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1 Journal of Life Science 2016 Vol. 26. No ~345 ISSN (Print) ISSN (Online) DOI : Antioxidant and Anti-inflammatory Effects of Extracts from the Flowers of Weigela subsessilis on RAW Macrophages Yung Choon Yoo 1, Gye Won Lee 2 and Young Ho Cho 2 * 1 Department of Microbiology, College of Medicine, Konyang University, Daejeon , Korea 2 Department of Pharmaceutics & Biotechnology, College of Medical Engineering, Konyang University, Daejeon , Korea Received November 13, 2015 /Revised December 16, 2015 /Accepted January 5, 2016 This study investigated the antioxidant and anti-inflammatory activity of ethanol extract from the flowers of Weigela subsessilis (WS-E) in lipopolysaccharide (LPS)-stimulated RAW macrophages. The total polyphenol and flavonoid content was ±0.04 μg tannic acid equivalents/ml and ±0.02 μg quercetin equivalents/ml, respectively. The antioxidant activities of WS-E were measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical scavenging activity. The antioxidant activities of WS-E increased markedly, in a dose-dependent manner. To screen for anti-inflammatory agents, the inhibitory effects of WS-E on the production of proinflammatory cytokines in the LPS-stimulated RAW macrophages was examined. WS-E had no effect on cell viability at a concentration of 100 μg/ml. Nitric oxide (NO) and interleukin (IL)-6 production were inhibited in a dose-dependent manner (p<0.05). WS-E had no effect on the production of tumor necrosis factor (TNF)-α at a concentration of μg/ml but induced TNF-α at a concentration of 100 μg/ml. Inducible nitric oxide synthase (inos) expression was also inhibited at lower concentrations (p<0.05). In addition, WS-E reduced the activation of nuclear factor (NF)-κB by inhibition of inhibitoy (I) κb phosphorylation in RAW macrophages upon stimulation with LPS (100 ng/ml) for 24 h but not that of mitogen-activated protein kinase (MAPK). These results suggest that WS-E may be a useful antioxidant and anti-inflammatory agent in functional cosmetics. Key words : Anti-inflammation, antioxidant, nitric oxide, nuclear factor-κb, Weigela subsessilis 서 염증반응은외부의물리화학적자극이나세균감염에대한 국소적인방어보호반응으로활성산소종 (reactive oxygen species, ROS), inflammatory cytokine, prostaglandin E 2 (PGE 2) 등다양한매개물질이관여하는것으로알려져있다 [6]. 염증반응에관여하는대표적인세포중하나인대식세포 는염증성매개물질인 interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α) 등과같은 pro-inflammatory cytokine 과 nitric oxide (NO), inducible nitric oxide synthase (inos), PGE 2 등의염증유발인자들을생성한다 [25]. 정상적인 NO 는 세균을죽이거나종양을제거하는중요한역할을하지만외부 요인에의한과도한 NO 의생성은염증을유발시켜신경손상, 조직손상및유전자변이를일으키는것으로알려져있다 [26]. Authors contributed equally. *Corresponding author *Tel : , Fax : * micael@konyang.ac.kr This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 론 한편강한항산화활성을가지는폴리페놀화합물은염증반응을매개하는 cytokine, inos, cyclooxygenase-2 (COX-2), NO 및 PGE 2 의활성을저해한다고보고되었다 [8]. 폴리페놀화합물은식물계에존재하는천연색소로서주로세포막에서유리형, 에스테르형또는결합형으로존재하며, 탄소수에따라페놀산, 탄닌, 리그난, 안토시아닌, 레스베라트롤및플라보노이드등의다양한물질로나뉠수있다 [21]. 특히, 플라보노이드는활성산소의소거및산화적스트레스를막는역할을함으로써항염증, 항산화, 항노화, 항암, 항고혈압및항당뇨등의여러생리적및약리적효능이있는것으로밝혀져, 최근에는다양한식물을대상으로 ROS를조절할수있는항산화활성과기능성소재로의연구가식품, 의약품, 화장품등여러분야에서진행되고있다 [4, 18, 20]. 병꽃나무 (Weigela subsessilis L. H. Bailey) 는인동과 (Caprifoliaceae) 에속하는낙엽관목으로주로한국과일본에분포하고있고, 높이 2~3 m, 잎은대생하고난형또는타원형으로길이 1~7 cm, 폭 1~5 cm이며양끝은뾰족하고, 가장자리에잔톱니가있으며양면에털이많이난다. 꽃은 5월에피고황록색이돌지만적색으로변하며 1~2개씩액생하고화엽에털이있으며, 꽃받침은선형으로서밑부분까지갈라진다. 열매는잔털이있고길이 10~15 mm로써 9월에익으며종자에날개가있다. 우리나라에는붉은병꽃나무 (Weigela florida

