본초학표지

大韓本草學會誌제 30 권제 4 호 (2015 년 7 월 ) Kor. J. Herbol. 2015;30(4):95-100 ISSN 1229-1765(Print), ISSN 2288-7199(Online) http://dx.doi.org/10.6116/kjh.2015.30.4.95. 산약의멜라노마세포 (B16F10) 에서 MITF, TRP-1, TRP-2, Tyrosinase, PKA, ERK 발현억제효과 이수연 #, 유단희, 주다혜, 이진영 * 호서대학교한방화장품과학과 Inhibitory Efficacy of Dioscoreae Rhizoma on MITF, TRP-1, TRP-2, Tyrosinase, PKA and ERK Expression in Melanoma Cells (B16F10) Soo-Yeon Lee #, Dan-Hee Yoo, Da-Hye Joo, Jin-Young Lee * Department of Herbal Cosmetic Science, Hoseo University, Chungnam, 336-795, Korea ABSTRACT Objectives : The purpose of this study was to research the whitening effects and developing by cosmetics of the extract from Dioscoreae Rhizoma, which is one of the most popular health-promoting herb in herbal medications. Methods : We performed tyrosinase inhibition assay, reverse transcription-polymerase chain reaction (RT-PCR) and western blot for whitening effects. Also we measured MTT assay for cell viability. Results : The results were obtained as follows : For whitening effect, tyrosinase inhibition rate of extract from Dioscoreae Rhizoma showed more than 42.28% at 1,000 μg / ml concentration. Cell toxicity effect on melanoma cells (B16F10) of extract from Dioscoreae Rhizoma showed 81.97% with toxicity at 50 μg / ml concentration. So we were measured at a concentrations of 5, 10 and 50μg / ml in all experiments involving cell. In addition, whitening related mrnas including microphthalmia associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), tyrosinase were reduced by Dioscoreae Rhizoma. We also found Dioscoreae Rhizoma transiently decreased protein kinase A (PKA) which is known to be upstream to the down regulation of MITF and tyrosinase. But phosphorylation of extracellular signal related kinase (perk) were increased by Dioscoreae Rhizoma. These results imply that Dioscoreae Rhizoma decrease melanogenesis via ERK activation and subsequent down regulation of MITF and tyrosinase. Conclusions : Therefore, all these findings suggested the potent usage of Dioscoreae Rhizoma as materials of functional cosmetics by confirming whitening activity related with melanin content. Key words : Dioscoreae Rhizoma, whitening, cosmetic ingredient, cosmeceutical 서론 1) 최근오존층의파괴로자외선의조사량이증가하고있는데이때자외선의영향을많이받는기관은피부이다. 