B16F10 mouse melanoma cells were kindly provided by Shandong Freda Biotechnology Co., Ltd. α-Arbutin (α-Ar; CAS no: 84380-01-8, purity ≥99%) and α- α-MSH (CAS no: 171869-93-5, purity ≥97%) were obtained from Shanghai Macklin Biochemical Technology. DMEM high glucose culture medium was obtained from M&C Gene Technology. Trypsin-EDTA digestion solution was obtained from Yisheng Biotechnology. Penicillin-streptomycin solution was obtained from HyClone; Cytiva. Fetal bovine serum (FBS) was obtained from Wuhan Pricella Biotechnology Co., Ltd. The CCK-8 was purchased from Biosharp Life Sciences. L-DOPA (CAS: 59-92-7, purity ≥98%) was sourced from Beijing Solarbio Science & Technology Co., Ltd. H89 (PKA inhibitor), IBMX (cAMP activator) and Forskolin (AC activator) were acquired from Beyotime Institute of Biotechnology. RNA extraction reagent, chloroform substitute, RNA dissolution solution, beta-actin (cat. no. GB11001) and HRB-conjugated Goat Anti-Rabbit IgG H&L secondary antibodies (cat. no. GB23303), primary antibody diluent (cat. no. G3337) were obtained from Wuhan Servicebio Technology Co., Ltd. RIPA buffer (purity ≥98%) was sourced from Beijing Solarbio Science & Technology Co., Ltd.

The GC-MS system 8890-5977B was purchased by Agilent. The LS-C0105 CO₂ incubator was purchased by NuAire Lab Equipment (https://www.nuaire.com/). The SW-CJ-2D type ultra-clean workbench was procured by Suzhou Hengda Purification Equipment. The HVE-50 high-pressure steam sterilizer pot was obtained from Xinhua Medical Equipment Co., Ltd. and the CKX53 inverted microscope was purchased from Olympus Corporation.

The PNEO was prepared in the Natural Products Research Center of Shandong Normal University. A total of 20 g of pine needle powder from Pinus tabuliformis Carrière was dissolved in distilled water, and the PNEO was extracted by using microwave-assisted extraction technology and stored at 4˚C (33).

GC-MS was used to analyze the constituent of PNEO. The analysis employed an Agilent HP-5MS column (30 mm x 0.25 mm x 0.25 µm). The temperature program of the column was initiated at 60˚C, maintained for 1 min, ramped up at a rate of 8˚C/min to 140˚C and held for 2 min the temperature was increased at 2˚C/min to 240˚C and held for 2 min, followed by a further ramp up at 8˚C/min to 280˚C, where it was held for 5 min. Helium (purity, ≥99.999%) was used as the carrier gas at a flow rate of 1.0 ml/min. The inlet temperature was set at 260˚C, and a split injection mode with a split ratio of 10:1 was employed, with an injection volume of 1 µl.

Mass spectrometry analysis was carried out by using instrument utilized an Electron Impact (EI) ionization mode with a voltage of 70 eV, a source and transfer line temperatures of 280˚C. The detection mode operated in full scan (SCAN) mode, nitrogen (N₂) (purity, ≥99.999%) was used as the carrier gas. A solvent delay of 3 min was implemented to optimize the analysis process.

B16F10 melanoma cells were cultivated in DMEM medium containing 10% FBS. The cells were maintained in a humidified 5% CO₂ incubator at 37˚C and were sub-cultured every 2-3 days to maintain logarithmic growth for subsequent experimentation. Experimental groups included: i) A control group; ii) PNEO treatment group (300 nM α-MSH + 12.5, 25, 50, 100, 200, 400, 800 µg/ml PNEO); iii) α-MSH model group (300 nM α-MSH); and iv) a positive control group (300 nM α-MSH + 100 µg/ml α-Ar).

