\u003ewith Tyrosinase and their binding energy.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) 3D structure of tyrosinase with docked Rg1, showing the binding site in a ribbon representation. Close-up view of the binding pocket highlighting Rg1 (green sticks) interactions within the active site of tyrosinase. (\u003cstrong\u003eB\u003c/strong\u003e) 2D interaction diagram of Rg1 depicting van der Waals forces, hydrogen bonds, and alkyl interactions with key amino acid residues. (\u003cstrong\u003eC\u003c/strong\u003e) Structural representation of tyrosinase with docked Rb1 and zoomed-in view of its binding orientation within the catalytic pocket. (\u003cstrong\u003eD\u003c/strong\u003e) 2D interaction map of Rb1 showing Pi-alkyl, hydrogen bonding, and hydrophobic interactions with surrounding amino acids.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/5abd74d0b6581b448b11839e.png"},{"id":87990580,"identity":"c8239d1b-959b-4a09-9e1d-9691296bb02f","added_by":"auto","created_at":"2025-07-31 08:23:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":299744,"visible":true,"origin":"","legend":"\u003cp\u003eProposed mechanism of WGEE-mediated inhibition of melanogenesis. WGEE suppresses tyrosinase activity, reduces MITF and TRP1/TRP2 expression, and enhances antioxidant defenses in B16F10 cells.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/25a8c893556e3264a4ae62a6.jpg"},{"id":89582154,"identity":"b079a4a1-f1ea-4bac-ada0-ab1e7fdfde51","added_by":"auto","created_at":"2025-08-21 14:17:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1838037,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/638b79ba-c6e4-46ac-b454-b3c05a2efdae.pdf"},{"id":87990224,"identity":"abb42296-90ec-4fde-833d-0363a5aef257","added_by":"auto","created_at":"2025-07-31 08:15:05","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35155,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/59726c77035c734ccd04c152.pdf"},{"id":87990234,"identity":"90d3df33-13b2-4fb6-8f2d-f1e988cc9082","added_by":"auto","created_at":"2025-07-31 08:15:06","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":978689,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata2.Westenblotimage.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7125633/v1/4928d12bb45d42d3621ca2df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"White Ginseng Ethanol Extract (WGEE) Inhibits Tyrosinase and Melanogenesis via MITF Downregulation in B16F10 Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlpha-melanocyte stimulating hormone (α-MSH) is a melanocortin peptide hormone derived from proopiomelanocortin (POMC), a proprotein produced in the pituitary gland \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. When exposed to ultraviolet (UV) radiation, α-MSH binds to the melanocortin 1 receptor (MC1R) found on melanocytes, leading to the activation of adenylyl cyclase and an increase in cyclic adenosine monophosphate (cAMP) levels \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This elevation in cAMP activates protein kinase A (PKA), which in turn phosphorylates Cyclic AMP-responsive element-binding protein (CREB), leading to the induction of Microphthalmia-associated transcription factor (MITF) expression, a key regulator of melanogenesis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The MITF protein regulates the production of key enzymes involved in melanin synthesis, such as tyrosinase and tyrosinase-related proteins (TRPs). Melanin exists in two primary forms: eumelanin (black or brown pigment) and pheomelanin (yellow or red pigment). Tyrosine is enzymatically converted into 3,4-dihydroxy-L-phenylalanine (L-DOPA) by the enzyme tyrosinase, subsequently leading to its conversion into dopaquinone \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Dopaquinone undergoes additional processing by TRP-1 and TRP-2 enzymes to generate eumelanin.\u003c/p\u003e\u003cp\u003eThere are multiple strategies available to inhibit melanin production, with primary emphasis on the inhibition of tyrosinase activity. Chemical compounds found in nature, such as hydroquinone and licorice extract, have been shown to impede the enzymatic conversion of tyrosine to L-DOPA, whereas antioxidants like vitamin C have been demonstrated to inhibit the oxidative processes involving tyrosine \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Additionally, niacinamide has the capability to inhibit the transfer of melanin to keratinocytes, and chemical bleaching agents are commonly utilized in treatments aimed at skin whitening \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Nevertheless, many of the agents mentioned above are closely linked to negative effects such as stinging, contact dermatitis, irritation, high toxicity, and sensitivity. As a result, current studies from cosmetic companies and research institutions have focused on creating new whitening agents that specifically block tyrosinase activity in order to diminish hyperpigmentation, without harming normal, healthy melanocytes.