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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 45(11), 1595~1603(2016) Lipopolysaccharide 로유도한대식세포의염증반응에서미나리에탄올추출물및분획물의항염증효과 장지훈 1 조현우 1 이보영 2 유강열 2 윤지영 2 1 한약진흥재단 2 전주농생명소재연구원 nti-inflammatory Effects of Oenanthe javanica Ethanol Extract and Its Fraction on LPS-Induced Inflammation Response Ji-Hun Jang 1, Hyun-Woo Cho 1, o-young Lee 2, Kang-Yeol Yu 2, and Ji-Young Yoon 2 1 National Development Institute of Korean Medicine 2 Jeonju groio-materials Institute STRCT The present study examined the anti-inflammatory effects of Oenanthe javanica ethanol extract (OJE) and its fraction on the lipopolysaccharide (LPS)-induced inflammatory response in RW macrophage cells. OJE remarkably reduced protein expression of inducible nitric oxide (inos) and cyclooxygenase-2 (COX-2), resulting in inhibition of production of nitric oxide (NO). In order to identify the anti-inflammatory effects of bioactive fractions, OJE was fractionated into hexane, dichloromethane, ethyl acetate, and n-butanol fractions. The results show that the ethyl acetate and dichloromethane fractions reduced production of NO without cytotoxicity. Especially, the ethyl acetate fraction effectively reduced protein expression of inos and COX-2. Proinflammatory cytokine production was also reduced by ethyl acetate fractions in LPS-induced RW cells. These data suggest that OJE and its fraction possess pharmacological activity and might be useful for development of anti-inflammatory agents or dietary supplements. Key words: Oenanthe javanica, anti-inflammatory, nitric oxide 서 체내의염증반응은세균감염과같은자극으로물리적인자극이나화학적인자극등다양한원인에의해발생하며손상부위를복구또는재생하려는기전중하나이지만, 만성적염증반응은오히려인간에게질병을일으키는원인이되기도한다 (1). Lipopolysaccharide(LPS) 는몇몇 gramnegative bacteria에서외표면에서찾을수있는 glycolipid 로서 in vitro 실험시에염증관련실험에서염증유도제로사용한다 (2). LPS는대표적으로대식세포또는단핵구의 toll-like receptor 4(TLR4) 를자극하며, tumor necrosis factor-α (TNF-α), interleukin(il)-1β 및 IL-6를포함한 pro-inflammatory cytokine, nitric oxide(no) 및 eicosanoid의분비를촉진한다. 이러한염증매개성물질들이급성으로일어나거나과도한반응이이어지게되면숙주에치명적인결 Received 12 September 2016; ccepted 31 October 2016 Corresponding author: Ji-Young Yoon, Jeonju groio-materials Institute, Jeonju, Jeonbuk 54810, Korea yjy@jami.re.kr, Phone: 론 과를초래한다고알려져있으며, 염증반응초기에는 NO와 cytokine을생산하여생체방어에중요한역할을한다 (3). 그중 NO는반응성이높은물질로 NO synthase에의해형성되며, 과잉생성된 NO는혈관투과성및부종등의염증반응을촉진하는것으로알려져있다 (4-6). 이러한활성조절은염증반응을조절하기위한핵심요소로알려져있으며, 이를조절함에따라염증반응을완화시키는천연소재를찾기위한연구가활발히진행되고있다. 미나리는우리식생활에서다량으로섭취되는식품으로독특한향미및약리작용으로인해기능성식품소재나향신료로서의활용도가높은식품원료이다. 또한, 예로부터숙취해소및배변활동을개선시키는작용이있다고알려져있다 (7). 최근연구에따르면미나리는미네랄중에서칼륨이많이함유되어있으므로소금을많이먹는우리식단에적합하며, 비교적풍부한 carotene, 엽록소와섬유질등을함유하고있어장의활동을촉진한다고보고되었고, 미나리추출물에서분리된 persicarin은간을보호하는작용이있는것으로알려져있다 (8). 최근보고에따르면미나리즙이과산화지질과알코올을급여한흰쥐에서혈중및간조직의지질함량을감소시키