피부내에존재하는멜라노사이트 (melanocyte) 는외부신호전달물질및호르몬수용체를가지고있어자외선등과같은외부환경이나호르몬, 사이토카인 (cytokine) 등에의해서멜라닌생성촉진인자 (MITF, TRP-1, TRP-2, tyrosinase) 와같은효소 들에영향을주어멜라닌 (melanin) 을형성한다 1,2). 이렇게형성된멜라닌 (melanin) 은기미와주근깨등과같은색소침착현상을야기시킨다. 멜라닌의생합성에서가장중요한단계는 tyrosinase 의촉매작용을통해일어나는초기반응 3) 으로 tyrosin 에 hydroxyl 기를부착시켜 DOPA를생성하는기작이다 4). 따라서최근까지미백기능성물질에관한연구는 tyrosinase 의활성을억제함으로써멜라닌생합성을억제하는 *Corresponding author : Jin-Young Lee. Department of Herbal Cosmetic Science, Hoseo University Tel : +82-41-540-9552 Fax : +82-41-540-9538 E-mail : jylee@hoseo.edu #First author : Soo-Yeon Lee. Department of Herbal Cosmetic Science, Hoseo University Tel : +82-41-540-9559 Fax : +82-41-540-9538 E-mail : suyun_14@naver.com Received:24 June 2015 Revised:10 July 2015 Accepted:12 July 2015

96 大韓本草學會誌 Vol. 30 No. 4, 2015 수준에서이루어져왔으며, 이러한 tyrosinase 저해제로많이알려져있는물질로는 hydroquinone, ascorbic acid, kojic acid, arbutin 등이있다 5). 이들은 tyrosinase 의활성을억제하고자외선으로인해생긴검버섯, 기미, 주근깨방지와같은목적으로의약외품및미백기능성원료로사용되어져왔다 6). 이러한원료들은경제성및화장품의부작용등과같은안전성에문제등으로최근화장품업계에서는화장품의기능성원료를천연소재로부터얻으려는연구가활발히진행되고있다. 산약 (Dioscoreae Rhizoma) 은백합목마과 (Dioscoreacea) 에속하는덩굴성다년생식물로서한방에서는뿌리줄기의주피를벗겨그대로쪄서말린것을말하며, 약용으로사용되어왔다 7). 산약의구성성분은식이섬유가대부분을차지하며, 탄수화물, 아미노산, 점액성당단백질, polyphenol성화합물등을함유하고있다 8). 최근연구보고에의하면산약에서분리추출한저장성단백질 dioscorin, 점액성다당류및 polyphenol 성화합물등이중금속 (Co, Cr, Cu) 흡착제거능과항산화성이뛰어나다고보고되었다 8,9). 산약의주요약리작용으로는혈당강화효과, 콜레스테롤저하작용, 면역조절효과, 세포독성저해작용등이있는것으로보고되고있다 10). 또한최근산약추출물이대식세포활성을억제하며, 특히산약의 diosgenin 에의해대식세포로부터염증매개물질분비를억제한다고보고되었다 11,12). 본연구에서는천연물질인산약추출물이화장품의기능성원료로서사용의가치가있는지알아보고자하였다. 재료및방법 1. 재료및시료추출본실험에사용한산약 (Dioscoreae Rhizoma) 은대전대학교부속한방병원에서구입정선하여사용하였으며원산지는한국경북청송군부남면이다. 산약 100 g에증류수 2,000 ml를가하여열탕추출기에서 2시간추출하여얻은액을흡입여과하여이를감압증류장치로농축하여, 이를다시동결건조기를이용하여완전건조한산약추출물을냉동보관 (-84 ) 하면서적당한농도로희석하여사용하였다. 2. 실험시약및기기실험에사용된시약인 tyrosinase mushroom, dimethyl sulfoxide (DMSO) 등은 Sigma Chemical Co. (St. Louis, MO, USA) 에서구입하였다. PCR에사용된 extracellular signal-regulated kinase (ERK), camp-response element binding protein (CREB), Microphthalmia-associated transcription factor (MITF), tyrosinase related protein 1 (TRP-1), tyrosinase related protein 2 (TRP-2), tyrosinase, GAPDH, primary antibody 와 mouse-anti-goat, rabbbit-anti-mouse 등 secondary antibody는 Santacruz (CA, USA) 에서구입하였다. 