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J Korean Med Ophthalmol Otolaryngol Dermatol 2015;28(4):51-61 pissn 1738-6640 eissn 2234-4020 http://www.ood.or.kr http://dx.doi.org/10.6114/jkood.2015.28.4.051 Original Article / 원저용담화에탄올추출물의항염증효과에관한연구 최형욱 1 인명희 2 리순화 3 김은주 4 임규상 1,6 우원홍 5,6* 1 원광대학교한의학전문대학원한약자원개발학과, 2 원광대학교한의학전문대학원 BK21-plus 팀, 3 건국대학교교육대학원미용교육학과, 4 열린사이버대학교뷰티건강디자인학과, 5 원광대학교한의과대학해부학교실, 6 원광대학교한국전통의학연구소 Study on Anti-inflammatory Effect of Ethanol Extract of Gentianae sino-ornata Hyung-Wook Choi 1 Myung-Hee In 2 Shun-Hua Lee 3 Eun-Joo Kim 4 Kyu-Sang Lim 1,6 Won-Hong Woo 5,6* 1 Dept of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang 2 BK21-plus Team, Professional Graduate School of Oriental Medicine, Wonkwang University 3 Dept. of Beauty Education, Graduate School of Education, Konkuk University 4 Dept of Beauty Health Design, Open Cyber University 5 Dept. of Anatomy, College of Oriental Medicine, Wonkwang University 6 Research Center of Traditional Korean Medicine, Wonkwang University Abstract Objective : This research has been conducted to comparative analysis of anti-inflammatory effects to suggest a usefulness of ethanol extracts from Gentianae sino-ornata (GSO) as a functional material for inflammatory activities. Methods : Cell viability was measured by neutral red (NR) assay, and nitric oxide (NO) production in RAW264.7 cells was monitored by measuring the nitrite content in culture medium. The expressions of cyclooxigenase-2 (COX-2) was determined by western blot analysis, and Inducible nitric oxide syntase (inos) and cytokine were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Results : When the GSO extract was added the concentration of 5-20 μg / μl, the viability of cells was maintained 90% or more at all levels. NO production was suppressed by the treatment of GSO in LPS-stimulated RAW264.7 cells. GSO inhibited the expression of inos, COX-2, IL-1β and IL-6 in LPS-stimulated RAW264.7 cells. Conclusions: From this results, we consider that GSO can be a useful therapeutic and preventive approach to various inflammatory diseases as a functional material for inflammatory activities. Key words : Gentuanae sino-ornata; LPS; COX-2; inos; IL-6 c 2015 the Society of Korean Medicine Ophthalmology & Otolaryngology & Dermatology This is an Open Access journal distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 51