2 1596 장지훈 조현우 이보영 유강열 윤지영 고, 간조직에서항산화효소의활성을증가시켜알코올성지방간및간보호효과에효능이있음을보고하였다 (9). 하지만항염증에관한연구는미흡함에따라본연구에서는미나리추출물이 LPS에의해염증반응이유도된대식세포에서항염증작용및생리활성에어떠한영향을미치는지를알아보고자하였다. 세포배양연구에사용한마우스대식세포주 RW cell은한국세포주은행 (KCL, Seoul, Korea) 으로부터분양받아 penicillin/streptomycin 100 unit/ml와 10% FS가함유된 DMEM 배지를사용하여 37 C, 5% CO 2 조건하에 incubator에서배양하였다. 재료및방법시료및추출미나리원시료는미나리재배농가인전주미나리작목반에서생산하는논미나리를 100 g 구입하여적용하였다. 구입한미나리 100 g을대형건열건조기 (SM-DR 1700, Samgongsa, Gyeonggi, Korea) 를이용하여 60 C에서 4일간건조를하였고, 그중건조된미나리 30 g을 micro hammer-cutter mill(mhk Trading Co., ucheon, Korea) 을이용하여 50 mesh로분쇄하였다. 분쇄된미나리분말을 100% 에탄올을이용해 70 C에서 3시간동안 3회추출한후, filter paper(whatman No. 2, Whatman, Maidstone, UK) 로여과하였고, 얻어진여과액은감압농축기 (N-1100 series, EYEL, Tokyo, Japan) 에서농축하여에탄올추출물 9 g을얻었다. 이추출물을증류수에현탁시킨후, 현탁액과헥산을 1:1 비율로분획깔때기에넣고헥산층과물층으로분획하였으며, 분획된헥산층을다시감압농축하여시료를얻었다. 이상의동일한과정을헥산, 메틸렌클로라이드, 에틸아세테이트, 부탄올로순차적으로가하여각각분획물을얻었고, 농축이완료된추출물은동결건조기 (Ilsin, Incheon, Korea) 를이용하여건조시료로제조하였다. 실험재료 Dulbecco's modified Eagle's medium(dmem) 과 fetal bovine serum(fs), penicillin, streptomycin은 Gibco/ RL(Eggenstein, Germany) 에서구입하였고, 3-(4,5-di- methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium(MTS, CellTiter 96 Queous One Solution Cell Proliferation ssay), Griess reagent system은 Promega(Madison, WI, US) 에서구입하였다. Tripure Isolation Reagent는 Roche(asel, Switzerland) 에서구입하였다. High Capacity cdn Reverse Transcription Kit, Taqman Universal Master Mix II, lexa Fluor 514 antibody는 Thermo Fisher Scientific (Waltham, M, US) 에서구입하였다. inos, COX-2, GPDH, Iκα, p-iκα antibodies 는 Cell Signaling Technology Inc.(Danvers, M, US) 에서구입하였다. Lipopolysaccharide(LPS), dimethyl sulfoxide(dmso) 및기타시약은 Sigma-ldrich Co.(St. Louis, MO, US) 에서구입하였다. 세포생존율분석미나리용매별추출물및분획물의 RW cell에대한세포독성효과를측정하기위해 MTS assay를실시하였다. 96 well plate에 cells/well로분주하고미나리용매별분획물을농도별로처리하였다. 24시간배양한후에각각세포배양액용량의 10% 의 MTS 용액을첨가한다음 37 C에서 2시간배양한후 microplate reader(infinite 200 pro, TECN, Grödig, ustria) 를이용하여 490 nm에서흡광도를측정하였다. NO 농도측정미나리용매별추출물및분획물을농도별로전처리하고 30분후에 LPS(500 ng/ml) 를각세포에처리하여 24시간후배양액에분비된 NO를측정하였다. 96 well plate에세포배양액과 Griess reagent(promega) 를 1:1로혼합하여넣고 10분동안반응시킨후 microplate reader(infinite 200 pro, TECN) 를이용하여 540 nm에서흡광도를측정하였다. Cytokine(TNF-α, IL-1β, IL-6) mrn 발현분석 RW 세포를 6 cm 배양용기에 cells/dish 씩분주한후 24시간배양하여세포를안정화시켰다. 미나리용매별분획물을농도별로세포에전처리하고 30분후에 LPS(500 ng/ml) 를처리한뒤 24시간동안배양한후, 세포를 PS로 2회세척하고 Tripure Isolation Reagent(Roche) 를이용하여 RN를분리하였다. 5 μg의 total RN를 High Capacity cdn Reverse Transcription Kit(Thermo Fisher Scientific) 을이용해 cdn로합성하였다. 합성된 cdn 1 μl를 Taqman primer 1 μl, Taqman Universal Master Mix II(Thermo Fisher Scientific) 10 μl, 3차증류수 8 μl와함께 real-time PCR을수행하였다. 정량중합효소반응에쓰인 TaqMan gene은 technologies.com에서주문후사용하였고, 분석하고자하는유전자특이적 gene의정보는 Table 1에나타내었다. 또한, real-time PCR 반응조건은 50 C에서 2분, 95 C에서 10분동안 1회수행하고, 변성온도 95 C에서 15초, 어닐링온도 60 C에서 15초인사이클을 40회반복수행하였다. Immunoblotting 분석미나리용매별추출물및분획물의항염증효과를확인하기위해 western blot을이용하여 inos와 COX-2, Iκα,

