ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2018; 16(2): R E S E A R C H

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1 ISSN (Print) ISSN (Online) Asian J Beauty Cosmetol 2018; 16(2): R E S E A R C H A R T I C L E Open Access Anti-wrinkle and Skin turnover Improvement Effects of Niacinamide-dipeptide Convergence Ga Yeon Kim 1, Seung Je Lee 1, Mi Ji Jeon 1, Bo Min Kim 1, Geun Tae Kim 1, Sang Moon Kang 2, Kee Young Lee 2, Eun Jin Shin 2, Sang Yong Kim 3, Young Min Kim 1* 1 Department of Biological Sciences and Biotechnology, Hannam University, Daejeon, Korea 2 A&PEP Affiliated R&D Center, Cheongju-si, Chuncheongbuk-do, Korea 3 Department of Food Science & Bio Technology, Shinansan University, Ansan-si, Gyeonggi-do, Korea * Corresponding author: Yong Min Kim, Department of Biological Sciences and Biotechnology, Hannam University, 70 Hannamro, Daedeok-Gu, Daejeon 34430, Korea Tel.: Fax: kym@hnu.kr Received December 7, 2017 Revised April 21, 2018 Accepted April 30, 2018 Published June 30, 2018 Abstract Purpose: This study aimed to determine the anti-wrinkle and skin turnover improvement effects of niacinamide-dipeptide convergence (N-peptide). Methods: The following methods were employed to assess the anti-wrinkle and skin turnover improvement effects of N-peptide: 3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing assay, chicken chorioallantoic membrane (CAM) assay, reverse transcription polymerase chain reaction (RT-PCR), and three-dimensional (3D) cell culture. Results: The MTT assay revealed that the N-peptide does not exert a cytotoxic effect in human keratinocytes (HaCaT). Moreover, the scratch wound healing and CAM assays showed that the N-peptide induced migratory properties and angiogenesis, respectively, in HaCaT cells. The expression of matrix metalloproteinases (MMP)-1, -2, -3, and -9 was measured using RT-PCR to evaluate the anti-wrinkle effect of N-peptides. The findings demonstrated that N-peptides were responsible for reducing the expression of the aforementioned MMPs. In addition, using western blotting, N-peptides were identified to increase energy metabolism activity. Furthermore, 3D cell culture analysis indicated improvement in the skin turnover rate because of the N-peptides. Conclusion: Taken together, these results suggest that N-peptides may be a source of cosmetic agents for the improvement of wrinkles and skin turnover. Keywords: Niacin-dipeptide convergence, Functional cosmetic, Anti-wrinkle, Skin barrier, Skin turnover rate Introduction 인체의약 16% 를차지하는피부는각질아래표피, 진피, 피하조직으로구성되어있으며, 외부유해인자들로부터인체를보호하는중요한역할을한다 (Yang et al., 2010). 그러나나이가들어감에따라각종오염물질및스트레스에의해피부세포들이손상을입게되고, 세포증식이억제되어피부의탄력성과윤택성이감소되고, 기미, 주근깨등피부색소침착및주름이생기게된다 (Yang et al., 2010; Lee et al., 2014a). 이와같은피부노화현상 (skin aging) 은피부각질층의구조적, 기능적변화로인한것으로, 그요인에따라외인성노화 (extrinsic aging) 와내인성노화 (intrinsic aging) 로구분할수있다 (Lee et al., 2014a; Yoo et al., 2010). 외인성노화는자외선및스트레스등과같은외부요인에의한피부노화를말하며, 내인성노화는나이가들어감에따라피부의생리적기능이저하되어자연스럽게나타나는현상이다 (Yoo et al., 2010). 피부의진피층은피부의물리, 화학적성질을결정하는역할을하며, 진피의대부분은콜라겐 (collagen) 과탄력섬유 (elastic fiber) 가차지한다 (Kim et al., 2000). 교원질즉콜라겐과탄력섬유는피부의기계적견고성, 조직의결합력및탄력성등에관여한다 (Jeon & Kang, 2009; Kim et al., 2000). 그중콜라겐은진피의 90% 이상을차지하고있으면서외부로부터의자극에대해피부를보호하는역할을하는데, 이러한콜라겐이내부또는외부로부터자극을받게되면구조변화, 생합성량감소등을 Copyright c Korea Institute of Dermatological Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Anti-wrinkle and Skin Turn over Effects of Niacinamide-dipeptide Convergence 겪는다. 