The skin comprises epidermal units responsible for melanin production and distribution, a process termed melanogenesis. These units consist of a melanocyte surrounded by keratinocytes and are regulated by a closed paracrine system (Videira et al., 2013). Melanin, predominantly produced by epidermal melanocytes, resides in the outermost layer of the skin, determining human skin color and providing protection against UVB, UVA, and blue visible light (Qian et al., 2020; Solano, 2020).

Melanin synthesis initiates with melanosomes and involves various intermediates and substrates in a sequential cascade. External stimuli, such as UV light, prompt melanocytes to generate eumelanin, imparting a black hue. The amino acid tyrosine and its hydroxylated DOPA product serve as the initial components in melanic pigment biosynthesis (Maranduca et al., 2019). Tyrosinase, a pivotal enzyme, catalyzes a crucial step in melanin synthesis. Downregulating tyrosinase stands as a prominent strategy for developing melanogenesis inhibitors (Pillaiyar et al., 2017). TRP-2 functions as a dopachrome tautomerase, catalyzing the rearrangement of dopachrome to form 5,6-dihydroxyindole-2-carboxylic acid (DHICA), while TRP-1 oxidizes DHICA to produce carboxylate indole-quinone (Kobayashi et al., 1994; Yokoyama et al., 1994). The tyrosinase family genes, TYR, TRP-1, and TRP-2, undergo tight regulation by the microphthalmia-associated transcription factor (MITF) (Levy et al., 2006). MITF, a critical transcription factor, governs TYR gene expression, impacting pigmentation, proliferation, and survival of melanocytes, thereby playing a pivotal role in melanogenesis (Buscà and Ballotti, 2000, Slominski et al., 2004). It binds to the M-box within the TYR promoter, upregulating TYR gene expression (Bentley et al., 1994). Upon exposure to UVB light, keratinocytes secrete α-melanocyte-stimulating hormone (α-MSH), which binds to the melanocortin-1 receptor (MC1R) on melanocytes (Imokawa et al., 1997). Activated MC1R triggers adenylate cyclase stimulation, elevating cyclic adenosine monophosphate (cAMP) levels, activating cAMP-dependent protein kinase (PKA), and phosphorylating the transcription factor cAMP response element-binding protein (CREB) (Hachiya et al., 2001). Phosphorylated CREB becomes active, binding to the cAMP response element (CRE) site within the MITF gene promoter (Buscà and Ballotti, 2000).

Piperine is a major alkaloid isolated from Piper nigrum or Piper retrofractum Vahl. Piperine has been used in traditional medicine for a long time, in treatment of various conditions: rheumatism and muscular aches, as a digestive tonic, in dyspepsia, in flatulence and indigestion, as antipyretic, for throat pain and cough; as antiseptic, bactericide, insecticide, diuretic, etc (Kim et al., 2011b).

There are reports suggesting that ointments containing Piper nigrum extract or piperine are effective in treating vitiligo, but there are no reports on the effects of piperine itself on melanin synthesis or its mechanisms of action (Merecz-Sadowska et al., 2022; Ozkan et al., 2022).

Therefore, we investigated the effects of piperine on melanin production in melanocytes and its mechanisms of action.

Materials

Pipirine (PPN), dimethyl sulfoxide (DMSO), phenylthiourea (PTU), L-tyrosine, mushroom tyrosinase, kojic acid, sodium monophosphate, sodium diphosphate, PD98059, MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

MITF, phospho ERK, phospho JNK, phospho p38, ERK, JNK, p38 primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). TYR, TRP-1, TRP-2 antibodies were provided from Vincent J. Hearing (National Institute of Health, Bethesda, MD, USA). β-actin primary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho MITF primary antibody was purchased from Invitrogen (Waltham, MA, USA). Goat anti-mouse IgG secondary antibody was purchased from Bio-Rad (Hercules, CA, USA). Donkey anti-rabbit IgG secondary antibody war purchased from Bethyl Laboratories (Montgomery, TX, USA).

