Dried stem lettuce (Lactuca sativa L. var. augustana) was purchased from J&Company (Today Food, Korea; origin: China). To ensure complete dryness, the material was dried in an oven at 55℃ for 1 h, then pulverized into a fine powder with a laboratory blender. A 30 g aliquot of the powder was extracted with 70% ethanol (300 mL) using an ultrasonic cleaner (Daihan Scientific, Korea) at 60℃ for 1 h. This extract was subsequently filtered twice through filter paper (F1002; CHMLAB Group, Spain) under reduced pressure. The resulting filtrate was concentrated using a vacuum system (40-100 mbar) and a rotary evaporator (Rotavapor^® R-300; BUCHI Korea, Korea) at 45℃ and 70 rpm for 3 h to obtain approximately 40 mL of concentrated extract. For final purification, the concentrated extract was filtered through a 0.2 μm syringe filter (Minisart^®; Sartorius, Germany), aliquoted (1 mL per tube) into 1.5 mL microcentrifuge tubes, and stored at −20℃ until experimental use. Throughout this study, this prepared stock solution was designated Lactuca sativa L. stem extract (LSSE).

B16F10 murine melanoma cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (WELGENE, Korea) containing high glucose [DMEM(1X), LM001-05], and supplemented with 10% fetal bovine serum (Bandio, USA) and 1% penicillin-streptomycin (PS) (Gibco, Australia). The cells were incubated at 37℃ in an incubator containing 5% CO₂.

To evaluate the cytotoxic effect of LSSE on B16F10 cell viability, a water-soluble tetrazolium salt (WST-1)-based assay was performed using the EZ-Cytox Cell Viability Assay Kit (DoGenBio, Korea). B16F10 cells were seeded into 96-well plates (3.0×10³ cells/well) and incubated for 24 h. The cells were then treated with LSSE at several concentrations (0% to 2.0%, v/v) in fresh medium for an additional 48 h. Next, 10 μL of EZ-Cytox reagent was added to each well and incubated for 30 min at 37℃. The absorbance at 450 nm was measured using a microplate reader (iMark; Bio-Rad, USA). Finally, cell viability was calculated by the following equation:

Cell viability (%)=((Absorbance of treated cells)/(Absorbance of control cells))×100

The direct enzymatic inhibitory effect of LSSE against mushroom tyrosinase was measured in a cell-free system using both L-dihydroxyphenylalanine (L-DOPA; Sigma-Aldrich, USA) and L-tyrosine (Sigma-Aldrich) as substrates. Briefly, 10 μL of mushroom tyrosinase solution (1000 U/mL; Sigma-Aldrich) was initially added to each well of a microplate. Next, LSSE (0.5%, 1.0%, and 2.0%, v/v) or kojic acid (10 μM; positive control) was then added, and the final reaction volume was adjusted with Dulbecco’s phosphate-buffered saline (D-PBS; WELGENE). For the substrate oxidation assays, 10 μL of either 10 mM L-DOPA (Sigma-Aldrich) or 10 μL of 20 mM L-tyrosine (Sigma-Aldrich) was finally added, and the mixture was incubated for 15 min at 37℃. The absorbance at 490 nm was measured using a microplate reader (iMark; Bio-Rad). The inhibitory activity against tyrosinase-mediated substrate oxidation (%) was calculated using the following equation (Sripathi & Lalitha, 2016):

Tyrosinase inhibition (%)=[1-((Absorbance of treated group)/(Absorbance of untreated group))]×00