세포독성측정에사용된세포주는마우스흑색세포종인 B16F10을 ATCC에서구입하여사용하였다. 세포배양을위해 dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin/streptomycin, trypsin 는 Thermo Scientific Hyclone (USA) 및 Gibco BRL Co. (Grand Island, USA) 에서구입하여사용하였다. 세포독성측정에사용된 Haemacytometer (Marienfeld, Germany), 3-[4,5-dimethylthiazol]-2-yl]-2,5-diphenyl -tetrazoliumbromide (MTT) 는 Sigma Chemical Co. (St. Louis, MO, USA) 에서구입하였다. 실험에사용된기기는 rotary vacuum evaporator (EYELA, Japan), centrifuge (Hanil Science Industrial Co. Korea), freeze drier (ILShin BioBase Co. Korea), microscope (Olympus, Japan), CO 2 incubator (Vision Scientific, Korea), vortex (Scientific Industries, INC, USA), ph meter (Mettler-Toledo AG, Swltzerland), BOD incubator (DAIHAN Scientific, Korea), autoclave (JS Research Inc, Korea), ELISA Reader (Tecan, Austria), PCR (C-100, Bio-Rad, USA) 을사용하였다. 3. Tyrosinase 저해활성측정 Tyrosinase 저해활성측정은 Yagi 등의방법 13) 에따라측정하였다. 반응구는 67 mm sodium phosphate buffer (ph 6.8) 80 µl에 10mM L-DOPA (Sigma, USA) 를녹인기질액 40 µl 및시료용액 40 µl의혼합액에 200 U/ml mushroom tyrosinase (Sigma, U.S.A) 40 µl을첨가하여 37 에서 10 분간반응시켜반응액중에생성된 DOPA chrome 을 492 nm에서측정하였다. Tyrosinase 저해활성은시료용액의첨가구와무첨가구의흡광도감소율로나타내었다. 저해율 (%) = ( 1 - 시료첨가군의흡광도 ) 100 무첨가군의흡광도 4. 세포배양 본실험에이용한각세포의배양은 10% FBS와 1% penicillin/streptomycin (100 U/ml) 을첨가한 DMEM 배지를사용하였으며, 37, 5% CO 2 incubator 에적응시켜계대배양하였다. 5. MTTassay 에의한세포생존율측정 세포독성측정은 Carmichael 14) 의방법에따라측정하였다. 멜라노마세포 (B16F10) 을 96 well plate에 5 10 4 cells/well 이되게 0.18 ml 분주하고, 시료를농도별로조제하여 0.02 ml 첨가한후 37, 5% CO incubator 에서 24 시간배양하였다. 대조군은시료와동량의증류수를첨가하여동일한조건으로배양하였다. 여기에 2.5 mg/ml 농도로제조한 MTT 용액 0.02 ml를첨가하여 4시간배양한후배양액을제거하고각 well당 DMSO 0.15 ml를가하여실온에서 30분간반응시킨뒤 ELISA reader로 540 nm에서흡광도를측정하였다. 세포독성측정은시표용액의첨가군과무첨가군의흡광도감소율로나타내었다.

산약의멜라노마세포 (B16F10) 에서 MITF, TRP-1, TRP-2, Tyrosinase, PKA, ERK 발현억제효과 97 세표독성 (%) = ( 1 - 시료첨가군의흡광도 ) 100 무첨가군의흡광도 6. Total RNA 분리및 cdna 합성 세포를 100 mm culture dish 에 cell seeding 한뒤 24 시간동안배양한후 α-msh (100 nm) 과 sample 을농도별로처리하여 24 시간동안배양하였다. 배지상등액을제거한후 trizol lysis buffer 를각 well 에 1 ml 씩분주하여세포를 lysis 한후 chloroform 200 µl 를분주하여 20 초간위아래로흔들어주었다. 그후 13,200 rpm 에서 20 분간원심분리하여상층액을 isopropanol 500 µl 이들어있는튜브에옮겨섞었다. 