\u003c/p\u003e\u003cp\u003eThe term Panax, originating from the botanical name of ginseng (Panax ginseng), translates in Greek to panacea \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Ginseng has historically been utilized for the prevention and treatment of a variety of illnesses, and contemporary studies have confirmed its broad spectrum of pharmacological benefits. Ginseng is categorized based on its processing technique, where white ginseng pertains to dried, unaltered ginseng, while red ginseng goes through a steaming and drying procedure \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Though there have been many studies investigating the pharmacological advantages of ginseng, there is a scarcity of research on white ginseng. Recent interest in skin whitening has led to ongoing research into the use of natural and synthetic substances for treating skin conditions like pigmentation.\u003c/p\u003e\u003cp\u003eIn this study, we employed an ethanol extract of white ginseng to examine its bioactive compound content, antioxidant properties, and effects on cell whitening in B16F10 cells. Moreover, our goal is to showcase the potential of processed white ginseng, which is a type of Panax ginseng cultivated in Korea, as a valuable ingredient in functional cosmetics for skin brightening, as well as its application in food products.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe concentration of polyphenols and flavonoids in ethanol extracts of white ginseng\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe concentration of 200 \u0026micro;g/ml was used to quantify the total polyphenol and flavonoid content of WGEEs. The WGEE contained a total polyphenol content of 33.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 mg/g at the specified concentration, and a total flavonoid content of 33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mg/g as indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eTotal phenolic and flavonoids contents of WGEE\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentration\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eWGEE\u003c/p\u003e\u003cp\u003e(2mg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal polyphenol (GAE mg/g)\u003csup\u003e1)\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e33.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal flavonoid (QE mg/g)\u003csup\u003e2)\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Total polyphenol content is expressed as gallic acid equivalents (GAE).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Total flavonoid content is expressed as quercetin equivalent (QE).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntioxidant Activity of WGEE\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe radical scavenging activity of WGEE was observed at concentrations ranging from 40 to 300 \u0026micro;g/mL, with scavenging rates increasing proportionally. Vit. C demonstrated a scavenging activity of 91.1%, significantly higher by 65.3% compared to the 300 \u0026micro;g/mL concentration of WGEE at 100 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe ABTS radical scavenging activity of WGEE was measured at various concentrations (10 \u0026micro;g/mL to 300 \u0026micro;g/mL), showing scavenging rates ranging from 5.3\u0026ndash;60.9%. Vit. C demonstrated a scavenging activity of 94.0%, exceeding 33.1% the activity observed in the 300 \u0026micro;g/mL concentration of WGEE at 100 \u0026micro;g/mL as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInhibitory Effects of WGEE on Melanogenesis and Tyrosinase Activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study assessed the anti-melanogenic properties of WGEE by analyzing melanin content and tyrosinase activity in B16F10 melanoma cells. Cytotoxicity assessment revealed a notable reduction in cell viability at concentrations equal to or greater than 60 \u0026micro;g/mL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), leading to the adoption of non-cytotoxic concentrations of 20 and 40 \u0026micro;g/mL for subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB indicate that treatment with 100 nM α-MSH led to a significant increase in melanin production, reaching 127.97% compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), thus confirming its melanogenic properties. Treatment with 20 \u0026micro;g/mL of WGEE resulted in 104.88% melanin content, indicating no significant alteration. In contrast, treatment with 40 \u0026micro;g/mL of WGEE led to a significant suppression of melanin synthesis to 94.31%, in comparison to both the α-MSH group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The findings demonstrate that the inhibitory effect of WGEE on melanin synthesis is dependent on the dose. Additionally, experiments conducted to assess tyrosinase inhibition in vitro showed that WGEE reduced L-tyrosinase activity in a dose-dependent manner ranging from 20 to 200 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The inhibition rates ranged from 9.