Natural dual inhibitor isorhamnetin-3-O-neohespeidoside targets tyrosinase and MC1R for skin pigmentation management

Abstract

Skin pigmentation disorders involve complex biological regulation, with tyrosinase (TYR) and melanocortin 1 receptor (MC1R) serving as key therapeutic targets. Through molecular docking screening of 389 natural compounds, we identified isorhamnetin-3-O-neohespeidoside as a potent dual inhibitor, demonstrating superior binding affinities (-8.001 kcal/mol for TYR and − 7.342 kcal/mol for MC1R) compared to arbutin (reference compound). Subsequent in vitro validation revealed that isorhamnetin-3-O-neohespeidoside (8 µM) significantly inhibited TYR activity by 44.42% (p < 0.0001) and reduced MC1R expression by 33.39% (p < 0.0001) in B16 melanoma cells, while maintaining > 85% cell viability (IC₅₀ = 52.22 µM). The compound also decreased melanin content by 38.7% (p < 0.0001) and upregulated LC3-II expression (2.1-fold vs. control, p < 0.0001), indicating enhanced autophagy. These results demonstrate that isorhamnetin-3-O-neohespeidoside, a flavonoid glycoside from Typhae Pollen, acts through multiple mechanisms - direct enzyme inhibition, receptor downregulation, and autophagy induction - making it a promising natural candidate for hyperpigmentation treatment. Our integrated approach combining computational screening with experimental validation provides a robust framework for identifying multi-target depigmenting agents.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-08485-2.

Introduction

Melanocytes (dendritic cells derived from the neural crest) and keratinocytes in the skin are the cells most involved in melanin production and transport¹. The epidermal melanin unit consists of one melanocyte and multiple adjacent keratinocytes (approximately 36), which cooperate to produce and distribute melanin². Melanin is synthesized in the melanosomes, the organelles of melanocytes, then transferred to neighboring keratinocytes through dendrites of melanocytes, and then the keratinocytes transport it to the skin surface, and the skin regulates the change of skin color through the generation and degradation of melanin^3–5. Molecular pathways of melanin production include Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, melanocortin 1 receptor (MC1R)/α-melanocyte-stimulating hormone (α-MSH) signaling pathway, mitogen-activated protein kinases (MAPK) signaling pathway, Endothelin 1 (EDN1) mediated signaling cascade and wingless-related integration site(Wnt)/β-Catenin signaling pathway. The molecular pathways mentioned above regulate melanin synthesis and degradation within melanocytes to maintain melanin balance in the skin, among which MC1R/α-MSH is one of the most important signaling pathways.

In the above MC1R/α-MSH signaling pathway, MC1R is the central determinant of pigmentation, while tyrosinase is a key enzyme in melanin synthesis. G-protein-coupled receptor, MC1R, one of the most important regulators of skin pigmentation, is expressed primarily on the cell membrane of melanocytes^6,7. After binding with endogenous melanocortin receptor agonist α-MSH, MC1R activates adenylate cyclase and then stimulates the production of cAMP, which further activates downstream PKA signaling. The activation of PKA stimulates the phosphorylation of cAMP response element binding protein (CREB) and MITF, and the expression of MITF increases and stimulates the transcription of TYR. Finally, melanin synthesis and melanosome transport were increased^7–9. Tyrosinase is a rate-limiting enzyme that regulates the synthesis of melanin. Under the action of tyrosinase, tyrosine is transformed into dopa, and then through a series of steps, it is finally transformed into melanin. After the activation of the molecular pathway of melanin synthesis, melanocyte related proteins such as TYR and tyrosinase-related protein 1/2 (TRP-1/2) are transcribed and translated, and at the same time, melanosome is formed in the cell. During the maturation of melanosome, TYR, TRP-1, TRP-2 and other enzymes and related complexes synthesized by lysosomes are selectively transported to melanosome, jointly promoting the production of melanin in melanosome^10,11.

With the spread of skincare knowledge on social media and the Internet, consumers are gradually becoming more aware of the risks of ingredients used in cosmetics, while also expecting whitening agents that are naturally sourced and have low side effects. Whitening agents medically applied to effectively lighten the skin tone of hyperpigmented lesions include corticosteroids, tretinoin and hydroquinone. With the development of the cosmetics industry, solving skin pigmentation is not only a medical problem, but also reflects consumers’ pursuit of beauty and positive attitude towards life. There are many whitening agents in the cosmetics industry, such as arbutin, kojic acid, nicotinamide, hydroquinone, resorcinol, 4-Methoxyphenol, 4-Ethoxyphenol, and ascorbic acid. However, hydroquinone can lead to several pathological conditions, such as erythema, dryness, and skin desquamation¹². Therefore, natural sources have gained more attention and interest from consumers and researchers than chemically synthesized melanin inhibitors.

Studies have shown that melanin production inhibitors extracted from herbal plants regulate melanin production through a variety of mechanisms, including Akt/GSK3β/β-catenin pathway, MAPK pathway, MC1R/cAMP/PKA pathway and proteins associated with autophagy such as beclin-1 and LC3-II^13,14. Andrographolide suppresses melanin synthesis through Akt/GSK3β/β-catenin signal pathway, and effectively suppress melanin content and TYR activity in B16-F10 cells, HEM cells and UVB-induced brown guinea pig skin by inhibiting β-catenin into the nucleus and decreasing the expression of MITF and TYR family¹⁵. Selaginellin (SEL) inhibits melanogenesis through inhibiting the MAPK signaling pathway, then down-regulating the expression of MITF, TYR and TRP-2¹⁶. Phenolic acids in Panax ginseng inhibit melanin production through bidirectional regulation of melanin synthase transcription via different signaling pathways. P. ginseng roots’ phenolic acid monomers can safely inhibit melanin production by bidirectionally regulating melanin synthase transcription. Furthermore, they reduced MITF expression via MC1R/cAMP/PKA signaling pathway and enhanced MITF post-translational modification via Wnt/mitogen-activated protein kinase signaling pathway¹⁷. Pterostilbene showed depigmenting activity by increasing LC3-II/p62 levels and Beclin-1/Bcl-2 ratio, decreasing ATG4B levels, and inducing melanocytes autophagy¹⁸. Ellagic acid showed anti-melanogenic activity by increasing LC3-II accumulation and enhancing autophagy, 3-MA (an autophagy inhibitor) pretreatment or LC3 silencing (siRNA transfection) of cells significantly reduced anti-melanogenic activity of Ellagic acid¹⁹.