그로인해피부진피층에존재하는세포외기질이손상되어피부주름이만들어지게된다 (Jeon & Kang, 2009). 콜라겐은전구체 (procollagen) 형태로합성된후세포밖으로배출되어 pn collagen, pc collagen 등의중간산물을거쳐합성이되며, 현재까지 19 종류의콜라겐이밝혀져있다 (Scharffetter-Kochanek et al., 2000; Lee et al., 2014b). 콜라겐의분해와관련된다양한메커니즘중하나는전사인자인 activating protein-1 (AP-1) 에의한것으로, AP-1에의해유도되는기질금속단백질분해효소 (matrix metalloproteinases, MMPs) 에의해서세포외기질과프로콜라겐및콜라겐으로분해된다 (Scharffetter-Kochanek et al., 2000; Yoo et al., 2010). MMPs는아연이온을필요로하는단백질분해효소로세포외기질단백질을분해하는효소이며지금까지 24 종류가알려져있다 (Pyun et al., 2012; So et al., 2008). MMPs 중 MMP-1 (collagenase) 은콜라겐타입 I, III 을분해하고, MMP-3 (stromelysin-1) 은콜라겐타입 IV을분해하며 MMP-9 (gelatinase B) 는콜라게나아제에의해분해된산물을더작게가수분해한다 (Pyun et al., 2012). 최근들어피부주름개선에효과가있는화장품에대한관심이높아지면서기능성화장품소재를탐색하기위한연구가활발히이루어지고있다 (Kim et al., 2009). 그중단백질가수분해물로부터얻어진펩타이드는아미노산서열이가지는배열및구조에따라생체내혹은피부에서생리활성을가지게되어생물학적제제로서의산업적응용가능성이높다 (Jung et al., 2015; Kim et al., 2009; Kim et al., 2016). 또한최근에는재조합과정을통해합성된펩타이드연구가활발히이루어지고있는추세이다 (Lupo & Cole, 2007). 따라서본연구에서는기능성화합물-펩타이드융합체를이용한신규기능성화장품소재를개발할목적으로항노화기능성물질-나이아신펩타이드융합체가주름개선및피부에너지대사에미치는영향을확인하고자하였다. Methods 1. 실험재료 1) 시약및기기실험에사용된 3-(4,5)dimethyl-2 thiazolyl)-2,5- diphny-2h-tetra-zolium bromide (MTT) 는 Sigma- Aldrich (USA) 에서구입하였으며, 94.5% 에탄올 (ethanol), 클로로폼 (chloroform), 포름알데히드 (formaldehyde) 는 SAMCHUN (Korea) 의제품을, 이소프로판올 (isopropanol) 은 JUNSEI CHEMICAL (Japan) 의제품을사용하였다. 시료의흡광도는 ELISA microplate reader (Model 680; Bio-Rad Laboratories, Japan) 를사용하여측정하였다. 2) 펩타이드융합체제조본실험에사용된나이아신디펩타이드융합체는 A&PEP (Korea) 에서공급받아사용하였다. N-peptide1 (molecular weight: Da) 과 N-peptide2 (molecular weight: Da) 는피부미백기능성물질로잘알려진나이아신아미드에 2종의 dipeptide를각각결합시킨신규융합펩타이드다. 3) 세포주배양본실험에서사용된 HaCaT (human keratinocyte cell line) 세포는 Dr. N. Fusenig (German Cancer Research Center, DKFZ, Germany) 로부터분양받았으며, human fibroblast 세포는 American Type Culture Collection (ATCC; Rockville, USA) 에서분양받아사용하였다. 세포는 10% FBS와 1% antibiotics가포함된 Dulbecco's Modified Eagle Medium (DMEM; Hyclone TM, USA) media를사용하였으며, 5% CO 2, 37 incubator에서배양하였다. 매 48 h 마다 trypsin-edta (Hyclone TM ) 를이용하여세포를부유상태로만든다음세포를 cells/ml로분주하여계대배양하였다. 2. 실험방법 1) MTT assay 12 well plate에 HaCaT 세포를 cells/well로분주한다음 5% CO 2, 37 incubator에서 24 h 동안배양하였다. 그후, 시료 N-peptide1과 N-peptide2를농도별 (100 μg/ml, 500 μg/ml, 1 mg/ml) 로처리한후 24 h 동안 CO 2 incubator 에서배양하였다. 5 mg/ml solution을각 well 당 40 μl씩최종농도가 200 μg/ml가되도록처리한후 30 min 동안반응시킨다음 media를제거하였다. 각 well 당 dimethyl sulfoxide (DMSO; SAMCHUN, Korea) 150 μl을첨가한후 96 well plates에 100 μl씩옮긴후 ELISA microplate reader (680; Bio-Red laboratories, Japan) 로 595 nm에서흡광도를측정하였다. 모두 3번이상측정하였으며, 이에따른평균값과표준오차는 Microsoft excel program을이용하여분석하였다. 2) Wound healing assay HaCaT 세포를 6 well plate에 cells/well로분주한다음세포가 confluent한상태가될때까지 5% CO 2, 37 incubator에서배양하여안정화를유도하였다. 그런다음 200 μl의 tip (Neptune Scientific, USA) 의종단을이용하여세포의창상을 1회유도하였으며 N-peptide1 및 N-peptide2를농도별 (100 μg/ml, 500 μg/ml, 1 mg/ml) 로처리후, 시간대별 (0, 12, 24 h) 로세포의이동정도를도립현미경 (Axiovert 244