다시 13,200 rpm 에서 20 분간원심분리하였고, 그상층액을제거한후 75% EtOH-diethylpyrocarbonate water 를각튜브에 1 ml 씩분주하여 13,200 rpm 에서 5 분간원심분리한뒤상층액을제거한뒤실온에서건조시켰다. DEPC 를 50 µl 씩분주하여녹인후 96 well plate 에 RNA 5 µl 와멸균수 195 µl 를첨가하여 260 nm, 280 nm 에서각각흡광도를측정하여 total RNA 양을측정하였다. Oligo (dt) 15primer (500 µg/ml) 1 µl, 추출한 RNA(2 µg) 와 nuclease free water 로 10 µl 를맞추고 75 에서 5 분간반응시킨후 5X reaction buffer, MgCl 2, PCR necleotide mix, rnasin inhibitor, reverse transcriptase, ruclease free water 를첨가하여 25 에서 5 분, 42 에서 60 분, 70 에서 15 분간반응시켜 cdna 를합성시켰다. 8. Western blot을통한단백질의발현측정 ERK, PKA 활성을보기위하여 cell line (B16F10) 을 100 mm tissue culture dish에 cell seeding 후 24시간동안배양하여 cell을안정화시켰다. 배지를제거한후 α -MSH (100 nm) 과추출물을농도별로처리한배지로 24~48시간배양한후다시배지를제거하고 PBS로 2번세척해주었다. RIPA buffer 10 ml에 complete mini 1 tab를가함 100 µl로용해해서 4 12,000 rpm에서 20분간원심분리하였다. 원심분리하여얻은상층액은 Bradford assay 로정량하여 30 µl의단백질을 10% 의 SDS-PAGE사에서전기영동하여분리하였다. 분리된단백질은 semi dry transfer cell 기기 (Hofer, USA) 를이용하여 PVDF membrane에옮긴다음실온에서 1시간 blocking buffer (5% skim milk in TBST) 에서 incubation 시켰다. 1차항체를희석하여 4 에서 over night한다음, 다시 10분간격으로 TBST로 3회세척하고 mouse anti-rabbit IgG HRP, bovine anti-goat IgG HRP의각각의 2차항체를 1 : 1,000로희석하여실온에서 2시간동안배양하였다. 3회세척한뒤 LAS 4,000 기기를이용하여밴드확인및정량하였다. 9. 통계처리각실험군결과값은 unpaired student's T-test 통계프로그램을사용하여통계처리하였으며, p < 0.05 이하의수준에서유의성검정을실시하였다. 7. Reverse transcription-polymerase chain reaction (RT-PCR) 미백인자인 Microphthalmia-associated transcription factor (MITF), tyrosinase related protein 1 (TRP-1), tyrosinase related protein 2 (TRP-2), tyrosinase 의 mrna 발현을알아보기위하여 polymerase chain reaction (PCR) 을실시하였다. 실험에사용한 primer sequences 는 Table 1 과같다. PCR tube 에 Go Flexi DNA polymerase, primer, 합성한 cdna 를첨가하여잘섞은후 PCR 을실행하였다. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 는 94 에서 30 초, 55 에서 45 초, 72 에서 45 초 (35 cycles), tyrosinase 는 94 에서 30 초, 60 에서 45 초, 7 2 45 초 (40 cycles), TRP-1, TRP-2, MITF 는 94 에서 30 초, 58 에서 45 초, 72 에서 45 초 (40 cycles) 을하였다. PCR 로합성시킨후 0.002% ethidium bromide 를첨가한 1.5% agarose gel 에 100 V 에서 40 분간전기영동후 LAS 4,000 을이용하여밴드를확인하여분석정량하였다. 결과 1. Tyrosinase 저해활성측정결과산약추출물의 tyrosinase 저해활성을측정한결과를 Fig. 1에나타내었다. 산약추출물의경우 1000 µg/ml의농도에서 42.3% 이상의효과를나타내었다. 