B16F10 melanoma cells were seeded into 60-mm culture dishes (1.5×10⁵ cells/dish) and incubated for 24 h. The cells were stimulated with 100 nM α-melanocyte-stimulating hormone (α-MSH) (Sigma-Aldrich) to induce melanogenesis and co-treated with LSSE (0.5%, 1.0%, and 2.0%, v/v) or arbutin (500 μM; positive control) for 48 h. The cells were then washed with 1 mL of D-PBS (WELGENE), harvested with a cell scraper. Cell pellets were collected by centrifugation (12,000 rpm, 4℃, 2 min), and the supernatants were discarded. The pellets were lysed in 240 μL of RIPA buffer, centrifuged (12,000 rpm, 4℃, 30 min), and the supernatants were collected. For the intracellular tyrosinase activity assay, 90 μL of cell lysate was mixed with 10 μL of 10 mM L-DOPA (Sigma-Aldrich) in a 96-well plate and incubated at 37℃ for 1 h. The absorbance at 490 nm was measured using a microplate reader (iMark; Bio-Rad). Total protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA) by incubating 10 μL of the cell lysate with 100 μL of BCA working solution (reagent A:B =50:1) at 37℃ for 30 min, followed by absorbance measurement at 595 nm. Intracellular tyrosinase activity was normalized to total protein content and calculated by the following equation:

Intracellular tyrosinase activity (%)=((Protein-normalized absorbance of treated group)/(Protein-normalized absorbance of untreated group))×100

B16F10 cells were seeded into 60-mm culture dishes (1.5×10⁵ cells/dish) and incubated for 24 h. The cells were stimulated with 100 nM α-MSH (Sigma-Aldrich) and co-treated with LSSE (0.5%, 1.0%, and 2.0%, v/v) or arbutin (500 μM; positive control) for 48 h. The cells were then washed with 1 mL of D-PBS (WELGENE), harvested with a cell scraper, and transferred to 1.5 mL microcentrifuge tubes. The cell pellets were photographed to document any visual changes in pigmentation. To extract the pigment, the pellets were resuspended in 160 μL of 1 N sodium hydroxide (NaOH; Ducksan, Korea), followed by vortexing and incubation at 90℃ for 5 min. This vortexing–incubation cycle was repeated five times. The solubilized samples were loaded into a 96-well plate (100 μL/well), and the absorbance was measured at 490 nm using a microplate reader (iMark; Bio-Rad). A BCA Protein Assay Kit (Thermo Fisher Scientific) was used to measure total protein concentrations for normalization using 25 μL of lysate and 200 μL of BCA working solution (reagent A:B =50:1). The samples were incubated for 30 min at 37℃, and the absorbance was measured at 595 nm. Melanin content was expressed relative to the untreated control using the following equation:

Melanin content (%)=((Protein-normalized absorbance of treated group)/(Protein-normalized absorbance of untreated group))×100

To determine the effects of LSSE on the expression of melanogenesis-related proteins, a western blot analysis was conducted. B16F10 melanoma cells were seeded into 60-mm culture dishes (2.5×10⁵ cells/dish) and incubated for 24 h. The cells were stimulated with 100 nM α-MSH (Sigma-Aldrich) and co-treated with LSSE (0.5, 1.0, and 2.0%, v/v) for 24 h. Next, the cells were harvested using a cell scraper and centrifuged (12,000 rpm, 4℃, 2 min). The cell pellets were lysed in 200 μL of RIPA buffer containing 1 μL of phenylmethylsulfonyl fluoride (PMSF; Biopure, Korea) and cleared by centrifugation (12,000 rpm, 4℃, 30 min). Total protein concentrations were determined using a BCA Protein Assay Kit by measuring the absorbance at 595 nm. After the protein concentrations were normalized across all groups with D-PBS (WELGENE), the lysates were mixed with 5× sample buffer (Bio-Rad), heated at 100℃ for 7 min, and immediately placed on ice for 20 min. Equal amounts of protein were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V for 1 h and transferred to nitrocellulose membranes at 100 V for 1 h. The membranes were blocked with 5% of skim milk (Biopure) for 1 h and incubated with primary antibodies overnight at 4℃. The antibody information is listed in Table 1. The membranes were washed three times with Tris-buffered saline with Tween 20 (TBST) and incubated with the corresponding secondary antibodies for 2 h at room temperature. The protein bands were detected using enhanced chemiluminescence (ECL; Thermo Fisher Scientific) and captured on a ChemiDoc Imaging System (Bio-Rad). Band intensities were quantified using ImageJ software (v1.52a).