2 Journal of Life Science 2016, Vol. 26. No (Bunge) A. DC.), 색병꽃나무 (Weigela florida for. Alba Rehder), 병꽃나무및골병꽃나무 (Weigela hortensis (Siebold & Zucc.) K. Koch) 등 4종이자생하고있는것으로보고되어있다. 민간에서는봄에채취하여반그늘에말리거나생것을간염, 황달, 소화불량및식중독치료에사용하였다 [15]. 현재까지병꽃나무잎과줄기에대한항산화연구는있었으나병꽃나무꽃에대한화학적성분연구나이를활용한활성연구는거의이루어지지않고있는실정이다. 따라서본연구에서저자들은병꽃나무꽃을에탄올로추출하여총플라보이드와폴리페놀함량을조사하고, 항산화및항염증효과를확인하여기능성화장품소재로의적용가능성을타진하였다. ml를가한후, 725 nm에서 1시간이내에흡광도 (UV/Vis spectrophotometer, Thermo Scientific, G10S, MA, USA) 를측정하였다. Tannic acid를표준물질로사용하였으며총폴리페놀함량은시료 1 ml 중의 μg tannic acid로나타내었다. 총플라보노이드함량측정총플라보노이드함량은 Moreno 등 [16] 의방법에의해측정하였다. 각시료 0.5 ml에 10% aluminum nitrate 0.1 ml, 1 M potassium acetate 0.1 ml 및 ethanol 4.3 ml를가하여혼합하고실온에서 40분동안정치한뒤 415 nm에서흡광도를측정하였다. Quercetin을표준물질로사용하였으며총플라보노이드함량은시료 1 ml 중의 μg quercetin으로나타내었다. 재료및방법시약및재료시약으로사용된 1,1-diphenyl-1-picrylhydrazyl (DPPH), xanthine, xanthine oxidase, nitro blue tetrazolium (NBT), potassium persulfate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) 등은 Sigma-Aldrich Chemical 사 (St. Louis, MO, USA) 에서각각구입하여사용하였다. 그외실험에사용된모든시약들은일급및특급시약을구입하여사용하였다. 본연구에사용한병꽃나무꽃은소망농원 (Seoul, Korea) 에서묘목을구입하여심고키워개화기에채집하여사용하였다. 시료제조본연구에사용한병꽃나무꽃에탄올추출물은묘목에서꽃잎만채취하여음건한후무게의 10배량 (w/v) 의 70% 에탄올을가하여 3시간씩 3회환류추출후냉침하여 Whatman 여과지 (No. 5) 로여과하였다. 여과된추출물을 40 이하에서감압농축및동결건조한후 DMSO에녹여시료 (WS-E) 로사용하였다. DPPH 라디칼소거능측정 DPPH 라디칼에대한소거활성은 Blois의방법 [2] 을변형하여측정하였다. 각시료 20 μl에 0.1 mm DPPH 180 μl를넣고 vortex한후, 30분동안방치한다음 microplate reader (Tecan, Sunrise, NC, USA) 를사용하여 560 nm에서흡광도를측정하였다. 시료를첨가하지않은대조군과비교하여시료의 DPPH 라디칼소거능을구하였다. Superoxide 음이온라디칼소거능측정 Superoxide 음이온라디칼소거활성은 Nishikimi의방법 [19] 을변형하여각시료의 xanthine-xanthine oxidase system 에의해생성된 supreroxide 음이온라디칼을소거하는효과를측정하였다. 3 mm xanthine, 0.6 mm NBT, 15 mm EDTA 및 50 mm potassium phosphate buffer (ph 7.4) 를혼합한혼합액에시료 30 μl를첨가하고 0.1 U xanthine oxidase를 50 μl 가하여 5분간전반응 (preincubation) 시킨후, 37 에서 20분간반응시켰다. 반응이끝난후 microplate reader (Tecan) 을사용하여 560 nm에서흡광도를측정하였다. 시료를첨가하지않은대조군과비교하여시료의 superoxide 음이온라디칼소거능을구하였다. 세포배양본연구에사용된마우스대식세포주인 RAW 264.7세포는한국세포주은행 (Seoul, Korea) 에서분양받아사용하였다. 세포는 10% fetal bovine serum (FBS) 과 1% antibiotics (penicillin-streptomycin) 을첨가한 DMEM 배지를사용하여 37, 5% CO 2 조건에서배양하였다. 총폴리페놀함량측정총폴리페놀함량은 Folin-Denis 방법 [5] 을변형하여측정하였다. 즉, 적당하게희석된시료 1 ml에 95% ethanol 1 ml와증류수 5 ml를첨가하고 1 N Folin-Ciocalteu s reagent 0.5 ml를넣어잘섞어주었다. 5분간방치한후, 10% Na 2CO 3 1 세포독성측정세포독성은 MTT 시약을이용하여세포생존율을측정하는 Mosmann의방법 [17] 을변형하여실시하였다. RAW 세포를 cells/well 농도로 96-well plate에분주한후시료를 0~100 μg/ml의농도가되도록세포배양배지로희석하여첨가하고 12시간전처리하였다. 그후 LPS (E. coli serotype 055:B5) 를 100 ng/ml로세포에첨가하여 24시간동안염증반응을유도하였다. MTT 용액 (5 μg/ml) 을첨가하고 4시간후원심분리하여상등액을제거하고 100 μl acid-isopropanol (0.04 N HCl in isopropanol) 을첨가한후, 생성된 formazan이용출되도록하여 microplate reader (Tecan) 로 560 nm에서흡광도를측정하였다. 생존율은대조군에대한흡광도의차를