Naturally active compounds mentioned above have previously been shown to have antioxidant or anti-inflammatory properties, and now more and more studies have revealed that they have anti-melanin production or enhance autophagy functions²⁰. In this study, we established a comprehensive library of 389 natural compounds sourced from TCMSP, CNKI, and other authoritative databases. Using structure-based virtual screening targeting both TYR and MC1R in the MC1R/α-MSH pathway, we identified isorhamnetin-3-O-neohespeidoside - a flavonoid glycoside originally characterized as the principal anti-nociceptive component of Typhae Pollen (the dried pollen of Typha angustifolia L. or T. orientalis Presl)²¹. Typhae Pollen, the dried pollen of Typha angustifolia L., Typha orientalis Presl or the same genus, which medicinal record began in the book entitled Shennong’s Classic of Materia Medica. Typhae pollen has the functions of removing blood stasis and promote blood circulation, but it has the effect of hemostasis after stir-fried²². At present, the pharmacological activity of isorhamnetin-3-O-neohespeidoside has been reported only in vitro antioxidant activity, and other activities have not been reported²³. We focused on MC1R and TYR in the MC1R/α-MSH signaling pathway of melanocytes, and used molecular docking technology to screen the natural components that inhibit MC1R and TYR, and investigate the effects of natural components on melanin synthesis and autophagy, in order to provide references for the development of natural melanin synthesis inhibitors.

Materials and methods

Materials

Cells

B16 melanoma cells, No. MZ-0024, was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. ( Ningbo City, Zhejiang Province, China).

Human immortalized epidermal keratinocytes (HaCaT), No. ZQ0044, was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd.( Shanghai, China), and originated from ScienceCell Corporation in the United States.

Active ingredients

Asiaticoside (purity ≥ 98%, #MUST-21032213), Notoginsenoside R1(purity ≥ 98%, #MUST-21011910), Ginsenoside Rb2(purity ≥ 98%, #MUST-21071202), Isobavachalcone(purity ≥ 98%, #MUST-20121110), Isorhamnetin-3-O-neohespeidoside(purity ≥ 98%, #MUST-21082104), Glabridin(purity ≥ 98%, #MUST-21010412), Baicalin(purity ≥ 98%, #MUST-21032414), Salvianolic acid A(purity ≥ 98%, #MUST-19112210), (EGCG)(-)-Epigallocatechin gallate(purity ≥ 98%, #MUST-20090214), Curcumin(purity ≥ 98%, #MUST-21041911), Asperuloside(purity ≥ 98%, #MUST-21051903), Capsaicin(purity ≥ 98%, #MUST-21103108), Ethyl ferulate(purity ≥ 98%, #MUST-22080110), α-Mangostin(purity ≥ 98%, #MUST-21042302), Neohesperidin(purity ≥ 98%, #MUST-21040707), Ononin(purity ≥ 98%, #MUST-21012314), Procyanidin(purity ≥ 98%, #MUST-20011303), Bavachinin A(purity ≥ 98%, #MUST-21022711), Isochlorogenic acid A(purity ≥ 98%, #MUST-21011110), Arbutin(purity ≥ 98%, #MUST-21070205), Neoandrographolide(purity ≥ 98%, #MUST-20111413), Palmitoyl tetrapeptide-7(purity ≥ 98%, #MUST-21041308), Platycodin D(purity ≥ 98%, #MUST-20051714), Ginsenoside Rg3(purity ≥ 98%, #MUST-21041512), Aucubin(purity ≥ 98%, #MUST-21011312), Isochlorogenic acid C(purity ≥ 98%, #MUST-21081010), Licochalcone A(purity ≥ 98%, #MUST-20112317), Isoorientin(purity ≥ 98%, #MUST-20122809), β-Sitosterol(purity ≥ 98%, #MUST-21022710), Isoliquiritin(purity ≥ 98%, #MUST-21050309), Aloin(purity ≥ 98%, #MUST-20092711), Bisdemethoxycurcumin(purity ≥ 98%, #MUST-20123011), Geniposide(purity ≥ 98%, #MUST-21031016), were purchased from Chengdu Must Bio-tech Co., Ltd (Chengdu, China). Benzene-1,4-diol(purity ≥ 99%, #H811112), Tranexamic acid(purity ≥ 98%, #A800982), vitamin C(purity ≥ 99%, #C12453039), Tea polyphenol(purity ≥ 99%, #T821916), Melitose(purity ≥ 99%, #D832616), Tannic acid(purity ≥ 98%, #T818845), were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Hexylresorcinol(purity ≥ 99%, #H-25485), Silybin(purity ≥ 80%, determined by UV, #S-22151), were purchased from Heowns Biochem Technologies Co., Ltd. (Tianjin, China). VE(purity ≥ 99%, #YZ-100051), was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Kojic acid(purity ≥ 98%, #P325008), was purchased from Shanghai chemxyz Co., Ltd. (Shanghai, China). α-MSH(purity ≥ 95%, #S48512), Argireline(purity ≥ 98%, #S80841), Matrixyl(purity ≥ 95%, #B28580), Palmitoyl tripeptide-1(purity ≥ 98%, #S88037), Nonapeptide-1(purity ≥ 99%, #B28680), Linolenic acid(purity ≥ 98%, #B21967), were purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China).

Reagents

1640 Medium (Dalian Meilun Biotechnology Co., Ltd, #MA0215), Complete medium specifically for B16 cell culture (Wuhan Pricella Biotechnology Co., Ltd., #WH2522A231), PBS buffer solution (1X), cell-grade(Dalian Meilun Biotechnology Co., Ltd, #MA0015), Trypsin-EDTA (Gibco by Life Technologies Corporation, 0.25%, #25200-072), Mouse Melanocortin 1 Receptor (MC1R) ELISA Kit(Jiangsu Meimian Industrial Co., Ltd., #MM-46019M1), DMSO (Suzhou Meilun Biotechnology Co., Ltd, #O1028A, cell culture grade, purity > 99.5%), L-DOPA(Shanghai Macklin Biochemical Technology Co., Ltd., #D807434,>99.0%), Mushroom Tyrosinase(9ding Chemical (Shanghai) Technology Co., Ltd., #L-WL431, purity > 99.0% ), Triton Cell Lysis Buffer(Shanghai Macklin Biochemical Technology Co., Ltd., #T885422), Thiazolyl Blue Tetrazolium Bromide( Shanghai Macklin Biochemical Technology Co., Ltd., #T6126, purity > 99.0%), Bovine Serum Albumin (BSA, Wuhan Servicebio Technology Co., Ltd., #GC305010), DAPI Staining Reagent(Wuhan Servicebio Technology Co., Ltd., #G1012), RIPA Lysate Buffer (Wuhan Servicebio Technology Co., Ltd., #G2002).

Docking-based screening of TYR/MC1R dual inhibitors

Construct a database of ligand molecules

The ingredients of traditional Chinese medicine that potentially inhibit skin pigmentation were obtained from databases such as Traditional Chinese Medicine Systems Pharmacology(http://tcmspw.com/), CNKI (https://www.cnki.net/), CAS SciFinder (https://www.cas.org/zh-hans/solutions/cas-scifinder-discovery-platform/cas-scifinder), EBSCO (https://search.ebscohost.com/), and PubMed (https://www.ncbi.nlm.nih.gov/pubmed). The 2D structures of all these active ingredients were downloaded from PubChem and saved in SDF format. These SDF files were then converted into SD files using Discovery Studio software. These SD files served as ligand molecules for molecular docking, totaling 389 ligand molecules.

