Biomolecules & Therapeutics 2024; 32(2): 231-239  https://doi.org/10.4062/biomolther.2023.103
Methylanthranilate, a Food Fragrance Attenuates Skin Pigmentation through Downregulation of Melanogenic Enzymes by cAMP Suppression
Heui-Jin Park1,†, Kyuri Kim1,†, Eun-Young Lee2, Prima F. Hillman2, Sang-Jip Nam2,* and Kyung-Min Lim1,*
1College of Pharmacy, Ewha Womans University, Seoul 03760,
2Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Republic of Korea
*E-mail: sjnam@ewha.ac.kr (Nam SJ), kmlim@ewha.ac.kr (Lim KM)
Tel: +82-2-3277-6805 (Nam SJ), +82-2-3222-3055 (Lim KM)
Fax: +82-2-3277-4546 (Nam SJ), +82-2-3277-3760 (Lim KM)
The first two authors contributed equally to this work.
Received: May 30, 2023; Revised: August 11, 2023; Accepted: August 28, 2023; Published online: February 1, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Methyl anthranilate (MA) is a botanical fragrance used in food flavoring with unexplored potential in anti-pigment cosmetics. MA dose-dependently reduced melanin content without affecting cell viability, inhibited dendrite elongation and melanosome transfer in the co-culture system of human melanoma cells (MNT-1) and human keratinocyte cell line (HaCaT), and downregulated melanogenic genes, including tyrosinase, tyrosinase-related protein 1 and 2 (TRP-1, TRP-2). Additionally, MA decreased cyclic adenosine monophosphate (cAMP) production and exhibited a significant anti-pigmentary effect in Melanoderm™. These results suggest that MA is a promising anti-pigmentary agent for replacing or complementing existing anti-pigmentary cosmetics.
Keywords: Methyl anthranilate, Skin pigmentation, Melanogenesis, Melanoderm™, Cyclic adenosine monophosphate, Anti-ppigmentary agent
INTRODUCTION

Numerous food-grade materials are also cosmetic ingredients, including retinol, ascorbic acid, adenosine, and niacinamide. Compared to synthetic chemicals or non-food botanical extracts, food materials offer additional safety advantages for cosmetic use. Methyl anthranilate (MA) is a naturally occurring fragrance found in various plant species, such as Vitis labrusca grapes, bergamot, black locust, champak, jasmine, lemon, mandarin orange, nero li, strawberry, and tuberose (Moio and Etievant, 1995; Wang and Luca, 2005; Pillet et al., 2017). MA is a prevalent flavor additive in candy, gum, soft drinks, and alcoholic beverages (Luo et al., 2019). Additionally, MA is utilized as a bird repellent in agriculture (Bernklau et al., 2019), and as an ingredient in medicines and perfumes (Irawan et al., 2018). In this study, we investigated the potential of MA as a novel skin brightening agent.

Skin pigmentation is regulated by genetic, environmental, and endocrine factors that control the amount, type, and distribution of melanin in the skin, hair, and eyes (Costin and Hearing, 2007). Melanocytes synthesize the biomolecular pigment melanin in melanosomes secreted and transmitted to keratinocytes (Goenka and Simon, 2020). While melanin is integral for protecting the skin from ultraviolet radiation and oxidative damage, excessive concentrations in the skin engender various medical conditions such as lentigo, melasma, and skin cancer (Brenner and Hearing, 2008).

Melanogenesis is a complex multi-step process that is catalyzed by one or more enzymes (D’Mello et al., 2016). Well-known enzymes involved in melanin production are tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) (Kim et al., 2013) that turn tyrosine into melanin in melanosomes. Melanogenesis is initiated by the binding of specific ligands including alpha-melanocyte stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), and endothelin-1 to receptors on the surface of melanocytes. Upon binding to their respective receptors, these ligands activate a signaling pathway that involves a cascade of intracellular signals, including the activation of protein kinase A (PKA) and the cAMP response element-binding protein (CREB). This pathway ultimately leads to the activation of the transcription factor microphthalmia-associated transcription factor (MITF), which plays a key role in regulating the expression of genes involved in melanin synthesis.

