Journal of Life Science 2010 Vol. 20. No. 12. 1838~1843 ISSN : 1225-9918 DOI : 10.5352/JLS.2010.20.12.1838 Anti-Wrinkle Effects of Korean Rice Wine Cake on Human Fibroblast Jung-Min Yoo, Yeo-Jin Kang, Hyeong-Bae Pyo 1, Eui Su Choung 2, Shin Young, Park 3, Ji Ho Choi 3, Gwi-Jung Han 3, Choong Hwan Lee 4 and Tack-Joong Kim* Division of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju 220-710, Korea 1 R&D Center, Hanbul Cosmetics Co., Umsung 369-834, Korea 2 R&D Center, DANJOUNGBIO Ltd. Co., Wonju 220-842, Korea 3 Rural Development Administiration, Fermention & Food Processing Division, Suwon 411-853, Korea 4 Department of Bioscience and Biotechnology and Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Korea Received August 31, 2010 /Accepted October 26, 2010 Skin aging is related to genetic and environmental factors (e.g., gene mutation and UV radiation respectively). To develop a new anti-wrinkle cosmetic or functional food by using Korean rice wine cake, we examined the effects of Korean rice wine cake, a brewery byproduct, on antioxidant effect, collagen synthesis and expression of MMP-1. Interestingly, we found that Korean rice wine cake has the ability to promote scavenging activity of DPPH radical. We also found that the cell proliferation and synthesis of collagen in HS27 cells was increased by Korean rice wine cake in a concentration-dependent manner. However, elastase inhibitory activity was not changed. In addition, the expression of MMP-1 was inhibited by Korean rice wine cake in a concentration-dependent manner. All these results suggest that Korean rice wine cake can be effectively used for the prevention of wrinkles in human skin. Key words : Korean rice wine cake, skin aging, anti-wrinkle cosmetics 서 최근현대인들의경제수준이향상되고현대의학의발전으로인하여평균수명이늘어나고여유로워짐에따라피부건강에도많은관심을가지게되었다 [9]. 건강한피부를유지하고자하는목적에따라많은미용관련화장품및식품이개발되고있고, 최근피부주름개선을위한연구가활발히진행되고있다 [16]. 피부노화 (skin aging) 는피부세포및조직의구조적, 기능적인변화로인한현상으로, 내인성노화 (intrinsic aging) 와외인성노화 (extrinsic aging) 로구분된다. 내인성노화는나이가들어감에따라자연스럽게생기는변화로유전적요인이가미된피부주름의발생을의미하고, 외인성노화의경우는자외선, 중력, 오염된공기와같은환경적요인에의한피부주름의발생을의미한다 [15,19,21]. 내인성노화와외인성노화는모두피부진피층에존재하는세포외기질 (extracellular matrix) 의구조적인변화로발생되는데 [3-4,15,22], 그구성요소중탄력섬유 (elastic fiber) 의변성과교원질 (collagen) 양의감소가피부의주름을야기한다 [6]. 노화된피부의대표적인증상은탄력저하에의해유발된잔주름및주름의발생이며, 이는피부진피조직의교원질에서피부탄력과관련된 collagen 단백질의현저한감소에의해 론 *Corresponding author *Tel:+82-33-760-2242, Fax:+82-33-760-2183 *E-mail : ktj@yonsei.ac.kr 서발생될수있다. Collagen은진피의 90% 이상을차지하고있으면서피부의장력과강도를부여하기때문에외부로부터의자극에대해피부를보호하고유지시킨다. 