Glossogyne tenuifolia Essential Oil Prevents Forskolin-Induced Melanin Biosynthesis via Altering MITF Signaling Cascade

Abstract

Glossogyne tenuifolia (Labill.) Cass. ex Cass (Compositae) is a herbaceous plant that is endemic to Taiwan. Traditional Chinese Medicine has utilized it as a treatment for fever, inflammation, and liver preservation. Recent research has unveiled its bioactivities, including anti-inflammation, anti-cancer, antiviral, antioxidant, anti-fatigue, hepatoprotection, and immune modulation elements. Nevertheless, its effect on skin health remains to be investigated. Thus, we investigated the impact of G. tenuifolia essential oil (GTEO) on forskolin (FRK)-induced melanin biosynthesis and its mechanisms in B16-F10 murine melanoma in vitro. Treatment of GTEO resulted in a substantial decrease in FRK-induced melanin production, accompanied by a significant decrease in tyrosinase mRNA and protein expression levels. Additionally, our data demonstrated that the decrease in tyrosinase expression resulted from the suppression of MITF, as indicated by the reduced movement of MITF into the cell nucleus. Moreover, GTEO prompted a prolonged ERK1/2 activation, leading to the decline of MITF through proteasomal degradation, and it was verified that GTEO had no inhibitory impact on MITF activity in ERK1/2 inhibitor-treated cells. Additional studies demonstrated that α-pinene and D-limonene, which are the primary components in GTEO, showed strong melanin and tyrosinase inhibitory effects, indicating that α-pinene and D-limonene may contribute to its anti-melanogenic effects. Collectively, these data presented compelling proof that GTEO, along with its primary components α-pinene and D-limonene, show great potential as natural sources for developing innovative skin-whitening agents in the field of cosmetics.

1. Introduction

Melanin is involved in pigmentation in the skin of a diverse range of animal species. Melanin is an inherent pigment that gives color to the skin, eyes, and hair while also playing a crucial function in safeguarding the skin from potential harm, including from damaging light like ultraviolet rays [1,2]. Despite its beneficial effects, abnormalities in melanin synthesis and regulation give rise to pigment-related conditions like albinism, vitiligo, melasma, freckles, and lentigo [3]. Various elements like intracellular pH shifts, prolonged exposure to UV radiation, and the aging process can disturb the usual synthesis of melanin, resulting in its buildup within the epidermis [1]. As an example, exposure to UV radiation activates melanocytes, leading to irregular melanin production and distribution [4].

The basal layer of the epidermis contains special cells called melanocytes, which are responsible for producing melanin. This progression entails a sequence of enzyme-driven actions, predominantly facilitated by enzymes like tyrosinase, TYRP-1, and TYRP-2 [5]. Among these, tyrosinase is crucial in the two fundamental stages of melanin production; first L-tyrosine is hydroxylated into L-3,4-dihydroxyphenylalanine (L-DOPA), which is oxidized into dopaquinone and transformed into dopachrome. Furthermore, TYRP-2 carries out the transformation of L-dopachrome into DHICA, and TYRP-1 facilitates the conversion of DHICA into IQCA. It is via this sequence of processes that eumelanin is synthesized [6]. It is possible to reduce skin pigmentation in several ways with the use of a depigmentation agent, including hindering the functioning of the tyrosinase family and (2) also preventing ROS generation when exposed to UV radiation [3,7]. Among them, inhibiting tyrosinase activity is a promising strategy for controlling excessive melanin production [8].

Plant volatiles, commonly referred to as essential oils, are commonly used in modern cosmetic products because they contain various active ingredients, potent fragrances, and appealing marketing images. Recently, essential oils and their constituents have garnered significant public interest, owing to their broad consumer acceptance and versatile functional applications [9,10]. Increasing scientific evidence supports the utilization of essential oils for addressing various skin disorders, including acne, premature skin aging, and hyperpigmentation, and offering a defense against UV radiation [11,12]. Essential oils have been identified for their strong in vitro anti-melanogenic effects [13,14].

