Introduction

Melanogenesis, the biological process of melanin synthesis, is intricately regulated by a network of signaling pathways influenced by both intrinsic and extrinsic factors1. Among these, oxidative stress and glycation have emerged as pivotal contributors to aberrant melanin production, leading to hyperpigmentation2,3. Oxidative stress, primarily mediated by reactive oxygen species (ROS), disrupts melanocyte homeostasis through multiple mechanisms4. Ultraviolet B (UVB) radiation and environmental pollutants induce ROS overproduction, which directly activates tyrosinase (TYR), the rate-limiting enzyme in melanin biosynthesis5. Additionally, ROS impair antioxidant defense systems, such as the Nrf2 pathway, further exacerbating oxidative damage and perpetuating melanin overproduction6. Glycation, on the other hand, arises from the non-enzymatic reaction between reducing sugars and proteins, leading to the accumulation of advanced glycation end products (AGEs)7. AGEs bind to their receptor (RAGE) on melanocytes, triggering pro-inflammatory signaling cascades that stimulate TYR expression8. However, current therapeutic strategies often focus on isolated targets—either antioxidants to neutralize ROS or antiglycation agents to inhibit AGE formation9,10. For instance, mono-target antioxidant-based products such as vitamin C serums primarily mitigate oxidative stress but offer limited efficacy against glycation-driven pigmentation11. Conversely, antiglycation-focused treatments may reduce AGE accumulation yet provide insufficient protection against ROS-induced pigmentation12. Therefore, developing therapeutic agents that simultaneously target both oxidative stress and glycation pathways may offer a more effective strategy to counteract hyperpigmentation.

Recent advances in dermatological research underscore the promise of combining antioxidant and antiglycation agents to address the multifactorial nature of melanogenesis. Ergothioneine, a naturally occurring amino acid and thiol antioxidant, exhibits superior antioxidant capacity through selective cellular uptake and ROS scavenging13. Tocopheryl glucoside is a stabilized vitamin E prodrug that is hydrolyzed by skin β-glucosidases to release active δ-tocopherol, thereby providing prolonged antioxidant protection against lipid peroxidation14. Decarboxy carnosine, an antiglycation agent and modified dipeptide, effectively traps reactive carbonyl species to prevent AGE formation15. Naringin, a citrus flavonoid, inhibits glycation by blocking lysine/arginine residues in human serum albumin, reducing AGE formation16. Thus, combining these antioxidant and antiglycation compounds may synergistically suppress melanogenesis by dual targeting oxidative stress and glycation, providing a more effective intervention for hyperpigmentation.

To date, there has been no systematic evaluation of a formulation designed to co-target oxidative stress and glycation within the integrated biological framework. Therefore, we hypothesized that a dual-targeting composition—simultaneously enhancing antioxidant defenses and inhibiting glycation—would more effectively suppress melanogenesis than single-target interventions. This study aims to develop and evaluate a novel dual-effect composition (DEC) that integrates antioxidant agents (ergothioneine and tocopheryl glucoside) and antiglycation agents (decarboxy carnosine and naringin). A multi-model approach was employed, incorporating UVB-induced and AGE-induced in vitro melanocyte models, a pigmented full-thickness human skin explant system, and a randomized clinical trial. This comprehensive strategy was utilized to systematically assess the efficacy of DEC in simultaneously neutralizing oxidative stress, inhibiting glycation-associated damage, and attenuating melanogenesis. By targeting both oxidative stress and glycation, this study provides a new approach for managing hyperpigmentation and demonstrates the potential of multifunctional formulations in dermatological and cosmetic applications.

Materials and methods

Samples

The dual-effect composition (DEC) was formulated by blending 4.53% ergothioneine, 0.3% tocopheryl glucoside, 5.94% decarboxy carnosine, and 11.76% naringin, and stored at 4℃. An oil-in-water serum incorporating DEC was prepared for clinical evaluation. The DEC-containing serum exhibited excellent pH stability, with a minimal deviation (∆pH ≤ 0.29) after 12 weeks of storage at 25℃.

In vitro study

Cell culture and cytotoxicity assay

Human melanoma A875 cell line (BeNa Culture Collection, BNCC, China) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. For cytotoxicity assessment, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and allowed to adhere overnight. Cells were then treated with serial dilutions of DEC (0.0078, 0.0157, 0.0313, 0.0625, 0.125, 0.25, 0.5 and 1%, v/v) or 10% PBS (control) for 24 h. Following treatment, MTT solution (0.5 mg/mL, Sigma, USA) was added to each well and incubated for 4 h at 37 °C. The formed MTT–formazan crystals were dissolved in DMSO and absorbance was measured at 490 nm using a Spark multimode microplate reader (Tecan, Switzerland). Cell viability was calculated as percentage relative to untreated controls.

