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Journal of the Korea Academia-Industrial cooperation Society Vol. 19, No. 3 pp. 229-242, 2018 https://doi.org/10.5762/kais.2018.19.3.229 ISSN 1975-4701 / eissn 2288-4688 발효돈태반과그주요다이펩타이드섭취가 UVB 조사에의한무모생쥐의피부주름생성에미치는효과 이지은 1, 박종일 1, 김종배 2, 정혜인 2, 황재성 1* 1 경희대학교생명공학원유전공학과 2 LG 생활건강기술연구원 The Effects of Orally Administered Fermented Porcine Placenta and Its Major Dipeptides on UVB-induced Wrinkle Formation in the Hairless Mice JiEun Lee 1, Jongil Park 1, Jongbae Kim 2, Hyein Jeong 2, JaeSung Hwang 1* 1 Department of Genetic engineering, Collage of Life Sciences, Kyung Hee University 2 LG household & Healthcare Research Park 요약본연구에서는발효돈태반 (fermented porcine placenta, FPP) 과그의주요다이펩타이드인 L-Leucyl-Glycine (Leu-Gly), Glycyl-L-Leucine (Gly-Leu) 의섭취가 UVB 조사에의한무모쥐의피부주름생성에미치는효능을알아보았다. Human Primary Dermal fibroblasts-neonatal (HDF-N) 세포에서세포독성을나타내지않는농도를설정하여평가하였을때, FPP, Leu-Gly, Gly-Leu 처리시 procollagen의증가및 MMP-1 (matrix metalloproteinase-1) 의감소를확인하였다. 또한무모생쥐에주 3회 UVB를조사하여광노화를유도하였고, FPP 10, 100, Leu-Gly 10, Gly-Leu 10 을매일총 8주간경구투여한후, 주름생성, 홍반및 MMPs의발현량을측정하였다. 8주간진행된동물실험결과 UVB만조사한군과비교하여 UVB를조사하고 FPP, Leu-Gly, Gly-Leu 을섭취시킨군에서주름생성과홍반이감소하였고피부수분함량과콜라겐생합성이증가하였다. 뿐만아니라 FPP, Leu-Gly, Gly-Leu 섭취군에서콜라겐분해효소인 MMP-3, MMP-13 의 mrna 발현량이감소하였고, MMP-2 와 MMP-9 의활성이감소하였다. 결과를종합하였을때, FPP 의주요다이펩타이드인 Leu-Gly 과 Gly-Leu 은자외선에의한주름생성을억제하고, 피부손상을회복시키는효능을갖는피부미용식품소재로서활용가능성이클것으로사료된다. Abstract The effects of orally administered fermented porcine placenta (FPP) and its major dipeptides, L-Leucyl-Glycine (Leu-Gly) and Glycyl-L-Leucine (Gly-Leu), on UVB-induced wrinkle formation of the skin in hairless mice was studied. Treatment with FPP, Leu-Gly or Gly-Leu increased type I procollagen synthesis and decreased MMP-1 (matrix metalloproteinase-1) in human dermal fibroblast cells (HDF-N). Hairless mice were also exposed UVB irradiation three times a week and fermented porcine placenta extract (FPP), Leu-Gly and Gly-Leu was administered once a day for eight weeks. Daily intake of FPP, Leu-Gly and Gly-Leu for eight weeks decreased wrinkles, erythema and thickness of the skin and increased skin hydration and synthesis of collagen relative to a UVB-control. Moreover, FPP, Leu-Gly or Gly-Leu intake decreased the expression of MMP-3 and MMP-13 mrna levels and inhibited activation of MMP-2 and MMP-9 induced by UVB irradiation in hairless mice skin. These results suggest that major dipeptides of the placenta, Leu-Gly and Gly-Leu have the potential for use as a functional food ingredient with anti-wrinkling properties. Keywords : Fermented porcine placenta extract, Gly-Leu, Leu-Gly, Matrix metalloproteinase, UVB, Wrinkle * Corresponding Author : Jae Sung Hwang(KyungHee Univ.) Tel: +82-10-3242-8148 email: jshwang@khu.ac.kr Received December 11, 2017 Revised (1st January 3, 2018, 2nd January 10, 2018) Accepted March 9, 2018 Published March 31, 2018 229

