The murine melanoma B16F10 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained as a monolayer culture in Dulbecco’s Modified Eagle Medium (DMEM; Lonza, Walkersville, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; GIBCO/Invitrogen, Carlsbad, CA), 100 units/ml penicillin, and 100 μg/ml streptomycin (Welgene, Korea) at 37°C in a humidified 5% CO2 incubator. For the preparation of murine hepatocytes, 6–8 weeks old female ICR mouse were purchased from Nara Bio animal center (Nara Biotech, Korea), and housed under standard conditions at a temperature of 24 ± 1°C and humidity of 55 ± 5%. The experimental procedures were approved by Korea Institute of Oriental Medicine Care and Use Committee with a reference number 12–093 and carried out in accordance with the Korea Institute of Oriental Medicine Care Committee Guidelines. Hepatocytes were isolated using a perfusion system with some modification . Cells suspended in the William’s E medium containing 10% FBS, 100 IU/ml insulin, 2 mM L-glutamine, 15 mM HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin were seeded on the culture plate coated with 10% gelatin/phosphate buffered saline (PBS), and incubated at 37°C in a humidified 5% CO2 incubator.
Antibodies and reagents
Alpha-melanocyte stimulating hormone (α-MSH), forskolin, synthetic melanin, mushroom tyrosinase, L-3,4-Dihydroxyphenylalanine (L-DOPA), and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Antibodies against tyrosinase, TRP-1, TRP-2, MITF, PKA, and phospho-PKA (Thr198) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-CREB, anti-phospho-CREB (Ser133), anti-p38, anti-phospho-p38 (Thr180/Tyr182), anti-extracellular signal-related kinase1/2 (ERK), anti-phospho-ERK (Thr202/Tyr204), anti-c-Jun-N-terminal kinase (JNK), anti-phopsho-JNK (Thr183/Tyr185), and anti-tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). All of the other chemicals and solvents used were analytical grade.
Preparation of Ssanghwa-tang (SHT)
SHT is composed of 9 medicinal herbs, the root of Paeonia lactiflora (28%), root of Angelica gigas (11.2%), root of Astragalus membranaceus (11.2%), root of Cnidium officinale (11.2%), preparata of root of Rehmannia glutinosa (11.2%), root of Glycyrrhiza glabra (8.4%), bark of Cinnamomum cassia (8.4%), root of Zingiber officinale (4.4%), and fruit of Zizyphus jujube (6.0%), which were purchased from Korea Medicinal Herbs Association (Yeongcheon, Korea). Identification of all herbs was confirmed by Prof. KiHwan Bae of the College of Pharmacy, Chungnam National University (Daejeon, Korea), and all voucher specimens were deposited in the herbal band in Korea Institute of Oriental Medicine (KIOM, Korea). SHT formula were extracted in distilled water by heating for 3 h at 115°C in an extractor (Cosmos-600 Extractor, Gyeonseo Co., Inchon, Korea) and then filtered using standard testing sieves (150 μm, Retsch, Haan, Germany). The freeze-dried SHT extract was dissolved in PBS, filtered (0.45 μm), and then kept at 4°C prior to use.
Cell viability assay
Cells, seeded at a density of 5 × 103 cells/well in 96-well culture plates, were cultured overnight and then treated with various concentrations of SHT (25, 50, 100, 250, 500, 1000, and 2000 μg/ml) or single herbal extract for the 48 h. After cells were incubated with 10 μl of MTT solution (5 mg/ml in PBS) for 4 h, the formazan precipitates were dissolved by dimethyl sulfoxide (DMSO) and then absorbance was measured at 570 nm with Infinite® M200 microplate reader (TECAN Group Ltd. Switzerland). Cell viability was presented as the percentage of viable cells compared with untreated, control cells.
Measurement of cellular melanin contents
Cellular melanin content was measured as described previously . Briefly, B16F10 cells seeded at a density of 3 × 105 cells on the 100 mm culture dishes were pre-treated with 250 and 500 μg/ml of SHT for 12 h, and then stimulated with 1 μM of α-MSH for additional 36 h. After harvest of cells, equal number of cells (1 × 107 cells/sample) were dissolved in 100 μl of 1 N NaOH/10% DMSO for 1 h at 80°C, and solubilized melanin was measured at 475 nm using Infinite® M200 microplate reader. Relative melanin content compared with untreated “control” cells was calculated from a standard curve using synthetic melanin.
