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042 Online ISSN 2234-7941 Print ISSN 1976-0442 Original Article: Bioactive Materials Whitening Effects of Solvent Fractions Isolated from Vitex rotundifolia Jae-Myo Yu Dong-Hee Kim Jun-Ho Son*

1 J Appl Biol Chem (2015) 58(3), Online ISSN Print ISSN Original Article: Bioactive Materials Whitening Effects of Solvent Fractions Isolated from Vitex rotundifolia Jae-Myo Yu Dong-Hee Kim Jun-Ho Son* 만형자용매분획물의미백개선효과 유재묘 김동희 손준호 * Received: 11 June 2015 / Accepted: 17 July 2015 / Published Online: 30 September 2015 The Korean Society for Applied Biological Chemistry 2015 Abstract Recently many effort focused to understand the mechanical insights of melanogenesis to develop the agent for hyper-pigmentation. So this study was performed to investigate the depigmentation of Vitex rotundifolia. With B16F10 mouse melanoma cell, we have seen inhibition of the tyrosinase, MITF, TRP-1, TRP-2, and melanin synthesis, which eventually were dose dependently decreased by Vitex rotundifolia. Specially, Vitex rotundifolia decreased the protein levels of tyrosinase and TRP-1. In conclusion, Vitex rotundifolia showed the whitening activity in all the experiments mentioned above and we expect that it can be used for preventing melanin synthesis. Keywords inhibition of melanin TRP-1 tyrosinase Vitex rotundifolia whitening effect 서 론 현대에있어서깨끗하고하얀피부는아름다움의척도가되었다. 특히생활수준의향상과수명의연장으로피부미용에대한관심이증가되었고, 환경오염에따른자외선노출증가로인해피부미백에대한관심을더욱커지게하였다. 이에따라피부 J.-M. Yu D.-H. Kim J.-H. Son Korea Promotion Institute for Traditional Medicine Industry, Gyeongsan , Republic of Korea. *Corresponding author (J.-H. Son: bio115@kotmin.kr) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( 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. 에안정적이고자극이없는미백소재를찾는연구가활발하게진행되고있다 (Lee 등, 2003). 피부미백에대한연구는 tyrosinase 의활성억제, 3,4-dihydroxyphenylalanine (DOPA) 산화억제, 자외선차단등에대해서행해지고있는데 keratinocyte 에있는멜라닌의양과분포에따라피부색및색소침착이결정되기때문에미백효과를검증하기위해서는멜라닌형성을억제하는지를확인하는것이중요하다 (Cho 등, 2003). 멜라닌은피부가자외선에노출되었을때 melanocyte 의 melanosome 에서 tyrosine 이 tyrosinase 라는효소에의해산화되어생성되는데이때 tyrosinase 의합성및전달에관여하는 dopachrome tautomerase, peroxidase, catalase, glutathione reductase 등의효소와 interferon, prostaglandin, cyclooxygenase 등과같은 mediator 및구리, 아연철과같은금속이온이관여하는것으로알려져있다 (Park 등, 2004b). 멜라닌은자연계에널리분포하는페놀류의고분자물질로서단백질과검은색소의복합체이다. 이것은인체내에서독성물질에대한제거작용을하여세포를보호한다. 하지만과잉생산시인체에기미, 주근깨를형성하고피부노화를촉진시키며피부암을유발하는것으로알려져있다. 이러한멜라닌의생합성경로를보면 tyrosine 이 tyrosinase 에의해서 DOPA 로전환되고다시 tyrosinase 에의해 DOPA quinone 으로전환되어최종적으로멜라닌을생성하게된다 (Hearing, 1999; Hwang 과 Lee, 2007). 이에따라멜라닌의생합성을억제하는물질을찾아내어미백효과등의효능을확인하는데적용시킬수있다 (Mauviel 등, 1994; Pentland 등, 1995; Park 등, 2004a). 만형자 (Vitex rotundifolia) 는순비기나무 (Vitex rotundifolia L.) 의성숙된열매를건조한것으로마편초과 (Verbenaceae) 에속하는약용식물자원이며우리나라중부이남의섬이나해안가에자생하는것으로알려져있다 (Lee, 1993; Yeeh 등, 1996). 또한진통, 진정, 소염의목적으로만성중이염, 습진, 이명, 난청, 두통, 신경통등에사용되어온생약으로 (Perry, 1980; Encyclopedia of Chinese herbal medicine, 1985), 동의보감에는풍두통 ( 風頭