Ⅰ. 서론 염증반응이란체내에외부로부터물리적, 화학적자극이나세균감염에대해면역세포가이를인지하여다양한염증매개물질을분비함으로써손상된조직을수복하거나재생하려는기전이다 1). 이과정은 Lipopolysaccharide(LPS) 와같은외부자극원또는 arachidonic acid 대사체같은내부자극원들을주요매개로하여 macrophages, granulocytes, fibroblasts 와같은염증성세포의염증부위로의유입과축적을주요특징으로한다 2). 그러나지속적으로또는과도하게발생된만성염증반응은조직의손상을유발하며이와관련한 Reactive oxygen species(ros) 와염증성 cytokine은내독소자극을포함한다양한질병의매개체로써중요한역할을한다 3). 특히염증반응은치매, 심혈관질환, 암, 비만, 대사성증후군등의만성질환의원인이되고 4), 이는염증반응을촉진하는인자들과신호달경로가염증반응뿐아니라만성질환에도깊숙이관여하고있기때문이며 5) 따라서염증반응을억제하는물질은만성질환을예방하거나억제하기위한기능성물질로사용될가능성이높다고볼수있다. 현재사용하고있는대부분의항염증제들은장기복용할경우출혈성소화관궤양, 신장기능저하및혈압상승등의부작용과함께심근경색및혈전형성등의순환계질환도유발할수있다는것이보고되었으며 6) 이에따라효능과안전성이우수한새로운항염증제의개발에대한관심이한층더높아졌으며천연물을이용한항염증제개발을위한많은연구들이수행되었다 7-10). 용담초 (Gentiana scabra Bunge) 는다년생초본으로용담의꽃을용담화 (Gentiana sino-ornata) 라고한 Corresponding author : Won Hong Woo, Department. of Anatomy, College of Oriental Medicine, Wonkwang University, 344-2, Sinyong-dong, Iksan-si, Jeollabuk-do, Korea(Tel : +82-63-850-6845, E-mail : whwoo@wku.ac.kr) Recieved 2015/9/24 Revised 2015/10/26 Accepted 2015/11/4 다. 한방에서사용되는뿌리의성미 ( 性味 ) 는고한 ( 苦寒 ) 으로간담 ( 肝膽 ) 의실화 ( 實火 ) 를사 ( 瀉 ) 하고청열 ( 淸熱 ), 조습 ( 燥濕 ), 하초 ( 下焦 ) 의습열 ( 濕熱 ) 을제거하는효능이있어두통, 협통 ( 脇痛 ), 습진, 피부및음낭소양 ( 搔痒 ), 황달, 만성위장질환에사용하며 11), 대장간균, 백선균, 피부진균에대하여억제작용이있는것으로알려져있다 12). 용담은 secoridoid 배당체를함유하고있는식물로서, 용담이가지는항염증효과에관한것으로는 Gentiana scabra Bunge에서분리된 5-(hydroxymethy1)-2-furfural 가 LPS로활성화시킨 RAW264.7 세포에서농도의존적으로 NO생성을저하시킨다는것을확인하였으며 gentiopicroside가 carrageenin 으로유발시킨흰쥐족적부종시험에있어서항염증효과가있고 13,14) 한방에서는용담이가지는차가운성질로인하여열을내리고염증을억제하는작용이있으며, 위와장의운동기능을항진시키고소화액의분비를촉진하는성질이있는것 15) 으로보고되었다. 본연구에서는항염증효과가우수한제품을개발하기위한기초연구의일환으로용담화추출물을처리한 RAW264.7 세포에 LPS를처리한후 COX 2와 inos 발현및전염증성 cytokines 들의생산량을조사하여 in vitro에서용담화추출물의항염증효과를조사하였다. Ⅱ. 재료및방법 1. 실험재료 1) 약재본연구에서실험에사용된용담화 (Gentiana sino ornata) 는중국티벳중약점 ( 西藏拉萨市藏源中药材公司 ) 에서구입하여사용하였다. 52

