Cells and cell culture

B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, USA) containing foetal bovine serum (FBS; HyClone), which was purchased from Gibco (USA), and the antibiotics penicillin and streptomycin, which were purchased from Invitrogen™ (USA). HaCaT cells were cultured at 37 °C in an incubator containing 5% CO₂ at 100% relative humidity.

Preparation of A. muricata L. extracts

Dried A. muricata L. tissues were pulverized and extracted in 10 volumes of 70% ethanol for 1 h in a sonicator. The mixture was then filtered with Whatman No. 2 filter paper, and the resulting filtrate was freeze-dried using a freeze-dryer (PVTFD-10R; IlShinBioBase, Dongducheon, Korea) and was stored at − 20 °C until analysis.

Cytotoxicity assays

Cytotoxic activities of A. muricata L. extracts were determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays as described previously (Mosmann 1983). Briefly, B16F10 cells were seeded into 96-well plates at 5 × 10³ cells/well and then cultured for 24 h. Culture solutions were then replaced with serum-free DMEM, and samples were treated with A. muricata L. extracts at various concentrations for 48 h. The cells were then washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific, USA), and MTT (Sigma-Aldrich, USA) solution was added to each well and incubated for 1–2 h at 37 °C. After removing the MTT solution, 200 μL of DMSO (Sigma-Aldrich, USA) was added to dissolve the formed formazan crystals and absorbance was determined using an ELISA reader at 540 nm.

Measurements of melanin contents

Melanin contents of cells were determined using the method described by Hosoi et al. (1985) with slight modifications. Briefly, cells were washed with PBS three times and then centrifuged to obtain cell pellets. The pellets were then placed in 200-μL aliquots of 1-N NaOH containing 10% DMSO and were incubated at 80 °C for 1 h. Subsequently, absorbance was measured at 405 nm using the ELISA reader, and total melanin contents were expressed as percentages of the control group.

In vitro tyrosinase activity assays

Tyrosinase is the rate-limiting enzyme in melanogenesis and catalyses the oxidation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and then to DOPA quinone. In previous studies of skin-whitening activities, melanin absorbance was measured before and after oxidation reactions to investigate the capacity of samples to inhibit tyrosinase activity (Kwon et al. 2014). Herein, 1.7-mM l-tyrosine was completely dissolved in 10-mM sodium phosphate buffer (pH 6.8). Subsequently, 5-μL aliquots of A. muricata L. extracts at various concentrations of up to 0.3 mg/mL were added to 450-μL aliquots of l-tyrosine. Mushroom tyrosinase (250 U/mL; 50 μL) was then added, mixed and incubated at 37 °C for 60 min, and absorbance was measured at 475 nm.

Tyrosinase activity assays

Tyrosinase activities were measured using the methods described by Choi et al. (1998). Briefly, treated cells were lysed in 100-μL aliquots of cell lysis solution and then centrifuged. Subsequently, 50-μL aliquots of the resulting supernatants were added to 450-μL solutions of 1.7-mM -tyrosine and 0.1-M PBS and incubated at 37 °C for 15 min prior to measurements of absorbance at 490 nm using an ELISA reader. Enzyme activities were expressed as percentages of those in the control group. Tyrosinase inhibitory activities of extracts were expressed as percentage absorbance decreases relative to the untreated control group.

qRT-PCR analysis

B16F10 cells were pretreated with extracts for 3 h and then incubated with 200-nM α-MSH for 48 h. After removing the culture solution, RNA extraction was performed using TRIzol (Invitrogen™, USA). RNA contents were then measured by a NanoDrop spectrophotometer (Thermo Scientific, USA), and equal amounts of RNA were used to synthesize cDNA with PrimeScript Reverse Transcriptase (Takara, Japan). Subsequently, gene expression was determined using qRT-PCR with the HOT FIREPol EvaGreen PCR Mix Plus (Solis BioDyne) and the primers presented in a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, USA). PCR was performed with denaturation at initial denaturation at 94 °C for 3 min, followed by 40 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 30 s) and polymerization (72 °C, 30 s). Data were analysed using the exclusive software program (ver. 2.0.6) provided by Applied Biosystems (USA).