한국산학기술학회논문지제 19 권제 3 호, 2018 1. 서론피부는신체를둘러싼가장큰조직으로외부자극과세균침입으로부터신체를방어하고체온조절, 감각기능및노폐물배출등을통해우리몸을보호하는역할을한다 [1]. 피부도다른신체기관과마찬가지로노화가진행되는데피부의노화는나이를먹음에따라인체기능의저하와호르몬등의생리적기능이변화되어나타나는내인성노화와자외선을비롯한여러환경적요인에지속적으로노출이됐을때나타나는외인성노화로나뉜다. 그중자외선으로인한광노화는가장직접적인외인성노화의원인이며, 피부건조, 주름발생, 피부탄력감소, 색소침착등이대표적인현상이다 [2-3]. 자외선은피부내에서콜라겐분해효소인 matrix metalloproteinases (MMPs) 의발현을증가시켜콜라겐의분해를유도하고피부수분을유지시키는히알루론산생합성을억제한다 [4]. 지속적으로자외선에노출된피부는이러한피부결합조직들의생리학적변화들을통해진피층의탄력이감소되어결국깊은주름을생성하게된다 [5-6]. 따라서광노화로인한피부손상과주름을예방하고개선하기위해 MMPs의합성을억제하면서콜라겐과히알루론산생합성을유도하는소재들이개발되고있다. 태반은임신중모체의자궁내막과태아를감싸는장막을연결하는기관으로모체와태아간의산소와탄산가스를교환하는역할을하고영양분제공과노폐물배출등태아의생존과성장에필수적인역할을하는기관이다 [7-8]. 태반추출물은항산화, 항염증, 면역력강화등에효과적인것으로알려져있기때문에기능성식품및약재로서의그쓰임이광범위하다 [9-11]. 이처럼태반에관한연구가다양하게이루어지고있으며, 식품의약품안전처고시 식품의기준및규격 에서는소, 돼지, 양, 말등가축유래태반만식품원료로사용이가능하다. [12]. 태반을이용한실험적연구로는태반추출물을처리로인한피부의상처치유개선효과와 [13], insulin-like growth factor-1 (IGF-1) 의단백질발현증가로인한쥐의모발성장증가효과 [14], 손상된간세포의재생을통한간섬유증억제효과 [15] 등이알려져있다. 또한, 돈태반추출물의마우스경구투여를통한 in vivo 실험결과진피층에서 MMP-2, 9 발현감소와 MMPs의억제제인 metalloproteinase-1 (TIMP-1) 과 metalloproteinase-2 (TIMP-2) 의발현증가로인한항주름효과가밝혀져있다 [16]. 또한발효돈태반추출물과그주요다이펩타이드인 Leu-Gly, Gly-Leu이염증과산화반응을억제하여항피로효능을갖는것으로보고되어있지만 [17-18], 발효돈태반과두가지다이펩타이드의피부주름에관한보고는밝혀진바가없다. 본연구는발효돈태반추출물과이에함유된다이펩타이드인 L-Leucyl-Glycine (Leu-Gly), Glycyl-L-Leucine (Gly-Leu) 의광노화로인한주름개선효능을알아보고자진행되었다. 이를위해 Human Primary Dermal fibroblasts-neonata (HDF-N) 세포를이용하여 procollagen과 MMP-1의발현을 ELISA로확인하였고, 광노화를유발한무모쥐 (hairless mouse) 동물모델에발효돈태반추출물 (FPP), Leu-Gly, Gly-Leu을경구투여한뒤주름생성, 피부두께, MMPs 발현및콜라겐합성등을측정하여그효능을확인하였다. 2. 실험방법 2.1 실험재료실험에사용한발효돈태반추출물 (Fermented Placenta Extract Powder A (K), HORUS Co., Ltd. Tokyo, Japan) 은 HORUS Co., Ltd. 로부터, Leu-Gly (G2495), Gly-Leu (M1460) 은 Bachem AG (Bubendorf, Switzerland) 로부터공급받아사용하였다. 2.2 세포배양 HDF-N 세포는 10% fetal bovine serum, 1% penicillin / streptomycin (WelGENE, Seoul, Korea) 을함유한 Dulbecco Modified Eagle Medium (DMEM) (WelGENE, Seoul, Korea) 배지를사용하여 37^, 5% CO 2 세포배양조건에서배양하였다. 2.3 세포독성시험 HDF-N 세포를 96 well plate에 1x10 4 cells/well로분주한다음세포배양조건에서 24시간배양하였다. 배지를제거하고 DPBS로세척한뒤검액및새로운배지를넣고배양하였다. 24시간뒤검액및배지를제거하고 Dulbecco's Phosphate-Buffered Saline (DPBS) 로세척한후, 세포의생존율을측정하기위해 EZ-Cytox (Daeil 230

발효돈태반과그주요다이펩타이드섭취가 UVB 조사에의한무모생쥐의피부주름생성에미치는효과 Lab Service, Seoul, Korea) 를포함하는배지를처리하여 1 시간동안반응시킨후, 450nm에서흡광도를측정하여대조군에대한세포생존율을백분율로표시하였다. 2.4 Procollagen 합성능평가시험 HDF-N 세포를 48 well plate에 1.2 10 4 cells/well로분주하고세포배양조건에서 24시간배양하였다. 그후, 배지를제거하고세포의기아상태를 24 시간유지한뒤시험물질을세포배양배지에희석하여세포에처리한후배양하였다. 24 시간후에배양된세포의배지를수거한뒤 procollagen type I c-peptide(pip) EIA kit (MK101; Takara, Shiga, Japan) 를사용하여 procollagen 양을측정하였다. 바닥에부착되어있는세포는 DPBS로세척한후 1 N NaOH로 lysis 시켜 BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, USA) 를이용하여총단백질양을측정하였고단백질당 procollagen 합성양을보정하여측정하였다. Guri, Korea) 를급여하였고. 물과식이는자유롭게섭취하도록하였다. 실험동물사육관리는 Guide for the Care and Use of Laboratory Animals" 를기준으로하였으며, 실험은경기과학기술진흥원 Institutional Animal Care and Use Committees (IACUC) 승인하에진행되었다. 2.7 실험군및실험물질경구투여 Reagan-Shaw et al (2008) 의논문을참조하여 FPP의일일섭취량을 animal dose로환산하였고 [19], Nam et al (2006) 의실험농도를참고하여 FPP는 10, 100 으로, Leu-Gly과 Gly-Leu은 10 으로결정하였다 [17]. 대조군, UVB군, UVB + FPP 10 군, UVB + FPP 100 군, UVB + Leu-Gly 10 군, UVB + Gly-Leu 10 군으로실험을진행하였다 (Table 1). 시료의투여는마우스 1 회경구투여적정량인 10 ml/kg으로하루에한번경구투여하였다. 2.5 MMP-1 저해활성평가시험 HDF-N 세포를 48 well plate에 1.2 10 4 cells/well로분주하고세포배양조건에서 24 시간배양하였다. 그후, 배지를제거하고세포의기아상태를 24 시간유지한뒤시험물질을세포배양배지에희석하여세포에처리한후배양하였다. 24시간후에배양된세포의배지를수거한뒤 Human total MMP-1 kit (DY901; R&D systems, Minneapolis, MN, USA) 를사용하여 MMP-1 양을측정하였다. 