In this study, various skin cell lines were utilized, including murine melanoma cell B16F10, human melanoma cell MNT-1, and human keratinocyte cell HaCaT, in order to evaluate the anti-melanogenic effects of MA. To further understand the mechanism of MA’s anti-melanogenic effect, an enzyme inhibition assay was performed, and the expression of melanogenic enzymes was analyzed at both the mRNA and protein levels. The effect of MA on cyclic adenosine monophosphate (c-AMP), a upstream signaling molecule for melanogenesis stimulation, was also observed. Finally, MA was evaluated in the pigmented human epidermal skin model, MelanodermTM, to ensure its effectiveness as a novel whitening agent.

MATERIALS AND METHODS

General experimental procedures

NMR spectra were acquired by containing Me4Si as internal standard on Varian Unity-Inova 500 MHz and 125 MHz spectrometers (Varian Inc., Palo Alto, CA, USA) using solvent chloroform-d (Cambridge Isotope Laboratories (CIL), Inc., Tewksbury, MA, USA). ESI (electrospray ionization) low-resolution LC-MS data were obtained with an Agilent Technologies 6120 quadrupole mass system (Agilent Technologies, Santa Clara, CA, USA) and Waters Alliance Micromass ZQ LC-MS system (Waters Corp, Milford, MA, USA) using reversed-phase column (Phenomenex Luna C18 (2) 100 Å, 50 mm×4.6 mm, 5µm) (Phenomenex, Torrance, CA, USA) at a flow rate 1.0 mL/min at the National Research Facilities and Equipment Center (NanoBioEnergy Materials Center) at Ewha Womans University (Seoul, Korea). Methyl anthranilate with purity 98.0% was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA).

Methyl anthranilate : colorless to pale yellow liquid; 1H NMR (500 MHz, CDCl3) : δH 7.85 (1H, dd, J=8.5, 1.7 Hz), 7.24 (1H, ddd, J=1.9 Hz, 7.4 Hz), 6.64 (1H, s), 6.63 (1H, m), 3.85 (1H, s, OCH3); 13C NMR (125 MHz, CDCl3) : δc 168.6, 150.5, 134.1, 131.2, 116.7, 116.2, 110.7, 51.5; LR-ESI-MS m/z : 151.17 [M+H]+.

Cell culture

B16F10 cells were obtained from ATCC (Manassas, VA, USA) and HaCaT cells were from AMOREPACIFIC Co (Seoul, Korea). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GE Healthcare Life Science, Piscataway Township, NJ, USA) high-glucose supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin- streptomycin (Hyclone). MNT-1 cells were maintained in minimum essential medium supplemented with 10% DMEM, 20% FBS, 1 M HEPES, and 1% streptomycin-penicillin. All cells were cultured in CO2 incubators at 37°C under a humidified atmosphere of 5% CO2. When the cell confluency reached 80%, the cells were detached using 0.05% trypsin (Hyclone).

Melanin assay

One day prior to an experiment, cells (2×104 cells/well) were seeded in 48-well plate for 24 h. For the measurement of melanin contents, cells were co-treated with various concentrations of MA in phenol-red culture medium containing 0.5% dimethyl sulfoxide (DMSO) and 0.2 µM α-MSH for 72 h. α-MSH was used to induce melanin synthesis in all experiments. The cells without α-MSH were used as negative control group, and with 50 µg/mL arbutin were used as positive control group. After medium was removed, the cells were dissolved in 200 μL of 1 N NaOH at 60°C for 1 h in the dark. The total melanin content was estimated by measuring the absorbance of cell lysis solution at 405 nm (Infinite M200 Pro microplate reader, Tecan Group Ltd., Mannedorf, Switzerland equipped at Ewha Drug Development Research Core Center. Morphology of cells was observed using a microscope (X400, ECLIPSE TS100, Nikon, Tokyo, Japan).

Cell viability assay

The cell viability was detected by 3-(4,5-dimethylthaizaol-2-yl)-2,5diphenyltetrazolium bromide (MTT) purchased from Sigma-Aldrich Co. LLC. Phenol-red free medium was used as solvent in 0.5 mg/mL MTT solution. The cells were treated with MTT for 2 h in the incubator. After removing MTT solution, 300 μL DMSO was treated to the cells with gentle shaking. After 30 min of dissolution, the absorbance of the supernatant of the solution was read at 540 nm.