최형욱외 5 인 : 용담화에탄올추출물의항염증효과에관한연구 2) 시약 Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, phosphate buffered saline solution (PBS), lipopolysaccharide (LPS), formaldehyde, Tween 20, Blocking buffer (BSA), Griess reagent, p-nirophenylphosphate (pnpp), diethyl pyrocarbonate (DEPC) 는 Sigma사 (St. Louis, MO, USA) 제품을, trizol reagent는 Invitrogen 사 (USA) 제품을, chloroform, isopropanol는 Biopure사 (Canada) 제품을, inos, IL-1β, IL-6 primer는 Bioneer사 (Korea) 제품을, trizol reagent 는 intron 사 (Biotechnology, Korea) 제품을, western blotting detection reagent는 Thermo 사 (USA) 제품을 M-MLV reverse transcriptase (Enzynomics, Korea), Acco Power Hot Start PCR premix 은 Bioneer사 (Korea) 제품을, super signal west pico solution은 Pierce사 (USA) 제품을, protease inhibitor cocktail 은 Roche 사 (Switzerland) 제품을, anti-cox-2 primary antibody 는 Cell signaling Tech사 (Beverly, Ma, USA) 제품을구입하여사용하였다. 3) 사용기기원심분리기는 Hanil 사 (Science Industrial, Korea) 제품을, clean bench는동의과학사 (Korea) 제품을, CO 2 incubator 는 Samyo 사 (Japan) 제품을, microplate reader 는 Synergy-HT 사 (BIO-TEK instruments, USA) 제품을, 감압농축기는 Eyela 사 (Japan) 제품을, icycler Q system 은 Bio-Rad사 (USA) 제품을, Nanodrop은 Nanodrop사 (USA) 제품을, ultraviolet transilluminator 는 Htech 사 (Korea) 제품을, 자동현상기는 QX-130II사 (Konica) 제품을사용하였다. 4) 세포주실험에사용된세포주인 RAW264.7 macrophage 는한국세포주은행 (Korea) 에서구입하여사용하였다. 2. 실험방법 1) 시료추출용담화는각시료중량에 10배의 95% 에탄올을가하여상온에서 72시간방치하고그추출액을 9,000 rpm으로 15분간원심분리하여얻은상층액을여과지 (Whatman No. 2) 로여과한후필터링한다음감압농축하여 3일동결건조한후그분말을본실험에사용하였다. 2) 세포배양분양받은 RAW264.7 macrophage 세포는 10% FBS, 1% antibiotic-antimycotic을첨가한 DMEM을사용하여 37, 5% CO 2 배양기에서배양하였다. 3) 세포생존율측정실험에서사용된시료의세포독성을나타내는농도범위는 neutral red (NR) assay 를이용하여확인하였다 16). 연구에사용한세포주는 RAW264.7 세포를사용하였으며, 96 well plate 에 well 당 3 10⁴세포로분주하여 24시간동안전배양하였다. 세포가 well plate에충분히부착된것을확인한후무혈청배지를이용하여용담화를 5, 10, 20, 50, 100 μg / ml로농도별로첨가하고 48시간동안 37, 5% CO 2 배양기에서배양하였다. 배양한세포는배양액을 NR solution이 1% 포함된무혈청배지로교환하여 3시간배양한다음현미경으로 NR의결정화유무를확인하였다. Formaldehyde 용액 10% 가첨가된 phosphate buffered saline (PBS) 을각 well에 100 μl로 20분처리하여세포를고정하였다. NR desorb solution (1% glacial acetic acid, 49% ethanol, 50% distilled water) 을각 well에 100 μl분주하여세포내의 NR을추출하고 540 nm에서흡광도를측정하였다. 세포생 53

존율은아래의식에따라계산하였다. 세포생존율 (%) = 시료첨가군흡광도 100 시료무첨가군흡광도 4) Nitric oxide (NO) 생성저해능측정 용담화추출물이 RAW264.7 세포배양에서 NO 생성에미치는영향을측정하기위해 Green 등의방법 17) 에따라세포배양액내 NO 양을 nitrite (NO - 2 ) 와 nitrate (NO - 3 ) 형태로측정하였다. RAW264.7 세포를 96 well plate 에 5 10⁴/well로분주하고 24시간동안전배양후배지를제거하고 LPS가 1 μg / ml이포함된무혈청배지에용담화를 1.25, 2.5, 5, 10, 20 μg / ml의농도별로가하여 48시간동안후배양하였다. 