5.1 Materials

Dulbecco’s Modified Eagle Medium (DMEM), penicillin and streptomycin (PC/SM), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were obtained from Life Technologies Corporation. Acarbose, α-glucosidase (from S.cerevisiae), p-nitrophenyl-a-D-glucopyranoside (pNPG), α-melanocyte-stimulating hormone (α-MSH), dimethyl sulfoxide (DMSO), L-DOPA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), LY294002, PD98059, and SB216763 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Easy-Blue™ Total RNA extraction kit was purchased from Intron Biotechnology, Inc. (iNtRON Biotechnology, Seongnam, Korea). The TaqMan® RNA-to-Ct™ 1-Step Kit was purchased from Applied Biosystems (USA). RIPA buffer was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Switzerland). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The ECL Western Blotting Analysis System was obtained from Amersham Biosciences (Piscataway, NJ, USA).

5.2 Extraction and isolation

The dried and pulverized aerial part of P. lobata (10 kg) were extracted under reflux with 30% ethanol at 90 °C for 3 h. The solvent was removed under reduced pressure to give a residue (2.02 kg), which was suspended in distilled water (DW) and partitioned with ethyl acetate (EtOAc) to obtain the EtOAc extract of the aerial part of P. lobata (APPL, 145 g). APPL, activated carbon and EtOAc were suspended at a ratio of 1:1.5:7, filtered, and then the solvent was removed under reduced pressure to obtain a decolored extract (GALM-DC, 58 g). Then, GALM-DC was suspended in DW and partitioned with CHCl3 to give the CHCl3 extract (10.1 g). The CHCl3 extract was subjected to column chromatography (CC) on silica gel eluting with a gradient of CHCl3-MeOH (18:1 to 0:1, v/v) to give fourteen fractions (Fr.1-Fr.14). Fr.C3 (1.79 g) was performed on a sephadex LH-20 CC eluted with CHCl3 to afford eleven subfractions (Fr.C3.1-C3.11). Fr.C3.11 (1.1 g) was firstly performed on a silica gel CC eluted with hexane–EtOAc (4:1, v/v) to get three sub-fractions (Fr.C3.11.1-Fr.C3.11.3). Then, Corylin (18.5 mg) was yielded from Fr.C3.11.3 (46.8 mg) by recrystallization and decolorization with methanol. Compounds isolated from Fr.C3.11.3 were identified as corylin from 1H- and 13C-NMR spectral data (Palo Alto, CA, USA).

Corylin—Light yellow powder; 1H-NMR (DMSO-d6, 400 MHz) δ: 10.79 (1H, s, 7-OH), 8.33 (1H, s, H-2), 7.98 (1H, d, J = 8.8 Hz, H-5), 6.95 (1H, dd, J = 8.8, 2.4 Hz, H-6), 6.87 (1H, d, J = 2.4 Hz, H-8), 6.80 (1H, d, J = 8.0 Hz, H-5’), 7.28 (1H, d, J = 2.0 Hz, H-2’), 7.31 (1H, dd, J = 8.0, 2.4 Hz, H-6’), 5.78 (1H, d, J = 9.6 Hz, H-3’’), 6.44 (1H, d, J = 10.0 Hz, H-4’’), 1.40 (6H, s, 2’’-2CH3); 13C-NMR (DMSO-d6,100 MHz) δ: 153.7 (C-2), 123.7 (C-3), 175.1 (C-4), 127.8 (C-5), 115.8 (C-6), 163.2 (C-7), 102.7 (C-8), 158.0 (C-9), 117.2 (C-10), 125.0 (C-1’), 127.5 (C-2’), 121.1 (C-3’), 152.8 (C-4’), 116.1 (C-5’), 130.2 (C-6’), 76.8 (C-2’’), 131.8 (C-3’’), 122.3 (C-4’’), 28.3 (C-5’’), 28.3 (C-6’’).

5.3 Cell culture and cell viability assay

B16F10 murine melanoma cells were obtained from ATCC (VA, USA), and cultured in DMEM supplemented with 10% FBS and 1% antibiotics in 5% CO2 at 37℃.

B16F10 cells (1 × 104 cells/well) were seeded into a 96-well plate and incubated for 24 h. Cells were treated with fraction and corylin for 48 h. The medium was suctioned and 1 mg/mL MTT solution was added and incubated for 2 h. After that, formazan was dissolved in DMSO and absorbance was read at 540 nm using a microplate reader (BioTek, VT, USA).

