LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 로알려져있다 (Förstermann and Sessa, 2012). TNF-α, IL-1 β및 IL-6 와같은전염증성 cytokine 및단백질의유전자발현은 mitoge

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1 Korean J. Plant Res. 31(5): (2018) Print ISSN Online ISSN Original Research Article LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 우현심 1, 이선민 2, 허정두 3, 이민성 4, 김영수 5, 김대욱 5 * 1 국립경상대학교응용생명과학부, 박사과정생, 2 안전성평가연구소, 연구원, 3 안전성평가연구소, 박사연구원, 4 국립백두대간수목원, 연구원, 5 국립백두대간수목원, 박사연구원 Anti-inflammatory Activity of Extracts of Hovenia dulcis on Lipopolysaccharides-stimulated RAW264.7 Cells Hyun Sim Woo 1, Sun Min Lee 2, Jeong Doo Heo 3, Min-Sung Lee 4, Yeong-Su Kim 5 and Dae Wook Kim 5 * 1 Ph.D. Graduate Student, Division of Applied Life Science, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju-si 52828, Korea 2 Assistant Researcher and 3 Ph.D. Senior Researcher, Gyeongnam Department of Environmental Toxicology and Chemistry, Toxicology Screening Research Center, Korea Institute of Toxicology, Jinju-si 52834, Korea 4 Assistant Researcher and 5 Ph.D. Senior Researcher, Plant Resource Industry Division, Forest Plant Industry Department, Baekdudaegan National Arboretum, Bonghwa-gun 26209, Korea Abstract - In this study, the anti-inflammatory activities of the extracts of different parts of Hovenia dulcis such as leaves, stems, and roots were investigated. Among them, the roots extract (RE) showed the most potent suppressive effect against pro-inflammatory mediators in LPS-stimulated mouse macrophage cells. RE induced dose-dependent reduction of inducible nitric oxide synthase (inos) and cyclooxygenase-2 (COX-2) and concomitantly reduced the production of NO and PGE 2. Additionally, pre-treatment with RE significantly suppressed the production of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, as well as mrna levels. Moreover, phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear translocation of nuclear factor-kappa B (NF-kB) were also strongly attenuated by RE in RAW264.7 cell. Furthermore, RE induced HO-1 expression through nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) and increase HO-1 activity in RAW264.7 macrophages. Therefore, these results indicate that RE strongly inhibits LPS-induced inflammatory responses by blocking NF-kB activation, inhibiting MAPKs phosphorylation, and enhancing HO-1 expression in macrophages, suggesting that RE of H. dulicis and a major component, 27-O-protocatechuoylbetulinic acid could be applied as a valuable natural anti-inflammatory material. Key words - Anti-inflammatory effect, Heme oxygenase-1, Hovenia dulcis Thunb., Pro-inflammatory cytokines, 27-O- Protocatechuoylbetulinic acid 서언 대식세포는염증반응과면역기능을조절하며, 항상성을유지하는데중요한역할을한다. 그람음성균의외막성분인 lipopolysaccharide (LPS) 는대식세포의감염초기에반응하 * 교신저자 : dwking@bdna.or.kr Tel These authors contributed equally to this work. 고숙주방어에중추적인역할을하나, 과도한 LPS 자극에의해활성화된대식세포는 tumor necrosis factor-α(tnf-α), interleukin (IL)-1β 및 IL-6 와같은전염성 cytokine 을분비시키며, nitric oxide (NO), prostaglandin E 2 (PGE 2 ) 등의염증매개물질을다량분비하게된다 (Jenog et al., 2014). NO 는대식세포가활성화되면 inducible NO synthase (inos) 로부터생산되며, 과도한 NO의생성은염증을유발시키게되며병리학적혈관확장, 세포독성, 조직손상등유해작용을일으키는것으 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. 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.