새로운 96 well plate 에배양된세포배양상층액 100 ml와 Griess reagent 100 ml을첨가하여차광된상태에서 10분간반응시키고 540 nm에서흡광도를측정하였으며 LPS 처리군과비교하여백분율로표시하였다. NO 생성저해능 (%) = 100 - 시료첨가군의 O.D. at 540 nm 100 시료무첨가군의 O.D. at 540 nm 5) 역전사중합효소연쇄반응 (Reverse transcription polymerase chain reaction, RT-PCR) 용담화추출물이 RAW264.7 세포내의염증사이토카인인 inos, IL-1β, IL-6 의발현억제효과를확인하기위하여 RT-PCR 을수행하였다. 1 RNA 추출단계 LPS 1 μg / ml와용담추출물 5, 20 μg / ml로처리된 RAW264.7 세포를수거한후 phenol 성분이첨가된 trizol reagent 를이용하여 RNA를추출하였다. 수거한 RAW264.7 세포가들어있는 tube에 1 ml trizol reagent를넣고현탁한 후상온에 5분간반응시켜세포를용해하였다. 여기에 0.2 ml chloroform (Biopure, Canada) 을첨가하고강하게 15초간흔들어준뒤 3분간상온에서반응시킨후 12,000 rpm, 4 의조건에서 20분간원심분리를실시하여단백질을포함하는하단부의 phenol층과 RNA를포함하는상단부의수용성층으로분리하였다. 상단부의수용성층만수거하여 0.5 ml isopropanol (Biopure, Canada) 을첨가하고상온에 10분간반응시킨후 12,000 rpm, 4 에서 15분간원심분리를실시하여 RNA를침전시켰다. 침전된 RNA는 75% ethanol로세척한후 ethanol 성분을제거하고상온에서건조하였다. 건조된 RNA는 DEPC water 로녹여실험에사용하였으며추출된 RNA는순도확인을위해 Nanodrop (Nanodrop, USA) 을이용하여 260/280 nm의파장에서흡광도를측정후 260/280 nm의 ratio를확인하여사용하였다. 비율이 1.8 이상이면단백질이함유되지않은것이기때문에비율 1.8 이상의 RNA만을실험에사용하였다. 2 cdna 합성및 polymerase chain reaction 단계 cdna 는 1 μg의 RNA를대상으로 M-MLV reverse transcriptase (Enzynomics, Korea) 을사용하여제조하였다. 1 μg RNA와 0.5 μg oligo dt 18 μg에 DEPC water 를첨가한 10 μl의용액을 70 에서 10분간반응시켜 RNA를 denaturation 시킨후여기에 buffer, 10 mm dntp, 200 unit/ μl reverse transcriptase 를첨가하여 37 에서 1시간반응시켜 cdna를제조하여실험에사용하였다. 확보한 cdna 는 Acco Power Hot Start PCR premix (Bioneer, Korea) 와 inos, IL-1β, IL-6 primer 를혼합하여총 volume 이 20 μl가되도록 DEPC 처리된 water 로조정한후실행하였다. PCR 은 icycler Q system (Bio-Rad, 54

최형욱외 5 인 : 용담화에탄올추출물의항염증효과에관한연구 USA) 로수행하였고 PCR products 는 ethidium bromide (EtBr) 이포함된 1.5% agarose gel에서전기영동을시행한후 ultraviolet transilluminator (Htech, Korea) 로확인하였다. 본연구에사용한 primer 는 housekeeping 유전자인 β-action 유전자를사용하였다 (Table 1). 6) Western blot 분석용담화추출물이 COX-2 발현에미치는영향을확인하기위하여 Western blot 방법을이용하여단백질발현양을측정하였다. LPS 처리에의하여활성화된 RAW264.7 세포에용담화추출물을 5, 20 μg / μl의농도로처리하고 3시간배양한세포를수거하여 PBS 로세척후 RIPA buffer (50 mm Tris-Cl (ph 7.5), 50 mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, proteaseinhibitor cocktail (Roche, Switzerland)) 로 30분간용해시킨후 12,000 rpm, 4 에서 30분간원심분리하여상등액을회수하였다. 회수된상등액은 SDS sample buffer (60 mm Tris (ph 6.8), 14.4 mm 2-mercaptoethanol, 25% glycerol, 2% SDS, 0.1% bromo-phenol blue) 를첨가하고 100 에서 5분간 denaturation 시킨후단백질을 SDS-PAGE 로분자량별로분리하였다. 