Glossogyne tenuifolia (Labill.) Cass. ex Cass is a herb that is indigenous to Penghu Island in Taiwan, and it is also present in various regions across Asia and Australia. Herbal teas made from this herb have been traditionally utilized by Penghu Islanders to treat conditions such as fever, hepatitis, and inflammation [15]. Recent scientific research has uncovered the diverse range of bioactive attributes present in different extracts of G. tenuifolia. These attributes include antimicrobial, anti-inflammatory, anti-fatigue, antioxidant, antiviral, anti-angiogenesis, anti-cancer, hepatoprotective, and immune-modulating effects [16,17,18,19,20,21,22]. Additionally, Chyau et al. [23] employed concurrent steam distillation and solvent extraction methods to isolate essential oil from G. tenuifolia, yielding 62 distinct compounds. The primary components of G. tenuifolia essential oil are primarily terpenes. In one of our recent studies, we extracted essential oil from G. tenuifolia using the steam distillation method, yielding 21 compounds which make up 95.95% of the entire oil. Among them, p-cymene, β-myrcene, β-cedrene, β-ocimene, α-pinene, and D-limonene were index compounds of GTEO with concentrations of 35.5%, 14.68%, 9.8%, 8.49%, 6.69%, and 5.17%, respectively. Furthermore, we demonstrated that G. tenuifolia essential oil (GTEO) possesses strong anti-inflammatory activity against lipopolysaccharide-induced inflammatory responses in macrophage cells [19]. Based on our current understanding, the effect of G. tenuifolia essential oil (GTEO) on skin health, including skin protection and hypo-pigmentary effects, are unexplored. Consequently, we aimed to examine the influence of GTEO on melanin synthesis induced by forskolin in murine melanoma (B16-F10) cells and to uncover the underlying mechanism.

2. Materials and Methods

2.1. Chemicals and Reagents

Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin were obtained from the Life Technologies Corporation (Grand Island, NY, USA). Forskolin (FRK) was acquired from Selleckchem (Houston, TX, USA. Tyrosinase (EC 1.14.18.1), L-tyrosine, L-3,4-dihydroxyphenylalanine (L-DOPA), 4′-6-diamidino-2-phenylindole (DAPI), and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), resveratrol, gallic acid, kojic acid (KA), and ascorbic acid (AA) were procured from Sigma–Aldrich, St. Louis, CA, USA. PD98059, an ERK1/2 inhibitor was obtained from Calbiochem (La Jolla, CA, USA). Antibodies targeting tyrosinase and GAPDH were acquired from Santa Cruz Biotechnology, Dallas, TX, USA. Cell Signalling Technology (Danvers, MA, USA) provided horseradish peroxidase (HRP)-linked antibodies against mouse IgG and rabbit IgG. All chemicals, except for those specified, were of high quality and provided by either Sigma–Aldrich or Merck (Darmstadt, Germany).

2.2. Cell Culturing and Viability Testing

A B16-F10 cell line was obtained from the American Type Culture Collection (ATCC) in Manassas, VA, USA. The cells were cultivated in RPMI-1640 and supplemented with 10% FBS, penicillin, and streptomycin and incubated in a humidified environment with 5% CO₂ at a temperature of 37 °C. Cell viability was evaluated using the MTT colorimetric test. Briefly, B16-F10 cells were placed in a 96-well culture plate at a concentration of 1 × 10⁴ cells/well. Following a 24 h incubation period, cells were exposed to increasing doses of GTEO (12.5–200 µg/mL) or pure compounds (25–200 μM) for an additional 24 h. The culture media was removed, and MTT (1 mg/mL) in 200 μL of fresh culture media was introduced. The MTT farmazon crystals were dissolved in 200 μL of DMSO. The optical density of the test samples was measured at a wavelength of 570 nm (A₅₇₀) using an ELISA microplate reader. The cell viability percentage (%) was determined by multiplying the ratio of the absorbance of treated cells to the absorbance of untreated cells by 100.

