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Synthesis of Tetrapeptide Derived from Skin Structural Protein Sequence and Identification of Skin Anti-aging Effect
Biomed Sci Letters 2021;27:231-238
Published online December 31, 2021;  https://doi.org/10.15616/BSL.2021.27.4.231
© 2021 The Korean Society For Biomedical Laboratory Sciences.

Sang Moon Kang1,†,*, Yong-Seung Joun1,**, Kee-Young Lee1,**, Hyun Kang2,*** and Sung-Gyu Lee2,***

1R&D Center, ANPEP Inc., Cheongju-si, Chungcheongbuk-do 28101, Korea
2Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si, Chungnam 31116, Korea
Correspondence to: *Research director, **Principal researcher, ***Professor.
Corresponding author: Sang Moon Kang. R&D Center, ANPEP Inc., 13 Oksansandan 1-ro, Oksan-myeon, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do 28101, Korea.
Tel: +82-43-241-3360, Fax: +82-43-241-3365, e-mail: smkang@anpep.com
Received November 8, 2021; Revised December 8, 2021; Accepted December 10, 2021.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Several structural proteins present in keratinocytes of the skin are known to play an important role in the formation of epidermal tissue and barrier function, and the absence of structural proteins in keratinocytes causes various skin diseases. In this study, 42 types of tetrapeptides derived from the sequence of Loricrin, a kind of terminally differentiating structural protein, were synthesized, and skin anti-aging properties were measured by measuring the elastase inhibition, proliferation of skin cells. The anti-aging efficacy was verified and, based on this, it succeeded in selecting one of the most excellent peptides. It is expected that the selected tetrapeptide can be used as a raw material for various cosmetics and quasi-drugs based on anti-aging and skin cell proliferation effects.
Keywords : Loricrin, Tetrapeptide, Peptide synthesis, Anti-oxidant, Cell proliferation
꽌 濡

뵾遺뒗 씤泥댁뿉꽌 媛옣 겙 議곗쭅쑝濡 뻼鍮쏆씠굹 臾쇰━, 솕븰쟻 옄洹뱀쑝濡쒕꽣 떊泥 궡遺瑜 蹂댄샇빐 二쇰뒗 湲곕뒫쓣 븯硫 씠뒗 씤媛꾩쓽 깮紐 쑀吏瑜 쐞빐 젅쟻쑝濡 븘슂븯硫 걡엫뾾씠 옱깮릺뼱 빆긽꽦쓣 쑀吏떆궓떎. 뵾遺뒗 諛붽묑履쎌쑝濡쒕꽣 몴뵾, 吏꾪뵾, 뵾븯吏諛 닚꽌濡 援ъ꽦릺뼱 엳쑝硫 媛옣 뻼 議곗쭅씤 몴뵾뒗 뵾遺쓽 蹂댁뒿怨 蹂댄샇瑜 떞떦븯뒗 以묒슂븳 湲곕뒫쓣 떞떦븯怨 엳쑝硫 닔遺 넀떎, 넀긽怨 꽭洹 移⑥엯 벑쓣 諛⑹븯뒗 뿭븷쓣 留↔퀬 엳떎. 뵾遺뒗 떊泥대 쟾遺 媛먯떥怨 엳뒗 議곗쭅쑝濡 떎뼇븳 湲곕뒫쓣 媛吏怨 엳떎. 떊泥 궡遺 쇅遺 궗씠뿉꽌 옣踰 湲곕뒫쓣 媛吏怨 엳쑝誘濡 옉寃뚮Ⅴ븳뒪꽟뿉 쓽븳 硫댁뿭 泥닿퀎瑜 媛뽰텛怨 엳怨 몴뵾뿉 쓽븳 닔遺 議곗젅 湲곕뒫쓣 媛吏怨 엳쓬. 삉븳, 쟻젅븳 뻼鍮쏆쑝濡 鍮꾪誘 D瑜 빀꽦븯怨 吏吏덉쓽 옣怨좎 깦쑝濡쒖쓽 諛곗꽕 湲곕뒫룄 닔뻾븳떎. 嫄닿컯븳 뵾遺뿉꽌쓽 젙긽쟻씤 遺꾪솕怨쇱젙뿉 쓽빐 깮꽦맂 媛곸쭏痢듭 뵾遺쓽 蹂댁뒿쓣 쑀吏븯怨 쇅遺 솚寃쎌쓽 옄洹뱀쑝濡쒕꽣 蹂댄샇븯뒗 湲곕뒫쓣 媛吏怨 엳떎. 씠윭븳 湲곕뒫쓣 뵾遺 옣踰 湲곕뒫씠씪 븯硫 몴뵾쓽 媛옣 以묒슂븳 뿭븷씠씪怨 븷 닔 엳떎.