The cell viability was determined by using the CCK-8 method. B16F10 cells were seeded in a 96-well plate at a density of 5x10⁴ cells/ml. Following a 24-h incubation period, cells were categorized based on Section 2.2.2 and cultured for an additional 24 or 48 h. A 10% CCK-8 working solution was prepared in DMEM medium, replacing the original medium with 100 µl of the working solution per well. Subsequently, cells were further incubated at 37˚C for 1 h before measuring the optical density (OD) at 450 nm using a microplate reader. Cell viability was calculated as follows: (A₁-A)/(A₀-A) x100%. A₁ represents the OD value of the group treated with drugs, including cells, CCK-8 solution and drug solution; A denotes the OD value of the control group with medium and CCK-8 solution but no cells; and A₀ signifies the OD value of the group containing cells, medium and CCK-8 solution.

The protein concentration was determined using the BCA Protein Assay Kit following the manufacturer's protocol.

Melanin content was quantified using the NaOH lysis method. B16F10 cells were seeded in 6-well plates at a density of 1.25x10⁵ cells/ml per well, with 2 ml of cell suspension added to each well and then incubated for 24 h. Subsequently, abandoning the original culture medium, 2 ml of fresh medium was added to each well according to the aforementioned grouping. Following an additional 48-h static incubation, cells were washed twice with PBS, harvested by scraping, and centrifuged at 13,201 x g for 5 min at 4˚C. The resulting supernatant was discarded, and 300 µl of 1 M NaOH solution, containing 10% DMSO, was added to each centrifuge tube. After thorough mixing and incubation at 80˚C to solubilize the melanin, vigorous vortex followed. B16F10 lysate containing melanin was then dispensed into a 96-well plate (100 µl per well, with 3 replicates per group), and the absorbance at 405 nm was measured for each well using a microplate reader to determine the relative melanin content. Relative melanin content was calculated as follows: A₁/A₀ x 100%. A₁ represents the OD value of the experimental group, and A₀ represents the OD value of the control group.

The TYR activity within B16F10 cells was assayed in terms of DOPA oxidase activity. The plating and drug administration processes were carried out as aforementioned. After continuous static culture for 48 h, the cells were washed twice with PBS. Then, 500 µl of 1% RIPA solution was added to each well, and the plate was quickly placed in a -80˚C refrigerator for freezing. After being taken out, it was placed at room temperature to thaw so that the cells were ruptured. This freeze-thaw process was repeated several times. Then the cells were collected and centrifuged at 4˚C, 9,167 x g for 5 min. The supernatant was reserved in an ice bath. In a 96-well plate, 50 µl of the supernatant was added, followed by 50 µl of 1 mM L-DOPA solution. After reacting at 37˚C for 1 h, the absorbance was measured at 492 nm. The TYR activity was preliminarily calculated according to the protein concentration, and then the relative activity was compared. Relative TYR activity was calculated as follows: C₁/C₀ x 100% (3). C₁ represents TYR value of the experimental group, and C₀ represents TYR value of the control group.

RNA Extraction Solution (cat. no. G3013) was sourced from Wuhan Seavil Biotechnology Co., Ltd. Reverse transcription (RT) kit (cat. no. G3337) was provided by Wuhan Saiwell Biotechnology Co., Ltd. Total RNA from B16F10 cells treated with PNEO at a mass concentration of 50 µg/ml and the positive control group α-Ar for 48 h was subjected to reverse transcription on a regular PCR instrument. The cDNA first-strand product obtained from reverse transcription (temperature protocol: 25˚C for 5 min, then 42˚C for 30 min, final 85˚C for 5 sec) served as the template, with various reaction components sequentially added to the tube for amplification on a fluorescent quantitative PCR instrument. SYBR green (obtained from Wuhan Saiwell Biotechnology Co., Ltd.) was used as fluorophore for qPCR. Thermocycling conditions were as follows: Stage 1, 95˚C, 30 sec pre-denaturing; Stage 2, 95˚C, 15 sec denaturation; 60˚C, 30 sec annealing/elongation; Stage 3, 65-95˚C. The expression levels of the Tyr, Trp-1, Trp-2, Mitf and Mc1r genes were analyzed using the 2^-ΔΔCq method. Primer sequences are included in Table I.