분리된단백질은 100 V의조건에서 1시간동안 nitrocellulose membrane (Whatman, UK) 으로전이 시키고 0.1% BSA 용액으로 blocking 시킨후 membrane 을 anti-cox-2 primary antibody 용액에담가 4 에서 18시간반응시켰다. TBS/T (10 mm Tris-Cl (ph 7.5), 150 mm NaCl, 0.2% Tween 20) 로 5분씩 3회세척하였다. 세척된 membrane 을화학적형광을낼수있는 horse radish peroxidase (HRP) 가결합되어있는 anti-mouse IgG antibody 용액에담가상온에서 2시간처리후다시 TBS/T 로 5분씩 3회세척하였다. 이차항체가처리된 membrane 은 super signal west pico solution (Pierce, USA) 을처리하고암실에서실험용필름 (Konica, Japan) 으로덮어필름에감광을유도한후자동현상기 (QX-130II, Konica) 를이용하여현상하였다. 현상된필름상에있는단백질 band는 densitometer program 인 Image J (NIH, USA) 를이용하여 band 크기의변화를측정하였다. 7) 통계처리모든실험은 3회반복하였으며, 분석수치는 Mean ± S.D로표시하였고SPSS (Statistical Package for the Social Science) WIN 17.0 프로그램을이용하였으며통계분석은 ANOVA Test 에의해분석하였다. 통계적유의성은 *p<0.01, **(##)p<0.001로표기하여유의성의차이가있는것으로판단하였다. Table 1. Lists of Primers Used in This Study Gene Forward primer Reverse primer inos CCCTTCCGAAGTTTCTGGCAGCAGC GGCTGTCAGAGCCTCGTGG CTTTGG IL-1β CAGGATGAGGACATGAGC CTCTGCAGACTCAAACTCCA ACC C IL-6 GTACTCCAGAAGACCAGA GG TGCTGGTGACAACCACGGCC β-actin GTGGGCCGCCCTAGGCACC GGAGGAAGAGGATGCGGCAG AG T 55

Ⅲ. 실험결과 1. Neutral Red (NR) 에의한세포생존율측정 1) RAW264.7 세포에서세포생존율 RAW264.7 세포에용담화에탄올추출물의세포독성을알아보기위해 5, 10, 20, 50, 100 μg / μl로농도별로처리하여 48시간동안배양한후 NR assay 를수행하였다. 용담화추출물의세포생존율을측정한결과세포에에탄올추출물을 5-20 μg / μl의농도로첨가하였을때, 추출물은대체로모든농도에서 90% 이상의생존율을유지하였다. 그러나농도가 50-100 μg / μl에서는세포의생존율이현격히줄어드는것을확인할수있다. 따라서향후실험은 20 μg / μl이내에서실시하였다 (Fig. 1). 5, 10, 20 μg / μl의농도로처리하였을때, LPS 처리군에비해농도의존적으로 NO 생성이억제되는것으로나타났다 (Fig. 2). 특히용담화추출물 20 μg / μl의농도에서 NO 생성이 80% 이상억제되는것으로나타나항염작용이매우우수한것으로나타났다. Fig. 2. Effect of Gentianae sino-ornata extract on NO production in RAW264.7 cells. RAW264.7 cells were treated with LPS (1 μg / μl ) and combined with GSO for 48h. Nitrite was measured by Griess reagent. The results were expressed as the average of triplicate samples with S.D. ##p<0.001 compared with control, and *p<0.01, **p<0.001 compared with LPS treated group. 3. 세포내 Cyclooxygenase-2 (COX-2) 발현저해능 Fig. 1. Effect of Gentianae sino-ornata extract on cell viability in RAW264.7 cells. Cells were plated at 3 10⁴cells/well and incubated in media containing from 5 to 100 μg / μl for 48 hours. Cell viabilities were measured by NR assay as described in materials and methods. The results were expressed as the average of triplicate samples with S.D. *p<0.01, **p<0.001 compared with control. 