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1% to 23.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7%, with Vit. C at a concentration of 200 \u0026micro;g/mL used as a positive control exhibiting 66.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% inhibition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSuppressive Effects of WGEE on MITF and Tyrosinase Expression\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo examine the impact of WGEE on melanogenesis, levels of key melanogenesis-related proteins like tyrosinase, TRP-1, and MITF were measured through western blot analysis in α-MSH-stimulated B16F10 melanoma cells as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. In comparison to the control group treated with α-MSH alone, administration of WGEE led to a notable reduction in the expression levels of tyrosinase, TRP-1, and MITF. Significantly, the group administered with 40 \u0026micro;g/mL white ginseng extract showed a reduction of approximately 19.05% in tyrosinase expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 40% reduction in TRP-1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 76.47% TRP-2 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 33.33% reduction in MITF expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) compared to the group that received only α-MSH. These results suggest a potent inhibitory effect of white ginseng extract on melanin biosynthesis machinery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of Ginsenosides Rg1 and Rb1 in WGEE and Evaluation of Their Bioactive Potential\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUPLC analysis enabled the quantitative determination of the ginsenosides (Rg1 and Rb1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Ginsenosides Rg1 and Rb1 were identified in a 1000 ppm WGEE at concentrations of 12.0252 ppm and 37.5906 ppm, respectively, indicating the presence of ginsenoside components in the extract. While other minor compounds were also observed on the chromatogram, Rg1 and Rb1 constituted the major peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro and in silico insights into tyrosinase inhibitors with Ginsenoside\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protein is shown as cartoon ribbons, with red and blue colors representing different subunits, and the ligands (Rg1, Rb1) are displayed as stick models. You can see interaction types like hydrogen bonding, Van der Waals forces, and hydrophobic interactions with Discovery Studio Visualizer. We used molecular docking simulations to predict how tyrosinase (PDB ID: 3NM8) interacts with ginsenosides Rg1 and Rb1, which are key active compounds found in Panax ginseng. Both the ligands had strong binding affinities and interacted well within the active site of tyrosinase. Ginsenoside Rg1 was placed inside the enzyme's hydrophobic pocket, creating several regular hydrogen bonds and Van der Waals interactions with nearby residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Important amino acids that participate in the binding of Rg1 are histidine A204, glutamate A208, glycine A216, and methionine A261, which are vital for an enzymatic function. The 2D diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB showed that the ligand is stabilized by interactions like pi-sigma and alkyl bonding with PRO A253 and GLY A209 in the active site, helping keep its shape stable. The binding of ginsenoside Rb1 with tyrosinase is 3D diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, ARG 75, TRP 269, TYR 72, THR 271, THR 272, PRO 273, PHE 262, ASN 249, TYR 250, GLN 242, ASN 278, TRP 238, MET 277, GLU 274, VAL 276, ASP 275, PRO 67, MET 266, ARG 70, GLU 71, TRP 68, LEU 74, ALA 64, HIS 69, LEU 66, LEU 282 and PHE 65 were identified as important interacting residues connected through hydrogen bonds, hydrophobic interactions, and amide-π stacking.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that WGEE exhibits significant anti-melanogenic properties, positioning it as a promising natural ingredient for hyperpigmentation treatment. WGEE effectively reduced melanin production in α-MSH-stimulated B16F10 cells and downregulated key melanogenesis proteins including MITF, tyrosinase, TRP-1, and TRP-2. The antioxidant capacity and molecular docking results suggest that WGEE's skin-whitening effects involve both indirect antioxidant mechanisms and direct tyrosinase inhibition.