3 미나리에탄올추출물및분획물의항염증효과 1597 Table 1. TaqMan gene information of cytokines for real-time PCR Gene symbol TNF-α IL-1β IL-6 Hprt1 Gene description Tumor necrosis factor-alpha Interleukin-1 beta Interleukin-6 Hypoxanthine-guanine phosphoribosyltransferase (reference gene) TaqMan gene expression assay number Mm _m1 Mm _m1 Mm _m1 Mm _m1 Reference sequence NM_ NM_ NM_ NM_ p-iκα 단백질의발현정도를분석하였다. 미나리용매별분획물을농도별로처리한실험군과대조군을 inos, COX- 2는 24시간배양 Iκα, p-iκα는 30분배양후 62.5 mm Tris-HCl(pH 6.8), 2% SDS, 5% β-mercaptoethanol, 2 mm phenyl-methylsulfonyl fluoride, protease inhibitors (complete TM, Roche), 1 mm Na 3VO 4, 50 mm NaF과 10 mm EDT를함유하는완충제를사용하여세포를용해시켰다. 세포용해액을 15,000 g로 4 C에서 30 분간원심분리하여단백질이포함된상층액을얻은후 radford method 를이용해정량하였다. 정량한단백질 20 μg을 10% SDS- PGE에전기영동시킨후 PVDF(polyvinylidene difluoride) membrane(io-rd, Hercules, C, US) 으로옮겼다. 그리고 membrane은 5% bovine serum albumin(s) 이함유된 T-TS(0.1% Tween 20+TS) 용액을사용하여상온에서 2시간동안 blocking을실시하였다. inos, COX-2, GPDH에대한 1차항체를 membrane에노출시키고 2차항체인 horseradish peroxidase-conjugated anti-rabbit 또는 anti-mouse IgG를반응시키고 ECL detection reagents(millipore, illerica, M, US) 를사용하여단백질의발현정도를확인하였다. Immunofluorescence 분석 Iκα의 degradation 정도를확인하기위하여 immunofluorescence 실험방법을이용하여 Iκα와핵을염색하여단백질의양을측정하였다. Cover slip에세포를분주하여 24시간배양후에샘플및 LPS(500 ng/ml) 를처리하고 30분이지난다음그룹별로 PS로세척을 3회하고, 3.7% formaldehyde로실온에서 20분동안고정시켰다. 다시 PS로세척을 3회하고, 형광물질이들어갈수있도록 0.5% triton X-100을 15분간처리한후, 3% S로실온에서 1시간동안 blocking을하였다. 그런후에 1% S에든 anti-iκα(1:100) 및 DPI를처리하고 4 C에서밤새반응하였다. 그후마지막으로 PS로 3회세척후 cover slip을 fluorescence solution(dakocytomation, Carpinteria, C, US) 으로고정한다음 fluorescence microscope(carl Zeiss, Oberkochen, Germany) 로관찰하였다. 통계처리본실험에서얻은결과에대해서는평균치 ± 표준편차 (mean±sd) 로나타내고, 각실험군과의유의성의차이는 SPSS(18.0, Statistical Package for Social Science Inc., Chicago, IL, US) 통계패키지프로그램을활용 Duncan's multiple range test로분석하여 P-value 값이 0.05 미만일때통계적으로유의한차이가있는것으로판정하였다. Table 2. Yield of ethanol extract and fractions from Oenanthe javanica Extract and fractions Oenanthe javanica ethanol extract Hexane fraction Methylene chloride fraction Ethyl acetate fraction utanol fraction 결과및고찰 미나리에탄올추출물및분획물추출수율추출전건조시료와동결건조가완전히진행된시료를대상으로수득률은 Table 2와같다. 미나리에탄올추출물이 RW cell에서세포독성및 LPS로유도된 NO 생성에미치는영향미나리에탄올추출물을농도별로처리하고 24시간후 MTS로세포생존율을관찰하였다. 그결과 500 μg/ml 이상에서세포독성이있는게관찰되었고, 1,000 μg/ml 농도에서는 20% 이상의독성이관찰되었다 (Fig. 1). NO의과도한증가는염증반응을유발하게될뿐만아니라조직의손상을가져오는것으로알려져있다 (10). 따라서추출물단독처리에의한 NO 생성정도를측정하기위해 RW 세포에미나리추출물을 62.5, 125, 250, 500, 1,000 μg/ ml의농도로세포에처리하고미나리추출물에의한 NO 생성억제효과를보기위해미나리추출물을 62.5, 125, 250, 500, 1,000 μg/ml의농도로세포에전처리한후 LPS 를 24시간동안처리하여이로인해생성되는 NO 양을측정하였다. LPS만처리한군에서는미나리에탄올추출물단독처리군과비교하여 NO의생성량이현저하게증가하였으며, 미나리에탄올추출물을전처리하고 LPS를처리한실험군에서는 62.5 μg/ml 이상에서농도의존적으로유의성있게 NO의생성을억제하였다 (Fig. 2). 이러한결과를통해미나 Dried powders (g) Freeze dried powders (g) Yield (%)

