Effect of MMP/TIMP Balancing of Cynoglossus Semilaevis Shell Extracts on Skin Protection

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Article Effect of MMP/TIMP Balancing of Cynoglossus semilaevis Shell Extracts on Skin Protection

Soo-Cheol Choi and In-Ah Lee *

Department of Chemistry, College of Natural Science, Kunsan National University, 558 Daehak-ro, Gunsan-si 54150, Jeollabuk-do, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-063-469-4574

Abstract: Cynoglossus semilaevis shell is a by-product of the Cynoglossus semilaevis, a species of fish mainly distributed along the west coast of Korea. As its skin is very tough and difficult to process, it is not useful as food. For this reason, most of it is discarded except for a small amount that is used as feed, which results in environmental pollution. Considering this, there is a need for research on the development of functional materials using Cynoglossus semilaevis shell. This study focused on the mechanism of in vitro expression function of Cynoglossus semilaevis shell extract (CSE) for skin tissue in human dermal fibroblasts that induced or did not induce wrinkles by UV-B irradiation and aims to use it as a functional material for human skin beauty or wrinkle improvement through extraction and purification. According to the ELISA results using human dermal fibroblast cells, CSE reduced MMP-1 and elastase activity by up to 21.89% and 12.04%, respectively, in a concentration-dependent manner, and increased PIP synthesis by up to 62.24% in a concentration- dependent manner. The RT-PCR test results using mRNA showed the MMP-1, 2, and 3 expression

levels were suppressed in the CSE-treated group compared to the UVB-induced group and caused a concentration-dependent increase in TIMP-1 in the CSE-treat group. These results suggest that CSE

Citation: Choi, S.-C.; Lee, I.-A. Effect can maintain and improve skin tissue conditions through MMP/TIMP balancing in human dermal of MMP/TIMP Balancing of ﬁbroblast cell lines and indicate its potential as a functional material for improving skin diseases and Cynoglossus semilaevis Shell Extracts suppressing photo-aging. on Skin Protection. Fishes 2021, 6, 34. https://doi.org/10.3390/ﬁshes Keywords: Cynoglossue semilaevis shell; MMPs; TIMP-1; collagen; anti-aging 6030034

Academic Editor: Bernardo Baldisserotto 1. Introduction Wrinkles are caused by a decrease in hormone levels with age, exposure to UV rays, or Received: 8 July 2021 physical causes such as repeated muscle use and facial expressions and are characterized by Accepted: 18 August 2021 Published: 24 August 2021 the thinning of the dermis, causing epidermis thinning due to a decrease in the proliferation rate of cells that occupy the basal cell layer of the epidermis [1,2].

Publisher’s Note: MDPI stays neutral The dermis is a thick layer of skin beneath the epidermis, which makes up about 90% with regard to jurisdictional claims in of our skin. It mainly consists of connective and elastic fibers composed of hyaluronic acid, published maps and institutional affil- collagen, and elastin, which improve skin elasticity [3–5]. The collagen and elastin in the iations. dermal layer are strong connective proteins that form the dermal matrix, which prevents our skin from being easily damaged by external shocks and is known to play a significant role in maintaining skin elasticity. As such, the decomposition of collagen and elastin in human connective tissue directly affects skin elasticity and wrinkle formation [6,7]. MMPs are transcriptionally regulated by cell growth factors, hormones, and cytokines Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and are essential enzymes for biological processes such as blastogenesis, organogenesis, This article is an open access article nerve growth, ovulation, angiogenesis, and tissue absorption and remodeling by degrading distributed under the terms and ECM [8–12]. However, an imbalance with TIMPs (endogenous inhibitors), which can be conditions of the Creative Commons caused by various in vivo factors, is known to lead to excessive increases in MMPs, which Attribution (CC BY) license (https:// can cause inflammatory skin diseases due to skin tissue damage [13]. creativecommons.org/licenses/by/ Of the various types of MMPs, MMP-1 is a proteolytic enzyme specific to collagen. 4.0/). Although the synthesis of type 1 collagen and MMP-1 (degrading enzyme) is balanced in

Fishes 2021, 6, 34. https://doi.org/10.3390/ﬁshes6030034 https://www.mdpi.com/journal/ﬁshes Fishes 2021, 6, x FOR PEER REVIEW 2 of 11

increases in MMPs, which can cause inflammatory skin diseases due to skin tissue damage Fishes 2021, 6, 34 [13]. 2 of 11 Of the various types of MMPs, MMP-1 is a proteolytic enzyme specific to collagen. Although the synthesis of type 1 collagen and MMP-1 (degrading enzyme) is balanced in normal skin, imbalances between type 1 collagen and MMP-1 appear in aging skin due to normal skin, imbalances between type 1 collagen and MMP-1 appear in aging skin due to internal and external factors [8,14–16]. internal and external factors [8,14–16]. As major inhibitorsinhibitors ofof MMP, MMP, four four homologous homologous TIMPs TIMPs have have been been identified identified thus thus far [ 17far]. [17].Among Among them, them, TIMP-1 TIMP-1 is an inhibitoryis an inhibitory molecule molecule that regulates that regulates the expression the expression of various of variousMMPs inMMPs the skin in andthe askin cytoprotective and a cytoprotective factor that factor inhibits that the inhibits breakdown the ofbreakdown intercellular of intercellularsubstances found substances in aging found and in cell aging damage and cell caused damage by aging caused and by exogenous aging and stimuli exogenous and stimuliis known and to is promote known cellto promote differentiation cell differen and havetiation anti-apoptotic and have anti-apoptotic effects [18–20 effects]. TIMP-1 [18– 20].is a TIMP-1 natural inhibitoris a natural of inhibitor a wide range of a wide of metal-dependent range of metal-dependent proteolytic proteolytic enzymes present enzymes in presentalmost allin livingalmost tissues, all living including tissues, human including skin, human and is skin, known and as is a biomoleculeknown as a biomolecule that inhibits thatECM inhibits degradation, ECM degradation, such as collagen, such elastin, as collag gelatin,en, elastin, fibronectin, gelatin, and fibronectin, keratin by and proteolytic keratin byenzymes proteolytic most enzymes prominent most in naturalprominent and in extrinsic natural aging and extrinsic [21–24]. aging [21–24]. The tonguetongue sole sole (Cynoglossus (Cynoglossus semilaevis semilaevis) is a) benthic is a fishbenthic belonging fish tobelonging the Cynoglossidae to the Cynoglossidaefamily in Pleuronectiformes, family in Pleuronectiformes, typically found typically on the muddyfound on bottoms the muddy along bottoms the coast along or in thebrackish coastor or freshwater in brackish areas. or Tonguefreshwater sole, areas. commonly Tongue known sole, as commonly the tonguefish, known are caughtas the tonguefish,around the westare caught coast of around Korea, the such west as Gunsan coast of and Korea, Seocheon. such as It isGunsan the largest and speciesSeocheon. in the It isCynoglossidae the largest familyspecies and in the is very Cynoglossidae useful from family an industrial and is very perspective useful becausefrom an of industrial its rapid perspectivegrowth rate [because25,26]. Tongue of its rapid sole skingrowth is a by-productrate [25,26]. of Tongue tongue solessole skin living is ina by-product the west coast. of tongueAlthough soles tongue living soles in the are west effective coast. for Althou bone healthgh tongue and preventing soles are effective osteoporosis for bone due tohealth their andhigh preventing content of vitamins,osteoporosis folic due acid, to potassium, their high and content calcium, of vitamins, their skin folic is tough acid, and potassium, difficult andto process, calcium, which their meansskin is ittough is not and useful difficult as food. to process, For this which reason, means most it of is it not is discarded, useful as food.except For for this a small reason, amount most of used it is asdiscarded, feed, which except results for ain small environmental amount used pollution. as feed, which Most resultsfish skins in areenvironmental composed almost pollution. solely ofMost collagen, fish skins thus thearecollagen composed extracted almost and solely refined of collagen,from fish thus skin the can collagen be used forextracted food and and medicine refined from (Figure fish1 ).skin This can study be used proposes for food the and use medicineof tongue (Figure sole skin 1). toThis develop study functionalproposes the materials use of tongue as a therapeutic sole skin to agent develop for skincare functional or materialstreating skin as a diseases. therapeutic agent for skincare or treating skin diseases.