Download the receptor proteins TYR and MC1R

The receptor protein MC1R (PDB:7F4D) was downloaded from the Protein Data Bank database.

The Uniprot database has not yet analyzed human TYR with a clear structure, so the TYR simulated and predicted based on the amino acid sequence of human TYR was downloaded from the Alpha Fold database(https://alphafold.ebi.ac.uk/), 2021/8). UCLA software (http://services.mbi.ucla.edu/, 2021/8) was used to analyze the reliability of the above TYR in terms of residual geometry, global geometry, location, and environment (such as alpha helix, beta sheet, ring, polarity, nonpolarity, etc.). The amino acid sequence in FASTA format (529 AA, ID: P14679) was retrieved from UniProt Knowledge database (http://www.uniprot.org/uniprot/P14679). Predict docking boxes for TYR using PlayMolecule (https://www.playmolecule.com/deepsite/). Save the confidence analysis of TYR and its docking box for the next step of molecular docking.

Ligand molecules were molecularly docked with the receptor proteins TYR and MC1R, respectively

TYR and MC1R were selected as the receptor proteins, and 389 ligand molecules were docked with the receptor proteins TYR and MC1R, respectively.

The specific operation of molecular docking is as follows: First, the Protein Preparation Wizard module of Schrodinger 18.1 software(2021/8/19-2021/8/20) was used to pretreat and optimize the receptor proteins TYR and MC1R, including hydrogenation, removal of water molecules, residue correction and molecular energy minimization. Secondly, 389 ligand molecules were optimized by the Ligand Preparation module of Schrodinger 18.1 software, encompassing hydrogenation, removal of water molecules, charge, metal coordination bonds and tautomer generation. Then, the Ligand Docking module was used to dock the ligand with the receptor TYR and MC1R respectively, and the docking accuracy was SP (standard precision), and the docking result retained an optimal conformation, and other parameters were the default values. The Docking Score was utilized as the criterion for scoring and ranking the molecular docking results.

Based on the above molecular docking results, using arbutin as a reference point, the active ingredients from the ligand molecule database were selected if they exhibited a Docking Score value superior to that of arbutin in their binding to both TYR and MC1R. Both arbutin and tranexamic acid demonstrated favorable binding affinity with the target proteins. Given that arbutin’s inhibitory activity against TYR has been extensively documented in numerous studies, and tranexamic acid has been confirmed as an active ingredient capable of activating melanocyte autophagy, these two compounds were selected as reference drugs for subsequent experiments.

Verify the screening results of molecular docking by B16 melanoma cells

Detect the effect of 49 ligands on B16 cells viability

In this study, we innovatively employed mammalian tyrosinase derived from B16 melanoma cells for activity determination, which provides more accurate reflection of the compound’s true inhibitory effects under physiological conditions compared to the commonly used mushroom tyrosinase (mTyr) screening systems. Previous studies have demonstrated significant differences in catalytic activity and substrate specificity between mTyr and mammalian tyrosinase²⁴, which may explain the low clinical translation rate of many mTyr-screened inhibitors.

Methyl thiazolyl tetrazolium(MTT) assay was used to detect the effect of 49 active ingredients on B16 melanoma cells viability. The discrepancy in compound numbers arises from the overlap of compounds showing strong binding affinity for both TYR and MC1R in molecular docking. Specifically, 30 compounds were selected for TYR inhibition assays and 30 for MC1R content analysis based on docking scores, with 11 compounds overlapping. Thus, the initial count of unique compounds for these biological assays was 49 (30 for TYR + 30 for MC1R − 11 overlaps). This integrated approach enabled us to explore the potential dual - targeting effects of these compounds on TYR and MC1R, crucial factors in the melanogenesis pathway. The specific experiments steps were as follows.

Moreover, a control group and an administration group were set up. The concentration gradients of the 49 active ingredients ranged from 0.5 µM to 32 µM (specifically, 0.5 µM, 1.0 µM, 2.0 µM, 4.0 µM, 8.0 µM, 16.0 µM, and 32 µM). B16 melanoma cells were seeded in 96-well plates and cultured to 60% confluency, remove medium, control group was added with 100 µL of medium without active ingredient, and other groups were added with 100 µL of medium with active ingredient, and continued to culture for 24 h. We used arbutin as positive controls.

At the end of the culturing period, the medium was removed, and the cells were washed twice with PBS. Then, 100 µL of MTT solution at a concentration of 0.5 mg/mL was added to each well. The plates were incubated for 3–4 h. Afterward, the MTT solution was removed, and 100 µL of DMSO was added to each well. The plates were allowed to stand for 10 min to fully dissolve the MTT formazan crystals. Absorbance was measured at 490 nm using a microplate reader (Bio Tek, USA), and cell viability was calculated based on the results. For cell viability: Cell viability (%) = (OD₄₉₀ of treatment group / OD₄₉₀ of control group) × 100.

Detect the effect of 30 ligands on TYR activity of B16 cells

Dopa oxidation assay was used to detect the effect of 30 ligands on TYR activity, and the specific steps were as follows. The TYR activity quantitative experiment was used to draw a fitting curve with enzyme activity value (U/mL) as Y axis and OD value at 490 nm as X axis.

According to the results of item 2.3.1, a control group and an administration group were set up (the concentration of arbutin and 29 active ingredients were 8.0 µM). B16 melanoma cells were seeded in 96-well plates and cultured to 60% confluency, removed medium. Control group was added with 100 µL of medium without active ingredient, and other groups were added with 100 µL of medium with active ingredient, and continued to culture for 24 h.

At the end of the culture period, the medium was removed, and the cells were washed twice with PBS. Then, Triton X-100 was added, followed by the introduce 10 µL of an L-DOPA solution with a concentration of 2 g/L.The mixture was allowed to incubate for 1 h. Subsequently, the absorbance was detected at 492 nm using a microplate reader (Bio Tek, USA), and calculate TYR activity based on the aforementioned fitting curve. For tyrosinase (TYR) inhibition: TYR inhibition (%) = (1 - mean enzyme activity of treatment group / mean enzyme activity of control group) × 100%.

Detect the effect of 30 ligands on MC1R content of B16 cells

Enzyme-linked immunosorbent assay (ELISA) was used to detect the effect of 30 ligands on MC1R content of B16 cells, and the specific steps were as follows.

The Grouping and administration steps are the same as those described in item 2.3.2.

At the end of the culture period, the medium was removed, and Triton X-100 was added. The samples were prepared and experiments were performed according to the instructions provided with the MC1R ELISA kit (Jiangsu Meimian Industrial Co., Ltd, Jiangsu Province, China). Then, the OD values were detected at 450 nm with a microplate reader (Bio Tek, USA). For MC1R downregulation: MC1R downregulation (%) = (1 - mean protein concentration of treatment group / mean protein concentration of control group) × 100%.

