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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 (7), 993~999(2015) 당뇨처방에근거한생약재의 αglucosidase 활성저해효과및이를활용한미백소재평가법 김미진 임경란 윤경섭 ( 주 ) 사임당화장품기술연구소 Effect of Medicinal Herb Prepared through Traditional Antidiabetic Prescription on αglucosidase Activity and Evaluation Method for AntiMelanogenesis Agents Using αglucosidase Activity Mi Jin Kim, Kyung Ran Im, and KyungSup Yoon R&D Center, Saimdang Cosmetics Co., Ltd. ABSTRACT For the purpose of investigating the in vitro antidiabetic activity of a medicinal herb and herb mixture extracts prepared through traditional antidiabetic prescription, this study examined αglucosidase inhibitory activity. Tyrosinase, a type Ⅰ membrane glycoprotein, is synthesized and glycosylated in the endoplasmic reticulum (ER) and Golgi. The enzyme is subsequently transported to melanosomes, where it participates in melanogenesis. Previous studies showed that disruption of early ER Nglycan processing by an αglucosidase inhibitor suppresses tyrosinase enzymatic activity and melanogenesis. According to the results, most oriental medicinal herbal extracts were stronger than acarbose and Nbutyldeoxynojirimycin, known as an αglucosidase inhibitor. Interestingly, ethyl acetate layer of enzyme hydrolyzed had an inhibitory effect on melanin synthesis in B16F1 cells, although it did not inhibit tyrosinase activity directly. Together, αglucosidase inhibition activity could be used to evaluate antimelanogenesis, although crosschecking with melanin inhibitory assay is recommended. Key words: αglucosidase inhibition activity, skin whitening, traditional antidiabetic prescription, herb 서 최근서구화된식생활습관과운동부족등으로인한비만인구의증가로당뇨병환자가매년 10% 이상증가하고있는추세이다 (1). 고열량식사가늘어나면서당뇨, 고지혈증등대사증후군질환의발병률이증가하고있으며, 이들대사증후군을식품의섭취를통하여개선하고자하는노력이지속적으로이루어지고있다. 당뇨병의치료방법에는식이요법, 운동요법과함께약물요법이있으며, 임상에서사용되고있는약물로는인슐린제제, sulfonylurea 제제, biguanide 제제, troglitazone 제제등이있지만저혈당, 유산증등의치명적부작용이보고되고있다 (2). 또다른혈당관리방법은섭취한식이중탄수화물의소화와흡수를지연시켜식후혈당및혈중인슐린의상승을저하시킴으로써당뇨병의치료효과를가져오는것이있으며, 이러한물질로는식이섬유 (3,) 와장내소화효소인 αglucosidase에대한억제제 (5) 가있다. Received 11 March 2015; Accepted 5 June 2015 Corresponding author: KyungSup Yoon, R&D Center, Saimdang Cosmetics Co., Ltd., Cheongju, Chungbuk , Korea ksyoonjh@hanmail.net, Phone: 론 αglucosidases는소장내에서탄수화물로부터 αglucose를방출하는 αglucosidic bond의분해를촉진하여포도당을흡수하는데관여하는생체내필수효소이다. 