媛곸쭏痢듭 쇅遺 솚寃쎌쑝濡쒕꽣 슦由 紐몄쓣 蹂댄샇븯怨 슦由 떊泥 궡쓽 泥댁븸씠 쇅遺濡 냼떎릺뒗 寃껋쓣 留됰뒗 理 쟾諛⑹쓽 諛⑹뼱留됱쑝濡쒖꽌쓽 湲곕뒫쓣 닔뻾븳떎. 媛곸쭏삎꽦꽭룷(keratinocytes)媛 遺꾪솕븯뒗 룞븞 洹 꽭룷留됱 '媛곸쭏꽭룷留(cornified envelope)'씠씪뒗 룆듅븳 援ъ“臾쇰줈 泥대맂떎. 媛곸쭏꽭룷留됱뿉뒗 뿬윭 遺덉슜꽦 떒諛깆쭏뱾씠 援먯감 寃고빀(cross-link)븯뿬 援ъ“泥대 삎꽦븯硫, 꽭룷 쇅遺쓽 吏吏덈쭑뿉 몮윭떥씤 긽깭뿉꽌 吏吏덈쭑쓣 援ъ꽦븯뒗 씪遺 吏吏덉꽦遺꾧낵 怨듭쑀 寃고빀쓣 씠猷⑥뼱 臾쇰━쟻씤 옣踰쎌쓣 삎꽦븯뿬 쇅遺쓽 쐞삊쑝濡쒕꽣 씤泥대 蹂댄샇븷 닔 엳寃 븳떎. 듅엳 媛곸쭏 議곗쭅 궡 議댁옱븯뒗 떎뼇븳 援ъ“ 떒諛깆쭏(Structural protein) 몴뵾 議곗쭅쓽 삎꽦怨 옣踰 湲곕뒫뿉 以묒슂븳 뿭븷쓣 븯뒗 寃껋쑝濡 븣젮졇 엳쑝硫 媛곸쭏꽭룷 궡 援ъ“ 떒諛깆쭏쓽 遺옱濡 씤빐 빞湲곕릺뒗 떎뼇븳 뵾遺 吏덊솚씠 議댁옱븳떎(Cho et al., 2005; Hwang et al., 2011).

Loricrin terminally differentiating structural protein쓽 씪醫낆쑝濡 媛곹솕(cornified)맂 뵾遺 쇅踰쎌쓽 70%瑜 李⑥븯硫 媛곸쭏痢듭쓽 뵾遺 蹂댄샇 옣踰 湲곕뒫쓣 湲곗뿬븯뒗 寃껋쑝濡 븣젮졇 엳떎. 깮泥 궡뿉꽌 Loricrin 룷쑀瑜섏쓽 紐⑤뱺 痢듭긽 긽뵾(stratified epithelia)뿉꽌 諛쒗쁽릺硫 떊깮븘 몴뵾, 援ш컯 諛 빆臾 젏留됱쓽 긽뵾, 떇룄, 룷뵾, 吏 諛 깦쓽 몴뵾 遺遺꾧낵 媛숈 뒿븳 議곗쭅뿉꽌 媛옣 넂 닔以쑝濡 諛쒗쁽맂떎. 듅엳 Loricrin 援ш컯 젏留됲븯 꽟쑀利(oral sub mucous fibrosis) 諛 諛깆깋뙋利(leukoplakia) 媛숈 옞옱쟻씤 븙꽦 옣븷쓽 珥덇린 떒怨꾩뿉꽌 留덉빱濡쒖꽌 솢슜릺怨 엳떎(Nithya et al., 2015).

렔씠뱶(peptide)뒗 2媛 씠긽쓽 amino acid濡 援ъ꽦맂 깮泥 궡 遺꾩옄濡쒖꽌 떒諛깆쭏怨 룞씪븳 떒쐞泥(monomer)濡 援ъ꽦릺吏留 떒諛깆쭏怨 쑀궗븯嫄곕굹 떎瑜 諛⑹떇쑝濡 깮泥 궡 궗옉슜뿉 愿뿬븯怨 엳쑝硫 듅엳 떒諛깆쭏뿉 鍮꾪빐 깮궛씠굹 蹂삎, 씠 옄쑀濡怨 뿴怨 pH 벑쓽 븞젙꽦씠 슦닔븯뿬 떎뼇븯寃 솢슜릺怨 엳뒗 臾쇱쭏씠떎(Bergmann and Zervas, 1932; McKay and Albertson, 1957; Carpino and Han, 1970; Chang and Meienhofer, 1978; Rich and Singh, 1979; Bayer and Rapp, 1986; Hojo et al., 2004). 듅엳 3李⑥썝 援ъ“(3D structure)媛 以묒슂븳 떒諛깆쭏怨 떖由 꽌뿴 젙蹂(sequence)뿉 쓽븳 1李 援ъ“뿉 쓽빐 꽦吏덉씠굹 깮由 솢꽦씠 醫뚯슦릺뼱 떎뼇븳 臾쇱쭏뱾怨쇱쓽 쑖빀 諛 샎빀 벑씠 슜씠븳 옣젏쓣 蹂댁씤떎. 삉븳 떒諛깆쭏쓽 솢꽦 遺쐞(active site)쓽 꽌뿴濡 빀꽦맂 렔씠뱶뒗 쟾泥 떒諛깆쭏쓽 솢꽦怨 쑀궗븳 솢꽦쓣 蹂댁씠湲곕룄 븳떎. 씠젃寃 솗蹂대맂 렔씠뱶뒗 떒諛깆쭏뿉 鍮꾪빐 돺寃 솗蹂닿 媛뒫븯怨 蹂닿, 솢꽦 쑀吏 벑뿉꽌 쑀由ы븯뿬 떎뼇븳 遺꾩빞뿉꽌 솢슜씠 媛뒫븳 옣젏씠 엳떎(Kim et al., 2011; Moh et al., 2011).