Detect the effect of isorhamnetin-3-O-neohespeidoside on B16 melanoma cells

Detect the effect of isorhamnetin-3-O-neohespeidoside on Hacat cells viability and B16 cells viability

To further confirm the effect and safety of isorhamnetin-3-O-neohespeidoside on the viability of normal skin cells, the MTT assay was employed to assess its impact on the viability of Hacat cells. The specific steps were as follows.

Set up the control group and the administration group, with isorhamnetin-3-O-neohespeidoside concentrations of 5µM, 10µM, 20µM, 40µM, 80µM, 160µM, and 320µM. The other specific steps were the same as those described in item 2.3.1.

MTT assay was used to detect the effect of isorhamnetin-3-O-neohespeidoside on B16 cells viability, and the specific steps were as follows.

Set the control group and the administration group (the concentration gradients of isorhamnetin-3-O-neohespeidoside were 4µM, 8µM, 16µM, 32µM, 64µM, 128µM, 256µM and 512µM). The other specific steps were the same as item 2.3.1.

The specific steps were the same as item 2.3.1. Finally, the IC₅₀ value of isorhamnetin-3-O-neohespeidoside for B16 cells was analyzed using GraphPad Prism. The administration concentration was set according to the IC₅₀ value, and the maximum concentration should be at 1/3 IC₅₀.

Evaluate the impact of isorhamnetin-3-O-neohespeidoside on TYR activity and MC1R expression in B16 cells

Dopa oxidation assay was used to detect the effect of isorhamnetin-3-O-neohespeidoside on TYR activity, and the specific steps were as follows.

According to the results of item 2.4.1, set the control group and the administration group (the concentration gradients of isorhamnetin-3-O-neohespeidoside were 0.5µM, 1µM, 2µM, 4µM, 8µM and 16µM). The other specific steps were the same as those in item 2.3.2.

The dopa oxidation assay steps were the same as in item 2.3.2.

ELISA was used to detect the effect of isorhamnetin-3-O-neohespeidoside on MC1R expression of B16 cells, and the specific steps were as follows. Grouping and administration. The specific steps refer to those in item 2.4.1. Detection of ELISA. The specific steps refer to those in item 2.3.3.

Detect the effect of isorhamnetin-3-O-neohesperidin on melanin content of B16 cells

NaOH method was used to detect the effect of isorhamnetin-3-O-neohespeidoside on melanin content of B16 cells, and the specific steps were as follows.

The Grouping and administration steps were the same as in item 2.4.1.

The remaining medium was discarded and cells were harvested after digestion with 0.25% trypsin solution. 1.0 mL of 1 mol/L NaOH was added to the centrifuge tube. Melanin-containing tubes were heated at 80℃ for 1 h, melanin particles in cells were dissolved in NaOH solution, centrifuged (2000 rpm, 10 min), and the OD values were detected at 405 nm with a microplate reader (Bio Tek, USA).

Detect the effect of isorhamnetin-3-O-neohespeidoside on autophagy in B16 cells

Immunofluorescence staining was performed for confocal laser scanning microscope analysis. Tranexamic acid was used as positive control, and the specific steps were as follows.

Set control, tranexamic acid (8µM) and isorhamnetin-3-O-neohespeidoside (8µM). B16 melanoma cells were seeded in 6-well plates containing glass coverslips and until they reached 60% confluency. The medium was then removed. The control group was treated with 2 mL of medium without active ingredient in the absence or presence of α-MSH (8µM), and other groups were added with 2 mL of medium with active ingredient in the absence or presence of α-MSH (8µM), and were continued to be cultured for 24 h.

At the end of the culture period, remove the medium, wash twice with PBS. B16 melanoma cells were fixed with 4% paraformaldehyde for 25–30 min at room temperature, permeabilized with 0.1% TritonX-100 for 20 min, wash three times with PBS, and blocked with 1% BSA for 30 min. After washing several times with PBS. B16 melanoma cells were incubated with anti-LC3 rabbit pAb (diluted 1:400 in PBS) purchased from Wuhan Servicebio Biotechnology Co., LTD. (Hunan Province, China) overnight at 4℃, and incubated with HRP conjugated goat anti-mouse IgG (diluted 1:500 in PBS) purchased from Wuhan Servicebio Biotechnology Co., LTD. (Hunan Province, China) at room temperature for 50 min. B16 melanoma cells were finally stained with DAPI for 10 min and observe the resulting images using a confocal laser scanning microscope (CLSM) (NIKON, Japan).

Statistical analysis

Data were analyzed by GraphPad Prism 8.0 software, and differences between groups were statistically analyzed by one-way ANOVA. The experimental data obtained were represented by‾X ± SD, and P<0.05 was considered statistically significant.

Results

Molecular docking

In MC1R/α-MSH signaling pathway, MC1R is the central determinant of pigmentation, while tyrosinase is a key enzyme in melanin synthesis^6,7.

The results of molecular docking are shown in Table 1. The ligands were ranked based on the absolute values of their SP Docking Scores. A higher absolute value of the SP Docking Score indicates a stronger binding affinity of the ligand for TYR. Compounds were ranked by their absolute SP Docking Scores (Table 1 ). Only ligands with superior binding affinity or equivalent to TYR compared to arbutin were selected (e.g., isorhamnetin-3-O-neohesperidin: TYR = − 8.001 kcal/mol).

Table 1.

Molecular Docking results of ligands and TYR.

Number	CAS	Ligands	Docking score (kcal/mol)
1	16830-15-2	Asiaticoside	−8.963
2	80418-24-2	Notoginsenoside R1	−8.386
3	11021-13-9	Ginsenoside Rb2	−8.137
4	20784-50-3	Isobavachalcone	−8.02
5	55033-90-4	Isorhamnetin-3-O-neohespeidoside	−8.001
6	123-31-9	Benzene-1,4-diol	−7.859
7	1197-18-8	Tranexamic acid	−7.813
8	59870-68-7	Glabridin	−7.776
9	50-81-7	Vitamin C	−7.743
10	21967-41-9	Baicalin	−7.725
11	96574-01-5	Salvianolic acid A	−7.714
12	989-51-5	(EGCG)(-)-Epigallocatechin gallate	−7.684
13	458-37-7	Curcumin	−7.668
14	14259-45-1	Asperuloside	−7.656
15	404-86-4	Capsaicin	−7.652
16	136-77-6	Hexylresorcinol	−7.64
17	4046-02-0	Ethyl ferulate	−7.523
18	6147-11-1	α-Mangostin	−7.38
19	13241-33-3	Neohesperidin	−7.329
20	486-62-4	Ononin	-7.324
21	4852-22-6	Procyanidin	-7.232
22	65666-07-1	Silybin	−7.226
23	19879-30-2	Bavachinin A	−7.215
24	7718-59-4	Vitamin E	−7.171
25	2450-53-5	Isochlorogenic acid A	−7.138
26	497-76-7	Arbutin	−7.137
27	84650-60-2	Tea polyphenol	−7.134
28	512-69-6	Melitose	−7.133
29	501-30-4	Kojic acid	−7.083
30	27215-14-1	Neoandrographolide	−7.046

The results showed that the absolute value of SP Docking Score of 25 ligands, including asiaticoside, notoginsenoside R1 and ginsenoside Rb2, among the tested ligands, were high when binding to the receptor protein TYR. A Dopa oxidation assay will be conducted to further verify their activities.

