RESEARCH ARTICLE B16F10 mouse melanoma 세포에서삼채 (Allium hookeri) 추출물의멜라닌형성억제효과에관한연구 최연선, 최태부 * 건국대학교공과대학생물공학과 Melanogenesis Inhibitory Effects of Allium hookeri Extract in B16F10 Mouse Melanoma Cell Yoensun Choe, Tae-Boo Choe* Department of Bioengineering, Konkuk University Abstract The effect of Allium hookeri extracts on the melanogenesis in mouse melanoma B16F10 was studied as a raw material of functional cosmetics. Allium hookeri has been associated with the traditional recipes of meiteis and also with the ethnotherapy of certain diseases by local herlers since the time immemorial, the plant has been regarded as ethnobotanic relevance to the Manipuris. The effect of Allium hookeri extracts has found that the secreted melanin in culture medium was reduced by 50% at the concentration of 0.064%. At this concentration, tyrosinase activity was reduced by 20%. On the other hand, Tyrosinase expression level was reduced by 93% at the same concentration comparing to the case of positive control. TRP1 and TRP2 expression levels were also reduced as much as 70%. The expression levels of the enzymes necessary for the melanogenesis were confirmed by western blotting. This is the first study about melanogenesis inhibitory effect of Allium hookeri extracts. However, it is not yet clear which compound is effective in anti-melanogenesis. Further studies are necessary to elucidate the detail mechanism. Keywords: Melanogenesis, Allium hookeri, Tyrosinase, Whitening, Functional cosmetics 동서고금을막론하고미백은아름다움의필수요소로여겨진 다. 미백기능성화장품에대한수요도끊임없이증가하는추세에있고그기능성소재에대한연구도활발히진행되고있다 (Solano et al., 2006). 현재식약청고시제2009-166호에고시된미백기능성화장품의주성분은닥나무추출물, 알부틴, 에칠아스코빌에텔, 유용성감초추출물, 아스코빌글루코사이드, 마그네슘아스코빌포스페이트, 아스코빌테트라이소팔미테이트의 9종이있다. 주목할것은최근의미백소재개발은주로미생물유래성분이나약용식물추출물등을대상으로진행 *Corresponding author: Tae-Boo Choe, Department of Bioengineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Republic of Korea Tel.: +82 2 450 3523, Fax: +82 2 3436 5594 E-mail: tbchoe@konkuk.ac.kr Received September 27, 2013; Revised March 13, 2014; Accepted April 22, 2014; Published April 30, 2014 되고있다는것이다 (Lee et al., 2012; Matsuda et al., 2011; Momtaz et al., 2008; Ryu et al., 2008). 미백기능성소재에대한연구가미생물과약용식물로대변되는천연물을그대상으로삼는이유는기존의기능성소재들이세포독성이나낮은활성등으로시장에서사라지거나기능성에의심을받고있기때문이다 (Brtko et al., 2004). 삼채 (Allium hookeri, A. hookeri) 는 Alliaceae과에속하는약용실물로서뿌리와맛이인삼을닮았다하여삼채 ( 蔘菜 ), 혹은쓴맛, 매운맛, 단맛 3가지맛이난다하여삼채 ( 三菜 ) 라고도불린다. 현재는국내에서도재배및판매가이루어지고있으며원산지는실론 (Ceylon), 그리스 (Greece), 윈난 (Yunnan), 남중국 (Southern China), 부탄 (Bhutan), 스리랑카 (Sri Lanka), 인도 (India) 지역이다 (Sharma et al., 2011). 인도에서는삼채특유의향과맛그리고치료효과때문에향신료로사용되기도하고감기나상처, 화상의치료에사용하기도한다 (Kala 2005). 식품으로서의삼채는뿌리와줄기, 잎을모두사용한다. 그러나삼채의효능에대한연구는같은과에속하는양파나마늘등에비해거의이루어지지않은상태이다. 국내에서는 2012년삼채 www.kosac.or.kr 163
