Materials. Dimethyl sulfoxide (DMSO), 2,6-di-tertbutylate hydroxytoluene (BHT), 3,4-dihydroxy-
L-phenylalanine (
L-DOPA), β-actin, 1,1-diphenyl-2-picryl hydrazyl (DPPH), tannic acid,
L-tyrosine, ascorbic acid, Folin-Ciocalteu’s phenol reagent, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), IBMX, MITF-M, and diethylene glycol reagent were obtained from Sigma Chemical Company (St. Louis, MO, USA). TRP-1 and TRP-2 were obtained from Amersham Company (Bucks, UK). Propylene glycol was purchased from Chemical Innovation Company (Seoul, Korea). Methanol extract from the fruit of
C. officinalis (serial number: 014-046) was obtained from the Korea Plant Extract Bank (Daejeon, Korea). This specimen was dissolved in DMSO before use.
Antioxidant activity analysis. The total polyphenol content of COME was determined with the Folin-Denis assay (15) . One milliliter of test agent dissolved in DMSO was placed into test tube followed by the addition of 1 mL of Folin-Ciocalteu’s phenol reagent. The tubes were allowed to stand for 3 min. One milliliter of 10% Na
2CO
3 was added, and the mixture was shaken vigorously. After the tubes stood for 60 min, absorbance at 760 nm was measured. A standard curve was prepared with tannic acid.
Total flavonoid content of COME was determined using the modified method of Davies
et al. (16) (
link). One milliliter of test agent was placed into test tubes followed by the addition of 10 mL diethylene glycol reagent and 1 mL 1 N NaOH. The mixture was shaken vigorously and reacted in hot water at 37℃ for 60 min before absorbance at 420 nm was measured. A standard curve was prepared with rutin.
DPPH radical scavenging effects were evaluated according to the method of Blois (17) (
link). COME was dissolved in DMSO to final concentrations of 100, 500, and 1,000 μg/mL. One milliliter of the test agents were placed into each test tube followed by the addition of 4 mL of 4 × 10
−4 M DPPH. The mixture was shaken vigorously and kept for 10 sec in hot water at 60℃ before absorbance at 525 nm was measured. BHT was used as the positive control. The free-radical-scavenging activity of each solution was then calculated as a percent of inhibition.
Cell culture. The melan-a cells used in this study were obtained from Dr. Dorothy Bennett (St. George’s Hospital, UK). These highly pigmented and immortalized cells were derived from C57BL/6 mice. The cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 200 nM 12-
O-tetradecanoylphorbol-13-acetate at 37℃ in an incubator with 10% CO
2 for 72 hr.
MTT assay. The melan-a cells were divided in a 96-well plate (0.5 × 10
4 cells/well) and grown in the incubator at 37℃ with 10% CO
2 for 24 hr. Then, 200 μL of COME diluted with RPMI-1640 medium to various concentrations (3.125, 6.25, 12.5, and 25.0 μg/mL) was placed in the wells, and the cells were grown in the incubator at 37℃ with 10% CO
2 for 48 hr. Then, the cells were placed in medium containing 0.5 μg/mL MTT and grown in the incubator at 37℃ with 10% CO
2 for 3 hr. After centrifuging the plate at 180 ×g for 10 min, the cells settled. The medium was removed, 200 μL of DMSO was added, and the cells were dissolved for 15 min on a plate-shaker. Absorbance was measured at 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader.
Melanin assay. The melan-a cells were divided in a 96-well plate (2 × 10
4 cells/well) and grown in an incubator at 37℃ with 10% CO
2 for 24 hr. Then, 200 μL of COME diluted with RPMI-1640 medium to concentrations of 1.563, 3.125, 6.25, and 12.5 μg/mL was put in the wells, and the cells were grown in the incubator with 10% CO
2 at 37℃ for 72 hr. After the cells washed, the treatment was repeated. Next, the cells were dissolved in 1 N NaOH, and optical density was measured at 490 nm (OD 490) with an ELISA reader. Melanin content was estimated as the OD 490 value/μg of protein and expressed as a percentage relative to the untreated control value (100%).
Tyrosinase activity assay. For intracellular tyrosinase activity assay, melan-a cells were seeded in 60-mm cell culture dishes (4 × 10
5 cells/well) for 24 hr and then treated with 5 mL COME (0~12.5 μg/mL) for 48 hr. The cells were washed with phosphate buffer solution, detached with 200 μL 1% Triton X-100, transferred to Eppendorf tubes, extracted on ice with agitation, and centrifuged at 18,000 ×g for 20 min at 4℃. Thereafter, 100 μL
L -DOPA was added, the mixture was incubated at 37℃ under 10% CO
2 for 1 hr, and OD 490 was measured with an ELISA reader. Tyrosinase activity was estimated as the OD 490/μg protein/min and expressed as a percentage of the untreated control value (100%). For cell-extracted tyrosinase activity assay, after centrifugation of cultured melan-a cells, 50 μL supernatant was mixed with 49 μL 0.1 M phosphate buffer solution (pH 6.8) and 1 μL COME (0~12.5 μg/mL). L-DOPA (0.2%, 100 μL) was added, the absorbance measured, and the percentage activation was calculated.
Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated with the Trizol-reagent (Life Technologies, CA, USA) according to the manufacturer’s instructions. Five micrograms of total RNA were reverse transcribed with 8 μL Moloney murine leukemia virus reverse transcriptase (M-MLV RT) 5 × buffer, 3 μL 10 mM deoxyribonucleotide triphosphates (dNTPs), 0.45 μL 40 U/μL RNase inhibitor, 0.3 μL 200 U/μL M-MLV RT (Promega, Madison, USA), and 1.5 μL 50 μM oligo dT (Bioneer, Cheongju, Korea) in a 40-μL volume. Single-stranded complementary DNA was then amplified via PCR with 4 μL 5 × green Go Taq Flexi buffer, 0.4 μL 10 mM dNTPs, 0.1 μL 5 U/μL Taq polymerase, 1.2 μL 25 mM MgCl
2 (Promega), and 0.4 μL 20 μM each specific sense and anti-sense primers of tyrosinase, TRP-1, TRP-2, MITF-M, or β-Actin.
The primer sequences used for PCR were as follows: 5'-CAT TTT TGA TTT GAG TGT CT-3' (forward), 5'-TGT GGT AGT CGT CTT TGT CC-3' (reverse) for tyrosinase; 5'-GCT GCA GGA GCC TTC TTT CTC-3' (forward), 5'-AAG ACG CTG CAC TGC TGG TCT-3' (reverse) for TRP-1; 5'-GGA TGA CCG TGA GCA ATG GCC-3' (forward), 5'-CGG TTG TGA CCA ATG GGT GCC-3' (reverse) for TRR-2; 5'-TAC AGA AAG TAG AGG GAG GAG GAC TAA G-3' (forward), 5'-CAC AGT TGG AGT TAA GAG TGA GCA TAG CC-3' (reverse) for MITF-M; 5'-ACC GTG AAA AGA TGA CCC AG-3' (forward), 5'-TAC GGA TGT CAA CGT CAC AC-3' (reverse) for βActin. The expected sizes of the PCR product for tyrosinase, TRP-1, TRP-2, MITF-M, and β-Actin, respectively, were 1192, 268, 1044, 326, and 528 base pairs.
The following PCR conditions were applied: tyrosinase and TRP-1, 28 cycles of denaturation at 94℃ for 60 sec, annealing at 56℃ for 60 sec, and extension at 72℃ for 60 sec; TRP-2, 28 cycles of denaturation at 94℃ for 60 sec, annealing at 64℃ for 60 sec, and extension at 72℃ for 60 sec; MITF-M, 30 cycles of denaturation at 94℃ for 30 sec, annealing at 54℃ for 30 sec, and extension at 72℃ for 30 sec; β-Actin, 30 cycles of denaturation at 94℃ for 30 sec, annealing at 51℃ for 30 sec, and extension at 72℃ for 60 sec. The PCR products were analyzed on 1.2% agarose gel. β-Actin was used as an internal control to evaluate the relative expression of tyrosinase, TRP-1, TRP-2, and MITF-M.
Western blot analysis. Cell lysates were prepared by sonicating melan-a cells in 0.1 M Tris-HCl (pH 7.2) buffer containing 1% Nonidet P-40, 0.01% sodium dodecyl sulfate, and a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration of cell lysates was measured using a Pierce Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL, USA) with bovine serum albumin as the standard. Equal amounts of protein (10 μg) were loaded onto each lane and separated with electrophoresis on a 10% polyacrylamide gel. After being transblotted onto nitrocellulose, the membranes were incubated with antibodies against tyrosinase/prolyl endoprotease-7 (PEP-7, 1:10,000 dilution), TRP-1/PEP-1 (1:10,000), and TRP-2/PEP-8 (1:10,000), which were kindly provided by Dr. Vincent J. Hearing (National Institutes of Health, USA). Next, the membranes were incubated with horseradish-peroxidase-conjugated anti-rabbit immunoglobulin G (1:1,000 dilution; Amersham Biosciences, Buckinghamshire, UK). Immunoreactive bands were detected with chemiluminescence using electrochemical luminescence reagents (Amersham Biosciences). β-Actin was used as an internal control for immunoblotting.
Statistical analysis. Differences in values between the groups were evaluated statistically using one-way analysis of variance followed by Duncan’s multiple range test for a post hoc comparison by using SPSS 21.0 for windows (IBM, Armonk, NY, USA). Statistical significance was defined as
p < 0.05.