Mushroom tyrosinase inhibition assay

Mushroom tyrosinase was used to measure direct inhibitory effect against tyrosinase, a key enzyme of melanogenesis. L-tyrosine and L-DOPA were used as substrates at different concentration, 0.3 mg/mL and 2 mg/mL in 0.1 M potassium phosphate. For reaction of tyrosinase, 180 μL substrates, 2 μL DMSO for blank and negative control or 2 μL MA, 30 μL 0.1 M potassium phosphate for blank or 30 μL mushroom tyrosinase (250 units for L-tyrosine, 50 units for L-DOPA) for positive control and MA, were added in sequence into a 96-well plate and mixed at 37°C with 350 rpm, using thermomixer for indicated time. The production of dopachrome was monitored by measuring the absorbance at 475 nm at regular interval times.

Cellular tyrosinase inhibition assay

B16F10 cells (2×105 cells/well) were seeded in 6-well plates for 24 h. For inducing cellular tyrosinase, the cells were treated with MA and 0.2 µM α-MSH for 24 h. For extracting the cellular tyrosinase, the medium was removed and washed with PBS for 2 times. Then, the cells were lysed with 1mL RIPA buffer (Sigma-Aldrich Co. LLC) at 4°C for 1 h in the dark. Lysed cells were scratched and centrifuge for 12000 rpm at 4°C for 30 min. The supernatant was used as cellular tyrosinase and 0.9 mg/mL L-DOPA was used as substrate for tyrosinase. In the 96-well plate, 40 μL L-DOPA and 40 μL cellular tyrosinase were added and mixed at 37°C with 350 rpm, using thermomixer for indicated time. The absorbance was read at 475 nm for indicated time. The tyrosinase activity was normalized by the total protein content which was determined by BCA assay kit (Thermo Scientific, MA, USA).

Real-time PCR

B16F10 cells were cultured for 24 h in 6-well and treated with MA and 0.2 µM α-MSH for 18 h and 24 h. The cells were lysed with TRIzol reagent (Invitrogen, CA, USA) at 4°C for 30 min. Chloroform added to lysed cells and centrifuge for 12000 rpm at 4°C for 10 min. The aqueous phase was mixed with isopropanol, and RNA pellets were condensed by centrifugation for 12000 rpm at 4°C for 15 min. RNA pellets were washed with 70% ethanol and dissolved in RNase-free, DEPC (diethylpyrocarbonate)-treated water (Invitrogen, Waltham, MA, USA). The RNA yield was measured by the optical density at 260 nm with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, INC., Wilmington, DE, USA). Relative expression levels of mRNA were determined by quantitative real-time PCR. cDNA was synthesized from 1250 ng of total RNA with oligo(dT) (Bioelpis, Seoul, Korea). SYBR Green PCR master mix and StepOnePlusTM Real-time PCR machine (Applied Biosystems, Warrington, UK) were used in every reaction. The mRNA levels of target genes were normalized to those of β-actin as a house-keeping gene.

The sequence of primers was as follows:

Forward β-actin 5’-AGG GAA ATC GTG CGT GAC AT-3’

Reverse β-actin 5’-GGA AAA GAG CCT CAG GGC AT-3’

Forward Tyrosinase 5’-GGG CCC AAA TTG TAC AGA GA-3’

Reverse Tyrosinase 5’-ATG GGT GTT GAC CCA TTG TT-3’

Forward TRP-1 5’-GTT CAA TGG CCA GGT CAG GA-3’

Reverse TRP-1 5’-CAG ACA AGA AGC AAC CCC GA-3’

Forward TRP-2 5’-TTA TAT CCT TCG AAA CCA GGA-3’

Reverse TRP-2 5’-GGG AAT GGA TAT TCC GTC TTA-3’

Western blot analysis

B16F10 cells were cultured for 24 h in 6-well plate and treated with MA and 0.2 µM α-MSH. After 48 h of exposure to the compound, B16F10 cells were homogenized in lysis buffer consisting of RIPA buffer, 1 mM phenyl methane sulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail (PIC) at 4°C for 20 min. Homogenized cells were scraped out and centrifuge for 12000 rpm at 4°C for 10 min. The supernatant of the homogenate was collected, and the protein concentration was determined by BCA assay. Prior to electrophoresis, the proteins were boiled to denature protein at 95°C for 5 min. Each of the same amount of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitro-cellulose membranes (Amersham, Buckinghamshire, UK). After being blocked with 5% BSA at room temperature for 1 h, the membranes were probed with primary antibodies against each target protein in 5% BSA at cold room for 18 h. HRP-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA) were used to detect bound primary antibodies, and the bands with antibody reaction were visualized using ECL western blotting detection reagents (Amersham Biosciences, Little Chalfont, UK) and the Amersham Imager 600 (GE Healthcare Life Science, Chicago, IL, USA). The β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a control.

cAMP ELISA assay

cAMP levels in cellular lysates of B16F10 cells were calculated using a complete ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA) based on the manufacturer’s instructions and previous reports (Goenka and Simon, 2020), following a non-acetylated substrate protocol. The concentration of cAMP was normalized by the total protein content which was determined by BCA assay kit.