의에탄올추출물의항염증효과에대한연구결과가발표되었다 ( 배기춘외, 2012). 식품의약품안전처홈페이지에는삼채의주성분을유황화합물 (sulphur compounds) 로소개하고있다. 삼채의주요성분중유황성분은마늘과비교해약 6배정도많은것으로알려져있다. 삼채는현재식용으로대부분소비되고있으며약리적기능성에대해서는연구된바가거의없다. 재배되는지역또한인도와미얀마의접경지대, 남중국일부로제한되어있고같은과의양파나마늘, 차이브에비해알려진바가거의없다 (Pandey et al., 2008; Kyung 2011; Saleheen et al., 2004). 2012년에국내연구진에의해항염효과가밝혀진바있고중국에서항균효과와항산화효과에대한연구가발표된바있다 (Lu 2009). 그외에삼채의약리적혹은기능성에대한연구는거의이루어지지않은상태이며특히기능성화장품원료로서의연구는전무하다. 천연물유래의기능성화장품원료에대한소비자의요구가높아지는가운데삼채와같은약용식물들에대한연구가국내외에서활발하게진행되고있다 ( 김은주외, 2013; 홍미선외, 2012; 한정선외, 2012). 특히삼채의경우는최근에국내에소개되었고전세계적으로그기능성에대한연구가거의전무한상태이다. 본연구에서는삼채의줄기와잎의에탄올추출물의미백기능성을밝혀새로운천연물기능성화장품소재로서의가능성을밝히고자하였다. 생존율을계산하였다. 3. 멜라닌함량측정세포배양용 6well plate에 B16F10 세포를 1 10 4 cells/well 농도로분주하고 24시간후에 200 nm α-msh와삼채추출물을농도별로처리하였다. 물질처리후 72시간후에멜라닌이생성되는것을확인하고세포를수확하였다. 수확한세포를원심분리하여상등액은버리고 cell pellet에 1 N NaOH를 200 µl 처리한후 100 에서 10분간끓이고얼음을이용하여식힌다음 405 nm에서흡광도를측정하였다 (Virador et al., 1999). 멜라닌함량은무처리군과비교하여백분율로표시하였다. 4. Tyrosinase 활성측정 Tyrosinase 활성은 DOPA oxidase 활성을측정한것으로나타내었다. 3번과같은방법으로세포를배양하고 lysis buffer 100 µl를첨가하여 shaking 하고세포를회수하여 4 에서 overnight하여세포를용해시켰다. Cell lysate를 20 µl 취하여 96well plate에넣고 0.1 M sodium phosphate buffer (ph 7.0) 에 2 mg/ml 농도로녹인 L-DOPA 용액을 80 µl 가하여 37 에 2시간동안방치하였다. 반응이끝난후 tyrosinase에의해 DOPA로부터형성되는 dopachrome의양을 490 nm에서측정하여상대적 tyrosinase 활성으로나타내었다. 1. 실험재료및세포배양실험에사용된 α-melanocyte stimulating hormone (α -MSH), L-3,4-dihydroxyphenylalanin (L-DOPA) 는 Sigma(USA) 사에서구입하였다. 삼채추출물은삼채의입과줄기를 70% 에탄올을이용하여추출한후증발시켜얻은분말을증류수에녹여사용하였다. B16F10 세포는한국세포주은행으로부터분양받았으며 10% fetal bovine serum (FBS, Gibco BRL, USA), 0.22% sodium bicarbonate를첨가한 Dulbecco`s modified Eagle`smedium (DMEM, Sigma, USA) 을기본배지로하여 37, 5% CO 2 습윤배양기에서배양하였다. Antiactin antibody는 Sigma (USA) 사, tyrosinase, TRP1, TRP2 는 Santacruz (USA) 사에서구입하여사용하였다. 2. 세포독성측정삼채추출물의세포독성측정과실험에사용될농도범위를결정하기위해 6 well plate에세포를 1 10 4 cells/well 농도로분주하고 24시간후에삼채추출물을농도별로처리하였다. 물질처리 72시간후에세포를회수하여계수하여세포농도와 5. Western blotting 삼채추출물에의한 tyrosinase와 TRP1, TRP2의세포내발현량변화를확인하기위해 western blotting을실시하였다. 6 well plate 에서세포를배양하고 trypsin (Sigma, USA) 을이용하여세포를수확하였다. 수확한세포는 PBS로수세한후원심분리하여상등액을제거하고 lysis buffer (150 mm NaCl, 50 mm Tris-Cl (ph7.4), 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, protease inhibitor cocktail (Roche) 를첨가해얼음에두고 5분마다 vortex하면서 30분간반응시켰다. 