Korean Society for Biotechnology and Bioengineering Journal 32(3): 233-237 (2017) http://dx.doi.org/10.7841/ksbbj.2017.32.3.233 ISSN 1225-7117 / eissn 2288-8268 Research Paper 큰비쑥추출물의멜라닌생성억제효과 김민진 1, 김서연 2, 현광희 3, 김덕수 2, 김승영 4 *, 현창구 2 * Antimelanogenic of Artemisia fukudo Makino Extract in Melanoma Cells Min-Jin Kim 1, Seoyeon Kim 2, Kwang Hee Hyun 3, Duk Soo Kim 2, Seung-Young Kim 4 *, and Chang-Gu Hyun 2 * Received: 21 July 2017 / Revised: 2 August 2017 / Accepted: 4 August 2017 2017 The Korean Society for Biotechnology and Bioengineering Abstract: Melanin is one of the most important factors affecting skin color. Melanogenesis is the bioprocess of melanin production by melanocytes in the skin and hair follicles and is mediated by several enzymes, such as tyrosinase, tyrosinase related protein (TRP)-1, and TRP-2, MITF. In this study, we investigated the effect of Artemisia fukudo Makino extracts on tyrosinase activity and melanin production as natural products of whitening functional cosmetics. Melanin content in murine B16F10 melanoma cells were decreased by Artemisia fukudo Makino extracts in a dose-dependently. In addition, the inhibition of tyrosinase activity of Artemisia fukudo Makino extracts showed to decrease tyrosinase activity as the concentration of α-msh was increased. Furthermore, western blot analysis revealed that Artemisia fukudo Makino extracts significantly downregulated the expression These two authors contributed equally. 1 국립낙동강생물자원관 1 Nakdonggang National Institute of Biological Resources, Gyeongbuk 37242, Korea 2 제주대학교화학코스메틱스학과 2 Department of Chemistry and Cosmetics, Jeju National University, Jeju 63243, Korea Tel: +82-64-754-3542; Fax: +82-64-756-3561 e-mail: cghyun@jejunu.ac.kr 3 헬리오스 3 Helios 16, San cheon dan dong-gil, Jeju 63243, Korea 4 선문대학교 BT 융합제약공학과 4 Department of BT Convergent Pharmaceutical Engineering, Sunmoon University, Chungnam 31460, Korea Tel: +82-41-530-2390; Fax: +82-41-530-2939 e-mail: sykim01@sunmoon.ac.kr of tyrosinase, TRP-1 which treat of α-msh induced melanogenesis in murine B16F10 melanoma cells. As a result, Artemisia fukudo Makino extract showed functionalities as an effective whitening agent to inhibit melanin formation. Keywords: Artemisia fukudo Makino, Melanin, MITF, TRP, Tyrosinase 1. INTRODUCTION 인간의멜라닌은알칼리및난용성으로흑갈색색소인 eumelanin 과적황색색소인 pheomelanin 의복합체이다. 따라서미백효과를검증하기위해서는멜라닌형성억제여부를증명하는것이중요하다 [1]. 