2.3. Quantification of Melanin Formation and Fontana-Masson Staining

A melanin formation assay and Fontana-Masson staining were conducted following previously established procedures [24]. Concisely, cells were placed in a 6-well cell culture plate with a density of 1 × 10⁵ cells/well. Following a 24 h incubation period, cells were exposed to FRK (20 μM) for 48 h either with or without the presence of GTEO, KA, α-pinene, p-cymene, and D-limonene. Following the treatment, the cells were gathered and washed two times with PBS and the melanin was dissolved in 1 N NaOH. The mixture was then heated at a temperature of 68 °C for 20 min. Cellular melanin content was determined by quantifying the optical density at a wavelength of 475 nm, measured using an ELISA microplate reader. To detect melanin pigment, B16F10 cells were cultured on an 8-well chamber slide (ibidi GmbH, Gräfelfing, Germany). Following 48 h of treatment, the cells were fixed with formalin. Subsequently, they were stained using the Fontana-Masson ammoniacal silver staining method. In summary, the cells were immersed in an ammoniacal silver solution overnight, followed by fixation in an acid-fixing solution and staining with Kernechtrot. Images were captured using an inverted microscope (Motic Electric Group, Xiaman, Fujian, China) at 20× magnification.

2.4. Determination of Cellular Tyrosinase Activity

To assess the activity of tyrosinase in cells, cells were seeded at 1 × 10⁵ per dish in 6 cm dishes. Following a 24 h incubation period, cells were exposed to FRK (20 μM) for 48 h, either with or without the presence of GTEO, KA, α-pinene, p-cymene, and D-limonene. Following treatment, the cells were lysed using a lysis buffer. After centrifugation at 16,000× g for 10 min, a clear liquid (supernatant) was obtained. A 96-well plate was filled with 90 μL of each lysate, which contained 100 μg of protein in total. Following that, 10 µL of 15 mM L-DOPA was added to every well. An ELISA microplate reader was used to quantify the production of dopachrome at 475 nm after a 20 min incubation time at 37 °C.

2.5. Mushroom Tyrosinase Inhibition Assay

L-tyrosine and L-DOPA were used as substrates for mushroom tyrosinase assay. We investigated the inhibitory activity of GTEO against the oxidation of L-tyrosine catalyzed by tyrosinase, as described in our previous research [24]. Concisely, 1.5 mM L-tyrosine substrate (40 μL) was mixed with 120 μL of 100 mM phosphate-buffer solution. In this mixture, 20 μL of various concentrations of GTEO (25–100 μg/mL) or kojic acid (KA, 40 μM) were introduced. Subsequently, mushroom tyrosinase 2000 U/mL in 20 μL was added to initiate the reaction. After that, the mixture was allowed to incubate for a duration of 15 min at 37 °C, and then the absorbance was measured at 475 nm, which indicates the formation of dopachrome. Furthermore, the effect of GTEO and KA on the activity of mushroom tyrosinase in the oxidation of L-DOPA was evaluated. A mixture containing 100 μL of 0.1 M phosphate buffer and 20 μL of various concentrations of GTEO (25–100 μg/mL) or KA (40 μM) was prepared. This mixture was incubated with 20 μL of mushroom tyrosinase (2000 U/mL in phosphate buffer). After five minutes of incubation at 37 °C, 40 μL of L-DOPA (4 mM in 0.1 M of phosphate buffer) was added to the mixture. The mixture was then incubated for a further 10 min at 37 °C, and the absorbance of the reaction mixture was measured at 475 nm. Using the following formula, the percentage of the inhibition of L-tyrosine or L-DOPA oxidation was determined: percentage inhibition = 100 − (B/A × 100), where A = ^ΔOD₄₇₅ over 10 min without the sample, and B = ^ΔOD₄₇₅ over 10 min with the tested sample.