Brightening assay with the pigmented human epidermis model, MelanodermTM

MelanodermTM (MatTek, MA, USA) is an 3D human epidermis consisting of normal human epidermal keratinocytes and normal human melanocytes. After pre-incubating MelanodermTM for 24 h, MA treated to the tissues every other day for 16 days. The brightening effect was measured by ΔL values of the tissues using Adobe Photoshop CC 2015 software (Adobe, San Jose, CA, USA). On the 16th day of experiment, the tissues were fixed by phosphate-buffered formalin and stained with hematoxylin and eosin (H&E) and fontana-masson (FM).

Statistics analysis

Data was presented as mean ± standard deviation (SD) of 3 times of independently replicated experiments. The statistical analysis was evaluated by a two-sided Student’s t-test. If p-value of the results was less than 0.05, the data was considered significant.

RESULTS

The effect of MA on melanogenesis and cell viability of B16F10 cells

B16F10 cells were treated with MA (chemical structure; Fig. 1) at concentrations ranging from 10 to 50 µg/mL for 72 h in the presence of α-MSH stimulation. Untreated cells were used as negative controls and arbutin (50 µg/mL) served as the positive control. The results showed that MA treatment did not exhibit significant cytotoxicity, with cell viability remaining above 80% (Fig. 2A). On the other hand, MA displayed a dose-dependent inhibition of melanin content in the cells (Fig. 2B), as observed through a reduction in melanin synthesis and dendrite extension stimulated by α-MSH (Fig. 2C) and confirmed by visual observation of the cell pellets (Fig. 2D). These results indicate that MA has the potential to be an effective anti-melanogenic agent without causing severe cytotoxicity.

Figure 1. The structure of methyl anthranilate.

Figure 2. The effect of MA on cell viability and melanin contents of α-MSH (0.2 µM)-treated MNT-1 cells. α-MSH-untreated cells were used as negative controls, and arbutin-treated cells were used as positive controls. MA (10-150 µg/mL) and arbutin (50 µg/mL) were treated to MNT-1 cells for 72 h. (A) Cell viability was determined by MTT assay. (B) Melanin contents were measured by melanin assay. (C) Melanogenesis was observed under optical microscopy (10×). Scale bar is 100 µm. Data are presented as the mean ± SD (n=3, ##p<0.01 compared with the control group; **p<0.01 and *p<0.05 compared with the α-MSH-treated group).

MA inhibited melanogenesis in MNT-1 human melanoma cells

The inhibitory effect of MA on melanogenesis was also examined in human melanocyte cell line, MNT-1. α-MSH-stimulated MNT-1 cells were treated with 10 to 150 µg/mL of MA for 72 h. MA reduced the melanin contents in MNT-1 cells in a dose-dependent manner without having a significant impact on cell viability up to 50 µg/mL (Fig. 3 A, B). The suppression of melanogenesis was also visually confirmed through observation under an optical microscope (Fig. 3C).

Figure 3. The effect of MA on cell viability and melanin contents of α-MSH (0.2 µM)-treated B16F10 cells. (A) Cell viability was determined by MTT assay. (B) Melanin contents were measured by melanin assay. (C) Melanin is stained dark black with FM stain. Red arrows indicate dendrite formation from melanocytes. Scale bar is 50 µm. (D) Macroscopic view of B16F10 cell pellets. Data are presented as the mean ± SD (n=3, #p<0.05 compared with the control group; ***p<0.001, **p<0.01 and *p<0.05 compared with the α-MSH-treated group).