1598 장지훈 조현우 이보영 유강열 윤지영 Fig. 1. Effect of Oenanthe javanica ethanol extract (OJE) on cell viability in RW 264.7 cells. RW 264.7 cells were incubated for 24 h in the presence or absence of OJE at indicated dose.

Different letters are significantly different at P<0.05 by Duncan's multiple range test. CON: control groups, LPS: LPS treated groups. 리에탄올추출물이 NO를효과적으로억제함으로써항염증기능에관여하는것을확인하였다. 미나리에탄올추출물이 RW 264.

또한, pro-oxidant나 pro-inflammatory stimuli에의해 Fig. 2. Effect of Oenanthe javanica ethanol extract (OJE) on LPS-induced NO production in RW 264.7 cells.

The culture supernatant was subsequently isolated and analyzed for LPS treated group. Data represent the mean±sd of triplicate determinations from three separate experiments.

4 1598 장지훈 조현우 이보영 유강열 윤지영 Fig. 1. Effect of Oenanthe javanica ethanol extract (OJE) on cell viability in RW cells. RW cells were incubated for 24 h in the presence or absence of OJE at indicated dose. Cell viability was evaluated by MTS assay as described in Materials and Methods. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test. CON: control groups, LPS: LPS treated groups. 리에탄올추출물이 NO를효과적으로억제함으로써항염증기능에관여하는것을확인하였다. 미나리에탄올추출물이 RW cell에서 LPS로유도된 inos 및 COX-2 발현에미치는영향 inos는 NO를합성하는데가장큰기여를하는효소이며이는세포내 L-arginine을 L-citrulline으로전환시키면서 NO를생성한다. 이렇게생성된 NO가과량생성되면염증성 cytokine의발현을유도하며조직의손상, 유전자변이, 신경세포손상등을유발하는염증반응을일으킨다 (11,12). 또한, pro-oxidant나 pro-inflammatory stimuli에의해 Fig. 2. Effect of Oenanthe javanica ethanol extract (OJE) on LPS-induced NO production in RW cells. fter pretreatment with the indicated concentration of OJE for 30 min, RW cells were treated with LPS (500 ng/ml) for 24 h. The culture supernatant was subsequently isolated and analyzed for LPS treated group. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test. CON: control groups, LPS: LPS treated groups. MEKK-1, NF-κ의활성화를경유하여생성되는 COX-2 는 prostaglandin 합성을증가시키기때문에염증반응에있어중추적인역할을한다 (13). NO 생성억제기작에관한 inos 단백질의관련성을조사하기위하여 Immunoblot 분석을이용하여세포질내의 inos 단백질발현량을조사하였다. LPS 처리시에는 inos 단백질이발현이강하게유도되었으나, LPS에미나리에탄올추출물을 62.5 μg/ml 이상처리한실험군에서 LPS에의한 inos의발현량이농도의존적으로현저히감소하는것을관찰할수있었다 (Fig. 3). 또한, LPS만처리한군에서 Fig. 3. Effect of Oenanthe javanica ethanol extract (OJE) on LPS-induced p-iκα, Iκα, inos, and COX-2 proteins expression in RW cells. fter pretreatment with the indicated concentration of OJE for 30 min, RW cells were treated with LPS (500 ng/ml) for 24 h. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with inos, COX-2, and GPDH antibodies. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test.

미나리에탄올추출물및분획물의항염증효과 1599 는 COX-2 발현량이강하게유도되었으나, 62.5, 125, 250, 500 μg/ml 미나리에탄올추출물전처리로 LPS에의한 COX-2 발현량이농도의존적으로현저히줄어들었다 (Fig. 3). 이를바탕으로미나리에탄올추출물이 COX-2 및 inos 발현을억제함으로써 NO의생성을억제함에따라항염증효과를나타낸다고생각한다.