동물에있어서멜라닌합성의이상에따라백반과색소침착과같은비정상적인멜라닌착색을일으키며, 나아가서피부암을유발하기도하는 tyrosinase 라는효소가밀접한관련이있다 [2] Tyrosinase 는멜라닌생성첫단계를일으키는효소로, 티로신이도파 (DOPA) 를거쳐도파퀴논 (do paquinone) 으로전환되고, 도파퀴논으로부터자동산화반응과효소반응으로도파크롬 (dopachrome) 을거쳐흑갈색의공중합체인멜라닌이생성되게된다. 현재미백제의개발에있어서생선된멜라닌색소를환원시켜탈색하는방법과멜라닌색소를형성하는효소인 tyrosinase 의활성을억제하는방법이알려져있다 [3,4]. α-melanocyte stimulating hormone (α-msh) 는뇌하수체중엽에서분비되며멜라닌세포에서만발현되는막수용체인 MCIR (melanocortin 1 receptor) 과결합하여 adenylyl cyclase 를활성화시키고, 세포내 camp 신호를증폭시켜, protein kinase A (PKA) 의활성화를유도하며, 세포내 camp response element binding protein (CREB) 를활성화시킴으로써 [5-7] 멜라닌세포에 basic helix-
234 Korean Society for Biotechnology and Bioengineering Journal 32(3): 233-237 (2017) loop-helix leucine zipper (b-hlh-zip) 전사인자인 microphthalmia-associated transcription factor (MITF) 의발현을증가시킨다 [8,9]. 이러한경로로멜라닌세포에서의멜라닌합성은 tyrosinase, tyrosinase related protein(trp)-1, TRP-2, MITF 등의효소가관여하는복잡한과정을통해이루어진다. 쑥은오랫동안널리우리나라에서널리이용되어있으며쑥에포함된유효성분을생약학적방법으로연구한결과, 쑥에포함된다양한유효성분들이혈액순환촉진, 이뇨작용. 강장작용, 지혈, 피부질환치료, 항암작용등의효과를나타내는것으로보고되고있다 [10,11]. 쑥의생리활성물질로는 scoparone, capillarisin, cirsilineol, cirsimaritin, rhamnocitrin, 수종의 flavonoids 등많은물질이보고되고있다 [10]. 큰비쑥 (Artemisia fukudo Makino) 은다년초식물로서, 국화과 (Compositae) 103 속, 쑥속 (Artemisia) 40 종에속하며갯쑥이라고도불린다 [12]. 이와같이쑥의다양한기능성과생리효과를바탕으로본연구에서는쑥속식물중바닷가에서식하는큰비쑥을대상으로그유효성분이세포의증식과함께 α-msh 과추출물을처리함과동시에세포내에서의 melanin 생합성저해효과및 tyrosinase 저해활성뿐만아니라멜라닌합성의주요효소인 tyrosinase, TRP-1, TRP-2, MITF 발현에있어서변화를관찰하였다. 2. MATERIALS AND METHOD 2.1. 실험재료및세포배양본실험에서사용된 α-melanocyte stimulating hormone (α- MSH), L-3,4-dihydroxyphenylalanin (L-DOPA) 는 Sigma-Aldrich (St. Louis, MO, USA) 에서구입하였다. B16F10 세포는 ATCC로부터분양받았으며 10% fetal bovine serum (FBS, Gibco BRL, USA), Dulbecco s modified Eagle s medium (DM- EM, Gibco BRL, USA) 을기본배지로사용하여 37 o C, 5% CO 2 항온기에서배양하였으며, 3일에한번씩계대배양하였다. Tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinaserelated protein-2 (TRP-2), microphthalmia-associated transcription factor (MITF), primary antibody와 mouse-anti-goat, rabbitanti-mouse 등 secondary antibody는 Santacruz (CA, USA) 에서구입하였다. 2.2. 세포생존능측정 MTT 시험은 B16F10 melanoma cell 을 24 well plate 에 2.0 10 4 cells/well 로분주하여 37 o C, 5% CO 2 조건하에서 24 시간동안배양후, α-melanocyte stimulating hormone (α-msh) 와 sample 을농도별로동시에처리하여 24 시간배양하였다. 이후 500 μg/ml 의농도로 MTT 를첨가하여 37 o C 에서 3~4 시간동안반응시킨후, MTT 용액을제거하였다. 여기에 DMSO 를가하여살아있는세포와반응하여생긴 formazan 침전물 을용해시킨다음, 이를 96 well plate 에옮긴후 ELISA reader 를이용하여 540 nm 에서흡광도를측정하였다. 2.3. B16F10 세포내의멜라닌생성량측정멜라닌함량측정은 24 well plate 에 2.0 10 4 cells/well 로세포를분주하고 37 o C, 5% CO 2 incubator 조건하에서 18 시간 37 o C, 5% CO 2 incubator 배양후배양액을제거하였다. 