\u003c/p\u003e\u003cp\u003ePolyphenols and flavonoids are important antioxidant compounds present in plants, serving as participants in redox reactions and demonstrating diverse physiological functions \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Various phenolic compounds, including salicylic acid \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, p-benzoxybenzoic acid, gentisic acid, vanillic acid, and ascorbic acid, have been identified in ginseng for their noteworthy antioxidant properties \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Moreover, research suggests that the overall flavonoid content may differ depending on the extraction technique \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and different organs (leaves, stems, and roots) utilized \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For example, Chung \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and colleagues identified variations in phenolic compounds and their levels based on the specific parts of the ginseng plant, the geographical location of its habitat, and the methods used for cultivation. Therefore, it is apparent that both polyphenol and flavonoid levels can vary not only depending on the extraction technique employed, even for identical samples, but also based on the geographical location of cultivation.\u003c/p\u003e\u003cp\u003eThe stability of the DPPH radical is affected by factors such as light exposure, temperature variations, and pH levels, making it a commonly utilized compound in assays for antioxidant activity \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This technique is based on the transformation of the purple DPPH radical into a yellow hue as a result of its interaction with antioxidants \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The ABTS assay is a commonly utilized indirect method recognized for its resistance to pH variations, especially in the evaluation of food and natural water-soluble phenolic substances \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. ABTS displays durability in the absence of phenolic compounds \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Nevertheless, it displays a pronounced reactivity towards hydrogen donors, such as phenolic compounds, resulting in the formation of colorless ABTS \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlthough WGEE demonstrated moderate melanin production inhibitory activity relative to vitamin C, its gradual inhibitory effect suggests the presence of bioactive phytochemicals, including protocatechuic acid and ginsenosides, which may synergistically modulate melanogenesis through antioxidant-mediated and melanin synthesis-suppressive mechanisms. Collectively, these findings suggest that WGEE successfully hinders melanin production by reducing both melanin synthesis and tyrosinase activity.\u003c/p\u003e\u003cp\u003eThe expression level of MITF, a crucial regulator of tyrosinase and TRP-1, was significantly decreased, indicating that white ginseng extract may downregulate melanogenesis by suppressing transcription. The inhibition of MITF signifies upstream control, as MITF triggers the transcription of melanogenic enzymes such as tyrosinase and TRP-1 \u003csup\u003e18\u003c/sup\u003e. Considering the confirmed role of MITF in pigmentation, it is probable that its decreased expression is responsible for the observed reduction in downstream enzymes, consequently impacting melanin synthesis. To summarize, the ethanol extract of white ginseng demonstrates inhibitory effects on melanin production by regulating MITF and its downstream genes, indicating its possible use as a botanical skin-lightening agent.\u003c/p\u003e\u003cp\u003eGinsenosides are major bioactive components of ginseng, known to possess diverse physiological effects, including antioxidant \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, anti-inflammatory \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and immunomodulatory activities \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These findings are consistent with previous studies that report the whitening effects of ginsenosides such as Rg1 and Rb1 via suppression of MITF and melanogenic enzymes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Rg1 and Rb1 exhibit whitening, antioxidant, and skin-protective effects, suggesting their potential application as functional cosmetic ingredients. The findings of this study suggest that white ginseng possess sufficient bioactivity to be utilized as a functional material.\u003c/p\u003e\u003cp\u003eThe results of the molecular docking analysis showed that Rg1 exhibited a stronger binding affinity (\u0026minus;\u0026thinsp;8.1 kcal/mol) compared to Rb1 (\u0026minus;\u0026thinsp;6.2 kcal/mol) at the active site of tyrosinase (PDB ID: 3NM8). This suggests Rb1 has better antioxidants and anti-melanogenic properties than Rg1 in living organisms. Additionally, the way both compounds interact with histidine and glutamate residues close to the copper center of tyrosinase might prevent the oxidation of phenolic substrates like L-DOPA through a chelating mechanism or steric hindrance. These results show that ginsenosides, especially Rb1, could work as natural tyrosinase inhibitors and show potential as functional ingredients in cosmetics or pharmaceuticals for treating hyperpigmentation and oxidative stress-related disorders.