하지만 LPS의자극에의해 Iκα가인산화 degradation 되면 NFκ 신호전달이활성화되고, NF-κ 가핵으로이동하여 COX- 2, inos, clxl, cips 등의전사를유도한다 (13). 본실험에서는 Iκα의 phosphorylated form을측정하여 Iκα degradation에의한 NF-κ 기전을확인하였다. RW 264.

이는미나리추출물이 Ikα의 phosphorylation에의한 degradation을감소시키고 NF-κ의 nuclear translocation을차단하며, 이에따라염증반응을억제하는것으로예상한다 (Fig. 4).

7 cell 에서세포독성및 LPS로유도된 NO 생성에미치는영향미나리에탄올추출물의항염증효과를규명함에따라미나리에탄올추출물의용매별분획물에대한항염증효과를조사하기위해세포독성및 NO 생성을분석하였다. 미나리용매별분획물을 62, 125, 250, 500, 1,000 μg/ml의농도로 RW 264.7 cell에처리하고 24시간후 MTS로세포생존율을관찰하였다.

5 미나리에탄올추출물및분획물의항염증효과 1599 는 COX-2 발현량이강하게유도되었으나, 62.5, 125, 250, 500 μg/ml 미나리에탄올추출물전처리로 LPS에의한 COX-2 발현량이농도의존적으로현저히줄어들었다 (Fig. 3). 이를바탕으로미나리에탄올추출물이 COX-2 및 inos 발현을억제함으로써 NO의생성을억제함에따라항염증효과를나타낸다고생각한다. 미나리에탄올추출물이 RW cell에서 LPS로유도된 Iκα 발현에미치는영향 inos나 TNF-α 유전자의발현과관련되어있는 NF-κ 는바이러스, 박테리아감염시유도되는염증반응에의해활성화되며, 정상상태에서는세포질에서비활성상태인 Iκ α, Iκβ, Iκε, p105, p100 등과결합하여존재한다. 하지만 LPS의자극에의해 Iκα가인산화 degradation 되면 NFκ 신호전달이활성화되고, NF-κ 가핵으로이동하여 COX- 2, inos, clxl, cips 등의전사를유도한다 (13). 본실험에서는 Iκα의 phosphorylated form을측정하여 Iκα degradation에의한 NF-κ 기전을확인하였다. RW 세포에 LPS 처리시 p-iκα 발현이증가하였고, 62.5, 125, 250, 500 μg/ml 미나리에탄올추출물을전처리했을때 LPS에의해증가한 p-iκα 발현량을억제시켰다 (Fig. 3). Iκα의 phosphorylated form과 total form의발현양상을살펴본결과 p-iκα의발현량이증가함에따라상대적으로 Iκα 발현량이감소하는것을관찰하였다. 이는미나리추출물이 Ikα의 phosphorylation에의한 degradation을감소시키고 NF-κ의 nuclear translocation을차단하며, 이에따라염증반응을억제하는것으로예상한다 (Fig. 4). 결과적으로미나리에탄올추출물은전사인자인 NF-κ 의활성화를감소시키고, downstream signaling molecule 인 inos와 COX-2의전사를억제하며, NO의생성을감소시켜항염증효과를가짐을유추할수있었다. 미나리용매별분획물이 RW cell 에서세포독성및 LPS로유도된 NO 생성에미치는영향미나리에탄올추출물의항염증효과를규명함에따라미나리에탄올추출물의용매별분획물에대한항염증효과를조사하기위해세포독성및 NO 생성을분석하였다. 미나리용매별분획물을 62, 125, 250, 500, 1,000 μg/ml의농도로 RW cell에처리하고 24시간후 MTS로세포생존율을관찰하였다. 미나리부탄올분획물은세포독성이없고생존율이올라가는경향을보였으며, 미나리헥산분획물은 125 μg/ml 농도이상에서세포독성이나타났으며, 메틸렌클로라이드와에틸아세테이트는 1,000 μg/ml 농도에서세포독성이 RW cell에서세포독성이관찰되었다 (Fig. 5). 미나리용매별분획물을 62.5, 125, 250, 500 μg/ml의 Fig. 4. Effect of Oenanthe javanica ethanol extract (OJE) on Iκα degradation in LPS-induced RW cells. fter pretreatment with the indicated concentration of OJE for 30 min, RW cells were treated with LPS (500 ng/ml) for 30 min. Degradation of Iκα was visualized with () immunoblotting and () immunofluorescence staining with anti-iκα antibody. Green: Iκα. lue: DPI (nuclei). Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test.

