Cells and Chemicals
Human keratinocytes (HaCaT) cell lines were obtained from the American-style culture collection (ATCC, Manassas, USA). The Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), antibiotics, trypsin-EDTA and phosphate-buffered saline (PBS) were acquired from HyClone, Logan, USA. In addition, hydrogen peroxide (H2O2), Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), methyl thiazolyl diphenyl-tetrazolium bromide (MTT) gallic acid, and quercetin were procured from Sigma Aldrich CO., USA.
Protein assay kit (Pierce, USA), polyvinylidene fluoride (PVDF) membrane (Neuroscience, Seoul, South Korea), Primary antibodies (MMP-1, Hsp70, COX-2, and β-actin), secondary antibodies, and Chemiluminescent substrate were procured from Thermo Scientific, (MA, USA). cDNA Synthesis and qPCR Master Mix were purchased from Promega (Madison, USA) and other fine chemicals and reagents utilized in the present experiment were analytical grades.
Non-fermentation and fermentation H. cordata water/ethanol extraction
H. cordata was provided by Stemforce Co., Inc. (Gyeongsan, South Korea). The dried plants were washed three times, then allowed to dry, weighed, and then crushed into the powder used in this study. H. cordata extracts of non-fermented or fermented with A. pullulans were prepared with hot-water extracts and 50% ethanol extract.
Briefly, dried H. cordata with about 200 g and water were combined in a ratio of 1:2 (w/v) and wetted. Next, add 0.6% peptone, and mix evenly with sterilizing for 15 min at 121 °C. After cooling, dextrin was added in an amount corresponding to 1.5% of the sample and combined well. Next, 2.3x107 CFU/mL of A. pullulans suspension was inoculated and fermented for 18 days at 30 °C (Shin et al. 2019; Xia et al. 2017). Finally, the water extract, sterilized purified water equivalent to 5 times the weight of the fixed non-fermented and fermented sample, was added and bathed for 24 h at 60 °C, followed by three extractions. First, the supernatant was filtered after centrifuging the sample at 8,000×g for 10 minutes. Then, again, the sample was centrifuged at 8000×g for 20 min. Next, the extracted sample was filtered via filter paper, and the dry extract sample was powder by a rotary evaporator. A dried extract sample was kept at 4°C for future use.
Determination of total phenolic and flavonoid content
The total phenol content of the H. cordata extract was analyzed by the Folin-Ciocalteu method . The sample was 1 g and dissolved in 10 ml of the deionized distilled water (ddH2O), followed by a centrifuge of the sample at 240×g for 5 min. The obtained supernatant was filtered using Whitman filter paper, and the filtrate was further centrifuged at 240×g for 10 min. Briefly, 200 μL of H. cordata extract mixed with 670 μL ddH2O. Then. Folin-Ciocalteu’s phenol reagent (30 μL) was added to the mixture and placed at room temperature for 5 minutes. Next, added sodium carbonate (600 μL) was then kept at room temperature for 30 minutes and absorbance was determined at 765 nm using a UV-Vis spectrophotometer (Optizen 2120UV, Republic of Korea). The total polyphenol content of the H. cordata extracts was represented as mg gallic acid equivalents per gram of extract sample (mg/g).
The total flavonoid content was estimated by a colourimetric assay . H. cordata extracts of 200 μL were mixed with ddH2O (790 μl), 10% aluminium chloride hexahydrate, 1M potassium acetate (30 μL), and 95% ethanol (450 μL) were mixed and kept to incubation for 40 minutes. After incubation, absorbance was measured by UV-Vis spectrophotometer at 510 nm. The total flavonoid content of the H. cordata extracts was represented as mg quercetin equivalents per gram of extract sample (mg/g).
DPPH and ABTS scavenging assay
The scavenging efficiency of H. cordata extracts was determined by Blois et al. . The 200 μL of H. cordata extracts were diluted for each concentration with 100 μL of DPPH dissolved in ethanol solution mixed with 200 μL of H. cordata extracts sample. The reaction was allowed for 30 minutes and absorbance was determined at 517 nm using an ELISA reader (InfiniteTM F200, Männedorf, Switzerland).
The ABTS radical scavenging of H. cordata extracts was determined by Re et al. . Briefly, 2.45 mM potassium persulfate and 7 mM ABTS were mixed at a 1:1 (v/v) ratio and kept darkroom for 24 h to generate ABTS radicals. The ABTS solution in which radicals were formed was diluted with ethanol, and the absorbance measured at 734 nm was adjusted to be 0.70 ± 0.02. H. cordata extracts sample, an equal volume of ABTS (150 μl) and the 0.2 % test sample was mixed well and allowed at room temperature for 6 minutes. Finally, the absorbance was determined at 734 nm using an ELISA reader. DPPH or ABTS+ results were compared with ascorbic acid as a positive control. DPPH or ABTS+ scavenging (%) was calculated using a formula depicted in our previous study .