Promoter activity assays

B16F10 cells were seeded into 60-mm culture dishes and cultured for 24 h and then transfected with a vector for the expression of the pGL-MITF reporter. Transfected B16F10 cells were then treated with the extracts for 24 h, and luciferase activities were measured according to the protocol provided by the manufacturer of the luciferase assay system. Inhibitory activities were expressed relative to controls that were treated with α-MSH.

Western blotting analysis

After cell treatments, culture media were removed and the cells were washed twice in ice-cold PBS (pH 7.4) and then incubated on ice for 20 min in a radioimmunoprecipitation assay buffer (Sigma-Aldrich, USA) containing protease inhibitors (Roche, Germany). Lysates were then centrifuged at 10,000g for 15 min at 4 °C, and protein concentrations of supernatants were measured using the Bradford method. After adjusting protein concentrations of all samples, equal quantities of protein were mixed with 5× sodium dodecyl sulphate (SDS) sample buffer (Sigma-Aldrich) and boiled at 100 °C for 5 min. SDS–protein samples were then electrophoresed on 8–10% SDS polyacrylamide gels using a Mini-PROTEAN system (Bio-Rad, USA). Protein bands were then transferred to cellulose membranes (GE Healthcare Life Sciences, UK) and blocked in Tris-buffered saline (TBST, pH 8.0) containing 0.1% Tween-20 and 5% skim milk at room temperature for 1 h. Membranes were probed with anti-MMP-1 and anti-type-1 procollagen, anti-MMP-3 (Santa Cruz Biotechnology, USA) or anti-β-actin (Sigma-Aldrich) primary antibodies in blocking solution at 4 °C for 18 h. Membranes were then washed in TBST and exposed to the following secondary antibodies for 2 h at room temperature: Horseradish peroxidase-conjugated anti-rabbit IgG and anti-goat IgG (Santa Cruz Biotechnology), anti-mouse IgG (Sigma-Aldrich) After washing, ECL solution (Pierce, USA) was sprayed onto the membranes, and bands were identified and quantified using an LAS-3000 Luminescent Image Analyser (Fujifilm, Japan).

Statistical analysis

Data are presented as means ± standard deviations, and differences were identified using Student’s t test. Differences were considered significant when p < 0.05.

Cytotoxicity assays of A. muricata L. extracts

MTT assays were used to determine cytotoxic activities and appropriate concentration ranges in B16F10 cells. In these experiments, survival rates were 90% or higher after treatments with A. muricata L. extracts at 60 μg/mL or less (Fig. 1a). Therefore, subsequent experiments were performed with extracts at ≤ 60 μg/mL.

Measurement of skin-whitening effects of A. muricata L. extracts

To assess the skin-whitening effects of A. muricata L. extracts, melanin contents of mouse melanoma cells were determined after treatment with A. muricata L. extracts at 60 μg/mL. In these experiments, α-MSH-induced melanogenesis was reduced to 24.42% in cells treated with A. muricata L. extracts compared to those treated with α-MSH only (Fig. 1b).

Changed tyrosinase activities and expression levels in the presence of A. muricata L. extracts

A. muricata L. extracts decreased tyrosinase activities to 23.24% at 60 μg/mL (Fig. 2a). To determine whether A. muricata L. extracts inhibit tyrosinase directly (Fig. 2b), we measured changes in the expression of tyrosinase mRNA and protein (Fig. 3) and showed 23.24 and 48.13% decrease, respectively, following treatment with A. muricata L. extracts. These data suggest that the effects of the present extracts on tyrosinase activities are transcriptional.

Changes in MITF transcription and protein expression in the presence of A. muricata L. extracts

To confirm the mechanisms by which A. muricata L. extracts inhibit melanogenesis, we determined MITF mRNA and protein expression levels using qRT-PCR and Western blotting analyses, respectively. These experiments demonstrated that treatments with A. muricata L. lead to decreased expression of MITF, likely leading to decreased transcriptional activation of tyrosinase (Fig. 4).