Cell model

A875 cells were treated for 2 h with either a positive control (PC) or DEC at concentrations of 0.0157%, 0.0313%, and 0.0625% (v/v). For UVB-induced oxidative stress model, the PC consisted of 110 µg/mL vitamin C (VC) and 7 µg/mL vitamin E (VE), followed by UVB irradiation at 300 mJ/cm². For AGE-modified bovine serum albumin (AGE-BSA; Amyjet Scientific, China)-induced glycation model, the PC contained 10 mM aminoguanidine hydrochloride, followed by treatment with 200 ng/mL AGE-BSA. In both conditions, cells were then incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO₂. Blank control (BC) cells received an equal volume of ultrapure water instead of any treatment, with no UVB exposure applied.

3D pigmented skin model

The MelaFulKutis™ 3D pigmented full-thickness human skin models (Guangdong BioCell Biotech) were cultured in 6-well plates (2 mL medium/well) and randomly divided into six groups with three replicates each: blank control (BC, fresh medium), NC (3 mM methylglyoxal [MGO]-containing medium), three positive controls (PC1: 3 mM MGO + aminoguanidine sulfate; PC2: 3 mM MGO + VC + VE; PC3: 3 mM MGO + kojic acid), and the experimental group (3 mM MGO + DEC-containing serum). After two days of cultivation, treatments were administered, followed by 24 h incubation (37 °C, 5% CO₂) and UVA/UVB irradiation (12 J/cm² UVA + 50 mJ/cm² UVB). This daily treatment-irradiation cycle was repeated for seven days, with fluorescence probes added 1 h before the final irradiation. After the last irradiation, samples were collected, washed with PBS, and dried with sterile cotton swabs.

Measurement of intracellular Nrf2 and SOD

The levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and superoxide dismutase (SOD) in A875 cells were quantified using commercial ELISA kits (mlbio, China), following the manufacturer’s protocol.

Measurement of intracellular ROS

Intracellular ROS levels in A875 cells were measured using DCFH-DA (ROS Assay Kit, Beyotime, China). Cells were seeded in a 24-well plate (2 × 10⁵ cells/well) and cultured for 24 h. The culture medium was replaced with serum-free DMEM containing 10 µM DCFH-DA (diluted 1:1000 from stock), followed by incubation at 37 °C for 30 min in the dark. Cells were then washed three times with PBS, and fluorescence intensity was measured by Image pro-Plus 6.0 (IPP 6.0). Images were observed under a fluorescence microscope (BZ-X810, Keyence, USA).

Fluorescence staining

Following incubation, skin models were excised, fixed in 4% paraformaldehyde (light-protected with aluminum foil), and cryosectioned. Sections were PBS-washed, after which selected models underwent repeat excision and fixation to prepare additional cryosections. After an 8-min PBS wash, sections were mounted with anti-fade medium (20 µL/section) and coverslipped. Fluorescence imaging was performed using a laser confocal fluorescence microscope.

Immunofluorescence staining

For cellular staining, A875 cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 2% BSA for 1 h, cells were incubated with primary antibodies against AGER/RAGE polyclonal antibody (1:200; 16346-1-AP, Proteintech, China) and anti-carboxymethyl lysine (CML, 1:200; ab125145, Abcam, UK) overnight at 4 °C. Following PBS washes, cells were incubated with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (1:500; ab150077, Abcam) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (1:500; ab150115, Abcam) for 1 h. Nuclei were counterstained with DAPI (Sigma). Coverslips were mounted with ProLong Gold Antifade Mountant (Invitrogen) and imaged using a fluorescence microscope (BZ-X810, Keyence). Image analysis was performed using ImageJ (NIH) with threshold-adjusted fluorescence intensity quantification.

