생약학회지 Kor. J. Pharmacogn. 45(1) : (2014) 포도잎으로부터분리된 Quercetin-3-O-glucuronide 의 LPS 로유도된 BV2 미세아교세포에서의항염증효과 윤치수 1,2# 김동철 1,2# 고원민 1,2 김경수 1,2 이동

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1 생약학회지 Kor. J. Pharmacogn. 45(1) : (2014) 포도잎으로부터분리된 Quercetin-3-O-glucuronide 의 LPS 로유도된 BV2 미세아교세포에서의항염증효과 윤치수 1,2# 김동철 1,2# 고원민 1,2 김경수 1,2 이동성 3 김대성 4 조형권 4 서정원 2 김성연 2 오현철 1,2,3* 김윤철 1,2,3 * 1 원광대학교약학대학천연물신소재은행, 2 원광대학교약품연구소, 3 원광대학교한방체액조절센터, 4 ( 유 ) 한풍제약 Anti-neuroinflammatory Effects of Quercetin-3-O-glucuronide Isolated from the Leaf of Vitis labruscana on LPS-induced Neuroinflammation in BV2 Cells Chi-Su Yoon 1,2#, Dong-Cheol Kim 1,2#, Won-Min Ko 1,2, Kyoung-Su Kim 1,2, Dong-Sung Lee 3, Dae-Sung Kim 4, Hyoung-Kwon Cho 4, Jungwon Seo 2, Sung Yeon Kim 2, Hyuncheol Oh 1,2,3 *, Kim Youn-Chul 1,2,3 * 1 Standardized Material Bank for New Botanical Drugs, College of Pharmacy, Wonkwang University, Iksan , Korea 2 Institute of Pharmaceutical Research and Development,College of Pharmacy, Wonkwang University, Iksan , Korea 3 Hanbang Body-Fluid Research Center, Wonkwang University, Iksan , Korea 4 Hanpoong Pharm & Foods Co., Ltd., Jeonju, , Korea Abstract Grapes has long been used for food, and reported as containing polyphenol which has antioxidant and anti-cancer effects. Neuroinflammation is chronic inflammation at the brain, lead to neurodegenerative diseases. In this study, quercetin- 3-O-glucuronide (QG) isolated from the leaf of Vitis labruscana has anti-neuroinflammatory effects. QG were investigated using MTT assay, western blot, nitric oxide (NO) assay, prostaglandin E 2 (PGE 2 ) assay, cytokine assay in lipopolysaccharide (LPS)-induced inflammation in BV2 cells. QG dose-dependently attenuated the expression of inducible nitric oxide synthase (inos) and cyclooxygenase-2 (COX-2), accordingly inhibited the production of NO and PGE 2. QG decreases the levels of proinflammatory cytokine such as tumor necrosis factor-α (TNF-α), interlukin-1β (IL-1β). Thereby, QG may offer therapeutic potential for treatment of neurodegenerative disease related to neuroinflammation. Key words Vitis labruscana leaf, Quercetin-3-O-glucuronide, Anti-neuroinflammation, BV2 cells 포도는갈대나무목 (Rhamnales) 포도과 (Vitaceae) 에속하는목본성덩굴식물로, 포도과에는 11 속, 700 여종의포도가있다. 