그렇기때문에 collagen의감소는피부노화및주름생성과매우깊은관계를가지고있다 [16]. Collagen의분해와관련된분자기작으로전사인자인 AP-1 (activating protein-1) 에의해유도되는 MMPs (matrix metalloproteinases) 에의해서세포외기질과 type I procollagen의분해가일어난다 [5,18]. MMP family 중 MMP-1은 collagen을분해시키는대표적인분해효소로알려져있고, 이와는반대로 TIMP-1 (tissue inhibitor of metalloproteinase) 은 collagen 분해효소의활성을억제시키는억제자로알려져있다 [5]. 즉, 피부주름의형성은두효소에의해영향을받게되고, 대부분주름형성의억제효과에관한연구는두인자의조절을목적에두고있다. 또한, 피부의노화를이끄는주요요인중에는 collagen의감소와관련하여자외선이나스트레스에의한활성산소의생성을들수있다. 피부에서의활성산소의생성은세포막을공격함으로써세포에손상을입혀그기능을상실하게한다 [8,23]. 피부세포로써의기능상실은피부의거친상태를형성하거나유지시키고, 윤기를없애는결과를초래함으로써피부의주름을유발하게된다 [16]. 결국, 생체내활성산소의생성은 MMPs 유전자와 collagen 분해효소의연쇄적인활성화를유도함으로써 collagen의구조적인파괴를유발하고, 피부의탄력을감소시키는피부주름의형성을이끌게된다 [16]. 따라
Journal of Life Science 2010, Vol. 20. No. 12 1839 서, 피부노화는 collagen 분해효소와활성산소의생성억제, collagen의합성을촉진시킴으로써피부노화의완화효과를나타낼수있다. 주박 (Korean rice wine cake) 은청주, 약주등의술을빚고남은양조부산물로써, 알코올, 효소, 유기물등을포함하는천연소재이다. 일본에서는식용, 합성소주와소주의향미액원료, 사료첨가제등으로일부사용되고있으나다른분야의기능성산업화에는미흡한실정이다. 양조과정중에첨가되는주박미생물들은균체자체로서풍부한영양분을함유하고있다 [13]. 주박은피부와연관이있는것으로생각되고있는데, 민간에서주류생산및주박을다루는사람들의손은일반인들에비해훨씬부드러운것으로알려져있다. 즉, 주박이피부주름에연관이있는것으로추측되고있지만, 현재까지피부에미치는효과기전의연구의보고는미비한실정이다. 이에본연구는, 피부섬유아세포를이용하여주박이피부에미치는항노화효과에관한연구를수행하였고, 피부노화의직접적인원인인 MMP-1의유전자발현과피부의주요구성인자인콜라겐의생합성촉진효과에대해검증하였고, 이를보고하고자한다. 재료및방법실험재료시료의추출은다음과같이추출하였다. 주박 50 g에 50% 주정 150 ml을첨가하여 150 rpm으로 48시간동안반응하여여과한후, 감압농축하여동결건조한분말을본실험의시료로사용하였다. 세포배양을위한배양액인 Dulbecco s Modified Eagle s Medium (DMEM) 은 Sigma-Aldrich (St. Louis, MO, USA) 에서구입하였고, Fetal Bovine Serum (FBS) 과 penicillin-streptomycin은 Lonza (Walkersville, MD, USA) 에서구입하였다. Collagen의생합성양을측정하기위해서사용한 Procollagen Type-I C-Peptide (PIP) EIA kit는 TAKARA Bio Inc. (Shiga, Japan) 에서구입하여실험을하였다. 세포배양인간섬유아세포인 HS27세포는정해영교수 ( 부산대학교 ) 로부터분양받았으며, 10% FBS와 100 unit/ml의 penicillin-streptomycin이첨가된 DMEM 배지에서 2-3일마다배양액을교환해주었으며, 배양환경은 37 로유지되는 5% CO 2 배양기에서배양하였다. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical 소거능측정주박의항산화효과는 DPPH를이용하여라디컬소거효과를측정하였다. 0.2 mm DPPH 알코올용액에주박을농도별로알코올에희석하여혼합한후, 37 에서 30분간반응시킨 후, microplate reader를이용하여 517 nm의흡광도에서측정하였다. 세포증식측정주박의세포증식효과는 Ez-cytox enhanced cell viability kit (Daeil Lab, Seoul, Korea) 을이용하여측정하였다. 96-well plate에각 well당 2,500세포가되도록심어준후, 24시간을배양하였다. 이후, 배지를제거하고주박을농도별로처리한후 23시간을배양한후, Ez-cytox enhanced cell viability kit를 total volume의 10% 의양으로첨가한후 1시간동안 37 배양기에서반응하였고, microplate reader를이용하여 450 nm의흡광도에서측정하였다. Elastase 저해활성측정주박의탄력섬유분해효소의억제효과를알아보기위해 elastase를이용하여측정하였다. 