또한당지질과당단백질의합성과같은다양한대사경로에도관여하므로이를조절함으로써대사질환의치료가능성을기대할수있다. αglucosidase inhibitor는제2형당뇨병의치료에이용되고있으며, Actinoplanes strain SE50에서분리한 acarbose는강력한 αglucosidase inhibitor로식후혈당을감소시키고인슐린의분비를촉진시키는것으로밝혀졌다 (6). 멜라닌생합성경로에서가장중요한초기속도결정단계에관여하는효소인 tyrosinase 는 type Ⅰ membrane glycoprotein으로 (7) asparagine의 side chain에있는 NH 2 기에당을붙이는 Nlinked glycosylation 과정과 serine이나 threonine의 side chain에있는 OH기에당을붙이는 O linked glycosylation 과정 (8) 중 Nlinked glycosylation 과정에의해만들어진다. 단백질의 Nlinked glycosylation 은 endoplasmic reticulum(er) 에서단백질합성번역과함께일어난다 (9). Glycan unit의절반인 1개짜리의당 (Glc 3Man 9GlcNAc 2) 은 ER막의바깥인세포질쪽에서만들어지며이후나머지절반은 ER 내에서만들어져단백질로

2 99 김미진 임경란 윤경섭 전이된다 (10). ER에서이런합성과정은 oligosaccharide 의말단끝에존재하는 mannose에서 3개의 glucose를 α glucosidase Ⅰ과 Ⅱ가끊어줌으로써시작된다 (11). 그러므로 αglucosidase를저해하게되면 tyrosinase의 glycosylation이저해되어구조는변형되고불활성형태로멜라노좀으로이동하게되어결과적으로 melanogenesis가억제된다 (121). 이와같은 tyrosinase glycosylation 저해제에대한보고들로는 Imokawa와 Mishima(15) 가 B16 melanoma 세포배양에서 glucosamine과 tunicamycin 같은물질들이 tyrosinase의 glycosylation 반응을저해하여멜라닌합성이완전히억제될수있음을보고하였고, Franchi 등 (16) 은 calcium DpantetheineSsulfonate가 tyrosinase의 glycosylation을저해하여 tyrosinase의안정도를저해하는역할을한다고보고하였다. 그리고 Petrescu 등 (12) 에의하면 ER processing enzyme인 αglucosidase Ⅰ, Ⅱ의저해제인 Nbutyldeoxynojirimycin(NB DNJ) 이 B16 melanoma 세포의활성을억제하며비처리군에비해 2% 의멜라닌만이합성되었다고보고하여 tyrosinase glycosylation 저해제에대한연구들이이루어지고있음을알수있다. 본연구에서는특허및논문등을통하여당뇨병에효과가있는약재중 αglucosidase 저해활성을보이는생약재를검색하였다. 이중미백관련특허가없고 αglucosidase 저해활성의 IC 50 값이 1 mg/ml 이하이며쉽게구할수있는귀전우 (Euonymus alatus Siebold)(17), 연자육 (Nelumbo nucifera Gaertner)(18), 자화지정 (Viola mandshurica W. Becker)(19), 적양 (Alnus japonica Steud)(20) 을선정하였다. 또한오늘날의당뇨병과같은소갈의원인과증상, 치료를다룬동의보감의소갈문에기재된복합처방단과단방처방중미백과관련된생약재를포함하지않으며쉽게구할수있는마인 (Cannabis Semen), 죽력 (tabasheer), 맥문동음자 ( 麥門冬飮子 ), 문동음자 ( 門冬飮子 ), 청심연자음 ( 淸心蓮子飮 ), 활혈윤조생진음 ( 活血潤燥生津飮 )(21) 을선정하였다. 선정한생약재및복합처방단의 αglucosidase 저해활성을알아보았으며, 이방법이미백소재스크리닝을위한유용한평가법인지를알아보았다. 재료및방법실험재료본실험에사용한생약재및복합처방단의약재들은 ( 주 ) 지유본초 (Chuncheon, Korea) 로부터구입하여사용하였으며, 죽력은죽림식품 (Damyang, Korea) 에서구입하였다. 복합처방단은동의보감을참고하였으며, 맥문동 (), 지모 (Anemarrhena asphodeloides Bunge), 천화분 (Trichosanthes kirilowii), 인삼 (Panax ginseng), 오미자 (Schizandrachinensis Baill.), 갈근 (Pueraria lobata), 복령 (Poria cocos), 생지황 (Rehmannia glutinosa), 감초 (), 지골피 (Lycii radicis Cortex), 연자육 (Nelumbo nucifera Gaertner), 황기 (Astragalus membranaceus Bunge), 황금 (Scutellaria baicalensis), 차전자 (Plantaginis Semen), 천문동 (Asparagus cochinchinensis (Lour.) Merr.), 과루인 (Trichosanthis Semen), 마자인 (Cannabis Semen), 당귀 (Angelica gigas Nakai), 숙지황 (Rehmannia glutinosa), 생지황 (Rehmanniae Radix) 으로구성되어있다 (Table 1). 