Dendrite formation in co-culture system of MNT-1 and HaCaT cells

The transfer of melanosomes, which contain melanin, from melanocytes to keratinocytes occurs through dendrites (Seiberg, 2001; Ando et al., 2012). In order to investigate the impact of MA on this transfer process, a co-culture of MNT-1 and HaCaT cells was established in a 1:5 ratio and treated with α-MSH to activate the dendrite formation. The co-culture was then treated with MA at concentrations of 10 to 50 µg/mL for 24 h. The extension of dendrites was visualized by staining with L-DOPA (1 mg/mL). The cell viability (Fig. 4A) and melanin content (Fig. 4B) were also assessed using the same methods described above for the monoculture. While α-MSH prolonged the dendrites from MNT-1 cells, the increasing concentration of MA reduced the dendrite extension (Fig. 4C).

Figure 4. Tyrosinase enzymatic activity assay. (A, B) The effect of MA on mushroom tyrosinase activity. Tyrosinase enzymatic activity was measured using mushroom tyrosinase assay with (A) L-tyrosine and (B) L-DOPA as substrates. (C) The effect of MA on cellular tyrosinase activity was measured using L-DOPA as substrate. The cells were treated with MA (10-50 µg/mL) and arbutin (50 µg/mL) for 24 h. Data are presented as the mean ± SD (##p<0.01 compared with the control group).

The effect of MA on tyrosinase inhibition

To determine the mechanism behind the anti-melanogenic effect of MA, we conducted a tyrosinase activity assay. Tyrosinase is a key enzyme involved in melanin formation, so we used both cell-free mushroom tyrosinase and cellular tyrosinase from B16F10 cells to test whether MA could regulate tyrosinase activity. As shown in Fig. 5A and 5B, MA was not a potent inhibitor of mushroom tyrosinase activity. Although MA reduced tyrosinase activity at 150 µg/mL when L-tyrosine was used as a substrate, this effect was not significant compared to arbutin, which served as a positive control. When L-DOPA was used as a substrate, both MA and arbutin were ineffective. The cellular tyrosinase assay (Fig. 5C) produced similar results to the mushroom tyrosinase assay using L-tyrosine as a substrate (Fig. 5A), indicating that MA does not directly inhibit tyrosinase activity.

Figure 5. Dendrite formation changes in MNT-1/HaCaT (1:5) co-culture cells. The cells were treated with MA (10-50 µg/mL) and arbutin (50 µg/mL) for 24 h. (A) Cell viability was determined by MTT assay. (B) Melanin contents were measured by melanin assay. (C) Co-culture of cells were stained with L-DOPA. Morphological changes were observed under optical microscopy (10×). Scale bar is 100 µm. Data are presented as the mean ± SD (n=3, **p<0.01 and *p<0.05 compared with the α-MSH-treated group).

The effect of MA on mRNA expression of melanogenic enzymes

In order to investigate the effect of MA on melanogenic enzyme transcription, the mRNA levels of tyrosinase, TRP-1, and TRP-2 were analyzed using quantitative real-time PCR. α-MSH-stimulated B16F10 cells were treated with 25 to 50 µg/mL of MA for either 18 or 24 h. The results showed that MA effectively downregulated the mRNA levels of the three enzymes in response to α-MSH stimulation (Fig. 6). Notably, the mRNA levels of TRP-1 and TRP-2 were significantly and strongly suppressed after both 18-h (Fig. 6B, 6C) and 24-h (Fig. 6E, 6F) treatment with MA. Interstingly, the mRNA level of tyrosinase was not significantly impacted by MA, as compared to the positive control (Fig. 6A, 6D).

Figure 6. The effect of MA on the mRNA level of B16F10 cells. mRNA expression levels of (A, D) tyrosinase; (B, E) TRP-1; (C, F) TRP-2 in B16F10 cells were determined by real-time PCR. The cells were treated with MA (25-50 µg/mL) and bisabolol (50 µM) for (A-C) 18 h and (D-F) 24 h. Data are presented as the mean ± SD (n=3, #p<0.05 compared with the control group; *p<0.05 compared with the α-MSH-treated group).

The effect of MA on melanogenesis-related proteins and cAMP

The reduction of melanogenic enzyme protein levels was further substantiated by western blot analysis. B16F10 cells stimulated by α-MSH were treated with MA at concentrations of 25 to 50 µg/mL for 48 h. As depicted in Fig. 7, the protein levels of TRP-1 and TRP-2 were significantly reduced in comparison to the group treated with α-MSH only. While the tyrosinase protein expression was reduced by bisabolol, a positive control, MA only had a minor impact on the tyrosinase protein level, consistent with the mRNA expression data.