바닥에부착되어있는세포는 DPBS로세척한후 1N NaOH로 lysis 시켜 BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, USA) 를이용하여총단백질양을측정하였고단백질당 MMP-1 합성양을보정하여측정하였다. 2.6 실험동물및사육관리생후 6 주령 20-25 g 정도의특정병원체부재 (Specific pathogen free, SPF) 암컷 hairless mice (SKH-1:HR-a) 를오리엔트바이오 (Seongnam, Korea) 로부터구입하여사용하였다. 검역과일주일의순화기간을거치고 10 마리씩 6그룹으로군을분리한이후에실험을진행하였다. 실험동물사육환경은온도 (24 ± 2 ), 습도 (55 ± 10%), 그리고명암주기 12 시간을유지하였다. 사료는마우스전용사료 (Feed Lab Korea, Table 1. Experimental group and dose design Group UVB Test Amount of material oral administration Con X water UVB O water UVB + FPP 10 O FPP 10 UVB + FPP 100 O FPP 100 UVB+ Leu-Gly 10 O Leu-Gly 10 UVB+ Gly-Leu 10 O Gly-Leu 10 2.8 UVB 조사 광노화에의한주름을생성하기위해대조군을제외한나머지군들은매주주 3회동일한시간에 UVB를조사하였다. 정확한조사량을위해 UV meter (VARIOCONTROL, Waldmann ver 2.03, Villingen- Schwenningen, Germany) 를이용하여 1 MED (minimal erythemal dose, 약 75 mj/ cm2 ) 의값을측정하고 UVB 램프 (TL 20W/12RS SLV, wave length 290 to 390 nm, peak emission 315 nm; Philips, Netherlands) 6개를부착하여 UVB 조사를하였다. 첫째주는 1 MED, 둘째주는 2 MED, 셋째주는 3 MED, 넷째주부터실험종료시까지 3.3 MED를조사하였으며, 피부상태및적절한주 231

한국산학기술학회논문지제 19 권제 3 호, 2018 름이형성되도록조사량을조절하였다. 2.9 이상반응관찰및체중측정매일동물의이상반응과사망여부를관찰하였고실험에사용한모든동물에대해주 1 회체중측정을하였다. Korea) 와 primer를사용해진행하였다 (Table 2). MMP-3 증폭은 95, 30 초 ; 56, 1 분 ; 72 1 분, 35 Cycle로진행하였고, MMP-13와 β-actin 은 95, 30 초 ; 56, 1 분 ; 72 1 분, 45 Cycle로진행하였다. 증폭된 cdna는 3 % agarose gel에전기영동후 Gel-doc (Bio-Rad, Hercules, USA) 를이용하여분석하였다. 2.10 피부의육안관찰및피부주형의분석동물의등쪽을디지털카메라 (EOS 600D, Canon, Japan) 로근접촬영하고, 주름정도분석및평가를위해실리콘폴리머 (SILFLO impression material, Flexico, England) 를이용하여레플리카 (replica) 을얻었다. 피부주형은빛의입사각을 20도로고정하고, 주름그림자명암영상을 Visioline 프로그램을이용하여사진을촬영하고이미지화한후, 컴퓨터분석시스템인 Skin Viosiometer SV600 software (Courage & Khazaka, Mathias-Brüggen, Germany) 를이용하여주름의면적, 수, 길이, 깊이값을측정하고주름평가를하였다. Table 2. The nucleotide sequences of forward and reverse primer sets for mouse genes Gene Primer Sequence β-actin MMP-3 MMP-13 Forward Reverse Forward Reverse Forward Reverse 5'-CCA GCC AGC CAC CAT CGC TC-3' 5'-TGA CCT TGG CCA GGG GTG CA-3' 5'-TAG CAG GTT ATC CTA AAA GCA-3' 5'-CCA GCT ATT GCT CTT CAA T-3' 5'-CAT CCA TCC CGT GAC CTT AT-3' 5'-GCA TGA CTC TCA CAA TGC GA-3' 2.11 조직병리학적분석 실험종료후모든동물의등쪽피부를떼어내고 10% 포르말린에넣은후, 조직병리학적변화를확인할수있는 hematoxylin and eosin (H&E) 염색과진피에존재하는콜라겐을확인할수있는 Masson's trichrome 염색을하였다. 염색한조직을 Nikon ECLIPSE Ti-E 전동드립형광현미경 (Nicon, Tokyo, Japan) 을이용하여사진을찍어이미지화한후, NIS-Elements BR 3.0 Software (Nkion, Tokyo, Japan) 를이용하여분석하였다. 2.12 피부홍반평가 피부홍반측정은동물의등쪽을색차계 (spectrophotometer, KONICA MINOLTA, Tokyo, Japan) 와 Corneometer (CK Electronics GmbH, Cologne, Germany) 을이용하여측정하였다. 2.13 역전사중합효소연쇄반응 (Reverse Transcription-polymerase chain reaction) 피부조직에 RNAiso Plus (TAKARA BIO, Seta3-4-1, Otsu, Shiga 520-2193, Japan) 넣고 RNA를추출한후 cdna 로 합성하고, PCR premix (ELPIS, Daejeon, 2.14 Gelatin zymography 방법을이용한 MMP-2, MMP-9 발현평가피부조직으로부터단백질을추출한후에 10 X Blue Juice (Invitrogen, Carlsbad, USA) 와혼합하여 Novex 10% Zymogram (Gelatin) Gel (Invitrogen) 에넣고 125 V의일정한전압으로 1.5 시간동안전기영동을하였다. 전기영동후겔을 Zymogram renaturing buffer (Invitrogen, Carlsbad, CA, USA) 에넣어 30 분간실온에교반후버리고 Zymogram Developing buffer (Invitrogen) 으로교체후 30 분간실온에서교반하였다. 이후새로운 Zymogram developing buffer로교체해주고 37 에서 36 시간동안두었다. 36 시간후에겔을 Simply Blue Safe Stain (Invitrogen, Carlsbad, CA, USA) 으로 2 시간염색후 distilled water로 1 시간단위로 2 번탈색시키고활성화된 MMP-2, MMP-9을확인하였다. 활성화된 MMP-2, MMP-9은 62 kda, 86 kda 에서확인하였다. 2.15 통계분석본실험에서얻은결과는 Independent student s t-test 를이용하여각그룹간통계적유의성을검정하였다. 각군의유의성은 p<0.05 수준으로검정하였다. 232