3 나이아신펩타이드융합체의항주름및피부턴오버개선효과 Table 1. Primer sequence for the amplification of target gene Gene Primer sequence (5'-3') Amplification size (bp) Annealing temperature ( ) For: ATGCTGAAACCCTGAAGGTG MMP-1 Rev: CTGCTTGACCCTCAGAGACC For: ATGACAGCTGCACCACTGAG MMP-2 Rev: TGATGTCATCCTGGGACAGA For: GCAGTTTGCTCAGCCTATCC MMP-3 Rev: GAGTGTCGGAGTCCAGCTTC For: GAGACCGGTGAGCTGGATAG MMP-9 Rev: TACACGCGAGTGAAGGTGAG For: GGACTTCGAGCAAGAGATGG GAPDH Rev: AGCACTGTGTTGGCGTACAG MMP-1, -2, -3, and -9, matrix metalloproteinase 1, 2, 3, ; Zeiss, Germany) 을이용하여관찰하였다. 3) Total RNA 추출및 cdna 합성 HaCaT 세포를각각 6 well plate에 cells/well로분주한다음 5% CO 2, 37 incubator에서 24 h 동안안정화시켰다. 그후, 시료 N-peptide1과 N-peptide2의최종농도가 500 μg/ml가되도록처리하여 24 h 동안배양하였다. 그다음 RiboEX total RNA Isolation Solution (GeneAll Biotechnology, Korea) 500 μl를각 well에처리하고상온에서 5 min 방치한후 Scraper를이용해세포를용해한후클로로폼, 이소프로판올, 70% 에탄올을각각첨가하는과정을통하여 total RNA를추출하였다. 그후의 cdna 합성과정은 DiaStar RT kit (SolGent, Korea) 을이용하여진행하였다. Total RNA 1 μl와 oligo (dt) 20 primer 9 μl를혼합한뒤 65 에서 5 min 동안반응시킨후에 8 mm dithiothreitol (DTT; SolGent) 1 μl, RNase (200 U/μL) 1 μl, 10 mm의 dntp가포함된 5X RT reaction buffer 4 μl, DEPC-DW 를첨가한다음 40 에서 1 h, 95 에서 5 min 반응을통하여 cdna를합성하였다. 4) 역전사중합효소연쇄반응 (RT-PCR) PCR은 Solg TM 2X Taq PCR Smart mix 1 (SolGent) 를이용하여실시하였다. PCR 조건은 95 에서 10 min 동안예비가열후, 95 에서 30 s denaturation, 각 specific primer sequences에맞는온도에서 30 s annealing, 72 에서 30 s extension 과정을 35회반복하였으며 72 에서 10 min 동안안정화과정을진행하였다. MMP-1, MMP-2, MMP-3, MMP-9, GAPDH의 specific primer sequences는 Table 1과같다. 5) Western blot 6 well plate에 HaCaT 세포를 cells/well로분주하여 24 h 동안 5% CO 2, 37 incubator에서배양한다음 N-peptide1과 N-peptide2를각각최종농도가 500 μg/ml 가되도록처리하여 24 h 동안배양하였다. 그다음 RIPA lysis buffer [25 mm Tris-Cl (ph 7.4), 1% NP40, 0.5% sodium deoxycholate, 150 mm NaCl, 1 mm PMSF] 를각 well에 150 μl씩첨가하여단백질을분리한뒤 14,000 rpm, 4 에서 20 min 동안원심분리하여상등액을취하였다. 분리한단백질은 ELISA-reader를이용하여 595 nm에서흡광도측정한후, 정량한다음 8%, 12% acrylamide gel을이용하여전기영동을실시하였다. 그다음 nitro cellulose membrane에 transfer하고 2% bovine serum albumin (BSA; Bovogen, Australia) 를이용하여 blocking 한후, 1차항체를 4 에서 18 h 반응시키고 2 차항체를결합시킨다음실험결과를측정하였다. 6) CAM assay 수정란을구입하여 70% 에탄올로소독한후에 37, 90% 습도가유지되는배양기에넣어 3 일동안배양후수정란의 air sac 쪽에직경약 2 cm 크기의원형창을만들고 air sac 아래의막은핀셋으로제거한다음 Tegaderm (3M Healthcare, USA) 을이용해원형창을막은후, 동일한조건에서 2일간더배양하였다. 5일째되는날, N-peptide1과 N-peptide2 500 μg/ml을떨어뜨린 disk를혈관위에올려놓은후 3일더배양한후에혈관신생정도를현미경 (Nikon SMZ 445; Nikon Instruments Inc., Japan) 으로관찰하였다. 7) Organotypic coculture을통한 human skin model 제작 Organotypic 3D culture는 Hiro Nakagawa (Li et al., 2011) 의방법을따랐다. SPL Insert Haging 6 well plate 245

4 Anti-wrinkle and Skin Turn over Effects of Niacinamide-dipeptide Convergence Figure 1. Cytotoxic effects of N-peptides in HaCaT cells. Cell viability was measured using MTT assay. Cells were treated with (A) N-peptide1 and (B) N-peptide2 for 24 h. Each experiment was performed in triplicates. Statistical analysis was performed using a t-test. N.S., not significant; N, negative control; Vitamin C, positive control; Vehicle, distilled water; N-peptide, niacinamide-dipeptide convergence; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide. (SPL Life Sciences, Korea) 에 Purecol Bovine Collagen type Ⅰ (Advanced BioMatrix, USA) 을 0.8 μm pore size 를갖는 inset 내부에 1 ml씩분주하여 acellular layer를만든다. 겔이굳은뒤배양한 human fibroblast ( cells/ well) 를 Matrigel Basement Membrane Matrix (Corning, USA) 와함께섞은후, acellular layer 층위에 3 ml을분주하여 cellular layer 층을형성한다음 37, 5% CO 2 조건의 incubator에서 5일간배양하였다. Human fibroblast를배양한 cellular layer 위에 DMEM media와 Ham's F12 media (HycloneTM) 를 3:1로섞은배지를넣어 gel matrix를안정화시킨다음, HaCaT 세포 ( cells/ml) 를 basement cell layer에 50 μl씩분주하고, 37, 5% CO 2 에서 2 h 동안안정화과정을거쳐 2주간배양하였다. 