2 267 J Appl Biol Chem (2015) 58(3), 痛 ) 과뇌명 ( 腦鳴 ) 에달여먹는다고기술되어있다 (Heo, 1991). 만형자에관한연구로는약 76 종의정유성분및 monoterpene 류 (Jang 등, 2002), flavonoids, phenol 화합물등이분리된바있으며 (Kang 등, 1994; Yoshioka 등, 2004), 또한만형자는모발성장촉진효과 (Shin 등, 2009), 염증및알레르기반응 (Yeon 등, 2013) 등에효능을나타내는것으로알려져있다. 본연구에서는 B16F10 mouse melanoma cell 을이용하여예로부터한방생약재로사용하며유용한성분및효과를나타내는만형자용매분획물을처리한후 melanin 정량과 western blotting 으로 MITF, tyrosinase, TRP-1, TRP-2 등의단백질발현을확인하여미백효과에대한효능을검증하였다. 재료및방법 시료. 본실험에사용한만형자는 2014 년 2 월초순에 ( 주 ) 휴먼허브 (Korea) 에서구입하여시료로사용하였다. 시료추출및용매분획. 건조된시료 300 g 을분쇄기를이용하여잘게파쇄한후 70% 에탄올을 3L 넣어상온에서 24 시간동안동일용매로 3 회반복추출한다음 filter paper (Whatman No. 2, Japan) 로여과한뒤감압농축기로에탄올을제거하여농축액 7.24 g 을얻었다. 이후용매추출법을이용하여극성도가낮은 n-hexane 으로추출한후, 다시 ethyl acetate 와 butanol 로순차적으로분획하였다. 각용매추출분획을 freeze drier (Ilsin, Korea) 로감압농축하여 n-hexane (VR-H) 0.65 g, EtOAc (VR- EA) 1.21 g, n-buoh (VR-B) 1.56 g, H 2 O (VR-W) 1.02 g 을각각얻었다. 시약및기기. 미백효과측정에사용된시약인 L-ascorbic acid, L-3,4-dihydroxyphenylalanine (L-DOPA) 등은 Sigma chemical Co. (USA) 에서구입하여사용하였다. 세포독성측정에사용된세포주는 mouse melanoma cell 인 B16F10 을 Korean cell line bank (Korea) 에서구입하여사용하였다. 세포독성측정시약은 dulbecco s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, 0.25% trypsin-edta, 0.4% trypan blue stain 은 Gibco BRL Co. (USA) 및 haemacytometer (Marienfeld, Germany) 에서구입하여사용하였으며, 3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT) 는 Sigma chemical Co. (USA) 에서구입하여사용하였다. 1 차항체인 tyrosinase, MITF, tyrosinase-related protein (TRP)-1, TRP-2 와 2 차항체는 Santa cruz biotechnology Inc. (USA) 에서구입하였다. 실험에사용한기기는 ELISA reader (Tecan, Switzerland), rotary vaccum evaporator (Rikakikai Co., Japan), centrifuge (Eppendorf, Germany), CO 2 incubator (Hanbaek, Korea) 를사용하였다. 세포배양. 본실험에이용한각세포의배양은 10% FBS 와 1% penicillin/streptomycin (100 U/mL) 을첨가한 DMEM 배지를사용하였으며, 37 o C, 5% CO 2 incubator 에적응시켜계대배양하였다. 세포확인시 inverted microscope (Nikon, Japan) 를사용하였다. B16F10 mouse melanoma cell 에시료용액을농도별로 1 시간전처리한후 α-melanocyte stimulating hormone (α-msh) 10 nm 를 48 시간처리하였다. MTT assay 에의한세포독성측정. 세포독성측정은 Carmichael 등 (1987) 의방법에따라측정하였다. B16F10 mouse melanoma cell 을 96 well plate 에 cells/well 이되 게 0.18 ml 분주하고, 시료를농도별로조제하여 0.02 ml 첨가한후 37 o C, 5% CO 2 incubator 에서 48 시간처리하였다. 