J Korean Soc Food Sci Nutr 한국식품영양과학회지 43(), 695~7(24) http://dx.doi.org/.3746/jkfn.24.43..695 한방발효주주박추출물의미백및피부주름개선효과 이수민 * 이상진,2 * 권이영 백상훈 김종식 3 손호용 4 신우창 ( 주 ) 국순당부설연구소, 2 숙명여자대학교세포운명조절센터 3 안동대학교생명과학과, 4 안동대학교식품영양학과 Skin Whitening and Anti-Wrinkle Effects of Extract from Jubak of Oriental Herbal Liquor Su-min Lee *, Sang-Jin Lee,2*, Yi-Young Kwon, Sang-Hoon Baek, Jong-Sik Kim 3, Ho-Yong Sohn 4, and Woo-Chang Shin Research Laboratories, Kooksoondang Brewery Co., Ltd. 2 Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University 3 Department of Biological Sciences and 4 Department of Food and Nutrition, Andong National University ABSTRACT Oriental herbal liquor (Yakju) is a type of Korean traditional alcoholic beverage that uses Nuruk and oriental herbs for fermentation. The purpose of this study was to develop cosmetic ingredients using Jubak, which is a by-product of alcoholic fermentation of oriental herbal liquor. To investigate antioxidant, whitening, and anti-aging effects of Jubak, we prepared extract of Jubak and its solvent fractions. Ethyl acetate fraction () showed the most prominent free radical [,-diphenyl-2-picrylhydrazyl (DPPH)] scavenging activity (SC 5:.75 mg/ml). KSD E4-3 significantly inhibited in vitro mushroom tyrosinase activity (IC 5:.82 mg/ml) and reduced the melanin contents in mouse melanoma melanocyte, B6F cells. down-regulated protein expression of tyrosinase related proteins (TRP)-, -2, which play key roles in melanogenesis. For anti-aging effects, inhibition of matrix metalloproteinase (MMPs) expression was evaluated using human keratinocyte, HaCaT cells. Treatment of HaCaT cells with reduced expression of MMP-, -2, -9 and inhibited proteolytic activities of MMP-2, -9. These results suggest that induces down-regulation of cellular melanogenesis and protects against photoaging induced by UVB-induced damage. Thus could potentially be a valuable cosmetic ingredient. Key words: antioxidant, anti-melanogenesis, anti-wrinkle, oriental herbal liquor, Jubak 서 피부노화는그요인에따라내인적노화 (intrinsic aging) 와외인적노화 (extrinsic aging) 로구분할수있다. 외인적노화는외부스트레스에의해노화현상이나타나는것으로광노화가주된원인이다. 자외선을받게되면교원질 (collagen) 및탄력섬유 (elastin fiber) 등의기질단백질이손상되어피부내교원질의양이부족해지고탄력섬유가변성되어주름을유발하며기미, 주근깨및검버섯이증가하는현상을보인다 (,2). 자외선에피부가노출되면색소침착이증가되는데, 이는멜라닌세포가자외선흡수에의한손상을방어하기위한목적으로멜라닌을생성하기때문이다 (3). 