포도는대체로아시아, 지중해군에속하는유럽종 (Vitis vinifera L.), 미국종 (Vitis labrusca L.) 및그들상호간의교배종 (Vitis labruscana B.) 의세종류로크게나눌수있다. 1) 포도잎은맛이시고떫어서식용으로사용하지않다가, 최근웰빙에대한관심이높아지며식용으로쓰이려는시도가나타나고있으며, 항산화작용과, 항당뇨효과또한발표되었다. 2) 중동이나동 서유럽에서는포도잎을절임이나통조림으로조제하여식용으로이용하고있고, 그리스에서는포도잎을이용한전통음식이개발되어소비되고 # These authors contributed equally to this work. * 교신저자 ( ) :yckim@wku.ac.kr, hoh@wku.ac.kr (Tel): 있다. 3) 그러나우리나라에서는포도잎에함유된생리활성성분에대한연구가미흡하며국민들의인식이낮아포도재배시수확량과당도를높이기위한순따기를통해대부분폐기되고있는실정이다. 4) 포도에는당분과무기질이다량으로함유되어있고, 대표적인성분으로잘알려진레스베라트롤 (resveratrol), 안토시아닌 (anthocyanin) 계, 탄닌 (tannin) 계, 쿼세틴 (quercetin) 계등의폴리페놀 (polyphenol) 성분을함유하는것으로알려져있다. 5,6) 포도의다양한폴리페놀성분은항암, 항산화, 항노화에효과가있는것으로알려져있으며, 7,8) 특히레스베라트롤은항산화, 항염증, 혈관보호, 항암효과가있다고보고되어있다. 9) 적포도주에서분리되었다고보고된 10) 바있는 quercetin-3-o-glucuronide(qg) 는뇌를타겟으로한알츠하이머개선효과와, 11) RAW264.7 세포와 human macrophage 에서의항염증효과, 12) AMPK 의경 17

2 18 Kor. J. Pharmacogn. 로로 enos 활성을통한혈관기능개선에효과가있다. 13) 그러나 QG 의뇌염증개선에대한연구는아직까지보고된바가없다. 최근사회의고령화가진행됨에따라퇴행성뇌질환환자가증가하고있으며질병의원인규명과이에대한약물개발이빠르게이루어지고있다. 14) 뇌와척수의신경세포들은그위치에따라매우다양한기능을하고있으며, 중추신경계 (central nerve system, CNS) 에존재하는미세아교세포 (microglial) 는뇌의염증, 면역과퇴행성에중요한역할을하므로, 이세포에서발생되는염증반응을조절하여퇴행성뇌질환을막을수있다. 15) 미세아교세포가활성화되면약물이나독소에의한이물질을제거하고신경성장인자를분비하여신경세포를보호및 CNS 의항상성유지하는데중요한역할을한다. 그러나뇌손상등으로지속적인활성화가되거나, lipopolysaccharide(lps), interferon-gamma (IFNα), tumor necrosis factor-alpha(tnf-α) 와같은자극으로인해과도하게활성화되면신경계에독성을보이는 nitric oxide(no), prostaglandin E2(PGE 2 ) 와같은전염증매개체 (proinflammatory mediators), 사이토카인 (cytokine), 활성산소종 (reactive oxygen species, ROS) 등이분비되어신경독성을유발하고, 뇌조직을파괴함으로써알츠하이머, 파킨슨씨병및크로이펠츠야콥병과같은퇴행성뇌질환의원인이된다 ) 염증반응으로분비되는 NO 와 PGE 2 의경우소량분비되면혈액순환촉진, 면역력증강등의긍정적인효과를가져오지만과분비될경우염증반응을촉진시켜다양한염증성질환을야기하게된다. 19,20) 이중 NO 와 PGE 2 는염증유발인자인 TNF-α, IL-1α, LPS 등에의해발현되는 inducible nitric oxide synthase(inos) 와 cyclooxygenase- 2(COX-2) 에의해생성되므로이들단백질조절이염증의조절에중요한기전으로인식되고있다. 21) 따라서, 본논문에서는포도잎으로부터분리한 quercetin-3-o-glucuronide 의 LPS 로유도된 BV2 세포에서항염증효과에대한연구를진행하고자하였다. 재료및방법 실험재료 본실험에사용된포도잎은 2013 년전북김제시백구면지역의포도재배농가에서재배된것을채취후건조하여사용하였으며, 원광대학교자원식물원장규관교수에게의뢰하여동정하였다. 확증표본 (WP ) 은원광대학교약학대학표본실에보관하였다. 정선된포도잎 (Vitis labruscana B. campbell early) 100 g 을 20 배의 50% 에탄올과 3% 초산 (acetic acid) 을넣고, 85~95 o C 에서 3 시간동안추출하였다. 추출액을 1µm 크기의마이크로필터를이용하여여과하고잔사를동일조건으로 2 회추출하고여과후 60 o C 이하에서감압농축하여건조엑스약 21.4 g 을얻었다 ( 수 득률 21.4%). 