$1600 장지훈 조현우 이보영 유강열 윤지영 C D Fig. 5. Effect of Oenanthe javanica fractions on cell viability in RW 264.$ 7 cells were incubated for 24 h in the presence or absence of () OJE-HX (n-hexane), () OJE-uOH (butyl alcohol), (C) OJE-MC

7 cells were incubated for 24 h in the presence or absence of () OJE-HX (n-hexane), () OJE-uOH (butyl alcohol), (C) OJE-MC

Cell viability was evaluated by MTS assay as described in Materials and Methods.

Different letters are significantly different at P<0.05 by Duncan's multiple range test.

$Effect of Oenanthe javanica fractions on LPS-induced NO production in RW 264.$

6 1600 장지훈 조현우 이보영 유강열 윤지영 C D Fig. 5. Effect of Oenanthe javanica fractions on cell viability in RW cells. RW cells were incubated for 24 h in the presence or absence of () OJE-HX (n-hexane), () OJE-uOH (butyl alcohol), (C) OJE-MC (methylene chloride), and (D) OJE-E (ethyl acetate) at indicated dose. Cell viability was evaluated by MTS assay as described in Materials and Methods. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test. CON: control groups, LPS: LPS treated groups. C D Fig. 6. Effect of Oenanthe javanica fractions on LPS-induced NO production in RW cells. RW cells were pretreated with the indicated concentration of () OJE-HX (n-hexane), () OJE-uOH (butyl alcohol), (C) OJE-MC (methylene chloride), and (D) OJE-E (ethyl acetate) for 30 min before being incubated with LPS (500 ng/ml) for 24 h. The culture supernatant was subsequently isolated and analyzed for LPS treated group. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test. CON: control groups, LPS: LPS treated groups.

미나리에탄올추출물및분획물의항염증효과 1601 농도로세포에처리하여생성되는 NO 양을측정하였을때 LPS 단독처리군은미나리용매별분획물단독처리군과비교하여 NO의생성량이현저하게증가하였으며, 미나리헥산분획물을전처리하고 LPS를처리한실험군에서는 62.5 μg/ml 이상에서농도의존적으로 NO의생성을억제하였지만세포독성에따른 NO의감소인것을확인하고추후실험에서제외하였다.

이를바탕으로미나리에탄올추출물의용매별분획물중메틸렌클로라이드분획물과에틸아세테이트분획물에서만항염증효능이있음을확인할수있었다. 미나리용매별분획물이 RW 264.

5 μg/ml 이상전처리한실험군에서 LPS에의한 inos의발현량이농도의존적으로현저히감소하는것을관찰할수있었다 (Fig. 7). 메틸렌클로라이드그룹에비해에틸아세테이트그룹이낮은농도에서도 inos의발현량을확연히감소시키는것을확인할수있었다. 그뿐만아니라 LPS 처리시증가한 COX-2 발현량이미나리에틸아세테이트분획물전처리그룹에서 62.