대조군은시료와동량의증류수를첨가하여동일한조건으로배양하였다. 여기에 5mg/mL 농도로제조한 MTT 용액 0.02 ml 를첨가하여 4 시간배양한후배양액을제거하고각 well 에 DMSO 0.15 ml 를가하여실온에서 30 분간반응시킨뒤 ELISA reader 로 540 nm 에서흡광도를측정하였다. 세포독성측정은시료용액의첨가군와무첨가군의흡광도감소율로나타내었다. Melanin 정량. 멜라닌양은 Hosoi 등 (1985) 의방법을변형하여사용하였다. B16F10 mouse melanoma cell 를 6 well 에 cells/well 이되도록분주하여 24 시간뒤, 각각의 well 에 VR-EA, VR-H, VR-B, VR-W 25 µg/ml 와사용전에제조한 vitamin C (Vit. C; Sigma, USA) 25 µg/ml 를 48 시간동안처리하였다. 처리후 phosphate buffered saline (PBS; Gibco, USA) 로 2 회세척한후 12,000 rpm, 4 o C, 30 분간원심분리하여세포침전물을만들었다. 10% dimethyl sulfoxide (DMSO; Duksan, Korea) 가첨가된 1N NaOH 용액을 150 µl 첨가하고 60 o C 에서 1 시간용해하였으며 405 nm 에서흡광도를측정한후실험군의멜라닌양은대조군의멜라닌양에대한백분율로계산하여나타내었다. Cellular tyrosinase 저해율측정. Cellular tyrosinase 저해율측정은 Choi 등 (1998) 의방법을변형하여측정하였다. B16F10 mouse melanoma cell 을 6 well plate 에 cells/well 이되도록접종하고각 well 에 VR-EA, VR-EA, VR-H, VR-B, VR-W 및 Vit. C (25 µg/ml) 를 48 시간동안처리하였다. 처리후 PBS 로 2 회세척한후각 well 의세포에 lysis buffer (1% triton X-100, 0.1 M sodium phosphate buffer, 50 mm phenylmethylsulfonyl fluoride, ph 6.8) 를가하였다. 얼음위에서세포를파괴시키고 12,000 rpm, 4 o C, 30 분간원심분리한후상층액만따로모아효소용액으로사용하였다. L-DOPA 를 2mg/mL 농도로 0.1 M sodium phosphate buffer (ph 6.8) 에녹여기질을준비하고기질 160 µl 에효소용액 40 µl 를가하고 37 o C 에서 1 시간가온하고생성된 DOPA chrome 의양을 490 nm 에서측정한후억제율을계산하였다. Western blot 을통한 tyrosinase, MITF, TRP-1, TRP-2 단백질의발현측정. B16F10 mouse melanoma cell 을 6 well plate 에 cells/well 에되도록분주하고 24 시간동안안정화하였다. 배지를제거한후 VR-EA 를농도별로 (5, 10, 20, 25 µg/ml) 48 시간처리후 PBS 로세척하였다. Lysis buffer 를 well 당 30 µl 를첨가하여세포를용해시키고원심분리하여 (12,000 rpm, 4 o C, 30 min) 세포막성분들을제거하였다. 원심분리하여얻은단백질은 bradford assay 로정량하였으며, 20 µl 의단백질을 10% 의 sodium dodecyl sulfate polyacrylamide gel electrophoresis 를이용하여전기영동한후, 항체의비특이적결합을억제시키기위해 PVDF membrane 에옮긴다음 60 V 에서 2 시간이상 transfer 하였다. Transfer 가끝나면 5% skim milk 에 1 시간동안방치하여 background 를제거시켰다. 1 TBST 로 3 회 washing 후 1 차 antibody (1:1000) 를 1 시간동안붙인후다시 2 차 antibody (1:1000) 를붙이고 ECL kit (Amersham Pharmacia, England) 를이용하여 film 에옮겨측정하였다. Band density 는 ImageQuant LAS-4000 (GE life sciences, Taiwan) 으로확인하였다.