그다음 HaCaT 세포의각질분화유도후, N-peptide1과 N-peptide2는 500 μg/ml, vitamin C는 10 μg/ml이되도록 7일동안배양하였다. 8) Histology Organotypic co-culture한샘플을 phosphate buffered saline (PBS; Hyclone TM ) 로 2-3회 washing 후 10% phosphate-buffered formaldehyde (SAMCHUN, Korea) 에넣고 1 h 동안고정하였다. 탈수과정을거친후 paraffin 에 embedding하여 5 μm 크기로 section을나누었다. 조직은 hematoxylin과 eosin으로염색하여피부세포의각질탈락주기를확인하였다. 3. 통계처리통계프로그램인 SPSS 22.0을사용하였고실험설계에대한분산분석은일원배치분산분석 (one-way analysis of variance, one-way ANOVA) 과독립표본 t-test (independent sample t-test) 를실시하여검정하였다. 각자료는 3번이상의반복된실험을통하여얻어진결과로검정하였고 p<0.05인경우통계적으로유의한것으로판정하였다. Results and Discussion 1. 나이아신펩타이드융합체의세포독성검사피부표피층의 90% 이상을구성하는피부각질형성세포 (HaCaT) 는피부미백, 피부재생등과관련된많은실험에서널리사용되고있는세포주이다 (Choung et al., 2013). 본연구에서는나이아신펩타이드융합체 N-peptide1과 N-peptide2가화장품원료로써피부를구성하는피부세포에대한독성여부를확인하기위해 MTT assay를실시하였다. MTT 분석은세포생존율을측정하는대표적인실험방법중하나로살아있는세포수에비례해서흡광도를나타낸다 (Jeon et al., 2011). HaCaT 세포에나이아신펩타이드융합체 N-peptide1과 N-peptide2 를각각 24 h 동안처리후에세포생존율을측정한결과, Figure 1에나타난바와같이 HaCaT 세포에서 N-peptide1과 N-peptide2 농도별 (100 μg/ml, 500 μg/ml, 1 mg/ml) 처리에의한세포생존율이 90% 이상으로나타나세포독성이나타나지않음을확인하였다. 2. 나이아신펩타이드융합체의피부재생효과 HaCaT 세포는화학적및물리적자극에의해손상을받게되면다양한케모카인 (chemokine) 과사이토카인 (cytokine) 을분비함과동시에피부손상부위로이동해세포의증식을유도한다 (Choung et al., 2013). 이러한 HaCaT의증식및이주활성은콜라겐과같은피부구성단백질의합성을촉진하여피부재생과 246

5 나이아신펩타이드융합체의항주름및피부턴오버개선효과 Figure 2. Skin regeneration effect of N-peptides in HaCaT cells. The wound healing assay was determined the migration of HaCaT cells. Wound was scratched at 100% confluence, and cells were treated with the indicated concentrations of (A) N-peptide1 and (B) N-peptide2 for 24 h. Images were obtained at 0, 12, and 24 h. N, negative control; Vitamin C, positive control; N-peptide, niacinamide-dipeptide convergence. 상처치유등의항노화를유도한다 (Choung et al., 2013). 따라서피부세포의성장및이동을시험하는 wound healing assay 를통해나이아신펩타이드융합체의세포재생유도효과를검증하고자하였다. Figure 2A, 2B와같이 HaCaT 세포에나이아신펩타이드융합체 N-peptide1과 N-peptide2를 100 μg/ml, 500 μg/ml, 1 mg/ml의농도로처리했을때, 시간의존적으로아무것도처리하지않은무처리군에비해세포성장및세포이동이감소함을확인하였다. N-peptide1의경우, 농도별로 55%, 50%, 32% 로 wound area가감소하였으며, N-peptide2의경우에는 55%, 49%, 34% 로감소하였다. 이러한결과를바탕으로 N-peptide1과 N-peptide2 두시료모두 1 mg/ml의농도에서세포가이동한면적이양성대조군 vitamin C와동등하게감소되었음을확인하였다. 따라서본실험을통하여나이아신펩타이드융합체의세포재생유도효과를확인할수있었다. 3. 나이아신펩타이드융합체의주름관련유전자발현억제효과피부세포의결합조직을구성하는성분중하나인콜라겐은피부진피층에존재하며피부를지지하는역할을한다. 이러한콜라겐의감소는피부노화를이끄는중요한요인이될수있다. 콜라겐은일반적으로전사인자 AP-1에의해유도되는기질금속단백질분해효소 (MMPs) 에의해서분해된다 (Yoo et al., 2010). MMPs는피부세포를비롯한많은세포들로부터분비되어세포외기질과기저막 (basement membrane, BM) 을구성하는대부분의단백질성분을분해하며주름과피부처짐의원인이되는것으로알려져있으며, domain에의해 28개 (MMP-1-MMP-28) 로나누어진다 (Lee & Lee, 2013; Yoo et al., 2010). MMP family 중 MMP-1은콜라겐타입 I을분해시키는대표적인분해효소로알려져있고, MMP-2는제 4와제5형교원질및탄력소를분해하고 MMP-3의경우, 라미닌 (laminin), 파이브로넥틴 (fibronectin), 프로테오글리칸 (proteoglycan) 등기질단백질을분해한다고알려져있다 (Kim et al., 2011; Lee & Lee, 2013). 또한 gelatinase로알려진 MMP-9 역시콜라겐을분해한다 (Hardie et al., 2012). 따라서본연구에서는나이아신펩타이드융합체 N-peptide1 과 N-peptide2 각각 500 mg/ml의농도로 HaCaT 세포에처리했을때의피부노화관련유전자의발현정도를알아보고자하였다. 그결과, N-peptide1과 N-peptide2를처리했을때 MMP-1, -2, -3, -9의발현이아무것도처리하지않은 negative control 군에비해감소되는것을확인하였으며, 특히 MMP-3의경우양성대조군 vitamin C와유사하게감소되는것을확인하였다 (Figure 3). 이를통해, 나이아신펩타이드융합체가주름및노화방지에효과가있음을알수있다. MMP-1 MMP-2 MMP-3 MMP-9 GAPDH N Vehicle Vitamin C N-peptide1 N-peptide2 Figure 3. Regulation of MMP-1, -2, -3, and -9 mrna expression levels in HaCaT cells by N-peptides. Cells were treated with vehicle, vitamin C (2 μg/ml), N-peptide1 and N-peptide2 (500 μg/ml) for 24 h. The expression level of MMP-1, -2, -3, and -9 was analyzed using RT-PCR. The GADPH was used as a loading control. N, negative control; Vehicle, distilled water; Vitamin C, positive control; N-peptide, niacinamide-dipeptide convergence; MMP-1, -2, -3, and -9, matrix metalloproteinase 1, 2, 3, and 9; RT-PCR, reverse transcription polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. 247