Figure 7. The effect of MA on melanogenic enzymes protein levels in B16F10 cells. Tyrosinase, TRP-1 and TRP-2 protein levels were determined by western blotting. (A) Tyrosinase, TPR-1 and TRP-2 protein levels; (B) Quantitation of tyrosinase, TRP-1 and TRP-2 protein level; The cells were treated with MA (25-50 µg/mL) and bisabolol (50 µM) for 48 h. The quantitation of the western blot band was analyzed by image J software. Data are presented as the mean ± SD (n=3, #p<0.05 compared with the control group; **p<0.01 and *p<0.05 compared with the α-MSH-treated group).

cAMP is a key regulating factor in the upstream of melanogenesis mechanism. The activation of α-MSH leads to an increase in cAMP levels via adenylate cyclase (AC), which in turn triggers the signaling pathway in melanocyte, leading to the activation of tyrosinase, TRP-1 and TRP-2 (Cheli et al., 2009). Therefore, we investigated the effect of MA on the cAMP level. α-MSH-stimulated B16F10 cells were treated with 10 to 50 µg/mL of MA for 72 h, and the results showed that MA significantly downregulated the cAMP level in a dose-dependent manner (Fig. 8).

Figure 8. cAMP levels in B16F10 cells. Cells were treated with MA (10-50 µg/mL) and bisabolol (50 µM) for 72 h. Concentration of cAMP was measured by ELISA. Data are presented as the mean ± SD (n=3, #p<0.05 compared with the control group; **p<0.01 compared with the α-MSH-treated group).

Anti-pigmentary effect on the pigmented human epidermal skin model, MelanodermTM

The pigmented human epidermal skin model is widely used to assess the anti-pigmentation effects and safety of cosmetic ingredients in a simulated in-vivo environment (Kim et al., 2017). In this study, Melanoderm™, an artificially reconstructed pigmented human epidermal skin model composed of normal human keratinocytes and melanocytes, was used to evaluate the anti-pigmentation effect of MA. The model was treated with MA and kojic acid (as a positive control) every other day for 16 days, and the color change of the tissues was photographed and the ΔL value (the change in brightness from Day 0) was measured. As depicted in Fig. 9A, the untreated tissue showed an increase in darkness by Day 16 as compared to Day 0. In contrast, MA treatment resulted in lighter tissue color and reduction in pigmentation, as indicated by the reduced ΔL value (Fig. 9B). On the final day of the experiment, the tissue was stained with hematoxylin and eosin (H&E) and Fontana-Masson (FM) (Fig. 9C). The relative area of melanocytes and the number of melanocytes within the stained tissue were also calculated (Fig. 9D), providing further support for the anti-melanogenic effects of MA.

Figure 9. The effect of the MA on the Melanoderm™, 3D human pigmented epidermis model. Melanoderm™ was treated with MA (2,500-5,000 µg/mL) and kojic acid (10,000 µg/mL) every other day for 16 days. (A) Color of the 3D human skin tissue model on Day 0 and Day 16; (B) Degree of lightness of the tissues was measured by the ∆L value compared with the lightness of tissues on Day 0. (C) Tissues on Day 16 were stained with H&E and FM. (D) The relative area of melanocytes and the number of melanocytes of stained tissues were calculated by image J software. Data are presented as the mean ± SD (n=3, ###p<0.001, ##p<0.01, and #p<0.05 compared with the control group).
DISCUSSION

Skin pigmentation is determined by the amount of melanin synthesized within melanosomes and transferred to keratinocytes (Hurbain et al., 2018). Abnormal regulation of melanin synthesis can lead to hyperpigmentation and result in various skin disorders (Yamaguchi and Hearing, 2014). Preventing excessive melanogenesis and controlling the distribution of melanosomes are crucial for avoiding pigmentation-related skin problems. The synthesis of melanin in melanocytes is controlled by the tyrosinase gene family of proteins, including tyrosinase, TRP-1, and TRP-2 (Hearing et al., 1998). Substances that can suppress the expression of melanogenic enzymes and related factors can therefore be used as skin-lightening agents.

In this study, we explored the potential of methyl anthranilate (MA) as a new cosmetic ingredient, with a primary focus on its anti-pigmentation effect. Both B16F10 cells and MNT-1 cells were used to determine the antipigmetary effect of MA, and the results showed that MA has the potential to reduce melanin contents with minimal effect on cell viability.