(A) (B)

Figure 1. CynoglossusCynoglossus semilaevis semilaevis(A(A),), a a by-product by-product of of CynoglossusCynoglossus semilaevis semilaevis shell(shellB (B).).

2. Reagents Reagents and and Methods Methods 2.1. Materials Materials and and Reagents Reagents Dulbecco’s Modified Modiﬁed Eagle’s Medium/F12Medium/F12 Nutrient Nutrient Mixture Ham (DMEM/F12(DMEM/F12 3:1) (Gibco, Waltham,Waltham, MA, MA, USA), USA), Anti-biotic Anti-biotic Antimycotic Antimycotic (Gibco, (Gibco, Waltham, Waltham, MA, USA), MA, Trypsin- USA), Trypsin-EDTAEDTA (Gibco, Waltham, (Gibco, Waltham, MA, USA), MA, Phosphate USA), Phosphate Buffered Saline Buffered (Gibco, Saline Waltham, (Gibco, MA,Waltham, USA), MA,Fetal BovineUSA), SerumFetal (FBS)Bovine (Gibco, Serum Waltham, (FBS) MA,(Gibco, USA), Waltham, 3-(4,5-Dimethylthiazol-2-yl)-2,5- MA, USA), 3-(4,5- Diphenyl-2H-tetrazolium Bromide (MTT) (Sigma-Aldrich, St Louis, MO, USA), TGF-β1 Dimethylthiazol-2-yl)-2,5 Diphenyl-2H-tetrazolium Bromide (MTT) (Sigma-Aldrich, St (Sigma-Aldrich, St Louis, MO, USA), Albumin from Bovine Serum (BSA) (Sigma-Aldrich, Louis, MO, USA), TGF-β1 (Sigma-Aldrich, St Louis, MO, USA), Albumin from Bovine St Louis, MO, USA), Bradford Reagent (BIORAD, Hercules, CA, USA), 1 M Tris-HCl pH 8.0 (Elpis-biotech, Daejeon, Korea), Triton X-100 (Yakuri Pure Chemicals, Kyoto, Japan), Dimethyl Sulfoxide (DMSO) (Amresco, Solon, OH, USA), Pro-collagen Type I Peptide (PIP) ELISA Kit (Takara, Shiga, Japan), Matrix Metalloproteinase-1 (MMP-1) Human ELISA kit Fishes 2021, 6, 34 3 of 11

(Abcam, Cambridge, MA, USA), Phosphoramidon (Sigma-Aldrich, St Louis, MO, USA), STANA (Sigma-Aldrich, St Louis, MO, USA)

2.2. Preparation of Cynoglossus semilaevis Shell Extract (CSE) After washing the Cynoglossus semilaevis shell, it was naturally dried and finely pul- verized to prepare Cynoglossus semilaevis shell powder. A 10-fold weight of 60% ethanol aqueous solution was added to the Cynoglossus semilaevis peel powder, extracted for 12 h at room temperature, and then filtered through Whatman filter paper to prepare a Cynoglossus semilaevis peel extract. The extracted CSE was stored in a −80 ◦C freezer for 1 day and then powdered using a rotary vacuum concentrator, and the powdered Cynoglossus semilaevis peel extract was diluted in DMSO at a concentration of 50 mg/mL and stored at −20 ◦C for use in experiments.

2.3. Amino Acid Composition Analysis To analyze the amino acid composition contained in CSE, 100 mg of a CSE sample was put in a glass tube, dissolved in 3 mL of 6 N-HCl solution, and acid hydrolyzed in a hot air dryer at 110 ◦C for 24 h. Thereafter, the CSE sample was derivatized with PITC (Phenyl isothiocyanate) and used as a sample for analysis. The amino acid composition of the sample for analysis was analyzed using HPLC (HPLC PI-CO-TAG system). The HPLC used for the analysis was a Water HPLC System (Waters 510 HPLC Pump), Waters 717 automatic sampler, Waters 996 photodiode array detector, Waters gradient controller, Millenium 2010 chromatography manager (Waters Co.), and the column was a free amino acid analysis column (3.9 mm × 300 mm, 4 m waters pi-co-Tag, Pico Rivera, CA, USA). The column temperature was 40 ◦C, and the ﬂow rate was 1 mL/min. As the mobile phase, 140 µM sodium acetate (6% acetonitrile) was used for buffer A, and 60% acetonitrile was used for buffer B.