피부미백과주름개선효능이있는다양한소재연구가활발하게 Received 9 June 24; Accepted 5 October 24 * These authors contributed equally to this work. Corresponding author: Woo-Chang Shin, Research Laboratories, Kooksoondang Brewery Co., Ltd., Seongnam, Gyeonggi 462-2, Korea E-mail: wcshin@ksdb.co.kr, Phone: +82-3-739-538 론 진행되고있으며특히웰빙트렌드확산으로안전하고우수한효능을함유한천연소재개발에초점을맞추고있다 (4, 5). 주박은쌀, 물, 누룩, 효모를이용한전통주발효과정을거쳐약주나탁주를거르고남은양조부산물로원료쌀의약 2% 정도로발생하며, 전분과단백질이외에도섬유소, 무기질, 비타민, 알코올과유기산, 효소, 효모등의영양성분과유용생리활성성분을다량함유하고있는것으로보고되어있다 (6,7). 실제로쌀을주원료로사용하는탁주주박의경우식량이부족했던시절에대체식량으로이용된바있으며, 최근에는화장품소재로도각광받고있다. 그러나현재국내에서의주박이용은주박내알코올을이용한식초제조, 향미성분을이용한야채절임류제조및일부친환경비료와동물사료에한정되어있고, 대부분은폐기처리되고있는실정이다. 수백년동안동양에서사용되었던한약재의발효추출물은대부분일정수준이상효능과안전성이인정된소재이다 (8,9). 그러한이유로천연물, 특히한방발효추출물을이용
696 이수민 이상진 권이영 백상훈 김종식 손호용 신우창 한화장품은꾸준히인기를끌고있다. 우리나라대표발효주인막걸리는피부생리활성효능이확인되어산업적으로이용되고있으나 (,), 한약재를함께적용한한방발효주에대한체계적인연구는현재까지는미비한실정이다. 미백, 항산화, 항노화효과가있는한약재를이용하여발효주를만들면기존의발효주보다피부의기능활성에도움을주는생리활성물질이풍부할것으로예상되며, 본연구에서는 2가지한약재가첨가된전통한방발효주제조과정에서생기는부산물인주박을이용하여추출물및분획물을제조하고, 이를화장품소재로활용하기위하여피부미백및주름에미치는영향을평가하였다. 재료및방법시약 Fetal bovine serum(fbs), Dulbecco's modified Eagle medium(dmem), penicillin, streptomycin, trypsin-edta 는 Gibco-BRL(Grand Island, NY, USA) 에서구입하였다. Dimethyl sulfoxide(dmso), mushroom tyrosinase, L- tyrosine, α-melanocyte stimulating hormone(α-msh),,-diphenyl-2-picrylhydrazyl(dpph) 은 Sigma-Aldrich Co.(St. Louis, MO, USA) 에서구입하여사용하였다. Matrix metalloproteinase(mmp)-, -2, -9, anti-mouse horseradish peroxidase(hrp) conjugated secondary antibody와 anti-rabbit HRP conjugated secondary antibody 는 Cell Signaling Technology(Beverly, MA, USA) 에서, tyrosine related protein(trp)-, -2는 Abcam(Cambridge, UK) 에서구매하였다. Polyvinylidene fluoride (PVDF) membrane은 Millipore(Billerica, MA, USA) 에서, enhanced chemiluminescence detection kit는 Amersham Bioscience(Pittsburg, PA, USA) 에서구입하였다. 시료제조본연구에는자사 ( 국순당 ) 에서생산하여시중에유통중인전통한방발효주생산과정에서발생하는주박을이용하여실험하였다. 이전통한방발효주는분쇄한쌀에누룩과효모, 2가지 ( 구기자, 감초, 오미자, 하수오, 홍삼, 산수유, 맥문동, 산사육, 진피, 고수, 건강, 수국 ) 한약재를첨가하여발효과정을거친한방발효주이다. 전통한방발효주주박에 95% 에탄올을가하여 3일간교반후 차추출하여 KSD E4- 샘플을준비하고, 이를 n-헥산으로분획하여 KSD E4-2 샘플을준비하며, 다시에틸아세테이트로분획하여 샘플을준비하였다. 각샘플을여과지 (Filter paper No.2, Whatman, Piscataway, NJ, USA) 로거른후 55 C에서감압건조하여전통한방발효주주박의유기용매분획물을준비하였다. 시료는 DMSO에적당한농도로녹여활성평가에사용하였다. 세포및배양방법 Mouse melanoma melanocytes(b6f cells), human keratinocytes(hacat cells) 세포주는 % FBS, U/ ml penicillin, mg/ml streptomycin을첨가한 DMEM 배지를사용하였다. 모든세포주는 37 C, 5% CO 2 를유지하며배양하였다. DPPH 라디칼소거능항산화활성은 DPPH를이용하여시료의라디칼소거효과를측정하는 Blois법 (2) 을활용하였다. 