발효돈태반과그주요다이펩타이드섭취가 UVB 조사에의한무모생쥐의피부주름생성에미치는효과 3. 결과 3.1 세포독성평가 FPP, Leu-Gly, Gly-Leu의세포독성을확인하기위해 HDF-N 세포에각시료가함유된배양액을첨가하고 24시간배양후세포생존율을평가하였다. 시험결과, FPP와 Leu-Gly은 HDF-N 세포에서 100 μg/ml 농도까지, Gly-Leu은 10 μg/ml까지세포독성이나타나지않았다 (Fig1. a~c). 3.2 FPP, Leu-Gly, Gly-Leu 이 Procollagen 합성에미치는영향 FPP와 Leu-Gly, Gly-Leu의 procollagen 합성효능을평가하기위해 HDF-N 세포에세가지시료를처리하였고, 피부콜라겐중대부분을차지하는 type 1 procollagen의발현을 ELISA로확인하였다. 실험결과, FPP, Leu-Gly, Gly-Leu 처리시유의한 type 1 procollagen 합성증가를확인하였다 (p<0.05, p<0.01) (Fig2. a~c). (c) Fig. 1. Effect of FPP, Leu-Gly and Gly-Leu on cell viability in HDF-N. Cell viability of FPP-treated, Leu-Gly-treated, Gly-Leu-treated (c) cells was measured using EZ-Cytox assay kit. The experiment was repeated three times. Data are indicated as mean±sd. (c) Fig. 2. Effects of FPP, Leu-Gly and Gly-Leu on procollagen synthesis in HDF-N. Type-I procollagen synthesis of FPP-treated, Leu-Gly-treated and Gly-Leu-treated (c) cells was quantified using a Procollagen Type I C-Peptide (PIP) EIA kit. All experiments were performed in triplicate. Data are indicated as mean±sd. Data were analyzed using the Student s t test for independent samples. *p < 0.05, **p <0.01, ***p <0.001 versus untreated control. 233

한국산학기술학회논문지제 19 권제 3 호, 2018 3.3 FPP, Leu-Gly, Gly-Leu이 MMP-1 저해활성에미치는영향세가지시료의 MMP-1 저해효능을평가한결과, FPP 0.1μg/ml 처리시 18%, 1μg/ml 처리시 20%, 10μ g/ml처리시 6% 감소하였다. 또한 Leu-Gly 100μg/ml 처리시 13%, Gly-Leu 10 μg/ml 처리시 17% 감소하여유의한수준으로 MMP-1 저해효능을보였다 (p<0.05, p<0.01, p<0.001) (Fig3. a~c). 3.4 체중변화및증상관찰시료섭취시이상반응이나타나는지확인하기위해매일관찰하고체중을주 1회측정하였다. 실험기간동안모든동물들의체중은증가한것을확인하였고특이증상및사망한동물은보이지않았다. 체중증가에대한유의한변화는보이지않았다 (Fig. 4). 모든동물희생후, 장기에특이한이상은보이지않았다. (c) Fig. 3. Effects of FPP, Leu-Gly and Gly-Leu on inhibition of MMP1 in HDF-N. MMP-1 synthesis of FPP-treated, Leu-Gly-treated and Gly-Leu-treated (c) cells was quantified with a Human Total MMP-1 DuoSet ELISA assay kit. All experiments were performed in triplicate. Data are indicated as mean±sd. Data were analyzed using the Student s t test for independent samples. *p < 0.05, **p <0.01, ***p <0.001 versus untreated control. Fig. 4. Experimental design. Mice were subjected to UVB-irradiation and provided with FPP, Leu-Gly and Gly-Leu for 8 weeks. At the 4 weeks and 8 weeks skin biopsies were obtained and analyzed. Body weight changes in hairless mice. Data are indicated as mean±sd of ten mice in each group. 234

발효돈태반과그주요다이펩타이드섭취가 UVB 조사에의한무모생쥐의피부주름생성에미치는효과 3.5 FPP, Leu-Gly, Gly-Leu이 UVB 조사로인한주름형성에미치는영향 FPP, Leu-Gly, Gly-Leu의섭취후주름지표인주름의면적, 수, 길이, 깊이등을분석한결과, UVB군이대조군에비해모든주름의지표가증가한것을확인하였고 FPP 및 Leu-Gly, Gly-Leu군은 UVB군에비해유의한감소를보였다 (p<0.05, p<0.01, p<0.001) (Fig. 5, 6a-d). (c) Fig. 5. Effects of FPP, Leu-Gly or Gly-Leu intake on UVB-induced wrinkle formation after 4 weeks and 8 weeks. (d) Fig. 6. Effects of FPP, Leu-Gly or Gly-Leu intake on wrinkle value of analyzed replica in UVB-irradiated hairless mice. Total wrinkle area number of wrinkles mean length (c) mean depth (d). Data are indicated as mean±sd of ten mice in each group. Data were analyzed using the Student s t test for independent samples. ###p<0.001, ##p<0.01, compared with non-uvb irradiated mice; ***p<0.001, **p<0.01, *P<0.05 compared with UVB-irradiated mice. 3.6 FPP, Leu-Gly, Gly-Leu이 UVB조사로인한홍반생성에미치는영향다음으로자외선에의한피부홍반반응에 FPP, 235