\u003c/p\u003e\u003cp\u003eFurther investigations are warranted to elucidate the molecular mechanisms underlying WGEE's melanogenesis inhibitory effects and to evaluate its safety profile as a natural alternative to synthetic whitening agents. In vivo studies are essential to validate these findings and assess clinical efficacy in human subjects. Future research should also optimize extraction methodologies and evaluate ginsenoside stability to establish standardized protocols for cosmetic applications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAs summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, WGEE inhibits melanogenesis in B16F10 cells by blocking tyrosinase activity, reducing MITF expression downregulating TRP1/TRP2 expression, and enhancing antioxidant activity, making it a promising candidate for natural skin-whitening applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eCrude extract methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhite ginseng was extracted with 70% ethanol at 40\u0026deg;C for 24 hours. A mixture of white ginseng and white ginseng fine roots, blended in a weight ratio of 6:4 (w/w), was subjected to two rounds of extraction using consistent conditions. The final extract was filtered and concentrated at reduced pressure, resulting in the production of the WGEE.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of total flavonoid and total polyphenol concentration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo measure the total polyphenol content, white ginseng extract (2 mg/mL) was mixed with 500 \u0026micro;L of 2N Folin\u0026rsquo;s phenol reagent and allowed to stand at room temperature for 3 min. Next, 400 microliters of a 7.5% sodium carbonate solution were added. Subsequently, the solution was transferred to a 96-well plate and allowed incubate in the absence of light at an ambient temperature for a duration of 1 hour. After incubation, the absorbance at 750 nm was quantified using a microplate reader, and the total polyphenol content was calculated using a standard curve. To determine the total flavonoid content, 100 \u0026micro;L of the same extract was mixed in a 96-well plate with 860 \u0026micro;L of 80% ethanol, 20 \u0026micro;L of 10% aluminum chloride, and 20 \u0026micro;L of 1M potassium acetate. Following a 40-minute incubation period at room temperature, the absorbance at 415 nm was recorded to determine the total flavonoid content through the utilization of a standard curve.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDPPH and ABTS assays were used to evaluate radical scavenging activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo conduct the DPPH assay, a solution of DPPH was created through the dilution of the DPPH reagent in pure methanol to ensure an absorbance reading of 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 at a wavelength of 517 nm. Samples were additionally diluted with 100% methanol to different concentrations. A 100 \u0026micro;L portion of the DPPH solution was combined with 100 microliters of the sample, and the resulting mixture underwent incubation at 37\u0026deg;C in the absence of light for a duration of 30 minutes. Following this, the absorbance was recorded at 517 nm utilizing a microplate spectrophotometer. ABTS\u0026thinsp;+\u0026thinsp;radicals were produced through the combination of 7.4 mM ABTS and 2.6 mM potassium persulfate in distilled water at a 1:1 ratio. The mixture underwent a reaction period of 16\u0026ndash;24 hours in darkness. The ABTS\u0026thinsp;+\u0026thinsp;solution was subsequently diluted with distilled water until reaching an absorbance of 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm. In a 96-well plate, 100 microliters of the diluted ABTS\u0026thinsp;+\u0026thinsp;solution were mixed with 100 microliters of the sample, and the absorbance was promptly measured at 734 nanometers using a microplate spectrometer.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell Culture and Cytotoxicity Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eB16F10 cells sourced from the Korean Cell Line Bank were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco BRL, Waltham, MA, USA) and 0.2% NaHCO\u003csub\u003e3\u003c/sub\u003e (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated at a temperature of 37\u0026deg;C in a controlled environment with 5% carbon dioxide. A cytotoxicity assay was conducted using MTT to evaluate cell viability. Cells were distributed into 96-well plates at a concentration of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well and left to incubate for 24 hours. Following this, the culture medium was exchanged with a fresh medium that contained different concentrations of white ginseng extract ranging from 10 to 300 \u0026micro;g/mL. Subsequently, the cells were exposed to 10 \u0026micro;L of 1.0 mg/mL MTT solution and allowed to incubate at 37\u0026deg;C for a duration of 2 hours. After incubation, the MTT solution was aspirated, and the cells were rinsed with D-PBS. The resulting formazan crystals were dissolved in 100 \u0026micro;L of DMSO for 30 minutes, and the absorbance was measured at 570 nm using a Synergy H1 reader.