Methods

Cell culture: Melan-A cell, immortalized normal melanocyte cell line was derived from C57BL/6 mice. Melan-A cell was provided from Dorothy Bennett (St. George’s Hospital, London, UK). Cells were cultured in RPMI640 (Welgene, Daegu, Korea) containing 10% Fetal Bovine Serum (Welgene), 1% penicillin/streptomycin and 200 nM Tetradecanoyl Phorbol Acetate (Sigma-Aldrich) at 37°C in 10% CO₂.

Cell viability assay: Melan-A cells were seeded on a 96 well plate (0.01×10⁶ cells/mL). After 24 h incubation at 37°C, in 10% CO₂. Cells were washed in DPBS (Welgene) and different PPN concentrations (25 μM, 50 μM, 75 μM, 100 μM) were applied to cells in triplicate. After 72 h, the media containing PPN were replaced with 10% EZ-CYTOX (Daeil Lab Service, Seoul, Korea) in RPMI. Then, the cells were incubated for 30 min at 37°C. Using a microplate reader (Tecan, Mannedorf, Switzerland), the absorbance of the well was determined at 450 nm.

Melanin assay: PPN was treated on Melan-A cells for 72h and washed with DPBS. The cells were then dissolved in 1N NaOH 100 μL to obtain cell lysate. The supernatant was measured at 490nm absorbance using a microplate reader. The BCA assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) was used to determine melanin contents compared to the total protein. We used Phenylthiourea (PTU), positive control known as tyrosinase inhibitor.

Tyrosinase inhibitory assay: Using tyrosine as a substrate, the level of oxidation reaction between tyrosine and mushroom tyrosinase was determined. Different concentrations of PPN were diluted in DMSO and placed on 96 wells by 1 μL in triplicate. After that, 25 μL of 1.5 mM L-tyrosine, 24 μL of pH6.8 sodium phosphate buffer and 50 μL of mushroom tyrosinase (2000 U/mL) were distributed in each well. After incubation at 37°C for 30 min, a 490 nm absorbance was used to measure the final amount of oxidized melanin.

Western blot analysis: The cells were seeded on a 6 well (0.3×10⁶ cells/mL) and washed with PBS after treatment. And then, the cells were lysed in RIPA buffer at 4°C for 30 min. The lysates were centrifuged at 13,000 rpm, 4°C for 20 min. After centrifugation, supernatant was extracted and BCA assay was performed for protein quantification. the lysates were seperated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then using transfer buffer, the lysates were transferred to a polyvinylidene difluoride membrane. After blocking with 5% BSA, the membrane was incubated with primary antibodies at 4°C overnight, then, incubated with secondary antibodies at room temperature for 2 h. Following X-ray film exposure of the membranes, the Supersignal West Pico Plus Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to identify the protein bands.

Statistical analysis: Statistical analysis of data was performed with the ANOVA test using the Statistical Packages for Social Sciences (SPSS) program (SPSS, Inc., Chicago, IL, USA).

The effect of piperine on the growth of Melan-A and melanin content

This is the result of a viability assay after treating PPN on Melan-A cell at different concentrations (Fig. 1). As a result, it was confirmed that the cells were significantly reduced due to the toxicity of PPN at 100 μM, and the subsequent experiments were conducted at 25 μM to 75 μM.

Figure 1. The effect of piperine on the growth of Melan-A. Melan-A cells were treated with different concentrations of PPN (25-100 μM) for 72 h. Using a microplate reader, the absorbance of the well was determined at 450 nm. Value are the means ± SD from three replicates (n=3). Data were analyzed with One-Way ANOVA. ***p<0.001 compared with the untreated cells.

Melanin assays were conducted to determine whether melanin content was reduced when treating PPN. PTU was used as a positive control (Fig. 2). When PTU and PPN were treated for 72 h at a concentration of 25, 50, 75 μM on Melan-A cell, it was confirmed that the melanin content decreased in a concentration-dependent manner.