2.4. Cell Line Selection and Cell Culture Human Dermal Fibroblasts (HDFs) cells were selected and used based on the efﬁcacy test data of pGuideline for Efﬁcacy Evaluation of Functional Cosmetics (II)y. HDFs (ATCC, Manassas, VA, USA) were primary cells isolated from adult skin and were used in which Mycoplasma, Hepatitis B, Hepatitis C, HIV-1, Bacteria, Yeast, and other fungi were not detected. HDFs were inoculated on the bottom of a culture dish and cultured in a 37 ◦C, 5% CO2 incubator (HERAcell 150i, Thermo, Waltham, MA, USA) by adding DMEM/F12 3:1 mixed medium containing 1% Antibiotic-Antimycotic and 10% FBS.

2.5. MTT Assay Single HDFs cells suspensions with a density of 1 × 106 cells/well were plated to a 24-well plate and cultured overnight. Then, the cells were treated with 1.56, 3.13, 6.25, 12.5, 25, and 50 mg/mL CSE. After 24 h, 10 µL MTT stock (5 mg/mL) was added to each well and incubated for 2 h. Then, the supernatants were discarded, and 100 µL DMSO was added to each well. After 10 min of shaking, the optical density at the wavelength of 570 nm (OD570) was measured on a SPARK microplate reader (Bio Tek, Winooski, VT, USA). All samples were tested in triplicate and repeated 3 times [27–29].

2.6. UV-B Irradiation HDFs were irradiated by UV-B lamp GL20SE (Sankyo denki, Japan) ﬁtted with a UV-B source designed for microplates. HDFs grown in microplates were irradiated at 20 mJ/cm2 UV-B dose. Cells were irradiated in phosphate-buffered saline (PBS) without the plastic lid. When the irradiation received matched the desired programmed energy, the UV-B irradiation stopped automatically, and subsequently, the cells were incubated with DMEM without FBS until analysis. Fishes 2021, 6, 34 4 of 11

2.7. Enzyme-Linked Immunosorbent Assay Release of MMP-1 and type I pro-collagen in HDFs were analyzed by ELISA. Cells (1 × 106 cells/well) were pre-incubated in 24-well plates for 24 h to reach >90% conﬂuence and washed with PBS. The cells were treated with or without different concentrations (0.2, 0.39, 0.78, 1.56, 3.13, and 6.25 mg/mL) of CSE for 24 h. Cell culture medium from each well was analyzed for its MMP-1 (Abcam, USA) and type I pro-collagen (Takara, Japan) contents per the manufacturer’s instructions of the ELISA kit.

2.8. Measurement of the Ability to Inhibit Elastase Activity HDFs were inoculated and cultured in a 100 mm culture dish at 5 × 104 cells/well, and the cultured cells were dissolved by adding 0.1% Triton X-100 0.2 M Tris-HCl (pH 8.0) solution, and this cell lysate was dissolved at 4 ◦C. After centrifugation at 10,000 rpm for 10 min, the supernatant was taken, and an enzyme solution containing elastase was used. The elastase enzyme solution was quantiﬁed, 100 µg each was added to a 96-well plate, and 0.2 M Tris-HCl (pH 8.0) buffer was added to make 80 µL. The test substance was diluted in buffer and added by 10 µL, and 10 µL of STANA, a substrate of elastase, was added to each well and reacted for 90 min in an oven at 37 ◦C. The absorbance was measured at 405 nm to evaluate the elastase activity. Phosphoramidon (ﬁnal treatment concentration 5.9 µg/mL) was used as a positive control.

2.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis Cells (1 × 106 cell/well) were grown to >90% confluence prior to UV-B irradiation. Following UV-B irradiation, cells were treated with or without CSE for 24 h. Total RNA was isolated from nonirradiated and irradiated (UV-B, 20 mJ/cm2) HDFs using AccuPrep® Universal RNA Extraction Kit (Bioneer, Daejeon, Korea) according to manufacturer’s instructions. Total RNA was treated with RNase-free DNase I (Thermo Fisher Scientific, Rockford, IL, USA). The cDNA synthesis from total RNA was performed using Cell Script All-in-One cDNA synthesis Master Mix (CellSafe, Yongin, Korea) following the producer’s directions. cDNA amplification was carried out with quantitative PCR in a Thermal Cycler Dice® Real-Time System TP800 (Takara Bio Inc., Ohtsu, Japan) following the manufacturer’s protocol. Briefly 20 µL of cDNA sample mixed with forward and reverse primers in nuclease-free water. The target cDNA was amplified using following forward and reverse primers; TNF-α, forward 50-GAG CTG AGA GAT AAC CAG CTG GTG-30, reverse 50-CAG ATA GAT GGG CTC ATA CCA GGG-30, IL-6, forward 50-ATG AAC TCC TTC TCC ACA AGC-30, reverse 50-GTT TTC TGC CAG TGC CTC TTT G-30, MMP-1, forward 50-TGA AAA GCG GAG AAA TAG TGG-30, reverse 50-GAG GAC AAA CTG AGC CAC ATC-30; MMP-2, forward 50-TTG CCA TCC TTC TCA AAG TTG TAG G-30, reverse 50-CAC TGT CCA CCC CTC AGA GC-30; MMP-3, forward 50-CAC TCA CAG ACC TGA CTC GGT T-30, reverse 50-AAG CAG GAT CAC AGT TGG CTG G-30; TIMP-1, forward 50-ATA CTT CCA CAG GTC CCA CAA C-30, reverse 50-GGA TGG ATA AAC AGG GAA ACA C-30; β-actin, forward 50-AGA TCA AGA TCA TTG CTC CTC CTG-30, reverse 50-CAA GAA AGG GTG TAA CGC AAC TAA G-30. The PCR amplification was carried out with an initial denaturation at 95 ◦C for 1 min, followed by 40 PCR cycles, each cycle consisting of 95 ◦C for 15 s and 60 ◦C for 30 s. β-Actin was used as an internal control [30–33]. PCR results were isolated and quantified by beta-actin for quantitative comparison (FluoroBOX, Cellgentek, Korea).