96-well plate에 100 mm Tris-HCl buffer (ph 8.0) 를넣은후농도별로준비된주박을첨가하였다. 2 mm N-Succinyl-(Ala)-3-p-nitroanilide (Sigma- Aldrich, St. Louis, MO, USA) 를각 well에분주하고 0.2 unit 로조제한 elastase 효소액 (Sigma-Aldrich, St. Louis, MO, USA) 을첨가하여섞어주었다. 이후, 37 배양기에서 30분간반응을시킨후얼음에서 5분간방치하여반응을중지시켰다. Microplate reader를이용하여 410 nm의파장에서흡광도를측정하였다. Collagen 생합성측정 HS27세포에주박을농도별로처리했을때 collagen type I의합성양을측정하기위해 Procollagen Type-I C-Peptide (PIP) EIA kit를이용하였다. 96-well plate에각 well당 10,000 세포가되도록심어준후 24시간을배양하였다. 이후, 배양된배지를제거하고주박을농도별로처리한후 24시간을배양하였다. 각 well로부터상층액을회수하여 Procollagen Type-I C-Peptide (PIP) EIA kit의각 well에첨가한후, 제조사의방법에따라 procollagen type I의총양을측정하였다. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) HS27세포는 100 mm-culture dish에 70% 가될때까지배양하였다. 배지를제거하고, phosphate-buffered saline (PBS) 로두번헹구어준뒤, 주박을농도별로처리하여 24시간배양하였다. Total RNA는 TRI Reagent (Sigma-aldrich, St. Louis, MO, USA) 을이용하여제조사의방법으로추출하였다. 추출된 total RNA는 spectrophotometer를이용하여정량하여, 1 μg의 RNA를이용하였고, cdna 합성및 PCR은 Accupower RT/PCR premix kit (Bioneer, Daejeon, Korea) 를이용하여 RT-PCR을수행하였다. PCR 조건은 94 1분, 48 1분, 72
1840 생명과학회지 2010, Vol. 20. No. 12 1분씩 22 cycles 수행하였고, PCR 산물은 2% agarose gel로 전기영동을한후, ethidium bromide을첨가하여밴드를가시 화하였다. 사용된 primer는다음과같다. MMP-1 forward 5'-TGGGAGCAAACACATCTGA-3'; MMP-1 reverse 5'- ATCACTTCTCCCCGAATCGT-3'; TIMP-1 forward 5'- TTCCGACCTCGTCATCAGGG-'3; TIMP-1 reverse 5'- ATTACGGCTATCTGGGACCGC-3'; β-actin forward 5'- GAGACCTTCAACACCCCAGCC-3'; β-actin reverse 5'-GGCCATCTCTTGCTCGAAGTC-3'. 통계처리모든실험결과는평균 ± 표준편차 (means±sem) 로나타내었다. 통계처리는 Dunnett s test로검정하였고정상대조군과비교하여 <0.05 이하의경우유의적인차이가있다고판정하였다. 결과및고찰주박에의한 DPPH (1,1-diphenyl-2-picrylhydrazyl) radical 소거효과피부에있어서활성산소의생성은과산화질소를생성하여세포변성및 DNA를손상시켜유전정보를변화시켜노화를촉진시키는것으로알려져있다 [8,16,23]. 이러한활성산소활성의지표로이용되는 DPPH는지질과산화반응중에전자를받아산화 환원반응에따라색을띄는 radical로, 일반적으로 free radical의소거반응을보기위한실험방법중하나이다 [16]. 주박의항산화효과를알아보기위해 DPPH를이용하여그소거능을확인하였다 (Fig. 1). 농도별로처리된주박의경우 250 μg/ml에서 43% 의 free radical 소거활성을, 500 μg/ml에서 40% 의소거활성을나타내었다. 본결과로부터주박은 DPPH radical의소거효과가있는것을확인하였다. 주박에의한피부섬유아세포증식효과피부섬유아세포에서주박의처리가세포증식에미치는영향을확인하기위하여 Ez-cytox enhanced cell viability kit를이용하여세포증식효과를조사하였다. Fig. 2의결과에서, 주박의처리에의해피부섬유아세포가농도의존적으로증가함을알수있었다. 즉, 피부섬유아세포에처리된주박은세포의독성이없다는것과동시에세포의증식을유도하는것을확인하였고, 궁극적으로는피부세포의분화및재생에관여되는것으로판단할수있었다. 