시약및기기 DMEM 배지, trypsin-ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS) 는 Gibco Laboratorie사에서구입하였다. L-glutamate, Trolox와 3'- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 는 Sigma사에서구입하였다. 96-Well tissue culture plates와기타 tissue culture dishes는 Nunc사제품을이용하였다. 흡광도는 BioRad사의 Microplate Reader 를이용하여측정하였다. NMR 스펙트럼은 JEOL JNM ECP-400 spectrometer(400 MHz for 1 H and 100 MHz for 13 C) 기기로 DMSO-d 6 와 CD 3 OD 용매를이용하여측정하였다. HPLC는 Younglin사의 YOUNGLIN-YL9100 기기를통하여측정하였고, column은 SHISHIDO사의 CAPCELL PAK C 18 (4.6 mmi.d 250 mm, 5 µm-particle size) 를이용하였다. 포도잎추출물로부터 QG의분리및 NMR Analysis 포도잎추출물 14 g을증류수로현탁시킨후 n-hexane, CH 2 Cl 2, EtOAc, BuOH 순으로분획한후각분획들을감압농축하여용매를휘발시킨뒤무게를측정하였다. n- hexane (1.1 g), CH 2 Cl 2 (0.7 g), EtOAc (0.9 g), BuOH (2.1 g), H 2 O (9.3 g) 을얻었고, BuOH 분획물 2.1 g을 Sephadex- LH20 column chromatography를이용하여 80% 와 100% MeOH의용매조건으로순차적인분획을나누어 4가지분획물을얻고감압농축하였다. 3번째분획물 0.27 g을두차례 reverse phase (RP-C 18 ) column chromatography를 50% MeOH의용매조건으로하여 quercetin-3-o-glucuronide (41 mg) 을얻었다. Quercetin-3-O-glucuronide: 1 H NMR data (400 MHz, CD 3 OD) δ 7.70 (1H, s, H-2'), 7.59 (1H, dd, J=2.0 Hz, 8.4 Hz, H-6'), 6.83 (1H, d, J=8.4 Hz, H-5'), 6.36 (1H, brs, H-8), 6.18 (1H, d, J=1.5 Hz, H-6), 5.31 (1H, d, J=7.3 Hz, H-1"), 3.74 (1H, d, J=9.0 Hz, H-5"), (3H, combine glucose peak, H-2",3",4"); 13 C NMR (100 MHz, DMSO-d 6 ) δ (s, C-4), (s, C-6"), (s, C-7), (s, C-5), (s, C-2), (s, C- 9), (s, C-4'), (s, C-3'), (s, C-3), (s, C-6'), (s, C-1'), (s, C-5'), (s, C-2'), (s, C-10), (s, C-1"), 98.7 (s, C-6), 94.2 (s, C- 8), 76.0 (s, C-3"), 75.5 (s, C-2"), 73.8 (s, C-5"), 71.3 (s, C-4"). 검량선작성 QG를 250 mg/ml, 125 mg/ml, 62.5 mg/ml, mg/ml, mg/ml의농도로조제하였다. HPLC 측정시 QG는각각 10 µl씩주입하였고, 5%-50%-100%(v/v) AcN을용매기울기법으로측정하였다. 이를이용하여외부검량선을얻었으며검량선의식은 Y=33.7X+400.2(R 2 = ) 이다. 여기서 Y는 HPLC를 UV/vis 330 nm에서분석한 peak area이며, X는 QG의양을의미한다. 얻어진검

3 Vol. 45, No. 1, 량선을이용하여포도잎추출물에서의 QG의농도를구한후, 함량을 %(w/w) 로환산하였다. 세포배양 BV2 미세아교세포 ( cells/well) 를 10% heat-inactivated FBS, penicillin G (100 IU/ml), streptomycin (100 µg/ml), L-glutamine (2 mm) 을함유한 DMEM 배지에분주하고 5% CO 2 배양기내에서 37 C의온도로배양하였다. 세포독성 본실험에서 BV2 미세아교세포에대한세포독성및실험시처리농도를결정하고, QG의세포독성을측정하기위해 MTT assay를사용하였다. 간단히기술하면먼저 96 well plate에 cells/well로동일하게분주하고 24시간동안배양하였다. 기존의배지를제거하고새로운배지를넣어준후 DMSO에녹인시료를다양한농도 (10, 20, 40, 80 µm) 로 DMEM 배지에희석하여첨가하였다. DMSO의처리농도는배지대비 0.1% 이하가되도록하였다. 이를다시 24시간배양한후에배지를제거하고 MTT 시약 (5 mg/ml) 을넣고, 4시간동안방치한후상등액을제거하였다. 