7 미나리에탄올추출물및분획물의항염증효과 1601 농도로세포에처리하여생성되는 NO 양을측정하였을때 LPS 단독처리군은미나리용매별분획물단독처리군과비교하여 NO의생성량이현저하게증가하였으며, 미나리헥산분획물을전처리하고 LPS를처리한실험군에서는 62.5 μg/ml 이상에서농도의존적으로 NO의생성을억제하였지만세포독성에따른 NO의감소인것을확인하고추후실험에서제외하였다. 또한, 미나리부탄올분획물을전처리하고 LPS를처리한실험군도 NO의감소가유의성있게나타나지않아추후실험에서제외하였다. 미나리메틸렌클로라이드분획물과에틸아세테이트분획물은 62.5 μg/ml 이상에서농도의존적으로유의성있게 LPS에의해증가한 NO의생성을감소시켰다 (Fig. 6). 이를바탕으로미나리에탄올추출물의용매별분획물중메틸렌클로라이드분획물과에틸아세테이트분획물에서만항염증효능이있음을확인할수있었다. 미나리용매별분획물이 RW cell에서 LPS로유도된 inos 및 COX-2 발현에미치는영향미나리용매별분획물이 NO 생성억제기작에관여하는지알아보기위해 immunoblot 분석을이용하여세포질내에 inos와 COX-2 단백질의발현량을조사하였다. LPS 처리시에는 inos 단백질의발현이높게증가하였고, LPS를처 리하기전미나리용매별분획물을 62.5 μg/ml 이상전처리한실험군에서 LPS에의한 inos의발현량이농도의존적으로현저히감소하는것을관찰할수있었다 (Fig. 7). 메틸렌클로라이드그룹에비해에틸아세테이트그룹이낮은농도에서도 inos의발현량을확연히감소시키는것을확인할수있었다. 그뿐만아니라 LPS 처리시증가한 COX-2 발현량이미나리에틸아세테이트분획물전처리그룹에서 62.5, 125, 250, 500 μg/ml 농도의미나리용매별분획물전처리에따라현저히감소하였다 (Fig. 7). 하지만미나리메틸렌클로라이드분획물전처리그룹은 LPS에의해증가한 COX- 2의발현량이감소하지않았다. 이러한결과를종합해볼때미나리용매별분획물중에틸아세테이트그룹이 inos 및 COX-2의발현을가장강하게저해함으로써 NO의생성을억제함에따라항염증효과를나타낸다고생각한다. 미나리에서분리한성분인 persicarin과 isorhamnetin- 3-O-galactoside는 septic shock를유도한마우스모델과 endothelial cell에서면역세포의 recruit 하는단백질을감소시켜염증을완화시키는것으로보고가되었다 (14,15). 하지만본연구에서처럼용매별분획물을이용하여 RW 세포에서항염증기전을조사한것은없는것으로알려져있다. Fig. 7. Effect of Oenanthe javanica ethanol fractions (OJE) on LPS-induced inos and COX-2 proteins expression in RW cells. fter pretreatment with the indicated concentration of () OJE-MC and () OJE-E for 30 min, RW cells were treated with LPS (500 ng/ml) for 24 h. Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with inos, COX-2, and GPDH antibodies. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test.

1602 장지훈 조현우 이보영 유강열 윤지영 D E C F Fig. 8. Effect of Oenanthe javanica methylene chloride and ethyl acetate extract on pro-inflammatory expressions in LPS-induced RW 264.7 cells.

8 1602 장지훈 조현우 이보영 유강열 윤지영 D E C F Fig. 8. Effect of Oenanthe javanica methylene chloride and ethyl acetate extract on pro-inflammatory expressions in LPS-induced RW cells. Cells were pretreated with the indicated concentrations of OJE-MC (methylene chloride) of TNF-α mrn (), IL-6 mrn () and IL-1β mrn (C) and OJE-E (ethyl acetate) of TNF-α mrn (D), IL-6 mrn (E) and IL-1β mrn (F) for 30 min before being incubated with LPS (500 ng/ml) for 24 h. Gene expressions of TNF-α, IL-1β, and IL-6 were measured by real time PCR. Data represent the mean±sd of triplicate determinations from three separate experiments. Different letters are significantly different at P<0.05 by Duncan's multiple range test. 미나리용매별분획물이염증성사이토카인 mrn 발현에미치는영향미나리용매별분획물이 RW cell에서 LPS로유도되는각종전염증성및염증성 cytokine들의발현에대한영향을조사하기위하여전염증성및염증성 cytokine의 mrn 발현에대한미나리용매별분획물의영향을관찰하였다. 미나리용매별분획물을전처리한후 LPS로자극하여 24시간후에세포를수집하고세포내 mrn를추출분리하여 real time-pcr 방법을이용하여 TNF-α, IL-1β, IL- 6 mrn 발현량을확인해보았다. 그결과미나리메틸렌클 로라이드분획물처리군에서 TNF-α는 LPS 단독처리군과비교하여차이가없었으며, IL-6는 500 μg/ml 농도에서유의성있게감소한것을확인할수있었고, IL-1β는 62.5 μg/ml 농도에서부터농도의존적으로상기의 mrn 발현량이현저히감소함을확인할수있었다. 미나리에틸아세테이트분획물처리군에서 TNF-α는 LPS 단독처리군과비교하여 125 μg/ml 농도에서부터농도의존적으로감소하였고, IL-6, IL-1β는 62.5 μg/ml 농도에서부터농도의존적으로상기의 mrn 발현량이현저히감소함을확인할수있었다 (Fig. 8). 실험결과에틸아세테이트분획물처리군에