씠윭븳 옣젏뱾濡 렔씠뱶뒗 쓽빟, 빟븰, 솕옣뭹 벑 떎뼇븳 遺꾩빞뿉꽌 솢슜릺怨 엳쑝硫 삉븳 듅젙 꽌뿴쓽 렔씠뱶뿉꽌 3李⑥썝 援ъ“ 紐⑤뜽留(3D structural modeling)쓣 넻빐 醫뜑 슚뒫怨 븞젙꽦쓣 媛쒖꽑븳 렔씠뱶瑜 꽕怨꾪븯뒗 렔떚룄誘몃찓떛(Peptidomimetic) 湲곗닠 벑쑝濡 怨좊룄솕릺怨 엳떎.

씪諛섏쟻쑝濡 렔씠뱶쓽 寃쎌젣쟻 슚슜媛移섎뒗 렔씠뱶 꽌뿴怨 湲몄씠뿉 쓽빐 醫뚯슦릺뒗뜲 븘誘몃끂궛 꽌뿴뿉 뵲씪 빀꽦쓽 궃씠룄 슚쑉뿉 李⑥씠媛 諛쒖깮븯硫 븘誘몃끂궛씠 렔씠뱶뿉 異붽媛 맆 븣留덈떎 鍮꾩슜 겕寃 利앷릺誘濡 쓽빟뭹슜씠굹 솕옣뭹슜 렔씠뱶쓽 꽌뿴 씪諛섏쟻쑝濡 3~6 븘誘몃끂궛뿉 吏묒쨷릺뼱 엳쑝硫 씠蹂대떎 湲 꽌뿴 寃쎌젣꽦쓽 臾몄젣濡 솢꽦씠 엯利앸릺뜑씪룄 쟻슜씠 옒뱺 寃쎌슦媛 떎닔 議댁옱븳떎(Kunz and Birnbach, 1984; Kunz and Schaumlöffel, 1985; Lloyd-Williams et al., 1997; Hojo et al., 2001; Hojo et al., 2003).

蹂 뿰援ъ뿉꽌뒗 뵾遺 援ъ“ 떒諛깆쭏씤 Loricrin쓽 꽌뿴쓣 諛뷀깢쑝濡 42醫낆쓽 렔씠뱶瑜 諛쒓뎬, 빀꽦븳 썑 떎뼇븳 솢꽦쓣 젏寃븯뿬 빐떦 렔씠뱶쓽 솢꽦怨 룆꽦쓣 솗씤븯硫 떎뼇븳 뵾遺 吏덊솚쓽 移섎즺 諛 뵾遺 븞떚뿉씠吏 솕옣뭹쓽 썝猷뚮줈꽌 솢슜 媛뒫꽦쓣 寃利앺븯怨좎옄 븳떎(Tesser and Balvert-Beers, 1975; Presland and Jurevic, 2002; Park et al., 2009; Park and Kim, 2010).

옱猷 諛 諛⑸쾿

떎뿕 옱猷

렔씠뱶 빀꽦뿉 븘슂븳 슜留(Trifluoroacetic acid; TFA, Ethanol, methanol, hexane, chloroform, ethyl acetate, n-butane) 븘誘몃끂궛 Sigma-Aldrich Co. (St. Louis, MO, USA)뿉꽌 援щℓ븯뿬 궗슜븯쑝硫 UV-visible spectrophotometer뒗 Varian (Belrose, Australia)궗쓽 Cary 50, pH 誘명꽣뒗 Hanna (Seoul, Korea)궗 젣뭹쓣 궗슜븯쑝硫, HPLC뒗 Shimadzu (Kyoto, Japan)궗쓽 Shim-pack VP-ODS C18 column (250 mm × 4.6 mm, 5 μm) 젣뭹쓣 궗슜븯쑝硫, LC/ESI-MS/MS뒗 Applied Biosystems (Sanpracisco, USA) 젣뭹쓣 궗슜븯떎.