형성된 formazan의각 well에 DMSO 20 µl를첨가한후 orbital shaker를이용하여녹이고, 30분후 595 nm 에서흡광도를측정하였다. 실험은 3회반복실시하여평균값을구하였으며, control의흡광도값을기준으로세포생존율을비교하였다. Western Blot Analysis BV2 세포를 60 mm dish에 cells/well 밀도로 24시간배양한후각각의시료를농도별로처리하였다. BV2 세포에 RIPA buffer를첨가한다음, 4 o C, 14,000 g에서 25분간원심분리하고상등액을튜브에옮겼다. 단백질정량은 BSA 단백질실험키트를이용하였고각각의시료를 12% SDS-polyacrylamide gel에서영동하고 nitrocellulose membrane(nc membrane) 으로전사하였다. 전사된 NC membrane 을 5% 무지방유가포함된신선한 blocking buffer(0.1% Tween 20 in Tris-buggered saline) 에서 blocking한후 inos, COX-2 antibody를 1:1000으로희석하여넣고 1시간동안반응시켰다. 다시 2차 antiboby (Anti-mouse IgG) 를 1:1000으로희석하여넣고 1시간동안반응한다음, ECL 용액을 1:1로잘섞어서 NC membrane 위에가하여발광시키고암실에서 X선필름에감광한후현상하였다. 같은방법으로 actin antibody를이용하여 actin 을측정한다. Nitrite Assay 배양된세포를 cells/well 수준으로 96 well plate에 100 µl 씩접종한다음 24시간동안배양하고, 24시간후 medium을제거한후 DMEM으로희석된각농도별시료처리후 LPS(1 µg/ml) 를처리하여 24시간후세포에서 media로분비되어나온 NO의양을 Griess시약 (0.1%(w/v) N-(1-naphathyl)-ethylenediamine and 1%(w/v) sulfanilamide in 5%(v/v) phosphoric acid) 을사용하여반응하였다. 반응후 ELISA micro plate reader(bio Rad Laboratories Inc., California, USA, Model 550) 를사용하여 540 nm 에서측정하였다. EIA 에의한 PGE 2 측정 PGE 2 의측정은항체 (lyophilized prostaglandin E2 conjugate to horseradish peroxidase) 를사용하여 prostglandin E2 enzymeimmunoassay system(eia, Amersham Pharmacia Biotech, Inc., Piscataway, NJ) 을이용하였다. TNF-α and IL-1β assay TNF-α 와 IL-1β 의측정은항체 (lyophilized TNF-α or IL-1β conjugate to horseradish peroxidase) 를사용하여 enzymeimmunoassay system kit (R&D Systems, Abingdon, UK) 을이용하여측정하였다. 통계처리 본실험의통계처리는 GraphPad Prism, version 3.03(GraphPad Software Inc., San Diego, CA) 을사용하였다. 각실험군간의결과는평균치와표준오차로나타내었으며, 각실험군간의결과는 ANOVA test 를사용하여분석하고유의적인차이가있는항목에대해서만검정하였다. 실험군간의차이는 95% 수준 (p<0.05) 에서유의성있는것으로하였다. 결과및고찰 염증반응이란외부자극이나질병에대하여다양한세포와여러인자들이관여하는일련의방어기전이다. 염증이발생하면염증부위에면역세포들이침투되고, 화학반응에의해사이토카인이나여러인자들이생성및분비된다. 염증초기에항원제공및탐식에관여하는대식세포는 NO, TNF-α 등을분비하여염증반응을일으켜생체를방어한다. 하지만과도한염증반응은세포손상등의병리적상태에이르게하기때문에염증반응을유발하는사이토카인이나염증매개체의조절하는것이염증성질환의치료법으로제시되고있다. 22,23) 뇌염증은신경병성장애로인한신경조직의병리적인상태이다. 24,25) BV2 세포는무한증식하는 murine 세포로소교세포의병리작용에관한실험에널리쓰이고있다. 미세아교세포는신경교의대식세포로중추신경계의 10-20% 정도를차지하며, 면역방어나조직재생의중요한역할을한다. 26,27) 본연구에서는선행연구를통해포도잎추출물이 LPS 로자극한 BV2 세포에서 NO 의생성을억제하는효과가있음을확인하였고, 이결과를바탕으로추출물 14 g 을분획하여얻은 n-hexane, CH 2 Cl 2, EtOAc, BuOH 분획물을 BV2 세포에서 NO assay 결과 BuOH 분획물에서 NO 억제효과를나타내어 BuOH 분획물의분리를진행하였다. BuOH 분획물 2.1g 을 Sephadex LH-20 column chromatography, reverse phase(rp-18) column chromatography 이용하여순수한화합물 quercetin-3-o-glucuronide 을얻었고, QG 의 1 H NMR 과 13 C NMR 을측정한후선행문헌 28,29) 과의비교를통해구조를동정하였다 (Fig. 1A). QG 를이용한포도잎추