For tissue staining, tissue specimens were fixed in 4% paraformaldehyde for 24 h, paraffin-embedded, and sectioned (5 μm). Sections were baked at 70 °C for 4 h, deparaffinized in xylene twice for 10 min each and graded ethanol (100%−75%, 5 min each), then washed in PBS thrice for 5 min each. Antigen retrieval was performed using 0.01 M sodium citrate buffer (pH 6.0) under high pressure. Endogenous peroxidase was blocked with 3% H₂O₂ for 30 min at room emperature, followed by blocking with homologous serum for 1 h at 37 °C. Primary antibodies were applied overnight at 4 °C: Nrf2 (1:300; 23013625, Proteintech), SOD (1:100; 1010382-1, Abcam), GSH-Px1 (1:100; A06AB903, Abcam), GLO-1 (1:100; 1127788-13, Abcam), CML (1:50; 1090190-1, Abcam), TYR (1:100; 3AE44A46, Invitrogen), and MITF (1:200; ANTIB0DY, Invitrogen). After PBS washes three times for 5 min each, secondary antibody (1:500) was applied for 1 h at room temperature. Sections were counterstained with 100 µL Hoechst for 5 min, mounted, and imaged by a laser confocal fluorescence microscope.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from A875 cells and skin tissue samples using the RNA-easy Isolation Reagent (Vazyme, China) and AG RNAex Pro Reagent (Accurate Biology), respectively. RNA was reverse-transcribed into cDNA with HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) on the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, USA) with the following primers: TYR (F: 5’-ACT GGG ATA GCG GAT GCC TCT-3’, R: 5’-GCC ACT GCT CAA AAA TAC TGT CA-3’), RAGE (F: 5’-GAC TCT TAG CTG GCA CTT GGA T-3’, R: 5’-GTC TCC TGG TCT GTT CCT TCA C-3’), and GAPDH (F: 5’-GGA AGC TTG TCA TCA ATG GAA ATC-3’, R: 5’-TGA TGA CCC TTT TGG CTC CC-3’). GAPDH served as the endogenous control for normalization of mRNA quantification. Reactions were conducted in triplicate under standard cycling conditions (95 °C for 3 min, 45 cycles of 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s). Relative expression was calculated using the 2−ΔΔCt method.

Measurement of melanin content

A875 cells were seeded in 24-well plates (2 × 10⁵ cells/well) and incubated for 24 h. Cells were centrifuged and the supernatant was removed. The pellet was dissolved in 0.5 mL of 1 M NaOH (containing 10% DMSO) at 80 °C for 1 h to solubilize melanin. The absorbance was measured at 405 nm using a Spark multimode microplate reader (Tecan).

Masson-Fontana staining

Skin tissue samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned. The sections were deparaffinized with xylene I and II for 8 min each, rehydrated through a graded ethanol series (absolute ethanol I and II for 6 min each, 95%, 85%, and 75% ethanol for 6, 6, and 5 min, respectively), and rinsed with distilled water. Melanin was stained using Fontana-Masson ammonia silver solution, followed by incubation at 37 °C for 1 h. Sodium thiosulfate solution was applied for 5 min, and the sections were washed twice with distilled water. Counterstaining was performed with neutral red for 15 min. Excess dye was removed by immersing in 95% ethanol for 10 s and absolute ethanol II for 5 s. Finally, the sections were cleared in xylene I, II, and III for 5 min each, mounted, and imaged under a microscope.

Apparent chromaticity evaluation

Skin specimens were observed under a stereomicroscope. The field of view was carefully adjusted to ensure optimal clarity and complete specimen visualization. Images were then captured for apparent chromaticity analysis. Skin lightness was assessed by measuring the L* value using a colorimeter. Briefly, each skin model was placed on a flat, rigid white surface with the stratum corneum oriented upward. The instrument probe was positioned perpendicular to the surface of the model to ensure consistent contact and optimal alignment. Three replicate measurements were taken at different sites on each model, and the mean value was calculated and recorded as the representative L* value for that model.

Statistical analysis

All in vitro experiments were performed in triplicate, and data were expressed as mean ± standard deviation (SD). Differences between groups were analyzed using one-way ANOVA followed by Tukey’s post hoc test (cell experiments) or two-tailed Student’s t-test (skin model experiments). Statistical analyses were conducted using the GraphPad Prism software (version 8.3.0, GraphPad Program, USA). The statistical significance was set at a threshold of P < 0.05.

Clinical study

Ethical approval

The clinical study was reviewed and approved by the SGS Ethics Committee for Clinical Research (SS-2024-073) and complied with the tenets of the Declaration of Helsinki. All subjects provided informed consent to participate in the clinical trial.

Antioxidant efficacy evaluation

The inclusion criteria were as follows: (1) Healthy, aged between 18 and 60 years; (2) The Individual Type Angle (ITA°) of the skin color at the test site should be no less than 28°; (3) No history of allergic diseases, and no history of allergy to cosmetics or other topical preparations; (4) No history of photosensitive diseases, and no recent use of drugs that affect photosensitivity; (5) The skin at the test site should be free from phenomena such as pigmentation, inflammation, scars, pigmented moles, and hirsutism; (6) Those who can accept the irradiation of the skin in the test area with artificial light sources; (7) Those who can understand the test process, voluntarily participate in the test, and sign a written informed consent form.

