Antimelanogenesis of quercetin-related compounds in B16 melanoma cells: the difference moieties of C-3 position and its effect on H2O2

Antimelanogenesis of quercetin-related compounds in B16 melanoma cells: the difference moieties of C-3 position and its effect on H2O2 | 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 Research Article Antimelanogenesis of quercetin-related compounds in B16 melanoma cells: the difference moieties of C-3 position and its effect on H 2 O 2 Nur Fitriana, Dian Juliadmi, Hiroya Ishikawa, Widya Fatriasari, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7328612/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 Quercetin ( compound 2 ), isolated from the dried skin of Allium cepa , and 3-prenyl luteolin ( compound 6 ), derived from the wood of Artocarpus heterophyllus , are polyphenolic compounds with demonstrated potential in regulating melanogenesis. This study investigated their anti-melanogenic activity and reactive oxygen species (ROS) scavenging effects, particularly against hydrogen peroxide (H₂O₂), in B16 melanoma cells. Both compounds significantly suppressed melanin synthesis, indicating potential as natural depigmenting agents. Quercetin ( 2 ) exhibited a half-maximal inhibitory concentration (IC₅₀) of 8.0 µg/mL, while 3-prenyl luteolin ( 6 ) showed an IC₅₀ of 20.1 µg/mL. For comparison, compound 1 displayed the strongest activity with an IC₅₀ of 3.0 µg/mL, whereas compounds 3 – 5 demonstrated no appreciable inhibitory effects. Additionally, both compounds reduced intracellular H₂O₂ levels, supporting their antioxidative role in mitigating oxidative stress-induced melanogenesis. These findings highlight the potential application of quercetin and 3-prenyl luteolin as novel active ingredients in skin-whitening formulations through dual mechanisms of melanin synthesis inhibition and ROS reduction. quercetin related compounds antimelanogenesis H2O2 B16 melanoma cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Melanin is a complex, heterogeneous biopolymer synthesized through the enzymatic oxidation and subsequent polymerization of tyrosine-derived precursors within melanosomes, specialized organelles in melanocytes [ 1 – 3 ]. Serving as a critical photoprotective pigment, melanin effectively absorbs ultraviolet (UV) radiation, thereby mitigating UV-induced oxidative damage to skin cells and contributing to skin coloration and protection [ 4 – 6 ]. The melanogenesis pathway is tightly regulated, beginning with the rate-limiting enzyme tyrosinase catalyzing the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and subsequent oxidation to dopaquinone. This intermediate undergoes multiple enzymatic transformations, involving tyrosinase-related proteins TRP-1 and TRP-2, which lead to the formation of eumelanin and pheomelanin pigments [ 7 – 9 ]. The microphthalmia-associated transcription factor (MITF) plays a pivotal role by regulating the expression of tyrosinase and related enzymes, thus ensuring coordinated melanocyte differentiation, survival, and pigment synthesis [ 7 , 10 – 12 ]. Reactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂), are generated endogenously during melanin biosynthesis via tyrosinase-mediated oxidation of L-DOPA and are also produced exogenously following UV exposure [ 13 – 16 ]. At physiological concentrations (~ 50–300 µM), H₂O₂ acts as a secondary messenger that promotes melanogenesis by activating melanocyte-specific transcription factors such as MITF through pathways involving cyclic AMP (cAMP)/protein kinase A (PKA) and the nuclear factor erythroid 2–related factor 2 (Nrf2), which regulates cellular antioxidant responses [ 17 – 23 ]. However, excessive or chronic accumulation of H₂O₂ can disrupt intracellular redox homeostasis, leading to deleterious effects such as endoplasmic reticulum (ER) stress, impaired trafficking and maturation of tyrosinase, melanocyte senescence, and ultimately pigmentary disorders including melasma, post-inflammatory hyperpigmentation, and premature skin aging [ 16 , 24 , 25 ]. These pathological conditions highlight the dual role of ROS in melanogenesis as both necessary signaling molecules and potential mediators of oxidative damage. Melanocytes are equipped with robust endogenous antioxidant systems, including catalase, glutathione peroxidase, and thioredoxin reductase, which cooperatively degrade H₂O₂ to maintain redox balance and prevent oxidative stress [ 24 , 26 – 31 ]. These antioxidant enzymes are transcriptionally regulated by the Nrf2 signaling pathway, which governs the expression of a battery of protective genes such as heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and enzymes involved in glutathione synthesis [ 21 , 32 – 35 ]. Despite these sophisticated defenses, chronic oxidative stress and overwhelming ROS production can impair these protective mechanisms, resulting in protein misfolding, ER dilation, defective melanogenesis, and the exacerbation of hyperpigmentation disorders [ 16 , 20 , 36 , 37 ]. Thus, controlling oxidative stress and ROS levels remains a critical strategy in the development of effective depigmenting agents. Quercetin, a naturally abundant bioflavonoid in various fruits and vegetables, has emerged as a promising natural compound for inhibiting melanogenesis due to its potent antioxidant, anti-inflammatory, and enzyme inhibitory properties [ 38 – 40 ]. In vitro experiments in human melanocytes and murine B16F10 melanoma cells have demonstrated that quercetin, particularly at concentrations ranging from 20 to 50 µM, significantly inhibits tyrosinase activity and melanin synthesis [ 41 ]. This effect is mediated by downregulation of MITF and its downstream effectors, tyrosinase-related proteins TRP-1 and TRP-2, and suppression of the cAMP/PKA signaling cascade, all achieved without notable cytotoxicity [ 39 , 42 ]. Additionally, quercetin mitigates H₂O₂-induced ER stress, preserves ER structure, and supports proper trafficking and expression of melanogenic enzymes, thereby maintaining melanocyte functionality under oxidative stress conditions [ 43 ]. Mechanistically, quercetin scavenges various ROS species, including peroxynitrite and superoxide radicals, elevates intracellular glutathione levels, and modulates catalase and HO-1 activities to restore cellular redox balance and inhibit melanogenesis [ 40 , 44 – 48 ]. Its derivatives have demonstrated enhanced anti-melanogenic effects by targeting multiple signaling pathways including cAMP/PKA/CREB/MITF and MAPK, further highlighting the therapeutic potential of this class of compounds [ 41 ]. Notably, quercetin’s effects exhibit dose dependency, as lower concentrations (< 20 µM) may paradoxically stimulate melanogenesis through post-translational stabilization of tyrosinase and TRP-2 proteins, highlighting the necessity for precise dose optimization in therapeutic applications [ 41 ]. Despite encouraging in vitro and preclinical results, clinical evidence for quercetin’s efficacy in treating hyperpigmentation remains insufficient. Challenges such as improving its bioavailability, developing optimized topical formulations, and conducting rigorous clinical trials comparing quercetin with established depigmenting agents like kojic acid, arbutin, and vitamin C are critical for its translation to clinical use [ 49 , 50 ]. Addressing these gaps will be essential to fully harness the therapeutic potential of quercetin in oxidative stress–related pigmentary disorders. The present study aims to investigate the anti-melanogenic effects of two compounds: quercetin ( 2 ), isolated from the dried skin of Allium cepa (onion), and 3-prenyl luteolin ( 6 ), isolated from the wood of Artocarpus heterophyllus (jackfruit). These compounds have been selected as novel candidates for anti-melanogenesis due to their unique structures and potential bioactivity. This research will explore their inhibitory activity on melanin formation, revealing their potential as novel depigmenting agents for skin-whitening cosmetics. In addition, we will evaluate the capability of these compounds to reduce ROS, especially H₂O₂, in B16 melanoma cells, which represent a significant source of oxidative stress associated with melanin overproduction. By targeting the oxidative pathways contributing to melanogenesis, these compounds could serve as promising candidates for the development of effective skin whitening products through the reduction of melanin overproduction. Materials and Methods Chemicals Sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), and rutin ( compound 3 ) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ethylenediaminetetraacetic acid (EDTA) was purchased from Dojindo Laboratories (Kumamoto, Japan). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was sourced from Sigma-Aldrich (St. Louis, MO, USA), and Eagle’s Minimum Essential Medium (EMEM) was obtained from Nissui Pharmaceutical Co., Ltd. (Osaka, Japan). Hyperin ( compound 4 ) was supplied by Tokiwa Phytochemical Co. (Chiba, Japan), luteolin ( compound 1 ) was purchased from Extrasynthese (Genay, France), and isoquercitrin ( compound 5 ) was obtained from Fluka Chemie GmbH (Steinheim, Germany). Quercetin ( compound 2 ) was isolated from the dried outer skin of Allium cepa , while 3-prenyl luteolin ( compound 6 ) was purified from the wood of Artocarpus heterophyllus . All other reagents used were of analytical grade and sourced from standard commercial suppliers. The chemical structures of the isolated compounds are illustrated in Fig. 1 . Cell culture The B16 murine melanoma cell line was obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 0.09 mg/mL theophylline. Cell cultures were maintained at 37°C in a humidified incubator with 5% CO₂ atmosphere. Inhibitory effect of melanin biosynthesis using cultured B16 melanoma cells The assay was performed following the method described by Arung et al. (2007), with slight modifications. Briefly, confluent B16 melanoma cells were washed with phosphate-buffered saline (PBS) and detached using 0.25% trypsin-EDTA solution. The cells were seeded into two 24-well culture plates—one designated for melanin content analysis and the other for cell viability assessment—at a density of 1 × 10⁵ cells per well. After 24 hours of incubation in complete medium to allow cell attachment, the culture medium was replaced with 998 µL of fresh EMEM, and 2 µL of dimethyl sulfoxide (DMSO), with or without the test compounds at various concentrations (n = 3), was added. Arbutin was included as a positive control. Cells were incubated for an additional 48 hours, after which the medium was refreshed and the same concentrations of test compounds were reapplied. Following a further 24-hour incubation, adherent cells were subjected to melanin content and cell viability assays as described below. Determination of melanin content in B16 melanoma cells Melanin content in the treated B16 melanoma cells was quantitatively evaluated following compound exposure. After removing the culture medium, cells were washed thoroughly with phosphate-buffered saline (PBS) to eliminate residual media components. The adherent cells were then lysed by incubation in 1.0 mL of 1 N sodium hydroxide (NaOH), which solubilized intracellular melanin. The resulting lysates were transferred to a 96-well microplate, and absorbance was measured at 405 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA). Melanin levels in the treated samples were normalized to those