20 Kor. J. Pharmacogn. Fig. 1. Chemical structure of QG (A) and effects of QG on cell viability (B). BV2 microglia were incubated for 24 h with various concentrations of QG (10-80 µm).

Effects of QG on protein inos (A) and COX-2 (B) expression in BV2 microglia stimulated with LPS. Cells were pretreated for 3 h with indicated concentrations of QG, and 24 h with LPS (1 µg/ml).

4 20 Kor. J. Pharmacogn. Fig. 1. Chemical structure of QG (A) and effects of QG on cell viability (B). BV2 microglia were incubated for 24 h with various concentrations of QG (10-80 µm). Cell viability was determined as described under Materials and methods. Data represent the mean values of three experiments ±SD. Fig. 2. Effects of QG on protein inos (A) and COX-2 (B) expression in BV2 microglia stimulated with LPS. Cells were pretreated for 3 h with indicated concentrations of QG, and 24 h with LPS (1 µg/ml). Western blot analysis (A, B) were performed as described in Materials and methods, and representative blots of three independent experiments are shown. *p<0.05 compared to the group treated with LPS. **p<0.05 compared to the group treated with compound. 출물의 HPLC 정량실험에서추출물에 QG 가 0.74% 함유되어있음을확인하였다. 포도잎으로부터분리한 QG 가미세아교세포에서항염증효과를갖는지검색하기위해, LPS 로유도된 BV2 미세아교세포에서의염증관련인자들을측정하였다. QG 의 BV2 세포에서의독성은 MTT assay 를통해확인하였고, QG 를농도별로 BV2 미세아교세포에처리하였을때, 세포독성을나타내지않은 10, 20, 40, 80 µm 을실험가능한농도로정하여실험을진행하였다 (Fig. 1B). 항염증효과를검색하기위해서 LPS 로유도한 BV2 세포에서 inos, COX-2 단백질의발현과이에따라나오는부산물인 NO, PGE 2 의생성을측정하였다. 먼저 BV2 미세아교세포에 QG 를 10, 20, 40, 80 µm 로 3 시간동안전처리한후, LPS 1 µg/ml 을처리하고 24 시간후에 inos 와 COX-2 의발현정도를 Western blot 을통해관찰하였다. QG 는농도의존적으로 inos 와 COX-2 의발현을감소시켰다 (Fig. 2). 다음으로, BV2 미세아교세포에 QG 를 10, 20, 40, 80 µm 로처리한후, 3 시간동안 incubator 에배양하고 LPS 1 µg/ml 을처리하였고, 24 시간후에 NO 와 PGE 2 의생성을확인하였다. QG 는농도의존적으로 NO 와 PGE 2 의생성또한감소시켰다 (Fig. 3). 다음으로는 BV2 미세아교세포에 QG 를 10, 20, 40, 80 µm 로처리한후, 3 시간동안 incubator 에배양하고 LPS 1 µg/ml 을처리하여, 염증반응에서생성되는사이토카인 TNF-α, IL-1β 들을측정하였다. QG 는 LPS 로유도된 BV2 세포에서사이토카인을농도의존적으로감소시

Vol. 45, No. 1, 2014 21 Fig. 3. Effects of QG on nitrite (A), PGE 2 (B) production in BV2 microglia stimulated with LPS.