이는 Jung등 15) 의논문에서오매및복분자가각각 81%, 63% 의활성을나타내었다는연구결과비교하면다소낮은활성이지만오가피 22%, 두충 17% 의활성을나타내었다는연구결과와비교하였을때산약추출물의 tyrosinase 저해활성이우수함을확인할수있었다. Table 1. Sequence of the primers used for RT-PCR Gene Primer Sequence(5' 3') MITF Forward Reverse AGC GTG TAT TTT CCC CAC AG TAG CTC CTT AAT GCG GTC GT TRP-1 Forward Reverse ACT TCA CTC AAG CCA ACT GC AGC TTC CCA TCA GAT GTC GT TRP-2 Forward Reverse GCT CCA AGT GGC TGT AGA CC AAT GCA GTG GCT TGG AAA TC Tyrosinase Forward Reverse GAC GGT CAC TGC ACA CTT TG GCC ATG ACC AGG ATG AC GAPDH sense anti-sense TGA AGG TCG GTG TGA ACG GAT TTG GC CAT GTA GGC CAT GAG GTC CAC CAC Fig. 1. Inhibition rate of Dioscoreae Rhizoma extract on tyrosinase. DR : Dioscoreae Rhizoma extracted with ethanol. Results are means ± S.D. of triplicate data. 2. MTT assay 에의한세포생존율측정결과 산약추출물의세포독성을측정한결과 Fig. 2 에나타낸결과와같이 50 µg/ml 의농도에서 82% 이상의세포생존율을보였음을확인할수있었다. 따라서이하멜라노마세포

98 大韓本草學會誌 Vol. 30 No. 4, 2015 (B16F10) 에서의미백관련인자측정은세포의농도를 5, 25, 50 µg/ml 의농도로하여실험을실시하였다. Fig. 2. Cell viability of Dioscoreae Rhizoma extract on melanoma cell (B16F10). After B16F10 cells (1 10 5 cells) were started in serum free medium for 1h the cells were treated of Dioscoreae Rhizoma extract for 24h. Results are means ± S.D. of triplicate data. 3. MITF, TRP-1, TRP-2, tyrosinase 의 mrna 발현측정결과 산약추출물의 melanin 합성에관계된주요효소인 MITF, TRP-1, TRP-2, tyrosinase 의 mrna 에미치는영향을알아보기위하여멜라노마세포 (B16F10) 에 5, 25, 50 µg/ml 의농도별로처리한후 24 시간뒤에 reverse transcription-polymerase chain reaction 을통해 mrna 발현량을측정하여 Fig. 3 과같이나타내었다. 이때세포의여러조건에서도그발현정도의차이가거의없는 house keeping gene 인 GAPDH 를 positive control 로사용하였다. Fig. 3 에서보는바와같이산약추출물을 5, 25, 50 µg/ml 의농도별로처리한 B16F10 군에서는 MITF, TRP-1, TRP-2, tyrosinase mrna 발현이산약추출물을처리하지않은군보다감소하는것을확인할수있었으며, 특히 MITF, TRP-1, tyrosinase mrna 발현이비교군인 50 µg/ml 의농도의 kojic acid 보다감소한것을확인할수있었다. 이는 Kim 등 16) 의연구에서짚신나물물추출물의경우 TRP-1 및 TRP-2 의 mrna 발현에변화가거의없었다고하는연구결과와비교하였을때산약추출물이멜라노마세포 (B16F10) 에대하여멜라닌색소생성억제및 MITF, TRP-1, TRP-2, tyrosinase 의억제효과를가지고있음을확인하였다. Fig. 3. MITF, TRP-1, TRP-2 and tyrosinase mrna expression rate of Dioscoreae Rhizoma extract on melanoma cell (B16F10). After B16F10 cells (1 10 6 cells) were started in serum free medium for 1h the cells were treated with 5, 25 and 50 μg / ml of Dioscoreae Rhizoma extract for 48h. Each values represents mean ± SD of three individual experiments. 4. Melanin 합성에관계되어진신호전달기전 검증 본연구에서 melanin 합성과관련된단백질의신호전달경로의저해활성을확인하기위하여 western blot 기법을통해 MITF, TRP-1, TRP-2, tyrosinase의상위단계유전자인 PKA 와 p-erk 의단백질발현을확인한결과 Fig. 4와같이나타내었다. 산약추출물을 5, 25, 50 µg/ml 의농도별로처리한 B16F10군에서 PKA의단백질발현량이감소한것을확인할수있었다. 