멜라닌의생합성을유도하기위하여 200 nm α-msh 를포함하는배양액으로교환한후, sample 을농도별로각각첨가하여 24 시간동안 37 o C, 5% CO 2 incubator 에서배양하였다. 이후배지를제거하고 PBS (phosphate buffered saline) 를 24well 바닥면이골고루펴지게일정량을분주한후바로세척하여, trypsin-edta 를처리하여세포를 micro tube 에회수하였다. 회수된세포는 1 N NaOH 200 μl 를첨가하고 80 o C water bath 에서 1 시간방치하여세포내의 melanin 을녹여낸다음 1 시간동안 Ice 에방치하였으며, 멜라닌함량을 ELISA 를이용하여 405 nm 에서흡광도를측정하였다. 그결과로각각의데이터를단백질의양으로보정하여나타내었다. 2.4. B16F10 세포내의 tyrosinase 활성측정 Tyrosinase 의효소활성은기질인 L-3,4-dihydroxy-phenylalanine (L-DOPA) 이산화되어검붉은색을띄는 dopachrome 이생성되는것을측정하였다. 앞에서언급되었던실험들에서와같은방법으로세포를배양하고배양이끝난후 cell lysis buffer 인 RIPA buffer (50 mm Tris-HCl, ph 7.5, 150 mm NaCl, 1% NP-40, 0.5% sodium deoxychlolate, 0.1% SDS 와 Roche Diagnostics 사의 EDTA free Cocktail protease inhibitors) 로 lysis 한후 20 μl 를취하여 0.1 M sodium phosphate buffer (ph 7.0) 에녹인 2 mg/ml L-DOPA 80 μl 를각각혼합한후 2 시간동안 37 o C 에서배양하였다. 반응이끝난후 490 nm 에서흡광도를측정하였다. 2.5. Western blot 분석 B16F10 melanoma cell 을 1 10 5 cells/well 개의세포를부착시킨후 18 시간전배양하였고, 시료처리후 24 시간동안배양하였다. 수확한 cells 을 13,000 rpm 에서 15 분간원심분리하여그상등액을취한후, 단백질농도는 BSA kit (Bio-rad) 를이용하여정량하였다. 여기서얻은상층액을이용하여 10% SDS-PAGE 로전기영동하고, nitrocellulose membrane 에 transfer 한후 5% skim milk 가함유된 TBS (tris buffered saline containing 0.1% + Tween 20) 에넣어상온에서 1 시간정도 blocking 시킨후 TBS 로 10 분간 3 회세척하였다. tyrosinase, TRP- 1, TRP-2, MITF 1 차항체를처리하여상온에서 1 시간반응시킨후 TBS 로 10 분간 3 회세척한다음 2 차항체로는 HRP (horseradish peroxidase) 가결합된 anti-rabbit IgG 을희석하여상온에서 1 시간반응시켰다. 그후 membrane 을 TBS 로 10 분간 3 회세척한다음 ECL kit 사용하여 imaging densitometer (model GS-700, Bio-rad, USA) 을통해측정하였다.
큰비쑥추출물의멜라닌생성억제효과 235 2.6. 통계처리결과값은 3 번의독립적인실험을통한평균과표준편차로나타내었다. 대조군과실험군사이의통계학적유의성검정은 t-test 를사용하면서 ANOVA 검정을적용하였으며, *p<0.05 수준에서유의성검정을실시하였다. 3. RESULTS AND DISCUSSION 3.1. B16F10 세포생존능평가 B16F10 mouse melanoma cell 에서큰비쑥추출물이멜라닌합성에미치는영향을알아보기위해 cell proliferation 과 viability 의 in vitro 분석에매우유용하게사용되고있는 MTT assay [13] 를사용하여세포독성을측정하였다. 큰비쑥추출물의농도별세포생존율을측정한결과 50, 100, 200 μg/ml 까지의모든농도에서무첨가대조군보다 100% 가넘는세포생존율이확인된것으로보아세포독성이없는것으로확인되었다 (Fig. 1). 이결과를바탕으로동일한농도범위에서다음실험을진행하였다. Fig. 1. Cell viability on extract of A. fukudo Makino in B16F10 cells. MTT assay was performed after incubation of B16F10 cells treated with extract of A. fukudo Makino for 24 h at 37 o C in a 5% CO 2 atmosphere. Absorbance was measured at 540 nm. 3.2. Melanin 생합성저해효과생체내에서의멜라닌합성은기질인 tyrosine 이 tyrosinase 에의해 quinone 과 indolquinone 화합물들의여러중간체를거쳐멜라닌이생성된다 [14]. α-msh 에의해유도된멜라닌생성조건에서큰비쑥추출물의억제효과를확인하기위하여 α-msh 와시료를농도별로처리하여멜라닌생성을실험하였다. 