3 340 생명과학회지 2016, Vol. 26. No. 3 백분율로표시하여비교분석하였다. Nitric oxide (NO) 및 cytokine 생성량측정 NO 생성량측정은 Lee 등 [14] 의방법에따라다음과같이측정하였다. RAW 세포 ( cells/well) 를 96-well plate에분주한후시료를농도별 (0.16, 0.8, 4, 20, 100 μg/ml) 로 12시간전처리한다음, LPS로 24시간자극하여염증을유발하였다. 배양액중에분비된 NO의농도는 NO 정량 kit (intron Biotechnology, Seongnam, Korea) 를이용하여측정하였다. 또한세포배양액중에분비된 cytokine의생성량은 enzyme linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) 를이용하여측정하였다. Western bot inos의세포내발현은 Western blot으로분석하였다. 시료를농도별로전처리한 RAW 세포 ( cells/well) 에 LPS (100 ng/ml) 로자극한후세포를회수하여 RIPA buffer (protease and phosphatase inhibitor cocktail; Thermo Scientific, Waltham, MA, USA) 로 4 에서 30분간처리하였다. LPS 자극은 inos 분석에는 6시간, mitogen-activated protein kinase (MAPK) 와 nuclear factor (NF)-κB 분석에는 15분간처리하였다. 원심분리한후상층액을얻고 BCA protein assay kit (Bio-Rad, Hercules, CA, USA) 를이용하여단백질양을정량하였다. 그후 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel에각 lane 당 30 μg의세포단백질을넣어전기영동하고, gel 내의단백질을 polyvinylidene fluoride membrane filter (PVDF, Bio-Rad) 막에 transfer 하였다. PVDF 막을 5% skim milk 용액으로 blocking 하고, IκB, p38, extracellular signal regulated kinase (ERK), c-jun-n-terminal kinase (JNK), phosphorylated IκB (p-iκb), phosphorylated p38 (p-p38), phosphorylated ERK (p-erk), phosphorylated JNK (p-jnk) 각각에대한항체 (R&D Systems) 를실온에서 2시간반응시켰다. 그후각항체에대한 2차항체인 horseradish peroxidase (HRP)-conjugated antibody (R&D Systems) 를반응시킨후 ECL kit (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) 를이용하여각 band를발현시켜관찰하였다. 각 band의발현은 densitometer를이용하여분석하였다. 통계처리모든결과는평균 ± 표준편차로나타내었으며, 유의성검사는 SPSS TM version 20.0 (SPSS Inc., Chicago, USA) 를이용하여 one-way ANOVA를실시하였다. 대조군에대한시료처리군의통계적유의성은 Duncan's multiple range test로검증하였다. Table 1. The total polyphenol and flavonoid contents of extracts from the flowers of Weigela subsessilis (WS-E) Sample 결과및고찰 총폴리페놀및플라보노이드함량변화폴리페놀계화합물은대부분식물들의항산화능을부여하는 2차대사산물로식물계에 8,000여개의구조를가진성분으로존재하며페놀성화합물이라고도한다. 한분자내에 2개이상의페놀성수산기가효소단백질과같은거대분자들과결합하는성질이있기때문에항산화작용, 항암등의다양한생리활성을가지는것으로알려져있다 [7]. 또한, 주된식물계폴리페놀물질인플라보노이드는항산화, 항균, 충치예방, 항암및항염증등이보고되어있다 [3]. WS-E에존재하는총폴리페놀및플라보노이드함량을측정하기위하여표준물질로각각 tannic acid와 quercetin을사용하였다. 그결과 Table 1에서와같이 WS-E의총폴리페놀함량은 ±0.04 μg TAE/ml로나타났다. 또한, 총플라보노이드함량은 ±0.02 μg QE/ml로나타났다. Jung 등 [8] 의보고에의하면소태나무잎추출물의폴리페놀과플라보노이드함량이각각 μg/mg과 μg/mg인것에비해본연구의 WS-E의폴리페놀과플라보노이드함량은매우높은것으로사료된다. 항산화활성천연유래항산화제의작용기작은유리기와반응하는것으로라디칼의소거활성은자유라디칼에전자를공여하여항산화효과나인체에서노화를억제하는척도로활용되고있다 [1]. WS-E의항산화활성은 DPPH 라디칼소거능과 superoxide 음이온라디칼소거능을가지고측정하였다. DPPH 라디칼소거능은안정한라디칼인 DPPH를소거시키는항산화물질의활성을측정하는것으로짙은자색을띄는 DPPH 라디칼이방향족아민류등에의해환원되어색이탈색되는것을이용하여항산화물질을검색하는데이용되고있다 [13]. WS-E의 DPPH 라디칼소거능을측정한결과 Fig. 1에나타낸바와같이 WS-E의 DPPH 라디칼소거활성은 31.2, 62.5, 125 μg/ml에서각각 26.27±2.31%, 57.25±3.14%, 83.44± 0.41% 로나타났으며, 농도의존적으로활성이증가하고있음을알수있었다. Kang [9] 이보고한합환피 (Albizziae cortex) Total polyphenol (μg/ml) 1) Total flavonoid (μg/ml) 2) WS-E ±0.04 3) ±0.02 1) Total polyphenol content (μg/ml) was equivalent as tannic acid as standard. 2) Total flavonoid content (μg/ml) was equivalent as quercetin as standard. 3) Each value is mean of triplicate determinations±sd.