2 LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 로알려져있다 (Förstermann and Sessa, 2012). TNF-α, IL-1 β및 IL-6 와같은전염증성 cytokine 및단백질의유전자발현은 mitogen-activated protein kinases (MAPKs) 와 nuclear factor kappa B (NF-kB) 에의해조절된다. 면역과염증반응에관계된유전자의발현에있어서 NF-kB 가활성화되면 NF-kB 와결합해있던 inhibitory kappa B α(ikbα) 가분해되면서 NF-kB 가세포원형질에서핵으로들어가게되며이후 TNF-α, IL-6 와같은 cytokine 발현의전사인자로서중요한역할을한다 (Jang et al., 2005, Majdalawieh and Ro, 2010). Heme oxygenase (HO) 의대사를조절하는효소인 HO-1 은세포의 heme 을분해하여 carbon monoxide (CO), ferrous iron 및 biliverdin 으로전환하게되고, biliverdin 은다시환원효소에의해 bilirubin으로환원된다 (Elbirt and Bonkovsky, 1999). HO-1과생성된부산물들은항염증및항세포사멸에관련된유전자발현과효소의활성에영향과산화적스트레스로부터세포와조직을보호하는효소로인식되고있다 (Gozzelino et al., 2010, Tsan et al., 1989). Bilirubin 은 inos 와 COX-2의발현및 NO의생성을억제하며 biliverdin 은전염성 cytokine 인 IL-6 와 IL-1β 의생성을억제시키는것으로보고되었다. 특히 LPS를처리한 RAW264.7 세포에서 HO-1은 NO의생성과 inos 유전자발현을억제함으로써항염증작용을통하여세포손상을억제한다는연구결과가보고되었다. 헛개나무 (Hovenia dulcis Thunb.) 는갈매나무과의낙엽활엽교목으로내한성과내음성이강하고맹아력이강한수종으로, 우리나라에서는설악산, 오대산, 지리산및한라산등에주로자라며, 중북부지방보다는남쪽지방에서잘생육한다. 헛개나무의많은화학성분이알려졌으며주요활성물질로는 saponins 계열의 Hovenoside I-VII, Hovacerboside A1, Hovenidulcioside A1, A2, B1, B2, Hoduloside III등과 Flavonoid 계열의 Quercetin, Kaempferol, (+) Ampelosin, Hovenitin I~III, Hovenodulinol 등이분리되어구조가연구되었다 (Park et al., 2015). 지금까지보고된약리활성으로단맛억제효과, 간세포보호효과, 근육이완억제효과, 이뇨작용, 항산화작용, 항암작용및당뇨치료효과등이보고되고있다 (Yand et al., 2013). 최근연구에서는헛개나무열매추출물의항염증효능및성분분석에대한문헌이보고되었다 (Park et al., 2016). 본연구에서는헛개나무부위별 ( 잎, 줄기, 뿌리 ) 추출물의 NO 생성억제효과에서탁월한뿌리추출물의항염증효과를확인하기위해 NO, PGE 2 의생성량과전염성 cytokine (IL-1β, IL-6, TNF-α) 분비를측정하였고, 그조절기전으로 HO-1과 MAPKs, Ik-Bα 를조사하였다. 또한, 헛개나무뿌리추출물로부터유효성분분리및정제하여분광학적방법으로화학구조동정에대한연구를진행하고자하였다. 재료및방법실험재료및추출물조제본실험에사용한헛개나무시료는 2017 년 8월에경남고성군일대의야산에서채취하였으며, 정확히감정한후에음건 세절하여실험에사용하였으며, 표본은국립백두대간수목원자원식물산업실에보관하고있다 (PE ). 분쇄된헛개나무잎, 줄기, 뿌리건조시료 1.5 kg을취하여 10 L 메탄올을가하여 30 에서 24시간추출한후원심분리하여상등액을 1차적으로회수하고다시침전물을재추출하여원심분리하여상등액을회수하여 1차상등액과혼합한후감압농축하여, -20 의냉동고에보관하면서항염증효과및 HPLC 분석을위한시료로사용하였다. 시약및기기 Dulbecco s modified Eagle s medium (DMEM) 과 fetal bovine serum (FBS) 등의세포배양용시약들은 Gibco BRL 사 (Grand Island, NY, USA) 에서구입하였다. Lipopolysaccharide (LPS) 와 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphnyltetrazolium bromide (MTT) 는 Sigma 사 (Louis, MO, USA) 에서구입하였다. 96-well tissue culture plates 와기타 tissue culture dishes 는 Falcon 사 (Corning, NY, USA) 제품을이용하였다. 실험에사용된일차항체인 inos, COX-2, 그리고 Lamin B는 Santa Cruz Biotechnology 사 (SantaCruz, CA, USA) 에서구입하였고, p65, phosphor-p65, IκBα, HO-1, Nrf2, phospho-erk, ERK, phospho-p38, p38, phosphor-jnk, JNK (Cell Signaling, Beverly, MA, USA) 에서구입하였다. 2차항체인 Horseradish peroxidase (HRP)-conjugated anti-rabbit or goat antibodies 는 Jackson ImmunoResearch사 (West Grove, PA, USA) 에서구입하였다. Griess Reagent System 은 Promega 사 (Madison, WI, USA) 에서구입하였고, ELISA kit 는 R&D systems (Minneapolis, MN, USA) 에서구입하였다. 세포배양마우스대식세포주인 RAW264.7 세포는 American Type Culture Collection (ATCC, Manassas, VA, USA) 에서동결상태로구입하여 10% feta bovine serum (FBS) 과 1% antibiotics -467-