The formation of dendrites in melanocytes is essential for the transfer of melanosomes to keratinocytes (Kim and Lim, 2023). Therefore, the inhibition of dendritic formation presents another strategy to validation the antipigmentary effect of MA (Lee et al., 2019). In this study, MA was applied to the MNT-1/HaCaT co-culture system to mimic in vivo like situation. Since the co-culture system contained fewer melanocytes, which are responsible for melanin production, the inhibitory effect on melanin content was lower as compared to the MNT-1 monoculture result. Notably, the presence of MA suppressed α-MSH-induced dendrites, suggesting that MA has an antipigmentary effect in MNT-1/HaCaT co-culture system. Tyrosinase is a pivotal melanogenic enzyme in melanin synthesis pathway. Therefore, we conducted the enzymatic activity assay to validate antipigmentary effect of MA. The results showed that MA inhibited cellular tyrosinase activity, while mushroom tyrosinase activity remained unaffected.

Real-time PCR and Western blot assays were performed to determine the effect of MA on the transcription and translation of melanogenic enzymes, including Tyrosinse, TRP-1, and TRP-2. In this study, well-known antipigmentation agents, arbutin, and α-Bisabolol, were used as positive control (Lee et al., 2010; Kim et al., 2021a). The real-time PCR results showed that MA had an inhibitory effect on the expression of three enzymes, but TRP-1 and TRP-2 were more effectively downregulated than tyrosinase. A dose-dependent inhibitory effect was observed at 18 h, with 25 µg/mL of MA having a similar effect to 50 µM of bisabolol. However, at 24 h, the dose-dependent effect was not observed for all enzymes, suggesting that the inhibitory effect had already reached saturation before 24 h. The results of the western blot assay, which was used to evaluate protein expression, were in line with those of the real-time PCR assay at 18 h. Despite not showing a clear impact on tyrosinase, MA demonstrated a more robust downregulation of TRP-1 and TRP-2 compared to the positive control, bisabolol. These results indicated that MA has potential antimelanogenesis activity superior to that of the positive controls.

α-MSH binds to the melanocyte melanocortin receptor type 1 (MCR1) leading to the activation of adenylyl cyclase (Cui et al., 2007; Lee et al., 2019). This elevates cAMP levels in melanocytes, and activates protein kinase A (PKA), cAMP-response element-binding protein (CREB), which culminates in upregulation of the microphthalmia associated transcription factor (MITF) (García-Borrón et al., 2014). Upregulation of MITF enhances the expression of melanogenic enzymes, including tyrosinase, TRP-1, and TRP-2. In this study, MA effectively reduced cAMP levels as compared to the positive control. These results suggest that MA is highly effective in inhibiting cAMP, which may explain the anti-melanogenic effects of MA.

MelanodermTM is a pigmented human epidermal skin model derived from combination of primary human melanocytes and keratinocytes, making it a commonly used model for studying skin whitening effects in various studies (Lee et al., 2019; Kim et al., 2021b). In this study, MA was applied on Melanoderm™ skin model over a period of 16 days, and the color of the skin tissue was lightened with the treatment of MA as compared to untreated skin model. This was further confirmed by a reduction in melanin levels in H&E and FM-stained tissue, as seen in the calculation of the melanin area and amount.

In summary, we have demonstrated the antipigmentary effect of MA for the first time, using both 2D cell lines and an artificial human epidermal skin model. MA was shown to decrease dendrite elongation and reduce the expression of melanogenic enzymes. Although the inhibitory effect on tyrosinase activity and gene expression was limited, MA demonstrated significant inhibitory effects on TRP-1 and TRP-2, as well as cAMP, which is a key upstream regulator in melanogenesis. While additional studies are needed to establish a clear link between the decrease in cAMP and the downregulation of tyrosinase, TRP-1, and TRP-2, the observed reduction in cAMP levels may have contributed to the inhibition of melanosome maturation. This is because cAMP plays a critical role in regulating the maturation process of melanosomes (Goenka and Simon, 2020). Collectively, our findings indicate that MA holds potential as a promising novel cosmetic ingredient for skin brightening.

ACKNOWLEDGMENTS

This work was supported by grants from National Research Foundation of Korea (Grant No. 2021R1A2C2013347, 2021R1A6C101A442 and MSIT 2018R1A5A2025286).

CONFLICT OF INTEREST

None.

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  • National Research Foundation of Korea
      10.13039/501100003725
      2021R1A2C2013347, 2021R1A6C101A442, MSIT 2018R1A5A2025286

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