5.4 Melanin content determination

B16F10 cells (1 × 105 cells/well) were seeded in a 6-well plate and exposed to α-MSH (100 nM) for 24 h. Cells were treated with a combination of α-MSH, fraction and corylin for 48 h. Cells were washed twice with PBS and lysed in 400 μL of 1N NaOH for 1 h at 90℃. The resulting melanin concentration was quantified by measuring the absorbance at 405 nm. Melanin production was expressed as a percentage of that from α-MSH-treated controls.

5.5 Measurement of cellular tyrosinase activity

B16F10 cells were exposed to α-MSH (100 nM) for 24 h and treated with a combination of α-MSH and corylin for 48 h. The pellet was obtained by washing with PBS. The cells were lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 5 mM EDTA, 1% Triton X-100, and 0.1% phenylmethylsulfonyl fluoride (PMSF) in ice for 30 min. After centrifugation of the lysate at 13,000 rpm for 30 min at 4 °C, cellular tyrosinase activity was measured in the resulting supernatant. Enzyme activity was normalized to protein concentration, as determined by the Bradford assay. The cellular tyrosinase and 0.1% L-DOPA reaction was performed in 0.1 M sodium phosphate buffer at 37 °C for 1 h. Tyrosinase activity was quantified by measuring the absorbance at 475 nm.

5.6 Western blot analysis

B16F10 cells were treated with corylin and then lysed with RIPA buffer to obtain a protein. 30 μg of proteins were electrophoresed by 7.5% SDS‑PAGE gels and transferred to nitrocellulose.

The membranes were blocked with 5% skim milk in PBST (PBS containing 0.1% Tween 20) at RT for 1 h, washed with PBST, incubated with primary antibodies (1:1,000) for 18 h at 4˚C, washed with PBST, incubated with HRP-conjugated secondary antibodies for 2 h at RT. The protein-antibody complex was visualized by an ECL system. Bands were quantified using the FluorChem E system image analyzer (Cell Biosciences, CA). β-actin was used as an internal control.

5.7 Measurement of MITF, TRP-1, and tyrosinase mRNA expression

The mRNA was quantified by ND-1000 spectrophotometer (NanoDrop Technology, USA). The relative ratio of target gene expression was calculated with the △Ct method. The transcripts of selected genes were quantified by RT-PCR using a TaqMan RNA-to-Ct 1-Step Kit according to the manufacturer’s instructions. Reverse transcription was performed at 48 °C for 15 min followed by the activation of AmpliTaq Gold DNA Polymerase at 95 °C for 10 min, and 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min on an ABI Step One Plus (Applied Biosystem, USA).

5.8 α-Glucosidase inhibition assays

The α-glucosidase enzyme inhibition assay was performed according to the method described by Si et al. [33]. Briefly, 10 μL of test samples at various concentrations (31.3- 250 μM), 20 μL of α-glucosidase (0.5 unit/mL), and 120 μL of 0.1 M phosphate buffer (pH 6.9) were mixed. After incubating at 37℃ for 15 min, 20 μL of pNPG (5 mM) was put into substrates, which were incubated for an additional 15 min. The reaction was terminated by the addition of 80 μL of 200 mM sodium carbonate (Na2CO3). The absorbance was measured at 405 nm. Acarbose was used as a positive control. The enzyme inhibitory rate was calculated as follows:

Activity (%) = Sample absorption/Control absorption × 100

5.9 Culture of reconstructed 3D skin model

A 3D human skin model (Neoderm-ME) was purchased from Tegoscience Co. (Seoul, Korea). In brief, Neoderm-ME was removed from the medium-containing agar and transferred onto 12-well plates for equilibration at 37 °C in 5% CO2 for one day. Treatment of vehicle or sample (corylin or arbutin), for 1 h before UVB (50 mJ/cm) irradiation for eight days. The medium was changed every day and the plate was incubated at 37 °C and 5% CO2. Then, the skin tissues were used to determine melanin content and subjected to Fontana-Masson (F-M) staining.

5.9.1 Fontana-masson staining

To observe the degree of skin pigmentation, F-M staining was performed. 3D human skin tissue blocks were fixed with 4% formalin for 12 h and embedded in paraffin. Sections cut to 5 µm thickness were stained using the F-M staining kit from IHC World (Woodstock, MD, USA).

F-M staining was performed according to the manufacturer’s protocol. The stained tissues were observed with a Leica phase contrast microscope (Leica Microsystems, Wetzlar, Germany) to measure the epidermal thickness. The thickness of the epidermis was evaluated using the average of three measurements from each tissue.

5.10 Statistical analysis

Statistical calculations were performed with SigmaPlot software, version 10.0 (Systat Software Inc., San Jose, CA, USA). The results are expressed as the mean ± SD. Student's t-test was used for statistical analyses; p-values < 0.05 were considered statistically significant.