The exclusion criteria were as follows: (1) Pregnant or lactating women, or those with a recent plan to conceive; (2) Those with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, severe acne, or those with other chronic systemic diseases; (3) Those who have taken or applied anti-inflammatory drugs such as corticosteroids orally or topically within the last month; (4) Those who have used antioxidant samples or drugs within the last three months; (5) Those who have participated in a similar test within the last three months or those who participated in a similar test more than three months ago but the erythema or darkening marks on the test - site skin have not completely faded; (6) Those who have participated in a cosmetic clinical trial within the last month; (7) Those who are considered unsuitable for the trial by other clinical evaluations.

A total of 20 healthy Asian female subjects aged 28 to 59 years (mean age: 40.2 ± 8.3 years) were enrolled and completed the antioxidant efficacy evaluation study.

Three test sites (5 cm × 6 cm each) were delineated on the upper back of each subject and randomly assigned to one of three treatment groups: a pre-minimal erythema dose (Pre-MED) area for MED determination, a sample-treated area, and a negative control (NC) area. The MED was defined as the lowest UV radiation dose or shortest exposure time that induced clearly visible erythema encompassing more than 50% of the irradiated area and extending to its borders. The MED was determined by pre-irradiation and assessed visually 16–24 h post-exposure. The DEC-containing serum was applied once daily to the designated sample-treated area at a dosage of 2.0 ± 0.1 mg/cm² for four consecutive days. On Day 4, the final application was performed 30 min prior to irradiation. Three UV exposure levels—1×MED, 2×MED, and 3×MED—were administered to all areas. Non-invasive measurements and expert visual assessments of erythema were conducted before product application and 16–24 h after the final irradiation.

The following instrumentation was used for objective assessment of skin erythema: a UV Solar Simulator Model 601 − 300 V2.5 (Solar Light Company, USA) was employed for controlled UV exposure; skin redness was quantified using a Konica Minolta CM-26dG spectrophotometer (Konica Minolta, Japan) for a* value measurement, and a Mexameter® MX18 (Courage + Khazaka, Germany) was used to determine the erythema index (EI). Lower values of both a* and EI indicate reduced erythema severity. In addition, trained evaluators performed standardized visual grading of erythema using a 0.0–7.0 scale with 0.5-point increments: 0.0 = no visible erythema; 1.0 = questionable erythema; 2.0 = faint erythema; 3.0 = indistinct margins with > 50% area involvement; 4.0 = clear pale-red erythema; 5.0 = pronounced erythema; 6.0 = intensified erythema with possible mild edema; 7.0 = dark red erythema with edema or vesicles, potentially extending beyond the test site.

Anti-glycation and skin brightening evaluation

The inclusion criteria were as follows: (1) Healthy, aged 18–60 years; (2) baseline facial skin sallowness score of ≥ 3, as determined by visual assessment; (3) No history of allergic diseases, no history of allergy to cosmetics or other topical preparations; (4) No history of photosensitive diseases, and no use of drugs affecting photosensitivity recently; (5) The skin at the test site should have no phenomena such as pigmentation, inflammation, scars, pigmented moles, and hirsutism; (6) Those who can understand the test process, voluntarily participate in the test, and sign a written informed consent form.

The exclusion criteria were as follows: (1) Those with a daily habit of taking health supplements such as carotenoids; (2) Pregnant or lactating women, or those with a recent plan to conceive; (3) Those with a history of skin diseases such as psoriasis, eczema, atopic dermatitis, severe acne, or those with other chronic systemic diseases; (4) Those who have taken or applied anti-inflammatory drugs such as corticosteroids orally or topically within the last month; (5) Those who have used anti - glycation products or drugs within the last three months; (6) Those who have used retinoid preparations or undergone medical aesthetic treatments such as chemical peeling, laser, or pulsed light on the test site within three months; (7) Those who have participated in a cosmetic clinical trial within the last month; (8) Those who are considered unsuitable for the trial by other clinical evaluations.

A total of 34 healthy Asian participants aged 31 to 60 years (mean age: 50.5 ± 7.5 years) were enrolled and completed the anti-glycation and skin brightening evaluation study.