9 미나리에탄올추출물및분획물의항염증효과 1603 서염증관련 mrn의발현이감소된것으로미루어보아에틸아세테이트분획물에서염증관련 mrn를감소시킬수있는생리활성물질이다량함유된것을확인할수있었다. 요 본연구에서는미나리에탄올추출물및그분획물의항염증효과를알아보기위해서 LPS에의해염증반응이유도된 RW 세포에대해추출물이미치는항염증효과를살펴보았다. 미나리에탄올추출물은 nuclear factor-κ (NF-κ) 의활성을억제함으로써 inos와 COX-2의단백질발현을감소시켰고, 결과적으로 NO 생성을억제하였다. 또한, 미나리에탄올추출물의헥산, 부탄올, 에틸아세테이트그리고메틸렌클로라이드분획물을처리했을때에틸아세테이트와메틸렌클로라이드분획물전처리실험군에서 LPS에의해유도된 NO의형성이현저히감소함을확인할수있었다. 그중미나리에틸아세테이트분획물은 inos와 COX-2의단백질발현량및전염증성사이토카인을현저히감소시킴에따라우수한항염증효과를확인하였다. 따라서본연구결과는미나리에탄올추출물, 그분획물중에틸아세테이트분획물이염증을완화시키는유효성분이많은것을규명하였고향후에틸아세테이트분획물에서어떠한유효성분이있는지에대해추후실험이필요할것으로보인다. 또한, 이러한항염증효능을규명함으로써향후기능성식품소재로의이용가능성을제시한다. 약 감사의글 본연구는전주시기능성식품등개발연구지원사업에의해수행된결과로연구비지원에감사드립니다. REFERENCES 1. Lee ST, Jeong YR, Ha MH, Kim SH, yun MW, Jo SK Induction of nitric oxide and TNF-α by herbal plant extract in mouse macrophage. J Korean Soc Food Sci Nutr 29: Moran P, Prendergast MM, ppelmelk J Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med Microbiol 16: Jeong J, Hong SC, Jeong HJ, Koo JS nti-inflammatory effects of ethyl acetate fraction from Cnidium officinale Makino on LPS-stimulated RW and THP-1 cells. Korean J Plant Res 25: Kim DH, Park SJ, Jung JY, Kim SC, yun SH nti-inflammatory effects of the aqueous extract of Hwangnyenhaedok-tang in LPS-activated macrophage cells. Kor J Herbol 24: Lee TH, Kwak H, Kim HH, Lee ZH, Chung DK, aek NI, Kim J Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (inos) gene expression by blocking NF-κ transactivation in LPS-activated RW cells. Mol Cells 23: McDaniel ML, Kwon G, Hill JR, Marshall C, Corbett J Cytokines and nitric oxide in islet inflammation and diabetes. Proc Soc Exp iol Med 211: Jo HW, Lee SH, Nam DH, Kim JY, Lim SK, Lee JS, Park JC ntioxidant activity and phytochemical study on the aerial parts of Oenanthe javanica. Korean J Pharmacogn 39: Park JC, Kim JY, Lee YJ, Lee JS, Kim G, Lee SH, Nam DH Protective effect of Oenanthe javanica extract on acetaminophen-induced hepatotoxicity in rats. Yakhak Hoeji 52: Lee E, Park YH, Lim SC Effects of Oenanthe javanica Sap on lipid composition, liver function and oxidative capacity in oxidized fat and ethanol fed rats. Korean J Plant Resour 18: Nathan C Nitric oxide as a secretory product of mammalian cells. FSE J 6: Lim H, Shin S Effects of the essential oil components from Ligusticum chuanxiong on proinflammatory mediators of RW264.7 macrophage cells. Nat Prod Sci 16: Yun HY, Dawson VL, Dawson TM Neurobiology of nitric oxide. Crit Rev Neurobiol 10: Kranzhöfer R, rowatzki M, Schmidt J, Kübler W ngiotensin Ⅱ activates the proinflammatory transcription factor nuclear factor-κ in human monocytes. iochem iophys Res Commun 257: Kim TH, Ku SK, ae JS nti-inflammatory activities of isorhamnetin-3-o-galactoside against HMG1-induced inflammatory responses in both HUVECs and CLP-induced septic mice. J Cell iochem 114: Kim TH, Ku SK, ae JS Persicarin is anti-inflammatory mediator against HMG1-induced inflammatory responses in HUVECs and in CLP-induced sepsis mice. J Cell Physiol 228:

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α α α α α α α α 太陰調胃湯加減方 dbdb 마우스 肝에 대한 아디포사이토카인 및 발현에 미치는 영향 SREBPs 와 섞어서 하고 마커 또한 하여 전기 영동을 하였다 전기영동을 한 후에 에 를 쪼여서 각 를 확인하였다 이 를 프로그램을 이용해 수치화하 여 분석하였다 肝 조직 동결 절편 분리한 조직은 로 시간 동안 고정 시킨 후 에 세척한 후 물기를