2. NO 생성억제효과 용담화에탄올추출물을이용하여 RAW264.7 cell 에서 LPS에의해유도되는 NO의생성을억제시키는지의여부를평가한결과용담화추출물 1.25, 2.5, 용담화추출물이 NO 생성과관련된주요효소인 COX-2 발현에미치는영향은 Western blot 분석으로조사하였다. 실험결과 RAW264.7 세포에서 LPS 를첨가하면 COX-2 의발현이증가하였고, 용담화추출물 5, 20 μg / μl의농도에서농도의존적으로감소하는경향을나타냈다 (Fig. 3). 이상의결과용담화추출물은 RAW264.7 세포에서 COX-2의발현을억제하여 NO 생성을감소시키는것으로보인다. 56

최형욱외 5 인 : 용담화에탄올추출물의항염증효과에관한연구 Fig. 3. Effect of Gentianae sino-ornata extract on protein expression level of COX-2 in RAW264.7 cells. RAW264.7 cells are treated with LPS (1 μg / μl ) and combined with Gentiane sino-ornata (GSO) extract. Cells were lysed and cellular proteins were then separated by SDS-PAGE, followed by Western blot analysis using antibody against COX-2. Actin was used as an internal control. Fig. 4. Effect of Gentianae sino-ornata extract on inos, IL-1β and IL-6 mrna expressions in RAW264.7 cells. RAW264.7 cells are treated with LPS (1 μg / μl ) and combined with Gentiane sino-ornata (GSO) extract. After 24 hours, cells were analyzed using RT-PCR as described in marerials and methods. 4. 세포내 inos 및 cytokine 발현억제능 용담화추출물이 NO 생성과관련된효소인 inos 와염증사이토카인인 IL-1β 및 IL-6의 mrna 발현에미치는영향은 RT-PCR을수행하여조사하였다. 실험결과 RAW264.7 세포에서 LPS 처리시 inos 의발현이현저히증가하였고, 또한용담화추출물 20 μg / μl의농도에서다소감소하였다. IL-1β와 IL-6는용담화추출물 20 μg / μl농도에서감소하는경향을나타냈으나그효과는크지않았다 (Fig. 4). 이상의결과용담화추출물은 RAW264.7 세포에서 inos, IL-1β 와 IL-6의발현을억제하여 NO 생성을감소시키는것으로사료된다. Ⅳ. 고찰염증반응 (inflammation) 은세균및바이러스등의미생물들과생체이물질들에의해활성화된면역세포혹은염증매개인자 (pro-inflammatory mediators) 들의발현에서부터시작되며, 병원균들의감염, 화학적자극및상처등과같은외부자극에대한생체조직의방어기작이다 18). Macrophages 는염증반응에관여하는대표적인면역세포로써 LPS와같은염증유발물질에의해활성화되며 19), 활성화된 macrophages 로부터분비되는각종염증물질들은염증반응을유발하여기관지천식, 기관지염, 류마티스관절염, 동맥경화증, 그리고알츠하이머병이나파킨슨병과같은퇴행성뇌질환과뇌졸중, 바이러스감염등의발병에관여한다 20,21). 따라서최근에는각종염증물질들을효과적으로감소시킬수있는항염증제제개발에대한연구가활발히진행되고있으며한약재에서얻은생리활성성분이염증치료물질이되고있다. 용담초 (Gentiana scabra Bunge) 는다년생초본으로초용담 ( 草龍膽 ), 지담초 ( 地膽草 ) 등여러가지이름으로불리고있으며용담의꽃을용담화 (Gentiana sino-ornata) 라고한다. 한방에서사용되는뿌리의성미 ( 性味 ) 는고한 ( 苦寒 ) 으로간담 ( 肝膽 ) 의실화 ( 實火 ) 를사 ( 瀉 ) 하고청열 ( 淸熱 ), 조습 ( 燥濕 ), 하초 ( 下焦 ) 의습열 ( 濕熱 ) 을제거하는효능이있어두통, 협통 ( 脇痛 ), 습진, 피부및음낭소양 ( 搔痒 ), 황달, 만성위장질환에 57