추출물.9 ml에동일한양의. mm DPPH 용액을가한후상온에서 3분간반응시켜 56 nm에서흡광도를측정하였다. Ascorbic acid는 positive control로사용하였고추출물이라디칼 5% 를소거하는농도를 SC 5 으로표현하였다. 각시료의라디칼소거능은아래의식에의해계산하여백분율로나타내었다. DPPH 라디칼소거능 (%) =(- 시료의흡광도 ) 대조군의흡광도 In vitro tyrosinase 저해활성평가 Tyrosinase 저해활성측정은 No 등 (3) 의방법을변형하여측정하였다. 반응구는. M phosphate buffer(ph 6.5) 와 mm L-tyrosine을녹인기질액 4 ml, 시료용액 ml의혼합액에 mushroom tyrosinase 2 ml를첨가하여 37 C에서 2분간반응시킨후 49 nm에서흡광도를측정하였다. Kojic acid는 positive control로사용하였으며 tyrosinase 저해활성 (%) 은아래식을이용하여계산하였다. Tyrosianse 저해활성 (%) 멜라닌생성억제능평가 =(- 시료의흡광도 ) 대조군의흡광도 B6F cells을.5 5 cells/ml로 6-well plate에분주하고 % FBS가함유된 DMEM 배지에서 24시간배양하였다. 을 α-msh ng/ml가포함된 DMEM 배지에첨가하여 48시간배양하였다. 배지를모두제거하고 trypsin을처리하여세포를회수한후회수한세포에 N NaOH(% DMSO 포함 ) 를첨가하여 6 C에서 3분방치하여멜라닌을녹였다. 이용액을 microplate reader(molecular Devies, Silicon Valley, CA, USA) 로 45 nm에서흡광도를측정하여멜라닌함량을구하였다. Western blot anaylysis UVB 2 mj/cm 2 를조사하고 을처리하여 24 시간을배양하였다. 세포를회수한후 lysis buffer를첨가하여얻은단백질을 Bradford법 (4) 을이용하여정량하였다. 단백질 2 μg을 % SDS-PAGE에전기영동을수행하고 gel의단백질을 PVDF membrane으로이동시킨후 5% non-fat milk로상온에서 시간동안 blocking 하고 pri-
한방발효주주박추출물의미백및피부주름개선효과 697 mary antibody(mmp-, -2, -9, TRP-, -2) 를가하여 4 C에서 overnight 시켰다. 마지막으로 PVDF membrane을.% TBST로세척하고 secondary antibody conjugated- HRP를희석하여상온에서 시간동안교반하고 enhanced chemiluminescence detection kit를사용하여단백질을확인하였다. Zymography HaCaT를 6 cells/ml가되도록 6 mm cell culture dish에분주하여 24시간배양후 UVB 2 mj/cm 2 를조사하고 을처리하여 24시간을배양하였다. 배지를수거하여 3, rpm에서 3분원심분리를수행하고 Bradford 법을이용하여단백질의양을정량하여 % SDS- PAGE( mg/ml gelatin 함유 ) 에서전기영동수행후,.% triton-x로 3분씩 2번세척한다음 developing buffer를넣어 3 C에서 8시간동안반응시키고 Coomassie Blue 로염색을하여관찰하였다. 통계학적분석모든실험은 3회반복실험을실시하였으며각시료들의실험결과는평균 ± 표준편차로나타내었고, 각실험결과의통계적유의성검토는시료가포함되지않은대조구와비교하여 Student's t-test에의하여판정하였으며 P 값이.5 미만일때유의성이있다고판단하였다. A DPPH scavening activity (%). B DPPH scavening activity (%).. 9. 8. 7. 6. 5. 4. 3. 2... 2 8 6 4 2-2 5 mg/ml KSD E4- KSD E4-2 Ascorbic acid. mg/ml. mg/ml.5 mg/ml mg/ml Fig.. Anti-oxidant activity of extract from Jubak of oriental herbal liquor in DPPH. (A) KSD E4-: ethanol extract of Jubak of oriental herbal liquor, KSD E4-2: n-hexane fraction of KSD E4-, : ethylacetate fraction. (B) DPPH radical scavenging activity of. 결과및고찰한방발효주주박추출물의항산화효과활성산소종은피부노화의원인으로인체내지질, 단백질등과반응하여생체의노화를촉진시킨다. DPPH radical 소거법은항산화물질의전자공여능을이용한항산화측정법으로이방법을이용하여전통한방발효주주박분획물의항산화효과를살펴보았다. 3개의한방발효주주박분획물은항산화력을나타냈으며그중에틸아세테이트분획물인 은 5 mg/ml에서 95.5%, mg/ml에서 67.3% (SC 5:.75 mg/ml) 의우수한항산화활성을나타내었다. 