주박에의한 elastase 활성저해효과 Elastin은피부진피층의약 3-4% 정도를차지하는기질단백질로써, 피부의탄력을유지하는기능을한다 [2]. Elastin의분해는인체의중성구과립구내에존재하는 elastase에의해 Fig. 1. Antioxidative effect of Korean rice wine cake. DPPH radical scavenging activity was measured in the reaction mixture containing 0.2 mm DPPH solution, ethanol, and Korean rice wine cake. The scavenging activity was determined by microplate reader in absorbance at 517 nm. The values shown represent means±sem of three different assays. * p<0.05, ** p<0.01 compared with absence of Korean rice wine cake. Fig. 2. Effect of Korean rice wine cake on proliferation of HS27 cell. HS27 cells were incubated for 24 hr in DMEM medium containing 10% FBS, were treated with various concentrations of Korean rice wine cake for 24 hr and cell viability was measured by Ez-cytox enhanced cell viability kit. The values shown represent means±sem of three different assays. * p<0.05, ** p<0.01 compared with absence of Korean rice wine cake. 일어난다. Elastase는 elastin과더불어중요기질단백질인 collagen도분해할수있는비특이적가수분해효소이다 [9]. 이러한 elastase의활성은기질단백질을분해시켜피부의세포기저층의그물망구조를끊어줌으로써주름을유발하는주원인효소로이상조직에서는그활성이높아져직접적으로조직을파괴하여피부의주름을유발하게된다 [2,9]. 따라서, elastase의활성억제효과를확인하기위해 Tris-HCl과혼합
Journal of Life Science 2010, Vol. 20. No. 12 1841 된다양한농도의주박을 elastase 효소와기질을결합하여 elastase의활성억제현상을측정하였다 (Fig. 3). 주박을처리하지않은비처리군의 elastase의활성과비교하였을때, 농도별로처리된주박처리군은비처리군과마찬가지로활성의억제효과를나타내지않았다. 따라서, 주박의효과는 elastase의활성억제기작에는연관이없음을알수있었다. 주박에의한 collagen 생합성촉진효과 피부를구성하는주성분인 collagen은피부, 골, 인대, 연골및치아등에높은농도로존재하고있으며, 특히, 섬유아세포에서세포외기질을구성하는중요한구성요소이다. Collagen 은피부의기계적견고성, 결합조직의저항력과조직의결합력, 세포접착의지탱, 세포분할과분화의유도등다양한기능을하고있다 [2]. Collagen은피부의주름과밀접한관계를가지고있기때문에, collagen 부족은주름의유발로이어진다. 그러므로 collagen의합성을촉진하는소재의발견은화장품원료로서의이용이매우크다고볼수있다. Collagen은전구체인 procollagen으로부터합성이시작되며, procollgagen은아미노말단과카복시말단에 propeptide라불리는 peptide sequence를가지고있다. Propeptide는소포체내에서 procollagen의 folding을도와줌과동시에 collagen 중합반응이일어날때 collagen 분자로부터분리된다 [3,11-12,22]. 따라서, collagen 생합성의양은 propeptide의양을측정함으로써그생합성효과를유추할수있다. Fig. 4의결과에서섬유아세포에주박을다양한농도로처리했을때, 농도의존적으로 procollagen의양이증가하는것을확인할수있었고, 비처리군의 procollagen의양을 100% 로했을때주박의최대농도처리군 Fig. 3. Effect of Korean rice wine cake on inhibition of elastase. Elastase inhibition assay was investigated in the mixture containing Tris-HCl (ph 8.0), elastase, substrate for elastase, and various concentrations of Korean rice wine cake. The activity was measured by microplate reader in absorbance at 410 nm. The values shown represent means±sem of three different assays. Fig. 4. Effect of Korean rice wine cake on collagen type I synthesis. HS27 cells were cultured in 