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntracellular Melanin Content Measurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIntracellular melanin content was measured using a modified Hosoi method \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. B16F10 cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in 60 mm plates and incubated at 37\u0026deg;C for 24 hours. Subsequently, cells were treated with α-MSH and 20 and 40 \u0026micro;g/mL α-MSH (100nM) targeting polypeptides for 48 hours at 37\u0026deg;C. The culture medium supernatant was then collected, and extracellular melanin content was measured at 490 nm. For each group, 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were lysed using radio immunoprecipitation assay (RIPA) buffer. The cellular pellets, acquired through centrifugation, were rinsed with alcohol before being dissolved in a 1 N NaOH solution containing 10% DMSO at a temperature of 90\u0026deg;C for a duration of 1 hour, followed by the measurement of absorbance at 490 nm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTyrosinase inhibition assay conducted in vitro\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe activity of tyrosinase inhibition was assessed through a modified methodology based on the approach outlined in the study by Tomita et al. (1990) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this experimental procedure, 100 microliters of a sample solution at a concentration of 1 milligram per milliliter was combined with 3.2 milliliters of a 0.1 molar sodium phosphate buffer at a pH of 6.5. Subsequently, 50 microliters of mushroom tyrosinase with an activity of 2000 units per milliliter, obtained from Sigma-Aldrich in the United States, was added to the mixture. After mixing, 100 \u0026micro;L of 1.5 mM L-tyrosine (Sigma-Aldrich, USA) was added. The solution was allowed to incubate at a temperature of 37\u0026deg;C for a duration of 15 minutes, followed by measurement of absorbance at a wavelength of 475nm using a UV/Vis spectrophotometer (OPTIZEN POP, Mecasys, Korea). For the control group, a 0.1 M sodium phosphate buffer with a pH of 6.5 was employed instead of the sample, and a Vitamin C (ascorbic acid) solution served as the positive control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eB16F10 cells were washed with PBS and then dissolved in 100 \u0026micro;L lysis buffer for 20 min with ice. After centrifugation at 12,000 rpm for 15min, the protein suspension was obtained by collecting the liquid supernatant. Then, 20\u0026micro;g proteins were loaded into 8% SDS-PAGE gel before being transferred to 0.45\u0026micro;m PVDF membranes. The membranes were blocked with 5% Bovine Serum Albumin (BSA) buffer for 2h, then washed with tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) three times, and incubated with MITF (sc-515925), TYR (sc-20035), TRP-1 (sc-166857), TRP-2 (sc-74439), and GADPH with TBST. After the reaction with the second antibody, an Chemiluminescence Imager detection SH-Cute 523 system was used to visualize the proteins (Shenhua Science Technology, Hangzhou city, Chinese). Densitometric analysis of the bands were performed using ImageJ (Version 1.54). Western blot results represented at least three independent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003eUPLC analysis conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAn analysis of WGEE for both qualitative and quantitative purposes was conducted utilizing a Thermo Dionex Ultimate 3000 system. The separation process utilized a Pronto SIL C18 column with dimensions of 150 \u0026times; 4.6 mm, particle size of 5 \u0026micro;m, and model number 120-5-C18 SH, manufactured by Bischoff Chromatography in Leonberg, Germany. The samples were dissolved in a solution of 70% methanol to achieve a concentration of 1000 ppm before being passed through a 0.45 \u0026micro;m filter. Ginsenoside Rg1 and Ginsenoside Rb1 purchased from ChemFaces (Cat. No. CFN99967 and CFN99964, respectively), located in Wuhan, Hubei, China, were employed as reference compounds (\u003cb\u003eSupplementary data\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Docking Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMolecular docking was conducted to investigate the binding interactions between ginsenosides (Rg1 and Rb1) and tyrosinase (PDB ID: 3NM8) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The 3D structure of tyrosinase was obtained from the Protein Data Bank, and the structure of ginsenoside Rg1 was retrieved from the PubChem database. Ligand structures were converted from SDF to PDB format using Open Babel (v3.1.1) and energy-minimized with the MMFF94 force field. Protein preparation involved removal of water molecules, addition of hydrogen atoms, and assignment of Kollman charges using AutoDock Tools. Docking simulations for Rg1 were performed using AutoDock Vina (v1.1.2), with the grid box centered on the active site and the exhaustiveness set to 8. Due to the large molecular size and high conformational flexibility of Rb1, docking using open-source tools like AutoDock or PyRx was not feasible. Therefore, BIOVIA Discovery Studio, a commercially licensed software, was used for conformer generation and docking analysis of Rb1. Ligand-protein complexes were visualized in 3D using PyMOL and Discovery Studio Visualizer, and 2D interaction maps were generated to examine hydrogen bonding, hydrophobic interactions, and key amino acid residues involved in binding.