Figure 2. Piperine regulates melanin synthesis and inhibits mushroom tyrosinase activity. (A) Melan-A cells were treated with different concentrations of PPN (25 μM, 50 μM, 75 μM) for 72 h. The supernatant was measured at 490 nm absorbance using a microplate reader. The BCA assay kit was used to determine melanin contents compared to the total protein. PTU was used as positive control. Value are the means ± SD from three replicates (n=3). Data were analyzed with One-Way ANOVA. ***p<0.001 compared with the untreated cells. (B) Using tyrosine as a substrate, the level of oxidation reaction between tyrosine and mushroom tyrosinase was determined. Different concentrations of PPN were diluted in DMSO and placed on 96 wells by 1 μL in triplicate. After that, 1.5 mM L-tyrosine, pH6.8 SPB and mushroom tyrosinase were distributed in each well. A 490 nm absorbance was used to measure the final amount of oxidized melanin. Value are the means ± SD from three replicates (n=3). Data were analyzed with One-Way ANOVA. ***p<0.001 compared with the wells untreated PPN.

The effect of piperine on tyrosinase activity in Melan-A

Since melanin content was reduced when treated with PPN, mushroom tyrosinase assay was conducted to determine whether PPN regulates the activity of tyrosinase. Kojic acid was used as a positive control (Fig. 2). As a result, it was confirmed that PPN significantly reduced tyrosinase activity in a concentration-dependent manner. In the case of 75 μM, a similar level of tyrosine activity was reduced to kojic acid.

The effect of piperine on expression level of MITF and melanogenesis-related proteins

To confirm the level of expression of melanogenesis-related protein and MITF by PPN, western blot analysis was performed after treating PPN for 72 h to a concentration of 25, 50, 75 μM on Melan-A cell (Fig. 3). PPN significantly reduced the protein expression levels of MITF, TYR and TRP-1 in a concentration-dependent manner, and in the case of expression levels of TRP2, although not concentration-dependent, they were significantly lowest at 75 μM.

Figure 3. Piperine decreases expression level of MITF and melanogenesis-related proteins. (A) After treatment with PPN for 72 h, expression levels of MITF, melanogenesis-related proteins (TYR, TRP-1, TRP-2) and β-actin were detected by Western blotting. (B) The band intensity was standardized to β-actin using Image J program (NIH, MA, USA). ***p<0.001 compared with β-actin.

The effect of piperine on expression level of phosphorylation MAPK

Based on previous study that the expression level of MITF is regulated by the MAPK family, the expression levels of the phosphorylation of ERK, JNK, and p38 were confirmed after treating PPN on Melan-A cell for different time (0 min, 2 min, 30 min, 1 h, 3 h, 6 h) at a concentration of 75 μM (Fig. 4). PPN did not affect the phosphorylation of p38, whereas the phosphorylation levels of ERK and JNK increased to the maximum at 10 min.

Figure 4. Piperine regulates phosphorylation level of MAPK. After treatment with PPN for 6 h, expression levels of ERK, phospho ERK, JNK, phospho JNK, p38, phospho p38 and β-actin were detected by Western blotting. The band intensity was standardized to β-actin using Image J program.

Piperine regulates MITF level by activating ERK pathway

Western blotting was performed by treating PPN (75 μM) with or without PD98059 (80 μM), an ERK inhibitor for 6 h to confirm whether PPN regulates melanogenesis through ERK signaling, based on the results of previous studies showing that plant extracts and alkaloids regulate melanogenesis through ERK activation. In the group treated with PPN, level of phosphorylation of ERK increased and the expression level of MITF decreased compared to the control, whereas in the group treated with PPN and PD98059, the increase in level of phosphorylation of ERK decreased compared to the group treated with PPN (Fig. 5A). Through this, it was confirmed that PPN regulates the expression level of MITF through activation of ERK. And the level of phosphorylation of MITF by PPN was also confirmed by previous results that activation of ERK induces phosphorylation of MITF in Ser73 to induce ubiquitination of MITF and subsequent degradation (Ko ^{and Cho}, 2018). It can be seen that the level of phosphorylation of MITF increased when treated with PPN as ERK was phosphorylated (Fig. 5B). Additionally, the melanin content assay confirmed that the decreased melanin content by PPN was significantly restored when PPN was treated with PD98059 (Fig. 5C).