of the untreated control group and expressed as a percentage of control values to assess the inhibitory effects of the test compounds on melanin synthesis. Cell Treated with HO Cells were seeded at a density of 1 × 10⁵ cells per well into three separate 24-well plates and incubated for 24 hours to allow adherence. Following incubation, the test compounds were administered at designated concentrations. To induce oxidative stress, hydrogen peroxide (H₂O₂) was added to each well at a final concentration of 0.25% at 1, 2, and 4 hours after compound treatment. Cell viability was subsequently assessed using the MTT assay as described below. Cell viability Cell viability was evaluated using the microculture tetrazolium (MTT) assay, which assesses mitochondrial metabolic activity based on the formation of formazan crystals in viable cells. B16 melanoma cells were seeded into 24-well plates at a density of 1 × 10⁵ cells per well. Following the designated treatments and incubation periods, 50 µL of MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL in PBS] was added to each well. Plates were incubated for 4 hours at 37°C in a humidified incubator with 5% CO₂ to allow for formazan formation. After incubation, the medium was aspirated, and 1.0 mL of isopropyl alcohol containing 0.04 N HCl was added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference wavelength of 630 nm using a microplate reader (BioTek Instruments, USA). The absorbance values were used to calculate cell viability relative to untreated controls. Results and Discussion Antimelanogenesis of quercetin-related compounds The anti-melanogenic properties of all isolated constituents were assessed using B16 melanoma cell assays. Among the tested flavonoids, no statistically significant differences were observed in the melanin inhibition rates of rutin ( 3 ), hyperin ( 4 ), and isoquercetin ( 5 ). In contrast, compounds 1, 2 , and 6 exhibited markedly higher inhibitory effects on melanogenesis compared to the remaining isolates and the reference agent, arbutin. A dose-dependent suppression of melanin synthesis and cell viability was observed with increasing concentrations of these compounds. Notably, compound 1, structurally identified as luteolin, exerted the most potent melanogenesis-inhibitory effect, surpassing the activities of quercetin ( 2 ) and 3-prenyl luteolin ( 6 ), as illustrated in Figs. 2 – 7 . Luteolin demonstrated the capacity to inhibit melanin synthesis by over 50% at a concentration of 5 µg/mL. At 25 µg/mL, luteolin achieved more than 80% inhibition of melanin formation, concomitant with a reduction in cell viability to below 30% (Fig. 2 ). In contrast, quercetin inhibited melanin production by less than 50% at 5 µg/mL and exceeded 65% inhibition at 25 µg/mL, while maintaining cell viability above 70% (Fig. 3 ). The inhibitory profile of 3-prenyl luteolin differed from these compounds, with melanin synthesis suppressed by over 60% only at 30 µg/mL (Fig. 7 ). The IC₅₀ values determined for luteolin, quercetin, and 3-prenyl luteolin were 3.0 µg/mL, 8.0 µg/mL, and 20.1 µg/mL, respectively, indicating varying degrees of anti-melanogenic efficacy. Luteolin, quercetin, and 3-prenyl luteolin have been classified as polyphenolic compounds, which are well-recognized for their significant roles and extensive applications in the cosmetic industry. Previous studies have demonstrated the anti-melanogenic properties of these compounds [ 48 , 51 , 52 ]. Among them, 3-prenyl luteolin exhibits lower anti-melanogenesis activity compared to luteolin and quercetin, consistent with previous research [ 48 , 51 ]. This variation in activity is attributed to differences in functional groups, as prior research indicated that the presence of an isoprenoid moiety in 3-prenyl luteolin diminishes its inhibitory effect on melanin synthesis in B16 melanoma cells [ 51 ]. Additionally, other studies have highlighted that the anti-melanogenic efficacy is highly dependent on the specific position of functional groups within the molecular structure [ 53 – 55 ]. Anti-melanogenic agents exert their effects through multiple mechanisms, including inhibition of tyrosinase mRNA transcription, disruption of tyrosinase maturation, suppression of tyrosinase enzymatic activity, promotion of tyrosinase degradation, and indirect modulation of tyrosinase function [ 56 ]. Among these, tyrosinase inhibition is widely regarded as the primary target for reducing hyperpigmentation [ 57 – 59 ]. Luteolin and quercetin inhibit melanin synthesis via distinct pathways. Luteolin reduces melanin production in B16 melanoma cells by decreasing intracellular cyclic adenosine monophosphate (cAMP) signaling [ 52 ]. The downregulation of cAMP leads to dephosphorylation of PKA/CREB, resulting in diminished MITF expression, impaired tyrosinase regulation, and subsequent inhibition of melanin synthesis [ 39 , 47 , 60 , 61 ]. In contrast, quercetin acts as a potent tyrosinase inhibitor, directly interfering with the enzyme’s catalytic activity and downstream signaling pathways to prevent melanin formation [ 62 ]. Effect of quercetin related compounds on B16 melanoma cless treated with H 2 O 2 Cell viability following hydrogen peroxide (H₂O₂) exposure was assessed using the MTT assay to establish an optimal concentration for inducing oxidative stress in B16 melanoma cells. Cells were treated with increasing concentrations of H₂O₂ (0.05%, 0.1%, 0.25%, and 0.5%), and a clear dose-dependent decrease in viability was observed (Fig. 8 ), consistent with prior reports demonstrating H₂O₂'s cytotoxicity through oxidative damage and mitochondrial dysfunction [ 2 , 63 – 65 ]. Notably, there was no statistically significant difference in cell viability between 0.25% and 0.5% H₂O₂ treatments (Fig. 8 ), indicating a saturation effect at higher concentrations where additional oxidative burden did not further reduce viability [ 66 ]. Based on these results, 0.25% H₂O₂ was selected for subsequent assays, as it induced substantial oxidative stress while avoiding excessive cytotoxicity that could confound downstream analyses. Similar concentrations have been used in previous studies to model oxidative stress conditions in melanocytes and melanoma cells, validating its use as a physiologically relevant dose [ 20 ]. These doses effectively mimic oxidative environments that contribute to melanocyte damage, melanogenesis alterations, and melanoma cell death, supporting their physiological relevance. The impact of quercetin-related compounds on B16 melanoma cell viability under oxidative stress is presented in Figs. 9 and 10 . Notably, luteolin demonstrated a pronounced ability to enhance cell survival following hydrogen peroxide (H₂O₂) exposure, outperforming other tested compounds in mitigating oxidative damage. This suggests that luteolin exerts stronger cytoprotective effects, likely due to its potent antioxidant activity and modulation of intracellular signaling pathways involved in cell survival and melanogenesis regulation. Luteolin and its derivative luteolin 7-sulfate inhibit melanin synthesis by downregulating tyrosinase (TYR) expression through CREB- and MITF-mediated signaling pathways, which are key regulators of melanogenesis in melanoma cells. Moreover, luteolin reduces cellular reactive oxygen species (ROS) levels by directly inhibiting oxidative enzymes such as xanthine oxidase, thereby protecting cells from oxidative damage [ 44 , 67 ]. Other quercetin derivatives also partially restored cell viability compromised by H₂O₂, indicating their potential to alleviate oxidative stress in melanocytes. These findings corroborate previous reports indicating that quercetin and its analogs reduce reactive oxygen species (ROS) levels and protect against oxidative injury by scavenging free radicals and activating endogenous antioxidant defenses [ 40 , 47 , 68 , 69 ]. At a concentration of 5 ppm, the quercetin-related compounds exhibited lower cytotoxicity compared to quercetin 3-O-β-D-glucuronide (Q-3-G) and synthetic quercetin glycosides, which have shown dose-dependent cytotoxic effects in various cell lines including HaCaT keratinocytes and B16F10 melanoma cells at 20 µM [ 70 , 71 ]. This differential toxicity profile highlights the advantage of naturally derived compounds, which may offer enhanced safety and efficacy for potential therapeutic or cosmetic applications targeting hyperpigmentation and oxidative stress-related skin disorders. Furthermore, luteolin protects cells through both direct antioxidant activity via the Nrf2/ARE pathway and by modulating melanogenesis-related signaling cascades such as CREB/MITF, making it a potent agent against oxidative stress and hyperpigmentation [ 44 , 67 ]. These pathways contribute to reducing oxidative damage and restoring melanocyte function, thereby inhibiting excessive melanogenesis. In the current study, polyphenolic compounds including luteolin, isoquercetin, quercetin, and rutin demonstrated significant ROS-scavenging activity in B16 melanoma cells, with luteolin exerting the most pronounced protective effect against H₂O₂-induced cytotoxicity. This aligns with previous findings that highlight the superior antioxidant capacity of luteolin in skin cells through multiple mechanisms such as direct radical scavenging [ 72 ]. Quercetin’s efficacy in mitigating ROS accumulation is consistent with its chemical structure, which features multiple hydroxyl groups facilitating electron donation and free radical neutralization [ 47 , 68 ]. Furthermore, quercetin’s metal-chelating properties inhibit Fenton and Haber-Weiss reactions, which otherwise generate highly reactive hydroxyl radicals from H₂O₂ and transition metals, thus breaking a critical cycle of ROS amplification [ 73 – 75 ]. Quercetin, a well-known flavonoid isolated from Allium cepa (onion) skin, exhibits potent antioxidant and antimelanogenic activities, largely attributed to its polyphenolic structure and multiple hydroxyl groups that efficiently scavenge reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and superoxide anions [ 48 , 76 ]. The generation of ROS during UV exposure or cellular metabolism is a critical trigger for melanogenesis, often resulting in hyperpigmentation due to melanin overproduction [ 28 ]. Beyond antioxidative effects, quercetin’s anti-inflammatory properties reduce melanocyte activation caused by inflammation, which is a key factor in post-inflammatory hyperpigmentation [ 71 , 77 ]. However, challenges remain in improving its bioavailability and skin penetration for clinical applications. Nonetheless, the comprehensive inhibitory effects of quercetin on oxidative stress and melanogenesis highlight its potential as a natural, safe, and effective ingredient for skin-whitening and photoprotective cosmetics derived from Allium cepa . This study uniquely isolates quercetin specifically from Allium cepa skin and comparatively evaluates it alongside 3-prenyl luteolin, a prenylated flavonoid derivative obtained from the wood of Artocarpus heterophyllus . Prenylation of flavonoids like luteolin is known to enhance their lipophilicity and cellular uptake, often improving bioavailability and biological activity [ 78 ]. This modification may increase the compound’s antioxidant capacity and its interaction with key signaling pathways involved in melanogenesis and oxidative stress response. Our findings demonstrate that 3-prenyl luteolin exhibits significant inhibitory effects on melanin