Measurement of tyrosinase activity
B16F10 cells seeded in 6-well plates (1 × 105 cells/well) were pre-treated with 250 and 500 μg/ml of SHT for 12 h, and then further incubated with 1 μM of α-MSH for 36 h. For the measurement of cellular tyrosinase activity, the cells were washed with ice-cold PBS and then lysed with 1% Triton X-100 in PBS by repeated freezing/thawing. Each lysate was centrifuged at 12000 rpm for 15 min at 4°C to obtain a supernatant as a source of tyrosinase, and then determined for protein concentration. The reaction mixture containing same amount of supernatant (or mushroom tyrosinase) compensated with 50 mM phosphate buffer (pH 6.8) up to 90 μl and 10 μl of 10 mM L-DOPA as a substrate for tyrosinase was incubated at 37°C in a 96-well plate. Following incubation, dopachrome formation from L-DOPA was monitored by measuring the absorbance at 475 nm using Infinite® M200 microplate reader, and relative tyrosinase activity was calculated from that of standard mushroom tyrosinase. Relative tyrosinase activity was expressed as a percentage compared with untreated, control cells.
Luciferase reporter assay
For the analysis of tyrosinase, CRE, and MITF promoter activity, semi-confluent cells grown in 12-well culture plates were transiently transfected with each luciferase reporter plasmid (pTyrosinase-luc, pCRE-luc, and pMITF-luc) and Renilla luciferase plasmid using TransIT®-2020 Transfection reagent (Mirus Bio LLC., Madison, WI) according to the manufacturer’s instructions. After incubation, cells were lysed in passive lysis buffer, and the luciferase activities were measured by luminescence microplate reader set (TriStar LB 941, Berthold Technologies GmbH and Co. KG, Bad Wildbad, Germany) using dual luciferase reporter assay system according to the manufacturer’s instructions (Promega, Madison, WI).
Western blot analysis
After washing cells twice with PBS, whole cell lysates were extracted in M-PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) by centrifugation (12000 g × 15 min, 4°C), and the protein concentration was determined using Bicinchoninic Acid (BCA) Kit (Sigma). Total protein (40–80 μg) was separated by electrophoresis on 10–12% SDS-polyacrylamide gels, and transferred to Immobilon®-P PVDF transfer membrane (Millipore, Bedford, MA). After immunoblotting, proteins were visualized using a PowerOpti-ECL Western blotting detection reagent (Animal Gentetics, Inc. Korea) and an ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ). Equal amount of proteins was analyzed by Western blotting using tubulin as a loading control and band intensities were quantified using ImageJ software (National Institutes of Health, USA).
Preparation of standard solutions and SHT and analytical chromatic conditions
For the qualitative analysis, 10 standard compounds, paeoniflorin, liquiritin, nodakenin, benzoic acid, nodakenetin, decursinol, cinnamyl alcohol, cinnam aldehyde, decursin, and decursinal angelate, were prepared by dissolving in 100% methanol as described previously . Analytical SHT sample was prepared by dissolving powder in 100% H2O at a concentration of 40 mg/mL followed by filtration through a 0.45 μm filter. The main components profile of SHT was analyzed at the 254 nm UV wavelength using the Elite Lachrom HPLC system (Hitachi High-Technologies Co., Tokyo, Japan) consisting of pump (L-2130), auto-sampler (L-2200), column oven (L-2350), and diode array UV/VIS detector (L-2455). System control and data analyses were executed by EZchrom Elite software (verson 3.3.1a) system. The chromatographic separation was conducted with RS-tech C18 column (Optimapak C18, 4.6 × 250 mm, 5 μm, Daejeon, Korea) at 40°C and the injection volume was 10 μl. The mobile phase was a gradient elution of 1% acetic acid and acetonitrile at a flow rate of 1 ml/min, commencing with 5% acetonitrile for 5 min, linear gradient to 100% acetonitrile was applied over 70 min, and then maintained at 100% for 10 min.
Data are presented as the mean ± SD values of at least 3 independent experiments, unless otherwise specified. Statistical significance was analyzed by the two-tailed student’s t-test in Sigma Plot 8.0 software (SPSS Inc., Chicago, IL) and a P value of less than 0.05 was considered statistically significant.