Subjects applied the test serum twice daily to the entire face for a consecutive period of 56 days. Efficacy assessments were conducted at baseline, Day 28, and Day 56. Facial images were captured using the VISIA-CR imaging system (Canfield Scientific, USA). Skin autofluorescence (SAF) was measured using the AGE Reader (Diagnoptics, The Netherlands); lower SAF values indicate reduced levels of cutaneous glycation. Skin color parameters—including L* (lightness), b* (yellowness), and ITA°—were evaluated using the Colorimeter® CL400 (Courage + Khazaka, Germany). Improvements in skin tone are reflected by increases in L* and ITA° values and a decrease in b* value. In addition, facial sallowness was assessed visually by trained evaluators using a 0–9 severity scale with 0.5-point increments: 0 = no visible sallowness; 1–3 = mild; 4–6 = moderate; 7–9 = severe.

Consumer satisfaction survey

A 56-day consumer use test was conducted to evaluate subjective satisfaction with the product. A total of 32 participants, aged 20 to 50 years (mean age: 36.5 ± 9.6), were enrolled. Upon completion of the test period, participants completed a structured self-assessment questionnaire based on a 5-point scale, where 1 indicated “very dissatisfied” and 5 indicated “very satisfied.” The overall satisfaction rate was calculated as follows: Satisfaction (%) = (Number of participants scoring > 3/Total number of participants) × 100%.

Statistical analysis

Instrumental data were analyzed using IBM SPSS Statistics software. The Shapiro–Wilk test was employed to evaluate the normality of the data distribution. Paired t-tests were performed for datasets that exhibited a normal distribution. Wilcoxon signed-rank test was utilized for non-normally distributed data or ordinal data. A P-value of less than 0.05 (α = 0.05) was considered indicative of statistical significance for all analyses conducted.

Results

Cytotoxicity of DEC

The cytotoxicity of DEC was evaluated in A875 melanoma cells at concentrations ranging from 0.0078% to 1% (v/v). The result demonstrated the safety range up to 0.0625% (v/v) (Fig. 1A), at which more than 90% of cells survived17. Therefore, we selected three concentrations of 0.0157, 0.0313, and 0.0625% (v/v) in the subsequent experiments.

Effect of DEC on UVB-induced oxidative stress and melanogenesis in A875 melanoma cells

To evaluate the antioxidative effects of DEC, A875 were treated with DEC and then exposed to UVB irradiation. Results showed that UVB irradiation induced oxidative stress in A875 cells, as indicated by significantly reduced Nrf2 and SOD levels along with elevated ROS (P < 0.01). Treatment with DEC effectively reversed these effects, normalizing Nrf2 and SOD levels and reducing ROS (P < 0.01) (Fig. 1B-E). Moreover, UVB irradiation markedly enhanced both melanin synthesis and TYR expression in A875 cells, whereas DEC treatment effectively suppressed these UVB-induced effects (P < 0.01) (Fig. 1F, G). These findings indicate that DEC could protect human melanocyte from UVB-induced oxidative stress and melanogenesis.

Effect of DEC on AGE-induced glycation damage and melanogenesis in A875 melanoma cells

To assess the protective effects of DEC against glycation, A875 were treated with DEC and AGE-BSA. Exposure to AGE-BSA induced significant glycation damage, as demonstrated by elevated levels of RAGE and CML (P < 0.01). DEC treatment effectively suppressed these glycation markers, restoring them to near-baseline levels (P < 0.01) (Fig. 2A-D). Furthermore, AGE-BSA stimulation significantly increased melanin accumulation and upregulated TYR expression, whereas DEC treatment markedly reduced melanin content and downregulated TYR levels (P < 0.01) (Fig. 2E, F). These findings suggest that DEC mitigates AGE-BSA-induced glycation and its downstream effects on melanogenesis in human melanocytes.

The antioxidant effect of DEC-containing serum on pigmented full-thickness human skin model

To evaluate the antioxidant potential of the DEC-containing serum under oxidative stress induced by MGO and UV irradiation, we analyzed ROS levels using fluorescence staining and measured key antioxidant markers—including Nrf2, SOD, and GSH-Px1—through semi-quantitative immunofluorescence in a pigmented full-thickness human skin model. The DEC-containing serum significantly reduced intracellular ROS levels compared to the negative control group (P < 0.01, Fig. 3A). Fluorescence intensity of Nrf2 was markedly increased in the DEC-containing serum-treated group (P < 0.01, Fig. 3B), indicating activation of the Nrf2-mediated antioxidant defense pathway. Similarly, fluorescence signals for SOD and GSH-Px1 were also significantly enhanced in the DEC-containing serum group (P < 0.01, Figs. 3C, D), suggesting improved antioxidant enzyme activity. It is noteworthy that the DEC-containing serum demonstrated a more potent efficacy in both ROS suppression and SOD elevation compared to the PC (VC and VE) (P < 0.05, Figs. 3A, C).