Journal of Life Science 2016, Vol. 26. No. 3 341 농도에서각각 26.06%, 33.13%, 43.17% 의소거효과를보였다는보고와비교할때 WS-E의 superoxide 음이온라디칼소거능은소리쟁이추출물보다우수한것으로사료된다. 세포독성 MTT assay를이용하여 WS-E의 RAW 264.

All values were expressed as the average of triplicate samples with SD. * p<0.05 compared with control. 추출물이 400 μg/ml에서 25.80±2.80% 의소거활성을보인것과비교할때 WS-E의 DPPH 라디칼소거능이우수함을알수있다.

2에나타낸바와같이 WS-E는투여농도의존적으로 superoxide 음이온라디칼소거작용을나타내었다. 즉, 31.2, 62.5, 125 μg/ml의농도에서각각 53.12±10.48%, 65.61±9.31%, 76.11±5.22% 의소거능을보였다. Rhim 등 [23] 의연구에서소리쟁이 (Rumex crispus) 추출물의항산화활성연구결과 0.1, 0.

7 대식세포에 WS-E를농도별로처리한후염증매개인자인 NO 생성정도를정량하였다. 그결과 Fig. 4에나타낸바와같이 WS-E 를농도별로 (0.16, 0.8, 4, 20, 100 μg/ml) 처리하였을때 8.98, 8.15, 7.42, 6.48, 4.71 μm로투여농도의존적으로 NO의생성을억제하였다 (p<0.05).