추출과정에사용된용매들은시약급을사용하였다. 추출물의제조생약재추출물의제조를위하여시료중량 10배의 75% EtOH 수용액을가하여 60~90 C에서 시간동안환류냉각하면서추출하고여과지 (Whatman No. 2, Whatman GE Healthcare, Westborough, MA, USA) 로여과하였다. 복합처방단추출물은 Table 1의약재구성및비율로제조하였다. 이추출과정을 2회반복하고 0 C에서감압농축하여활성측정및분획물제조용시료로사용하였다 (75% EtOH Ext.). 이추출물에증류수를가하여가용화한후동량의메틸렌클로라이드 (CH 2Cl 2) 와에틸아세테이트 (EtOAc) 를순차적으로첨가하여용매분획을실시하여분획물을얻었다 ( 각각, ). Pectinase, cellulase 등의효소들은효모, 세균또는일부고등식물등에서분리되며, 일반적으로식물조직의세포벽이나세포사이를연결해주는펙틴을분해하여과실이나야채의조직을연화시키고당을생산및분해하거나생산수율을높이는데주로사용된다 (22). 이에청심연자음의효소처리에틸아세테이트분획물 (hydrolyzed ) 은 를 10% EtOH 수용액으로용해한뒤당분해효소인 Viscozyme L(Novozymes, Copenhagen, Denmark) 을처리하여 50~60 C에서 20시간동안반응시킨뒤여과한후감압농축하였다. 이추출물에증류수를가하여가용화한후동량의에틸아세테이트를첨가하고상층부를농축하여 hydrolyzed 를얻었다. αglucosidase 활성저해 αglucosidase가 pnitrophenyl(pnp) glycoside의 glycoside 부분을기질로인식하여 pnp와 glycoside를효소반응으로끊어주고, 여기에서끊어져나온 pnp의양을 05 nm에서흡광도를측정하여이것으로 αglucosidase 활성을간접적으로측정하였다 (23). 0.1 M phosphate buffer(ph 6.8) 에시료와 αglucosidase(1 unit/ml) 를넣고 37 C에서 5분간반응시켰다. 여기에 2 mm nitrophenyl αdglucopyranoside를첨가해 37 C에서 10분간효소반응을시킨후 1 M Na 2CO 3 을넣어반응을정지시키고생성된 pnp의양을 microplate reader(synergy, BioTek Instruments, Inc., Winooski, VT, USA) 로 05 nm에서흡광도를측정하였다.

3 당뇨처방에근거한생약재의 αglucosidase 활성저해효과및이를활용한미백소재평가법 995 Table 1. Composition of traditional prescriptions for diabetes Prescriptions Composition Dose (g) Maekmundongeumja Mundongeumja Hwalhyeolyunjosaengjineum Anemarrhena asphodeloides Bunge Trichosanthes kirilowii Panax ginseng Schizandra chinensis Baill. Pueraria lobata Poria cocos Rehmannia glutinosa Schizandra chinensis Baill. Panax ginseng Lycii radicis Cortex Poria cocos Nelumbo nucifera Gaertner Poria cocos Panax ginseng Astragalus membranaceus Bunge Scutellaria baicalensis Plantaginis Semen Lycii radicis Cortex Asparagus cochinchinensis (Lour.) Merr. Schizandra chinensis Baill. Trichosanthis Semen Cannabis Semen Angelica gigas Nakai Rehmannia glutinosa Rehmanniae Radix Trichosanthes kirilowii 세포주및세포배양세포주는마우스흑색종세포주인 B16F1 melanoma cell 을 ATCC(American Type Culture Collection, Manassas, VA, USA) 에서분양받아사용하였으며, 세포배양에사용된배지 (Dulbecco's modified Eagle's medium; DMEM) 는 10% fetal bovine serum(hyclone Lab., Logan, UT, USA), 1% antibiotic antimycotic(100 U/mL penicillin and 50 μg/ml streptomycin, Invitrogen, Carlsbad, CA, USA) 을혼합한배지를사용하여 37 C, 5% CO 2 incubator 에서배양하고 70~80% 의 cell culture confluency 를보일때실험을실시하였다. 