The anti-glycation effect of DEC-containing serum on pigmented full-thickness human skin model

Exposure of the skin model to MGO and UV irradiation induced a marked upregulation of CML, a well-characterized biomarker for AGEs formation18. Treatment with DEC-containing serum significantly suppressed CML accumulation (P < 0.01, Fig. 4A, B). To further investigate the DEC-containing serum’s mechanism of action, we examined its effect on RAGE using qRT-PCR. The results demonstrated that MGO and UV exposure significantly elevated RAGE mRNA expression levels; however, treatment with the DEC-containing serum effectively downregulated RAGE transcription (P < 0.01, Fig. 4C). The inhibitory effect of the DEC-containing serum on RAGE expression was significantly greater than that of the PC (aminoguanidine sulfate) (P < 0.01, Fig. 4C). These findings suggest that the DEC-containing serum exerts its anti-glycation effects by attenuating CML deposition and suppressing RAGE expression, thereby protecting against glycation-induced skin damage.

Effect of DEC-containing serum on melanogenesis in a pigmented full-thickness human skin model

In a pigmented full-thickness human skin model subjected to combined MGO and UV stimulation, immunofluorescence analysis was performed. The results showed that the DEC-containing serum significantly reduced the fluorescence intensities of MITF and TYR by 63.37% and 80.39%, respectively, compared to the NC group (P < 0.01, Fig. 5A, B). These findings suggest that the DEC-containing serum effectively suppresses the expression of key regulatory proteins involved in melanin synthesis. Further support was provided by Masson-Fontana staining. In the NC group, histological analysis revealed a dense accumulation of melanin granules throughout the epidermis, resulting in pronounced dark pigmentation. DEC-containing serum treatment exhibited a significant reduction in the number of melanin granules per unit area of skin tissue (P < 0.01, Fig. 5C). The DEC-containing serum demonstrated significantly greater efficacy in reducing both TYR expression and melanin content compared to PC (kojic acid) (P < 0.01, Figs. 5B, C). Representative chromaticity images showed visibly lighter skin in the DEC-containing serum treatment group, which was quantitatively confirmed by a significant increase in the L* value compared to NC (P < 0.01, Fig. 5D). These results indicate that the DEC-containing serum significantly inhibits melanin production and deposition, thereby attenuating skin pigmentation through the downregulation of MITF and TYR expression.

Effect of DEC-containing serum on skin brightness and susceptibility to UV-induced erythema

As shown in Fig. 6A, DEC-containing serum significantly attenuated UV-induced skin responses across all three MED irradiation intensities (1×, 2×, and 3×MED) when compared to negative controls (P < 0.05). The most pronounced photoprotective effect was observed at 1×MED irradiation, with reductions of 60.32% in Δa* value, 64.47% in ΔEI, and 76.92% in Δerythema grade (ΔEG) value (P < 0.05). These quantitative findings were corroborated by visual assessments (Fig. 6B), which clearly showed diminished UV-induced erythema. These findings demonstrate that the DEC-containing serum effectively protects against UV-induced damage and possesses antioxidant properties.

As shown in Table 1, continuous application of DEC-containing serum led to a statistically significant and progressive improvement in skin dullness and sallowness. After 8 weeks (56 days), the ITA° value, an objective measure of skin brightness, increased by 8.18% (P < 0.001), while the SAF value, which reflects levels of AGEs, decreased by 14.23% (P < 0.001). Expert visual assessments indicated an 11.38% reduction in facial sallowness (P < 0.001). These results suggest that the DEC-containing serum effectively reduces skin glycation and enhances overall skin luminosity and clarity.

In the 56-day consumer use study, 97% of participants reported improvements in skin radiance, 97% noted a more even skin tone, and 97% perceived reduced sallowness (Table 2).

Discussion

Melanogenesis is a complex process regulated by oxidative and glycation pathways, both of which are activated by environmental stressors such as UV radiation8,19. Current therapies for hyperpigmentation typically target either ROS neutralization or AGEs inhibition, but often overlook the synergistic interaction between these pathways3,20. This study presents a novel DEC that concurrently targets oxidative stress (via ergothioneine and tocopheryl glucoside) and glycation (via decarboxy carnosine and naringin), demonstrating its efficacy in suppressing melanogenesis across clinical, cellular, and 3D skin models. Our findings revealed that DEC not only attenuated UV-induced erythema and glycation-associated skin damage but also suppressed melanin synthesis by downregulating MITF and TYR expression, thereby reducing melanin deposition in pigmented skin models. These results demonstrate that DEC possesses dual antioxidant and anti-glycation activity, offering a promising therapeutic strategy for hyperpigmentation.