The protein expression levels associated with melanin biosynthesis in B16F10 cells were assessed through western blot (WB) analysis. B16F10 cells were cultured for 24 h based on distinct groupings, exposed to PNEO at a concentration of 50 µg/ml for 48 h, rinsed twice with PBS, desiccated, harvested, lysed, and the resulting lysates were transferred into 1.5-ml centrifuge tubes containing an appropriate volume of RIPA lysis buffer. Cell lysis was carried out on ice for 30 min to ensure complete cellular disruption. Subsequent steps included centrifugation at 4˚C, 12,000-16,000 x g for 10 min, determination of protein concentration, denaturation, SDS-PAGE electrophoresis (10% acrylamide and 25-30 µg protein loaded per lane), PVDF membrane transfer, incubation at room temperature for 30 min for blocking (5% skim milk), addition of primary antibody, and overnight agitation at 4˚C. The membrane underwent three 5-min washes with TBST (0.1% Tween-20), followed by incubation at room temperature for 30 min with a secondary antibody diluted in TBST at a ratio of 1:5,000. After three additional rapid 5-min washes with TBST, the membrane was exposed, and the original image was preserved for subsequent data analysis. An immunoblot image analysis software based on artificial intelligence learning, launched by Wuhan Servicebio Technology Co., Ltd., was used.

Each experiment was replicated three times, and the results are presented as the mean ± standard deviation (X ± SD). Data analysis was conducted using GraphPad Prism 9.0.0 software (Dotmatics), employing one-way analysis of variance for comparisons among multiple groups. P#x003C;0.05 was considered to indicate a statistically significant difference.

The total ion chromatogram of the PNEO obtained by microwave-assisted extraction is shown in Fig. S1 (33), and a summary of its components and relative peak areas is presented in Table SI. By conducting GC-MS analysis of the PNEO extracted using the microwave-assisted method, a total of 332 substances were identified. Peaks with matching degree exceeding 80% were considered as candidate compounds, and after qualitative comparison by retrieving the NIST spectral library (https://webbook.nist.gov/chemistry/), 103 chemical compounds were identified, accounting for 47.39% of the total composition. The relative percentage content of each component in the PNEO was calculated using area normalization. The results showed that the PNEO mainly contained alcohols (11.01%), hydrocarbons (11.04%), esters (9.3%) and terpenes (2.997%). These included Thunbergol (PubChem CID: 5363523; 3.938%), Verticillol (PubChem CID: 5377475; 3.597%), Hentriacontane (PubChem CID: 12410; 2.189%), α-Terpinyl acetate (PubChem CID: 111037; 1.901%), Methyl dehydroabietate (PubChem CID: 14697; 1.842%), α-cadinene (PubChem CID: 12306048; 1.534%), Isopimara-8 (PubChem CID; 13783133) (14), 15-dien-19-saeure-methylester (PubChem CID: 13710744; 1.517%), Cembrene (PubChem CID: 6430770; 1.139%) and α-Terpinene (PubChem CID: 7462; 0.825%), among others.

The CCK-8 results are demonstrated in Fig. 1A. Treatment of cells with different concentrations of PNEO for 24 h showed a significant impact on cell viability when the concentration exceeded 400 µg/ml. At concentrations #x003C;400 µg/ml, cell morphology was normal, and the cell viability was >85%, with no significant difference compared with the control group (P>0.05). After 48 h of treatment, cell viability decreased significantly with increasing concentration of PNEO. At concentrations #x003C;50 µg/ml, PNEO did not affect cell viability (P>0.05). However, when the PNEO concentration was 100 µg/ml, the cell viability significantly decreased to 81.89±3.27% (P#x003C;0.01). Subsequently, PNEO concentrations of 12.5, 25 and 50 µg/ml were selected for further experiments.

After 48 h of intervention with 12.5, 25 and 50 µg/ml of PNEO, the melanin content in B16F10 cells was measured. As can be observed in Fig. 1B, when the cells were stimulated by α-MSH, the relative melanin content was 156.25±1.70%, indicating that the high melanin expression cell model was successfully developed. PNEO dose-dependently inhibited melanin content, with melanin inhibition rates of 24.81±1.31%, 31.63±1.31% and 54.36±0.67% in the low, medium and high dose groups, respectively. At 50 µg/ml, PNEO was able to achieve inhibition effects similar to the α-Ar positive control.