4 Journal of Life Science 2016, Vol. 26. No 농도에서각각 26.06%, 33.13%, 43.17% 의소거효과를보였다는보고와비교할때 WS-E의 superoxide 음이온라디칼소거능은소리쟁이추출물보다우수한것으로사료된다. 세포독성 MTT assay를이용하여 WS-E의 RAW 대식세포에대한세포독성을측정한결과 100 μg/ml의농도까지세포의증식에별다른영향을주지않는안전한농도인것으로확인되었다 (Fig. 3). 따라서전혀독성을나타내지않는안전한농도인 100 μg/ml이하의농도로 WS-E를처리하여다음실험을수행하였다. Fig. 1. Scavenging activity of WS-E on DPPH radicals. All values were expressed as the average of triplicate samples with SD. * p<0.05 compared with control. 추출물이 400 μg/ml에서 25.80±2.80% 의소거활성을보인것과비교할때 WS-E의 DPPH 라디칼소거능이우수함을알수있다. Superoxide 음이온라디칼소거능은 xanthine-xanthine oxidase 반응으로 uric acid가생성되는과정에생성되는 superoxide 음이온라디칼을 WS-E와반응시켜 NBT로확인하는방법이다 [12]. WS-E의 xanthine oxidase에의해생성되는 superoxide 음이온라디칼소거능을측정한결과 Fig. 2에나타낸바와같이 WS-E는투여농도의존적으로 superoxide 음이온라디칼소거작용을나타내었다. 즉, 31.2, 62.5, 125 μg/ml의농도에서각각 53.12±10.48%, 65.61±9.31%, 76.11±5.22% 의소거능을보였다. Rhim 등 [23] 의연구에서소리쟁이 (Rumex crispus) 추출물의항산화활성연구결과 0.1, 0.5, 1 mg/ml NO 생성억제 NO는 L-arginine을기질로하여 NO 합성효소에의해생성되는무기유리체로지속적인염증의진행에의한종양형성촉진, 면역반응, 혈관이완등여러생물학적인과정에관여하는인자중의하나로인식되고있다 [10]. WS-E의항염증활성을측정하기위하여먼저 LPS로전처리한 RAW 대식세포에 WS-E를농도별로처리한후염증매개인자인 NO 생성정도를정량하였다. 그결과 Fig. 4에나타낸바와같이 WS-E 를농도별로 (0.16, 0.8, 4, 20, 100 μg/ml) 처리하였을때 8.98, 8.15, 7.42, 6.48, 4.71 μm로투여농도의존적으로 NO의생성을억제하였다 (p<0.05). 또한염증반응의자극에의해유도되는 NO를생합성하는효소로알려진 inos의세포내발현량을 Western blot으로측정하여 NO 분비억제에관한작용기전을확인하였다. 그결과 WS-E를전처리한경우에있어서 inos의세포내발현이현저하게억제되었으며, 이억제효과는 WS-E의농도에의존하는것으로나타났다 (Fig. 5). 또한 Fig. 2. Scavenging activity of WS-E on superoxide anion radicals. All values were expressed as the average of triplicate samples with SD. * p<0.05 compared with control. Fig. 3. Effect of WS-E on cytotoxicity in RAW macrophages. RAW cells ( cells/well) were treated with the indicated doses of WS-E for 12 hr prior to 24 hr incubation with LPS (100 ng/ml). All values were expressed as the average of triplicate samples with SD.

342 생명과학회지 2016, Vol. 26. No. 3 대한 WS-E의 NO 생성억제는 NO 생합성효소인 inos의세포내발현억제에의한것으로사료된다. Fig. 4. Inhibitory effect of WS-E on NO production from LPSstimulated RAW 264.7 macrophages.