반응액을 4, 12,000 rpm에서 30분간원심분리하여상등액만을취하였다. 상등액은 Bradford solution (Bio-Rad, USA) 을이용하여단백질정량하였다. 이로부터동량의단백질을취하여 sample buffer (12 mm Tris-Cl (ph6.8), 5% glycerol, 0.4% SDS, 7 mm 2-β-mercaptoethanol, 0.02% bromophenol blue) 와혼합한후 100 에서 5분간끓여전기영동샘플을준비하였다. 전기영동은 12% SDS-PAGE gel을이용하여실시하였다. 전기영동이끝난단백질은 cold transfer buffer (25 mm Tirs, 192 mm glycine, 20% methanol) 을이용하여 100 V로한시간동안 transfer 하여 nitro cellulose membrane (Protran Nitro-cellulose membrane, UK) 으로이동시켰다. 이동이끝난 membrane은 blocking solution (5% skim milk 164 www.kosac.or.kr
삼채추출물의멜라닌형성억제효과 6.00 120.0 Viable cell density (1E6 c/ml) 5.00 4.00 3.00 2.00 1.00 0.00 0.128% 0.064% 0.032% CTL Concentration of A. hookeri Figure 1. Cytotoxicity of A. hookeri extracts on B16F10. Relative melanin contents (%) 100.0 80.0 60.0 40.0 20.0 0.0 0.064% 0.032% (+)Ctl Concentration of A. hookeri in TBS-T (10 mm Tirs-Cl, ph 7.5), 150 mm NaCl, 0.05% Tween 20) 에넣고한시간동안교반시켰다. Blocking이끝난 membrane은 primary antibody가첨가된용액 (5% skim milk in TBS-T) 에담가 4 에서 18시간동안교반하였다. Primary antibody 처리후 membrane을 TBS-T로 4회수세한후 secondary antibody가첨가된용액에담가상온에서 2 시간동안반응시켰다. 반응후 TBS-T로 4회수세하고 ECL (Electrochemiluminescence) solution (Super signal west pico solution, Pierce, USA) 을처리한다음 Las 3000 (Fuji, Japan) 에감광하여분석하였다. Figure 2. The effect of A. hookeri extracts on melano-genesis of B16F10. Relative tyrisinase activyty (%) 120.0 100.0 80.0 60.0 40.0 20.0 0.0 0.064% 0.032% (+)Ctl Concentration of A. hookeri 1. 삼채추출물의세포독성삼채추출물을 0.128%, 0.064%, 0.032% 로처리한결과 0.128% 로처리한실험군에서대조군대비세포의증식이약 46% 정도억제되는것을확인하였고 0.064% 부터는대조군과비교하여세포의증식에영향이없는것을확인하였다 (Figure 1). 따라서실험은이농도범위내에서진행하였다. 2. 삼채추출물에의한멜라닌함량변화삼채추출물에의한 B16F10세포의멜라닌함량변화는 Figure 2와같다. 삼채추출물을처리한경우 0.064% 에서멜라닌함량이최대 50% 이상감소하는것을확인하였고삼채추출물의농도가높을수록멜라닌의함량도낮아지는것을확인할수있었다. B16F10 세포의멜라닌함량조절은세포내멜라노좀이형성되는단계에서첫번째로시작되고두번째로 tyrosinase, TRP1, TRP2와같은멜라닌합성에관여하는효소의활성과발현량에좌우된다 (Ito, 2003; Nelson et al., 2009; Brenner et al., 2008). 삼채추출물의경우에도위 2가지경 Figure 3. The effect of A. hookeri extracts on the tyrosi-nase activity of B16F10. 로를통한멜라닌합성억제기전을통할것으로생각되며이를확인하기위해 tyrosinase의활성변화를측정하고 western blotting을통해 tyrosinase와 TRP1, TRP2의발현변화를확인하였다. 3. 삼채추출물에의한 tyrosinase 의활성변화 Tyrosinase 는멜라닌합성에가장핵심적인역할을하는단 백질로서멜라닌합성억제기전이가장많이밝혀져있기도하다 (Ando et al., 2007; Chang 2009). Tyrosinase는멜라닌합성의출발물질인 tyrosine을 DOPA-quinone으로전환한다. 때문에 tyrosinase의활성이올라가면멜라닌합성이증가하고반대로활성이감소하면멜라닌의양도감소한다. 삼채추출물에따른 tyrosinase의활성변화는 Figure 3과같다. 0.064% 처리군에서대조군대비약 77% 의활성을보였고 0.032% 처리군 www.kosac.or.kr 165