Cell culture and cytotoxicity assay
The HaCaT cells were grown in DMEM supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin) at 37 °C in a 5% CO2 incubator. Next, the cell lines were subcultures at 2 ~ 3-day intervals.
The HaCaT (5×103) were seeded into a 96-well plate at 37 °C in a 5% CO2 incubator. For 24 hours. Next, different concentrations of HCE, HCW, HCFE, and HCFW (10, 100 μg/ml) were added and then incubated at 37 ℃ in a 5% CO2 incubator for 24 hours. Then cells were induced with UVA (3 J/cm2)/H2O2 (1 mM) for 24 hours (table. 1). Herein, the old medium was removed, and add fresh DMEM (100 μL) and MTT (20 μL) solution and incubated for 3 hours in 5% CO2 at 37 °C. Afterwards, the sample optical density was measured at an ELISA reader using a 96-well plate (InfiniteTM F200, Switzerland).
Protein expression by Western blot
They were treated with H. cordata extract samples and then UVA or H2O2 exposed to HaCaT cells. After the incubation, the media was replaced by the PBS buffer. Protein extraction was performed by adding protein lysis buffer with an inhibitor cocktail. The sample was centrifuged at 12,000 rpm at 4 °C for 15 minutes and the supernatant was collected. 30 μg of protein was electrophoresed on 7.5% SDS-PAGE and then attached to a PVDF membrane. After blocking the protein-attached PVDF membrane with NEO western enhanced buffer (Neuroscience, Seoul, Korea), the PVDF membrane was treated with primary antibodies COX-2, Hsp70, MMP-1 and β-actin shaken overnight at 4 °C, then washed 3 times for 5 minute each with TBST buffer. Next, the blot was incubated in secondary antibodies for one hour. Finally, the membranes were washed 3 times with TBST for 5 minutes, and protein bands were visualized by Chemiluminescent substrate (Thermo Scientific, MA, USA). After the secondary antibody reaction, the PVDF membrane was developed using the photosensitive film in the dark. The relative intensity of a specific protein band was quantified using Imager (SLB, Seoul, Korea) and lab works software (Upland, CA, USA).
RNA isolation and qRT-PCR analysis
According to the manufacturer's instructions, total RNA was isolated from all treated groups samples, using a Tri-RNA isolation kit (Favorgen Biotech, Ping-Tung, Taiwan). NanoDrop (Thermo Scientific, MA, USA) determined RNA content and purity. For cDNA Synthesis, a GoScript™ reverse transcription mix, oligo (dT) kit was used. Nuclease-free water, GoScript™ reaction buffer, oligo(dT), and GoScript™ enzyme mix were mixed with the reaction product so that the final volume was 10 μL. cDNA was synthesized as follows: 3 μg of RNA heated at 65 °C for 5 minutes, and Nuclease-Free water was added to the reaction product to make the final volume of the development of 20 μL. Then the reaction sample was repeated 1 at 25 °C for 5 minutes, 42 °C for 60 minutes, and 70 °C for 15 minutes, and then stored at -20 °C.
For real-time quantitative PCR (qPCR), cDNA and primers were added to a qPCR master mix (Promega, Madison, USA) kit. All the primer sequences were designed from the GenBank database and the BLAST pick primers program (https://www.ncbi.nlm.nih. gov/tools/primer-blast/) was shown in Table 2. Average 50 pmoles of primers for each gene, template (300 ng/2 μL), and qPCR master mix were added to each PCR tube, and Nuclease-free water was added to make the final reaction product 20 μL. The qPCR conditions were pre-denaturation at 95 °C for 1 minutes followed by 40 cycles of 10 s at 95 °C (denaturation), 15 s at 60 °C (annealing), and 20 s at 72 °C (extension). It was repeated twice, and the melting curve was analyzed by scanning at 60~95 °C for 1 °C each for 1 s. The relative expression of the genes was calculated using the 2-ΔΔCt methods. Then, ΔΔCt was calculated by the treatment group (Ct target gene - Ct GAPDH gene) and control group (Ct target gene−Ct GAPDH gene) estimated. Finally, the optimal GAPDH gene was used as an internal control.
All data were analyzed through one-way analysis of variance (ANOVA), the software SPPSS (SPSS program, USA). The statistical significance was determined using a value of p< 0.05.