큰비쑥추출물을같이처리한경우에는 α-msh 단독처리한경우에비하여농도가증가할수록멜라닌생성이유의적으로억제되는것으로나타났다. 특히농도 200 μg/ml 에서는 55% 로멜라닌생합성이억제되는것으로확인되었다 (Fig. 2). 또한큰비쑥추출물에의한억제효과는농도 100 μg/ ml 부터기존의 tyrosinase inhibitor 로알려진양성대조군 arbutin 보다뛰어난것으로보여진다. Fig. 2. Inhibitory effect on melanogenesis of A. fukudo Makino extract in B16F10 cells. B16F10 cells (2.0 10 4 ) were pre-incubated for 18 h and melanin content was performed after incubation of B16F10 cells treated with α-msh (200 nm), arbutin (100 μm) and extract of A. fukudo Makino (50, 100, and 200 μg/ml) for 72 h at 37 o C in a 5% CO 2 atmosphere. Absorbance was measured at 405 nm with ELISA. 3.3. Tyrosinase 효소활성 Melanin 은세포내의소기관인 ribosome 에서 tyrosinase 라는효소에의해서합성된다. 이효소의작용으로아미노산의일종인 tyrosine 에서몇단계의합성을거쳐, 기저층의 melanin 세포의 melanoma site 라는흑색소포표면에침착하여검고갈색의작은 melanin 입자가만들어진다 [15]. 본실험에서는큰비쑥추출물에대한 tyrosinase 효소활성을알아보기위해 tyrosinase 효소활성유도물질인 α-msh 200 nm 을각각첨가하여효소활성을측정하였다. 큰비쑥추출물 200 μg/ml 농도에서는 50 및 100 μg/ml 농도와는달리 tyrosinase 효소활성이 48.98% 로조금증가된것으로관찰되었으나, α-msh 만이처리된 ( 큰비쑥추출물무첨가 ) 대조군과비교했을때큰비쑥추출물이첨가된모든농도에서 tyrosinase 효소활성이약 50% 이상저해되는유의한결과를확인할수있었다 (Fig. 3). 3.4. 멜라닌합성관련단백질발현량측정멜라닌생합성에관여한인자로는 tyrosinase, dopachrome conversion factor, prostaglandin (PG), interferon (IFN), melanocyte stimulating hormone (MSH), Vitamin D3, histamine 등이보고되어있으며, 현재 tyrosinase 저해제로서 kojic acid와 albutin 이알려져있으나세포독성및돌연변이유발등의부작용이보고되고있다 [16,17]. 큰비쑥추출물이 α-msh 자극에의해유도된멜라닌생성을억제시켰으므로이러한생성억제효과가멜라닌관련단백질인 TRP-1, TRP-2, Tyrosinase, MITF 의발현억제에기인한것인지확인하기위하여 Western blot을수행하였다. α-msh 을단독처리한경우각단백질의발현이증가하는것을확인할수있었으며, a-msh에의해증가된 tyrosinase와 TRP-1의발현은큰비쑥추출물에의해농도의존적으로감소되는것을
236 Korean Society for Biotechnology and Bioengineering Journal 32(3): 233-237 (2017) Fig. 3. Inhibitory effect on intracellular tyrosinase activity of A. fukudo Makino extract in B16F10 cells. B16F10 cells (2.0 10 4 ) were pre-incubated for 18 h and melanin content was performed after incubation of B16F10 cells treated with α-msh (200 nm) and extract of A. fukudo Makino (50, 100, and 200 μg/ml) for 72 h at 37 o C in a 5% CO 2 atmosphere. 농도가증가함에따라멜라닌생성이유의적으로감소하는경향을보였다. 특히 100 μg/ml 이상농도에서는양성대조군이 arburin 보다높은멜라닌생합성저해효과를나타내었다. Tyrosinase 활성저해효과에는추출물의농도별로미비하게 tyrosinase 활성이증가하는것으로나타났으나, α-msh 처리한군보다훨씬낮은 tyrosinase 활성을나타내었다. 또한, 미백관련인자인 MITF, TRP-1, TRP-2, tyrosinase 의단백질및유전자발현억제효과를 western blot 을이용하여측정한결과미백관련인자 TRP-1, tyrosinase 단백질인자발현이억제됨으로써미백효능이있음을확인할수있었다. 