3 Korean J. Plant Res. 31(5) : 466~477(2018) (100 U/ ml penicillin G, 100 μg / ml streptomycin) 을첨가한 DMEM 배지로 37, 5% CO 2 환경에서배양하였다. 세포생존율측정뿌리추출물 (RE) 의세포독성유무를알아보기위하여 Cell Counting Kit-8 (Dojindo, Japan) 을이용하여측정하였다. RAW264.7 세포를 96 well plate 에 2x10 4 cells/ ml로분주하고 24시간배양한다음, 뿌리추출물 (RE) 를농도별 (0, 10, 20, 그리고 40 μg / ml ) 로처리하여 24시간배양하였다. 각 well 당 10 μl의 CCK-8 용액을첨가하여 37, 5% CO 2 조건에서 2시간반응시킨후 microplate Reader (BioTek, Winooski, VT, USA) 을이용하여 450 nm에서흡광도를측정하였다. Nitric oxide (NO) 및 PGE 2 의측정 LPS 자극에의해 RAW264.7 세포에서유도된염증반응에대한각부위별추출물의항염증효과를측정하기위한일환으로, 세포배양액중의 NO를정량하여비교하였다. NO 소거활성을측정하기위하여세포를 96-well plate 에 2x10 4 cells/ ml로분주하여 24시간배양한후 RE을 0, 10, 20, 그리고 40 μg / ml의농도로전처리하여 1시간동안배양한후, LPS를 1 μg / ml의농도로처리하여 24시간동안배양하였다. 배양상층액 50 μl와동량의 Griess reagent (Promega, Madison, WI, USA) 를혼합하여반응시킨뒤 540 nm에서 microplate reader (BioTek, Winooski, VT, USA) 를사용하여흡광도값을측정하였다. Sodium nitrite (NaNO 2 ) 의농도별표준곡선을이용하여 NO값을산출하였다. 그리고세포배양액내의 PGE 2 level은 PGE 2 ELISA assay kit (R&D System, Minneapolis, MN, USA) 를사용하여측정하였다. Cytokine (IL-1β, IL-6, TNF-α) 측정 NO 측정과같은방법으로세포를배양한후, 각 well 에서세포배양액을회수하였다. 세포배양상층액내의 TNF-α, IL-1 β, IL-6 농도는 enzyme linked immunosorbent assay (Elisa) kit (R&D system, MN, USA) 를이용하여측정하였다. 정량중합효소반응 real-time reverse transcription polymerase chain reaction (RT-PCR) RAW264.7 세포를 6-well plate 에 6x10 5 cells/ ml로분주하여 24시간배양한뒤 RE을 1시간동안처리한후, LPS를 1 μg / ml의농도로 24시간동안반응하였다. 이후 Trizol (Invitrogen, USA) 시약을이용하여 total RNA 를분리하였다. Total RNA 를정량하고, RT-PreMix (Promega, Madison, WI, USA) 를이용하여 1 μg의각 total RNA 로부터각각의 cdna 를합성하였다. 각 cdna 의 template와 inos, COX-2, IL-6, IL-1β, TNF-α 및 β-actin 의 primer 는 Table 1에타내었고, 이들의 mrna 발현을확인하였다. Table 1. Primer sequences used in this study Gene inos COX-2 IL-6 IL-1β TNF-α HO-1 β-actin Primer sequence 5 -CCC TTC CGA AGT TTC TGG CAG CAG C 5 -GGC TGT CAG AGC CTC GTG GCT TTG G 5 -ACT CAC TCA GTT TGT TGA GTC ATT C 5 -TTT GAT TAG TAC TGT AGG GTT AAT G 5 -CAA GAA AGA CAA AGC CAG AGT CCT T 5 -TGG ATG GTC TTG GTC CTT AGC C 5 -TGC AGA GTT CCC CAA CTG GTA CAT C 5 -GTG CTG CCT AAT GTC CCC TTG AAT C 5 -ACA AGC CTG TAG CCC ACG 5 -TCC AAA GTA GAC CTG CCC 5 -AAG ATT GCC CAG AAA GCC CTG GAC 5 -AAC TGT CGC CAC CAG AAA GCT GAG 5 -AGT GTG ACG TTG ACA TCC GTA AAG A 5 -GGA CAG TGA GGC CAG GAT GG -468-

4 LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 Western blot analysis RAW264.7 세포를 RE 및 LPS 로처리한후 HO-1 억제제 (SnPP) 는 1시간전에전처리하였다. Ice-cold PBS로 2회세척한후, RIPA buffer [25 mm Tris HCl ph 7.6, 150 mm NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitors] 를넣어 ice에서 30분간반응시키고 Whole cell lysate 를제조하여 20분간원심분리하여상층액을모았다. 각시료의단백질정량은 Bradford protein assay (Bio-Rad, Hercules, CA, USA) 를사용하여 595 nm에서흡광도를측정하여실시하였다. 동일한양의단백질 (30 μg ) 을 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel 로분리한후 iblot 2 Blotting System (Thermo Fisher, Waltham, MA USA) 을이용하여 transfer하였다. 이 PVDF membrane은 5% non-fat milk block solution 에서 1시간동안반응시킨후, 일차항체를 (1:1000 dilution) 를 4 에서 overnight 시켰다. TBST 용액으로 3번세척한후, 이차항체는 horseradish peroxidase (HRP)- conjugated anti-rabbit, or anti-mouse IgG (1:10000 dilution) 로실온에서 1시간반응시켰다. 이어서 3회세척후단백질은 ChemiDoc Imaging Systems (Bio-Rad, Hercules, CA, USA) 을이용하여발현을확인하였다. 부위별헛개나무추출물 HPLC 분석 HPLC 기기는 pump, auto sample, column oven, DAD (Agilent 1260 infinity HPLC system, Agilent Technologies, USA) 를사용하였으며, 분석에사용된모든용매는 J.T. Baker (Phillipsburg, NJ, USA) 로부터구입한 HPLC 급용매를사용하였다. 이동상으로 0.1% formic acid 를포함한물 ( 용매 A) 와아세토나이트릴 ( 용매 B) 를사용하였고, 1 ml /min 유속으로시료 10 μl를주입하여 254 nm파장에서 gradient condition 으로분석하였으며, 분석조건은 Table 2와같다. 