The mechanistic superiority of DEC stems from its dual modulation of oxidative and glycation stress. In UVB-exposed melanocytes, DEC restored Nrf2-mediated antioxidant defenses, significantly increasing the levels of SOD and GSH-Px1 while reducing ROS level. The antioxidant activity of DEC is largely attributed to ergothioneine, which effectively scavenges ROS in a thiol-dependent manner21. In parallel, tocopheryl glucoside, a stable, water-soluble derivative of vitamin E, contributes to sustained cellular antioxidant defense by promoting the regeneration of intracellular vitamin E14. By reinforcing endogenous antioxidant defenses, DEC suppresses the ROS-driven melanocyte activation. Simultaneously, DEC effectively suppressed AGE-BSA-induced RAGE overexpression and CML accumulation, interrupting the AGE-RAGE signaling axis that drives pro-inflammatory activation of TYR22. The anti-glycation effect is primarily attributed to decarboxy carnosine, which blocks AGE cross-linking reactions and protects target proteins from modification23. Meanwhile, naringin suppresses NF-κB-mediated inflammation downstream of RAGE activation, thereby modulating the associated pro-inflammatory signaling24. DEC exhibited superior therapeutic potential due to the coordinated action of its components, resulting in a marked 76.92% decrease in UV-induced erythema and an 8.18% increase in skin brightness.

The 3D pigmented skin model provided valuable insights into the translational potential of DEC by recapitulating the complex architecture of the epidermis and the functional interactions between melanocytes and keratinocytes, thereby offering a more physiologically relevant system25. Under conditions of oxidative stress and glycation induced by MGO and UV irradiation, DEC-containing serum significantly suppressed melanin synthesis and downregulated key melanogenic regulators, including MITF and TYR. These in vitro findings were corroborated by clinical observations, where subjects treated with DEC-containing serum exhibited significant improvements in skin lightness and reduced yellowness, with a high participant satisfaction rate of 97%. Furthermore, the model confirmed the potent antioxidant capacity of DEC, as evidenced by reduced ROS levels and upregulated Nrf2 expression, suggesting effective dermal penetration and activation of endogenous protective mechanisms.

These molecular and clinical benefits position DEC as a promising alternative to conventional depigmenting agents like hydroquinone and arbutin, which may carry risks of cytotoxicity or limited efficacy26. By simultaneously targeting upstream stressors (ROS and AGEs) instead of solely inhibiting tyrosinase activity, DEC addresses the key drivers of hyperpigmentation with a favorable safety profile. However, this study has several limitations. The clinical cohort, consisting exclusively of Asian females with moderate pigmentation, may not fully represent broader demographic variability, particularly individuals with higher Fitzpatrick skin types or severe hyperpigmentation. Furthermore, while the 3D skin model effectively recapitulates epidermal melanin dynamics, its lack of immune and vascular components may underestimate in vivo inflammatory cross-talk between glycation and oxidative stress. Future studies are necessary to validate DEC in diverse populations and assess its impact on secondary glycation markers to confirm its broad-spectrum antiglycation capacity and potential for wider dermatological applications.

Conclusion

This study demonstrates that DEC comprising ergothioneine, tocopheryl glucoside, decarboxy carnosine, and naringin, effectively inhibits melanogenesis by simultaneously targeting oxidative stress and glycation (Fig. 7). In vitro experiments revealed that DEC significantly reduced intracellular ROS levels and enhanced Nrf2-mediated antioxidant defenses in melanocytes, while suppressing AGE-RAGE signaling. DEC-containing serum significantly reduced melanin deposition in pigmented skin models and improved clinical outcomes, including enhanced skin lightness and reduced sallowness, with a high participant satisfaction rate (97%). The dual-action mechanism of DEC not only attenuates UV-induced erythema and glycation-associated damage but also enhances endogenous antioxidant defenses. From a practical standpoint, the use of stable, commercially available ingredients supports robust and scalable formulation development. The synergistic action of DEC may allow lower concentrations of individual components, potentially improving cost-effectiveness compared to multi-ingredient regimens or high-dose monotherapies. These findings underscore the potential of dual-targeting strategies in managing hyperpigmentation and highlight the importance of comprehensive approaches in dermatological treatments.