A A. hookeri (% ) 0 0 0.032 0.064 α-msh (200 nm) - + + + TYR TRP1 TRP2 β-actin B Relative band intensity (β-actin normalized, % of control) 120 100 80 60 40 20 0 α-msh A. hookeri - + + + - - 0.03% 0.06% C Relative band intensity (β-actin normalized, % of control) 160 140 120 100 80 60 40 20 0 α-msh A. hookeri - + + + - - 0.03% 0.06% D Relative band intensity (β-actin normalized, % of control) 140 120 100 80 60 40 20 0 α-msh A. hookeri - + + + - - 0.03% 0.06% Figure 4. The effect of A. hookeri extracts on tyrosinase, TRP1 and TRP2 expression level of B16F10. A: western blotting, B: tyrosinase, C: TRP1, D: TRP2 Table 1. Melanogenesis inhibitory effects and mechanisms of extracts from various plants Name Compound Mechanism Reference A. hookeri EtOH extracts Allium cepa (Onion) Quercetin - Tyrosinase activity inhibition - Reduce expression level of tyrosinase, TRP-1 and TRP-2 - Tyrosinase activity inhibition - Reduce expression level of TRP-1 and TRP-2 N/A Yang et al., 2011 Sapindus mukorossi MeOH extracts EA extracts - Tyrosinase activity inhibition Chen et al., 2010 Anastatica hierochuntica MeOH extracts - Tyrosinase activity inhibition Nakashima et al., 2010 Broussonetia kazinoki 1,3-diphenylpropanes - Tyrosinase activity inhibition Baek et al., 2009 MeOH: Methanol, EtOH: Ethanol, EA: Ethyl acetate 에서는약 82% 의활성을보였다. 멜라닌함량의변화와마찬가지로농도가높을수록활성의감소가컸다. 4. 삼채추출물에의한 tyrosinase와 TRP1, TRP2의발현량변화멜라닌의합성이줄어드는이유로 tyrosinase의활성감소와함께발현량의변화도확인하고 tyrosinase와함께멜라닌합성에관여하는 TRP1과 TRP2의발현량도확인하기위해 western blotting을실시하였다. Western blotting 결과 tyrosinase의활성이떨어지는것과같은패턴으로발현도감소하는것을확인하였고 TRP1과 TRP2역시 tyrosinase와같은패턴으로감소하는것을확인하였다 (Figure 4). 3가지단백질가운데 tyrosinase의발현이약 93% 정도감소하였으며 TRP1, TRP2도약 70% 이상감소하는것을확인하였다. 멜라닌의함량변화, tyrosinase 활성변화와같은패턴으로 3가지단백질모두처리한삼채추출물의농도가높을수록발현량감소의정도가컸다. 이로써삼채추출물에의한멜라닌합성억제는 166 www.kosac.or.kr
삼채추출물의멜라닌형성억제효과 tyrosinase의활성과발현의억제, TRP1과 TRP2의발현억제에의한것임을확인하였다. 본연구에서는미백기능성화장품의천연원료로서삼채추출물의가능성을확인하고자삼채추출물이 B16F10세포의멜라닌합성이미치는영향에대해분석하였다. 삼채추출물을각각 0.064%, 0.032% 처리하여전체멜라닌의함량변화를측정하고 tyrosinase의활성변화, tyrosinase와 TRP1, TRP2의발현량변화를측정하였다. 삼채추출물을처리한결과전체멜라닌함량은약 50% 까지감소하였고 (Figure 2) tyrosinase의활성도약 23% 감소하였다 (Figure 3). Western blotting결과 tyrosinase와 TRP1, TRP2 모두발현이감소하였으며특히 tyrosinase 는약 93% 이상감소하는것을확인하였다 (Figure 4). 삼채추출물에의한멜라닌합성억제기전은우선 tyrosinase 의활성감소와발현감소그리고 TRP1, TRP2의발현량감소에의한것으로확인되었다. Tyrosinase 와 TRP1, TRP2가관여하는멜라닌합성 pathway에는 MITF (Microphthalmia-associated transcription factor) 라는 leucine zipper 구조의전사인자가중심에있다. MITF 는 melanocyte의증식과분화에모두관여하며 tyrosinase와 TRP1, TRP2의발현을높이거나낮추는역할을한다 (Englaro et al., 1995; Goding 2000; Cervantes et al., 2001). 