To assess transcriptional changes in melanogenic enzymes, RT-PCR analysis was performed. B16F10 cells were seeded into 60-mm culture dishes (2.5×10⁵ cells/dish) and incubated for 24 h. The cells were stimulated with 100 nM α-MSH (Sigma-Aldrich) and co-treated with LSSE (0.5%, 1.0%, and 2.0%, v/v) for 24 h. Total RNA isolation was conducted using RiboEx Total RNA Isolation Solution (GeneAll Biotechnology, Korea). Lysates were phase-separated by adding chloroform, centrifuged, and the aqueous layer was collected. RNA was precipitated by adding an equal volume of isopropanol, incubated at -20℃ for 10 min, washed with 80% ethanol, and dissolved in nuclease-free water (Sigma-Aldrich). RNA concentration and purity were determined using a MaestroNano Micro-volume Spectrophotometer (MN-913; Maestrogen, USA). Complementary DNA (cDNA) synthesis was carried out using oligo(dT) primers (Bionics, Korea), dNTPs (Takara, Japan), DTT (Invitrogen, USA), and M-MLV reverse transcriptase (Invitrogen, USA). Target genes were amplified in a final volume of 20 μL. PCR amplification consisted of denaturation at 94℃ for 2 min, followed by cycles of denaturation (94℃ for 30 s), annealing (62℃ for Gapdh and 60℃ for Tyr, Trp-1, Trp-2, and Mitf for 30 s), extension (72℃ for 30 s), with a final extension step at 72℃ for 5 min. Gapdh was amplified for 28 cycles, whereas Tyr, Trp-1, Trp-2, and Mitf were subjected to 35 cycles. The resulting PCR products were separated on 1% agarose gels and visualized using a Fusion Solo 6S imaging system (Vilber Lourmat, France). The primer sequences are listed in Table 2.

All experiments were performed independently in triplicate. All experimental differences are presented as the mean±standard deviation (SD). Group comparisons and variance evaluations were calculated using GraphPad Prism software by a one-way or two-way analysis of variance (ANOVA), followed by Sidak’s multiple comparisons test. Statistical differences were considered significant at p<0.05.

To determine the effects of LSSE on the viability of B16F10 melanoma cells, the WST-1 assay was performed. LSSE treatment at concentrations ranging from 0.01% to 2.0% for 48 h resulted in significant cytotoxicity compared with the untreated control (Figure 1A). Therefore, all subsequent experiments were conducted using LSSE at concentrations of up to 2.0%.

To determine the inhibitory effects of LSSE on melanogenesis, a melanin content assay was conducted. B16F10 cells were stimulated with 100 nM α-MSH to induce melanogenesis and co-treated with LSSE (0.5%, 1.0%, and 2.0%) for 48 h. Arbutin (500 μM) was positive control. Melanin content was significantly increased to approximately 160% in the α-MSH-treated cells compared with the untreated control. Treatment with 0.5%, 1.0%, or 2.0% LSSE decreased melanin content by 12.5%, 15.0%, and 26.9%, respectively, in a dose-dependent manner compared with the α-MSH-treated group. The inhibitory effect progressively increased with increasing LSSE concentrations, with the greatest reduction occuring at 2.0% LSSE. At a 2.0% LSSE, the inhibitory effect was comparable to or greater than that of arbutin (Figure 1B). As discussed above, tyrosinase is a key rate-limiting enzyme in melanin biosynthesis and catalyzes the initial steps of melanin production; therefore, intracellular tyrosinase activity was further examined. To determine whether the reduction in melanin content was associated with the inhibition of intracellular tyrosinase activity, a tyrosinase activity assay was performed. LSSE significantly reduced intracellular tyrosinase activity by 18.8%, 26.9%, and 36.9% at 0.5%, 1.0%, and 2.0%, respectively, compared with the α-MSH-treated group (Figure 1C). Similar to the results of the melanin contents assay, intracellular tyrosinase activity exhibited a dose-dependent decrease. These results suggest that the reduction in melanin production by LSSE is associated with the suppression of intracellular tyrosinase activity.