The results of TYR activity measurement are demonstrated in Fig. 1C. Compared with the control group, the TYR activity in the α-MSH group was 185.22±1.58% (P#x003C;0.0001), indicating the success of the model. Different mass concentrations of PNEO and the α-Ar positive control group inhibited TYR activity in B16F10 cells, showing significant differences compared with the α-MSH model group. In the α-Ar positive control group, the inhibition rate of TYR in B16F10 cells reached 31.36±17.25%, while PNEO at a mass concentration of 50 µg/ml exhibited the strongest inhibition of TYR activity, with an inhibition rate of 17.77±8.57%. This suggests that PNEO could effectively inhibit TYR activity in α-MSH-induced B16F10 cells.

MITF, TYR, TRP-1 and TRP-2 are important signaling molecules in the process of melanogenesis. Based on the results of melanin content and TYR activity, 50 µg/ml of PNEO achieved the optimal inhibitory effect. Therefore, the concentration of PNEO was set at 50 µg/ml in RT-qPCR and WB detection. The gene expression levels of Tyr, Trp-1, Trp-2, Mitf and Mc1r are revealed in Fig. 2. After treatment with PNEO for 48 h, the expression of Tyr, Trp-1, Mitf and Mc1r genes in B16F10 cells was significantly inhibited compared with the α-MSH model group. However, the expression level of Trp-2, which is related to melanin production, showed no significant impact.

Through WB experiments, the effect of PNEO on the proteins MITF, TYR, TRP-1 and TRP-2, as well as the phosphorylation levels of proteins in the cAMP/PKA signaling pathway was examined to elucidate the mechanism of action by which PNEO inhibits melanin production in B16F10 cells (Fig. 3). Treatment of B16F10 cells with 50 µg/ml PNEO for 48 h resulted in decreased expression of TYR, TRP-1, TRP-2, MITF and MC1R proteins.

The CREB molecule is one of the phosphorylation substrates of various protein kinases. The expression level of p-CREB in the nucleus can indirectly reflect the activity of PKA in the cytoplasm. CREB-regulated transcription coactivator 1 (CRTC1) is dephosphorylated and enters the nucleus, forming a CREB/CRTC1 heterodimer in the nucleus, which enhances the transcription levels on the MITF-M promoter. To verify whether PNEO acts on the cAMP/PKA signaling pathway, the results are shown in Fig. 4. Treatment with PNEO resulted in decreased expression of Crtc1 and Prkaca. After treatment of B16F10 cells with PNEO for 48 h, the protein levels of p-CREB and p-PKA were significantly inhibited (Fig. 5). It was hypothesized that the cAMP/PKA signaling pathway is involved in the process by which PNEO inhibits melanin production.

To further investigate the molecular mechanism underlying the inhibitory effect of PNEO on melanin production, the effects of the PKA inhibitor H89, the cAMP activator IBMX, and the AC activator Forskolin on the signaling pathways related to melanin production were studied in the context of PNEO-mediated melanin inhibition. The results, as shown in Fig. 6A, demonstrate that the addition of PNEO or the H89 inhibitor significantly reduced melanin content, with a synergistic effect observed between the two, resulting in a melanin content of 88.93±1.07%. In Fig. 6B, the results of cellular TYR activity identified that treatment with α-MSH significantly increased TYR activity, while the addition of PNEO or H89 inhibitors decreased TYR activity to 83.55±1.44% and 77.41±0.24%, respectively. Additionally, treatment with PNEO in combination with IBMX or Forskolin activators resulted in increased melanin content (Fig. 6C) and TYR activity (Fig. 6D), with Forskolin activator showing a more significant promotion effect, reaching a TYR activity of 235.18±4.50%. In the groups treated with α-MSH, IBMX, or Forskolin activators in combination with PNEO, both melanin content and TYR activity decreased. These findings validate that PNEO regulates melanin production through the cAMP/PKA signaling pathway.