2.10. Statistical Analysis All data were expressed as mean value ± standard deviation, and statistical analysis was performed using SPSS® Package Pro-gram (IBM, Armonk, NY, USA). All results were the results of 3 or more independent experiments, and statistical significance was verified at the independent samples t-test p < 0.05 significance level. Fishes 2021, 6, 34 5 of 11

3. Results 3.1. Amino Acid Composition Analysis Table1 shows the results of analyzing the amino acids and free amino acids of CSE by HPLC. Unlike other proteins, collagen contains hydroxylated amino acids hydroxyproline and hydroxylysine and is a three-dimensional macromolecular form in which three polypeptide chains are twisted together in a helical structure. The triple helix region of collagen is composed of a repeating amino acid sequence of Gly-XY, and proline and hydroxyproline are frequently located at the positions of X and Y. The amino acid contents of glycine, proline, and hydroxyproline conﬁrm the presence or absence of collagen in the Cynoglossus semilaevis shell extract. In Table1, CSE contains 17 amino acids such as proline, and glycine accounts for about 20% of the total amino acids as 3.905 µg/mL, and proline accounts for about 7.4% as 1.432 µg/mL. In addition, when checking the free amino acid content of CSE, it contains 20 kinds of free amino acids, including hydroxyl-L-proline, and it was detected high content that occupied more than 85% of total free amino acid. These results suggest that glycine, proline, and hydroxyproline, which constitute the repetitive Gly-X-Y amino acid sequence in the triple helix region of collagen, are contained in a large amount in CSE, and the collagen component contained in a large amount was estimated to have a positive effect on the skin (please see the attachment).

Table 1. Amino acid composition in Cynoglossus semilaevis shell extract (please see the attachment).

Cynoglossus semilaevis Shell Extract Amino Acid Physiological (Free) Amino Acid Content Content Kind Kind (µg/mL) (µg/mL) Aspartic acid (Asp) 1217 o-Phosphoserine 4 Threonine (Thr) 447 Taurine 33 Serine (Ser) 807 o-Phosphoethanolamine 1 Glutamic acid (Glu) 1980 Sarcosine 0 Glycine (Gly) 3905 L-2-Aminoadipic Acid 2 Cysteine (Cys) 4537 L-Citruline 8 Alanine (Als) 30 DL-2-Aminobutyric 0 Valine (Val) 582 LCystathionine 10 Methionine (Met) 417 β-Alanine 8 Isoleucine (Ile) 225 DL-3-Aminoisobutyric Acid 7 Leucine (Leu) 621 4-Aminobutyric acid 1 Tyrosine (Tyr) 177 2-Aminoethanol 133 Phenylalanine (Phe) 398 DL-plusallo-δ-Hydroxylysine 0 Lysine (Lys) 709 L-Ornithine 3 Histidine (His) 235 L-1-Methylhistidine 0 Arginine (Arg) 1562 L-3-Methylhistidine 0 Proline (Pro) 1432 L-Anserine 0 L-Carnosine 0 hydroxyl-L-proline 1554 Total 19,281 Total 1764

3.2. Effect of CSE on HDF Cells Viability In order to assess the cytotoxic effect of CSE on HDF cells, an MTT assay was carried out. Various concentrations of CSE, including 1.56, 3.13, 6.25, 12.5, 25, and 50 mg/mL, were chosen to treat HDF cells for 24 h. As shown in Figure2, compared to control cells treated with 0.00001 mg/mL TGF-β1, the cell viabilities of the following treatments with 25 and 50 mg/mL CSE were 45% and 50%, respectively, which indicated that HDF cells viability was not affected signiﬁcantly below 12.5 mg/mL CSE for 24 h. Therefore, the concentration range of CSE for subsequent wrinkling improvement activity screening was determined to be within 12.5 mg/mL (Figure2). Fishes 2021, 6, x FOR PEER REVIEW 6 of 12

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Lysine (Lys) 709 L-Ornithine 3 Histidine (His) 235Lysine (Lys)Lysine (Lys) L-1-Methylhistidine 709 709 0 L-Ornithine L-Ornithine 3 3 Lysine (Lys) 709 L-Ornithine 3 Arginine (Arg) 1562Histidine Histidine (His) (His) L-3-Methylhistidine 235 235 L-1-Methylhistidine 0 L-1-Methylhistidine 0 0 Histidine (His) 235 L-1-Methylhistidine 0 Proline (Pro) 1432Arginine Arginine (Arg) (Arg) L-Anserine 1562 1562 L-3-Methylhistidine 0 L-3-Methylhistidine 0 0 Arginine (Arg) 1562 L-3-Methylhistidine 0

Proline (Pro)Proline (Pro)Fishes L-Carnosine 2021, 6, x FOR 1432 PEER REVIEW 1432 0 L-Anserine L-Anserine 0 0 7 of 12 Proline (Pro) 1432 L-Anserine 0