96-well plates with DMEM medium containing 10% FBS and then the medium was replaced with serum free medium containing the various concentrations of Korean rice wine cake. After incubation for 24 hr, the supernatant was collected from each well and type I procollagen was determined by EIA kit. The values shown represent means±sem of three different assays. ** p<0.01 compared with absence of Korean rice wine cake. 인 500 μg/ml의농도에서 42.5% 증가함을확인할수있었다. 따라서, 주박은섬유아세포의 collagen 합성촉진작용을하고, 이러한결과는 Fig. 2에서보여졌듯이세포의증식효과가일어난것과어느정도연관성을가진다고판단된다. 주박에의한 MMP-1 유전자발현의억제 피부세포의결합조직을구성하는성분중 collagen은약 90% 정도를차지하는중요한구성단백질이다. Collagen은 trypsin 등의단백질분해효소에는영향을받지는않지만, collagenase의영향에의해분해된다 [9]. Collagen을분해하는주요효소중에는전사인자인 AP-1에의해유도되는 MMP-1 에의해일어나는것이일반적이다 [8-9]. MMP-1은 type I collagenase 로도불리며, 세포외기질을분해할뿐만아니라특이적으로 type I procollagen을분해함으로써 collagen의합성을억제하는분해인자로알려져있다 [7,10,14,20]. 그러므로, MMP-1 활성의억제는곧 collagen 분해를감소시켜피부탄력의유지및주름형성을억제할수있으리라판단되어 mrna level에서의 MMP-1 유전자의발현변화를측정하였다 (Fig. 5). 그결과, 섬유아세포에처리된주박의농도가증가할수록비처리군에비해 MMP-1의유전자발현이농도의존적으로억제되는것을확인할수있었다. 또한, 조직특이적억제자로알려진 TIMP는특이적으로 MMPs를불활성화시켜생물학적으로평형상태를유지시킨다고알려져있다 [1]. TIMP-1은 MMP-1의억제자로서 MMP-1의활성을조절하고
1842 생명과학회지 2010, Vol. 20. No. 12 감사의글 본논문은농촌진흥청농업과학기술개발공동연구사업 ( 과제번호 PJ006761201003) 의연구비지원에의하여연구되었으며, 이에감사드립니다. References Fig. 5. Effect of Korean rice wine cake on MMP-1 gene expression. HS27 cells were cultured in 100-mm dish were treated with various concentrations of Korean rice wine cake for 24 hr and then total RNA was isolated. MMP-1 and TIMP-1 mrna level were determined by RT-PCR. The data from triplicate experiments were quantified by densitometry. ** p<0.01 compared with absence of Korean rice wine cake. 내인성파괴시스템으로부터 collagen과 elastin을보호하는역할을하고있는것으로알려져있다 [17]. 그러나본연구결과에서 collagen 분해효소억제유전자로알려진 TIMP-1의유전자발현에는영향이없었다. 따라서, 주박은 collagen의직접적인분해요소인 MMP-1의발현을 mrna level에서억제함으로써, 피부주름의형성을억제하는것으로판단되고, 이는주박을이용한피부주름개선화장품의개발의가능성을제시한다. 그러나, 정확한주박의피부개선분자기작에대한연구는미흡하며, 앞으로구체적인관련연구가더진행되어야할것이다. 본연구는양조부산물인주박이피부의주름억제즉, 피부의항노화효과를알아보기위해진행하였다. 그결과주박은항산화활성효능에서약 40-43% radical 소거능을보임으로써항산화활성의효과를나타내었다. 또한, 주박은세포의증식및피부의구성인자인 collagen의합성을촉진하는것을알수있었다. 세포증식의경우주박의비처리군에비해약 26% 의증식율을보였고, collagen 합성의경우비처리군에비해약 42.5% 의증가를나타내었다. 마지막으로 RT-PCR을이용한 collagen의직접적인분해인자인 MMP-1의 mrna level에서의발현을측정했을때, 농도의존적억제의결과를확인할수있었다. 따라서, 본연구를통해서주박은피부의주름예방및완화할수있는효과를입증하였고, 주박을이용한피부주름개선을위한천연소재로서의가능성을입증하였다. 1. Berneburg, M., H. Plettenberg, and J. Krutmann. 