한국산학기술학회논문지제 19 권제 3 호, 2018 Leu-Gly, Gly-Leu의섭취가미치는영향을확인하였다. UVB를조사한군에서홍반이유발되었고 FPP, Leu-Gly, Gly-Leu 섭취군의경우 UVB군에비해유의한수준으로홍반이감소한것을확인할수있었다 (p<0.001) (Fig. 7). Fig. 7. Effects of FPP, Leu-Gly or Gly-Leu intake on skin redness in UVB-irradiated hairless mice. Data are indicated as mean±sd of ten mice in each group. Data were analyzed using the Student s t test for independent samples. ###p<0.001 compared with non-uvb irradiated mice; *P<0.05, **p<0.01, ***p<0.00 compared with UVB-irradiated mice. 3.7 FPP, Leu-Gly, Gly-Leu 의섭취가광노화로인한피부두께변화와콜라겐감소에미치는영향 FPP, Leu-Gly, Gly-Leu의섭취가 UVB가조사된피부의두께와콜라겐형성에어떠한영향을미치는지확인하기위해조직염색을실시하였다. Hematoxylin and Eosin (H&E) 염색결과, UVB군이대조군에비해표피의두께가증가하였고, FPP, Leu-Gly, Gly-Leu 섭취군은 UVB군에비해표피두께가감소하였다 (Fig. 7a). NIS-Elements BR 3.0 Software를이용하여피부두께를측정한결과세가지시료를섭취시 UVB 조사에의해증가된피부두께가유의하게감소하는것을확인하였다 (p<0.01, p<0.001) (Fig. 7b). Masson's trichrome 염색결과, 대조군과비교하여 UVB군의콜라겐형성이현저하게감소하였지만 FPP, Leu-Gly, Gly-Leu 섭취시 UVB 조사에의해감소되었던콜라겐발현이회복된것을확인하였다 (Fig. 8). Fig. 8. Hematoxylin and Eosin (H&E) staining from the dorsum of the hairless mice skin. Magnification 400. Scale bars = 100 µm. Data are indicated as mean±sd of ten mice in each group. Data were analyzed using the Student s t test for independent samples. ###p<0.001 compared with non-uvb irradiated mice; ***p<0.001, **p<0.01 compared with UVB-irradiated mice. 3.8 UVB 조사로유도된 MMP-3 및 MMP-13 의발현억제에대한 FPP, Leu-Gly, Gly-Leu 의효과 FPP, Leu-Gly, Gly-Leu 섭취에따른 MMP-13과 MMP-3 의 mrna 발현을 RT-PCR 을이용하여측정하 236

발효돈태반과그주요다이펩타이드섭취가 UVB 조사에의한무모생쥐의피부주름생성에미치는효과 였다. 실험결과, 대조군에비하여 UVB군에서 MMP-13 과 MMP-3의 mrna 발현이증가하였고, FPP 100, Leu-Gly과 Gly-Leu 10 섭취시 UVB 조사에의해증가했던 MMP-13 과 MMP-3의발현이유의한수준으로감소하였다 (p<0.001) (Fig. 9a-c). Fig. 9. Masson s trichrome staining of collagen fibers from the dorsum of the hairless mice skin. Masson trichrome staining of the skin tissue showed tissue morphology. Magnification 400. Scale bars = 100 µm. 3.9 UVB 조사로인해증가된 MMP-2 및 MMP-9 의활성억제에대한 FPP, Leu-Gly, Gly-Leu의효과 Gelatin zymography로 FPP. Leu-Gy, Gly-Leu의섭취가 UVB 조사에의해활성화된 MMP-2, MMP-9 에대하여억제효과를갖는지확인하였다. 실험결과, FPP. Leu-Gy, Gly-Leu 섭취시 UVB 조사에의해증가된 MMP-9 (86kDa) 과 MMP-2 (62 kda) 의발현이감소하였다 (p<0.05, p<0.01, p<0.001) (Fig. 10a-b). 따라서, FPP, Leu-Gy, Gly-Leu의섭취가콜라겐분해효소인 MMP-2 및 MMP-9 의활성을감소시키는것을확인할수있었다. (c) Fig. 10. Effects of FPP, Leu-Gly or Gly-Leu intake on MMP-3 and MMP-13 mrna expression level in UVB-irradiated hairless mice. RT-PCR analysis and percentage of MMP-13 and MMP-3 (c) mrna expression. Data are indicated as mean±sd of ten mice in each group. Data were analyzed using the Student s t test for independent samples. ###p<0.001 compared with non-uvb irradiated mice; ***p<0.001 compared with UVB-irradiated mice. 237