hydroxyl-L-proline 1554L-Carnosine L-Carnosine 0 0 L-Carnosine 0

Total 19,281 Total 1764hydroxyl-L-proline hydroxyl-L-proline 1554 1554 hydroxyl-L-proline 1554 CSE-exposed cells released substantially decreased levels of MMP-1 compared to the Total Total 19,281 19,281non-exposed groupTotal (Figure Total 3B). The base MMP-1 1764 level 1764 in the HDF culture medium was Total 19,281 Total 3.2. Effect 1764 of CSE on HDF Cells Viability 4.3 ng/mL, and the presence of CSE suppressed the increase in MMP-1 levels in a In order to assess the cytotoxic effect3.2. Effectof CSE3.2. of onCSE Effect HDF on of HDF cells,CSE Cells onanconcentration-dependent HDFMTT Viability Cells assay Viability was carried manner. The MMP-1 levels significantly reduced the 3.2. Effect of CSE on HDF Cells Viability out. Various concentrations of CSE, includingIn order 1.56, 3.13,toIn assessorder 6.25, tothe 12.5, assess concentration-dependentcytotoxic 25, theand cytotoxic 50effect mg/mL, of CSEeffect were on of collagenaseHDF CSE cells,on HDF an activity MTTcells, assay anin cellsMTT was betweenassay carried was 12.41~21.89% carried compared to In order to assess the cytotoxic effect of CSE on HDF cells, an chosenMTT assay to treat was HDF carried cells for 24 h. As shownout. Various inout. Figure concentrationsVarious 2, compared concentrationssolvent of to CSE, control controls including of CSE, cells after includingtreated 1.56, CSE 3.13, (0 1.56,.39~6.25 6.25, 3.13, 12.5, mg/mL) 6.25, 25, and 12.5, treatment. 50 25, mg/mL, and 50 weremg/mL, were out. Various concentrations of CSE, including 1.56, 3.13, 6.25, 12.5, 25,with and 0.00001 50 mg/mL, mg/mL were TGF- β1, the cellchosen viabilities tochosen treat of the HDF to following treat cells HDF for treatments 24cells h. Asfor shown24 with h. As in25 shown Figure and in 2, Figure compared 2, compared to control to cells control treated cells treated chosen to treat HDF cells for 24 h. As shown in Figure 2, compared50 to mg/mL control CSE cells were treated 45% and 50%, respectively,with 0.00001with which mg/mL0.00001 indicated TGF-mg/mLβ 1,that TGF-the HDF cellβ1, viabilities cellsthe cellviability viabilities of the following of the following treatments treatments with 25 andwith 25 and with 0.00001 mg/mL TGF-β1, the cell viabilities of the following treatmentswas not affected with significantly25 and below 12.550 mg/mLmg/m50L CSE mg/mL forwere 24 CSE 45%h. Therefore, were and 45%50%, theand respectively, concentration 50%, respectively, which indicated which indicated that HDF that cells HDF viability cells viability 50 mg/mL CSE were 45% and 50%, respectively, which indicated rangethat HDF of CSE cells for viability subsequent wrinklingwas improvement not affectedwas not significantlyaffectedactivity screeningsignificantly below was 12.5 below determinedmg/m 12.5L CSE mg/m forL 24 CSE h. Therefore,for 24 h. Therefore, the concentration the concentration was not affected significantly below Fishes12.5 mg/m2021, 6,L 34 CSE for 24 h. Therefore,to be within the concentration 12.5 mg/mL (Figure 2). range of rangeCSE for of subsequentCSE for subsequent wrinkling wrinkling improvement6 of improvement 11 activity screeningactivity screening was determined was determined range of CSE for subsequent wrinkling improvement activity screening was determined to be withinto be 12.5 within mg/mL 12.5 (Figure mg/mL 2). (Figure 2). to be within 12.5 mg/mL (Figure 2).

Figure 3. Effect of CSE on the cellular release of type Iα1 pro-collagen (A) and MMP-1 (B) in HDFs. The contents of type Iα1 pro-collagen and MMP-1 were calculated from HDF culture media

following 24 h incubation with different concentrations of CSE using ELISA. The data are expressed Figure 2.2. Cell viability in human dermal ﬁbroblasts.fibroblasts.Figure 2. CellFigure The viability data 2. Cell are in viability expressed humanas % in dermal of asashuman Solvent %% ofof fibroblasts. solventsolventdermal control control controlfibroblasts. (MeanThe data ± SD areThe ( nexpressed data = 3). are * p expressed< as 0.05, % of Cynoglossus solvent as % of control solvent semilaevis control shell extract vs. Solvent Figure 2. Cell viability in human dermal fibroblasts. The data are expressed(Mean(Mean as % ±± ofSDSD solvent (n(n= = 3) 3),control,* p*

. Figure 3. Effect of CSE on the cellular release of type Iα1 pro-collagen (A) and MMP-1 (B) in HDFs. The contents of α type I 1 pro-collagen andFigure MMP-1 3. Effect were of calculated CSE on the from cellular HDF release culture of media type Iα following1 pro-collagen 24 h incubation(A) and MMP-1 with ( differentB) in HDFs. concentrations of CSE usingThe ELISA.contents The of datatype areIα1 expressedpro-collagen as %and of SolventMMP-1 controlwere calculated (Mean ± fromSD ( nHDF= 3). culture * p < 0.05,media Cynoglossus semilaevis shellfollowing extract vs.24 h Solvent incubation control). with different: Measures concentrations in each group, of CSE :using Mean ELISA. value The of untreated data are expressed group, : Mean value of CSE treatmentas % of Solvent group, control: Mean (Mean value ± of SD positive (n = 3). control. * p < 0.05, Cynoglossus semilaevis shell extract vs. Solvent control). ). ). : Measures in each group : Mean value of untreated group, : Mean value of CSE treatment group, : Mean value of positive control.

3.4. Effect of CSE on the Elastase The treatment of various concentrations (0.2, 0.39, 0.78, 1.56, 3.13, and 6.25 mg/mL) of CSE in HDF cells reduced elastase levels compared to the group that did not treat CSE (Figure 4). Based on 100% of the basic elastase activities of HDF culture medium, treatment of CSE has concentration-dependent inhibition of an increase in elastase levels. Elastase levels significantly reduced concentration-dependent elastase activity in cells by 12.41% to 21.89%, compared to solvent controls after CSE (0.39 to 6.25 mg/mL) treatment.