6 Anti-wrinkle and Skin Turn over Effects of Niacinamide-dipeptide Convergence A p-mtor mtor p-ampk AMPK p-akt Akt N Vehicle Vitamin C N-peptide1 N-peptide2 B Relative band intensity (folds) N Vehicle Vitamin C N-peptide1 N-peptide2 p-mtor p-ampk p-akt Figure 4. Regulation of energy metabolism-related protein expression levels in HaCaT cells by N-peptides. Cells were treated with vehicle, vitamin C (2 μg/ml), N-peptide1 and N-peptide2 (500 μg/ml) for 24 h. (A) The expression of mtor, AMPK, and Akt was analyzed by western blotting. (B) Relative band intensity of energy metabolism-related proteins. The β-actin was used as a loading control. N, negative control; Vehicle, distilled water; Vitamin C, positive control; N-peptide, niacinamide-dipeptide convergence; p-mtor, phosphorylated mammalian target of rapamycin; p-ampk, phosphorylated AMPactivated protein kinase; p-akt, phosphorylated protein kinase B. 4. 나이아신펩타이드융합체가피부세포의에너지대사관련단백질발현에미치는영향나이아신펩타이드융합체처리시, 에너지대사관련단백질에영향을미치는정도를알아보고자 western blotting을실시하였다. AMP-activated protein kinase (AMPK) 는생체내의에너지인식및항상성조절, 당과지방의대사에있어서다른여러대사관련효소들을직접인산화시켜세포내에너지균형및영양분대사조절에중요한역할을하며, mammalian target of rapamycin (mtor) 는세포내 ATP 수준을직접적으로감지하여에너지수준과성장요소를제어한다 (Son et al., 2005). 또한 serine/threonine kinase인 Akt는세포밖성장인자에의한신호를세포안으로전달하며, 세포의증식과생존, 사멸, 당대사를조절한다 (Yoon et al., 2007). 따라서본연구에서는나이아신펩타이드융합체가피부세포의에너지대사관련단백질발현에미치는영향을알아보기위하여 western blotting을실시하였다. 그결과, Figure 4A, 4B와같이나이아신펩타이드융합체 N-peptide1과 N-peptide2를 HaCaT 세포에 500 μg/ ml의농도로처리했을때, 에너지대사관련단백질 p-mtor, p-ampk, p-akt의발현이증가하는것을확인하였으며, N-peptide1, 2 모두양성대조군 vitamin C에비해에너지대사관련단백질의발현이증가되는것을확인하였다. 이를통해나이아신펩타이드융합체처리시, 활발한에너지대사가이루어지며이는피부재생과항노화에효과가있는것으로여겨진다. 5. 나이아신펩타이드융합체의혈관신생작용을통한피부재생효과혈관신생은상처치유뿐만아니라암화과정, 초기발생과 정등정상적인상태에서도중요한역할을담당하며, 피부에있어영양공급을담당한다고알려져있다 (Song & Kim, 2009). 또한내인성노화의경우, 혈관의감소가영양공급감소를유발하여피부노화의원인이된다는연구결과가있다 (Yoon et al., 2007). 따라서나이아신펩타이드융합체 N-peptide1 과 N-peptide2 500 μg/ml 처리에의한혈관신생촉진효과를확인하기위해 ex vivo CAM assay를실시한결과, 혈관신생작용이활발한것을확인하였으며양성대조군으로사용한 vitamin C와비례하여혈관신생을촉진하는것을확인하였다 (Figure 5). 이를통해나이아신펩타이드융합체 N-peptide1 과 N-peptide2로인한피부재생효과및항노화효과가있는것으로여겨진다. Figure 5. Effect of N-peptides on angiogenesis. 5-day chick embryos were treated with vehicle, vitamin C, N-peptide1 and N-peptide2 (500 μg/ml) for 72 h. Arrows indicate angiogenic vessels. N, negative control; Vehicle, distilled water; Vitamin C, positive control; N-peptide, niacinamide-dipeptide convergence; CAM, chicken choriallantoic membrane