헛개나무뿌리추출물활성성분분리및구조동정헛개나무부위별추출물중가장우수한항염증효과를가진뿌리추출물을 Recycling preparative HPLC system 을사용하여분리하였다. JAIGEL-ODS AP column (20 x 500 mm ) 에추출물 1 g을주입하고혼합용매 (Water:ACN=1:2, v/v) 를분당 10 ml / 씩용출시킨다음 UV detector 를사용하여흡광도 254 nm에서측정하였으며, 6개의소분획 (HR1-HR6) 으로나누었다. 이중 HR4 를 EtOAc:MeOH(30:1) 의혼합용매로 preparative TLC 를실시하고 Sephadex LH-20 (95% MeOH) 로정제하여화합물 1 (30 mg ) 을얻었다. 최종정제된뿌리추출물의주요성분은 Q Exactive plus Orbitrap LC-MS/MS (Thermo Fisher Scientific, MA, USA) 와 900 MHz Bruker AVANCE II NMR (Bruker, MA, USA) 을통하여얻은화학구조상의수소및탄소골격에대한정보를얻어정제된주요성분의구조를동정하였다. Table 2. HPLC conditions for separation of different part extract and major compound from H. dulcis Parameter Condition Column ZORBAX Eclipse plus C 18 ( mm, 5 μm ) Flow rate 1.0 ml /min Injection volume 10 μl UV detection 254 nm Run time 50 min Gradient z 0.1% formic acid in water. y 0.1% formic acid in acetonitrile. Time (min) % A z % B y

5 Korean J. Plant Res. 31(5) : 466~477(2018) 화합물 1 1 H-NMR (900 MHz, Acetone-d 6 ) δ1.68 (1H, m, H-1), 1.01 (1H, m, H-1), 1.59 (2H, m, H-2), 3.16 (1H, m, H-3), 0.8 (1H, m, H-5), 1.54 (1H, m, H-6), 1.35 (1H, m, H-6), 1.63 (1H, m, H-7), 1.48 (1H, m, H-7), 1.42 (1H, m, H-9), 1.55 (1H, m, H-11), 1.19 (1H, m, H-11), 1.79 (1H, m, H-12), 0.91 (1H, m, H-12), 2.54 (1H, m, H-13), 1.96 (1H, m, H-15), 1.49 (1H, m, H-15), 2.23 (1H, m, H-16), 1.35 (1H, m, H-16), 1.83 (1H, m, H-18), 3.09 (1H, m, H-19), 2.07 (1H, m, H-21), 1.40 (1H, m, H-21), 1.94 (1H, m, H-22), 1.49 (1H, m, H-22), 0.91 (3H, s, H-23), 0.90 (3H, s, H-24), 0.77 (3H, s, H-25), 1.05 (3H, s, H-26), 4.72 (1H, d, J = Hz, H-27), 4.55 (1H, d, J = Hz, H-27), 4.77 (1H, s, H-29), 4.63 (1H, s, H-29), 1.74 (3H, s, H-30), 7.53 (1H, d, J = 2.0 Hz, H-2 ), 6.92 (1H, d, J = 8.17 Hz, H-5 ), 7.46 (1H, dd, J = 2.15, 8.31 Hz, H-6 ); 13 C-NMR (225 MHz, Acetone-d 6 ) δ38.8 (C-1), 27.2 (C-2), 77.7 (C-3), 38.7 (C-4), 55.5 (C-5), 18.2 (C-6), 35.4 (C-7), 41.5 (C-8), 52.0 (C-9), 37.4 (C-10), 21.1 (C-11), 25.4 (C-12), 39.1 (C-13), 45.8 (C-14), 24.2 (C-15), 32.4 (C-16), 55.7 (C-17), 49.4 (C-18), 46.9 (C-19), (C-20), 30.4 (C-21), 36.4 (C-22), 27.7 (C-23), 15.4 (C-24), 16.4 (C-25), 16.1 (C-26), 62.8 (C-27), (C-28), (C-29), 18.9 (C-30), (C-1 ), (C-2 ), (C-3 ), (C-4 ), (C-5 ), (C-6 ), (C-7 ). 통계처리모든실험결과는 3회이상실시하여그평균값을기초로 Mean±SE 로나타내었으며, 실험결과에대한통계처리는 GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA) 를이용하여 Two-way ANOVA에준하였고 p-value 가 0.05 미만일경우유의한것으로판정하였다. 결과및고찰 NO, PGE 2 생성및 inox, COX-2 발현에미치는 RE 의효과헛개나무열매추출물이항염증효과를나타낸보고가있으나잎, 줄기, 뿌리추출물의항염증효과에대한연구는전무한실정이다. 헛개나무부위별추출물의항염증효과를비교하기위하여 lipopolysaccharide (LPS) 로염증반응을유도한 RAW264.7 세포내부위별추출물을동시에처리하여염증의지표인 nitric oxide (NO) 생성억제효과를측정한결과 Table 3과같이헛개나무뿌리추출물 (RE) 에서탁월한억제효과를나타내었다. 세포독성이나타내지않는농도인 10, 20, 그리고 40 μg / ml에서 RE을이용하여항염증효과및조절기전을관찰하였다. 초기염증반응은 inducible nitric oxide synthase (inos) 와 cyclooxygenase-2 (COX-2) 와이들단백질에의한염증매개체들 (NO, PGE 2 ) 이유도된다. 본연구에서는 RE의항염증효과를알아보기위하여 RAW264.7 세포에 RE 10, 20, 그리고 40 μg / ml을 1시간동안전처리한후 1 μg / ml LPS를 24시간동안처리하여 NO의생성량에미치는영향을같은방법으로분석결과, 10 μg / ml에서는 53.1%, 40 μg / ml에서는 94.7% 감소시켰다 (Fig. 1A). 또한, LPS에의해유도되는 PGE 2 의생성에미치는영향을분석결과, 10 μg / ml에서는 47.2%, 40 μg / ml에서는 90.1% 감소시켰다 (Fig. 1B). RE의항염증효과에대한작용기전을알아보기위하여체내염증발현효소 (inos, COX-2) 의 mrna 발현을조사하였다. inos 에의해서생성된 NO는면역반응에서유용한역할을하지만, 지속적인 NO의생성은만성염증질환을일으키는중요한요인이된다. 