production and effectively reduces intracellular ROS levels, although its cytoprotective efficacy under H₂O₂-induced oxidative stress was somewhat lower compared to luteolin and quercetin. This comparative analysis provides novel insights into how structural modifications influence the bioactivity of flavonoids in melanocyte models. Furthermore, the study emphasizes sustainable utilization of agricultural by-products as valuable sources of natural bioactives, bridging environmental considerations with practical applications in the development of effective and safe skin-whitening agents. This integrative approach advances the understanding of quercetin-related compounds and prenylated flavonoids as multifunctional depigmenting agents, highlighting their potential for cosmeceutical innovation beyond what has been previously reported. Conclusions Quercetin ( compound 2 ), isolated from the dried skin of Allium cepa , and 3-prenyl luteolin ( compound 6 ), derived from the wood of Artocarpus heterophyllus , are promising natural polyphenolic compounds with potent antioxidant and antimelanogenic activities. Their ability to scavenge reactive oxygen species and reduce oxidative stress is likely a key mechanism underlying their inhibition of melanogenesis. These properties make them attractive candidates for developing novel skin-whitening and depigmenting agents. However, further studies focusing on safety and efficacy are essential before their application in humans can be realized. Overall, these compounds offer valuable potential for managing hyperpigmentation and oxidative stress-related skin disorders through natural, multifunctional mechanisms. Declarations Funding Declaration This research was supported by the Japan Society for the Promotion of Science (JSPS) under the Postdoctoral Fellowship Program for Foreign Researchers. This research is also supported by the National Research and Innovation Agency (BRIN) and a research grant from BRIN (Grant number: B-1146/II.7/HK 01.00/5/2025) with the title “Pusat Kolaborasi Riset Kosmetik Berteknologi Nano Berbasis Biomassa”. Author Contribution NF, DJ, RAE, ETA : Conception or design of the workNF, DJ, WF, RAE, YUK, KS, ETA; or the acquisition, analysis, and interpretation of data;NF, DJ, HI, WF, RAE, YUK, KS, ETA : drafted the work or revised it critically for important intellectual content;NF, DJ, HI, WF, RAE, YUK, KS, ETA : approved the version to be published Acknowledgement The authors acknowledge the support and facilities provided by the Japan Society for the Promotion of Science and the National Research and Innovation Agency (BRIN). References Maranduca MA, Branisteanu D, Serban DN, Branisteanu DC, Stoleriu G, Manolache N, et al. Synthesis and physiological implications of melanic pigments (review). Oncology Letters. 2019;17(5):4183–7. Guo L, Li W, Gu Z, Wang L, Guo L, Ma S, et al. Recent Advances and Progress on Melanin: From Source to Application. International Journal of Molecular Sciences. 2023;24(5). Mahajan SG, Nandre VS, Kodam KM, Kulkarni M V. One-pot synthesis of black biopolymeric eumelanin pigment by indigenous salt-tolerant Pseudomonas stutzeri SGM-1. Materials Advances. 2024;5(4):1462–7. Yardman-Frank JM, Fisher DE. Skin pigmentation and its control: From ultraviolet radiation to stem cells. Experimental Dermatology. 2021;30:560–71. Zamudio Díaz DF, Busch L, Kröger M, Klein AL, Lohan SB, Mewes KR, et al. Significance of melanin distribution in the epidermis for the protective effect against UV light. Scientific Reports. 2024;14(1):1–14. Manganelli M, Stabile G, Scharf C, Podo Brunetti A, Paolino G, Giuffrida R, et al. Skin Photodamage and Melanomagenesis: A Comprehensive Review. Cancers. 2025;17(11):1–20. Lv J, Meng D, Zhang H, Xu W, An X, Yin C, et al. Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs. Frontiers in Pharmacology. 2025;16(April):1–16. Putri SA, Maharani R, Maksum IP, Siahaan TJ. Peptide Design for Enhanced Anti-Melanogenesis: Optimizing Molecular Weight, Polarity, and Cyclization. Drug Design, Development and Therapy. 2025;19(January):645–70. D’Mello SAN, Finlay GJ, Baguley BC, Askarian-Amiri ME. Signaling pathways in melanogenesis. International Journal of Molecular Sciences. 2016;17(7):1–18. Kawakami A, Fisher DE. The master role of microphthalmia-associated transcription factor in melanocyte and melanoma biology. Laboratory Investigation. 2017;97(6):649–56. Chen T, Zhao B, Liu Y, Wang R, Yang Y, Yang L, et al. MITF-M regulates melanogenesis in mouse melanocytes. Journal of Dermatological Science. 2018;90(3):253–62. Hu S, Bai S, Dai Y, Yang N, Li J, Zhang X, et al. Deubiquitination of MITF-M Regulates Melanocytes Proliferation and Apoptosis. Frontiers in Molecular Biosciences. 2021;8(June):1–11. Rodboon T, Palipoch S, Okada S, Charoenchon N, Nakornpakdee Y, Suwannalert P. Oxyresveratrol inhibits cellular tyrosinase-related oxidative stress-induced melanogenesis in B16 melanoma cells. Journal of Applied Pharmaceutical Science. 2020;10(4):8–13. Premi S, Wallisch S, Mano CM, Weiner AB, Bacchiocchi A, Wakamatsu K, et al. Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure. Science. 2015;347(6224):842–7. Zhang X, Li H, Liu C, Yuan X. Role of ROS‑mediated autophagy in melanoma (Review). Molecular Medicine Reports. 2022;26(4):1–9. Denat L, Kadekaro AL, Marrot L, Leachman S, Abdel-Malek ZA. Melanocytes as Instigators and Victims of Oxidative Stress L. Physiology & behavior. 2017;176(1):100–6. Kim HE, Lee SG. Induction of ATP synthase β by H2O2 induces melanogenesis by activating PAH and cAMP/CREB/MITF signaling in melanoma cells. International Journal of Biochemistry and Cell Biology. 2013;45(7):1217–22. Jessen C, Kreß JKC, Baluapuri A, Hufnagel A, Schmitz W, Kneitz S, et al. The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression. Oncogene. 2020;39(44):6841–55. Ko H, Kim MM. H 2 O 2 promotes the aging process of melanogenesis through modulation of MITF and Nrf2. Molecular Biology Reports. 2019;46(2):2461–71. Kaminski K, Kazimierczak U, Kolenda T. Oxidative stress in melanogenesis and melanoma development. Wspolczesna Onkologia. 2022;26(1):1–7. Qiu L, Song Z, Setaluri V. Oxidative stress and vitiligo: The Nrf2-ARE signaling connection. Journal of Investigative Dermatology. 2014;134(8):2074–6. Marrot L, Jones C, Perez P, Meunier JR. The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis. Pigment Cell and Melanoma Research. 2008;21(1):79–88. Eom S, Lee S, Lee J, Yeom HD, Lee SG, Lee J. DDX3 Upregulates Hydrogen Peroxide-Induced Melanogenesis in Sk-Mel-2 Human Melanoma Cells. Molecules. 2022;27(20):1–11. Xing X, Dan Y, Xu Z, Xiang L. Implications of Oxidative Stress in the Pathogenesis and Treatment of Hyperpigmentation Disorders. Oxidative Medicine and Cellular Longevity. 2022;2022. Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, et al. Tyrosinase depletion prevents the maturation of melanosomes in the mouse hair follicle. PLoS ONE. 2015;10(11):1–25. Nandi A, Yan LJ, Jana CK, Das N. Role of Catalase in Oxidative Stress- and Age-Associated Degenerative Diseases. Oxidative Medicine and Cellular Longevity. 2019;1–19. Liu HM, Cheng MY, Xun MH, Zhao ZW, Zhang Y, Tang W, et al. Possible Mechanisms of Oxidative Stress-Induced Skin Cellular Senescence, Inflammation, and Cancer and the Therapeutic Potential of Plant Polyphenols. Vol. 24, International Journal of Molecular Sciences. 2023. Lu Y, Tonissen KF, Di Trapani G. Modulating skin colour: Role of the thioredoxin and glutathione systems in regulating melanogenesis. Bioscience Reports. 2021;41(5):1–12. Schallreuter KU, Wood JM. Thioredoxin reductase - Its role in epidermal redox status. Journal of Photochemistry and Photobiology B: Biology. 2001;64(2–3):179–84. Diehl C. Melanocytes and Oxidative Stress. Journal of Pigmentary Disorders. 2014;01(04):1–5. Kim G. Antioxidant Properties of Melanocytes and their Potential Implication in Neurodegenerative Disorders. 2024;14(9):239–45. Jian Z, Li K, Song P, Zhu G, Zhu L, Cui T, et al. Impaired activation of the Nrf2-ARE signaling pathway undermines H 2 O 2-induced oxidative stress response: A possible mechanism for melanocyte degeneration in vitiligo. Journal of Investigative Dermatology. 2014;134(8):2221–30. Yuan J, Lu Y, Wang H, Feng Y, Jiang S, Gao XH, et al. Paeoniflorin Resists H2O2-Induced Oxidative Stress in Melanocytes by JNK/Nrf2/HO-1 Pathway. Frontiers in Pharmacology. 2020;11(536):1–11. Jin JH, Jiang YY, Wang Y, Meng ZW, Li DH, Zhang L, et al. Oxyresveratrol-induced Activation of Nrf2/HO-1 Signaling Pathway Enhances Ability of Resveratrol to Inhibit UVB-induced Melanin. International Journal of Dermatology and Venereology. 2021;4(3):152–62. Romano-Lozano V, Cruz-Avelar A, Peralta-Pedrero ML. [Translated article] Nuclear Factor Erythroid 2-Related Factor 2 in Vitiligo. Actas Dermo-Sifiliograficas. 2022;113(7):T705–11. Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, et al. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(47):18525–30. Manga P, Bis S, Knoll K, Perez B, Orlow SJ. The unfolded protein response in melanocytes: Activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding. Pigment Cell and Melanoma Research. 2010;23(5):627–34. Hoinoiu T, Dumitrascu V, Pit D, Schipor DA, Jabri-Tabrizi M, Hoinoiu B, et al. Quercetin as a Potential Therapeutic Agent for Malignant Melanoma—A Review of Current Evidence and Future Directions. Medicina (Lithuania). 2025;61(4):1–16. Choi MH, Shin HJ. Anti-melanogenesis effect of quercetin. Vol. 3, Cosmetics. MDPI AG; 2016. Alharbi HOA, Alshebremi M, Babiker AY, Rahmani AH. The Role of Quercetin, a Flavonoid in the Management of Pathogenesis Through Regulation of Oxidative Stress, Inflammation, and Biological Activities. Biomolecules. 2025;15(1). Yang YM, Son YO, Lee SA, Jeon YM, Lee JC. Quercetin inhibits α-MSH-stimulated melanogenesis in B16F10 melanoma cells. Phytotherapy Research. 2011;25(8):1166–73. Karadeniz F, Oh JH, Seo Y, Yang J, Lee H, Kong CS. Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation. International Journal of Molecular Sciences. 2023;24(4). Guan C, Xu W, Hong W, Zhou M, Lin F, Fu L, et al. Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes. Molecular Medicine Reports. 2015;11(6):4285–90. Liu-Smith F, Meyskens F. Molecular mechanisms of flavonoids in melanin synthesis and the potential for the prevention and treatment of melanoma. Mol Nutr Food Res. 2016;60(6):1264–74. Xu Z, Xie Y, Song J, Huang J, Shi W. Mechanism of Action of a Chinese Herbal Compound Containing Quercetin, Luteolin, and Kaempferol in the Treatment of Vitiligo Based on Network Pharmacology and Experimental Verification. Evidence-based Complementary and Alternative Medicine. 2022;2022. Harris Z, Donovan MG, Branco GM, Limesand KH, Burd R. Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy. Frontiers in Nutrition. 2016;3(October). Kim BH, Choi JS, Yi EH, Lee JK, Won C, Ye SK, et al. Relative antioxidant activities of quercetin and its structurally related substances and their effects on NF-κB/CRE/AP-1 signaling in murine macrophages. Molecules and Cells. 2013;35(5):410–20. Arung ET, Furuta S, Ishikawa H, Kusuma IW, Shimizu K, Kondo R. Anti-melanogenesis properties of quercetin- and its derivative-rich extract from Allium cepa. Food Chemistry. 2011;124(3):1024–8. Li H, Li M, Fu J, Ao H, Wang W, Wang X. Enhancement of oral bioavailability of quercetin by metabolic inhibitory nanosuspensions compared to conventional nanosuspensions. Drug Delivery. 