삼채추출물이 tyrosinase와 TRP1, TRP2의발현과활성을억제하는방식으로멜라닌의합성을억제한다면 MIFT를중심으로한기전일것으로판단된다. 이와유사하게삼채와가장가까운양파 (Allium cepa) 에서추출한 quercetin은 tyrosinase 의 monophenolase와 diphenolase 활성을억제하고 TRP-1 과 TRP-2의발현을억제해서멜라닌합성을저해한다는보고가있다 (Yang et al., 2011; Arung et al., 2011). 그외삼채와유사한형태로멜라닌합성을억제하는식물로무환자나무 (Sapindus mukorossi), 여리고장미 (Anastatica hierochuntica), 닥나무 (Broussonetia kazinoki) 등이있으며 methanol이나 ethyl acetate를이용한추출물에서 tyrosinase 의 monophenolase 혹은 diphenolase활성이나 mrna 발현을억제하는방식으로멜라닌합성을저해한다 (Table 1). 이러한식물추출물들의경우멜라닌합성에효과를보이는단일물질을규명한경우가대부분이지만삼채의경우식물자체에대한연구가거의전무한상태이며특히나멜라닌합성억제에관한연구는국내외에서시도된바가없다. 따라서어떤성분이직접적인효능이있는지, 구체적으로어떤경로를통하는지등은추가적인연구를통해밝혀야할것이다. 김은주, 문지선, 최태부. 상백피추출물의생물전환과티로시나제저해효과의비교. 대한피부미용학회지, 11: 845-854, 2013. 배기춘, 배대열. 국내산삼채에탄올추출물의항염증효과. 대한본초학회, 27: 55-61, 2012. 한정선, 이동희. 솔잎발효액의항산화활성멜라닌생성억제효과. 대한피부미용학회지, 10: 619-624, 2012. 홍미선, 윤영민, 안성관, 안인숙, 변부형. 인간진피섬유아세포에서천연성분쿼세틴의작용기전. 대한피부미용학회지, 10: 571-579, 2012. Ando H, Kondoh H, Ichihashi M, Hearing VH. Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase, J. Invest. Dermatol., 127: 751-761, 2007. Arung ET, Furuta S, Ishikawa H, Kusuma W, Shimizu K, Kondo R. Anti-melanogenesis properties of quercetin- and its derivative-rich extract form Allium cepa. Food Chemistry, 124: 1024-1028, 2011. Baek YS, Ryu YB, Curtis-Long MJ, Ha TJ, Rengasamy R, Yang MS, Park KH. Tyrosinase inhibitory effects of 1,3-dipheylpropanes from Broussonetia kazinoki. Bioorg. Med. Chem., 17: 35-41, 2009. Brenner M, Hearing VJ. Modifying skin pigmentationapproaches through intrinsic biochemistry and exogeneous agents. Drug. Discov. Today. Dis. Mech., 5: 189-199, 2008. Brtko J, Rondahl L, Fickova M, Hudecova D, Eybl V, Uher M. Kojic acid and its derivatives : history and present state of art. Cent. Eur. J. Publ. Health., 12: 16-18, 2004. Cervantes CJ, Esparza MM, Perez C, Daum N, Solano F, Borron JCG. Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor. J. Cell. Sci., 114: 2335-44, 2001. Chang TS. An updated review of tyrosinase inhibitors. Int. J. Mol. Sci., 10: 2440-2475, 2009. Chang TS. Natural mealnogenesis inhibitors acting through the down-regulation of tyrosinase activity. Materials, 5: 1661-1685, 2012. Chen CY, Kou PL, Chen YH, Huang JC, Ho ML, Lin RJ, www.kosac.or.kr 167
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