이때 positive control로사용한 ascorbic acid는. mg/ ml에서 96.8%(SC 5:.5 mg/ml) 의항산화활성을나타내었다 (Fig. A, B). 이것은 Cho 등 (5) 이막걸리침전물로부터의열수추출물이 mg/ml 농도에서 48% 의 DPPH 라디칼소거능을보인다고보고한것보다우수한효과이다. 효모, 곰팡이, 유산균등유익한미생물에의한발효공정은천연물의독성을낮추거나새로운물질을생성또는유효물질을전환하여생리활성을증가시켜이를통한기능성향상연구가시도되고있다 (6). 따라서 Ahn 등 (7) 의연구결과와같이발효및추출에의해기존천연물이가지고있는유효성분및생리활성물질의수율이극대화또는새로생성되어이러한효과가나타나는것이라생각되며, 이후 실험에서는항산화효과가높은 을이용하여실험하였다. 한방발효주주박추출물의피부미백효과멜라닌생합성에관여하는효소로 tyrosinase, TPR-, TRP-2가잘알려져있다. Tyrosinase는피부기저층에있는 melanocyte의 melanosome에서 L-tyrosine과 3,4- dihydroxyphenylalanine(dopa) 을기질로하여피부의색소성분인 melanin을생합성하는핵심효소이다 (8). 또한 TRP-은 5,6-dihydroxyindole-2-carboxylic acid(dhica) 를 indole-5,6-quinone-2-carboxylic acid로산화시키고, TRP-2는 dopachrome을 DHICA로이성화시켜멜라닌형성에관여한다 (9). 기질인 L-tyrosine과효소인 mushroom tyrosinase에의해산화되어생성되는멜라닌의흡광도측정을통해 의 tyrosinase 저해활성을알아본결과, 은농도의존적으로 tyrosinase 저해활성을나타내었으며 mg/ml에서 65.3% 의저해력을나타내었다 (Fig. 2). 멜라닌생성세포인멜라노사이트를이용하여 이세포에서멜라닌형성을저해하는지살펴보았다. B6F cells에 ɑ-msh와 을처리한후세포내멜라닌양을측정한결과, 은농도의존적으로 melanin 합성을저해하였으며 2 mg/ml에서 3.3%, mg/ml에서 25.8% 의 melanin 합성저해력을
698 이수민 이상진 권이영 백상훈 김종식 손호용 신우창 Tyrosinase inhibitory activity (%). 2 8 6 4 2..5 Concentration (mg/ml) Kojic acid. Fig. 2. Inhibitory effect of against tyrosinase activity. 나타내었다 (Fig. 3A). 이러한결과가멜라닌생성에관련된단백질발현과도연관성이있는지를확인하기위해 TRP-, TRP-2의항체를이용한 western blot을실시하였다. KSD E4-3은 TRP-과 TRP-2의단백질발현을저해하였다 (Fig. 3B). 선행연구에서미백효과가우수한 4가지약재 ( 감초, 구기자, 상백피및삼백초 ) 를첨가한한방이화주 ( 한방막걸리 ) 는일반이화막걸리보다미백효과가더우수함을확인한바있다 (2). 또한실험에사용한한방발효주의약재에는구기자, 감초등미백, 항산화효과가우수한약재들이포함되어있다 (2-25). 따라서 의 tyrosinase 활성저해와 TRP-과 TRP-2의발현저해를통한 melanin 생합성저해효과는약재에서유래된다양한유효성분이발효에의해증강된효과라사료된다. 한방발효주주박추출물의주름개선효과노화에따른진피의조직학적변화로는진피의감소를들 수있으며진피의주요구성물질인교원질의결핍이주름살형성의주요원인으로작용한다. 광노화된피부에서는교원질을분해하는 MMP-, -2, -9와같은 MMPs의발현증가로교원질의양이감소되어주름이형성된다 (27). KSD E4-3이 UV에의해발현이증가되는 MMP-에미치는영향을알아보기위하여 western blot analysis를수행하였다. 그결과 은농도의존적으로 MMP-의발현저해효과를나타내었다 (Fig. 4A). 또 UV 조사하에서 KSD E4-3이 keratinocytes인 HaCaT 세포주가분비하는 MMP-2 와 MMP-9에미치는영향을 zymography로평가하였다. 은 MMP-2와 MMP-9의효소적활성을저해하였으며 (Fig. 4B), western blot analysis를통해세포내단백질발현에미치는영향을평가한결과, 은 MMP-2와 MMP-9의단백질발현을농도의존적으로저해하였다 (Fig. 4C). 은노화가진행되면서피부탄력을유지하는구성단백질파괴에관여하는 MMPs의효소적활성과발현을저해하는것을확인하였다. 이는 KSD E4-3이피부탄력을유지하여노화에따른주름생성을억제할수있는가능성이높을것으로사료된다. 요약본연구에서는한방발효주주박의피부생리기능활성을알아보기위해주박추출물및분획물을준비하고항산화, 미백및주름개선효과를조사하였다. 그중한방발효주주박의에틸아세테이트분획물 () 이가장우수한항산화효과를나타내었으며이는다양한약재의발효에의한효과로사료된다. 