따라서체내에과도한 NO를생성하는 inos 의발현에대한 RE의영향을조사하기위해 RT-PCR 을시행하였다. LPS를처리한군에서 inos, COX-2 mrna 는유의적으로증가하였고, RE 를처리한군에서는 LPS 처리군과비교하여 inos, COX-2 mrna 의발현량이유의적으로감소함으로 inos, COX-2 mrna 저해효과는 NO, PGE 2 생성억제효과가유사한 Table 3. Cell viability and in vitro decrease of nitric oxide production of different part of extract in LPS-stimulated RAW264.7 cells Sample Cytotoxicity IC 50 ( μg / ml ) Inhibition of NO Production IC 50 ( μg / ml ) Methanol extracts of leaf >40 >40 Methanol extracts of stem > ± 0.73 z Methanol extracts of root > ± O-protocatechuoylbetulinic acid > ± 0.61 z Values are expressed as mean ± SD of the three replicates

6 LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 Fig. 1. Effects of RE on NO and PGE 2 production, inos mrna and COX-2 mrna expression in LPS-stimulated RAW264.7 cells. RAW264.7 cells were stimulated with the indicated concentrations of RE in the presence or absence of LPS (1 μg / ml ) for 24 h: (A) amounts of NO were determined using the Griess reaction in culture medium. (B) PGE 2 levels in the cell culture medium were detected by ELISA assay. (C and D) The inos and COX-2 protein levels were determined 6 h after LPS stimulation. GAPDH was used as an internal control for the real-time PCR assay (n = 4). The data represent the mean ± S.D of three experiments. *p<0.05, **p<0.001 compared to LPS alone. 경향을나타내었다 (Fig. 1C and B). 그러므로 RE는 inos 와 COX-2 발현을억제함으로써염증억제효과를가짐을확인할수있었다. 전염증성 cytokines 생성과 mrna 발현에미치는 RE의효과염증반응이유도되는과정에서 NO 및 PGE 2 와같은염증매개물의생성과면역반응을통한염증성 cytokine 의생성이동반되며, 염증을나타내는중요한지표이다 (Horwood et al., 2006). 특히대식세포에서는 LPS 등의여러자극인자에의하여활성화가되면전염증성 cytokine (TNF-α, IL-6 및 IL-1β) 을증가시키고, 이들은 inos 의발현을유도한다. 실험결과, LPS 를처리한군에서는 TNF-α, IL-6, IL-1β 발현이유의성있게증가되었고, RE 를처리한군에서 TNF-α 는 pg/ ml, L-6 는 pg/ ml, IL-1β 는 pg/ ml로 LPS 처리군과비교하여염증인자들을농도의존적으로강력하게감소하는확인하였다 (Fig. 2A). 위와같은결과에착안하여 mrna 수준에서도전염성인자들을억제하는지알아보기로하 였다. TNF-α, IL-6, IL-1β 의 mrna 발현을정량적중합효소반응방법으로측정한결과 LPS 자극은 mrna 의발현을유의성있게증가시켰고, RE 를전처리한실험군에서 TNF-α, IL-6 및 IL-1βmRNA 발현이모두농도의존적으로억제되었다 (Fig. 2B). 이결과들로통해 RE는 TNF-α, IL-1β 및 IL-6 의생성을감소시킴으로써 inos 의발현을조절하여 NO 생성을억제하는항염증효과를나타낸다는것으로확인하였다. 모든실험에사용된 RE의농도별세포독성여부를 MTT assay 를통하여알아보았으며사용된모든농도에서 90% 이상의생존율을보여 RE 은 RAW264.7 세포에서독성을나타내지않는것을확인하였다 (Fig. 2C). Ik-B 발현량에미치는 RE 의효과 RAW264.7 세포나 LPS에의하여염증이일어나게되면다양한신호전달기전에의하여염증성매개물질들을분비하게되는데, 대표적인경로인 NF-kB (Nuclear Factor-kappa B) 가있다. NF-kB 는모든세포에서발현되는유도성전사인자로세포외부자극에대한방어작용과면역세포의활성화를일으키 -471-

7 Korean J. Plant Res. 31(5) : 466~477(2018) Fig. 2. Effects of RE on LPS-induced pro-inflammatory cytokine expression in RAW264.7 cells. Cells were treated with the indicated concentration of RE and/or LPS (1 μg / ml ) for 24 h (for mrna): (A) the protein levels of IL-6, IL-1β, and TNF-αin the culture medium were measured using ELISA. (B) The mrna levels of IL-6, IL-1β, and TNF-αwere determined using RT-PCR with specific primers. (C) Cell viability after treatment with RE for 24 h was evaluated by CCK-8 kit assay, and the results are expressed as percentages relative to the untreated control. Date represent the mean ± S.D of three experiments. *p<0.05, **p<0.001 compared to LPS alone. 며세포의염증반응에관련된다양한유전자의발현을조절하는것으로알려져있다 (Ghosh et al., 2012; Kim et al., 2018). LPS 에의하여세포막에존재하는 toll like receptor 4 (TLR4) 와결합하게되고, 이에의해세포질에있는전사인자 NF-kB 가활 성화되면결합해있던 inhibitory kappa Ba (Ik-Ba) 가분해되고, 그에따라 NF-kB 가세포질에서핵내로이동하여 COX-2, inos 등의전사를유도한다 (Kim et al., 2013). 본연구에서는 RE 처리에의해서감소되는염증매개인자들의생성억제가 -472-