2.6. Protein Extraction and Immunoblotting

Cellular protein was extracted using RIPA buffer (Pierce Biotechnology, Rockford, IL, USA), and the protein concentration was measured using the Bradford dye-binding method with Bio-Rad reagents. Afterward, to separate protein samples (100 µg), 8–12% SDS-PAGE was used, followed by transfer onto a PVDF membrane. Following the transfer, protein membranes were blocked with 5% non-fat skim milk at room temperature for 30 min, then left to incubate overnight with particular primary antibodies at 4 °C. The membranes were then probed for 2 h using HRP-conjugated anti-rabbit or anti-mouse antibodies. Enhanced chemiluminescence (ECL) reagents (Advansta Inc., San Jose, CA, USA) were used to visualize immunoblots, and the ChemiDoc XRS⁺ docking system was utilized to capture pictures. Image lab software version 6.0.1 from Bio-Rad Laboratories was used to perform a quantitative analysis of the protein bands.

2.7. RNA Extraction and Q-PCR Analyses

The GeneMark Total RNA Purification Kit (GeneMark, New Taipei City, Taiwan) was used to extract total RNA. A First-Strand Synthesis Kit (SuperScript^TM IV; Invitrogen, Waltham, MA, USA) was used to transcribe 2 μg of isolated total RNA into cDNA. Then, employing the Applied Biosystems Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and Power SYBR Green Master Mix (Applied Biosystems), the levels of mRNA expression were measured. The qPCR reaction was carried out as follows: initial denaturation at 96 °C for 3 min was followed by 40 cycles of denaturation at 96 °C for 1 min, annealing at 50 °C for 30 s, and extension at 72 °C for 90 s. The qPCR primer sequences for each gene were as follows: Tyrosinase—forward primer (F), 5′-TATTGAGCCTTACTTGGAAC-3′; reverse primer (R), 5′-AAATAGGTCGAGTGAGGTAA-3′, and GAPDH—forward primer (F), 5′-TCAACGGCACAGTCAAGG-3′; reverse primer (R), 5′-ACTCCACGACATACTCAGC-3′. The quantity of each transcript was assessed by computing the relative copy number, which was adjusted based on the GAPDH copy number. Using the ^2ΔCt cycle threshold method, the relative abundance of the target mRNA in each sample was determined by calculating the ^ΔCt values of the target and the endogenous reference gene GAPDH.

2.8. Statistical Analysis

The results are shown as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism version 6.0 for Windows (GraphPad Software, La Jolla, CA, USA). Statistical assessment was conducted using a one-way ANOVA followed by Dunnett’s test for multiple comparisons. p values of less than 0.05 *, 0.01 **, and 0.001 *** were considered statistically significant for the FRK vs. sample treatment groups. Additionally, p values of less than 0.001 ^$$$ were considered statistically significant when evaluating the FRK in comparison to the control group.

3. Results

3.1. The Effect of GTEO on Melanin Biosynthesis

Before examining GTEO’s anti-melanogenic properties, the cytotoxicity of murine melanoma (B16-F10) was examined. Cells were treated with various doses of GTEO (12.5, 25, 50, 100, and 200 μg/mL) for 48 h and cell viability was determined by MTT testing. GTEO did not show any detectable reduction in cell viability with doses of 100 μg/mL, while 200 μg/mL demonstrated a substantial decrease in cell viability (Figure 1A). Therefore, the non-cytotoxic concentrations of GTEO (12.5, 25, 50, and 100 μg/mL) were subjected to further studies. Next, the melanin inhibitory effect of GTEO was examined through FRK-induced melanogenesis in B16-F10 cells.

The cells were treated with various concentrations of GTEO (25–100 µg/mL) or 40 μM KA in combination with FRK (20 μM) for 48 h. The intracellular melanin content was determined by the colorimetric method. Figure 1B demonstrates the cellular melanin content in FRK-stimulated cells, which exhibited a significant increase from 3.1 µg/mL (baseline 100%) to 8.54 µg/mL (246%). However, when treated in combination with GTEO, there was a significant decrease in the melanin content in B16-F10 cells caused by FRK, which was reduced in a dose-dependent manner. The reduction was observed at concentrations of 5.22 µg/mL (172%), 3.84 µg/mL (165%), and 2.56 µg/mL (82%) with GTEO doses of 25 µg/mL, 50 µg/mL, and 100 µg/mL, respectively. Notably, the melanin inhibition produced by GTEO (100 μg/mL) was highly comparable with KA (40 μM), a known melanin inhibitor. Additionally, microscopic observation of B16-F10 cells stained with the Fontana-Masson (FM) method highlights increased intracellular melanin accumulation in FRK-induced cells, as indicated by unstained dark regions. However, melanin content in B16-F10 cells was considerably and dose-dependently decreased by co-treatment with GTEO. This result further supports the notion that GTEO inhibits melanin biosynthesis and dendrite formation stimulated by FRK (Figure 1C).