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Lysine (Lys)Lysine (Lys) 709 709 L-Ornithine L-Ornithine 3 3 Lysine (Lys) 709 L-Ornithine 3 HistidineHistidine (His) (His) 235 235 L-1-Methylhistidine L-1-Methylhistidine 0 0 Histidine (His) 235 L-1-Methylhistidine 0 ArginineArginine (Arg) (Arg) 1562 1562 L-3-Methylhistidine L-3-Methylhistidine 0 0 Arginine (Arg) 1562 L-3-Methylhistidine 0 Proline (Pro)Proline (Pro)Fishes 2021, 6, x FOR 1432 PEER REVIEW 1432 L-Anserine L-Anserine 0 0 7 of 12 Proline (Pro) 1432 L-Anserine 0 L-CarnosineL-Carnosine 0 0 L-Carnosine 0 hydroxyl-L-prolinehydroxyl-L-proline 1554 1554 hydroxyl-L-proline 1554 CSE-exposed cells released substantially decreased levels of MMP-1 compared to the Fishes 2021, 6, 34 Total Total 19,281 19,281non-exposed groupTotal 7(Figure of 11Total 3B). The base MMP-1 1764 level 1764 in the HDF culture medium was Total 19,281 Total 1764 4.3 ng/mL, and the presence of CSE suppressed the increase in MMP-1 levels in a 3.2. Effect3.2. of CSE Effect on of HDF CSE Cellsonconcentration-dependent HDF Viability Cells Viability manner. The MMP-1 levels significantly reduced the 3.2. Effect of CSE on HDF Cells Viability concentration-dependent collagenase activity in cells between 12.41~21.89% compared to 3.4. Effect of CSE on the Elastase In order toIn assessorder tothe assess cytotoxic the cytotoxic effect of CSEeffect on of HDF CSE cells,on HDF an MTTcells, assayan MTT was assay carried was carried In order to assess the cytotoxic effect of CSE on HDF cells, an MTT assay was carried out. Variousout. concentrationsVarious concentrationssolvent of CSE, controls including of CSE, after including 1.56, CSE 3.13, (0 1.56,.39~6.25 6.25, 3.13, 12.5, mg/mL) 6.25, 25, and 12.5, treatment. 50 25, mg/mL, and 50 weremg/mL, were The treatment of various concentrations (0.2, 0.39, 0.78, 1.56, 3.13, and 6.25 mg/mL) out. Various concentrations of CSE, including 1.56, 3.13, 6.25, 12.5, 25, and 50 mg/mL, were chosen tochosen treat HDF to treat cells HDF for 24cells h. Asfor shown24 h. As in shown Figure in 2, Figure compared 2, compared to control to cells control treated cells treated of CSE in HDF cells reduced elastase levels compared to the group that did not treat CSE chosen to treat HDF cells for 24 h. As shown in Figure 2, compared to control cells treated with 0.00001with mg/mL0.00001 TGF-mg/mLβ1, TGF-the cellβ1, viabilities the cell viabilities of the following of the following treatments treatments with 25 andwith 25 and (Figure4). Based on 100% of the basic elastase activities of HDF culture medium, treatment with 0.00001 mg/mL TGF-β1, the cell viabilities of the following treatments with 25 and 50 mg/mL50 CSE mg/mL were CSE 45% were and 45%50%, and respectively, 50%, respectively, which indicated which indicated that HDF that cells HDF viability cells viability of CSE has concentration-dependent inhibition of an increase in elastase levels. Elastase 50 mg/mL CSE were 45% and 50%, respectively, which indicated that HDF cells viability was not affectedwas not significantlyaffected significantly below 12.5 below mg/m 12.5L CSE mg/m forL 24 CSE h. Therefore,for 24 h. Therefore, the concentration the concentration Fishes 2021, 6, x FOR PEER REVIEW levels signiﬁcantly reduced concentration-dependent elastase activity in cells by 12.41%8 of to12 was not affected significantly below 12.5 mg/mL CSE for 24 h. Therefore, the concentration range of rangeCSE for of subsequentCSE for subsequent wrinkling wrinkling improvement improvement activity screeningactivity screening was determined was determined 21.89%, compared to solvent controls after CSE (0.39 to 6.25 mg/mL) treatment. range of CSE for subsequent wrinkling improvement activity screening was determined to be withinto be 12.5 within mg/mL 12.5 (Figure mg/mL 2). (Figure 2). to be within 12.5 mg/mL (Figure 2).

Figure 3. Effect of CSE on the cellular release of type Iα1 pro-collagen (A) and MMP-1 (B) in HDFs. The contents of type Iα1 pro-collagen and MMP-1 were calculated from HDF culture media