한국산학기술학회논문지제 19 권제 3 호, 2018 Fig. 11. Effect of FPP, Leu-Gly or Gly-Leu intake on MMP-2 and MMP-9 activity in UVB-irradiated hairless mice. MMP-2 and MMP-9 expression level. Data are indicated as mean±sd of eleven mice in each group. ###p<0.001, ##p<0.01, #p<0.05 compared with non-uvb irradiated mice; ***p<0.001 compared with UVB-irradiated mice. 4. 결론및논의 현대인들의외모에대한관심이증가하면서피부노화 에대한관심또한높아지고있다. 광노화로인한주름생 성은피부노화의지표로사용되는만큼변화가뚜렷하 고화장으로도잘가려지지않기때문에이미생성된주 름을없애거나생성을미리예방할수있는식품들이각 광받고있다. 자외선은피부내활성산소종 (Reactive Oxygen Species; ROS) 생성을유발하고생성된 ROS는피부세포의산화를일으키며 MMPs의활성을증가시켜콜라겐분해를유도한다. Park et al. (2015) 의연구에따르면, 돈태반추출물이 Human keratinocytes (HaCaT) 세포에서 UVB 조사에의해증가된세포내 ROS 생성을억제 시키고, MMP-2의 mrna 발현을감소시킨다 [21]. 따라서이와같은돈태반추출물을이용한기존의연구들을바탕으로 FPP의광노화로인해유발되는피부주름개선소재로서의가능성을예측하였고 [16,21], FPP 와그의주요다이펩타이드인 Leu-Gly과 Gly-Leu의효능을알아보기위해본연구를진행하게되었다. 먼저 FPP, Leu-Gly, Gly-Leu의작용기전을알아보기위한세포실험결과, 세가지시료는 HDF-N 세포에서 procollagen의합성을증가시켰다. 체내의 MMP family 중 MMP-1은주로콜라겐을분해하는효소로자외선에노출된피부는 MMP-1의발현이증가하여콜라겐합성을저해하는것으로알려져있다 [22]. 따라서 MMP-1 활성억제평가를진행한결과, 세가지시료모두 UVB 에의해증가된세포내 MMP-1의활성을억제시키는효능을보였다. FPP, Leu-Gly, Gly-Leu의섭취시그효능을알아보기위한동물실험결과, FPP, Leu-Gly, Gly-Leu의섭취는 UVB에의해생성된무모쥐피부의주름의면적, 수, 길이, 깊이및홍반생성을감소시켰고, 피부수분량을증가시켰다. 자외선조사는표피의두께를증가시키고콜라겐분해효소를활성화시켜진피층의콜라겐섬유를파괴한다. 따라서 FPP, Leu-Gly, Gly-Leu의섭취가 UVB가조사된피부의두께와콜라겐형성에어떠한영향을미치는지확인하기위해조직염색을실시한결과, 자외선에의해증가된표피두께를감소시켰고콜라겐생합성감소를막는것을확인하였다. MMP family 중 MMP-13은특이적인 type Ⅱ 콜라겐분해작용을하고 MMP-3는 type Ⅳ 콜라겐을분해하면서 pro-mmp-9을활성화시키고 proteoglycan, fibronectin과같은여러가지기질단백질들을분해시킨다 [23-24]. MMP-2와 MMP-9 은 UVB 조사에의해서활성화되고 type Ⅳ 와 type Ⅴ 콜라겐, fibronectin 및엘라스틴섬유를분해하는활성을갖는다 [25]. FPP, Leu-Gly, Gly-Leu 섭취시광노화로인한콜라겐감소를회복시킬수있다는것을확인하였기때문에 FPP, Leu-Gly, Gly-Leu이콜라겐분해효소인 MMPs 발현에어떠한영향을미치는지확인하였다. 실험결과, FPP, Leu-Gly, Gly-Leu의섭취가자외선에의해증가되는 MMP-13과 MMP-3의 mrna 발현을감소시키고 MMP-2와 MMP-9의활성을억제를나타내었다. 또한 Leu-Gly과 Gly-Leu 다이펩타이드가 FPP와비슷한수준의효능을보이는것을확인할수있었다. 238

발효돈태반과그주요다이펩타이드섭취가 UVB 조사에의한무모생쥐의피부주름생성에미치는효과 Table 3. Comparison of total wrinkle, number of wrinkle, mean length, mean depth and redness in the hairless mice skin at 4 weeks after FPP, Leu-Gly and Gly-Leu intake. 1) Total Wrinkle area [mm 2 ] Con UVB UVB + FPP 10 UVB + FPP 100 Total Wrinkle area [mm 2 5.64 ± 1) 3.03 38.79± 13.12 23.62 ± 9.55 9.22 ± 4.80 ] significance ### 2) ** 3) *** Number of Wrinkles Con 3.18 ± 1) 2.06 232.60 ± 90.12 UVB 21.02 ± 8.40 707.60 ± 81.12 UVB + FPP 10 10.08 ± 8.68 503.60 ± 100.88 UVB + FPP 100 9.82 ± 05.84 significance ### 2) *** 3) *** Number of Wrinkles 146.40 ± 56.68 441.00 ± 72.65 317.10 ± 41.83 261.30 ± 57.97 significance ### *** *** Mean Length 0.23 ± 0.04 0.33 ± 0.06 0.25 ± 0.05 0.27 ± 0.06 [mm] significance ### *** ** Mean Depth [μm] 34.12 ± 2.56 38.41 ± 2.45 35.26 ± 3.54 35.25 ± 2.53 significance ## * * Redness 4.13 ± 0.95 6.73 ± 0.68 4.61 ± 0.53 4.59 ± 0.51 significance ### *** *** UVB + Leu-Gly 10 UVB + Gly-Leu 10 Total Wrinkle area [mm 2 ] 10.22 ± 5.43 9.59 ± 3.93 Number of Wrinkles 306.00± 37.24 300.00 ± 38.67 Mean Length [mm] 0.26 ± 0.02 0.27 ± 0.02 significance *** ** Mean Depth [μm] 35.90 ± 1.82 35.52 ± 2.88 significance * * Redness 4.22 ± 0.72 4.20 ± 0.30 significance ### 2) Mean±SD, ##p<0.01, ##p<0.001 compared with non-uvb irradiated mice, 3) * p<0.05, **p<0.01, ***p<0.001 compared with UVB-irradiated mice Table 4. Comparison of total wrinkle, number of wrinkle, mean length, mean depth, redness in the hairless mice skin at 8 weeks after FPP, Leu-Gly and Gly-Leu intake. 