세포생존율시험본실험에서 B16F1 melanoma cells에대한시료의처리농도를결정하기위해 MTT[3(,5dimethythiazol2 yl)2,5diphenytetrazolium bromide, SigmaAldrich Co., St. Louis, MO, USA] assay를 Mosmann(2) 의방법을변형하여실시하였다. 이분석법은노란색의수용성기질인 MTT를진청색의비수용성 formazan으로변환시키는살아 있는세포의 mitochondria dehydrogenase의능력을이용한방법이다. 생성된 formazan의양은살아있는세포수에비례한다. B16 melanoma cells을 cells/well 농도로 96 well plate에분주하여 37 C, 5% CO 2 조건하에서 2시간동안배양하였다. 배양후배양액을제거하고시료를농도별로배지에희석하여교체한후최종 200 nm αmsh(αmelanocyte stimulating hormone) 가되도록첨가하여 8 시간동안더배양하였다. 배양후 MTT assay를통하여세포생존율을확인하였다. 멜라닌양측정멜라닌양측정은 Oka(25) 의방법을변형하여사용하였다. B16F1 melanoma cells을 6well plate에 cells/well이되게준비한후 2시간동안 37 C, CO 2 항온기에서배양하였다. 배양액을제거하고시료를농도별로배지에희석하여교체한후최종 200 nm αmsh가되도록첨가하여 8시간동안더배양하였다. 대조군은 αmsh만첨가한것으로하였으며양성대조군으로는알부틴을사용

4 996 김미진 임경란 윤경섭 하였다. 배양후배양액을제거하고 PBS(phosphate buffered saline) 로세척한후 10% DMSO(dimethyl sulfoxide) 가함유된 1 N NaOH를첨가한후 50 C 항온조에서세포내멜라닌을용해시켰다. 이액을 microplate reader 를이용하여 90 nm에서흡광도를측정하였으며총단백질량으로보정하였다. Tyrosinase 저해활성측정 Tyrosinase에대한활성은각농도별시료 0.9 ml, 0.1 M 인산완충액 (ph 6.8) 1.0 ml, 1.5 mm Ltyrosine 용액 1.0 ml를넣은후, 37 C에서 10분간반응시킨뒤 mushroom tyrosinase(1,500 units/ml) 0.1 ml를첨가하여 37 C에서 10분간반응시킨후 microplate reader를사용하여 75 nm에서흡광도를측정하였다. DOPA(3,dihydroxyphenylalanine) oxidation 저해활성측정 DOPA oxidation 저해활성은각농도별시료 100 μl, 0.1 M 인산완충액 (ph 7.0) 800 μl, 5 mm LDOPA 용액 50 μl를넣은후 37 C에서 3분간반응시킨다. Mushroom tyrosinase(1,500 units/ml) 50 μl를첨가하여 37 C에서 1분간반응시킨후 ice에서반응을종결시킨다음 microplate reader를이용하여 90 nm에서흡광도를측정하였다. 결과및고찰 αglucosidase 활성저해효과 Tyrosinase는다양한조절경로중생체내당단백질합성과정인 glycosylation 과정을통해만들어진다. 이과정중에문제가생겨 tyrosinase의당부분에이상이생기면 tyrosinase는세포내멜라닌생합성장소인 melanosome 으로이동하지못하거나이동하더라도 tyrosinase의활성을나타내지못하게되고따라서멜라닌이생성되지않는다. αglucosidase는 glycosylation 과정에관여하는많은효소중하나로 αglucosidase 활성억제를통해미백소재를 스크리닝하고자하였다. 한의학과민간에서당뇨의개선및치료효과가우수하다고알려진복합처방및생약재들을 75% EtOH 수용액으로추출하고그추출물들의 αglucosidase 저해활성을평가하였다. 그결과죽력, 귀전우, 적양, 연자육이 100 μg/ml 농도에서 90% 이상으로뛰어난활성저해효과를나타내었다. 이는 αglucosidase 활성저해제로알려져있는 acarbose( 약 29% 저해 ) 와 Nbutyldeoxynojirimycin( 약 19% 저해 ) 보다탁월한효과를나타낸것이다. 다음으로마인은 57.28±2.15% 저해효과를보였으며, αglucosidase 활성저해효과가알려진자화지정은 2.71±0.21% 로위의측정농도에서는활성저해효과가거의나타나지않았다. 복합처방단인청심연자음은 37.2±2.26% 의저해효과를보였으며, 활혈윤조생진음 (11.28±7.19% 저해 ), 맥문동음자 (13.23 ±2.62% 저해 ), 문동음자 (7.26±3.77% 저해 ) 의 75% EtOH Ext. 에서는 αglucosidase 활성저해효과가거의나타나지않았다 (Table 2). Table 2의생약추출물중우수한 αglucosidase 활성저해효과가나타난생약재추출물에함유되어있는활성물질분획물을얻고자 를제조하고이를에틸아세테이트, 메틸렌클로라이드로분획하였다. 