8 LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 Fig. 3. Effects of RE on the activation of the NF-kB pathway in LPS-stimulated RAW264.7 cells. Cells were pretreated for 30 min with the indicated concentrations of RE before LPS (1 μg / ml ) treatment for 5 min. Nuclear and cytosolic proteins were subjected to Western blot analysis with the indicated antibodies. β-actin was used as internal controls for the cytosolic and nuclear fractions, respectively. Date represent the mean ± S.D of three experiments. *p<0.05, **p<0.001 compared to LPS alone. NF-kB 활성과의관계를조사하기위해분석한결과는 Fig. 3과같다. LPS 를처리한군에서 NF-kB 의 subunit 인 p65의핵안으로의이동이무처리군과비교하여크게감소하였고, RE 를처리한군에서는 LPS 처리군과비교하여농도의존적으로발현이감소하였다. 또한 LPS를처리한군에서 Ik-Bα 의 degradation 이무처리군과비교하여뚜렷이나타났으며, RE 에서는 LPS 처리군과비교하여 IkB-α 의 degradation 의농도의존적으로발현을감소하였다. 결과적으로 RE은전사인자인 NF-kB 의활성화를감소시키고, downstream signaling molecule인 inos 와 COX-2 의전사를억제하며, NO 의생성을감소시켜항염증효과를나타냄을유추할수있었다. MAPK 활성에미치는 RE 의효과 MAPK (Mitogen Activated Protein Kinase) 의신호전달경로는염증반응의활성화에중요한역할을한다. 특히, LPS에의해유도된 cytokine 의분비에 p38 MAPK 가중요한역할을하고, 폐와관련된염증질환에서는 p38, JNK, ERK 신호경로가중요한역할을한다고알려져있다 (Lee and Kang, 2018; Yang et al., 2015). 특히대식세포에서 LPS는표면에 TLR4 를자극하여하부세포신호전달경로인 MAPK 의활성화를유도하며활성화된신호전달경로는전염증성사이토카인, NO, PG 와같은여러가지염증성매개인자들의발현을유도한다 (Kim et al., 2010). 본연구에서는 RE의항염증효과가 MAPK 분자조절에의한것인지를분석결과는 Fig. 4와같다. RE 처리에따른대식세포내인산화된 MAPKs 발현량에미치는영향을 Western blot으로분석한결과, LPS 로유도된 RAW264.7 세포에는 MAPKs 에속한단백질이모두활성화되었음을알수있었다. RE의첨가량이높아질수록인산화된 ERK, JNK, p-38의발현이 LPS 처리군에비해감소하는것을확인할수있었다. 따라서 RE는 MAPK 의활성화를억제함으로써항염증성 cytokine, NO, prostaglandin (PG)s 와같은여러염증성매개인자들의발현을억제하는효과를나타내었다. HO-1 발현유도에미치는 RE 의효과 / Nrf2 활성에미치는 HDRE 의효과 Hemo oxygenase-1 (HO-1) 은 heme 을산화시켜 biliverdin, 인산화탄소 (CO), free iron 등을생성에관여하는효소로서산화적스트레스에대한보호작용을하여염증반응을약화시킨다 (Kaur et al., 2003). 활성화된대식세포에 HO-1을발현시키거나 CO를처리하면염증촉진사이토카인의생산이저해되며, 또한염증촉진매개체의생산을억제한다. HO-1 단백질발현의전사조절인자인 nuclear factor-e2-related factor 2 (Nrf2) 의핵내로의발현이매우중요한인자로작용한다. RE 가 Nrf2 의전사여부를조사하기위하여 RAW264.7 세포에 RE를처리한후 HO-1발현과 Nrf2 활성화를 Western blotting 을이용하여분석하였다. RE 의농도에따라 Nfr2 는점점감소하는반면, 핵내부의 Nrf2 는증가하는양상을보이는것을보아 Nrf2 의핵내로의전사가이루어졌음을확인할수있었다 (Fig. 5). LPS 를단독처리하였을때보다 RE를전처리하여 LPS를처리하였더니 HO-1의발현이증가하는것으로나타났다. Fig. 5에서전사조절인자 Nrf2 의세포질과핵내발현양을확인하였더니세포질 -473-

9 Korean J. Plant Res. 31(5) : 466~477(2018) Fig. 4. Effect of RE on LPS-stimulated MAPKs activation in RAW264.7cells. Phosphorylation and total protein expression of ERK, p38, and JNK were extracts prepared from the cells RE treated with LPS-stimulated analyzed by western blot. Date represent the mean ± S.D of three experiments. *p<0.05, **p<0.001 compared to LPS alone. Fig. 5. Effect of RE on Cytosolic Nrf2, Nuclear Nrf2 and HO-1 expression in LPS-stimulated RAW264.7cells. Cells were pretreated for 1 h with various concentrations of RE (10-40 μg / ml ) and stimulated with LPS (1 μg / ml ) for 24 h. The Cytosolic Nrf2, Nuclear Nrf2 and HO-1 protein expression were analyzed by western blot analysis. The mrna level was analyzed by real-time PCR. Date represent the mean ± S.D of three experiments. *p<0.05, **p<0.001 compared to LPS alone. 에서는감소하였으나, 핵내에서증가하는결과를보였다. 그러므로 RE는 Nrf2 활성을증가시킴으로 HO-1를유도한다고결론지을수있다. RE 의항염증효과와 HO-1의발현간의직접적 인관계를알아보기위하여, HO-1 억제제인 Snpp를이용하여실험하였다. SnPP 를 RE (40 μg / ml ) 와함께 12시간동안처리한실험에서 RE에의해억제되었던 inos 와 COX-2 가 SnPP를병 -474-