332.20 ± 88.12 significance ### *** *** 1) Mean Length [mm] 0.24 ± 0.02 0.32 ± 0.05 0.28 ± 0.01 0.25 ± 0.05 significance ### * ** Mean Depth [μm] 34.17 ± 2.86 42.68 ± 1.24 38.00 ± 2.82 35.65 ± 2.75 significance ### *** *** Redness 3.81 ± 0.82 5.89 ± 0.40 5.13 ± 0.35 4.78 ± 0.28 significance ### *** *** Epidermis Thickness 258.99 ± 16.81 654.99 ± 91.16 452.38 ± 46.70 382.82 ± 29.63 significance ### ** *** MMP-13(%) 100.00 ± 0.00 271.22 ± 0.03 626.89 ± 0.06 179.90 ± 0.01 significance ### *** *** MMP-3 (%) 100.00 ± 0.05 169.49 ± 0.03 206.13 ± 0.02 44.38 ± 0.01 significance ### *** *** MMP-2 (%) 100.00 ± 29.62 575.19 ± 52.65 145.86 ± 69.26 340.76 ± 55.05 significance ### *** ** MMP-9 (%) 100.00 ± 58.91 411.46 ± 81.63 215.69 ± 145.44 266.53± 145.44 significance ## * NS UVB + Leu-Gly 10 UVB + Gly-Leu 10 Total Wrinkle area [mm 2 ] 12.23 ± 4.24 16.23 ± 12.78 significance *** ** Number of Wrinkles 394.40 ± 37.68 315.30 ± 106.52 Mean Length [mm] 0.27 ± 0.04 0.26 ± 0.04 significance * * Mean Depth [μm] 37.16 ± 2.41 37.25 ± 3.64 Redness 4.88 ± 0.21 4.82 ± 0.34 Epidermis Thickness 416.55 ± 42.31 402.58 ± 38.58 MMP-13(%) 283.63 ± 0.04 200.00 ± 0.02 MMP-3 (%) 80.63 ± 0.04 0.98 ± 0.00 MMP-2 (%) 143.06 ± 67.76 187.84 ± 79.62 significance *** ** MMP-9 (%) 213.14 ± 47.54 109.07 ± 34.63 significance * ** 2) Mean±SD, #p<0.05, ##p<0.01, ##p<0.001 compared with 3) non-uvb irradiated mice, * p<0.05, **p<0.01, ***p<0.001 compared with UVB-irradiated mice, 4) NS: not significance 239

한국산학기술학회논문지제 19 권제 3 호, 2018 Glycine, leucine 및 proline은 type Ⅰ collagen을구성하는주요아미노산으로이중 glycine은콜라겐합성과피부장력을증가시켜상처치유효능이있는것으로알려져있다 [26-27]. 홍어류껍질을가수분해하여 MMP-1 저해활성을보인펩타이드를분리정제한연구를통해이러한펩타이드들이소수성아미노산인 leucine과 valine으로구성되어있으며소수성아미노산잔기가 MMP-1의 p1 효소활성부위에특이적으로결합하여저해활성을갖는것을확인하였다 [28]. 또한 Park et al (2017) 의연구에서돈태반의 Leu-Gly, Gly-Leu 다이펩타이드가엘라스틴분해효소인 elastase 활성감소를나타냈다 [29]. Elastase의효소작용은엘라스틴뿐만아니라 type 1 collagen을분해하기때문에피부탄력을저하시켜피부처짐과주름형성에중요한요인이된다. 본연구에서 Leu-Gly과 Gly-Leu 다이펩타이드의주름개선효능은 glycine과 leucine의이러한기능들을토대로효능을보인것으로생각된다. Park et al. (2010) 의연구에서 UVB가조사된무모생쥐에가시오가피 (A. senticosus, AS) 추출물을경구투여한결과자외선에의한주름생성과콜라겐분해가감소되는효과가보고된바있다 [30]. 또한 Tilapia fish 에서얻은콜라겐펩타이드의섭취가 UVB 조사에의해증가된 MMPs의발현을감소시켰고, 손상된콜라겐과 filaggrin 단백질을회복시켜주름생성을억제하고피부손상을보호하는효과를나타내었다 [31]. 위의결과들과본연구의식이섭취량을비교하였을때, FPP (10, 100 ), Leu-Gly (10 ), Gly-Leu (10 ) 의효과가가시오가피추출물 (400 ) 과 Tilapia fish 추출물 (545, 1010 ) 의효과와비슷한유의성을보였다. 따라서발효돈태반추출물과그의주요다이펩타이드인 Leu-Gly, Gly-Leu이향후광노화로인한피부주름을개선할수있는미용기능식품소재로서의활용가능성이높음을알수있다. References [1] M. Sand, T. Gambichler, D. Sand, M. Skrygan, P. Altmeyer and F.G. Bechara, "MicroRNAs and the skin: tiny players in the body's largest organ", J. Dermatol. Sci., vol. 53, no. 3, pp. 169-175, 2009. DOI: https://doi.org/10.1016/j.jdermsci.2008.10.004 [2] M. El Domyati, S. Attia, F. Saleh, D. Brown, D.E. Birk, F. Gasparro, H. Ahmad and J. Uitto, "Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin", Exp. Dermatol., vol. 11, no. 5, pp. 398-405, 2002. DOI: https://doi.org/10.1034/j.1600-0625.2002.110502.x [3] J.Y. Lee, Y.K. Kim, J.Y. Seo, C.W. Choi, J.S. Hwang, B.G. Lee, I.S. Chang and J.H. Chung, "Loss of elastic fibers causes skin wrinkles in sun-damaged human skin", J. Dermatol. Sci., vol. 50, no. 2, pp. 99-107, 2008. DOI: https://doi.org/10.1016/j.jdermsci.2007.11.010 [4] K.K. Dong, N. Damaghi, S.D. Picart, N.G. Markova, K. Obayashi, Y. Okano, H. Masaki, S. Grether Beck, J. Krutmann and K.A. Smiles, "UV induced DNA damage initiates release of MMP 1 in human skin", Exp. Dermatol., vol. 17, no. 12, pp. 1037-1044, 2008. DOI: https://doi.org/10.1111/j.1600-0625.2008.00747.x [5] C.E. Griffiths, T.S. Wang, T.A. Hamilton, J.J. Voorhees and C.N. Ellis, "A photonumeric scale for the assessment of cutaneous photodamage", Arch. Dermatol., vol. 128, no. 3, pp. 347-351, 1992. DOI: https://doi.org/10.1001/archderm.128.3.347 [6] G.L. Grove, M.J. Grove and J.J. Leyden, "Optical profilometry: an objective method for quantification of facial wrinkles", J. Am. Acad. Dermatol., vol. 21, no. 3, pp. 631-637, 1989. DOI: https://doi.org/10.1016/s0190-9622(89)70230-9 [7] S. Togashi, N. Takahashi, M. Iwama, S. Watanabe, K. Tamagawa and T. Fukui, "Antioxidative collagen-derived peptides in human-placenta extract", Placenta, vol. 23, no. 6, pp. 497-502, 2002. DOI: https://doi.org/10.1053/plac.2002.0833 [8] M. Yeom, H. Lee, G. Kim, I. Shim, H. Lee and D. Hahm, "Therapeutic effects of Hominis placenta injection into an acupuncture point on the inflammatory responses in subchondral bone region of adjuvant-induced polyarthritic rat", Biological and Pharmaceutical Bulletin, vol. 26, no. 10, pp. 1472-1477, 2003. DOI: https://doi.org/10.1248/bpb.26.1472 [9] T.K. Biswas, B. Auddy, N.P. Bhattacharya, S. Bhattacharya and B. Mukherjee, "Wound healing activity of human placental extracts in rats.", Acta Pharmacol.Sin., vol. 22, no. 12, pp. 1113-1116, 2001. [10] M. Kawakatsu, Y. Urata, S. Goto, Y. Ono and T. Li, "Placental extract protects bone marrow-derived stem/progenitor cells against radiation injury through anti-inflammatory activity", J. Radiat.Res., vol. 54, no. 2, pp. 268-276, 2012. DOI: https://doi.org/10.1093/jrr/rrs105 [11] S. Togashi, N. Takahashi, Y. Kubo, A. Shigihara, K. Higashiyama, S. Watanabe and T. Fukui, "Purification and identification of antioxidant substances in human-placenta extracts", J. Health Sci., vol. 46, no. 2, pp. 117-125, 2000. DOI: https://doi.org/10.1248/jhs.46.117 [12] MFDS. Food Code. Ministry of Food and Drug Safety, Cheongju, Korea 2016. [13] J.W. Hong, W.J. Lee, S.B. Hahn, B.J. Kim and D.H. Lew, "The effect of human placenta extract in a wound healing model", Ann. Plast. Surg., vol. 65, no. 1, pp. 96-100, 2010. 240

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한국산학기술학회논문지제 19 권제 3 호, 2018 박종일 (Jong il Park) [ 준회원 ] < 관심분야 > 피부노화, 피부미백 2006 년 3 월 ~ 2012 년 2 월 : 경희대학교유전공학과 ( 학사 ) 2012 년 3 월 ~ 2014 년 2 월 : 경희대학교생명공학원 ( 이학석사 ) 2014 년 3 월 ~ 현재 : 경희대학교생명공학원박사과정 황재성 (Jae Sung Hwang) [ 정회원 ] < 관심분야 > 피부노화, 피부미백 1994 년 2 월 : 서울대학교대학원 ( 석사 ) 2003 년 8 월 : 아주대학교의과대학피부과학 ( 의학박사 ) 1994 년 3 월 ~ 2009 년 2 월 : 아모레퍼시픽기술연구원수석연구원 2009 년 3 월 ~ 현재 : 경희대학교유전공학과교수 김종배 (Jong bae Kim) [ 정회원 ] 2008 년 2 월 : 한양대학교식품영양학과 ( 이학사 ) 2013 년 2 월 : 한양대학교식품영양학과 ( 이학석사 ) 2012 년 12 월 ~ 현재 : LG 생활건강기술연구원 < 관심분야 > 건강기능식품, 기능성소재개발 정혜인 (Hye in Jeong) [ 정회원 ] 2009 년 3 월 ~ 2013 년 2 월 : 서울대학교농업생명과학대학식품생명공학과 ( 농학사 ) 2013 년 3 월 ~ 2015 년 2 월 : 서울대학교농업생명과학대학바이오모듈레이션전공 ( 농학석사 ) 2015 년 1 월 ~ 현재 : LG 생활건강기술연구원 < 관심분야 > 피부노화, 피부미백 242