2021;28(1):1226–36. Joseph A, Shanmughan P, Balakrishnan A, Maliakel B, Krishnakumar IM. Enhanced Bioavailability and Pharmacokinetics of a Natural Self-Emulsifying Reversible Hybrid-Hydrogel System of Quercetin: A Randomized Double-Blinded Comparative Crossover Study. ACS Omega. 2022;7(50):46825–32. Arung ET, Shimizu K, Tanaka H, Kondo R. 3-Prenyl luteolin, a new prenylated flavone with melanin biosynthesis inhibitory activity from wood of Artocarpus heterophyllus. Fitoterapia. 2010;81(6):640–3. Choi MY, Song HS, Hur HS, Sim SS. Whitening activity of luteolin related to the inhibition of cAMP pathway in α-MSH-stimulated B16 melanoma cells. Archives of Pharmacal Research. 2008;31(9):1166–71. Fujii T, Saito M. Inhibitory effect of quercetin isolated from rose hip (Rosa canina L.) against melanogenesis by mouse melanoma cells. Bioscience, Biotechnology and Biochemistry. 2009;73(9):1989–93. Yamauchi K, Mitsunaga T, Batubara I. Novel quercetin glucosides from Helminthostachys zeylanica root and acceleratory activity of melanin biosynthesis. Journal of Natural Medicines. 2013;67(2):369–74. Yamauchi K, Mitsunaga T, Inagaki M, Suzuki T. Synthesized quercetin derivatives stimulate melanogenesis in B16 melanoma cells by influencing the expression of melanin biosynthesis proteins MITF and p38 MAPK. Bioorganic and Medicinal Chemistry. 2014;22(13):3331–40. Ando H, Kondoh H, Ichihashi M, Hearing VJ. Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase. Journal of Investigative Dermatology. 2007;127(4):751–61. D’Ischia M, Wakamatsu K, Napolitano A, Briganti S, Garcia-Borron JC, Kovacs D, et al. Melanins and melanogenesis: Methods, standards, protocols. Pigment Cell and Melanoma Research. 2013;26(5):616–33. Gillbro JM, Olsson MJ. The melanogenesis and mechanisms of skin‐lightening agents - existing and new approches. International Journal of Cosmetic Science. 2011;33:210–21. Xie L, Chen Q, Huang H, Wang H, Zhang R. Inhibitory effects of some flavonoids on the activity of mushroom tyrosinase. Biokhimiya. 2003;68(4):598–602. Cao HH, Tse AKW, Kwan HY, Yu H, Cheng CY, Su T, et al. Quercetin exerts anti-melanoma activities and inhibits STAT3 signaling. Biochemical Pharmacology. 2014;87(3):424–34. Su TR, Lin JJ, Tsai CC, Huang TK, Yang ZY, Wu MO, et al. Inhibition of melanogenesis by gallic acid: Possible involvement of the PI3K/Akt, MEK/ERK and Wnt/β-catenin signaling pathways in B16F10 cells. International Journal of Molecular Sciences. 2013;14(10):20443–58. Kim YJ. Hyperin and Quercetin Modulate Oxidative Stress-Induced Melanogenesis. Biol Pharm Bull. 2012;35(11):2023–7. Bhatti JS, Bhatti GK, Reddy PH. Mitochondrial dysfunction and oxidative stress in metabolic disorders — A step towards mitochondria based therapeutic strategies. Biochimica et Biophysica Acta - Molecular Basis of Disease. 2017;1863(5):1066–77. Ho HJ, Shirakawa H. Oxidative Stress and Mitochondrial Dysfunction in Chronic Kidney Disease. Cells. 2023;12(1):1–29. Chen J, Liu Y, Zhao Z, Qiu J. Oxidative stress in the skin: Impact and related protection. Vol. 43, International Journal of Cosmetic Science. John Wiley and Sons Inc; 2021. p. 495–509. Tochigi M, Inoue T, Suzuki-Karasaki M, Ochiai T, Ra C, Suzuki-Karasaki Y. Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation. International Journal of Oncology. 2013;42(3):863–72. Lee SW, Kim JH, Song H, Seok JK, Hong SS, Boo YC. Luteolin 7-sulfate attenuates melanin synthesis through inhibition of CREB- and MITF-mediated tyrosinase expression. Antioxidants. 2019;8(4). Qi W, Qi W, Xiong D, Long M. Quercetin: Its Antioxidant Mechanism, Antibacterial Properties and Potential Application in Prevention and Control of Toxipathy. Molecules. 2022;27(19). Nishimura FDCY, De Almeida AC, Ratti BA, Ueda-Nakamura T, Nakamura CV, Ximenes VF, et al. Antioxidant effects of quercetin and naringenin are associated with impaired neutrophil microbicidal activity. Evidence-based Complementary and Alternative Medicine. 2013;2013. Mitsunaga T, Yamauchi K. Effect of quercetin derivatives on melanogenesis stimulation of melanoma cells. Journal of Wood Science. 2015 Aug 7;61(4):351–63. Ha AT, Rahmawati L, You L, Hossain MA, Kim JH, Cho JY. Anti-inflammatory, antioxidant, moisturizing, and antimelanogenesis effects of quercetin 3-o-β-d-glucuronide in human keratinocytes and melanoma cells via activation of nf-κb and ap-1 pathways. International Journal of Molecular Sciences. 2022 Jan 1;23(1). Yan M, Liu Z, Yang H, Li C, Chen H, Liu Y, et al. Luteolin decreases the UVA-induced autophagy of human skin fibroblasts by scavenging ROS. Molecular Medicine Reports. 2016;14(3):1986–92. de Castilho TS, Matias TB, Nicolini KP, Nicolini J. Study of interaction between metal ions and quercetin. Food Science and Human Wellness. 2018;7(3):215–9. Lomozová Z, Catapano MC, Hrubša M, Karlíčková J, Macáková K, Kučera R, et al. Chelation of Iron and Copper by Quercetin B-Ring Methyl Metabolites, Isorhamnetin and Tamarixetin, and Their Effect on Metal-Based Fenton Chemistry. Journal of Agricultural and Food Chemistry. 2021;69(21):5926–37. Kasprzak M, Erxleben A, Ochocki J. Properties and applications of flavonoid metal complexes. RSC Publishing. 2013;00(1–3):1–25. Kim JS, Lee EB, Choi JH, Jung J, Jeong UY, Bae UJ, et al. Antioxidant and Immune Stimulating Effects of Allium cepa Skin in the RAW 264.7 Cells and in the C57BL/6 Mouse Immunosuppressed by Cyclophosphamide. Antioxidants. 2023;12(4). Ren Q, Qu L, Yuan Y, Wang F. Natural Modulators of Key Signaling Pathways in Skin Inflammageing. Clinical, Cosmetic and Investigational Dermatology . 2024;17(December):2967–88. Mukai R. Prenylation enhances the biological activity of dietary flavonoids by altering their bioavailability. Bioscience, Biotechnology and Biochemistry. 2018;82(2):207–15. Additional Declarations No competing interests reported. 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luteolin in B16 melanoma cells\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/57538665d2866b72180b07ba.png"},{"id":90265857,"identity":"5540ca98-fcd5-4e05-8936-a7637c23877c","added_by":"auto","created_at":"2025-08-31 17:07:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21759,"visible":true,"origin":"","legend":"\u003cp\u003eAntimelanogenesis effect of quercetin in B16 melanoma cells\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/1ea722facd990ffa067f4491.png"},{"id":90265806,"identity":"e65552cc-112e-492a-a0b4-aa891643fb50","added_by":"auto","created_at":"2025-08-31 16:59:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21308,"visible":true,"origin":"","legend":"\u003cp\u003eAntimelanogenesis effect of rutin in B16 melanoma 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cells\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/09df4c4ce92efad9818e60e7.png"},{"id":90265860,"identity":"13fcaa6e-8f99-4fb6-97df-114c749eb683","added_by":"auto","created_at":"2025-08-31 17:07:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":20807,"visible":true,"origin":"","legend":"\u003cp\u003eAntimelanogenesis effect of 3-prenyl luteolin in B16 melanoma cells\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/ddc76cf0245a1da5e89f34f4.png"},{"id":90265859,"identity":"c878e9dc-3c4a-4266-ab14-eb4055463c47","added_by":"auto","created_at":"2025-08-31 17:07:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":33994,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in B16 melanoma cells\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/c51719db93b6efe3a51db49f.png"},{"id":90265819,"identity":"ce220d89-1cbd-408f-b437-fb892d8004fb","added_by":"auto","created_at":"2025-08-31 16:59:21","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":37823,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Quercetin related compounds in B16 melanoma cells treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (concentration : 0.25%)\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/d7d35eca733af31b2ae69c7c.png"},{"id":90265822,"identity":"9f991450-85e7-4698-af46-72a0f9c81b9b","added_by":"auto","created_at":"2025-08-31 16:59:21","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":26150,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Quercetin related compounds in B16 melanoma cells treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (concentration : 0.25%)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/555044abef26591ba4524420.png"},{"id":90702077,"identity":"3d998855-278f-464e-9b0e-9f77d066ec9a","added_by":"auto","created_at":"2025-09-06 01:16:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":887380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7328612/v1/1eb17313-1f05-4729-a08d-46a676fdc5a0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAntimelanogenesis of quercetin-related compounds in B16 melanoma cells: the difference moieties of C-3 position and its effect on H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMelanin is a complex, heterogeneous biopolymer synthesized through the enzymatic oxidation and subsequent polymerization of tyrosine-derived precursors within melanosomes, specialized organelles in melanocytes [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Serving as a critical photoprotective pigment, melanin effectively absorbs ultraviolet (UV) radiation, thereby mitigating UV-induced oxidative damage to skin cells and contributing to skin coloration and protection [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The melanogenesis pathway is tightly regulated, beginning with the rate-limiting enzyme tyrosinase catalyzing the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and subsequent oxidation to dopaquinone. This intermediate undergoes multiple enzymatic transformations, involving tyrosinase-related proteins TRP-1 and TRP-2, which lead to the formation of eumelanin and pheomelanin pigments [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The microphthalmia-associated transcription factor (MITF) plays a pivotal role by regulating the expression of tyrosinase and related enzymes, thus ensuring coordinated melanocyte differentiation, survival, and pigment synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eReactive oxygen species (ROS), particularly hydrogen peroxide (H₂O₂), are generated endogenously during melanin biosynthesis via tyrosinase-mediated oxidation of L-DOPA and are also produced exogenously following UV exposure [\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At physiological concentrations (~\u0026thinsp;50\u0026ndash;300 \u0026micro;M), H₂O₂ acts as a secondary messenger that promotes melanogenesis by activating melanocyte-specific transcription factors such as MITF through pathways involving cyclic AMP (cAMP)/protein kinase A (PKA) and the nuclear factor erythroid 2\u0026ndash;related factor 2 (Nrf2), which regulates cellular antioxidant responses [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, excessive or chronic accumulation of H₂O₂ can disrupt intracellular redox homeostasis, leading to deleterious effects such as endoplasmic reticulum (ER) stress, impaired trafficking and maturation of tyrosinase, melanocyte senescence, and ultimately pigmentary disorders including melasma, post-inflammatory hyperpigmentation, and premature skin aging [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These pathological conditions highlight the dual role of ROS in melanogenesis as both necessary signaling molecules and potential mediators of oxidative damage.