10 LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 Fig. 6. Effect of RE on inos, COX-2, HO-1 and Nrf2 expression in LPS-stimulated RAW264.7 cells. Cell were pretreated with 10 µm SnPP for 1h prior to with various concentrations of RE (10-40 μg / ml ) and stimulated with LPS (1 μg / ml ) for 24 h. The inos, COX-2, HO-1 and Nrf2 protein expression were analyzed by western blot analysis. Date represent the mean ± S.D of three experiments. *p<0.05, **p<0.001 compared to LPS alone. Fig. 7. Representative HPLC chromatogram detected at 254 nm for different parts extracts (A) and major compound (B) of H. dulcis (insert) Structure of 27-O-protocatechuoylbetulinic acid (1). 용투여함으로써부분적으로증가되었다 (Fig. 6). 이와같은결과로부터 RE에의한 HO-1 발현이 NO와 PGE 2 의생성을억제하는것을확인하였다. 헛개나무부위별성분분석및주요성분의구조동정부위별헛개나무추출물의주요성분차이를비교하기위하 여 HPLC 를이용하여분석하였다. 그결과잎, 줄기추출물에비해뿌리추출물에서일정한주요피크 (29.5 min) 를확인할수있었다 (Fig. 7). 본연구에서는뿌리추출물에서추출되는주요성분을 Recycling preparative HPLC system 를이용하여분리및정제하였다. 화합물 1 (White powder) 는 obitrap/ms 분석을수행하였으며, m/z [M] + 의분자이온 peak가관측되 -475-

11 Korean J. Plant Res. 31(5) : 466~477(2018) 어분자량을 로결정하였다. 1 H NMR spectrum (in Acetone-d 6, 900 MHz) 에서 δ1.05, 0.91, 0.90 및 0.77 에서 4개의 angular methyl group 과 δ3.16에서 multiplet으로나타나는 triterpene 의통상적인 3번 oxymethine proton 을확인할수있었다. δ1.74에서 sp 2 carbon 에결합하고있는또하나의 methyl group 을확인하였고, δ4.77 및 4.63 에서각각 singlet 으로나타나는두개의 olefin proton을확인하여이화합물이 isopropenyl group 을가지고, δ4.72, 4.55 에서 doublet 으로나타나는하나의 oxygenate methylene group과 aromatic 영역의 δ7.53, 6.92, 7.46 에서 ABX system 의 proton coupling 으로 protocatechuoyl group 을함유하고있는 lupane 계열의 triterpenoid 임을추정할수있었다. 13 C-NMR spectrum (in Acetone-d 6, 225 MHz) 에서는총 37개의 signal 을확인하여 triterpene 임을확인하였다. δ27.7, 15.3, 16.3, 16.1 및 18.8 에서 5개의 methyl 기를확인하였고, δ77.7 에서 3번의 oxymethine carbon 을, δ149.9 및 109.4에서 isopropenyl group 의 olefin carbons 을확인하였고, δ62.8에서 oxygenated methylene carbon의전형적인 signal임을다시한번확인할수있고, δ165.9 는 protocatechuoyl group 의 carboxy carbon 을확인할수있었다. 따라서 NMR 분광분석및 MS 분석에의하여헛개나무뿌리주요성분의화학구조가 lupane 계의 triterpenoid인 27-O-protocatechuoylbetulinic acid 임을규명할수있었다 (Kang et al., 2017). 적요본연구는헛개나무잎, 줄기, 뿌리로부터얻어진메탄올추출물의항염증효과를구명하기위해서수행되었다. LPS 로염증이유도된 RAW264.7 세포내부위별추출물을동시에처리하여염증매개성물질인 NO 생성량분석결과 40 μg / ml농도에서뿌리 (94.7%) 와줄기 (42.6%) 에서는효과가있는반면잎에서는효과가없는것으로확인되었다. 이중탁월한효과를보인뿌리추출물 (RE) 의항염증효과및관련분자적기전을확인하였다. 그결과, 염증반응의주요경로인 NF-kB 및 MAPK 신호전달경로에서 RE가 LPS 로유도된 NF-kB 의핵이동을억제하고, ERK, JNK, p38의인산화를억제함으로써 inos, COX-2 의발현이감소되고, NO 와 pro-inflammatory cytokine (IL-6, IL-1β, TNF-α) 의생성이억제됨을확인하였다. 또한 RE는대식세포에서 Nrf2 를활성화시켜항염증성단백질인 HO-1 발현을유도하여항염증효과를나타내었다. 또한, RE 로부터주요성분을 분리한후 NMR 과 MS 기기를이용하여구조동정된 27-Oprotocatechuoylbetulinic acid 화합물에서도높은항염증효과를확인하였다. 이러한연구결과는헛개나무뿌리와그주요성분은의약품소재및기능성식품등의기능성소재로활용될수있는기초적인정보를제공할것으로생각된다. 사사이논문은한국수목원관리원국립백두대간수목원조성사업지원과한국기초과학지원연구원 2018선도장비이용자프로그램사업지원에의하여연구되었으며, 이에감사드립니다. References Elbirt, K.K. and H.L. Bonkovsky Heme oxygenase: recent advances in understanding its regulation and role. Proc. Assoc. Am. Physicians 111: Förstermann, U. and W.C. Sessa Nitric oxide synthases: regulation and function. Eur. Heart J. 33: Horwood, N.J., T.H. Page, J.P. McDaid, C.D. Palmer, J. Campbell, T. Mahon, F.M. Brennan, D. Webster and B.M. Foxwell Bruton s tyrosine kinase is required for TLR2 and TLR4-induced TNF production, but not IL-6, production. J. Immunol. 