따라서위의결과는큰비쑥추출물이독성과부작용이적은미백효과를가진천연기능성재료로서가능성이매우높음을제시한다. Acknowledgements 이논문은 2017 년교육부와한국연구재단의사회맞춤형산학협력선도대학 (LINC+) 육성사업의지원을받아수행된연구입니다. REFERENCES Fig. 4. Inhibitory effect of A. fukudo Makino extract on the protein level of TRP-1, TRP-2, Tyrosinase and MITF in B16F10 cells. B16 F10 cells (1.0 10 5 ) were pre-incubated for 18 h and the cells were stimulated with α-msh (200 nm) in the presence of arbutin (100 μm) and extract of A. fukudo Makino (50, 100, and 200 μg/ml) for 24 h. Protein level was determined using immunoblotting method. 확인할수있었다. 그러나 TRP-2 와 MITF 의발현에는아무런영향을주지않았다 (Fig. 4). 이결과를통해 tyrosinase 와 TRP-1 의감소는결과적으로멜라닌합성이억제된것으로보여진다. 이러한결과는 tyrosinase 와 TRP-1 단백질발현양은감소하지만 TRP-2 단백질발현양의변화가없다고하는선행연구가있다 [18]. 4. CONCLUSION 본연구에서는큰비쑥추출물의미백에대한세포실험을실시하여그효능을입증하고기능성화장품소재로서의가능성을검토하고자하였다. MTT 방법으로세포생존율을측정한결과, 큰비쑥추출물은 50, 100, 200 μg/ml 농도에서세포생존율이감소되지않고오히려대조군보다세포증식이증가하였다. 멜라닌생합성저해효과에서는큰비쑥추출물의 1. Brown, D. A. (2001) Skin pigmentation enhancers. J. Photochem. Photobiol. B: Biol. 63: 148-161. 2. Cabanes, J., S. Chazarra, and F. Garxia-Carmona (1994) Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. J. Pharm. Pharmacol. 46: 982-985. 3. Vincent, J. H. and J. Mercedes (1987) Mammalian tyrosinase-the critical regulatory control point in melanocyte pigmentation. Int. J. Biochem. 19: 1141-1147. 4. Chang, Y. H., C. Kim, M. Jung, Y. H. Lim, S. Lee, and S. Kang (2007) Inhibition of melanogenesis by selina-4(14), 7(11)-diene-8- one isolated from Atractylodis Rhizoma Alba. Biol. Pharm. Bull. 30: 719-723. 5. Chakraborty, A. K., Y. Funasaka, A. Slominski, G. Ermak, J. Hwang, J. M. Pawelek, and M. Ichihashi (1996) Production and release of proopiomelanocortin (POMC) derived pep regulation by ultraviolet B. Biochim. Acta 1313: 130-138. 6. Bertolotto, C., P. Abbe, T. J. Hemesath, K. Bille, D. E. Fisher, J. P. Ortonne, and R. Ballotti (1998) Microphthalmia gene product as a signal transducer in camp-induced differeniation of melanocytes. J. Cell Biol. 142: 827-835. 7. Cheli, Y., F. Luciani, M. Khaled, L. Beuret, K. Bille, P. Gounon, J. P. Ortonne, C. Bertolorro, and R. Ballotti (2009) Alpha-MSH and Cyclic AMP elevating agents control melanosome ph through a protein kinase A-independent mechanism. J. Biol. Chem. 284: 18699-18706. 8. Price, E. R., M. A. Horstmann, A. G. Wells, K. N. Weilbaecher, C. M. Takemoto, M. W. Landis, and D. E. Fisher (1998) Alpha-melanocyte-stimulating hormone signaling regulates expression of mic-
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