5 342 생명과학회지 2016, Vol. 26. No. 3 대한 WS-E의 NO 생성억제는 NO 생합성효소인 inos의세포내발현억제에의한것으로사료된다. Fig. 4. Inhibitory effect of WS-E on NO production from LPSstimulated RAW macrophages. The level of NO was determined by NO detection kit. All values were expressed as the average of triplicate samples with SD. * p<0.05 compared with control. Western blot에의한결과를정량적으로관찰하기위해 densitometer를이용하여분석한결과에서도 inos의발현이현저하게억제되는것으로관찰되었다. Kim 등 [11] 은 NO 저해활성이높게나타난왕쥐똥나무 (Ligustrum ovalifolium) 잎에탄올추출물이 inos의발현을효과적으로저해한다고보고하였으며, 이는 WS-E의 NO 생성억제와 inos 발현저해양상과유사한결과로사료된다. 따라서 LPS로자극한대식세포에 TNF-α 및 IL-6 측정주로단핵세포와대식세포에서생산되는 TNF-α는면역반응과염증반응을유도하는염증유도매개 cytokine 으로세포의성장과분화, apoptosis, necrosis 등의기능에관여하며, 또한대식세포와 T-세포에의해서생산되는 IL-6는 inflammatory와 pro-inflammatory cytokine으로알려져있다 [27]. 본연구에서는염증반응에서대식세포로부터분비되는중요한 inflammatory cytokine으로알려진 TNF-α와 IL-6의분비에대한 WS-E의억제활성을조사하였다. 그결과 LPS로자극된대식세포로부터분비되는 TNF-α에대해서는 WS-E 전처리에의한억제효과가관찰되지않았다 (Fig. 6). WS-E가 NO의분비를억제하는활성을보임에도불구하고 TNF-α의분비를억제하지않는이유를분석하기위하여 WS-E가대식세포에대해 TNF-α의생성을유도하는가를조사하였다. 그결과비교적높은농도인 100 μg/ml의농도에서부터 WS-E는대식세포로부터 TNF-α의분비를유도하는것으로관찰되었다 (data not shown). 결과적으로 WS-E의항염증활성에서 TNF-α의분비 A A B B Fig. 5. Effect of WS-E on inos expression in LPS-stimulated RAW macrophages. Total cellular proteins were obtained at 6 hr after LPS stimulation for detection of inos. The level of expression of inos was measured by Western blot (A). Actin was used as an internal control in a densitometric analysis (B). This result is the representative of almost same results from two independent experiments. Fig. 6. Inhibitory effect of WS-E on TNF-α and IL-6 production from LPS-stimulated RAW macrophages. The level of TNF-α (A) and IL-6 (B) in the supernatants of RAW cells was determined by ELISA kits. All values were expressed as the average of triplicate samples with SD. * p<0.05 compared with control.

Journal of Life Science 2016, Vol. 26. No. 3 343 유의하게억제되는것을확인하였다 (Fig. 6). 이상의결과로부터 WS-E는 LPS에의한대식세포의염증반응에있어서 NO와 inflammatory cytokine인 IL-6의분비를억제하는활성이인정되었으나 TNF-α의분비에대해서는억제효과가없는것으로확인되었다. Fig. 7.

Phosphorylation of p38, ERK, and JNK was determined by Western blot. This result is the representative of almost same results from two independent experiments.

6 Journal of Life Science 2016, Vol. 26. No 유의하게억제되는것을확인하였다 (Fig. 6). 이상의결과로부터 WS-E는 LPS에의한대식세포의염증반응에있어서 NO와 inflammatory cytokine인 IL-6의분비를억제하는활성이인정되었으나 TNF-α의분비에대해서는억제효과가없는것으로확인되었다. Fig. 7. Effect of WS-E on MAPK activation in LPS-stimulated RAW macrophages. Total cellular proteins were harvested at 15 min after LPS stimulation. Phosphorylation of p38, ERK, and JNK was determined by Western blot. This result is the representative of almost same results from two independent experiments. 억제가인정되지않았던것은 WS-E가단독으로 TNF-α를유도하는것과관련이있는것으로사료된다. 한편, 또다른 inflammatory cytokine인 IL-6의경우에는 NO에서와동일하게 WS-E의전처리에의해 IL-6의분비가 MAPK 및 NF-κB 억제활성 LPS 자극에의해대식세포가염증작용을일으킬때에는다양한세포내신호전달을통해염증이유도된다. 특히 p38, JNK 및 ERK와같은 MAPK는이러한염증반응에있어서매우중요한신호전달분자로서이들분자의인산화에의해염증반응이유발되는것으로알려져있다 [24]. 따라서 WS-E의항염증활성이 MAPK의조절여부에의해나타나는지알아보기위하여평가한결과, Fig. 7에나타난바와같이 LPS 자극에의한 p38, ERK 및 JNK 등의 MAPK 활성을억제하지못하는것으로나타났다. 한편 NF-κB는염증반응에있어서 inos 및 COX-2의발현을조절하는전사조절인자로 LPS 자극에의한대식세포의염증유발기전에는 NF-κB 의활성화에의한핵내전위가중요하다. 평상시 NF-κB는세포질에서 IκB와결합되어핵내전위가불가능한불활성화상태로존재하나염증성자극에의하여 IκB kinase가활성화되어 IκB가인산화될경우 IκB로부터유리되어핵내로이동하여 inos, COX-2 및 inflammatory cytokine의합성을촉진한다고알려져있다 [22]. 따라서 WS-E A B C Fig. 8. Inhibitory effect of NF-κB activation by WS-E in LPS-stimulated RAW macrophages. NF-κB activation in LPS-stimulated RAW cells was measured by IκB phosphorylation. Equal amounts of total protein were resolved by SDS-PAGE (A). Actin was used as an internal control in a densitometric analysis (B; piκb/actin, C; IκB/Actin). This result is the representative of almost same results from two independent experiments.