$J Appl Biol Chem (2015) 58(3), 266 271 268 Fig. 1 Cytotoxicity of mouse melanoma cell (B16F10) of solvent fractions isolated from Vitex rotundifolia.$

3 J Appl Biol Chem (2015) 58(3), Fig. 1 Cytotoxicity of mouse melanoma cell (B16F10) of solvent fractions isolated from Vitex rotundifolia. VR-H: Vitex rotundifolia hexane fraction; VR-EA: Vitex rotundifolia ethyl acetate fraction; VR-B: Vitex rotundifolia butanol fraction; VR-W: Vitex rotundifolia water fraction; Con: α-msh induced group. Fig. 2 Inhibition melanin synthesis of solvent fractions isolated from Vitex rotundifolia on mouse melanoma cell (B16F10). VR-H: Vitex rotundifolia hexane fraction; VR-EA: Vitex rotundifolia ethyl acetate fraction; VR-B: Vitex rotundifolia butanol fraction; VR-W: Vitex rotundifolia water fraction; Vit. C: L-ascorbic acid; Nor: α-msh not induced group; Con: α-msh induced group. Result are means ± SD of triplicate data. Fig. 3 Inhibition cellular tyrosinase of solvent fractions isolated from Vitex rotundifolia on mouse melanoma cell (B16F10). VR-H: Vitex rotundifolia hexane fraction; VR-EA: Vitex rotundifolia ethyl acetate fraction; VR-B: Vitex rotundifolia butanol fraction; VR-W: Vitex rotundifolia water fraction; Vit. C: L-ascorbic acid; Nor: α-msh not induced group; Con: α-msh induced group. Result are means ± SD of triplicate data. 결과및고찰 MTT assay 에의한세포독성측정. 만형자분획물에의한 B16F10 mouse melanoma cell 의독성을확인하기위해분획물을적정농도로 48 시간동안처리한후 MTT assay 를실시하였다. 세포독성은 VR-EA 이농도 5, 10, 25 µg/ml 에서각각 98, 91, 85% 의생존율을보여처리하지않은대조군에비하여세포생존율이크게감소하지않는것으로보아세포에대한독성을나타내지않는것을확인할수있었다 (Fig. 1). Melanin 정량. 멜라닌은동물, 식물, 미생물에널리분포하는색소로서사람의경우피부색을결정하는동시에유해한자외선이나유리기 (free radical) 로부터인체를보호하는역할을담당하고있다. 이색소는피부의기저층에존재하는멜라닌생성세포가자외선및자극에의해활성화되어만들지고표피의가장위층인각질층에존재한다. 멜라닌색소의생합성은 tyrosinase 효소를비롯하여다양한효소들에의해조절되고있는것으로알려져있다 (Aroca 등, 1993; Paval, 1993; Jimenez- Cervantes 등, 1994). 색소침착을치유하기위해멜라닌생성을억제하는페놀유도체나 kojic acid, arbutin, glucosamine 등이개발되어있으나, 피부에대한자극이나안정성에문제가있어서제한된양만사용되고있다. 멜라닌생합성저해정도를확인한결과, Vit. C 의경우 25 µg/ml 를처리하였을때멜라닌을 58% 저해하였으며, 만형자분획물의경우 VR-EA, VR-B, VR-H, VR-W 의순서로높게나타났고각각 35, 27, 19, 17% 의억제효과를보였다 (Fig. 2). 반면에삼백초에탄올추출물은동일한농도에서약 21% 의억제효과를나타내어 (Park 과 Lee, 2008) VR-H, VR-W 보다는높은효과를보였지만 VR-EA, VR-B 의효과에는미치지못하였다. Cellular tyrosinase 저해율측정. 멜라닌생성에있어서가장

$269 J Appl Biol Chem (2015) 58(3), 266 271 Fig. 4 Tyrosinase protein expression rate of ethyl acetate fraction isolated Fig.$ $5 MITF protein expression rate of ethyl acetate fraction isolated Fig.$ DOPA quinone 으로바뀌고, 이들은효소의작용및자동산화반응에의해 DOPA chrome, indole carboxylic acid, indole quinine 등으로대사되어최종적으로멜라닌을합성한다 (Lin 등, 2002).

DOPA quinone 으로바뀌고, 이들은효소의작용및자동산화반응에의해 DOPA chrome, indole carboxylic acid, indole quinine 등으로대사되어최종적으로멜라닌을합성한다 (Lin 등, 2002).

VR-EA 는 25 µg/ ml 에서 31% 의저해능을나타낸반면, Vit.

4 269 J Appl Biol Chem (2015) 58(3), Fig. 4 Tyrosinase protein expression rate of ethyl acetate fraction isolated Fig. 6 TRP-1 protein expression rate of ethyl acetate fraction isolated Fig. 5 MITF protein expression rate of ethyl acetate fraction isolated Fig. 7 TRP-2 protein expression rate of ethyl acetate fraction isolated 중요한역할을하는효소는 tyrosinase 로서 melanosome 내의 tyrosine 을산화시켜 DOPA, DOPA quinone 으로바뀌고, 이들은효소의작용및자동산화반응에의해 DOPA chrome, indole carboxylic acid, indole quinine 등으로대사되어최종적으로멜라닌을합성한다 (Lin 등, 2002). 따라서피부내에서멜라닌중합체생합성을효과적으로저해할수있는 tyrosinase 저해활성을측정하기위하여 cellular tyrosinase 활성측정결과, 모든분획물에서 tyrosinase 저해효과를보여주었다. VR-EA 는 25 µg/ ml 에서 31% 의저해능을나타낸반면, Vit. C 는 25 µg/ml 에서 58% 의저해능을나타내었으며 VR-B 는 19%, VR-H 는 18% 그리고 VR-W 의경우 10% 의저해효과를보여주었다 (Fig. 3). 동일한실험에서감귤추출물 (70% EtOH) 의경우같은농도에서약 4% 의효과를나타내어 (Kim 등, 2007) 상대적으로효능이뛰어난것을알수있었다. Western blot 을통한 tyrosinase, MITF, TRP-1, TRP-2 단백질의발현측정. α-msh 는 tyrosinase 의활성을자극시키고 (Abdel-Maldk 등, 1995), 멜라닌합성에관여하는효소인 TRP- 1, TRP-2 에작용하여 eumelanin 을합성하도록유도한다 (Fuller