사용하며 11), 대장간균, 백선균, 피부진균에대하여억제작용이있는것으로알려져있다 12). Lipopolysaccharide(LPS) 는병원균의내독소로서그람음성세균의막구조물로다당류, 인지질및소량의단백질로구성되어있으며, 여러종류의염증세포및조직구성세포들이생산하는 cytokine들의생산을촉진한다 22). 또한 Raw 264.7 cell 과같은 macrophage 또는 monocyte 에서는 tumor necrosis factor-α(tnf-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) 와같은 pro-inflammatory cytokine들을증가시키는것으로알려저있다 23,24). Nitric oxide (NO) 는아르기닌 (L-arginine) 으로부터산화질소합성효소 nitric oxide synthase (NOS) 에의하여생성되는물질로 NO synthases (NOS) 인 inducible nitric oxide synthase (inos), endothelial nitric oxide synthase (enos) 및 constitutive nitric oxide synthase (cnos or nnos) 에의해생성된다. cnos 는내피세포, 신경세포, 심근세포와같은세포내에상존하며칼슘에의하여활성화되어소량의 NO를계속생성한다. 반면 inos 는간세포, 혈관평활근세포, 섬유아세포, 마우스의대식세포와같은세포들에서면역학적자극이나염증성자극에의해합성되고이들세포에서는다량의 NO를생성한다. 또한혈관계에존재하는 enos 는염증을억제하지만 inos 는염증을촉진하는것으로알려져있다. 과잉생산된 NO는그자체로도유전자및단백질에독성을나타내지만활성산소의하나인 superoxide anion과반응해맹독성을가진 peroxynitrite 를생성하므로더욱강력한독성물질로변화되어암형성과진행에중요한역할을하는것으로보고되고있다 25). Cyclooxygenase (COX) 는 COX-1 과 COX-2 의두가지이성체가존재하는데 26), 세포가병적스트레스를받으면세포막에서지방산의일종인 Arachidon 산이유리되고이를원료로하여 COX-1 효소와 COX-2 효소가스트레스를해결하기위하여염증매개물질인각 prostaglandins (PGs) 을만들어낸다. COX-1 은 혈관, 위, 신장의정상조직에서발견되며, 혈류의유지나점액과중탄산염의생성, 신장기능조절과같은신체의항상성유지에관여하는반면 COX-2 는염증반응뿐만아니라혈액응고, 신장기능, 혈관조절및면역반응등에관여하고, 염증성 cytokine이분비되면 COX-2 가활성화된다 27). 또한 COX-2 는염증조직, 악성종양조직에서정상세포에비해많은양의 prostaglandin (PG) 의생성을유도하여혈관생성을촉진하고세포의증식을도울뿐아니라면역능력을억제함으로써암세포성장에좋은환경을제공하여또다른질병의병원성과의직접적인연관성을시사하고있다 28). COX-2 가발현되면류마티스관절염같은만성염증질환과대장, 두경부, 폐, 방광, 전립선, 위등인체에악성종양이발견된연구결과들이보고되었다 29,30). COX 에의해생성되는 PGs는염증, 유사분열생식, 세포의유착, 면역감시기구등에서중요한기능을수행하고세포분열이나증식에영향을줌으로서유방암, 대장암, 위암, 폐암등각종인체암의발생및진행에역할을한다는실험적증거들이보고 31) 되고있다. 이상의연구결과용담화에탄올추출물은 LPS로활성화시킨 RAW264.7 세포에서농도의존적으로 NO생성을저하시키고염증반응에관련된사이토카인의발현을억제시켜높은항염증효능을나타냈으며, 향후항염증전략으로개발하기위한기초자료로응용될수있을것으로기대한다. Ⅴ. 결론용담화에탄올추출물의항염증효과를알아보기위하여 RAW264.7 세포를활용하여세포독성과세포내 NO (Nitric Oxide) 생성량을측정하였다. Western blot 분석으로단백질발현양을측정하였으며아울러 RT-PCR을수행하여 RAW264.7 세포내에서용담화추출물의염증사이토카인발현억제효 58