following 24 h incubation with different concentrations of CSE using ELISA. The data are expressed Figure 4. 4. ElastaseElastase activity activity in ina cell-free a cell-free system. system.Figure The 2. The daCellFigureta data areviability 2 expressed. areCell expressedin viability human asas % % inofdermal as of humansolvent %Solvent of fibroblasts. solvent dermalcontrol control control fibroblasts.(mean (MeanThe data ± SD areThe ( nexpressed data = 3). are * p expressed< as 0.05, % of Cynoglossus solvent as % of control solvent semilaevis control shell extract vs. Solvent Figure 2. Cell viability in human dermal fibroblasts. The data are expressed±(mean SD as ( n%± = of3).SD solvent * (n p < = 0.05, 3) control.* pCynoglossus< 0.05, Cynoglossus semilaevis(Mean semilaevis shell ±extract SD(Meanshell (n =vs. extract 3), ± solventSD * p (n< vs. 0.05, = solventcont3), Cynoglossus*control) rol).p < control).0.05, ). . ).Cynoglossus : ).Measures semilaevis: : Measures in semilaevis eachshell inin eachextract shellgroup vs. extract solvent : Mean vs. control). solvent value ofcontrol). :untreated Untreated : group,Untreated : Mean value of CSE (Mean ± SD (n = 3), * p < 0.05, Cynoglossus semilaevis shell extract vs. solventgroupeach control). group, : Mean : Untreated Meanvalue of value untreated of untreated group, group, : Mean group,: value: Mean CSE treatmentof value CSE: CSE of treatmen CSEtreatment group,treatment treatmentt group, group, group, group : Positive : Mean , : : Mean Mean:control.value Positive value control. of positive control. group, : CSE treatment group, : Positive control. ofvalue positive of positive control. control. 3.3. Effect3.3. of CSE Effect on of the CSE Type on Ithe Pro-Collagen Type I Pro-Collagen and MMP-1 and MMP-1 3.3. Effect of CSE on the Type I Pro-Collagen and MMP-1 3.5. Effect Effect of CSE on the UV-B Mediated Expression CSE treatmentCSE treatment at various at concentrationsvarious concentrations (0.2, 0.39, (0.2, 0.78, 0.39, 1.56, 0.78, 3.13, 1.56, and 3.13, 6.25 andmg/mL) 6.25 mg/mL) UV-B-irradiated HDF cells were treated with CSE at different3.4. concentrations Effect of CSE to on analyze the Elastase CSE treatment at various concentrations (0.2, 0.39, 0.78, 1.56, 3.13,UV-B-irradiated and 6.25 mg/mL) HDF cells werein treated HDF cellsin wi HDFth showed CSE cells at increasedshowed different increased synthesis concentrations synthesis of type to Iof pro-collagen type I pro-collagen compared compared to untreated to untreated mRNA expression levels related to inflammation and photo-aging. In the UV-B-treated in HDF cells showed increased synthesis of type I pro-collagen analyzecompared mRNA to untreated expression levels relatedgroups. to inflammation Thegroups. basic The type and basic I photo-aging.pro- typecollagenThe I pro- treatment Incollagenlevel the ofUV-B- of HDF levelvarious cultureof HDFconcentrations wasculture 132.32 was (0.2, ng/mL, 132.32 0.39, and ng/mL,0.78, the 1.56, and 3.13, the and 6.25 mg/mL) HDF cells, inflammation-related genes of TNF-alpha and IL-6 were strongly increased, and groups. The basic type I pro-collagen level of HDF culture was treated132.32 ng/mL,HDF cells, and inflammation-relatedthe presence genespresence of of CSETNF-alpha ofincreased CSE of and increasedCSE theIL-6 in type HDFwere the cellsI stronglypro-collagentype reduced I pro-collagen elastase synthesis levels synthesis depending compared depending toon the the group on thatthe did not treat CSE it was confirmed that the concentration-dependent reduction of inflammation when CSE presence of CSE increased the type I pro-collagen synthesisincreased, depending and onit wasthe confirmed concentration.that theconcentration. concentration-dependent Type I pro-collagen Type(Figure I pro-collagen 4). levels reductionBased increased levelson 100%of increased significantly of the significantly basic from el astase29.69% from activities to 29.69% 62.24% toof 62.24%HDF culture medium, was treated by concentration (Figure5A). concentration. Type I pro-collagen levels increased significantly inflammationfrom 29.69% towhen 62.24% CSE was treated byconcentration-dependent concentrationconcentration-dependent (Figure 5A). comparedtreatment compared ofto CSEsolvent has to co concentration-dependentsolventntrol after control treatment after treatment of CSE inhi (0.20bition of CSE to of 6.25 (0.20an increase to 6.25 in elastase levels. Furthermore, in photon-aging-related gene expressions such as MMP-1,2 and TIMP-1 concentration-dependent compared to solvent control after treatment ofFurthermore, CSE (0.20 to in 6.25 photon-aging-related mg/mL) gene inmg/mL) expressionsHDF cells. in HDF such cells.Elastase as MMP-1,2 levels andsignificantly TIMP- reduced concentration-dependent elastase activity in cells by in HDF cells where UV-B was investigated, TIMP-1 and COL1A1 mRNA expressions were mg/mL) in HDF cells. 1 in HDF cells where UV-B was investigatCSE-exposeded, TIMP-1CSE-exposed and cells COL1A1 released12.41% cells mRNAreleasedsubstantially to 21.89%, expressions substantially compared decreased decreased to levels solvent of levelsMMP-1 controls of compared MMP-1after CSE compared to(0.39 the to 6.25to the mg/mL) treatment. reduced, and MMP-1 and MMP-2 mRNA expressions were strongly increased. UV-B in- CSE-exposed cells released substantially decreased levels of MMP-1were reduced, compared and to MMP-1 the and MMP-2non-exposed mRNAnon-exposed expressions group (Figure weregroup strongly3B). (Figure The increased. 3B).base The MMP-1 base UV- levelMMP-1 in thelevel HDF in theculture HDF medium culture mediumwas was duced changes in the MMP-1,2 and TIMP-1 were ameliorated by CSE (1, 5, and 10 mg/mL) non-exposed group (Figure 3B). The base MMP-1 level in the HDFB inducedculture mediumchanges wasin the MMP-1,2 and4.3 TIMP-1ng/mL, wereand theameliorated presence byof CSECSE (1, suppressed 5, and 10 the increase in MMP-1 levels in a treatment in a concentration-dependent manner.4.3 In ng/mL, HDF cells and treated the presence with CSE, of the CSE mRNA suppressed the increase in MMP-1 levels in a 4.3 ng/mL, and the presence of CSE suppressed the increase mg/mL)in MMP-1 treatment levels inin aa concentration-dependen t manner. In HDF cells treated with CSE, expression of MMP-1 and 2 increasedconcentration-dependent by UV-Bconcentration-dependent irradiation was significantlymanner. manner.The reduced, MM The andP-1 MMlevelsP-1 significantlylevels significantly reduced reducedthe the concentration-dependent manner. The MMP-1 levels significantlythe mRNA reduced expression the of MMP-1 and 2 increased by UV-B irradiation was significantly reduced,the mRNA and expression the mRNA of TIMP-1 expression decreased of TI byMP-1 UV-B decreased irradiation wasby UV-B significantly irradiation increased. was significantlyAs the above increased. result, the As amount the above of type result, Iα1 pro-collagenthe amountbiosynthesis of type Iα1 was pro-collagen increased through the control of MMP/TIMP by treatment by the concentration of CSE (Figure5B). biosynthesis was increased through the control of MMP/TIMP by treatment by the concentration of CSE (Figure 5B).

Fishes 2021, 6, x FOR PEER REVIEW 8 of 11

3.5. Effect of CSE on the UV-B Mediated Expression UV-B-irradiated HDF cells were treated with CSE at different concentrations to analyze mRNA expression levels related to inflammation and photo-aging. In the UV-B- treated HDF cells, inflammation-related genes of TNF-alpha and IL-6 were strongly increased, and it was confirmed that the concentration-dependent reduction of inflammation when CSE was treated by concentration (Figure 5A). Furthermore, in photon-aging-related gene expressions such as MMP-1,2 and TIMP- 1 in HDF cells where UV-B was investigated, TIMP-1 and COL1A1 mRNA expressions were reduced, and MMP-1 and MMP-2 mRNA expressions were strongly increased. UV- B induced changes in the MMP-1,2 and TIMP-1 were ameliorated by CSE (1, 5, and 10 mg/mL) treatment in a concentration-dependent manner. In HDF cells treated with CSE, the mRNA expression of MMP-1 and 2 increased by UV-B irradiation was significantly reduced, and the mRNA expression of TIMP-1 decreased by UV-B irradiation was significantly increased. As the above result, the amount of type Iα1 pro-collagen Fishes 2021, 6, 34 8 of 11 biosynthesis was increased through the control of MMP/TIMP by treatment by the concentration of CSE (Figure 5B).