Dimethyl sulfoxide (DMSO) 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazol-ium Bromide (MTT), Elastase뒗 Sigma-Aldrich Chemical Co. (St. Louis, MO, USA)濡쒕꽣 援ъ엯븯뿬 궗슜븯떎. 꽭룷 諛곗뼇뿉 궗슜맂 떆빟쑝濡 fetal bovine serum (FBS), penicillin怨 RPMI 1640 諛곗뒗 Gibco BRL Co. (Grand Island, NY, USA)뿉꽌 援ъ엯븯뿬 궗슜븯떎. 떎뿕뿉 궗슜븳 꽭룷뒗 HDF (Human dermal fibroblast)뒗 neonatal (ATCC No. PCS-201-010) ATCC뿉꽌 援щℓ븯쑝硫 紐⑥쑀몢꽭룷(Dermal Papilla; DP)뒗 Promocell GmbH (Heidelberg, Germany)궗뿉꽌 씤媛 keratiocyte씤 HaCaT 꽭룷二쇰뒗 븳援꽭룷二쇱뻾(Seoul, Korea)뿉꽌 遺꾩뼇 諛쏆븘 궗슜븯떎.

렔씠뱶 씪씠釉뚮윭由 솗蹂

꽌뿴쓣 넻빐 珥 42醫낆쓽 렔씠뱶 꽌뿴쓣 룄異쒗븯쑝硫 빐떦 렔씠뱶쓽 꽌뿴 Table 1뿉꽌 솗씤븷 닔 엳떎. Table 1쓽 렔씠뱶瑜 빀꽦븯湲 쐞븳 렔씠뱶 씪씠釉뚮윭由щ 援ъ텞븯湲 쐞빐 19媛쒖쓽 nsc-븘誘몃끂궛(nsc-Ala, nsc-Arg (pbf), nsc-Asp (OtBu), nsc-Asn (trt), nsc-Gly, nsc-Glu (OtBu), nsc-Gln (trt), nsc-His (trt), nsc-Ser (tBu), nsc-Thr (tBu), nsc-Tyr (tBu), nsc-Trp (Boc), nsc-Leu, nsc-Ile, nsc-Val, nsc-Phe, nsc-Met, nsc-Lys (Boc), nsc-Pro)씠 遺李⑸맂 겢濡쒕줈 듃由ы떥 겢濡쒕씪씠뱶 젅吏(Chloro trityl chloride resin: CTL resin, Novabiochem Cat No. 01-64-0021)쓣 96쎇 뀒봽濡 諛섏쓳湲곗뿉 떆由ъ쫰 蹂꾨줈 媛 19 씪씤뿉 50 mg쓣 꽔怨 硫뷀떥젋 겢濡쒕씪씠뱶(MC) 1 mL瑜 媛븯뿬 3遺꾧컙 援먮컲븯떎. 슜븸쓣 젣嫄고븯怨 뵒硫뷀떥룷由꾩븘留덉씠뱶(DMF) 1 mL瑜 꽔뼱 3遺꾧컙 援먮컲븳 썑 떎떆 슜留ㅻ 젣嫄고븯떎. 젣議곕맂 듃씪꽖궪궛-렔떚뵜 젅吏꾩 DMF, MC 諛 硫뷀깂삱濡 媛곴컖 3踰덉쓣 꽭泥숉븯怨, 吏덉냼 怨듦린瑜 泥쒖쿇엳 쓽젮 嫄댁“븳 썑, P2O5 븯뿉꽌 吏꾧났쑝濡 媛먯븬븯뿬 셿쟾엳 嫄댁“븯떎. 젣議곕맂 젅吏꾩뿉 깉猷⑥슜븸[듃由ы뵆濡쒕줈솕 珥덉궛(Trifluroacetic acid; TFA) 81.5%, 利앸쪟닔 5%, 떚삤븘땲議(Thioanisole) 5%, 럹 5%, EDT (1,2-Ethanedithiol) 2.5% 諛 TIS (Triisopropylsilane) 1% 룷븿] 30 mL쓣 꽔 썑 긽삩뿉꽌 媛걫 쓷뱾뼱二쇰ʼn 뼹쓬 닔議 븞뿉꽌 1떆媛 諛섏쓳쓣 쑀吏븯떎. 젅吏꾩쓣 뿬怨쇳븯怨, 냼웾쓽 TFA 슜븸쑝濡 꽭泥숉븳 썑 紐⑥븸怨 빀븯떎. 씠 썑 뀒듃씪 렔씠뱶 꽌뿴 렔씠뱶瑜 닔뱷븯떎. 렔씠뱶 빀꽦뿉 븳 쟾泥댁쟻씤 紐⑥떇룄뒗 Fig. 1뿉 굹궡뿀떎.