그결과자화지정은 에비해 에서 αglucosidase 활성을 50% 저해하는농도인 IC 50 값이 13.3±8 μg/ml로향상되었으며, 복합처방단인청심연자음은 EtOAc layer,, hydrolyzed 에서 IC 50 값이각각.6±1.05, 37.2±2.26, 2.0±0.33 μg/ml로효과가향상되었다. 귀전우, 적양, 연자육은 에서 IC 50 값이각각 0.99±0.06, 1.76±0.20, 1.57±0.23 μg/ml 로 에비해유사하거나약간향상된결과를나타내었다. 반면귀전우, 적양, 연자육의 에서 IC 50 값이각각 6.20±.96, 26.6±0.99, 12.3±1.33 μg/ ml로 에비해활성저해효과가감소하였다. 그러나 αglucosidase 저해제로알려진 acarbose 및 Nbutyldeoxynojirimycin에비해서는월등한효과를보였다 (Table 3). Table 2. Inhibitory effects of 75% EtOH extracted medicinal herbs on αglucosidase Medicinal herbs Inhibition rate of αglucosidase Tabasheer Euonymus alatus Siebold Alnus japonica Steud Nelumbo nucifera Gaertner Cannabis Semen Viola mandshurica W. Becker Hwalhyeolyunjosaengjineum Maekmundongeumja Mundongeumja Acarbose NButyldeoxynojirimycin 1) IC 50: concentration of the sample required for 50% the activity to be inhibited. IC 50 1) =21.77±2.32 μg/ml IC 50=1.76±0.15 μg/ml IC 50=2.71±0.07 μg/ml IC 50=1.91±0.20 μg/ml 57.28±2.15% inhibition at 0.1 mg/ml 6.31±3.21% inhibition at 1.0 mg/ml 37.2±2.26% inhibition at 0.1 mg/ml 11.28±7.19% inhibition at 0.1 mg/ml 13.23±2.62% inhibition at 0.1 mg/ml 7.26±3.77% inhibition at 0.1 mg/ml IC 50=7.73±18.90 μg/ml IC 50=569.80±15.80 μg/ml

5 당뇨처방에근거한생약재의 αglucosidase 활성저해효과및이를활용한미백소재평가법 997 Table 3. Inhibitory effects of solvent fractions from medicinal herbs on αglucosidase Medicinal herbs Acarbose Euonymus alatus Siebold Alnus japonica Steud Nelumbo nucifera Gaertner Viola mandshurica W. Becker Hydrolyzed Inhibition rate of αglucosidase (IC 1) 50, μg/ml) 7.73 ± ± ± ± ± ± ± ± ±8.6± ±0.33 1) IC 50: concentration of the sample required for 50% the activity to be inhibited. 멜라닌생성저해효과 앞선연구에따르면 mushroom tyrosinase 활성저해효과는없으면서 αglucosidase 활성저해효과가우수한연자육추출물이 tyrosinase의 Nglycosylation 과정을저해함으로써멜라닌합성을저해한다는보고가있으며, αglucosidase 활성저해효과가우수한적양추출물의멜라닌생성저해효과도연구된바있다 (26,27). 이에본연구에서는연자육을포함하며 αglucosidase 활성저해효과를나타낸청심연자음추출물의멜라닌생성저해효과에대해알아보았다. MTT assay를통하여청심연자음 hydrolyzed EtOAc layer는 100 μg/ml 농도까지세포생존에영향을주지않는것을확인하였다 (data 미첨부 ). Fig. 1에서와같이청심연자음 hydrolyzed 는 20, 50, 100 μg/ml 농도에서각각 26.13±1.78, 32.17±12.71, 9.13±3.92% 의 Melanin (%) ** ** 0 μg/ml 0 μg/ml 20 μg/ml 50 μg/ml 100 μg/ml * 200 nm αmsh Fig. 1. Inhibitory effect of of viscozyme hydrolyzed 75% EtOH extract of on melanin synthesis in B16F1 cells. The cells were treated with at indicated concentration for 8 h. The absorbance was measured at 90 nm and corresponding total protein content was used to normalize absorbance. The results were expressed as the mean±sd from the three independent experiments. * P<0.05 and ** P<0.01 vs. control group treated with αmsh. 