The results of the molecular docking experiments are shown in Table 2. The ligands are ranked according to the absolute values of their SP Docking Scores. The greater the absolute value of the SP Docking Score, the more firmly the ligand binds to MC1R. Compounds were ranked by their absolute SP Docking Scores (Table 2). Only ligands with superior binding affinity or equivalent to MC1R compared to arbutin were selected (e.g., isorhamnetin-3-O-neohesperidin: MC1R = − 7.342 kcal/mol).

Table 2.

Molecular docking results of ligands and MC1R.

Number	CAS	Ligands	Docking score (kcal/mol)
1	581-05-5	α-MSH	-10.703
2	616204-22-9	Argireline	-9.509
3	214047-00-4	Matrixyl	-9.199
4	221227-05-0	Palmitoyl tetrapeptide-7	-8.843
5	147732-56-7	Palmitoyl tripeptide-1	-8.129
6	11021-13-9	Ginsenoside Rb2	-8.108
7	16830-15-2	Asiaticoside	-8.057
8	158563-45-2	Nonapeptide-1	-7.858
9	58479-68-8	Platycodin D	-7.842
10	14197-60-5	Ginsenoside Rg3	-7.836
11	2450-53-5	Isochlorogenic acid A	-7.82
12	479-98-1	Aucubin	-7.786
13	80418-24-2	Notoginsenoside R1	-7.773
14	96574-01-5	Salvianolic acid A	-7.735
15	13241-33-3	Neohesperidin	-7.693
16	32451-88-0	Isochlorogenic acid C	-7.523
17	58749-22-7	Licochalcone A	-7.494
18	55033-90-4	Isorhamnetin-3-O-neohespeidoside	-7.342
19	4261-42-1	Isoorientin	-7.268
20	989-51-5	(EGCG)(-)-Epigallocatechin gallate	-7.197
21	1401-55-4	Tannic acid	-6.995
22	83-46-5	β-Sitosterol	-6.989
23	486-62-4	Ononin	-6.958
24	5041-81-6	Isoliquiritin	-6.956
25	4852-22-6	Procyanidin	-6.933
26	512-69-6	Melitose	-6.901
27	463-40-1	Linolenic acid	-6.865
28	1415-73-2	Aloin	-6.747
29	33171-05-0	Bisdemethoxycurcumin	-6.727
30	24512-63-8	Geniposide	-6.707

The results indicated that the absolute values of the SP Docking Scores for 30 ligands, including α-MSH, argireline, and matrixyl, were high when binding to the receptor protein MC1R. An ELISA assay will be conducted to verify their activities.

Verification results of molecular docking

The effect of 49 ligands on B16 cells viability

The MTT assay was used to detect the effect of 49 active ingredients on cell viability. The results were shown in Figure S1(see Supplementary Materials 1 ). When the concentration exceeded 8µM, ginsenoside Rg3, bisdemethoxycurcumin, tannic acid, ethyl ferulate, α-mangostin and other active ingredients inhibited cell viability, resulting in a cell viability of less than 85%. According to the ISO 10993-5 standard, in vitro cell viability greater than 80% is considered non-cytotoxic. To ensure a margin of safety, a more stringent 85% threshold was selected. The 8 µM concentration served as a critical threshold: it demonstrated significant growth inhibition for some compounds while maintaining safety profiles (cell viability > 85%) for others. Consequently, 8 µM was established as the standardized test concentration for all subsequent experiments to ensure consistent evaluation and avoid artifacts related to cytotoxicity.

The effect of 30 ligands on TYR activity of B16 cells

Compounds were ranked by their absolute SP Docking Scores (Tables 1 and 2). Only ligands with superior binding affinity or equivalent to both TYR and MC1R compared to arbutin were selected (e.g., isorhamnetin-3-O-neohespeidoside: TYR = − 8.001 kcal/mol, MC1R = − 7.342 kcal/mol). The final 30 ligands were prioritized based on: (i) Commercial availability (purity ≥ 98%, Chengdu Must Bio-tech Co., Ltd.); (ii) Cell viability > 85% at 8 µM (Figure S1), excluding cytotoxic compounds.

The inhibitory effect of 30 ligands such as asiaticoside and notoginsenoside R1 on TYR activity were verified by dopa oxidation assay. The fitting curve between TYR activity (Y axis, U/mL) and OD_490nm (X axis) was Y = 108.13 × ² + 46.275X-2.1163.

TYR activity and inhibition rates were calculated based on the above fitted curve equation and OD_490nm values, and the results are presented in Table 3. The top six ingredients that inhibited TYR activity were, in descending order: isorhamnetin-3-O-neohespeidoside, glabridin, vitamin C, asiaticoside, baicalin, and α-mangostin. Compared with the arbutin group, isorhamnetin-3-O-neohesperidin significantly inhibited TYR activity (P < 0.0001). The inhibition rate of isorhamnetin-3-O-neohespeidoside was 44.42%.

Table 3.

Effect of 30 ligands on TYR activity (X  ± SD, n = 6).

Number	Ligands	TYR activity (U/mL)	Inhibition (%)
	Control	30.33 ± 0.61	0
1	Isorhamnetin-3-O-neohespeidoside	16.82 ± 0.14****	44.42
2	Glabridin	18.45 ± 0.61****	39.01
3	Vitamin C	18.96 ± 0.35****	37.35
4	Asiaticoside	19.11 ± 0.20****	36.86
5	Baicalin	19.66 ± 0.32****	35.02
6	α-Mangostin	20.64 ± 0.30*	31.79
7	Notoginsenoside R1	20.90 ± 0.36	30.92
8	Ginsenoside Rb2	20.99 ± 0.44	30.63
9	Curcumin	21.21 ± 0.45	29.9
10	Salvianolic acid A	21.66 ± 0.50	28.43
11	Isobavachalcone	21.68 ± 0.27	28.36
12	Asperuloside	21.79 ± 0.23	27.32
13	Arbutin	22.07 ± 0.65	27.06
14	Isochlorogenic acid A	22.37 ± 0.19	26.06
15	Ononin	22.46 ± 0.51	25.76
16	Benzene-1,4-diol	22.53 ± 0.34	25.54
17	(EGCG)(-)-Epigallocatechin gallate	22.80 ± 0.27	24.64
18	Hexylresorcinol	22.85 ± 0.30	24.49
19	Neohesperidin	22.94 ± 0.33*	24.19
20	Vitamin E	23.05 ± 0.52**	23.81
21	Silybin	23.22 ± 1.17**	23.25
22	Capsaicin	23.35 ± 0.43***	22.83
23	Tranexamic Acid	23.42 ± 0.16****	22.6
24	Neoandrographolide	23.49 ± 0.31****	22.37
25	Bavachinin A	23.56 ± 0.43****	22.14
26	Melitose	23.74 ± 0.20****	21.54
27	Kojic acid	24.00 ± 0.43****	20.69
28	Procyanidin	24.14 ± 0.31****	20.23
29	Ethyl ferulate	24.16 ± 0.33****	20.16
30	Tea polyphenol	25.26 ± 0.31****	16.52

Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with arbutin, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

The effect of 30 ligands on MC1R content of B16 cells

The inhibitory effects of 30 ligands on MC1R content were verified by ELISA. The results are shown in Table 4. The top six ingredients that inhibited MC1R in B16 cells were, in descending order of inhibitory effect: licochalcone A, salvianolic acid A, linolenic acid, isochlorogenic acid A, melitose, and isorhamnetin-3-O-neohespeidoside. The corresponding inhibition rates were 40.99%, 39.58%, 38.97%, 38.44%, 34.13%, and 33.39%, respectively.

Table 4.

Effect of 30 ligands on MC1R content (X  ± SD, n = 6).

Number	Ligands	MC1R (ng/L)	Inhibition (%)
	Control	4.389 ± 0.022	0
1	Licochalcone A	2.590 ± 0.006	40.99
2	Salvianolic acid A	2.652 ± 0.006	39.58
3	Linolenic acid	2.679 ± 0.018	38.97
4	Isochlorogenic acid A	2.703 ± 0.025	38.44
5	Melitose	2.892 ± 0.025	34.13
6	Isorhamnetin-3-O-neohespeidoside	2.924 ± 0.025	33.39
7	Isochlorogenic acid C	3.178 ± 0.019	27.60
8	Platycodin D	3.187 ± 0.018	27.40
9	Palmitoyl tripeptide-1	3.238 ± 0.022	26.25
10	Ginsenoside Rg3	3.285 ± 0.006	25.18
11	Ginsenoside Rb2	3.351 ± 0.007	23.43
12	Matrixyl	3.409 ± 0.003	22.35
13	Aucubin	3.418 ± 0.029	22.15
14	Neohesperidin	3.441 ± 0.018	21.61
15	Argireline	3.471 ± 0.005	20.93
16	Nonapeptide-1	3.569 ± 0.029	18.71
17	Asiaticoside	3.592 ± 0.011	18.17
18	Isoorientin	3.666 ± 0.030	16.49
19	Notoginsenoside R1	3.719 ± 0.006	15.28
20	β-Sitosterol	3.746 ± 0.020	14.67
21	Isoliquiritin	3.758 ± 0.011	14.41
22	Ononin	3.902 ± 0.018	11.11
23	Procyanidin	3.911 ± 0.024	10.91
24	Palmitoyl tetrapeptide-7	3.920 ± 0.011	10.70
25	Aloin	3.923 ± 0.040	10.64
26	(-)-Epigallocatechin gallate (EGCG)	3.926 ± 0.025	10.57
27	Bisdemethoxycurcumin	4.033 ± 0.093	8.14
28	Tannic acid	4.133 ± 0.018	5.86
29	Geniposide	4.287 ± 0.028	2.36
30	α-MSH	4.582 ± 0.015	-4.38

Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with arbutin, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

The verification experiment focused primarily on the inhibitory effect of active ingredients on TYR, followed by their inhibitory effect on MC1R. Therefore, based on the results of Sect. The effect of 30 ligands on TYR activity of B16 cells and The effect of 30 ligands on MC1R content of B16 cells, isorhamnetin-3-O-neohespeidoside was determined as the research object for subsequent experiments. isorhamnetin-3-O-neohespeidoside (chemical structure shown in Fig. 1) is a flavonoid glycoside compound consisting of an isorhamnetin aglycone (3’-methoxy-3,4’,5,7-tetrahydroxyflavone) linked to a neohesperidose moiety via a glycosidic bond.

Fig. 1.

Chemical structure of isorhamnetin-3-O-neohespeidoside.

The effect of isorhamnetin-3-O-neohespeidoside on B16 melanoma cells

The effect of isorhamnetin-3-O-neohespeidoside on Hacat and B16 cells viability

Figure 2(a) displays the effect of various concentrations of isorhamnetin-3-O-neohespeidoside on the viability of normal skin cells, specifically Hacat cells. At concentrations ranging from 10µM to 40µM, it exhibits a proliferative effect on Hacat cells (P < 0.05).

Fig. 2.

Effects of test active ingredients on Hacat viability and B16 cells viability inhibition(x̅±SD, n = 6); (a) Effects of isorhamnetin-3-O-neohespeidoside on Hacat cells viability. Following 24-hour treatment with gradient concentrations (0.5–320 µM), isorhamnetin-3-O-neohespeidoside maintained > 85% (vs. untreated control, p > 0.05) cell viability in HaCaT cells. These results validate the non-toxic nature of the active compounds within concentrations (0.5–320 µM) in HaCaT cells. Compared with the control group, *P< 0.05. (b) Effects of isorhamnetin-3-O-neohespeidoside on B16 cells inhibition. MTT-based viability assays performed across a 4-512 µM concentration range (24 h exposure) yielded classical sigmoidal dose-response curves, enabling accurate IC₅₀ determination through four-parameter logistic modeling.

Figure 2(b) IC₅₀ value of isorhamnetin-3-O-neohespeidoside was 52.22µM. According to the IC₅₀ value, the maximum administration concentration of isorhamnetin-3-O-neohespeidoside in subsequent experiments was 16µM.

The effect of isorhamnetin-3-O-neohespeidoside on TYR activity and MC1R content of B16 cells

The inhibitory effect of isorhamnetin-3-O-neohespeidoside on TYR activity were detected by dopa oxidation assay (Fig. 3(a)). Compared with control group, isorhamnetin-3-O-neohespeidoside at 4µM,8µM and 16µM significantly inhibited TYR activity (P < 0.0001 for all groups). Compared with positive control arbutin (8µM) group, 8µM isorhamnetin-3-O-neohespeidoside (P < 0.01) and 16µM isorhamnetin-3-O-neohespeidoside (P < 0.0001) significantly inhibited TYR activity.

Fig. 3.