은 tyrosinase 활성억제 A B α-msh ( nm) - + + + + + (μg/ml) 5 2 TRP- 4 TRP-2 Melanin contents (%). 2 8 6 4 2 * * * ** Control α-msh 5 2 (µg/ml) + α-msh.8.6.4.2 TRP- TRP-2 Control α-msh 5 2 (µg/ml) + α-msh Fig. 3. Inhibitory effects of on melanin biosynthesis. (A) Melanin contents were determined in B6F cells treated with or α-msh for 48 hr. (B) B6F cells were treated with or α-msh for 48 hr. After immunoblotting, the expression levels of TRP-, -2 were identified by their antibodies. Data represent mean±sd of three independent observations performed in triplicate. * P<.5 and ** P<. compared to α-msh.
한방발효주주박추출물의미백및피부주름개선효과 699 A UV (2 mj/cm 2 ) - + + + + (μg/ml) 5 MMP- B UV (2 mj/cm 2 ) - + + + + (μg/ml) 5 MMP-9 MMP-2.8.6.4.2 UV - 5.8.6.4.2 MMP-9 MMP-2 UV + (µg/ml) C UV (2 mj/cm 2 ) - + + + + (μg/ml) 5 UV - 5 UV + (µg/ml) MMP-9 MMP-2 MMP-9 MMP-2.8.6.4.2 UV - 5 Fig. 4. Inhibitory effect of on MMPs. HaCaT cells were treated with, followed by exposure to 2 mj/cm 2 of UV. (A) After immunoblotting, the expression levels of MMP- were identified by its antibodies. (B) Gelatin zymography was performed for determination of MMP-2, -9 activities of. (C) After immunoblotting, the expression levels of MMP-2, -9 were identified by their antibodies. UV + (µg/ml) 및 tyrosine을기질로 melanin이형성되는 pathway에관여하는주요한인자인 TRP-과 TRP-2를저해하는작용기전을통해피부색소침착의주요원인물질인 melanin 생합성을농도의존적으로저해하는것을확인하였다. 또한 KSD E4-3은피부의 keratinocyte가생성, 분비하는 MMP-, -2, -9의발현을억제하여 UV 조사에의한피부광노화에따른피부주름생성을억제할수있음을확인하였다. 이상의결과를종합하면한방발효주주박에서추출한 KSD E4-3은피부미백및주름개선에우수한효과를나타내고있으므로기능성화장품의주요한소재로이용가치가높을것으로사료된다. 감사의글본연구는 22년도농림수산식품부고부가가치식품기술개발사업 ( 과제번호 273-3) 에의해수행되었으며, 이에 감사드립니다. REFERENCES. Gilchrest BA. 989. Skin aging and photoaging: an overview. J Am Acad Dermatol 2: 6-63. 2. Kang KS, Kim ID, Kwon RH, Heo YY, Oh SH, Kim MA, Jung HJ, Kang HY, Ha BJ. 27. The evaluation of anti-wrinkle effects in oriental herb extract. J Life Sci 7: 47-5. 3. Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH. 26. Cosmetic applications of selected traditional Chinese herbal medicines. J Ethnopharmacol 6: 353-359. 4. Lee BG, Kim JH, Ham SG, Lee CE. 24. Study on biological activities of extracts for cosmeceutical development from Lagerstroemia indica L. branch. Korean J Plant Res 27: 29-34. 5. Kim DS, Kim DH, Oh MJ, Lee KG, Kook MC, Park CS. 2. Antiaging and whitening activities of ethanol extract
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