멜라닌생성저해효과를나타내었다. 이러한결과로보아청심연자음 hydrolyzed 의 αglucosidase 활성저해효과가멜라닌생성에영향을준것으로생각된다. Tyrosinase 활성및 DOPA oxidation 저해효과본연구의생약추출물들에서 in vitro 미백효능스크리닝을위한평가법으로널리이용되는 tyrosinase 활성저해및 DOPA 자동산화억제효과가있는지알아보았다. Tyrosinase는멜라닌생합성경로에서가장중요한초기속도결정단계에관여하는효소이며, DOPA oxidation은멜라닌합성과정에서 tyrosine이 DOPA를거쳐 DOPAquinone으로전환되고, DOPAquinone으로부터자동산화반응과효소반응으로 DOPAchrome을거쳐멜라닌이생성되는데관여하는 ** Table. Inhibitory effects of medicinal herb extracts on tyrosinase (at 1 mg/ml) Medicinal herbs Inhibition rate of tyrosinase Inhibition rate of DOPA oxidation Arbutin (at 0.5 mg/ml) 31.5±1.52% Euonymus alatus Siebold Alnus japonica Steud Nelumbo nucifera Gaertner Viola mandshurica W. Becker Hydrolyzed 21.75±2.25% 15.50±1.35% 12.98±2.25% 33.85±3.06% 2.17±0.28% 7.3±2.1% 13.06±2.27% 9.92±2.35% IC 50=0.21±0.00 mg/ml 3.02±1.57% 36.73±0.50% 28.60±2.17% 8.15±3.27% 28.0±1.2% 37.0±.90% 30.86±6.08% 37.0±0.62% 17.90±1.63% 10.9±0.62% 52.7±3.86% 21.81±1.89% IC 50=0.93±0.09 mg/ml 59.26±.82%

6 998 김미진 임경란 윤경섭 단계이다 (28). αglucosidase 활성효과가양호하였던생약재추출물들의 tyrosinase 활성측정결과는 Table 에나타내었다. 비교한결과 arbutin은 500 μg/ml 농도에서 31.5% 의저해효과가나타났으며, 귀전우및자화지정추출물에서는저해활성이나타나지않았다. 그외추출물에서는 arbutin 과유사하거나낮은저해효과를나타내었으며, 청심연자음의 에서는 IC 50=0.21±0.00 mg/ml로양호한저해효과가나타났으나청심연자음 hydrolyzed EtOAc layer에서는 tyrosinase 활성저해효과가나타나지않았다. DOPA 자동산화저해활성측정결과자화지정추출물에서는효과가나타나지않았으며, 그외의추출물군에서는유사한저해효과가나타났다. 이러한결과로볼때청심연자음 hydrolyzed 는 tyrosinase 활성억제제가아닌 tyrosinase의 glycosylation inhibitor로작용함으로써멜라닌생성저해효과를나타낸것으로사료되며, 이러한작용기전을확인하기위해서는청심연자음 hydrolyzed 에서의 tyrosinase Nglycosylation 저해효과에대해알아봐야할것이다. 요약본연구에서는선정한생약재및복합처방단의 αglucosidase 저해활성을알아보았으며, 이방법이미백소재스크리닝을위한유용한평가법인지를알아보았다. 한의학과민간에서당뇨의개선및치료효과가우수하다고알려진생약재및처방중죽력, 귀전우, 적양, 연자육, 마인, 청심연자음의 αglucosidase 활성저해효과는식후혈당조절제인 acarbose와비교하여볼때우수한효과를나타내었다. 미백효과가알려진연자육을함유한청심연자음 hydrolyzed 는 100 μg/ml 농도에서약 50% 멜라닌생성저해효과를보였다. 또한청심연자음 hydrolyzed EtOAc layer는 αglucosidase 활성저해효과가우수하였으나 mushroom tyrosinase 활성저해효과는나타나지않았다. 이로써청심연자음 hydrolyzed 는 αglucosidase 활성을저해시켜 tyrosinase의 glycosylation을저해함으로써멜라닌생성억제효과가나타나는것으로생각된다. 이상의결과로볼때 αglucosidase 활성억제효과가있으면서당뇨병에효과가있는생약재들은 Nlinked glycoprotein인 tyrosinase의 glycosylation을저해하여 tyrosinase의세포내이동이나활성을억제함으로써멜라닌생성을억제할것으로사료되며, 본연구에서선정된생약재들은당뇨병치료를위한목적뿐만아니라화장품에서새로운미백소재로서의활용가치가있을것으로판단된다. 또한미백에효과가있는소재스크리닝을위해현재널리사용되고있는 mushroom tyrosinase 활성저해효과와다른접근방법으로써 αglucosidase 활성측정방법도하나의평가법으로유용할것으로생각된다. 