7 344 생명과학회지 2016, Vol. 26. No. 3 의항염증활성이 NF-κB 활성화억제에의한것인지를알아보기위하여 WS-E를전처리하고, LPS로자극한대식세포에서 IκB의인산화에대한영향을측정하였다. 그결과 Fig. 8에나타낸바와같이 WS-E 는 LPS에의한 IκB의인산화를농도의존적으로억제하는것으로확인되었다. 이상의결과를종합해보면병꽃나무꽃에탄올추출물인 WS-E의항염증활성은 MAPK 억제보다는 NF-κB의활성억제, 그리고 inos의세포내발현억제를통한 NO 생성의저해, inflammatory cytokine인 IL-6의억제등과같은일련의기전을통해일어나는것으로추정되었다. 향후 TNF-α의분비에관한활성에대해면밀한검토가필요할것으로사료된다. 감사의글 본연구는지식경제부와한국산업기술진흥원의지역산업기술개발사업 (No. A ) 으로수행된연구결과입니다. 또한본연구는부분적으로 2014년도중소기업청기술개발사업 (No. C ) 의지원으로수행되었습니다. References 1. Ahn, S. I., Heuing, B. J. and Son, J. Y Antioxidative activity and nitrite scavenging abilities of some phenolic compounds. Kor. J. Food Cookery Sci. 23, Blois, M. S Antioxidant determination by the use of a stable free radical. Nature 181, Cha, J. Y., Kim, S. Y., Jeong, S. J. and Cho, Y. S Effects of hesperetin and naringenin on lipid concentration in oratic acid treated mice. Kor. J. Life Sci. 9, Chung, Y. A. and Lee, J. K Antioxidative properties of phenolic compounds extracted from black rice. J. Kor. Soc. Food Sci. Nutr. 32, Follin, A. D. and Denis, W A colorimetric method for the determination of phenols and phenol derivatives in urine. J. Biol. Chem. 22, Han, S., Lee, J. H., Kim, C., Nam, D., Chung, W. S., Lee, S. G., Ahn, K. S., Cho, S. K., Cho, M. and Ahn, K. S Capillarisin inhibits inos, COX-2 expression, and proinflammatory cytokines in LPS-induced RAW macrophages via the suppression of ERK, JNK, and NF-κB activation. Immunopharmacol. Immunotoxicol. 35, Jeong, H. J., Park, S. B., Kim, S. and Kim, H. K Total polyphenol content and antioxidative activity of wild grape (Vitis coignetiae) extracts depending on ethanol concentrations. J. Kor. Soc. Food Sci. Nutr. 36, Jung, Y. S., Eun, C. S., Jung, Y. T., Kim, H. J. and Yu, M. H Anti-inflammatory effects of Picrasma quassioides (D. DON) Benn leaves extracts. J. Life Sci. 23, Kang, H. L Effects of Albizziae cortex extracts on the elastase activity and DPPH and NO scavenging activities. Kor. J. Ori. Physiol. Pathol. 25, Kim, J. Y., Jung, K. S. and Jeong, H. G Suppressive effects of the kahweol and cafestol on cyclooxygenase-2 expression in macrophages. FEBS Lett. 569, Kim, Y. S., Lee, S. J., Hwang, J. W, Kim, E. H., Park, P. J. and Jenong, J. H Anti-inflammatory effects of extracts from Ligustrum ovalifolium H. leaves on RAW264.7 macrophages. J. Kor. Soc. Food Sci. Nutr. 41, Kuppusamy, P. and Zweieer, J. L Characterization of free radical generation by xanthine oxidase. J. Biol. Chem. 264, Lee, S. G., Jeong, H. J., Lee, E. J., Kim, J. B. and Choi, S. W Antioxidant and anti-inflammatory activities of ethanol extracts from medicinal herb mixtures. Kor. J. Food Sci. Technol. 43, Lee, S. J., Kim, E. K., Kim, Y. S., Hwang, J. W., Lee, K. H., Choi, D. K., Kang, H., Moon, S. H., Jeon, B. T. and Park, P. J Purification and characterization of a nitric oxide inhibitory peptide from Ruditapes philippinarum. Food Chem. Toxicol. 50, Lee, Y. S. and Lee, S. T Modern plant taxonomy. pp , Woosenong Press, Seoul. 16. Moreno, M. I., Isla, M. I. Sampeiettro, A. R. and Vatuone, M. A Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. J. Enthnopharmacol. 71, Mosmann, T Rapid colorimetric assay for the cellular growth and survival: Application to proliferation and cytotoxic assay. J. Immunol. Methods 65, Nakatani, N Recent advances in the study on natural antioxidants. Nippon Shokuhin Kogyo Gakkaishi. 37, Nishikimi, M Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system. Biochem. Biophy. Res. Commun. 63, Nozaki, K Current aspect and future condition of phytogenic antioxidants. Fragrance J. 6, Park, S. W., Jung, Y. S. and Ko, K. C Quantitative analysis of anthocyanins among mulberry cultivars and their pharmacological screening. J. Kor. Soc. Hort Sci. 38, Pruett, S. B., Fan, R. and Zheng, Q Characterization of glucocorticoid receptor translocation, cytoplasmic IkappaB, nuclear NFkappaB, and activation of NFkappaB in T lymphocytes exposed to stress-inducible concentrations of corticosterone in vivo. Int. Immunopharmacol. 3, Rhim, T. J., Choi, M. Y. and Park, H. J Antioxidative activity of Rumex crispus L. extract. Kor. J. Plant Res. 25, Robinson, M. J. and Cobb, M. H Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9, Storck, M., Schilling, M., Burkhardt, K., Prestel, R., Abendroth, D. and Hammer, C Production of proinflammatory cytokines and adhesion molecules in ex-vivo xenogeneic kidney perfusion. Transpl. Int. 7, S647-S Stuehr, D. J., Cho, H. J., Kwon, N. S., Weise, M. F. and Nathan, C. F Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc. Natl. Acad.