To determine whether the anti-melanogenic activity of LSSE involves direct enzymatic inhibition of tyrosinase, a mushroom tyrosinase assay was performed using L-DOPA and L-tyrosine as substrates (Shim, 2019). LSSE significantly inhibited mushroom tyrosinase activity in a dose-dependent manner using both substrates (Figure 2A). When L-DOPA was used as the substrate, treatment with 0.5%, 1.0%, and 2.0% LSSE reduced tyrosinase activity by 5.17%, 10.98%, and 17.42%, respectively, compared with the L-DOPA-treated control. For L-tyrosine as the substrate, LSSE treatment reduced tyrosinase activity by 6.76%, 18.24%, and 34.46%, respectively, compared with the L-tyrosine-treated control. For both substrates, the inhibitory effect gradually increased with increasing LSSE concentrations, indicating a dose-dependent inhibition of activity. At a concentration of 2.0%, the inhibitory effect of LSSE was comparable to that of 10 μM kojic acid, the positive control. These results indicate that LSSE directly inhibits tyrosinase activity in a cell-free system.

To identify the anti-melanogenic mechanism of LSSE, we measured the mRNA and protein expression levels of melanogenesis-related enzymes, including tyrosinase, TRP-1, and TRP-2. Cells were stimulated with α-MSH to induce melanogenesis and co-treated with LSSE (0.5%, 1.0%, and 2.0%) for 24 h. Western blot analysis revealed that α-MSH markedly increased the expression of the tyrosinase, TRP-1, and TRP-2 proteins, whereas LSSE treatment dose-dependently suppressed the expression of all three proteins (Figure 2B). The suppressive effect of LSSE increased concomitantly with increasing concentrations, indicating a dose-dependent reduction in melanogenic enzyme expression. At a concentration of 2.0%, LSSE reduced the levels of the Tyrosinase, TRP-1, or TRP-2 proteins by 46.5%, 78.5%, and 78.2%, respectively, compared with the α-MSH-treated group. Consistently, RT-PCR revealed that LSSE treatment dose-dependently decreased the levels of Tyr, Trp-1, and Trp-2 mRNA (Figure 2C). The mRNA expression patterns were consistent with those observed at the protein level and exhibited a decrease with increasing LSSE concentrations. At a concentration of 2.0%, LSSE reduced the expression of Tyr, Trp-1, and Trp-2 by 77.4%, 91.0%, and 56.4%, respectively, compared with the α-MSH-treated group. These results indicate that LSSE suppresses melanogenic enzyme expression at the transcriptional and protein levels in a dose-dependent manner.

To identify the upstream regulatory mechanism responsible for the suppression of melanogenic enzymes, the expression of MITF was assessed. α-MSH treatment significantly increased MITF protein and mRNA levels, whereas LSSE treatment reduced MITF expression in a dose-dependent manner (Figure 3A, B). At a concentration of 2.0%, MITF protein and mRNA expression levels were reduced by 59.5% and 84.9%, respectively, compared with the α-MSH-treated group. To identify the upstream signaling pathway regulating MITF expression, the PKA-CREB signaling pathway was examined. Although total CREB and PKA levels remained unchanged, p-CREB and p-PKA were markedly decreased following LSSE treatment in a dose-dependent manner (Figure 3C). At a concentration of 2.0%, p-CREB and p-PKA levels were reduced by 41.2% and 50.3%, respectively, compared with the α-MSH-treated group. These results indicate that dose-dependent decreases in p-PKA, p-CREB, and MITF are accompanied by the suppression of melanogenic enzyme expression, which suggests that LSSE-mediated inhibition of melanogenesis is associated with the downregulation of the PKA-CREB-MITF signaling cascade.