감사의글 본연구는 2013년중소기업기술혁신개발사업 ( 과제번호 SA112778) 의연구비일부에의하여이루어진것으로지원에감사드립니다. REFERENCES 1. Xu ML, Wang L, Xu GF, Wang MH Antidiabetes and angiotensin converting enzyme inhibitory activity of Sonchus asper (L) Hill extract. Kor J Pharmacogn 2: Kim TW, Kwon YB, Lee JH, Yang IS, Youm JK, Lee HS, Moon JY A study on the antibodiabetic effect of mulberry fruits. Korean J Seric Sci 38: Torsdottir I, Alpsten M, Andersson H, Einarsson S Dietary guar gum effects on postprandial blood glucose, insulin and hydroxyproline in humans. J Nutr 119: Blackburn NA, Redfern JS, Jarjis H, Holgate AM, Hanning I, Scarpello JH, Johnson IT, Read NW The mechanism of action of guar gum in improving glucose tolerance in man. Clin Sci (Lond) 66: Puls W, Krause HP, Müller L, Schutt H, Sitt R, Thomas G Inhibitors of the rate of carbohydrate and lipid absorption by the intestine. Int J Obes 8: Puls W, Keup U, Krause HP, Thomas G, Hoffmeister F Glucosidase inhibition. A new approach to the treatment of diabetes, obesity, and hyperlipoproteinaemia. Naturwissenschaften 6: BranzaNichita N, Negroiu G, Petrescu AJ, Garman EF, Platt FM, Wormald MR, Dwek RA, Petrescu SM Mutations at critical Nglycosylation sites reduce tyrosinase activity by altering folding and quality control. J Biol Chem 275: Goochee CF Bioprocess factors affecting glycoprotein oligosaccharide structure. Dev Biol Stand 76: Petrescu AJ, Butters TD, Reinkensmeier G, Petrescu S, Platt FM, Dwek RA, Wormald MR The solution NMR structure of glucosylated Nglycans involved in the early stages of glycoprotein biosynthesis and folding. EMBO J 16: Kornfeld R, Kornfeld S Assembly of asparaginelinked oligosaccharides. Annu Rev Biochem 5: Winchester B, Fleet GW Aminosugar glycosidase inhibitors: versatile tools for glycobiologists. Glycobiology 2: Petrescu SM, Petrescu AJ, Titu HN, Dwek RA, Platt FM Inhibition of Nglycan processing in B16 melanoma cells results in inactivation of tyrosinase but does not prevent its transport to the melanosome. J Biol Chem 272: BranzaNichita N, Petrescu AJ, Dwek RA, Wormald MR, Platt FM, Petrescu SM Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alphaglucosidase Ⅱ. Biochem Biophys Res Commun 261: Wang Y, Androlewicz MJ Oligosaccharide trimming plays a role in the endoplasmic reticulumassociated degradation of tyrosinase. Biochem Biophys Res Commun 271: Imokawa G, Mishima Y Functional analysis of tyrosinase isozymes of cultured malignant melanoma cells during the recovery period following interrupted melano