The concentration of nitrite (A), PGE 2 were determined as described under Materials and methods. Data represent the mean values of three experiments ±SD. *p<0.

5 Vol. 45, No. 1, Fig. 3. Effects of QG on nitrite (A), PGE 2 (B) production in BV2 microglia stimulated with LPS. Cells were pre-treated for 3 h with indicated concentrations of QG, and stimulated 24 h with LPS (1 µg/ml). The concentration of nitrite (A), PGE 2 were determined as described under Materials and methods. Data represent the mean values of three experiments ±SD. *p<0.05 compared to the group treated with LPS. **p<0.05 compared to the group treated with compound. Fig. 4. Effects of QG on TNF-α (A), and IL-1β (B) production in BV2 microglia stimulated with LPS. Cells were pre-treated for 3 h with indicated concentrations of QG, and stimulated 24 h with LPS (1 µg/ml). The concentration of TNF-α (A), and IL-1β (B) were determined as described under Materials and methods. Data represent the mean values of three experiments ±SD. *p<0.05 compared to the group treated with LPS. **p<0.05 compared to the group treated with compound. 켰다 (Fig. 4). 한편, QG 는 human macrophage 에서도항염증효과를나타낸다는사실이보고되어있어, 12) QG 가본연구에서사용한 BV2 에서항염증효과를나타내는것을보완해주는결과라고판단된다. 사 본연구는산업통상자원부와한국산업기술진흥원의지역특화산업육성사업 (R ) 으로수행된연구결과입니다. 사 결 론 인용문헌 본연구에서는포도잎추출물에서분리한 QG 의항염증효과를확인하고자하였고, 그결과 QG 는유의적인항염증효과를나타냈다. QG 는 inos 와 COX-2 의발현억제와, 그의부산물인 NO, PGE 2 의생성을억제시켰고, TNF-α, IL- 1β 와같은사이토카인의생성을억제하는뚜렷한항염증효과를나타냈다. 이러한결과와선행연구들을토대로포도잎과그주요성분인 QG 는앞으로퇴행성뇌질환약물이나항염증제로개발가능할것으로사료된다. 1. Hwang, I. W., Lee, H. R., Kim, S. K., Zheng, H. Z., Choi, J. U., Lee, S. H., Lee, S. H. and Chung, S. K. (2008) Proanthocyanidin content and antioxidant characteristics of grape seeds. Korean J. Food 15: Orhan, N., Aslan, M., Orhan, D. D., Ergun, F. and Yeilada, E. (2006) In-vivo assessment of antidiabetic and antioxidant activities of grapevine leaves (Vitis vinifera) in diabetic rats. J. Ethnopharmacol. 108: Simopoulos, A. P. (2003) The traditional diet of Greece and cancer. Eur. J. Cancer. Prev. 13:

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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,