최형욱외 5 인 : 용담화에탄올추출물의항염증효과에관한연구 과를확인하여다음과같은결론을얻었다. 1. 용담화추출물은 5-20 μg / μl의농도로첨가하였을때, 대체로모든농도에서 90% 이상의생존율을유지하여세포독성이없는것을확인하였다. 2. 용담화에탄올추출물은 20 μg / μl의농도에서 NO 생성이 80% 이상억제되는것으로나타나항염작용이매우우수한것으로나타났다. 3. 용담화에탄올추출물은 COX-2, inos, IL-1β, IL-6의발현을농도의존적으로감소시켰다. 이상의결과용담화추출물은본실험에서세포에대해독성을나타내지않으면서항염효능을가지고있는것으로보아항염증제품을개발하기위한기초연구의일환으로활용할수있을것이라사료된다. References 1. Willoughby DA. Human arthritis applied to animal models. Towards a better therapy. Ann Rheum Dis. 1975;34:471-8. 2. Trowbridge HO, Emling RC. Inflammation: a review of the process, 5th ed. Chicago: Quintessence Pub.Co. 1997;3-9. 3. Chung EK, Seo EH, Park JH, Shim JH, Kim KH, Lee BR. Anti-inflammaatory and anti-allergic effect of extracts from organic soybean. Korean J Organic Agric. 2011;2:245-53. 4. Medzhitov R. Origin and physiological roles of inflammation. Nature. 2008;454:428-35. 5. Chung HY, Cesari M, Anton S, Marzetti E, Giovannini S, Seo AY et al. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev. 2009;8:18-30. 6. Boumpas DT, Chrousos GP, Wilder RL, Cupps TR, Balow JE. Glucocorticoid therapy ofimmune-mediated diseases:basic and clinical correlates. AnnInternMed. 1993;119:1198-208. 7. Barton CC, Barton EX, Ganey PE, Kunkel SL, Roth RA. Bacterial lipopolysaccharide enhances aflatoxin B1 hepatotoxicity in rats by a mechanism that depends on tumor necrosis factor-α. Hepatology. 2001;33:66-73. 8. Byun BH. Effects of Inonotus obliquus EthanolExtract on Cytokine Expression in Raw 264.7Cell. Kor.J.Herbology. 2005;20(2): 55-60. 9. Lee E. Anti-inflammatory effect of Scutellariae Radix. Korean J.Plant Res. 2007;20(6):548-52. 10. Yun HJ, Heo SK, Yi HS, Kim CH, Kim BW, Park SD. Anti-inflammatory effect of Injinho-tang in RAW 264.7 Cells. Kor.J.Herbology. 2008;23(2):169-78. 11. Seo JW. The anti-oxidative activities and separation of the Gentiana scabra var. buergeri extracts. Wonkwang University. 2008. 12. Kim MS, Kim SC, Jee SY, Hwang SY, Cho WJ, Lee JR et al. Inhibitory effect of Gentianae Radix MeOH extract on pro-inflammatory mediator production in lipopolysaccharide activated Raw264.7 cells. J Korean Med Ophthalmol Otolaryngol Dermatol. 2008;21(1):28-38. 13. Kim NY, Kang TH, Kim DH, Kim YC. Anitric oxide synthesis inhibitor from the roots of Gentiana scabra in RAW 264.7 cells. J Kor Pharmacogn. 1999;30(2):173-6. 14. Zhu YP. Harwood academic publishers. 59

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최형욱외 5 인 : 용담화에탄올추출물의항염증효과에관한연구 Carcimogenesis and Its Selective Inhibition for Cancer Chemoprevention. Molecules and cells. 2002;13(4):8-17. 61