7 당뇨처방에근거한생약재의 αglucosidase 활성저해효과및이를활용한미백소재평가법 999 genesis induced by glycosylation inhibitors. J Invest Dermatol 83: Franchi J, Coutadeur MC, Marteau C, Mersel M, Kupferberg A Depigmenting effects of calcium DpantetheineSsulfonate on human melanocytes. Pigment Cell Res 13: Kim TJ, Song HJ, So JN, Lee JH Preventive effect of Euonymus alatus extract on experimental diabetes. Korean J Immunol 18: Mani SS, Subramanian IP, Pillai SS, Muthusamy K Evaluation of hypoglycemic activity of inorganic constituents in Nelumbo nucifera seeds on streptozotocininduced diabetes in rats. Biol Trace Elem Res 138: Lee BB, Park SR, Han CS, Han DY, Park EJ, Park HR, Lee SC Antioxidant activity and inhibition activity against αamylase and αglucosidase of Viola mandshurica extracts. J Korean Soc Food Sci Nutr 37: Kwak TH, Joo JH, Park MG Active extracts from natural plants showing antiobesity and antidiabetes. Korea Patent 523, Heo J Japbyeongpyeon. In Donguibogam. 3rd ed. Yeogang, Seoul, Korea. p Park MK, Kim CH Extraction of polyphenols from apple peel using cellulase and pectinase and estimation of antioxidant activity. J Korean Soc Food Sci Nutr 38: Angelov A, Putyrski M, Liebl W Molecular and biochemical characterization of αglucosidase and αmannosidase and their clustered genes from the thermoacidophilic archaeon Picrophilus torridus. J Bacteriol 188: Mosmann T Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: Oka M, Ichihashi M, Chakraborty AK Enhanced expression of protein kinase C subspecies in melanogenic compartments in B16 melanoma cells by UVB or MSH. J Invest Dermatol 106: Lee JY, Im KR, Jung TK, Yoon KS The inhibitory effects of Alnus Japonica Steud. extract on melanogenesis. J Soc Cosmet Scientists Korea 39: Lee JY, Im KR, Jung TK, Yoon KS The inhibitory effects of Nelumbo nucifera Gaertner extract on melanogenesis. KSBB J 28: Alaluf S, Heath A, Carter N, Atkins D, Mahalingam H, Barrett K, Kolb R, Smit N Variation in melanin content and composition in type Ⅴ and Ⅵ photoexposed and photoprotected human skin: the dominant role of DHI. Pigment Cell Res 1:

Statistical Data of Dementia.

Screening of Prolyl Endopeptidase Inhibitors from Medicinal Plants. Lee Si-young, Lee Jung-han, Paik Young-Sook College of Environment and Applied Chemistry, Kyung Hee University. Statistical Data of Dementia.