Figure 5. Effects of the CSE on the expression of inflammatory and photo-aging in HDFs cells. RT- Figure 5. Effects of the CSE on the expression of inflammatory and photo-aging in HDFs cells. RT- PCR analysis of mRNA expression levels of inflammatory (A) and photo-aging (B). Total RNA was PCR analysis of mRNA expression levels of inflammatory (A) and photo-aging (B). Total RNA was isolated from the HDFs cells of the normal, UV-B, and CSE (1, 5 and 10 mg/mL) groups for RT-PCR isolated from the HDFs cells of the normal, UV-B, and CSE (1, 5 and 10 mg/mL) groups for RT-PCR analysisanalysis at at 24 24 h. h. Total Total RNA RNA was was isolated isolated and and quanti quantifiedfied by by beta-actin beta-actin for for quantitative quantitative comparison. comparison. 4. Discussion 4. Discussion Photo-aging and wrinkles are caused by the production of reactive oxygen species in skinPhoto-aging tissue exposed and to wrinkles UV light are and caused by the by imbalance the production between of reactive MMP/TIMP oxygen in species activated in skinfibroblasts, tissue exposed which weakens to UV light the dermal and by layer’s the imbalance ability to between regenerate MMP/TIMP cells and the in factors activated that control skin elasticity. The epidermis generates ROS when it is exposed to external stimuli, and the generated ROS stimulates keratinocytes to produce and secrete interleukins, such as IL-1α, IL-1β, IL-3, IL-6, and IL-8, as well as colony-stimulating factors and cytokines, such as TNF-α

(tumor necrosis factor—alpha) [28,30,34,35]. When the cytokines produced and secreted by keratinocytes affect the dermal tissue in an autocrine or paracrine manner, the macrophages in the dermis react and cause complex inflammatory and immune responses or bind to each cytokine receptor in the fibroblasts to activate systems of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this reason, balancing MMPs and TIMPs is one of the most important mechanisms for improving skin diseases and inhibiting photo-aging [12,23,36]. This study evaluated the in vitro efficacy of CSE on skin cells by linking the biological expression levels of MMPs and TIMPs with skin diseases, such as wrinkles and photo-aging, in HDF cells that induced or did not induce skin diseases using UVB. Prior to the experiment, various types of amino acids among the main components contained in CSE were analyzed through amino acid composition analysis. Among them, the presence or absence of collagen components in CSE was determined by detecting glycine, proline, and hydroxyproline constituting collagen, CSE contains various kinds of Fishes 2021, 6, 34 9 of 11

amino acids and contains a high content of glycine (3905 µg/mL), proline (1432 µg/mL), and hydroxyproline (1554 mg/mL). These results showed that CSE contains a large amount of collagen, thus it was expected that the collagen component contained in a large amount would have a positive effect on the skin, and it was used as a basic research result to proceed with the follow-up study. Through measuring CSE cell viability using MTT assay in HDF cells, it was determined that there was no toxicity to the cells at a concentration of 12.5 mg/mL or less, thus the subsequent tests were performed by setting the concentration to this level. Collagen protects the skin from external stimuli or force by giving it strength and tension, and a decrease in collagen is closely related to skin aging, thus the synthesis and degradation of ECM extracellular matrix, such as collagen, needs to be controlled properly. However, excessive exposure to stimuli, such as UV rays, promotes the expression of MMPs, which are proteolytic enzymes that degrade proteins such as extracellular matrix (ECM) and basement membrane (BM), and the activated MMPs cause various skin diseases by disrupting skin tissue balance through breaking down collagen in the dermal layer. This study investigated the changes in the physiological activity of skin cells according to CES concentration (0.2 mg/mL, 0.39 mg/mL, 0.78 mg/mL, 1.56 mg/mL, 3.13 mg/mL, and 6.25 mg/mL) in HDF cell lines through MMP-1, elastase, and PIP ELISA tests. CES inhibited MMP-1 in a concentration-dependent manner by 21.89% compared to the solvent control at a concentration of 6.25 mg/mL. In the elastase inhibition assay test, CSE inhibited elastase activity in a concentration-dependent manner by 12.04% compared to the solvent control at a concentration of 6.25 mg/mL. These results suggest that CSE could prevent skin tissue decomposition and maintain and improve skin tissue conditions, and the PIP test results showed that CSE enhanced the synthesis of collagen by 62.24% at a concentration of 6.25 mg/mL by inhibiting ECM degradation. In an experiment using RT-PCR, UV-B roll was irradiated to HDF cells to measure the efficacy of CSE treated with different concentrations (1, 5, and 10 mg/mL) on inflammation and photo-aging. CSE dose-dependently decreased UV-B-enhanced inflammatory cytokines TNF-α and IL-6 in skin cells. In addition, CSE regulated the mRNA expression of MMP-1, MMP-2, and TIMP-1 in the imbalance of MMPs and TIMPs caused by cytokines increased by the inflammatory response and increased the production of type Iα1 pro-collagen. This result showed that CES could maintain and improve the condition of normal skin tissue in HDF cell lines not induced by UV-B. Furthermore, the results of inhibiting ECM degradation, such as elastin and collagen, through MMP/TIMP balancing by inhibiting and promoting MMPs and TIMPs in HDFs cell lines, where skin diseases such as photo- aging and wrinkles were induced by UV-B, confirmed the potential of CSE as a functional material that can play a significant role in skin health by inhibiting photo-aging and treating skin diseases in skin tissues damaged or undamaged by UV-B.

5. Conclusions Today, fish are used not only as a variety of food but also as a health-promoting food. However, this industrial use is still limited. In particular, it is known that the nutrient components, including collagen, are distributed in the fish skin, but the development of a product using the fish skin in the industry has not been activated. In this study, it was confirmed that CSE treatment reduced TNF-α and IL-6 in a dose- dependent manner after UV-B irradiation on HDF cells induced a strong inflammatory response and photo-aging. In addition, CSE inhibited the activity of MMP-1 and 2 related to skin diseases such as wrinkles and photo-aging, and increased the activity of TIMP-1, thereby inhibiting elastin degradation and increasing COL1A1 activity through the balance between MMP and TIMP. Through these results, we would like to suggest that commercially valuable substances can be obtained using fish skins discarded as waste, and in particular, they can be used as beneficial materials for skincare. Further studies will be designed to evaluate the efficacy Fishes 2021, 6, 34 10 of 11

as a functional material through the separation and puriﬁcation of Cynoglossus semilaevis shell peptide components.

Author Contributions: Conceptualization, S.-C.C. and I.-A.L.; methodology, S.-C.C. and I.-A.L.; software, S.-C.C. and I.-A.L.; validation, S.-C.C. and I.-A.L.; formal analysis, S.-C.C. and I.-A.L.; investigation, S.-C.C. and I.-A.L.; resources, S.-C.C. and I.-A.L.; data curation, S.-C.C. and I.-A.L.; writing—original draft preparation, S.-C.C. and I.-A.L.; writing—review and editing, S.-C.C. and I.-A.L.; visualization, S.-C.C. and I.-A.L. All authors have read and agreed to the published version of the manuscript. Acknowledgments: This research was supported by research funds of Kunsan National Univer- sity(18B18843131) and National Research Foundation (NRF-2019R1A4A1026423). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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