Sequence of Tetrapeptide derived from Loricrin

Number Sequence Molecular weight Number Sequence Molecular weight
1 MSYQ 455.19443 22 FSSG 324.15394
2 QKKQ 458.3071 23 GQLE 373.221047
3 PTPQ 369.2118 24 ELPE 414.236377
4 PPVD 354.20081 25 EQQE 460.20236
5 DCVK 391.19943 26 LELP 398.292184
6 VKTS 361.2431 27 GQLK 372.273417
7 KTSG 319.19614 28 KHLE 453.294887
8 SGGS 234.10698 29 EHQE 469.20269
9 GSGY 310.13828 30 EGQL 373.221047
10 GQVS 317.18049 31 LEVP 384.262197
11 GYVS 352.18524 32 PEEQ 429.19655
12 SQQV 388.21761 33 QMGQ 390.17911
13 TQTS 363.18597 34 GQDP 343.15966
14 CAPQ 345.15766 35 QLKY 478.315287
15 QPSY 421.20671 36 YLEQ 479.262917
16 YGGG 280.12771 37 EQQE 460.20236
17 SSGG 234.10698 38 TKGE 361.20671
18 GGSG 204.09641 39 VLLP 368.318004
19 GSSG 234.10698 40 VEHQ 439.22851
20 GCFS 340.1311 41 QQKQ 458.27071
21 CFSS 370.14167 42 EVQW 488.24892


Fig. 1. Solid phase synthesis of tetrapeptides. The protecting groups for the amino groups used in the peptide synthesis are 2-(4-Nitrophenyl) sulfonylethoxycarbonyl (Nsc); using N-terminal and t-butyloxycarbonyl (Boc); using R group.

렔씠뱶 遺꾩꽍

빀꽦맂 42醫낆쓽 렔씠뱶쓽 닚룄 諛 遺꾩옄웾쓣 솗씤븯湲 쐞빐 HPLC 遺꾩꽍쓣 吏꾪뻾븯떎. 媛 렔씠뱶瑜 waters궗쓽 HPLC (waters 2695) waters쓽 C18 (Waters Xterra MS C18 column (L: 250 mm, LD: 4.6 mm, 5 μm)) 而щ읆쓣 씠슜븯뿬 遺꾩꽍븯떎. 씠븣 HPLC쓽 寃異쒓린쓽 뙆옣 216 nm 뙆옣쓣 씠슜븯떎. HPLC쓽 씠룞긽쓽 슜留ㅻ줈뒗 0.1% 궪遺덉냼 븘꽭듃궛(Trifluoroacetic acid, TFA)씠 룷븿맂 븘꽭넗땲듃由(Acetonitile, ACN)怨 0.1% TFA媛 룷븿맂 water瑜 씠슜븯쑝硫, 떆媛꾩뿉 뵲씪 ACN쓽 냽룄瑜 蹂솕떆궡쑝濡쒖꽌 렔씠뱶쓽 닚룄 遺꾩꽍쓣 愿李고븯떎. 렔씠뱶 臾쇱쭏 遺꾩꽍뿉 븘슂븳 議곌굔 븘옒 Table 2怨 媛숇떎.

HPLC condition of Tetrapeptide derived from Loricrin

Condition of HPLC analysis
Column Shim-pack VP-ODS C18 column (L: 250 mm, LD: 4.6 mm, 5 μm)
Detector UVD 170s DIONEX
Detection wavelength 216, 254 nm
Flow rate 1.0 mL/min
Injection volume 50 μL
Mobile phase conditions for HPLC gradient-elution Program order Time (min) 0.1% TFA1) in D.W (%) 0.1% TFA1) in 50% ACN2) (%)
1 0 100 0
2 10 75 25
3 20 50 50
4 30 25 75
5 40 0 100
6 45 100 0


뀒듃씪 렔씠뱶쓽 Elastase 솢꽦 뼲젣 떆뿕

빀꽦맂 뀒듃씪 렔씠뱶 42醫낆쓽 뵾遺 二쇰쫫 媛쒖꽑 슚怨쇰 솗씤븯湲 쐞빐 뿕씪뒪젣 뼲젣 떆뿕쓣 吏꾪뻾븯떎. 湲곗쭏濡쒖꽌 N-succinyl-(L-Aal)3-p-nitroanilide瑜 궗슜븯뿬 37℃뿉꽌 30遺꾧컙 p-nitroanilide쓽 깮꽦웾쓣 痢≪젙븯떎. 媛 떆뿕 렔씠뱶 쓬꽦, 뼇꽦議곌뎔쓣 100 μg/mL뵫 떆뿕愿뿉 痍⑦븯怨 pancreatic solution (Type I: from porcine pancreas 쑀옒, 0.6 unit/mL, Sigma Aldrich, USA)슜븸 50 μg/mL쓣 媛븳 썑 湲곗쭏濡 50 mM Tris-HCl buffer (pH 8.6)뿉 끃씤 N-succinyl-(LAla)3-p-nitroanilide (1 mg/mL)쓣 100 μg/mL쓣 泥④븯뿬 30遺꾧컙 諛섏쓳떆궎怨 microplate reader瑜 씠슜븯뿬 410 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. Elastase 빐 솢꽦 떆猷뚯슜븸쓽 泥④援ъ 臾댁꺼媛援ъ쓽 씉愿묐룄 媛먯냼쑉濡 굹궡뿀떎.