3.2. GTEO Inhibits Mushroom Tyrosinase Activity

Tyrosinase serves as a crucial enzyme controlling the pace of melanin biosynthesis. Thus, we proceeded to investigate whether GTEO influences the activity of mushroom tyrosinase in a system devoid of cells, using L-tyrosine and L-DOPA as substrates. As depicted in Figure 2A, when mushroom tyrosinase was incubated with L-tyrosine, it resulted in high enzyme activity, as indicated by rapid browning of the mixture, whereas co-incubation with GTEO significantly reduced the activity of mushroom tyrosinase in a dosage-dependent manner. Additionally, mushroom tyrosinase with L-DOPA produced high enzyme activity that was significantly and dose-dependently inhibited by GTEO (Figure 2B). Indeed, the mushroom tyrosinase inhibitory activity of GTEO was highly comparable with ascorbic acid (AA). Taken together, these data suggest that GTEO could inhibit tyrosinase during both monophenolase and diphenolase activities. This finding indicates that GTEO possessed a strong tyrosinase inhibitory effect.

3.3. GTEO Inhibits Cellular Tyrosinase Activity and Expression

Several anti-melanogenic agents that inhibit melanin production have been found to directly inhibit tyrosinase and disrupt melanin production in melanocytes. To further elucidate the mechanism of inhibition, GTEO was tested on FRK-stimulated cellular tyrosinase activity. Figure 3A demonstrates that the cellular tyrosinase activity increased significantly to 302.5% after FRK stimulation. Nevertheless, co-administration of GTEO caused a reduction in cellular tyrosinase activity to 230.0%, 167.9%, and 127.7% at doses of 25, 50, and 100 µg/mL, respectively. Moreover, GTEO exhibited a similar inhibitory effectiveness as kojic acid (KA). These findings suggest that GTEO effectively inhibits intracellular tyrosinase activity. To further comprehend how GTEO inhibits melanin production and tyrosinase activity, we sought to evaluate its impact on protein expression level of tyrosinase. As depicted in Figure 3B,C, after FRK stimulation, tyrosinase expression notably surged by 7.6-fold compared to control cells. Nonetheless, co-treatment of GTEO substantially decreased tyrosinase expression, nearly returning to basal levels (1.9-fold) at a concentration of 100 µg/mL. To delve deeper into understanding how GTEO reduces tyrosinase protein expression, we continued by examining the mRNA expression levels of tyrosinase using q-PCR. Figure 3D shows that the tyrosinase mRNA level was markedly increased after FRK-stimulation to 5.1-fold. Interestingly, co-treatment with GTEO altered the mRNA expression level of tyrosinase. Indeed, the positive drug control galic acid (GA) significantly inhibited tyrosinase in FRK-stimulated cells, which is highly comparable with GTEO. These findings indicate that GTEO could disrupt tyrosinase at both transcriptional and translational stages.