7 나이아신펩타이드융합체의항주름및피부턴오버개선효과 Figure 6. Improvement effect of N-peptides on skin turnover. N-peptide1 and N-peptide2 regulated hyperkeratosis in organotypic culture of HaCaT cells. 3D organotypic skin co-cultures were established. The organotypic model was maintained for 14 days and treated for 7 days. Representative H & E stained sections were photographed using a Leica DM750 microscope at 10x magnification. N, negative control; Vitamin C, positive control; N-peptide, niacinamide-dipeptide convergence; H & E, hematoxylin and eosin. 6. 3D cell culture 최근각질형성세포의 3 차원적인배양을통하여여러가지연구 가진행되고있는데, 이러한 3차원배양은기존에수행하던단층배양의단점을극복해피부의특징인각질화를형성하여표피의분화연구, 피부독성검사등많은피부연구에이용되고있다 (Kim et al., 2000; Ravi et al., 2015; Yoon et al., 2007). 인체표피중 90% 이상을차지하는각질형성세포는대략 28일을주기로진피층에서표피층으로밀려나가각질층으로부터탈락되며, 이러한세포의교체를턴오버 (tern-over) 라고한다 (Kim, 2007; Ko et al., 2009). 피부의턴오버주기는노화가진행될수록각질형성세포의기능이저하되어더길어지는데, 이로인해두꺼워진각질층에의해피부가거칠어지고주름이늘어나게된다 (Min & Bae, 2013). 따라서 N-peptide1 및 N-peptide2 500 μg/ml 처리에의한피부턴오버주기개선효과를확인하기위해 3D cell culture를실시한결과, 피부의각질층이양성대조군인 vitamic C의경우 px, 나이아신펩타이드융합체 N-peptide1의경우 px, N-peptide2의경우 px로두시료모두양성대조군 vitamin C와유사하거나더감소함을확인하였다 (Figure 6). 이를통하여나이아신펩타이드융합체가피부턴오버주기개선효과를가지고있음을알수있다. Conclusion MTT assay를통하여나이아신펩타이드융합체의세포독성검사를실시한결과, N-peptide1과 N-peptide2 처리에따른세포독성은나타나지않았다. 나이아신펩타이드융합체의피부재생효능을확인하기위하여 wound healing assay와 CAM assay를실시한결과, N-peptide1과 N-peptide2이양성대조 군 vitamin C와유사하게 HaCaT 세포의이동을유도하는것이관찰되었으며, CAM model에서역시혈관신생작용이활발하게이루어짐을확인하였다. 노화의대표적인원인인콜라겐감소에관여하는 MMPs 발현측정에서는 N-peptide1과 N-peptide2 처리에의해 MMP-1, -2, -3, -9의발현이감소하였으며, 이를통해 N-peptide1 및 N-peptide2가주름유발관련유전자의발현을억제시키는것을확인하였다. 또한피부세포의에너지대사에나이아신펩타이드융합체가미치는영향을확인하기위하여 western blot을실시하였을때, 에너지대사에관여하는단백질들의발현이양성대조군 vitamin C와유사하거나보다증가함을확인하였다. 마지막으로나이아신펩타이드융합체의피부주름개선효능외에전반적인피부턴오버개선효과를확인하기위해 3D cell culture 후, hematoxylin & eosin (H & E) 염색방법을이용해세포층단면을현미경으로관찰한결과, N-peptide1과 N-peptide2 처리군에서피부재생유도효과및턴오버개선효과가양성대조군 vitamin C와유사하거나더높게확인되었다. 결론적으로피부세포에유해하지않은나이아신펩타이드융합체는단순주름개선효능만이아닌피부의턴오버주기개선을통한피부의과각질화예방효능또한있는것으로보아주름개선및피부개선을위한기능성화장품소재로서이용될수있을것으로사료된다. Acknowledgements 본연구는 2016년도중기청융복합사업 ( 과제번호 : S ) 에의해수행되었음. References Choung MG, Hwang YS, Kim GP, Ahn GK, Shim HS, Hong SB, Choi JH, Yu CY, Chung IM, Kim SH, et al. Antimelanogenic effect and whitening of antocyanin Rich Fraction from seeds of Liriope platyphylla. Korean Journal of Medicinal Crop Science, 21: , Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nature Reviews. Molecular Cell Biology, 13: , Jeon MH, Park MR, Park YS, Hwang HJ, Kim SG, Lee SH, Kim M. Effect of pine (Pinus densiflora) needle extracts on antioxidant activity and proliferation of osteoclastic RAW cells. Journal of the Korean Society of Food 249

8 Anti-wrinkle and Skin Turn over Effects of Niacinamide-dipeptide Convergence Science and Nutrition, 40: , Jeon YS, Kang SM. Influence of collagen intake upon facial-skin wrinkles. Asian Journal of Beauty and Cosmetology, 7: 79-93, Jung HS, Song MY, Kim HS, Seo HH, Lee JH, Lee KR, Hong I, Moh SH. Development of anti-wrinkle materials using galloyl-peptide derivatives. Journal of Korea Academia- Industrial Cooperation Society, 16: , Kim DY, Hwang DI, Yoon MS, Choi IH, Lee HM. Effect of hydrosol extracted from Chrysanthemum boreale Makino flower on proliferation and migration in human skin keratinocyte. Journal of the Society of Cosmetic Scientists of Korea, 42: , Kim EA. Phytochemicals and beauty food. Food Science and Industry, 40: 3-9, Kim ID, Kwon RH, Heo YY, Jung HJ, Kang HY, Ha BJ. Supercritical extraction of oriental herb: Anti-aging and anti-wrinkle effects. Korean Society for Biotechnology and Bioengineering Journal, 23: , Kim JK, Lee JH, Yang MS, Seo DB, Lee SJ. Beneficial effect of collagen peptide supplement on anti-aging against photodamage. Korean Journal of Food Science and Technology, 