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eGraphPad Prism version 8.0 was used for all statistical analyses (GraphPad Software, SanDiego, CA, USA). The researchers utilized one-way factorial analysis of variance to assess the variation. The findings are presented as the mean value along with the standard error of the mean or standard deviation. A p-value lower than 0.05 was deemed statistically significant for all tests.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article (and its supplementary information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. RS-2022-NR075631), the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Science and ICT (No. RS-2024-00403488), and the Korea Institute of Oriental Medicine grant number KSN2412012.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.J.K., Y.J.Y., J.W.H., H.N.C., C.U.L., S.H.J., H.H.K., G.S.K., Y.H.K., J-H.Y. and K.I.P. searched and collected the literature, summarized the contents, and described the articles. M.J.K., Y.J.Y., J.W.H. and H.H.K. organized the tables and created the figures. S.H.J., G.S.K., J.H.Y., Y.H.K. and K.I.P. provided valuable suggestions during manuscript preparation and critically revised the manuscript accordingly. G.S.K., J-H.Y., and K.I.P. conceptualized and wrote the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have approved the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSlominski, A., Tobin, D. 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Med.\u003c/em\u003e \u003cb\u003e77\u003c/b\u003e, 939\u0026ndash;952. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11418-023-01719-5\u003c/span\u003e\u003cspan address=\"10.1007/s11418-023-01719-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidant, B16F10 Cell, MITF, Tyrosinase, White Ginseng","lastPublishedDoi":"10.21203/rs.3.rs-7125633/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7125633/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHyperpigmentation disorders such as melasma and freckles are common cosmetic concerns that have increased interest in safe, naturally occurring compounds for melanin production control. This study investigated white ginseng ethanol extract (WGEE) from the Geonsam cultivar of Panax ginseng as a potential functional ingredient for skin-whitening applications. WGEE was prepared by extracting white ginseng with 70% ethanol at 40\u0026deg;C for 24 hours. Chemical composition was analyzed using UPLC and colorimetric assays. Antioxidant capacity was evaluated via DPPH and ABTS assays, and tyrosinase inhibition was measured spectrophotometrically. B16F10 melanoma cells were assessed for cytotoxicity, melanin content, and melanogenesis-related proteins using MTT assay and Western blot analysis. Molecular docking evaluated ginsenosides Rg1 and Rb1 binding with tyrosinase. WGEE showed antioxidant activity with DPPH and ABTS radical scavenging of 18.18% and 48.54% at 200 \u0026micro;g/mL. In α-MSH-stimulated B16F10 cells, WGEE at 40 \u0026micro;g/mL reduced melanin production by 33.65% and downregulated MITF, tyrosinase, TRP-1, and TRP-2 expression similar to arbutin. Molecular docking revealed binding energies of -8.1 kcal/mol for Rg1 and \u0026minus;\u0026thinsp;6.2 kcal/mol for Rb1, suggesting direct tyrosinase inhibition. WGEE demonstrates potential as a natural cosmetic ingredient for skin whitening and pigmentation control.\u003c/p\u003e","manuscriptTitle":"White Ginseng Ethanol Extract (WGEE) Inhibits Tyrosinase and Melanogenesis via MITF Downregulation in B16F10 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 08:15:00","doi":"10.21203/rs.3.rs-7125633/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5234e2b7-51fb-42f3-a8b6-fa3e08752491","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52276999,"name":"Biological sciences/Biochemistry"},{"id":52277000,"name":"Biological sciences/Biotechnology"},{"id":52277001,"name":"Biological sciences/Drug discovery"},{"id":52277002,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-08-21T14:08:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-31 08:15:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7125633","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7125633","identity":"rs-7125633","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}