Effects of test active ingredients on TYR activity, MC1R expression and melanin synthesis inhibition of B16 cells (x̅±SD, n = 6). (a) Effects of isorhamnetin-3-O-neohespeidoside on TYR activity of B16 cells; After 24-hour treatment with compounds, tyrosinase (TYR) activity was measured using the dopa oxidation assay. Quantification of TYR activity via dopa oxidation revealed that isorhamnetin-3-O-neohespeidoside (8 µM, 16 µM) exhibited significantly lower activity than arbutin (8 µM; P < 0.01 by ANOVA with Tukey’s test). (b) Effects of isorhamnetin-3-O-neohespeidoside on MC1R expression of B16 cells. Following 24-hour treatment, MC1R protein levels were quantified by ELISA. Quantification of MC1R expression revealed dose-dependent suppression by isorhamnetin-3-O-neohespeidoside (4 µM, 8 µM, 16 µM), outperforming arbutin (8 µM; P < 0.01 by ANOVA with Tukey’s test). (c) Effects of isorhamnetin-3-O-neohespeidoside on melanin synthesis inhibition of B16 cells. After 24-hour treatment with compounds, melanin content was quantified using the NaOH lysis method. Quantification of melanin content revealed dose-dependent inhibition by isorhamnetin-3-O-neohespeidoside (8 µM, 16 µM) significantly exceeding arbutin’s effect (8 µM, P < 0.01). Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with arbutin, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

The inhibitory effect of isorhamnetin-3-O-neohespeidoside on MC1R expression were verified by ELISA (Fig. 3(b)). Compared with control group or positive control arbutin (8µM) group, isorhamnetin-3-O-neohespeidoside at 4µM, 8µM and 16µM significantly inhibited MC1R expression (P < 0.0001 for all groups).

The effect of isorhamnetin-3-O-neohespeidoside on melanin content of B16 cells

The effect of isorhamnetin-3-O-neohespeidoside on melanin synthesis inhibitory were verified by NaOH method (Fig. 3(c)). The melanin synthesis inhibition rate of 4µM, 8µM and 16µM isorhamnetin-3-O-neohespeidoside were significantly higher than that in control group (P < 0.0001 for all groups). And melanin synthesis inhibition rate of 8µM and 16µM isorhamnetin-3-O-neohespeidoside were significantly higher than that in arbutin (8µM) group.

The effect of isorhamnetin-3-O-neohespeidoside on autophagy in B16 cells

The results of immunofluorescence staining were shown in Fig. 4. Without 8µM α-MSH, Fig. 4(a) shows that the LC3-II protein labeled with green fluorescence in the TXA and isorhamnetin-3-O-neohespeidoside groups was higher than that in the control group. Figure 4(c) shows that the mean gray value ratio of LC3-II content for 8µM TXA (P < 0.0001) and 8µM isorhamnetin-3-O-neohespeidoside (P < 0.0001) was significantly higher than that in the control group. Additionally, the mean gray value ratio of LC3-II content for isorhamnetin-3-O-neohespeidoside was significantly higher than that for TXA (P < 0.05).

Fig. 4.

LC3-II expression in B16 cells were observed by CLSM(n = 3, ×600). (a) Basal conditions (α-MSH-absent) and (b) α-MSH-stimulated conditions (8 µM). Cells were treated with 8 µM TXA or isorhamnetin-3-O-neohespeidoside for 24 h. (c) Mean gray value ratio of LC3-II content in B16 cells (x̅±SD, n = 3). Autophagy induction by test compounds was evaluated through LC3-II immunofluorescence. Without α-MSH stimulation, both TXA andisorhamnetin-3-O-neohespeidoside (8 µM) significantly increased LC3-II fluorescence intensity versus control (P < 0.0001), with isorhamnetin-3-O-neohespeidoside showing superior efficacy (P < 0.05 vs. TXA). This enhancement persisted under α-MSH challenge (8 µM), where isorhamnetin-3-O-neohespeidoside maintained stronger LC3-II induction than TXA (P < 0.05). Compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Compared with TXA, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

With 8µM α-MSH, Fig. 4(b) shows that the LC3-II protein labeled with green fluorescence in the TXA and isorhamnetin-3-O-neohespeidoside groups was also higher than that in the control group. Figure 4(c) further indicates that the mean gray value ratio of LC3-II content for 8µM TXA (P < 0.001) and 8µM isorhamnetin-3-O-neohespeidoside (P < 0.0001) was significantly elevated compared to the control group. Moreover, the mean gray value ratio of LC3-II expression for isorhamnetin-3-O-neohespeidoside remained significantly higher than that for TXA (P < 0.05).

Discussion

In our study, molecular docking identified isorhamnetin-3-O-neohespeidoside as a dual inhibitor of TYR and MC1R, with binding energies of -8.001 kcal/mol (TYR) and − 7.342 kcal/mol (MC1R), surpassing the reference compound arbutin. Subsequent in vitro validation confirmed these findings: at 8 µM, the compound significantly inhibited TYR activity by 44.42% (p < 0.0001) and reduced MC1R expression by 33.39% (p < 0.0001) in B16 cells. The strong agreement between docking predictions and experimental results underscores the reliability of our screening approach. Further, the compound’s ability to simultaneously target both TYR and MC1R highlights its potential as a multi-mechanistic agent for pigmentation management. Previous studies have reported that isorhamnetin exhibits significant inhibitory effects on mushroom tyrosinase²⁵. However, in our molecular docking results, isorhamnetin showed a lower binding affinity for human tyrosinase (TYR) compared to the reference compound arbutin and thus was excluded from the candidate list (TYR docking score: below threshold; MC1R: -6.956 kcal/mol). This suggests that while isorhamnetin demonstrates notable activity against mushroom tyrosinase, its affinity for human TYR may be suboptimal, though it exhibits moderate binding to MC1R.

Notably, isorhamnetin-3-O-neohespeidoside (chemical structure shown in Fig. 1), a flavonoid glycoside derived from isorhamnetin (3’-methoxy-3,4’,5,7-tetrahydroxyflavone) linked to a neohesperidose moiety via a glycosidic bond, emerged as a potent dual inhibitor in our study. The introduction of the glycosidic bond may enhance its binding affinity for human TYR, as evidenced by its superior docking scores (TYR: -8.001 kcal/mol; MC1R: -7.342 kcal/mol) compared to arbutin. This structural modification could explain the improved inhibitory efficacy against human tyrosinase, highlighting the importance of glycosylation in optimizing ligand-receptor interactions for melanogenesis-related targets.

Future studies will explore structural optimizations to enhance binding affinity and efficacy. While the current study focuses on efficacy validation, future work will systematically investigate structure-activity relationships through molecular dynamics simulations and 3D-QSAR modeling to elucidate the structural determinants of MC1R/TYR inhibition.