3.4. GTEO Inhibits Melanin Biosynthesis via MITF Signaling Pathway

MITF, a transcription factor, is responsible for transcribing tyrosinase and its associated genes [25]. Thus, our subsequent objective was to investigate whether GTEO influences MITF at transcriptional and translational levels. Our study revealed that increased MITF mRNA expression was observed in FRK-stimulated cells. However, GTEO treatment resulted in a significant and dose-dependent decrease in MITF expression (Figure 4A). Additionally, treatment with GTEO significantly and dose-dependently reduced the FRK-induced MITF protein levels (Figure 4B). These data suggest that GTEO could affect MITF transcriptional activity. Therefore, we sought to examine whether GTEO modulates the nuclear translocation of MITF through the use of immunofluorescence. Figure 4C demonstrates that FRK-stimulated cells exhibited enhanced nuclear exportation of MITF, as indicated by the increased nuclear accumulation of MITF in FRK-stimulated cells. Remarkably, the concurrent administration of GTEO effectively prevented the FRK-mediated nuclear traslocation of MITF. To clarify the impact of GTEO-mediated suppression of MITF and melanin biosythesis, MITF siRNA was transiently transfected into cells for 6 h. Subsequently, the cells were exposed to FRK for 48 h, either with or without the presence of GTEO. FRK-treated cells had a substantial reduction in melanin synthesis following MITF siRNA transfection. Cells treated with GTEO also showed a comparable inhibition, which was further reduced when combined with GTEO and MITF-specific siRNA (Figure 4D). These findings indicate that GTEO hinders the process of melanogenesis by inhibiting the MITF signaling pathway.

3.5. GTEO Suppress MITF Activity through ERK1/2 Activation

ERK1/2 is a crucial factor in melanogenesis, as it regulates the activation of MITF. ERK1/2 activation results in the degradation of MITF through ubiquitination, which is crucial for the suppression of melanogenesis. Consequently, we tried to investigate whether GTEO controls MITF activity via the ERK1/2 pathway. The application of GTEO resulted in a dose-dependent and substantial increase in ERK1/2 phosphorylation, as demonstrated by Western blot analysis (Figure 5A). Based on these observations, we speculate that MITF degradation by proteasomes may be triggered by GTEO-mediated ERK1/2 phosphorylation. As anticipated, GTEO failed to inhibit FRK-stimulated MITF activity in PD98059, a pharmacological inhibitor of ERK1/2 pre-treated cells (Figure 5B). To further clarify, we investigated the impact of GTEO on FRK-induced melanin synthesis under ERK1/2 inhibition. ERK1/2 inhibition exhibited a notable augmentation in melanin synthesis, surpassing that of FRK-treated cells. Surprisingly, the addition of GTEO did not prevent the increase in melanin synthesis caused by FRK in cells treated with an inhibitor of ERK1/2 (Figure 5C).

3.6. Effect of Major Compounds of GTEO on Melanin Production and Cellular Tyrosinase Activity

Initially, the cytotoxic effects of α-pinene, β-cedrene, β-myrcene, β-ocimene, p-cymene, and D-limonene were quantified by MTT assay. Indeed, p-cymene or α-pinene did not display a cytotoxicity against B16-F10 cells with a maximum tested dose (100 μM) for 48 h, whereas β-cedrene and β-ocimene displayed cytotoxicity starting from 25 μM. At the same time, β-myrcene and D-limonene exhibited cytotoxicity after 50 μM (Figure 6A). Due to the strong cytotoxicity, β-myrcene, β-cedrene, and β-ocimene were excluded for further studies. In order to assess the effect of GTEO’s bioactive components on melanogenesis, we investigated cellular tyrosinase activity and melanin biosynthesis in B16-F10 cells stimulated with FRK. It is interesting to note that D-limonene (100 μM) and α-pinene (100 μM) greatly prevented the FRK-mediated rise in melanin formation. Indeed, these compounds decreased the melanin formation from 307.2% (FRK-treatment) to 111.5% and 74.2%, respectively (Figure 6B,C), However, p-cymene treatment did not change the FRK-stimulated melanin production. Further, we studied the effect of p-cymene, α-pinene, and D-limonene on cellular tyrosinase activity. The cellular tyrosinase activities exhibited a resemblance to the melanin content (Figure 6D,E) while p-cymene did not inhibit cellular tyrosinase activity.