Elastase inhibition rate%=Absorbance of controlabsorbance of sampleAbsorbance of contro1100

42醫낆쓽 빀꽦 렔씠뱶瑜 泥섎━븳 뮘 뿕씪뒪젣 솢꽦 뼲젣 슚怨쇰 痢≪젙븯떎.

꽭룷二 諛곗뼇

DP 꽭룷 HaCaT 꽭룷뒗 Dulbeccós Modified Eagle Medium (DMEM; Gibco BRL, Gaithersberg, USA) 諛곗뿉 10% FBS (Gibco BRL) 1% penicillin-streptomycin (Gibco BRL)쓣 泥④븯뿬 湲곕낯 諛곗濡 궗슜븯怨, 37℃, 5% CO2 議곌굔쓽 諛곗뼇湲곗뿉꽌 2~3씪뿉 븳 踰덉뵫 怨꾨 諛곗뼇 븯떎.

MTT assay

DP 꽭룷瑜 48 well-plate뿉 1×104媛쒕줈 遺꾩<븯뿬 24떆媛 諛곗뼇븯떎. 諛곗뼇꽭룷뿉 GQVS瑜 0, 25, 50, 100, 200 μg/mL濡 泥섎━븯뿬 72떆媛 諛곗뼇븯怨, HaCaT 꽭룷뒗 諛곗뼇 썑 48 well-plate뿉 1×104媛쒕줈 遺꾩<븯뿬 24떆媛 諛곗뼇 썑 諛곗뼇꽭룷뿉 GQVS瑜 0, 25, 50, 100, 200 μg/mL濡 泥섎━븯뿬 24떆媛 諛곗뼇븯떎. 媛 꽭룷二 蹂꾨줈 諛곗뼇 썑 5 mg/mL MTT (Sigma) 떆빟쓣 10 μL 媛븯怨 4떆媛꾩쓣 諛곗뼇븯떎. 洹 썑 긽痢듭븸쓣 젣嫄고븯怨 삎꽦맂 formazan쓣 DMSO 100 μL濡 끃뿬 ELISA reader濡 550 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎.

넻怨꾪븰쟻 遺꾩꽍

議곌뎔怨 렔씠뱶 泥섎━援 寃곌낵뿉 븳 넻怨꾩쿂由щ뒗 Student's t-test濡 鍮꾧탳븯쑝硫, 넻怨꾩쿂由 썑 P 媛믪씠 0.05 誘몃쭔씪 寃쎌슦 넻怨꾩쟻씤 쑀쓽꽦씠 엳떎怨 뙋젙븯떎.

寃곌낵 諛 怨좎같

렔씠뱶 씪씠釉뚮윭由 빀꽦 諛 HPLC 遺꾩꽍

怨좎껜긽 빀꽦(solid phase synthesis)瑜 넻빐 솗蹂대맂 42醫낆쓽 뀒듃씪 렔씠뱶쓽 빀꽦 寃곌낵뒗 Table 3뿉 몴湲고븯떎. 빀꽦맂 뀒듃씪 렔씠뱶쓽 닔쑉 렔씠뱶쓽 꽌뿴뿉 李⑥씠뿉 뵲瑜 臾쇱꽦 李⑥뿉 쓽빐 긽씠븯굹 왂 20% 쓽 룊洹 닔쑉쓣 蹂댁씠怨 엳떎. 빀꽦맂 렔씠뱶뒗 HPLC 遺꾩꽍쓣 넻빐 닚룄瑜 솗씤븯쑝硫 而щ읆 遺꾨━ 떆 elution 떆媛꾨룄 뿭떆 긽씠븯굹 5~7遺꾨뿉꽌 뵾겕瑜 솗씤븷 닔 엳뿀떎. Fig. 2 媛숈씠 90% 씠긽쓽 닚룄瑜 媛吏 뀒듃씪 렔씠뱶濡 솗蹂대릺뿀떎. 렔씠뱶 以 씪遺쓽 HPLC 遺꾩꽍 李⑦듃瑜 Fig. 3뿉 몴湲고븯떎.