Figure 5. Piperine regulates MITF level by activating ERK pathway. (A) After treatment with PPN for 6 h with or without PD98059, expression levels of total ERK, phosphor ERK, MITF and β-actin were detected by Western blotting. The intensity of phosphor ERK was standardized to total ERK using Image J program. And the intensity of MITF was standardized to β-actin using Image J program. (B) After treatment with PPN for different time (0 min, 2 min, 10 min, 30 min, 1 h, 3 h, 6 h), expression levels of MITF were detected by Western blotting. The intensity was standardized to β-actin using Image J program. (C) Additionally, melanin assay was performed under the same conditions as in the previous experiment method. Value are the means ± SD from three replicates (n=3). Data were analyzed with One-Way ANOVA. **p<0.01, ***p<0.001, ^###p<0.001 compared with the untreated cells or the wells treated PPN.

Piperine decreases MITF level by proteasomal degradation

In order to confirm whether proteasomal degradation by phosphorylation of ERK, MG132, a proteasomal inhibitor, was treated with PPN to confirm the expression level of MITF through Western blot (Fig. 6A). After cycloheximide (50 μg/mL), an inhibitor of protein biosynthesis, was treated with Melan-A cell for 2 h, PPN (75 μM) with or without MG132 (100 nM) was treated for 6 h. In the group treated with PPN, the expression level of MITF was decreased, and in the group treated with PPN and MG132, the expression level of MITF was recovered compared to the group treated with PPN, indicating that PPN degrades MITF by proteasomal degradation. Additionally, the melanin content assay confirmed that the decreased melanin content by PPN was significantly restored when PPN was treated with MG132 (Fig. 6B).

Figure 6. Piperine decreases MITF level by proteasomal degradation. (A) After starvation, cells were pre-treated with CHX for 2 h. And then, Melan-A cells were treated with PPN for 72 h with or without MG132. The expression levels of MITF and β-actin were detected by Western blotting. The band intensity was standardized to β-actin using Image J program. (B) Then, melanin assay was performed under the same conditions as in the previous experiment method. Value are the means ± SD from three replicates (n=3). Data were analyzed with One-Way ANOVA. **p<0.01, ***p<0.001, ^###p<0.001 compared with the untreated cells or the wells treated PPN.

To lighten the skin, whitening chemicals such hydroquinone, ascorbic acid, and retinoic acid were used. But they have a lot of negative impacts on the health of the body and the skin, such skin inflammation (Zhao et al., 2022). Consequently, there is increasingly focusing on harnessing the benefits of botanical extracts and natural ingredients to address the rising demand for skin-whitening without compromising on safety and skin health (Takizawa et al., 2004).

We found Piper retrofractum Vahl. extract inhibit melanin synthesis in Melan-a cells and found PPN was the main compound for the inhibition of melanin synthesis. Interestingly, there have been reports the topical application of Piper nigrum extract induced pigmentation in vitiligo patients (Merecz-Sadowska et al., 2022). But PPN was not effective as compared to extract of Piper nigrum. Therefore, the therapeutic effects on vitiligo may be due to other components, and no in vitro studies on the effects of piperine on melanin production have been published

Therefore, we are trying to elucidate the effect of PPN on the inhibition of melanin biosynthesis and its mechanism of action.