4. Discussion

There are several chemically synthesized compounds employed in the cosmetic sector for skin whitening purposes. Many of these compounds directly inhibit tyrosinase enzyme activity; however, these agents have severe drawbacks, such as high cytotoxicity, limited dermal absorbance, and reduced efficacy. One example is kojic acid, which is a synthetic substance that is commonly used in cosmetics to suppress the enzyme tyrosinase and achieve skin lightening effects, which has been linked to various adverse effects, including erythema and contact dermatitis. Hence, there is a pressing need to identify safe, biocompatible, and naturally derived skin-whitening agents [26]. Recently, public attention has significantly increased towards essential oils and their components. This is due to their widespread acceptance among consumers and their ability to be used in various functional applications. Their biological activities span a wide range, including antibacterial, antifungal, antiviral, antiseptic, analgesic, anti-inflammatory, and dermato-protective effects [27]. Moreover, their distinct and pleasing aromas make them suitable for use in cosmetic products. Mounting scientific data substantiates the use of essential oils in treating diverse skin conditions such as acne, premature skin aging, hyperpigmentation, and protecting against UV radiation [28]. Multiple essential oils have been researched for their anti-melanogenic properties, demonstrating robust inhibition of melanin biosynthesis by either suppressing tyrosinase activity or modifying melanogenic signaling pathways [13,14]. Previously, we conducted a study where we recorded the anti-inflammatory properties of essential oils derived from G. tenuifolia [19]. Furthermore, other researchers have observed the powerful antioxidant properties of GTEO. Based on this information, we suggest that the essential oils derived from G. tenuifolia may also possess anti-melanogenic characteristics.

B16-F10, a murine melanoma cell line that is well regarded for its melanin-producing capabilities, serves as a common model for investigating the anti-melanogenic effects of both synthetic and natural agents. Various substances can induce melanogenesis in these melanoma cells, including α-MSH and FRK, which are identified as cAMP activators, stimulating melanin biosynthesis [29]. In the present study, FRK was employed to stimulate melanin biosynthesis and evaluate the effect of GTEO and its major bioactive compounds on melanogenesis. GTEO exhibited the ability to hinder melanin biosynthesis in B16-F10 cells. The melanin and cell viability assays revealed that treating B16-F10 cells with 25–100 µM GTEO resulted in a reduction in melanin biosynthesis while maintaining a cell viability of above 90%. Indeed, the GTEO’s potency was highly comparable with a known skin-lightening agent, kojic acid. The process of melanogenesis is tightly controlled by specific enzymes, including tyrosinase, TRP-1, and TRP-2. Hence, we aimed to investigate whether GTEO has the potential to regulate the activity of the tyrosinase enzyme. The mushroom tyrosinase inhibitory assay is commonly employed to assess the skin-whitening potential of candidate substances in a cell-free system, as tyrosinase serves as the key enzyme in melanin biosynthesis. Employing this assay, we examined the direct tyrosinase inhibitory properties of GTEO, where L-tyrosine and L-DOPA served as substrates. Indeed, our results revealed a significant inhibition of mushroom tyrosinase enzyme activity upon co-incubation with GTEO, notably observed in the presence of either L-tyrosine or L-DOPA substrates. L-tyrosine and L-DOPA are commonly used as substrates in mushroom tyrosinase assays because they serve as precursors in the biosynthesis of melanin. Tyrosinase catalyzes the conversion of L-tyrosine to L-DOPA (monophenolase) and further converts L-DOPA to dopaquinone (diphenolase), which is a key step in melanin synthesis [30].

Numerous natural and synthetic compounds demonstrate dual capabilities in directly inhibiting tyrosinase and altering cellular signaling pathways, effectively suppressing melanin production [31,32]. As anticipated, the treatment with GTEO demonstrated a potent tyrosinase inhibitor, which aligns with others’ findings indicating that various essential oils possess the ability to inhibit cellular tyrosinase activity. This extended to the inhibition of tyrosinase both transcriptionally and translationally by GTEO.