Result of Tetrapeptides derived from Loricrin

Number Sequence Molecular weight Purity (%) Yield (%) Number Sequence Molecular weight Purity (%) Yield (%)
1 MSYQ 455.19443 92.54 32.51 22 FSSG 324.15394 97.25 29.21
2 QKKQ 458.3071 95.21 30.25 23 GQLE 373.221047 95.12 30.54
3 PTPQ 369.2118 93.21 29.21 24 ELPE 414.236377 93.21 27.14
4 PPVD 354.20081 92.54 28.01 25 EQQE 460.20236 95.88 28.65
5 DCVK 391.19943 98.21 27.98 26 LELP 398.292184 95.17 32.99
6 VKTS 361.2431 97.22 31.07 27 GQLK 372.273417 94.36 30.17
7 KTSG 319.19614 95.21 30.98 28 KHLE 453.294887 95.87 30.54
8 SGGS 234.10698 94.20 28.14 29 EHQE 469.20269 94.06 31.01
9 GSGY 310.13828 95.83 27.14 30 EGQL 373.221047 95.08 27.14
10 GQVS 317.18049 96.24 29.66 31 LEVP 384.262197 95.37 29.69
11 GYVS 352.18524 97.21 30.17 32 PEEQ 429.19655 96.33 27.09
12 SQQV 388.21761 96.21 33.01 33 QMGQ 390.17911 95.22 26.39
13 TQTS 363.18597 96.55 28.74 34 GQDP 343.15966 99.71 30.72
14 CAPQ 345.15766 93.18 29.44 35 QLKY 478.315287 94.21 29.66
15 QPSY 421.20671 95.68 28.09 36 YLEQ 479.262917 95.36 27.17
16 YGGG 280.12771 97.21 29.36 37 EQQE 460.20236 96.21 22.01
17 SSGG 234.10698 96.12 31.14 38 TKGE 361.20671 94.21 30.74
18 GGSG 204.09641 97.25 29.47 39 VLLP 368.318004 96.21 17.14
19 GSSG 234.10698 96.33 27.21 40 VEHQ 439.22851 97.01 29.71
20 GCFS 340.1311 94.18 21.88 41 QQKQ 458.27071 95.36 30.54
21 CFSS 370.14167 95.36 30.07 42 EVQW 488.24892 94.06 28.14


Fig. 2. HPLC analysis of Tetrapeptide. (A) Tetrapeptide No. 10, Sequence; GQVS, (B) Tetrapeptide No. 34, Sequence; GQDP, Peptides were identified by HPLC. HPLC Simadzu (Japan) Shim-pack VP-ODS C18 column (250mm * 4.6mm, 5μm).

Fig. 3. The Effect of GQVS on the cell proliferation in DP and HaCaT cells. DP (A) and HaCaT cells (B) were treated with GQVS (25, 50, 100, and 200 μg/mL) for 72 h and 24 h, respectively. The proportion of survival cells was measured by MTT assay. Experiments were repeated in triplicate and the results were expressed as the mean ± SD.

뿕씪뒪젣 솢꽦 뼲젣 슚怨

뀒듃씪 렔씠뱶쓽 뵾遺 솢꽦쓣 솗씤븯湲 쐞빐 뿕씪뒪젣 솢꽦 뼲젣 떆뿕쓣 吏꾪뻾븯떎. 뿕씪뒪젣(Elastase)뒗 뵾遺 吏꾪뵾 꽭룷 궡 肄쒕씪寃(collagen), 뿕씪뒪떞(elastin)쓣 遺꾪빐븯뒗 슚냼濡 뵾遺꽭룷쓽 利앹떇 벑뿉 븘슂븳 슚냼씠굹 怨쇰웾 諛쒗쁽븯硫 吏꾪뵾 痢 궡 援ъ“ 떒諛깆쭏쓣 媛먯냼떆耳 뵾遺 二쇰쫫쓽 썝씤씠 릺뒗 슚냼씠떎. 뵲씪꽌 뿕씪뒪젣쓽 솢꽦쓽 뼲젣瑜 넻빐 뵾遺쓽 二쇰쫫쓣 뼲젣븷 닔 엳쑝硫 留롮 뵾遺 끂솕 諛⑹ 臾쇱쭏뱾쓽 寃쎌슦 뿕씪뒪젣 솢꽦 뼲젣뒫쓣 蹂댁씠怨 엳떎. 蹂 떎뿕뿉꽌 빀꽦맂 뀒듃씪 렔씠뱶쓽 寃쎌슦 42醫낆쓽 렔씠뱶 以 씪遺쓽 렔씠뱶뿉꽌 삉븳 뿕씪뒪젣 빐뒫(%)씠 슦닔븳 빀꽦 뀒듃씪 렔씠뱶媛 솗씤릺뿀쑝硫(Table 4)쓽 寃쎌슦뒗 湲곗〈쓽 뿕씪뒪젣 뼲젣 湲곕뒫꽦 냼옱씤 Ursonic acid뿉 鍮꾪빐꽌룄 쑀궗븳 寃껋쑝濡 뙆븙릺뿀떎 씠윭븳 寃곌낵濡쒕꽣 뵾遺뿉 二쇰쫫 깮꽦쓣 쑀諛쒖떆궎뒗 슂씤씠 쟻슜릺뿀쓣 븣 빀꽦맂 뀒듃씪 렔씠뱶媛 뿕씪뒪젣 뼲젣 슚怨쇰 넻빐 二쇰쫫 깮꽦쓣 뼲젣빐 뵾遺 끂솕 諛⑹뿉 슚怨쇨 엳쓣 寃껋쑝濡 궗猷뚮맂떎. Table 4뒗 뀒듃씪 렔씠뱶쓽 援ъ껜쟻씤 뿕씪뒪젣 솢꽦 빐 뒪겕由щ떇 寃곌낵瑜 굹궡뿀떎.