\u003c/p\u003e\u003cp\u003eMelanocytes are equipped with robust endogenous antioxidant systems, including catalase, glutathione peroxidase, and thioredoxin reductase, which cooperatively degrade H₂O₂ to maintain redox balance and prevent oxidative stress [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These antioxidant enzymes are transcriptionally regulated by the Nrf2 signaling pathway, which governs the expression of a battery of protective genes such as heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and enzymes involved in glutathione synthesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Despite these sophisticated defenses, chronic oxidative stress and overwhelming ROS production can impair these protective mechanisms, resulting in protein misfolding, ER dilation, defective melanogenesis, and the exacerbation of hyperpigmentation disorders [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, controlling oxidative stress and ROS levels remains a critical strategy in the development of effective depigmenting agents.\u003c/p\u003e\u003cp\u003eQuercetin, a naturally abundant bioflavonoid in various fruits and vegetables, has emerged as a promising natural compound for inhibiting melanogenesis due to its potent antioxidant, anti-inflammatory, and enzyme inhibitory properties [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In vitro experiments in human melanocytes and murine B16F10 melanoma cells have demonstrated that quercetin, particularly at concentrations ranging from 20 to 50 \u0026micro;M, significantly inhibits tyrosinase activity and melanin synthesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This effect is mediated by downregulation of MITF and its downstream effectors, tyrosinase-related proteins TRP-1 and TRP-2, and suppression of the cAMP/PKA signaling cascade, all achieved without notable cytotoxicity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, quercetin mitigates H₂O₂-induced ER stress, preserves ER structure, and supports proper trafficking and expression of melanogenic enzymes, thereby maintaining melanocyte functionality under oxidative stress conditions [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMechanistically, quercetin scavenges various ROS species, including peroxynitrite and superoxide radicals, elevates intracellular glutathione levels, and modulates catalase and HO-1 activities to restore cellular redox balance and inhibit melanogenesis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Its derivatives have demonstrated enhanced anti-melanogenic effects by targeting multiple signaling pathways including cAMP/PKA/CREB/MITF and MAPK, further highlighting the therapeutic potential of this class of compounds [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Notably, quercetin\u0026rsquo;s effects exhibit dose dependency, as lower concentrations (\u0026lt;\u0026thinsp;20 \u0026micro;M) may paradoxically stimulate melanogenesis through post-translational stabilization of tyrosinase and TRP-2 proteins, highlighting the necessity for precise dose optimization in therapeutic applications [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Despite encouraging in vitro and preclinical results, clinical evidence for quercetin\u0026rsquo;s efficacy in treating hyperpigmentation remains insufficient. Challenges such as improving its bioavailability, developing optimized topical formulations, and conducting rigorous clinical trials comparing quercetin with established depigmenting agents like kojic acid, arbutin, and vitamin C are critical for its translation to clinical use [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Addressing these gaps will be essential to fully harness the therapeutic potential of quercetin in oxidative stress\u0026ndash;related pigmentary disorders.\u003c/p\u003e\u003cp\u003eThe present study aims to investigate the anti-melanogenic effects of two compounds: quercetin (\u003cb\u003e2\u003c/b\u003e), isolated from the dried skin of \u003cem\u003eAllium cepa\u003c/em\u003e (onion), and 3-prenyl luteolin (\u003cb\u003e6\u003c/b\u003e), isolated from the wood of \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e (jackfruit). These compounds have been selected as novel candidates for anti-melanogenesis due to their unique structures and potential bioactivity. This research will explore their inhibitory activity on melanin formation, revealing their potential as novel depigmenting agents for skin-whitening cosmetics. In addition, we will evaluate the capability of these compounds to reduce ROS, especially H₂O₂, in B16 melanoma cells, which represent a significant source of oxidative stress associated with melanin overproduction. By targeting the oxidative pathways contributing to melanogenesis, these compounds could serve as promising candidates for the development of effective skin whitening products through the reduction of melanin overproduction.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals\u003c/h2\u003e\u003cp\u003eSodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), and rutin (\u003cb\u003ecompound 3\u003c/b\u003e) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ethylenediaminetetraacetic acid (EDTA) was purchased from Dojindo Laboratories (Kumamoto, Japan). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was sourced from Sigma-Aldrich (St. Louis, MO, USA), and Eagle\u0026rsquo;s Minimum Essential Medium (EMEM) was obtained from Nissui Pharmaceutical Co., Ltd. (Osaka, Japan). Hyperin (\u003cb\u003ecompound 4\u003c/b\u003e) was supplied by Tokiwa Phytochemical Co. (Chiba, Japan), luteolin (\u003cb\u003ecompound 1\u003c/b\u003e) was purchased from Extrasynthese (Genay, France), and isoquercitrin (\u003cb\u003ecompound 5\u003c/b\u003e) was obtained from Fluka Chemie GmbH (Steinheim, Germany). Quercetin (\u003cb\u003ecompound 2\u003c/b\u003e) was isolated from the dried outer skin of \u003cem\u003eAllium cepa\u003c/em\u003e, while 3-prenyl luteolin (\u003cb\u003ecompound 6\u003c/b\u003e) was purified from the wood of \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e. All other reagents used were of analytical grade and sourced from standard commercial suppliers. The chemical structures of the isolated compounds are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe B16 murine melanoma cell line was obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were cultured in Eagle\u0026rsquo;s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 0.09 mg/mL theophylline. Cell cultures were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂ atmosphere.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eInhibitory effect of melanin biosynthesis using cultured B16 melanoma cells\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe assay was performed following the method described by Arung et al. (2007), with slight modifications. Briefly, confluent B16 melanoma cells were washed with phosphate-buffered saline (PBS) and detached using 0.25% trypsin-EDTA solution. The cells were seeded into two 24-well culture plates\u0026mdash;one designated for melanin content analysis and the other for cell viability assessment\u0026mdash;at a density of 1 \u0026times; 10⁵ cells per well. After 24 hours of incubation in complete medium to allow cell attachment, the culture medium was replaced with 998 \u0026micro;L of fresh EMEM, and 2 \u0026micro;L of dimethyl sulfoxide (DMSO), with or without the test compounds at various concentrations (n\u0026thinsp;=\u0026thinsp;3), was added. Arbutin was included as a positive control. Cells were incubated for an additional 48 hours, after which the medium was refreshed and the same concentrations of test compounds were reapplied. Following a further 24-hour incubation, adherent cells were subjected to melanin content and cell viability assays as described below.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eDetermination of melanin content in B16 melanoma cells\u003c/h3\u003e\n\u003cp\u003eMelanin content in the treated B16 melanoma cells was quantitatively evaluated following compound exposure. After removing the culture medium, cells were washed thoroughly with phosphate-buffered saline (PBS) to eliminate residual media components. The adherent cells were then lysed by incubation in 1.0 mL of 1 N sodium hydroxide (NaOH), which solubilized intracellular melanin. The resulting lysates were transferred to a 96-well microplate, and absorbance was measured at 405 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA). Melanin levels in the treated samples were normalized to those of the untreated control group and expressed as a percentage of control values to assess the inhibitory effects of the test compounds on melanin synthesis.\u003c/p\u003e\n\u003ch3\u003eCell Treated with HO\u003c/h3\u003e\n\u003cp\u003eCells were seeded at a density of 1 \u0026times; 10⁵ cells per well into three separate 24-well plates and incubated for 24 hours to allow adherence. Following incubation, the test compounds were administered at designated concentrations. To induce oxidative stress, hydrogen peroxide (H₂O₂) was added to each well at a final concentration of 0.25% at 1, 2, and 4 hours after compound treatment. Cell viability was subsequently assessed using the MTT assay as described below.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell viability\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCell viability was evaluated using the microculture tetrazolium (MTT) assay, which assesses mitochondrial metabolic activity based on the formation of formazan crystals in viable cells. B16 melanoma cells were seeded into 24-well plates at a density of 1 \u0026times; 10⁵ cells per well. Following the designated treatments and incubation periods, 50 \u0026micro;L of MTT solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL in PBS] was added to each well. Plates were incubated for 4 hours at 37\u0026deg;C in a humidified incubator with 5% CO₂ to allow for formazan formation. After incubation, the medium was aspirated, and 1.0 mL of isopropyl alcohol containing 0.04 N HCl was added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm with a reference wavelength of 630 nm using a microplate reader (BioTek Instruments, USA). The absorbance values were used to calculate cell viability relative to untreated controls.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eAntimelanogenesis of quercetin-related compounds\u003c/h2\u003e\u003cp\u003eThe anti-melanogenic properties of all isolated constituents were assessed using B16 melanoma cell assays. Among the tested flavonoids, no statistically significant differences were observed in the melanin inhibition rates of rutin (\u003cb\u003e3\u003c/b\u003e), hyperin (\u003cb\u003e4\u003c/b\u003e), and isoquercetin (\u003cb\u003e5\u003c/b\u003e). In contrast, compounds \u003cb\u003e1, 2\u003c/b\u003e, and \u003cb\u003e6\u003c/b\u003e exhibited markedly higher inhibitory effects on melanogenesis compared to the remaining isolates and the reference agent, arbutin. A dose-dependent suppression of melanin synthesis and cell viability was observed with increasing concentrations of these compounds. Notably, compound 1, structurally identified as luteolin, exerted the most potent melanogenesis-inhibitory effect, surpassing the activities of quercetin (\u003cb\u003e2\u003c/b\u003e) and 3-prenyl luteolin (\u003cb\u003e6\u003c/b\u003e), as illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eLuteolin demonstrated the capacity to inhibit melanin synthesis by over 50% at a concentration of 5 \u0026micro;g/mL. At 25 \u0026micro;g/mL, luteolin achieved more than 80% inhibition of melanin formation, concomitant with a reduction in cell viability to below 30% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, quercetin inhibited melanin production by less than 50% at 5 \u0026micro;g/mL and exceeded 65% inhibition at 25 \u0026micro;g/mL, while maintaining cell viability above 70% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The inhibitory profile of 3-prenyl luteolin differed from these compounds, with melanin synthesis suppressed by over 60% only at 30 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The IC₅₀ values determined for luteolin, quercetin, and 3-prenyl luteolin were 3.0 \u0026micro;g/mL, 8.0 \u0026micro;g/mL, and 20.1 \u0026micro;g/mL, respectively, indicating varying degrees of anti-melanogenic efficacy.