The primary transcription factor responsible for directly regulating the transcription of tyrosinase, TRP-1, and TRP-2 is MITF. At the transcriptional level, CREB, a cAMP-dependent transcription factor, induces the transcription of MITF upon stimulation [33]. We found that FRK, functioning as a cAMP activator, elevated MITF at both transcriptional and translational levels, whereas GTEO treatment notably suppressed this elevation. The nuclear translocation of MITF signifies a hallmark event in MITF’s transcriptional activities. The administration of GTEO effectively hinders the FRK-induced nuclear translocation of MITF and subsequently suppresses its transcriptional activation. In a previous study, it was noted that the majority of melanoma cells exhibit hyper-activated MAPKs, resulting in MITF phosphorylation and subsequently leading to ubiquitin-mediated proteasomal degradation [34]. Additionally, we previously reported that essential oils can impede the protein stability of MITF by activating ERK [29]. To confirm this hypothesis, cells were treated with ERK1/2 inhibitor prior to FRK and GTEO treatment, after which melanin production was assessed. In this study, we found that the application of GTEO did not hinder FRK-induced melanin formation in cells treated with the ERK inhibitor (PD98059). However, notable reductions in melanin formation were observed in cells treated solely with GTEO. These data align with a notable increase in ERK phosphorylation induced by GTEO. Other researchers have also reported a comparable anti-melanogenic mechanism [35].

Chyau et al. [23] utilized a combined approach of steam distillation and solvent extraction methods to isolate essential oil from G. tenuifolia, resulting in the identification of 62 distinct compounds, predominantly terpenes, which are the primary constituents of G. tenuifolia essential oil. Similarly, in our previous work, we employed the steam distillation method to extract essential oils from G. tenuifolia, identifying 21 compounds. Monoterpenoids and sesquiterpenoids comprise the majority of these constituents. Among these, p-cymene, β-myrcene, β-cedrene, β-ocimene, α-pinene, and D-limonene were identified as key compounds with concentrations of 35.5%, 14.68%, 9.8%, 8.49%, 6.69%, and 5.17%, respectively [19]. The cell cytotoxicity assay conducted with these primary compounds indicated that the treatment with β-myrcene, β-cedrene, and β-ocimene resulted in significant cytotoxicity toward B16-F10 melanoma cells. This observation aligns with our earlier findings, where these compounds demonstrated cytotoxic effects on the murine macrophage (RAW 264.7) cell line [19]. Subsequent investigations showed that the primary compound, p-cymene, did not exhibit any significant effect on melanin inhibition. This finding is consistent with our previous study, which indicated that p-cymene isolated from the essential oil of Alpinia nantoensis did not demonstrate notable melanin inhibition even at a concentration of 100 µM [29]. Indeed, α-pinene and D-limonene markedly suppressed FRK-induced melanin formation, subsequently inhibiting cellular tyrosinase activity. This finding was strongly supported by previous observations indicating that α-pinene exhibits potent melanin inhibitory properties [36]. Furthermore, our earlier research also documented that D-limonene impedes FRK-induced melanin formation in B16-F10 cells [29]. These findings indicate that α-pinene and D-limonene present in GTEO may be accountable for its potent anti-melanogenic properties.

5. Conclusions

In this study, we have illustrated that the essential oils derived from G. tenuifolia, as well as their bioactive constituents α-pinene and D-limonene, diminish melanin biosynthesis in cultured B16-F10 melanoma cells. We propose a dual mechanism of action for GTEO. Firstly, through a competitive inhibition model, GTEO binds to tyrosinase, the enzyme responsible for catalyzing its oxidation. This binding impedes the oxidation of tyrosine, slowing down melanogenesis. Secondly, GTEO alters cellular signaling pathways, thereby intercepting melanin biosynthesis. Presumably, GTEO reduces tyrosinase at both the transcriptional and translational levels by limiting MITF transcriptional activity and subsequently inducing protein instability, achieved through ERK activation. Subsequent investigations revealed that the anti-melanogenic properties of GTEO were attributed to the presence of α-pinene and D-limonene. Also, other secondary metabolites present in smaller proportions may indeed act as adjuvants to the main metabolites. To our knowledge, this is the first in vitro examination of the essential oils of G. tenuifolia regarding their impact on tyrosinase and melanin biosynthesis in melanocytes. These essential oils exhibit potential in regard to the treatment of skin hyperpigmentation disorders; however, further exploration through in vivo testing is essential to ascertain their efficacy and potential side effects. Furthermore, these natural products may provide an alternative to synthetic compounds that have demonstrated significant adverse effects.