Chemicals
CCD-986sk human skin fibroblasts were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from HyClone Laboratories, Inc. (Logan, UT, USA), while fetal bovine serum (FBS), penicillin-streptomycin were obtained from Gibco (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) obtained from Sigma Chemical Co. (St. Louis, MO, USA) and enzyme-linked immunosorbent assay (ELISA) was performed using ROS-Glo™ H2O2 assays (Promega, Madison, WI, USA) and pro-Collagen type 1 C-peptide protein Elisa kit (Takara, Shiga, Japan) and human FGF basic Quantikine ELISA kit (R&D Systems., Minneapolis, MN, USA). All the antibodies used were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA)
Identification of the purified bioactive compound
The 1H-NMR (CDCl3, 600 MHZ) spectrum showed a profile of 6.234 (1H, d, J=2.1 Hz, H-6), 6.485 (1H, d, J=2.1 Hz, H-8), 7.505–7.561 (3H, m, H-3′, H-4′,5′), 8.156 (2H, dd, J=1.5 and 8.0 Hz, H-2′,6′), and the 13C-NMR (CDCl3, 62.9 MHz) spectrum showed a profile of 176.18 (C-4), 164.14 (C-7), 160.49 (C-5), 156.40 (C-9), 145.64 (C-2), 137.02 (C-3), 130.92 (C-1), 129.89 (C-4), 128.48 (C-2′, C-6′), 127.51 (C-3′, C-5′), 103.16 (C-10), 98.22 (C-6), 93.53 (C-8). The purified compound, a yellow-green powder, was spread out in TLC which the color was developed into yellow color when heated with 10% H2SO4 sprayed. In the 1H-NMR spectrum, two protons were identified to be coupling in doublet at 8.156 ppm (2H, dd, J=1.7,9.7 Hz, H-2′,6′). 6.485 ppm (1H, d, J=2.1 Hz, H-8) and 6.234 ppm (1H, d, J=2.1 Hz, H-6) was found to be coupling in J=2.1 Hz. 13 Carbon signals were detected in the 13C-NMR spectrum but an overlapping peak of 128.48 ppm (C-2′-,6′) and 127.51 ppm (C-3′,5′) was detected which totals 15 carbons. With these results, purified compound from Alpinia officinarum Hance was identified as galangin.
Cell culture
Cells were plated in 75 cm2 culture flasks and grown in DMEM supplemented with 10% FBS and 5% penicillin-streptomycin in a 5% CO2 incubator at 37 °C. For the treatment, cells were cultured in fresh medium for 24 h. After overnight incubation, the cells were washed with phosphate buffered saline (PBS; Gibco, Grand Island, NY, USA) and pretreated by UVB irradiation. After UVB irradiation, PBS was removed, and the cells were incubated in serum-free medium with galangin-treated for further 48 h more.
Elastase and collagenase inhibition assay
The elastase inhibitory activity was evaluated by the method of [19] with minor modifications. Simply, 1.0 U/mL porcine pancreatic elastase and the sample or distilled water were added to the mixed solution of 0.2 M Tris-HCl buffer (pH 8.0) and 0.8 mM N-succinyl-(Ala)3-\( {\uprho} \)-nitroanilide, then the absorbance was measured at 410 nm.
The collagenase inhibition activity was proceeded by the method of [20] with minor modifications. Briefly, 0.2 mg/mL collagenase and sample or distilled water were added to the mixed solution of 0.1 M Tris-HCl buffer (pH 7.5) and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (0.3 mg/mL), then the absorbance was measured at 320 nm. The inhibition rate of both elastase and collagenase was calculated as the following formula: Inhibition (%) = 1 − (absorbance of sample/absorbance of control) × 100.
Cell viability
The toxicity of the galangin in skin fibroblasts was measured by using a MTT colorimetric assay. Briefly, \( 5 \times 10^{3} \) cells were seeded in 48-well plate and incubated for 24 h. Skin fibroblasts were subsequently exposed to UVB (20 mJ/cm2) then incubated in the presence of galangin at a concentration of 5, 10, 20, 25, 50 µg/mL for 48 h in serum-free medium. After incubation, cell medium was replaced with MTT reagent and incubated for additional 4 h. Then, all the medium was removed and dimethyl sulfoxide (DMSO) was added to dissolve insoluble formazan crystals. Cell viability was then measured at an absorbance of 540 nm with a micro-plate reader.
Measurement of ROS
Skin fibroblasts were seeded in 96-well plates and after 24 h incubation, the culture medium was replaced and pretreated with UVB (20 mJ/cm2) for 1 min. After UVB irradiation, they were treated with 5, 10, 25 µg/mL of galangin and incubated for 48 h in serum-free medium. The production of ROS was quantified using hydrogen peroxide assay kit (ROS-Glo™ H2O2, Promega).
Enzyme-linked immunosorbent assay (ELISA)
Skin fibroblasts were seeded in plates and cultured for 24 h. They were pretreated with UVB and 5, 10, 25 µg/mL of galangin then incubated for 48 h in serum-free medium. The production of type 1 procollagen was quantified according to manufacturer’s introductions (Takara, Shiga, Japan) so do fibroblast growth factor 2 (FGF-2) (R&D Systems Inc., Minneapolis, MN, USA). Then detected by using ELISA plate reader.
Western blot analysis
Skin fibroblasts were seeded in 6-well plates and grown for 24 h, followed by galangin treatment for 48 h. Skin fibroblasts were then lysed in radio immune precipitation assay (RIPA) lysis buffer (Pierce, Rockford, IL, USA) with protease inhibitor cocktails (100X). To obtain a supernatant, the lysate was centrifuged at 12,000 rpm for 20 min at 4 °C and protein concentration was determined with a bicinochoninic acid (BCA) protein assay kit (Thermo scientific, USA). 20 µg of each protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immediately placed in blocking buffer (5% skim milk with TBST) and blocked for 1 h. Subsequently, PVDF membranes were incubated at 4 °C with primary antibodies against IkB, phosphor-IkB, nucleus NF-kB, cytoplasm NF-kB, c-Fos, c-Jun, MMP-1, MMP-3, MMP-9, p-38, phosphor-p-38, ERK, phospho-ERK, 4-HNE, β-actin (1:1000 dilution) for 24 h. To detect primary antibodies, horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000 dilution) were incubated for 2 h. Signals were detected using the Super Signal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) under a LAS 4000 image analyzer (Fuji Film Life Science, Tokyo, Japan).
Statistical analysis
All the results in this study were obtained from the average of triplicate experiment data, and the mean values and standard deviations were analyzed using one-way ANOVA in SPSS 23 program (Statistical Package for Social Science, Chicago, IL, USA). Analysis of variance Duncan’s multiple range test and one-way ANOVA were used to compare the significance of differences between the samples at P < 0.05 level. Differences among the means were determined by a t-test, and values of P < 0.05 and P < 0.01 were considered statistically significant.