41: , Kim SC, Hong NP, Choo JH, Park SH, Hong SC, Cha CI. The tree-dimensional co-culture of cholesteatomatous keratinocyte and fibroblast. Korean Journal of Otorhinolaryngology Head and Neck Surgery, 43: , Kim YC, Park EY, Kim SN, Yoo YG, Park MS, Lee GY, Lee SJ, Chan BS. Inhibitory effects of nude pack containing black tea water extract on skin wrinkle formation in hairless mice. Applied Microscopy, 41: , Ko JY, Choi KH, Kim YC. Effect of Scirpi rhizoma ethanol extract on skin whitening in an animal model of brown guinea pigs. Environmental Health and Toxicology, 24: , Lee BG, Kim JH, Ham SG, Lee CE. Study on biological activities of extracts for cosmeceutical development from Lagerstroemia indica L. branch. Korean Journal of Plant Reources, 27: 29-34, 2014b. Lee KO, Kim SN, Kim YC. Anti-wrinkle effects of water extracts of teas in hairless mouse. Toxicological Research, 30: , 2014a. Lee SY, Lee JY. Inhibitory efficacy of Smilax china L. on procollagen type-1 activity and MMP-1 gene expression in fibroblasts (CCD-986sk). Journal of Life Science, 23: , Li L, Fukunaga-Kalabis M, Herlyn M. The three-dimensional human skin reconstruct model: a tool to study normal skin and melanoma progression. Journal of Visualized Experiments, 54: 2937, Lupo MP, Cole AL. Cosmeceutical peptides. Dermatologic Therapy, 20: , Min JA, Bae HS. The effect of Opuntia humifusa and microneedle therapy system on the women s facial skin. Asian Journal of Beauty and Cosmetology, 11: , Pyun HB, Kim M, Park J, Sakai Y, Numata N, Shin JY, Shin HJ, Kim DU, Hwang JK. Effects of collagen tripeptide supplement on photoaging and epidermal skin barrier in UVB-exposed hairless mice. Preventive Nutrition and Food Science, 17: , Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FD. 3D cell culture systems: advantages and applications. Journal of Cellular Physiology, 230: 16-26, Scharffetter-Kochanek K, Brenneisen P, Wenk J, Herrmann G, Ma W, Kuhr L, Meewes C, Wlaschek M. Photoaging of the skin from phenotype to mechanisms. Experimental Gerontology, 35: , So SH, Lee SK, Hwang EI, Koo BS, Han GH, Chung JH, Lee MJ, Kim NM. Mechanisms of Korean red ginseng and herb extracts (KTNG0345) for anti-wrinkle activity. Journal of Ginseng Research, 32: 39-47, Son UD, Hwang JS, Chang IS. Approach of antiaging care in cosmetics. Journal of the Society of Cosmetics Scientists of Korea, 31: , Song JS, Kim YA. A study on the future market prospect of domestic functional cosmetics industry: focused on the cosmeceutical products. Journal of the Korean Society of Design Culture, 15: , Yang WS, Kim YM, Kim EH, Seu YB, Yang YJ, Kim HW, Kang SC. Anti-wrinkle effect of cosmetics containing Duchesnea indica extract. Journal of the Society of Cosmetic Scientists of Korea, 36: , Yoon HJ, Shin JW, Kim YK, Kim JE, Cho KH. The effect of vitamin C and E on the expression of the cutaneous basement membrane components. Korean Journal of Investigative Dermatology, 14: 87-98,

9 나이아신펩타이드융합체의항주름및피부턴오버개선효과 Yoo JM, Kang YJ, Pyo HB, Choung ES, Park SY, Choi JH, Han GJ, Lee CH, Kim TJ. Anti-wrinkle effects of Korean rice wine cake on human fibroblast. Journal of Life Science, 20: ,