5 J Appl Biol Chem (2015) 58(3), 와 Meyskens, 1981). TRP-1 은 DHICA 를 indole-5,6-carboxylic acid 로전환시키는효소로서 DHICA oxidase 라고도하며 tyrosinase 의변성을막는데작용하여 tyrosinase 의활성에기여하는효소로간접적으로멜라닌생성을조절한다 (Hearing 등, 1992; Takechi 등, 1996). 따라서미백소재의개발에있어서멜라닌생성을억제하는기전과이미생성된멜라닌의분해를촉진시키는단계에서작용하는효소의조절이중요하며멜라닌생성에관련된단백질발현과연관성이있는지보기위해 tyrosinase, MITF, TRP-1, TRP-2 의항체를이용한 western blotting 으로관련단백질의발현양변화에대하여확인하였다. B16F10 mouse melanoma cell 에 VR-EA 를 5, 10, 20, 25 µg/ml 처리한후 48 시간뒤에 tyrosinase, MITF, TRP-1, TRP-2 protein 발현을 western blotting 으로확인하였다. 또한세포에서발현정도의차이가거의없는 house keeping gene 인 α-tubulin 을이용하여 tyrosinase, MITF, TRP-1, TRP-2 protein 을정량하였다. VR- EA 를농도별로처리하였을때 25 µg/ml 을처리한군에서는 tyrosinase protein 이 53.2% 감소하였고, MITF 는 34.7%, TRP- 1 은 88.4%, TRP-2 의경우 23.6% 의감소율을보여주었다 (Fig. 4-7). 상대적으로노간주나무추출물 (70% EtOH) 은동일농도에서 tyrosinase, MITF, TRP-1, TRP-2 의순서로약 12, 10, 9, 17% 의효과를나타내어 (Lee 등, 2013) VR-EA 보다 protein 발현의억제율이낮게나타났다. 초 록 최근에는 hyper-pigmentation 에대한제품개발을위해 melanogenesis 의경로에대한이해를위해많은노력을하고있다. 이에따라본연구에서는만형자 (Vitex rotundifolia) 의 depigmentation 에대한효과를조사하였다. B16F10 mouse melanoma cell 을이용하여 tyrosinase, MITF, TRP-1, TRP-2 그리고 melanin synthesis 의저해를확인하였고, 만형자 ethyl acetate 분획물 (VR- EA) 을처리하였을때농도의존적으로감소한다는것을알수있었다. 특히, VR-EA 는 tyrosinase 와 TRP-1 의경우각각 53.2, 88.4% 의저해율을보여미백에있어서효과적인활성을보여주었다. 따라서만형자는 melanin synthesis 를효과적으로막아주어미백소재로서활용가능성이충분하다고사료된다. Keywords 만형자 미백효과 멜라닌형성저해 TRP-1 tyrosinase 감사의글이연구는산업통상자원부지역연고산업육성사업 (R , 경북화장품산업육성사업 ) 의일환으로수행하였음. References Abdel-Maldk ZA, Swope NB, Suzuki I, Harriger D, Boyce ST, Urabe K et al. (1995) Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. 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6 271 J Appl Biol Chem (2015) 58(3), melanosomal membrane protein is a cell surface tarket for melanoma therapy. Clin Cancer Res 2, Yeeh Y, Kang SS, Chung HG, and Chung MS (1996) Genetic and clonal diversity in Korean populations of Vitex rotundifolia (Verbenaceae). J Plant Research 109, Yeon KJ, Jung HA, and Roh SS (2013) The Effects of Vitex rotundifolia Linne fil. Extract on the Inflammatory and Allergic Reactions. Daejeon journal of the institute of oriental medicine 22, Yoshioka T, Inokuchi T, Fujioka S, and Kimura Y (2004) Phenolic compounds and flavonoids as plant growth regulators from fruit and leaf of Vitex rotundifolia. Z Naturforsch 59,

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,