또한 ERK의단백질발현이농도별로증가하는것을확인할수있었으며대조군인 kojic acid보다발현량이증가한것을확인할수있었다. 산약추출물에의해 ERK 가 activation됨으로써 ERK 신호전달경로를조절하여색소침착저해활성을가짐으로써산약추출물의미백효과를확인할수있었다 17,18). camp 활성에의해활성화되어지는 Protein kinase A (PKA) 를활성화하며 PKA는핵내로들어가 CREB의단백질을인산화하여 MITF의활성을촉진시키므로산약추출물에의한미백효과는멜라닌생성에있어서분자적기작의상위에자리잡고있는신호전달을억제함으로써기인하는것으로생각되어진다. Fig. 4. PKA and ERK protein expression rate of Dioscoreae Rhizoma extract on melanoma cell (B16F10). After B16F10 cells (1 10 6 cells) were started in serum free medium for 1h the cells were treated with 5, 25 and 50 μg / ml of Dioscoreae Rhizoma extract for 48h. Each values represents mean ± SD of three individual experiments. 고찰 최근화장품에첨가되는천연물원료중에는대부분식물성소재가사용되어지고, 화장품으로사용되는약재의유효성분은주로주근깨, 검버섯등과같은황갈반을방지하는작용이있다. 그래서본연구에서는백합목마과에속하는덩굴성다년생식물인산약을추출하여기능성화장품중미백화장품의첨가물로서의사용가능성을알아보고자하였다. Tyrosinase 는멜라닌을형성하는데있어서주요단백질로서피부색소조절과정연구에서중요한지표로여겨지며티로신 (tyrosine) 으로부터도파퀴논 (DOPA quinone) 의전환과정에관여하고유멜라닌 (eumelanin) 의중간다사물인 DHI를 indole-5,6-quinone 으로전환시키는과정에관여하는단백질이다 19). 따라서산약추출물의멜라닌생성에있어주요역할을하는 tyrosinase 의저해활성을측정한결과산약추출물이 1000 µg/ml의농도에서약 42.3% 이상의저해활성을나타낸것을확인할수있었다. 멜라노나세포 (B16F10) 의세포생존율을측정하기위해

산약의멜라노마세포 (B16F10) 에서 MITF, TRP-1, TRP-2, Tyrosinase, PKA, ERK 발현억제효과 99 사용된 MTT 검색법은 96-well plate를사용하여그결과를 ELISA reader(multiwell microplate reader) 를이용하여많은시료를단단하게판독할수있어세포독성및증식을검색하는법으로서흔히사용되고있는분석방법이다 20). MTT는 mitochondrial succinate dehydrogenase 에의해노란색수용성 MTT tetraxolium 을자주색을띄는비수용성의 MTT formazan 으로환원시켜그환원되는정도를측정하는것으로세포독성을평가한다 21). 이방법을이용하여산약추출물의세포생존율을측정하였으며그결과 80% 이상의세포생존율을보인 5, 25, 50 µg/ml의농도에서이하미백관련된세포실험을진행하였다. 멜라닌 (melanin) 은흑갈색의유멜라닌 (eumelanin) 과적황색의페오멜라닌 (pheomelanin) 이있으며, melanin 합성효소중티로시나아제 (tyrosinase) 는두가지멜라닌합성에필요하고, TRP-1 과 TRP-2 는유멜라닌합성에더많은관여를하는것으로알려져있다 22). 멜라닌합성신호전달체계에는매우다양한신호전달물질이관여하고있는데그중 camp/ PKA 경롸멜라닌합성의주요경로로써자외선에피부가노출되었을때멜라닌세포의 camp가증가되고하류신호전달물질인 PKA를활성화시키며, CREB를거쳐 MITF의발현을증가시킨다 23-25). 또한주로세포의증식, 분화에관여하는것으로알려진 ERK 신호는 MITF의인산화를유도하여 MITF의 ubiquitination 이이뤄져 proteosomal degradation 을일으키게됨으로써멜라닌합성을감소시키는것으로알려져있다 26,27). 산약추출물을 5, 25, 50 µg/ml의농도별로처리한 B16F10군에서는 MITF, TRP-1, TRP-2, tyrosinase mrna 발현이산약추출물을처리하지않은군보다감소하는것을확인할수있었으며, 특히 MITF, TRP-1, tyrosinase mrna 발현이비교군인 50 µg/ml의농도의 kojic acid 보다감소한것을확인하였으며 PKA의단백질발현량이감소한것을확인할수있었다. 또한 ERK의단백질발현이대조군인 kojic acid보다발현량이증가한것을확인할수있었다. 