\u003c/p\u003e\u003cp\u003eLuteolin, quercetin, and 3-prenyl luteolin have been classified as polyphenolic compounds, which are well-recognized for their significant roles and extensive applications in the cosmetic industry. Previous studies have demonstrated the anti-melanogenic properties of these compounds [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Among them, 3-prenyl luteolin exhibits lower anti-melanogenesis activity compared to luteolin and quercetin, consistent with previous research [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This variation in activity is attributed to differences in functional groups, as prior research indicated that the presence of an isoprenoid moiety in 3-prenyl luteolin diminishes its inhibitory effect on melanin synthesis in B16 melanoma cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additionally, other studies have highlighted that the anti-melanogenic efficacy is highly dependent on the specific position of functional groups within the molecular structure [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnti-melanogenic agents exert their effects through multiple mechanisms, including inhibition of tyrosinase mRNA transcription, disruption of tyrosinase maturation, suppression of tyrosinase enzymatic activity, promotion of tyrosinase degradation, and indirect modulation of tyrosinase function [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Among these, tyrosinase inhibition is widely regarded as the primary target for reducing hyperpigmentation [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Luteolin and quercetin inhibit melanin synthesis via distinct pathways. Luteolin reduces melanin production in B16 melanoma cells by decreasing intracellular cyclic adenosine monophosphate (cAMP) signaling [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The downregulation of cAMP leads to dephosphorylation of PKA/CREB, resulting in diminished MITF expression, impaired tyrosinase regulation, and subsequent inhibition of melanin synthesis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In contrast, quercetin acts as a potent tyrosinase inhibitor, directly interfering with the enzyme\u0026rsquo;s catalytic activity and downstream signaling pathways to prevent melanin formation [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEffect of quercetin related compounds on B16 melanoma cless treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eCell viability following hydrogen peroxide (H₂O₂) exposure was assessed using the MTT assay to establish an optimal concentration for inducing oxidative stress in B16 melanoma cells. Cells were treated with increasing concentrations of H₂O₂ (0.05%, 0.1%, 0.25%, and 0.5%), and a clear dose-dependent decrease in viability was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), consistent with prior reports demonstrating H₂O₂'s cytotoxicity through oxidative damage and mitochondrial dysfunction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Notably, there was no statistically significant difference in cell viability between 0.25% and 0.5% H₂O₂ treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), indicating a saturation effect at higher concentrations where additional oxidative burden did not further reduce viability [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Based on these results, 0.25% H₂O₂ was selected for subsequent assays, as it induced substantial oxidative stress while avoiding excessive cytotoxicity that could confound downstream analyses. Similar concentrations have been used in previous studies to model oxidative stress conditions in melanocytes and melanoma cells, validating its use as a physiologically relevant dose [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These doses effectively mimic oxidative environments that contribute to melanocyte damage, melanogenesis alterations, and melanoma cell death, supporting their physiological relevance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe impact of quercetin-related compounds on B16 melanoma cell viability under oxidative stress is presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Notably, luteolin demonstrated a pronounced ability to enhance cell survival following hydrogen peroxide (H₂O₂) exposure, outperforming other tested compounds in mitigating oxidative damage. This suggests that luteolin exerts stronger cytoprotective effects, likely due to its potent antioxidant activity and modulation of intracellular signaling pathways involved in cell survival and melanogenesis regulation. Luteolin and its derivative luteolin 7-sulfate inhibit melanin synthesis by downregulating tyrosinase (TYR) expression through CREB- and MITF-mediated signaling pathways, which are key regulators of melanogenesis in melanoma cells. Moreover, luteolin reduces cellular reactive oxygen species (ROS) levels by directly inhibiting oxidative enzymes such as xanthine oxidase, thereby protecting cells from oxidative damage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Other quercetin derivatives also partially restored cell viability compromised by H₂O₂, indicating their potential to alleviate oxidative stress in melanocytes. These findings corroborate previous reports indicating that quercetin and its analogs reduce reactive oxygen species (ROS) levels and protect against oxidative injury by scavenging free radicals and activating endogenous antioxidant defenses [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt a concentration of 5 ppm, the quercetin-related compounds exhibited lower cytotoxicity compared to quercetin 3-O-β-D-glucuronide (Q-3-G) and synthetic quercetin glycosides, which have shown dose-dependent cytotoxic effects in various cell lines including HaCaT keratinocytes and B16F10 melanoma cells at 20 \u0026micro;M [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This differential toxicity profile highlights the advantage of naturally derived compounds, which may offer enhanced safety and efficacy for potential therapeutic or cosmetic applications targeting hyperpigmentation and oxidative stress-related skin disorders. Furthermore, luteolin protects cells through both direct antioxidant activity via the Nrf2/ARE pathway and by modulating melanogenesis-related signaling cascades such as CREB/MITF, making it a potent agent against oxidative stress and hyperpigmentation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. These pathways contribute to reducing oxidative damage and restoring melanocyte function, thereby inhibiting excessive melanogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the current study, polyphenolic compounds including luteolin, isoquercetin, quercetin, and rutin demonstrated significant ROS-scavenging activity in B16 melanoma cells, with luteolin exerting the most pronounced protective effect against H₂O₂-induced cytotoxicity. This aligns with previous findings that highlight the superior antioxidant capacity of luteolin in skin cells through multiple mechanisms such as direct radical scavenging [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Quercetin\u0026rsquo;s efficacy in mitigating ROS accumulation is consistent with its chemical structure, which features multiple hydroxyl groups facilitating electron donation and free radical neutralization [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Furthermore, quercetin\u0026rsquo;s metal-chelating properties inhibit Fenton and Haber-Weiss reactions, which otherwise generate highly reactive hydroxyl radicals from H₂O₂ and transition metals, thus breaking a critical cycle of ROS amplification [\u003cspan additionalcitationids=\"CR74\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eQuercetin, a well-known flavonoid isolated from \u003cem\u003eAllium cepa\u003c/em\u003e (onion) skin, exhibits potent antioxidant and antimelanogenic activities, largely attributed to its polyphenolic structure and multiple hydroxyl groups that efficiently scavenge reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and superoxide anions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. The generation of ROS during UV exposure or cellular metabolism is a critical trigger for melanogenesis, often resulting in hyperpigmentation due to melanin overproduction [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond antioxidative effects, quercetin\u0026rsquo;s anti-inflammatory properties reduce melanocyte activation caused by inflammation, which is a key factor in post-inflammatory hyperpigmentation [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. However, challenges remain in improving its bioavailability and skin penetration for clinical applications. Nonetheless, the comprehensive inhibitory effects of quercetin on oxidative stress and melanogenesis highlight its potential as a natural, safe, and effective ingredient for skin-whitening and photoprotective cosmetics derived from \u003cem\u003eAllium cepa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis study uniquely isolates quercetin specifically from \u003cem\u003eAllium cepa\u003c/em\u003e skin and comparatively evaluates it alongside 3-prenyl luteolin, a prenylated flavonoid derivative obtained from the wood of \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e. Prenylation of flavonoids like luteolin is known to enhance their lipophilicity and cellular uptake, often improving bioavailability and biological activity [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. This modification may increase the compound\u0026rsquo;s antioxidant capacity and its interaction with key signaling pathways involved in melanogenesis and oxidative stress response. Our findings demonstrate that 3-prenyl luteolin exhibits significant inhibitory effects on melanin production and effectively reduces intracellular ROS levels, although its cytoprotective efficacy under H₂O₂-induced oxidative stress was somewhat lower compared to luteolin and quercetin. This comparative analysis provides novel insights into how structural modifications influence the bioactivity of flavonoids in melanocyte models. Furthermore, the study emphasizes sustainable utilization of agricultural by-products as valuable sources of natural bioactives, bridging environmental considerations with practical applications in the development of effective and safe skin-whitening agents. This integrative approach advances the understanding of quercetin-related compounds and prenylated flavonoids as multifunctional depigmenting agents, highlighting their potential for cosmeceutical innovation beyond what has been previously reported.