10 Anti-wrinkle and Skin Turn over Effects of Niacinamide-dipeptide Convergence 국문초록 나이아신펩타이드융합체의항주름및피부턴오버개선효과 김가연 1, 이승제 1, 전미지 1, 김보민 1, 김근태 1, 강상문 2, 이기영 2, 신은진 2, 김상용 3, 김영민 1* 1 한남대학교생명시스템과학과, 대전, 한국 2 에이앤펩부설연구소, 충청북도청주시, 한국 3 신안산대학교식품생명과학과, 경기도안산시, 한국 목적 : 본연구에서는나이아신펩타이드융합체의항주름및피부턴오버개선효과를입증하고기능성화장품소재로서이용가능성을검증하고자하였다. 방법 : 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing assay, chicken choriallantoic membrane (CAM) assay, reverse transcription polymerase chain reaction (RT-PCR), 3D cell culture 를이용하여나이아신펩타이드융합체의항주름및피부턴오버개선효과를측정하였다. 결과 : 나이아신펩타이드융합체는 HaCaT 세포에대해독성이없었고, 세포이동능력회복효과가있으며, 이로인해피부재생에도움을줄수있음을확인하였다. 또한나이아신펩타이드융합체의혈관신생효능을확인한결과, 양성대조군 vitamin C와유사한것으로확인되었다. 주름표지인자인 MMP-1, -2, -3, -9의발현양에미치는효과를측정한결과, MMPs 발현이감소함을확인하였다. 피부에너지대사와관련하여 Western blotting을실시한결과, 나이아신펩타이드융합체에의해피부에너지대사활성이증가함을확인하였다. 마지막으로피부턴오버개선효과를확인한결과, 양성대조군인 vitamin C와유사하거나우수한피부턴오버개선효능이있음을확인하였다. 결론 : 본연구결과를통하여나이아신펩타이드융합체의항주름및피부턴오버개선효능을확인하였으며, 기능성화장품소재로서이용가능성이높을것으로사료된다. 핵심어 : 나이아신펩타이드융합체, 기능성화장품, 항주름, 피부장벽, 피부턴오버 본연구는 2016 년도중기청융복합사업 ( 과제번호 : S ) 에의해수행되었음. 참고문헌 고주영, 최경화, 김영철. 기니아피그동물모델에서삼릉에탄올추출물의미백효과. 환경독성보건학회지, 24: , 김도윤, 황대일, 윤미소, 최인호, 이환명. 산국화 (Chrysanthemum boreale Makino) 유래 Hydrosol의피부각질형성세포증식및이주유도활성에미치는효과. 대한화장품학회지, 42: , 김석천, 홍남표, 추재학, 박수홍, 홍석찬, 차창일. 진주종각질형성세포와섬유아세포의삼차원적분리공배양. 대한이비인후과학회지두경부외과학, 43: , 김영철, 박은예, 김상남, 유용기, 박미순, 이귀영, 이석준, 장병수. 홍차추출물함유누드팩의 Hairless 마우스피부주름형성억제효과. 한국현미경학회지, 41: , 김은아. 식물생리활성영양소와미용기능식품. 식품과학과산업, 40: 3-9, 김인덕, 권륜희, 허예영, 정혜진, 강환열, 하배진. 한방원료의초임계추출을이용한항노화및주름개선효과. 한국생물공학회지, 23: , 김정기, 이지해, 양미숙, 서대방, 이상준. 콜라겐펩타이드의피부광노화예방효과. 한국식품과학회지, 41: , 민정아, 배현숙. 천년초손바닥선인장추출물을적용한 MTS관리가여성피부개선에미치는영향. 아시안뷰티화장품학술지, 11: ,

11 나이아신펩타이드융합체의항주름및피부턴오버개선효과 소승호, 이성계, 황의일, 구본석, 한경호, 정진호, 이민정, 김나미. 홍삼생약복합물 (KTNG0345) 의피부주름개선에관한작용기전. 고려인삼학회지, 32: 39-47, 손의동, 황재성, 장이섭. 항노화화장품연구개발동향. 대한화장품학회지, 31: , 송지성, 김영아. 국내기능성화장품산업의향후시장전망에관한연구 : 코스메슈디컬제품을중심으로. 한국디자인문화학회지, 15: , 양웅석, 김용민, 김이화, 서영배, 양윤정, 김현우, 강세찬. 사매추출물을함유하는화장품의주름개선효과. 대한화장품학회지, 36: , 유정민, 강여진, 표형배, 정의수, 박신영, 최지호, 한귀정, 이충환, 김택중. 양조부산물인주박의주름개선효과. 생명과학회지, 20: , 윤호준, 신정원, 김연경, 김지은, 조광현. 피부의기저막구성성분의발현에미치는비타민 C와비타민 E의효과. 대한피부연구학회지, 14: 87-98, 이병근, 김종협, 함상경, 이창언. 화장품개발을위한배롱나무 (Lagerstroemia indica Linnaeus) 가지추출물의생리활성에관한연구. 한국자원식물학회지, 27: 29-34, 2014b. 이수연, 이진영. 섬유아세포 (CCD-986sk) 에서청미래덩굴의 pro-collagen type-1 활성및 MMP-1의유전자발현저해능. 생명과학회지, 23: , 전민희, 박미라, 박용수, 황현정, 김성구, 이상현, 김미향. 적송잎추출물이항산화활성및파골세포의증식에미치는영향. 한국식품영양과학회지, 40: , 전영선, 강상모. 콜라겐섭취가안면피부주름에미치는영향. 아시안뷰티화장품학술지, 7: 79-93, 정명근, 황영선, 김기쁨, 안경근, 심훈섭, 홍승범, 최재후, 유창연, 정일민, 김승현, 임정대. 맥문동종실안토시아닌분획물의멜라닌생성억제및미백효과. 한국약용작물학회지, 21: , 정해수, 송미영, 김형식, 서효현, 이정훈, 이경록, 홍일, 모상현. 갈릭산펩타이드유도체를이용한주름개선소재개발. 한국산학기술학회지, 16: ,

12 Anti-wrinkle and Skin Turn over Effects of Niacinamide-dipeptide Convergence 中文摘要 烟酰胺二肽融合体的抗皱和皮肤周期改善效果 金佳姸 1, 李昇帝 1, 全美智 1, 金甫敃 1, 金根泰 1, 姜相汶 2, 李起英 2, 申銀珍 2, 金相龍 3 1*, 金榮民 1 韩南大学生命科学科, 大田, 韩国 2 A&PEP 附属研究所, 忠淸北道淸州市, 韩国 3 新安山大学食品生命科学科, 京畿道安山市, 韩国 目的 : 探讨烟酰胺二肽融合体的抗皱以及皮肤周期改善效果, 确认作为功能性化妆品原料的应用可行性 方法 : 利用 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound healing assay, chicken choriallantoic membrane (CAM) assay, reverse transcription polymerase chain reaction (RT-PCR), 3D cell culture 测定烟酰胺二肽融合体的抗皱以及皮肤周期改善活性 结果 : 烟酰胺二肽融合体对 HaCaT 细胞不具有细胞毒性, 并具有恢复细胞迁移能力, 从而确认烟酰胺二肽融合体有助于皮肤再生 另外, 烟酰胺二肽融合体的的血管生成潜力被证实与阳性对照群维生素 C 相似 测定皱纹标记因子 MMP-1,-2,-3 和 -9 的表达水平结果显示, 降低了 MMPs 的表达水平 蛋白质印迹与皮肤能量代谢表明, 烟酰胺二肽融合体增加了皮肤能量代谢活性 最后, 确认皮肤周期改善效果与阳性对照组维生素 C 相似, 或者皮肤周期改善效果优异 结论 : 通过以上研究, 确认烟酰胺二肽融合体的抗皱和皮肤周期改善效果, 并认为其作为功能性化妆品原料充分具有利用可行性 关键词 : 烟酰胺二肽融合体, 功能性化妆品, 抗皱, 皮肤屏障, 皮肤周期改善 254