결론 최근천연한방소재를첨가한화장품연구개발이증가하고있어본연구에서는산약추출물을첨가하였을때기존의효과보다향상된효과를갖는화장품원료로서의사용가능성을검토하였다. 1. Tyrosinase 의저해활성을측정한결과산약추출물이 1000 µg/ml의농도에서약 42.3% 이상의저해활성을나타낸것을확인하였다 2. 산약추출물을 5, 25, 50 µg/ml의농도별로처리한 B16F10 군에서는 MITF, TRP-1, TRP-2, tyrosinase mrna 발현이산약추출물을처리하지않은군보다감소하는것을확인할수있었으며, 특히 MITF, TRP-1, tyrosinase mrna 발현이비교군인 50 µg/ml의농도의 kojic acid 보다감소한것을확인하였다. 3. PKA의단백질발현량이감소하였으며 ERK의단백질 발현이대조군인 kojic acid보다발현량이증가한것을확인하였다. 따라서본연구에서천연한방소재인산약추출물을이용하여미백활성에관한연구를한결과우수한미백활성을확인하였고, 아울러향후이를첨가한미백기능성화장품등의기능성원료로서충분한사용가치가있다고판단된다. References 1. Libert A, Ghanem G, Arnould R, Lejeune FJ. Use of an alpha-melanocyte-stimulating hormone analogue to improve alpha-melanocyte-stimulating hormone receptor binding assay in human melanoma. Pigment Cell Res. 1989 ; 2 : 510-8. 2. Busca R, Ballotti R. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res. 2000 ; 13 : 60-9. 3. Hearing VJ, Tsukamoto K. Enzymatic control of pigmentation in mammals. FASEB J. 1991 ; 5 : 2902-9. 4. Jimenez-Cervantes C, Solano F, Kobayashi T, Urabe K, Hearing VJ, Lozano JA, Garcia-Borron JC. A new enzymatic function in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1 (TRP1). J Biol Chem. 1994 ; 269 : 17993-8000. 5. Han JY, Sung JH, Kim DJ, Jeong HS, Lee JS. Inhibitory effect of methanol extract and its fractions from grape seeds on mushroom tyrosinase. J Korean Soc Food Sci Nutr. 2008 ; 37 : 1679-83. 6. Qiu F, Komatsu K, Saito K, Kawasaki K, Yao X, Kano Y. Pharmacological properties of traditional medicines. XXII. Pharmacokinetic study of mulberroside A and its metabolites in rat. Biol Pharm Bull. 1996 ; 19 : 1463-7. 7. Jang SM, Noh SH, Park SD. Botany of herbal medicine resources. Seoul : Hakmun Publishing Ltd. 1999 : 299. 8. Chung YC, Chiang BH, Wei JH, Wang CK, Chen PC, Hsu CK. Effects of blanching, drying and extraction processes on the antioxidant activity of yam (Dioscorea alata). Int J Food Sci Technol. 2008 ; 43 : 859-64. 9. Ha YD, Lee SP, Kwak YG. Removal of heavy metal and ACE inhibition of yam mucilage. J Korean Soc Food Sci Nutr. 1998 ; 27 : 751-5. 10. Yang SM, Shon MY, Sung NJ. Effects of sujungro on blood glucose and lipid level in streptozotocindiabetic rats. Food Nurt. 2004 ; 9 : 40-4. 11. Jin MH, Suh SJ, Yang JH, Lu Y, Kim SJ, Kwon

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