On the other hand, it is worth mentioning that the performance of isorhamnetin-3-O-neohespeidoside in terms of cell viability came as a surprise to us. Isorhamnetin-3-O-neohespeidoside displayed particularly interesting dual characteristics (Fig. 2). In HaCaT keratinocytes, it not only maintained > 85% viability across an exceptionally wide concentration range (0.5–320 µM), but actually stimulated proliferation at 10–40 µM (P < 0.05), suggesting potential cytoprotective effects. In contrast, dose-response analysis in B16 melanoma cells revealed classical concentration-dependent inhibition with an IC₅₀ of 52.22 µM. The selection of 16 µM (approximately 30% of IC₅₀) for subsequent studies represents a balanced approach that maximizes potential efficacy while maintaining safety in normal cells.

These findings highlight the importance of cell-type specific cytotoxicity assessments. The differential effects observed between normal and cancerous cell lines, particularly the proliferative versus inhibitory responses to isorhamnetin-3-O-neohespeidoside, warrant further investigation into the underlying molecular mechanisms.

We focused on MC1R and TYR within the MC1R/α-MSH signaling pathway of melanocytes of melanocytes, and utilised molecular docking technology to screen the components from plant and herbal sources that inhibit MC1R and TYR, currently. As we all know, when searching for brightening ingredient, we are familiar with the screening of tyrosinase inhibitors, however, MC1R represents a potential target for the treatment of pigmentation disorders⁸. Some studies have reported that some natural components inhibit melanin synthesis formation partly by down-regulating MC1R, MITF and TYR family, and molecular docking results show that they bind stably to MC1R and TYR proteins. The 100 µg/mL epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), theaflavine-3,3’-digallate (TFDG), and theasinensin A (TSA) inhibited melanin synthesis by downregulating the protein expression of MC1R, MITF, and TYR, as evidenced by their stable binding to MC1R protein²⁶. The results revealed that 150 µg/mL Sargassum cristaefolium (SCE) ethanol extract effectively inhibited the production of melanin content and intracellular tyrosinase activity, and that the bioactive compound of SCE putative kaurenoic acid showed a strong binding affinity against TYR (− 6.5 kcal/mol) and MC1R (− 8.6 kcal/mol)²⁷. Different from the above studies, we used molecular Docking to screen natural source components, compared the absolute value of SP Docking Score, screened 30 components with excellent binding ability to MC1R and TYR, and then further verified the results of molecular docking screening by using cell models. The advantage of this research model is that more whitening candidates can be selected from a large number of natural ingredients. According to the experimental results, the natural components stably bound to TYR or MC1R proteins have different degrees of inhibition on the proteins regulating melanin synthesis. In this study, only isorhamnetin-3-O-neohespeidoside was studied, while other natural components such as salvianolic acid A, isochlorogenic acid A, notoginsenoside R1, and ginsenoside Rb2, whose molecular mechanism of inhibiting melanin synthesis is worthy of further investigation.

In addition to inhibiting melanin synthesis, promoting the metabolism of produced melanin synthesis is also a key means to solve pigmentation. Melanosomes, as melanin carriers, are transported to neighboring keratinocytes to regulate pigmentation. This process of melanin transport can be divided into several modes. However, the role of autophagy in regulating skin pigmentation has attracted extensive attention.

Autophagy induction was reported to regulate skin pigmentation through melanosome degradation²⁸. The research showed that PTPD-12 (the synthetic autophagy inducer) increased autophagic flux of melanocytes and keratinocytes containing transferred melanosomes, which contribute to melanosome degradation and visibly lightened cell pellets of both melanocytes and keratinocytes color. In contrast, inhibition of autophagic flux resulted in marked attenuation of PTPD-12-induced melanosome degradation²⁹. Melasolv, brightening agent in cosmetics for decades, which can activates autophagy to promote melanosome degradation and inhibit skin pigmentation^30,31.

Although autophagy may contribute to skin pigmentation by regulating melanin synthesis and melanosomes degradation, their mechanism involved has not been clearly defined³². In this study, we showed that isorhamnetin-3-O-neohespeidoside increase the expression of LC3-II associated with autophagy. In addition, we found that treatment with isorhamnetin-3-O-neohespeidoside reduces melanin content of B16 cells. Taken together, our results demonstrate that isorhamnetin-3-O-neohespeidoside inhibits skin pigmentation through multiple mechanisms: direct inhibition of MC1R expression and TYR activity, reduction of melanin content, and induction of LC3-II-mediated autophagy.Notably, although MC1R can be activated by multiple ligands including α-MSH, ACTH and β-defensins, our current study did not specifically examine the interaction between isorhamnetin-3-O-neohespeidoside and α-MSH signaling. Future studies should include: (1) competitive binding assays with α-MSH, (2) measurement of downstream cAMP production, and (3) evaluation of MITF expression to fully characterize the mechanism of MC1R expression downregulation.

Conclusions

In this study, isorhamnetin-3-O-neohespeidoside was found to be a potential whitening ingredient. Isorhamnetin-3-O-neohespeidoside exhibits a proliferative effect on Hacat cells at concentrations ranging from 10µM to 40µM (P < 0.05). Within the tested concentration range of our work, it is non-cytotoxic to both Hacat cells and B16 cells. It can stably bind to MC1R and TYR proteins at the same time, inhibit the expression of MC1R and activities of TYR, and effectively reduce melanin production. In addition, isorhamnetin-3-O-neohespeidoside also promotes the autophagy process of B16 cells cells, which offers more possibilities for its application in whitening or skin care products. In the future, we will further study the mechanism of isorhamnetin-3-O-neohespeidoside in order to provide more scientific basis for the development of new whitening or skin care products. It needs further study whether isorhamnetin-3-O-neohespeidoside promotes the degradation of melanin and melanosome by increasing autophagy. It needs further study whether B16 cells treated with isorhamnetin-3-O-neohespeidoside suppressed α-MSH increased melanin content and activated autophagy. It needs further study whether depletion of the ATG5 gene associated with autophagy resulted in significant suppression of isorhamnetin-3-O-neohespeidoside-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16 cells.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

TYR

Tyrosinase

MC1R

Melanocortin 1 receptor

ELISA

Enzyme-Linked Immunosorbent Assay

LC3-II

Microtubule-associated protein 1 light chain 3 (MAP1LC3)

α-MSH

α-melanocyte-stimulating hormone

PI3K

Phosphoinositide 3-kinase

AKT

Protein kinase B

MAPK

Mitogen-activated protein kinases

EDN1

Endothelin 1

Wnt

Wingless-related integration site

MTT

Methyl thiazolyl tetrazolium

Author contributions

R.D. processed the experiments, analyzed the data and drafted the manuscript. S.Z.and S.X. designed the research study, performed the research and reviewed the data. G.H. and Z.O. reviewed the manuscript and contributed essential reagents or tools. Z.S. contributed by reviewing the data analysis and critically revising the manuscript for improving the content. All authors have read and approved the final manuscript.Conflict of interest and funding statementThe authors declare that they have no conflict of interest and funding to the paper submitted.

Data availability

The datasets generated and analyzed during the current study are not publicly available due to proprietary restrictions (the data are part of an ongoing research project with potential future patents/commercial applications) but are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.