It is well recognized that tyrosinase is a rate-limiting enzyme required for the production of melanin (Ando et al., 2007). Thus, melanin synthesis suppression is associated with a reduction in tyrosinase activity (Boissy et al., 2005). Several previous studies have reported cases in which components obtained from natural sources were shown to be able to inhibit tyrosinase activity, which in turn caused a decrease in the produce of melanin (Chang, 2012). Treatment of PPN with 75 μM on Melan-A Treatment with 75 μM PPN on Melan-A cells not only decreased melanin content but also attenuated tyrosinase activity. There was a dramatic cell death at 100 µM piperine, but no effect was observed below 75 µM. Similar results were obtained when the experiment was repeated multiple times. Therefore, while it is not possible to completely rule out the potential impact of 75 µM piperine on cell viability, the concentration-dependent results observed in melanin production and the expression of melanogenic proteins at lower concentrations suggest that the findings are consistent and reliable. PPN reduced melanin content to a similar level as PTU and inhibited tyrosinase activity to a similar level as kojic acid. Furthermore, PPN reduced melanogenesis by downregulating the expression of melanogenesis-related proteins. Notably, PPN treatment led to a decrease in the expression level of MITF, a key transcription factor in regulating melanogenesis-related proteins, TYR, TRP-1 and TRP-2. This suggests that PPN inhibits the main signaling system of melanin synthesis.

Previous studies indicate that melanogenesis is influenced by the phosphorylation of the MAPK family (Sale et al., 1995; Alesiani et al., 2009; Chung et al., 2017). Additionally, post-translational modifications, including phosphorylation, sumoylation, and ubiquitination, affect MITF in addition to transcriptional regulation (Hsiao and Fisher, 2014). MITF expression is regulated by various signaling pathways, including phosphorylation by extracellular signal-regulated ERK and Ribosomal S6 kinase (RSK) (Kim et al., 2011a). Activation of ERK induces MITF phosphorylation at Ser73, resulting in ubiquitination and degradation. RSK-1, a downstream kinase of ERK, phosphorylates MITF at Ser409, leading to MITF degradation (Hsiao and Fisher, 2014; Wellbrock and Arozarena, 2015).

It has been investigated how natural materials or active substances phosphorylate MITF and cause proteasome degradation. Extracts from α-mangosteen, a tropical evergreen tree from Southeast Asia, degrade MITF in B16F10 rat melanoma cells through ERK/GSK3β signaling pathways (Zhou et al., 2021). [6]-Shogaol, the main biologically active component of ginger, induces the degradation of MITF through ERK/AKT signaling (Huang et al., 2014). Phytol, a precursor for the synthesis of vitamin E and vitamin K₁, inhibits melanin synthesis by inducing proteasome degradation of MITF through ERK signaling induced by reactive oxygen species (Ko and Cho, 2018).

In this study, we observed an increase in ERK phosphorylation following PPN treatment. ERK phosphorylation induced MITF degradation via proteasomal degeneration through ubiquitination. Restoration of MITF degradation by PD98059 (ERK inhibitor) and MG132 (proteasomal inhibitor) supports the claim that PPN-induced ERK phosphorylation leads to MITF degradation through the proteasomal pathway. Future investigations are necessary to determine whether PPN-induced JNK phosphorylation affects melanogenesis. PPN could exerts a synergistic effect by inhibiting tyrosinase activity and promoting MiTF degradation. However, the reversal of whitening effects upon inhibition of MITF degradation by both ERK and proteasomal inhibitors indicates that the contribution of tyrosinase inhibition may be minimal.

In summary, PPN downregulates the degradation of MITF through phosphorylation of ERK in Melan-A cell. In addition to down-regulating melanin-related proteins by the regulation of MITF, melanin production is suppressed by inhibiting the activity of tyrosinase (Fig. 7). These results suggest that PPN can be used as a skin-whitening material or as a potential natural material to treat skin pigmentation disorders.

Figure 7. Scheme of the mechanism of action of piperine.

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HP23C0001).

The authors have declared no conflicting interests.