2000. Photoaging of human skin. Photodermatol. Photoimmunol. Photomed. 16, 239-244. 2. Cheon, S. J., M. J. Jang, Y. A. Jang, E. Y. Choi, D. H. Jun, Y. H. Kim, W. A. Cho, Y. S. Jeong, H. B. Kwon, T. H. Kim, K. I. Choi, J. R. Do, C. E. Lee, and J. T. Lee. Anti-wrinkle Effect of Cambodian Phellinus Linteus Extracts. 2008. J. Life Sci. 18, 1718-1722. 3. Dumont, S., L. Cattuzzato, G. Trouvé, N. Chevrot, and C. Stoltz. 2010. Two new lipoaminoacids with complementary modes of action: new prospects to fight out against skin aging. Int. J. Cosmet. Sci. 32, 9-27. 4. Imokawa G. 2008. Recent advances in characterizing biological mechanisms underlying UV-induced wrinkles: a pivotal role of fibrobrast-derived elastase. Arch. Dermatol. Res. 300, 7-20. 5. Kang, T. H., H. M. Park, Y. B. Kim, H. Kim, N. Kim, J. H. Do, C. Kang, Y. Cho, and S. Y. Kim. 2009. Effects of red ginseng extract on UVB irradiation-induced skin aging in hairless mice. J. Ethnopharmacol. 123, 446-451. 6. Kim, E. J., M. K. Kim, X. J. Jin, J. H. Oh, J.E. Kim, and J. H. Chung. 2010. Skin aging and photoaging alter fatty acids composition, including 11,14,17-eicosatrienoic acid, in the epidermis of human skin. J. Korean Med. Sci. 25, 980-983. 7. Kim, H. H., S. Cho, S. Lee, K. H. Kim, K. H. Cho, H. C. Eun, and J. H. Chung. 2006. Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo. J. Lipid Res. 47, 921-30. 8. Kim, M. J., J. Y. Kim, T. K. Jung, S. W. Choi, and K. S. Yoon. 2006. Skin anti-aging effect of Forsythia viridissima L. extract. Korean J. Biotechnol. Bioeng. 21, 444-450. 9. Kim, S. H., H. J. Yong, C. Shin, and S. G. Ko. 2008. Research of traditional herbal medicines for anti-aging, inhibition effect of wrinkle and whitening effect in the skin. Korean J. Ori. Physiol. & Pathol. 22, 691-698. 10. Kim, S. Y., S. J. Kim, J. Y. Lee, W. G. Kim, W. S. Park, Y. C. Sim, and S. J. Lee. 2004. Protective effects of dietary soy isoflavones against UV-induced skin-aging in hairless mouse model. J. Am. Coll. Nutr. 23, 157-162. 11. Kim, Y. H., C. B. Chung, J. G. Kim, K. I. Ko, S. H. Park, J. H. Kim, S. Y. Eom, Y. S. Kim, Y. I. Hwang, and K. H. Kim. 2008. Anti-wrinkle activity of ziyuglycoside I isolated from a Sanguisorba officinalis root extract and its application as a cosmeceutical ingredient. Biosci. Biotechnol. Biochem. 303-311. 12. Kivirikko, K. I. and J. Myllyharju. 1998. Prolyl 4-hydrox-
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