8 Journal of Life Science 2016, Vol. 26. No Sci. USA 88, Yang, H., Oh, K. H. and Yoo, Y. C Anti-inflammatory effect of hot water extract of Aronia fruits in LPS-stimulated RAW macrophages. J. Kor. Soc. Food Sci. Nutr. 44, 초록 :RAW 대식세포에미치는병꽃나무꽃추출물의항산화및항염증효과유영춘 1 이계원 2 조영호 2 * ( 1 건양대학교의과대학미생물학교실, 2 건양대학교의료공과대학제약생명공학과 ) 본연구에서는병꽃나무꽃추출물의항산화효과및 LPS로자극된 RAW 대식세포에서항염증효과를조사하였다. 병꽃나무꽃추출물의총폴리페놀과플라보노이드함량을측정한결과총폴리페놀함량과플라보노이함량은각각 ±0.04 μg/ml, ±0.02 μg/ml로나타났다. 항산화활성을확인하기위하여 DPPH 라디칼과 superoxide 음이온라디칼소거활성을측정한결과투여농도의존적으로항산화활성이증가하는것으로나타났다. 또한항염증활성을확인하기위하여염증매개물질인 NO와 inflammatory cytokine인 IL-6와 TNFα의생성량을측정하였다. 그결과 NO와 IL-6의생성을투여농도의존적으로저해하였지만, TNF-α의경우병꽃나무꽃추출물의농도가 20 μg/ml 이하에서는 TNF-α의생성을억제하지않았고 100 μg/ml 이상에서는오히려분비를유도하는것으로나타났다. 또한, 병꽃나무꽃추출물의염증반응과관련된 inos 발현과 MAPK 및 NF-κ B의활성에미치는영향을측정한결과 inos는농도유의적으로그발현이감소되었으며, MAPK의발현및인산화에는별다른영향을미치지못하는것으로나타났다. 반면에 IκB의인산화를효과적으로감소시켜 NF-κB의활성을억제하여항염증활성을나타내는것을확인할수있었다. 이상의결과로부터병꽃나무꽃추출물은항산화및항염증활성이우수한기능성소재로활용이가능할것으로사료된다.

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,