176: Ghosh, G., V.Y. Wang, D.B. Huang and A. Fusco NF-κB regulation: lessons from structures. Immunol. Rev. 246: Gozzelino, R.,V. Jeney and M.P. Soares Mechanisms of cell protection by heme oxygenase-1. Annu. Rev. Pharmacol. Toxicol. 50: Jang, B.C., J.H. Paik, S.P. Kim, D.H. Shin, D.K. Song, J.G. Park and S.I. Suh Catalase induced expression of inflammatory mediators via activation of NF-kappaB, PI3K/ AKT, p70s6k, and JNKs in BV2 microglia. Cell. Signal. 17: Jeong, D.H., K.B. Kim, M.J. Kim, B.K. Kang and D.H. Ahn Anti-inflammatory activity of methanol extract and n-hexane fraction mojabanchromanol b from Myagropsis myagroides. Life Sci. 114: Kang, K.B., J.B. Jun, J.W. Kim, H.W. Kim and S.H. Sung Ceanothane-and lupine-type triterpene esters from the roots of Hovenia dulcis and their antiproliferative activity on HSC-T6 Cells. Phytochemistry 142: Kaur, G, M.N. Hughes, C.J. Green, P. Naughton, R. Foresti and -476-

12 LPS 로유도된 RAW264.7 대식세포에대한헛개나무 (Hovenia dulcis) 추출물의항염증효과 R. Motterlini Interaction of bilirubin and biliverdin with reactive nitrogen species. FEBS Lett. 543: Kim, B.H., Y.T. Lee and K.H. Kang Codonopsis lanceolate inhibits inflammation through regulation of MAPK in LPS stimulated RAW264.7 cells. Korean J. Oriental Physiol. Pathol. 24: Kim, D.H., J.H. Chung, J.S. Yoon, Y.M. Ha, S. Bae, E.K. Lee, K.J. Jung, M.S. Kim, Y.J. Kim and M.K. Kim Ginsenoside Rd inhibits the expressions of inos and COX-2 by suppressing NF-κB in LPS-stimulated RAW264.7 cells and mouse liver. J. Ginseng Res. 37: Kim, H.N., S.B. Park, G.H. Park, H.J. Eo, J.H. Song, H.Y. Kwon and J.B. Jeong Anti-inflammatory effects of the root extracts from Hibiscus syriacus in LPS-stimulated RAW264.7 cells. Korean J. Plant Res. 31: Lee, J.W. and Y.J. Kang Anti-inflammatory effects of Abeliopyllum distichum flower extract and associated MAPKs and NF-kB pathway in Raw264.7 cells. Korean J. Plant Res. 31: Majdalawieh, A. and H.S. Ro Regulation of IκBα function and NF-κB signaling: AEBP1 is a novel proinflammatory mediator in macrophages. Mediators Inflamm. 2010: Park, J.S., I.S. Kim, S.U. Rehman, C.S. Na and H.H. Yoo HPLC determination of bioactive flavonoids in Hovenia dulcis fruit extracts. J Chromatogr Sci. 54: Park, J.Y., J.Y. Moon, S.D. Park, W.H. Park, H. Kim and J.E. Kim Fruits extracts of Hovenia dulcis Thunb. suppresses lipopolysaccharide-stimulated inflammatory responses through nuclear factor-kappab pathway in Raw264.7 cells Asian Pac J Trop Med. 9: Tsan, M.F., J.E. White and C.L. Rosano Modulation of endothelial GSH concentrations: effect of exogenous GSH and GSH monoethyl ester. J. Appl. Physiol. 66: Yang, H., K.H. Oh and Y.C. Yoo Anti-inflammatory effect of hot water extract of Aronia fruits in LPS-stimulated RAW264.7 macrophages. J Korean Soc Food Sci. Nutr. 44:7-13. Yang, J., S. Wu and C. Li High efficiency secondary somatic embryogenesis in Hovenia dulcis Thunb. through solid and liquid cultures. Sci. World J :1-6. (Received 29 May 2018 ; Revised 7 August 2018 ; Accepted 18 September 2018) -477-