Result of Elastase inhibition efficacy of Tetrapeptides

Number Sequence Elastase inhibition (%) Number Sequence Elastase inhibition (%)
1 MSYQ 79.4±5.6 22 FSSG 69.1±4.4
2 QKKQ 88.7±6.3 23 GQLE 73.2±3.3
3 PTPQ 69.3±4.9 24 ELPE 90.5±4.2
4 PPVD 72.5±5.3 25 EQQE 53.6±4.9
5 DCVK 118.4±3.3 26 LELP 78.2±3.3
6 VKTS 66.3±4.8 27 GQLK 56.0±5.2
7 KTSG 44.5±6.2 28 KHLE 79.3±6.4
8 SGGS 49.6±5.8 29 EHQE 66.3±6.3
9 GSGY 56.3±3.3 30 EGQL 69.7±4.9
10 GQVS 33.9±5.4 31 LEVP 70.6±5.5
11 GYVS 78.5±5.5 32 PEEQ 64.0±6.4
12 SQQV 49.3±4.6 33 QMGQ 55.6±5.7
13 TQTS 59.0±5.0 34 GQDP 34.5±5.1
14 CAPQ 77.2±4.6 35 QLKY 60.5±5.2
15 QPSY 63.5±5.3 36 YLEQ 72.3±3.9
16 YGGG 67.1±4.4 37 EQQE 64.2±4.0
17 SSGG 77.0±6.2 38 TKGE 59.3±4.2
18 GGSG 103.5±3.2 39 VLLP 60.7±5.3
19 GSSG 58.3±5.1 40 VEHQ 55.7±4.9
20 GCFS 55.2±5.6 41 QQKQ 72.3±5.4
21 CFSS 47.4±4.4 42 EVQW 49.5±4.8
- Control - 100±5.2 + Control (Ursonic acid) - 28.8±3.6


뵾遺꽭룷 利앹떇 珥됱쭊 슚怨

븵꽑 뿕씪뒪젣 솢꽦 빐 떎뿕쓣 넻빐 媛옣 슦닔븳 寃곌낵瑜 蹂댁뜕 tetrapeptide No. 10, GQVS媛 DP 諛 HaCaT 꽭룷쓽 利앹떇뿉 誘몄튂뒗 쁺뼢쓣 솗씤븯湲 쐞븯뿬 떆猷뚮 냽룄 蹂(25, 50, 100, 200 μg/mL)濡 泥섎━븯怨 媛곴컖 72떆媛꾧낵 24떆媛 썑 MTT assay瑜 씠슜븯뿬 痢≪젙븯떎. 떎뿕 寃곌낵, DP 꽭룷쓽 寃쎌슦 媛곴컖쓽 냽룄뿉꽌 72떆媛 썑 議곌뎔(100%)뿉 鍮꾪빐 110.23 (±2.21)%, 147.37 (±1.24)%, 160.23 (±5.21)%, 164.21 (±3.24)%쑝硫, HaCaT 꽭룷쓽 寃쎌슦 24떆媛 썑 121.14 (±3.11)%, 140.70 (±1.57)%, 144.32 (±1.27)%, 140.15 (±5.07)%濡 굹궗떎. 蹂 寃곌낵瑜 넻빐 Tetrapeptide GQVS뒗 냽룄쓽議댁쟻쑝濡 2醫낆쓽 꽭룷, DP, HaCaT 꽭룷쓽 利앹떇쓣 珥됱쭊븯뒗 寃껋쑝濡 솗씤릺뿀떎.

蹂 뿰援щ 넻빐 뿰援ъ쭊 援ъ“ 떒諛깆쭏쓽 씪醫낆씤 Loricirn쓽 꽌뿴뿉꽌 룄異쒕맂 42醫낆쓽 tetrapeptide쓽 빀꽦뿉 꽦怨듯븯뿬 씪씠釉뚮윭由щ 셿猷뚰븯쑝硫 씠뱾 tetrapepide쓽 뿕씪뒪젣 뼲젣뒫, 뵾遺꽭룷 利앹떇 珥됱쭊 슚뒫쓣 솗씤븯떎. 寃곌낵쟻쑝濡 솗蹂대맂 GQVS 꽌뿴쓽 tetrapepide뒗 DP 꽭룷 利앹떇怨 HaCaT 꽭룷 利앹떇쓽 珥됱쭊 媛곴컖 紐⑤컻쓽 꽦옣怨 뵾遺 二쇰쫫 媛쒖꽑뿉 쁺뼢쓣 誘몄튌 寃껋쑝濡 뙋떒릺硫, 깉紐 諛 二쇰쫫 媛쒖꽑 냼옱 媛쒕컻쓽 湲곗큹옄猷뚭 맆 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

This research was supported by the Ministry of Trade, Industry & Energy (MOTIE), Korea Evaluation Institute of Industrial Technology (KEIT) through the Encouragement Program for Bio industry core technology development project (Project NO: 20009105).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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