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eQuercetin (\u003cb\u003ecompound 2\u003c/b\u003e), isolated from the dried skin of \u003cem\u003eAllium cepa\u003c/em\u003e, and 3-prenyl luteolin (\u003cb\u003ecompound 6\u003c/b\u003e), derived from the wood of \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e, are promising natural polyphenolic compounds with potent antioxidant and antimelanogenic activities. Their ability to scavenge reactive oxygen species and reduce oxidative stress is likely a key mechanism underlying their inhibition of melanogenesis. These properties make them attractive candidates for developing novel skin-whitening and depigmenting agents. However, further studies focusing on safety and efficacy are essential before their application in humans can be realized. Overall, these compounds offer valuable potential for managing hyperpigmentation and oxidative stress-related skin disorders through natural, multifunctional mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Japan Society for the Promotion of Science (JSPS) under the Postdoctoral Fellowship Program for Foreign Researchers. This research is also supported by the National Research and Innovation Agency (BRIN) and a research grant from BRIN (Grant number: B-1146/II.7/HK 01.00/5/2025) with the title “Pusat Kolaborasi Riset Kosmetik Berteknologi Nano Berbasis Biomassa”.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNF, DJ, RAE, ETA : Conception or design of the workNF, DJ, WF, RAE, YUK, KS, ETA; or the acquisition, analysis, and interpretation of data;NF, DJ, HI, WF, RAE, YUK, KS, ETA : drafted the work or revised it critically for important intellectual content;NF, DJ, HI, WF, RAE, YUK, KS, ETA : approved the version to be published\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the support and facilities provided by the Japan Society for the Promotion of Science and the National Research and Innovation Agency (BRIN).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaranduca MA, Branisteanu D, Serban DN, Branisteanu DC, Stoleriu G, Manolache N, et al. Synthesis and physiological implications of melanic pigments (review). Oncology Letters. 2019;17(5):4183\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eGuo L, Li W, Gu Z, Wang L, Guo L, Ma S, et al. Recent Advances and Progress on Melanin: From Source to Application. International Journal of Molecular Sciences. 2023;24(5). \u003c/li\u003e\n\u003cli\u003eMahajan SG, Nandre VS, Kodam KM, Kulkarni M V. One-pot synthesis of black biopolymeric eumelanin pigment by indigenous salt-tolerant Pseudomonas stutzeri SGM-1. Materials Advances. 2024;5(4):1462\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eYardman-Frank JM, Fisher DE. Skin pigmentation and its control: From ultraviolet radiation to stem cells. Experimental Dermatology. 2021;30:560\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eZamudio D\u0026iacute;az DF, Busch L, Kr\u0026ouml;ger M, Klein AL, Lohan SB, Mewes KR, et al. Significance of melanin distribution in the epidermis for the protective effect against UV light. Scientific Reports. 2024;14(1):1\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eManganelli M, Stabile G, Scharf C, Podo Brunetti A, Paolino G, Giuffrida R, et al. Skin Photodamage and Melanomagenesis: A Comprehensive Review. Cancers. 2025;17(11):1\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eLv J, Meng D, Zhang H, Xu W, An X, Yin C, et al. Apigenin promotes melanogenesis and melanosome transport through the c-KIT/Raf-1/MAPK/CREB pathway in HEMCs. Frontiers in Pharmacology. 2025;16(April):1\u0026ndash;16. \u003c/li\u003e\n\u003cli\u003ePutri SA, Maharani R, Maksum IP, Siahaan TJ. Peptide Design for Enhanced Anti-Melanogenesis: Optimizing Molecular Weight, Polarity, and Cyclization. Drug Design, Development and Therapy. 2025;19(January):645\u0026ndash;70. \u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Mello SAN, Finlay GJ, Baguley BC, Askarian-Amiri ME. Signaling pathways in melanogenesis. International Journal of Molecular Sciences. 2016;17(7):1\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eKawakami A, Fisher DE. The master role of microphthalmia-associated transcription factor in melanocyte and melanoma biology. Laboratory Investigation. 2017;97(6):649\u0026ndash;56. \u003c/li\u003e\n\u003cli\u003eChen T, Zhao B, Liu Y, Wang R, Yang Y, Yang L, et al. MITF-M regulates melanogenesis in mouse melanocytes. Journal of Dermatological Science. 2018;90(3):253\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eHu S, Bai S, Dai Y, Yang N, Li J, Zhang X, et al. Deubiquitination of MITF-M Regulates Melanocytes Proliferation and Apoptosis. Frontiers in Molecular Biosciences. 2021;8(June):1\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eRodboon T, Palipoch S, Okada S, Charoenchon N, Nakornpakdee Y, Suwannalert P. Oxyresveratrol inhibits cellular tyrosinase-related oxidative stress-induced melanogenesis in B16 melanoma cells. Journal of Applied Pharmaceutical Science. 2020;10(4):8\u0026ndash;13. \u003c/li\u003e\n\u003cli\u003ePremi S, Wallisch S, Mano CM, Weiner AB, Bacchiocchi A, Wakamatsu K, et al. Chemiexcitation of melanin derivatives induces DNA photoproducts long after UV exposure. Science. 2015;347(6224):842\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eZhang X, Li H, Liu C, Yuan X. Role of ROS‑mediated autophagy in melanoma (Review). Molecular Medicine Reports. 2022;26(4):1\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eDenat L, Kadekaro AL, Marrot L, Leachman S, Abdel-Malek ZA. Melanocytes as Instigators and Victims of Oxidative Stress L. Physiology \u0026amp; behavior. 2017;176(1):100\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eKim HE, Lee SG. Induction of ATP synthase \u0026beta; by H2O2 induces melanogenesis by activating PAH and cAMP/CREB/MITF signaling in melanoma cells. International Journal of Biochemistry and Cell Biology. 2013;45(7):1217\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003eJessen C, Kre\u0026szlig; JKC, Baluapuri A, Hufnagel A, Schmitz W, Kneitz S, et al. The transcription factor NRF2 enhances melanoma malignancy by blocking differentiation and inducing COX2 expression. Oncogene. 2020;39(44):6841\u0026ndash;55. \u003c/li\u003e\n\u003cli\u003eKo H, Kim MM. H 2 O 2 promotes the aging process of melanogenesis through modulation of MITF and Nrf2. Molecular Biology Reports. 2019;46(2):2461\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eKaminski K, Kazimierczak U, Kolenda T. Oxidative stress in melanogenesis and melanoma development. Wspolczesna Onkologia. 2022;26(1):1\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eQiu L, Song Z, Setaluri V. Oxidative stress and vitiligo: The Nrf2-ARE signaling connection. Journal of Investigative Dermatology. 2014;134(8):2074\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eMarrot L, Jones C, Perez P, Meunier JR. The significance of Nrf2 pathway in (photo)-oxidative stress response in melanocytes and keratinocytes of the human epidermis. Pigment Cell and Melanoma Research. 2008;21(1):79\u0026ndash;88. \u003c/li\u003e\n\u003cli\u003eEom S, Lee S, Lee J, Yeom HD, Lee SG, Lee J. DDX3 Upregulates Hydrogen Peroxide-Induced Melanogenesis in Sk-Mel-2 Human Melanoma Cells. Molecules. 2022;27(20):1\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eXing X, Dan Y, Xu Z, Xiang L. Implications of Oxidative Stress in the Pathogenesis and Treatment of Hyperpigmentation Disorders. Oxidative Medicine and Cellular Longevity. 2022;2022. \u003c/li\u003e\n\u003cli\u003ePaterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, et al. Tyrosinase depletion prevents the maturation of melanosomes in the mouse hair follicle. PLoS ONE. 2015;10(11):1\u0026ndash;25. \u003c/li\u003e\n\u003cli\u003eNandi A, Yan LJ, Jana CK, Das N. Role of Catalase in Oxidative Stress- and Age-Associated Degenerative Diseases. Oxidative Medicine and Cellular Longevity. 2019;1\u0026ndash;19. \u003c/li\u003e\n\u003cli\u003eLiu HM, Cheng MY, Xun MH, Zhao ZW, Zhang Y, Tang W, et al. Possible Mechanisms of Oxidative Stress-Induced Skin Cellular Senescence, Inflammation, and Cancer and the Therapeutic Potential of Plant Polyphenols. Vol. 24, International Journal of Molecular Sciences. 2023. \u003c/li\u003e\n\u003cli\u003eLu Y, Tonissen KF, Di Trapani G. Modulating skin colour: Role of the thioredoxin and glutathione systems in regulating melanogenesis. Bioscience Reports. 2021;41(5):1\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eSchallreuter KU, Wood JM. Thioredoxin reductase - Its role in epidermal redox status. Journal of Photochemistry and Photobiology B: Biology. 2001;64(2\u0026ndash;3):179\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eDiehl C. Melanocytes and Oxidative Stress. Journal of Pigmentary Disorders. 2014;01(04):1\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eKim G. Antioxidant Properties of Melanocytes and their Potential Implication in Neurodegenerative Disorders. 2024;14(9):239\u0026ndash;45. \u003c/li\u003e\n\u003cli\u003eJian Z, Li K, Song P, Zhu G, Zhu L, Cui T, et al. Impaired activation of the Nrf2-ARE signaling pathway undermines H 2 O 2-induced oxidative stress response: A possible mechanism for melanocyte degeneration in vitiligo. Journal of Investigative Dermatology. 2014;134(8):2221\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eYuan J, Lu Y, Wang H, Feng Y, Jiang S, Gao XH, et al. Paeoniflorin Resists H2O2-Induced Oxidative Stress in Melanocytes by JNK/Nrf2/HO-1 Pathway. Frontiers in Pharmacology. 2020;11(536):1\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eJin JH, Jiang YY, Wang Y, Meng ZW, Li DH, Zhang L, et al. Oxyresveratrol-induced Activation of Nrf2/HO-1 Signaling Pathway Enhances Ability of Resveratrol to Inhibit UVB-induced Melanin. International Journal of Dermatology and Venereology. 2021;4(3):152\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eRomano-Lozano V, Cruz-Avelar A, Peralta-Pedrero ML. [Translated article] Nuclear Factor Erythroid 2-Related Factor 2 in Vitiligo. Actas Dermo-Sifiliograficas. 2022;113(7):T705\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eMalhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW, et al. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(47):18525\u0026ndash;30. \u003c/li\u003e\n\u003cli\u003eManga P, Bis S, Knoll K, Perez B, Orlow SJ. The unfolded protein response in melanocytes: Activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding. Pigment Cell and Melanoma Research. 2010;23(5):627\u0026ndash;34. \u003c/li\u003e\n\u003cli\u003eHoinoiu T, Dumitrascu V, Pit D, Schipor DA, Jabri-Tabrizi M, Hoinoiu B, et al. Quercetin as a Potential Therapeutic Agent for Malignant Melanoma\u0026mdash;A Review of Current Evidence and Future Directions. Medicina (Lithuania). 2025;61(4):1\u0026ndash;16. \u003c/li\u003e\n\u003cli\u003eChoi MH, Shin HJ. Anti-melanogenesis effect of quercetin. Vol. 3, Cosmetics. MDPI AG; 2016. \u003c/li\u003e\n\u003cli\u003eAlharbi HOA, Alshebremi M, Babiker AY, Rahmani AH. The Role of Quercetin, a Flavonoid in the Management of Pathogenesis Through Regulation of Oxidative Stress, Inflammation, and Biological Activities. Biomolecules. 2025;15(1). \u003c/li\u003e\n\u003cli\u003eYang YM, Son YO, Lee SA, Jeon YM, Lee JC. Quercetin inhibits \u0026alpha;-MSH-stimulated melanogenesis in B16F10 melanoma cells. Phytotherapy Research. 2011;25(8):1166\u0026ndash;73. \u003c/li\u003e\n\u003cli\u003eKaradeniz F, Oh JH, Seo Y, Yang J, Lee H, Kong CS. Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation. International Journal of Molecular Sciences. 2023;24(4). \u003c/li\u003e\n\u003cli\u003eGuan C, Xu W, Hong W, Zhou M, Lin F, Fu L, et al. Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes. Molecular Medicine Reports. 2015;11(6):4285\u0026ndash;90. \u003c/li\u003e\n\u003cli\u003eLiu-Smith F, Meyskens F. Molecular mechanisms of flavonoids in melanin synthesis and the potential for the prevention and treatment of melanoma. Mol Nutr Food Res. 2016;60(6):1264\u0026ndash;74. \u003c/li\u003e\n\u003cli\u003eXu Z, Xie Y, Song J, Huang J, Shi W. Mechanism of Action of a Chinese Herbal Compound Containing Quercetin, Luteolin, and Kaempferol in the Treatment of Vitiligo Based on Network Pharmacology and Experimental Verification. Evidence-based Complementary and Alternative Medicine. 2022;2022. \u003c/li\u003e\n\u003cli\u003eHarris Z, Donovan MG, Branco GM, Limesand KH, Burd R. Quercetin as an Emerging Anti-Melanoma Agent: A Four-Focus Area Therapeutic Development Strategy. Frontiers in Nutrition. 2016;3(October). \u003c/li\u003e\n\u003cli\u003eKim BH, Choi JS, Yi EH, Lee JK, Won C, Ye SK, et al. Relative antioxidant activities of quercetin and its structurally related substances and their effects on NF-\u0026kappa;B/CRE/AP-1 signaling in murine macrophages. Molecules and Cells. 2013;35(5):410\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eArung ET, Furuta S, Ishikawa H, Kusuma IW, Shimizu K, Kondo R. Anti-melanogenesis properties of quercetin- and its derivative-rich extract from Allium cepa. Food Chemistry. 2011;124(3):1024\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eLi H, Li M, Fu J, Ao H, Wang W, Wang X. Enhancement of oral bioavailability of quercetin by metabolic inhibitory nanosuspensions compared to conventional nanosuspensions. Drug Delivery. 2021;28(1):1226\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003eJoseph A, Shanmughan P, Balakrishnan A, Maliakel B, Krishnakumar IM. Enhanced Bioavailability and Pharmacokinetics of a Natural Self-Emulsifying Reversible Hybrid-Hydrogel System of Quercetin: A Randomized Double-Blinded Comparative Crossover Study. ACS Omega. 2022;7(50):46825\u0026ndash;32. \u003c/li\u003e\n\u003cli\u003eArung ET, Shimizu K, Tanaka H, Kondo R. 3-Prenyl luteolin, a new prenylated flavone with melanin biosynthesis inhibitory activity from wood of Artocarpus heterophyllus. Fitoterapia. 2010;81(6):640\u0026ndash;3. \u003c/li\u003e\n\u003cli\u003eChoi MY, Song HS, Hur HS, Sim SS. Whitening activity of luteolin related to the inhibition of cAMP pathway in \u0026alpha;-MSH-stimulated B16 melanoma cells. Archives of Pharmacal Research. 2008;31(9):1166\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eFujii T, Saito M. Inhibitory effect of quercetin isolated from rose hip (Rosa canina L.) against melanogenesis by mouse melanoma cells. Bioscience, Biotechnology and Biochemistry. 2009;73(9):1989\u0026ndash;93. \u003c/li\u003e\n\u003cli\u003eYamauchi K, Mitsunaga T, Batubara I. Novel quercetin glucosides from Helminthostachys zeylanica root and acceleratory activity of melanin biosynthesis. Journal of Natural Medicines. 2013;67(2):369\u0026ndash;74. \u003c/li\u003e\n\u003cli\u003eYamauchi K, Mitsunaga T, Inagaki M, Suzuki T. Synthesized quercetin derivatives stimulate melanogenesis in B16 melanoma cells by influencing the expression of melanin biosynthesis proteins MITF and p38 MAPK. Bioorganic and Medicinal Chemistry. 2014;22(13):3331\u0026ndash;40. \u003c/li\u003e\n\u003cli\u003eAndo H, Kondoh H, Ichihashi M, Hearing VJ. Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase. Journal of Investigative Dermatology. 2007;127(4):751\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Ischia M, Wakamatsu K, Napolitano A, Briganti S, Garcia-Borron JC, Kovacs D, et al. Melanins and melanogenesis: Methods, standards, protocols. Pigment Cell and Melanoma Research. 2013;26(5):616\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eGillbro JM, Olsson MJ. The melanogenesis and mechanisms of skin‐lightening agents - existing and new approches. International Journal of Cosmetic Science. 2011;33:210\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eXie L, Chen Q, Huang H, Wang H, Zhang R. Inhibitory effects of some flavonoids on the activity of mushroom tyrosinase. Biokhimiya. 2003;68(4):598\u0026ndash;602. \u003c/li\u003e\n\u003cli\u003eCao HH, Tse AKW, Kwan HY, Yu H, Cheng CY, Su T, et al. Quercetin exerts anti-melanoma activities and inhibits STAT3 signaling. Biochemical Pharmacology. 2014;87(3):424\u0026ndash;34. \u003c/li\u003e\n\u003cli\u003eSu TR, Lin JJ, Tsai CC, Huang TK, Yang ZY, Wu MO, et al. Inhibition of melanogenesis by gallic acid: Possible involvement of the PI3K/Akt, MEK/ERK and Wnt/\u0026beta;-catenin signaling pathways in B16F10 cells. International Journal of Molecular Sciences. 2013;14(10):20443\u0026ndash;58. \u003c/li\u003e\n\u003cli\u003eKim YJ. Hyperin and Quercetin Modulate Oxidative Stress-Induced Melanogenesis. Biol Pharm Bull. 2012;35(11):2023\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eBhatti JS, Bhatti GK, Reddy PH. Mitochondrial dysfunction and oxidative stress in metabolic disorders \u0026mdash; A step towards mitochondria based therapeutic strategies. Biochimica et Biophysica Acta - Molecular Basis of Disease. 2017;1863(5):1066\u0026ndash;77. \u003c/li\u003e\n\u003cli\u003eHo HJ, Shirakawa H. Oxidative Stress and Mitochondrial Dysfunction in Chronic Kidney Disease. Cells. 2023;12(1):1\u0026ndash;29. \u003c/li\u003e\n\u003cli\u003eChen J, Liu Y, Zhao Z, Qiu J. Oxidative stress in the skin: Impact and related protection. Vol. 43, International Journal of Cosmetic Science. John Wiley and Sons Inc; 2021. p. 495\u0026ndash;509. \u003c/li\u003e\n\u003cli\u003eTochigi M, Inoue T, Suzuki-Karasaki M, Ochiai T, Ra C, Suzuki-Karasaki Y. Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation. International Journal of Oncology. 2013;42(3):863\u0026ndash;72. \u003c/li\u003e\n\u003cli\u003eLee SW, Kim JH, Song H, Seok JK, Hong SS, Boo YC. Luteolin 7-sulfate attenuates melanin synthesis through inhibition of CREB- and MITF-mediated tyrosinase expression. Antioxidants. 2019;8(4). \u003c/li\u003e\n\u003cli\u003eQi W, Qi W, Xiong D, Long M. Quercetin: Its Antioxidant Mechanism, Antibacterial Properties and Potential Application in Prevention and Control of Toxipathy. Molecules. 2022;27(19). \u003c/li\u003e\n\u003cli\u003eNishimura FDCY, De Almeida AC, Ratti BA, Ueda-Nakamura T, Nakamura CV, Ximenes VF, et al. Antioxidant effects of quercetin and naringenin are associated with impaired neutrophil microbicidal activity. Evidence-based Complementary and Alternative Medicine. 2013;2013. \u003c/li\u003e\n\u003cli\u003eMitsunaga T, Yamauchi K. Effect of quercetin derivatives on melanogenesis stimulation of melanoma cells. Journal of Wood Science. 2015 Aug 7;61(4):351\u0026ndash;63. \u003c/li\u003e\n\u003cli\u003eHa AT, Rahmawati L, You L, Hossain MA, Kim JH, Cho JY. Anti-inflammatory, antioxidant, moisturizing, and antimelanogenesis effects of quercetin 3-o-\u0026beta;-d-glucuronide in human keratinocytes and melanoma cells via activation of nf-\u0026kappa;b and ap-1 pathways. International Journal of Molecular Sciences. 2022 Jan 1;23(1). \u003c/li\u003e\n\u003cli\u003eYan M, Liu Z, Yang H, Li C, Chen H, Liu Y, et al. Luteolin decreases the UVA-induced autophagy of human skin fibroblasts by scavenging ROS. Molecular Medicine Reports. 2016;14(3):1986\u0026ndash;92. \u003c/li\u003e\n\u003cli\u003ede Castilho TS, Matias TB, Nicolini KP, Nicolini J. Study of interaction between metal ions and quercetin. Food Science and Human Wellness. 2018;7(3):215\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eLomozov\u0026aacute; Z, Catapano MC, Hrub\u0026scaron;a M, Karl\u0026iacute;čkov\u0026aacute; J, Mac\u0026aacute;kov\u0026aacute; K, Kučera R, et al. Chelation of Iron and Copper by Quercetin B-Ring Methyl Metabolites, Isorhamnetin and Tamarixetin, and Their Effect on Metal-Based Fenton Chemistry. Journal of Agricultural and Food Chemistry. 2021;69(21):5926\u0026ndash;37. \u003c/li\u003e\n\u003cli\u003eKasprzak M, Erxleben A, Ochocki J. Properties and applications of flavonoid metal complexes. RSC Publishing. 2013;00(1\u0026ndash;3):1\u0026ndash;25. \u003c/li\u003e\n\u003cli\u003eKim JS, Lee EB, Choi JH, Jung J, Jeong UY, Bae UJ, et al. Antioxidant and Immune Stimulating Effects of Allium cepa Skin in the RAW 264.7 Cells and in the C57BL/6 Mouse Immunosuppressed by Cyclophosphamide. Antioxidants. 2023;12(4). \u003c/li\u003e\n\u003cli\u003eRen Q, Qu L, Yuan Y, Wang F. Natural Modulators of Key Signaling Pathways in Skin Inflammageing. Clinical, Cosmetic and Investigational Dermatology . 2024;17(December):2967\u0026ndash;88. \u003c/li\u003e\n\u003cli\u003eMukai R. Prenylation enhances the biological activity of dietary flavonoids by altering their bioavailability. Bioscience, Biotechnology and Biochemistry. 2018;82(2):207\u0026ndash;15.\u003c/li\u003e\n\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":"quercetin related compounds, antimelanogenesis, H2O2, B16 melanoma cells","lastPublishedDoi":"10.21203/rs.3.rs-7328612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7328612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eQuercetin (\u003cstrong\u003ecompound 2\u003c/strong\u003e), isolated from the dried skin of \u003cem\u003eAllium cepa\u003c/em\u003e, and 3-prenyl luteolin (\u003cstrong\u003ecompound 6\u003c/strong\u003e), derived from the wood of \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e, are polyphenolic compounds with demonstrated potential in regulating melanogenesis. This study investigated their anti-melanogenic activity and reactive oxygen species (ROS) scavenging effects, particularly against hydrogen peroxide (H₂O₂), in B16 melanoma cells. Both compounds significantly suppressed melanin synthesis, indicating potential as natural depigmenting agents. Quercetin (\u003cstrong\u003e2\u003c/strong\u003e) exhibited a half-maximal inhibitory concentration (IC₅₀) of 8.0 µg/mL, while 3-prenyl luteolin (\u003cstrong\u003e6\u003c/strong\u003e) showed an IC₅₀ of 20.1 µg/mL. For comparison, compound 1 displayed the strongest activity with an IC₅₀ of 3.0 µg/mL, whereas compounds \u003cstrong\u003e3\u003c/strong\u003e–\u003cstrong\u003e5\u003c/strong\u003e demonstrated no appreciable inhibitory effects. Additionally, both compounds reduced intracellular H₂O₂ levels, supporting their antioxidative role in mitigating oxidative stress-induced melanogenesis. These findings highlight the potential application of quercetin and 3-prenyl luteolin as novel active ingredients in skin-whitening formulations through dual mechanisms of melanin synthesis inhibition and ROS reduction.\u003c/p\u003e","manuscriptTitle":"Antimelanogenesis of quercetin-related compounds in B16 melanoma cells: the difference moieties of C-3 position and its effect on H2O2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-31 16:59:16","doi":"10.21203/rs.3.rs-7328612/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":"3cdff858-cff4-4b20-9bde-d580c2194680","owner":[],"postedDate":"August 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-06T01:08:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-31 16:59:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7328612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7328612","identity":"rs-7328612","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}