Fig. 1
figure 1

Effect of dual-effect composition (DEC) on UVB-induced oxidative stress and melanogenesis in A875 melanoma cells. (A) Cytotoxicity of DEC in A875 cells. (B, C) Nrf2 and HO-1 protein levels were measured by ELISA. (D, E) Fluorescence staining and quantification for reactive oxygen species (ROS). Scale bar = 50 μm. (F) Relative melanin contents in A875 cells. (G) qRT-PCR analysis of TYR mRNA expression levels. Data are expressed as the mean ± SD (n = 3). ##P < 0.01 vs. blank control (BC). **P < 0.01 vs. UVB. Positive control (PC) was treated with a combination of vitamin C and vitamin E.

Full size image
Fig. 2
figure 2

Effect of dual-effect composition (DEC) on AGE-induced glycation damage and melanogenesis in A875 melanoma cells. (A, C) Immunofluorescence staining and quantification for RAGE. (B, D) Immunofluorescence staining and quantification for CML. Scale bar = 50 μm. (E) Relative melanin contents in A875 cells. (F) qRT-PCR analysis of TYR mRNA expression levels. Data are expressed as the mean ± SD (n = 3). ##P < 0.01 vs. blank control (BC). *P < 0.05 and **P < 0.01 vs. AGE-BSA. Positive control (PC) was treated with aminoguanidine hydrochloride.

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Fig. 3
figure 3

Antioxidant effects of dual-effect composition (DEC)-containing serum in a pigmented full-thickness human skin model exposed to methylglyoxal (MGO) and UV irradiation. (A) Fluorescence staining for reactive oxygen species (ROS). Immunofluorescence staining for (B) nuclear factor erythroid 2-related factor 2 (Nrf2), (C) superoxide dismutase (SOD), and (D) glutathione peroxidase 1 (GSH-Px1). Scale bar = 50 μm. Data are expressed as the mean ± SD (n = 3). ##P < 0.01 vs. blank control (BC; untreated baseline). **P < 0.01 vs. negative control (NC; MGO + UV irradiation). ^P < 0.05 and ^^P < 0.01; ns, not significant (P > 0.05). Positive control (PC) was treated with a combination of vitamin C and vitamin E.

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Fig. 4
figure 4

Anti-glycation effects of dual-effect composition (DEC)-containing serum on a pigmented full-thickness human skin model exposed to methylglyoxal (MGO) and UV irradiation. (A, B) Immunofluorescence staining of N-carboxymethyllysine (CML) expression. Scale bar = 50 μm. (C) qRT-PCR analysis of RAGE mRNA expression levels. Data are expressed as the mean ± SD (n = 3). ##P < 0.01 vs. blank control (BC; untreated baseline). and **P < 0.01 vs. negative control (NC; MGO + UV irradiation). ^^P < 0.01; ns, not significant (P > 0.05). Positive control (PC) was treated with aminoguanidine sulfate.

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Fig. 5
figure 5

Effect of dual-effect composition (DEC)-containing serum on melanogenesis in a pigmented full-thickness human skin model exposed to methylglyoxal (MGO) and UV irradiation. Immunofluorescence staining analysis of (A) MITF and (B) TYR expression. (C) Masson-Fontana staining for the qualitative assessment of melanin content. Scale bar = 50 μm. (D) Apparent chromaticity images of skin models. Data are expressed as the mean ± SD (n = 3). ##P < 0.01 vs. blank control (BC; untreated baseline). *P < 0.05 and **P < 0.01 vs. negative control (NC; MGO + UV irradiation). ^^P < 0.01; ns, not significant (P > 0.05). Positive control (PC) was treated with kojic acid.

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Fig. 6
figure 6

Evaluation of dual-effect composition (DEC)-containing serum in modulating cutaneous erythema responses to UV irradiation at varying intensities. (A) Δa* (skin redness), ΔEI (erythema index), and ΔEG (erythema grade) values were quantified. Δvalue = post-irradiation measurement after 4 days of serum application − baseline measurement before serum application. Change (%) = (Δvalueserum − ΔvalueNC)/ΔvalueNC × 100%. *P < 0.05 vs. negative control (NC). (B) Clinical photograph demonstrating photoprotective effects against UV-induced erythema in a 34-year-old female subject (upper back).

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Fig. 7
figure 7

A schematic picture illustrating the inhibition of melanogenesis by dual-effect composition (DEC) via a dual-targeting antioxidant and anti-glycation strategy. Red arrows represent the regulatory molecular mechanisms of DEC; black arrows indicate the previously reported molecular mechanisms.

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Table 1 Changes in skin brightness and glycation-related parameters after dual-effect composition (DEC)-containing serum application.
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Table 2 Results of the consumer questionnaire satisfaction survey.
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