Korean J. Plant Res. 31(4): (2018) Print ISSN Online ISSN Original Research Artic

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1 Korean J. Plant Res. 31(4): (2018) Print ISSN Online ISSN Original Research Article 정용환 *, 함영민, 윤선아, 오대주, 김창숙, 윤원종 ( 재 ) 제주테크노파크생물종다양성연구소 Antioxidant and Anti-inflammatory Effects of Ficus erecta var. sieboldii Leaf Extract in Murine Macrophage RAW Cells Yong-Hwan Jung*, Young-Min Ham, Seon-A Yoon, Dae-Ju Oh, Chang-Suk Kim and Weon-Jong Yoon Biodiversity Research Institute, Jeju Technopark (JTP), Seogwipo 63608, Korea Abstract - In this study, a preliminary evaluation of the antioxidant and anti-inflammatory activity of the Ficus erecta var. sieboldii (Miq.) King (FES) leaf extract has been performed to assess its potential as a natural resource for food and medicinal materials. FES was extracted using 70% EtOH and then fractionated sequentially using n-hexane, CH 2 Cl 2, EtOAc, and n-buoh. To screen for antioxidant and anti-inflammatory agents effectively, the inhibitory effect of the FES extracts on the production of oxidant stresses (DPPH, xanthine oxidase, and superoxide) and pro-inflammatory factors (NO, inos, COX-2, PGE 2, IL-6, and IL-β) in the murine macrophage cell line RAW activated with lipopolysaccharide (LPS) was examined. Among the sequential solvent fractions of FES, the CH 2 Cl 2 and EtOAc fractions showed decreased production of oxidant stresses (DPPH, xanthine oxidase and superoxide), and the hexane and CH 2 Cl 2 fractions of FES inhibited the production of pro-inflammatory factors (NO, inos, COX-2, and PGE 2 ). The CH 2 Cl 2 fraction also inhibited the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β). These results suggest that FES has a significant effects on the production of oxidant stresses and pro-inflammatory factors and may be used a natural resource for antioxidant and anti-inflammatory agents. Key words - Antioxidant, Anti-inflammatory, Ficus erecta var. sieboldii, Oxidant stresses, Pro-inflammatory factors 서언 생체내항산화시스템은외부의산화적스트레스로부터야기되는여러가지질환을억제함으로서인체를보호하지만, 산화적스트레스를완전하게방어할수는없기에이를보완및대체할수있는항산화소제연구가꾸준하게이루어지고있다 (Carocho and Ferreira, 2013; Jang et al., 2016; Sim et al., 2017). 특히, 인체는물질의대사및에너지생산을위하여필수적으로산소를이용하고있으며정상적으로는물과이산화탄소를배출한다. 그러나이중약 4% 가량은완전히환원되지못하 * 교신저자 : yhjung@jejutp.or.kr Tel 여반응성이강하고유해한활성산소가된다 (Halliwell and Gutteridge, 1993). 유해산소로알려져있는활성산소는노화나암등의여러가지성인병의원인이되고있다 (Talalay and Benson, 1982; Wefers et al., 1984; Bae, 2002). 최근에노화및성인병질환의원인이활성산소종에기인된것이라는연구가보고됨에따라이를조절하거나제거할수있는항산화제에대한연구가활발히진행되어많은항산화제의개발연구가보고되고있다 (Corl, 1974; Coleman et al., 2003). 그러나, 현재 BHA (butylated hydroxyanisole) 및 BHT (butylated hydroxytoluene) 와같은합성항산화제는효과가우수하고경제성때문에많이이용되고있지만생체효소, 지방의변이및독성으로인해인체에암을유발할수있다는안전성 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 Korean J. Plant Res. 31(4) : 303~311(2018) 문제로 phenolics, flavonoids, carotenoids 등과같은천연항산화제의개발이요구되고있다 (Branen, 1975; Williams et al., 1990) 염증은외부자극에대한생체조직의방어반응의하나로서손상된조직을재생하려는기작이다. 그러나지속적인염증반응은만성염증질환및종양등의유발원인이되기도한다 (Ismaki and Punnonen, 1997). 염증반응에관여하는대표적인세포중하나인대식세포는염증성매개물질인 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 (Storck et al., 1994). 염증반응의지표물질인 NO는 L-arginine에서 NO synthase (NOS) 에의해합성된다. NOS 에는 endothelial NOS, neuronal NOS, inducible NOS (inos) 의세가지형태가있으며, 이들중 inos 에의한 NO 생성은병리학적으로중요한역할을한다 (Nathan, 1992). 일반적인 NO의형성은박테리아를죽이거나종양을제거하는면역반응의역할을한다 (Moncada et al., 1991). 그러나 lipopolysaccharide (LPS) 또는염증성 cytokine 등에의해발현이유도된 inos 에의한과다한 NO 생성은염증반응을심화시켜조직의손상, 유전자변이및신경손상등을일으키는것으로알려져있다 (Stuehr et al., 1991; McCartney et al., 1993; Weisz et al., 1996; Yun et al., 1996). 좁은잎천선과나무 (Ficus erecta var. sieboldii (Miq.) King) 는무화과속에속하는식물로서젖꼭지나무, 가는잎천선과등의이명을가지며, 우리나라에서도제주도와남해안지방특히제주도에주로자생하는식물이다. 바닷가산기슭에서자라며높이는 2 4 m 정도로자라는낙엽관목으로수피는평활하며가지는회백색이고털이없으며, 잎은오생하며피침형이고, 길이는 cm로서가장가리가밋밋하나맹아에톱니가있으며양면에털이없으나표면에털이약간있는것도있고엽맥이뚜렷하게돌출하며엽병은길이가 1 3 cm이다 (Lee, 1980). 민간에서는오래전부터보중, 익기, 건비, 화습, 강근장골, 소종, 활혈, 해독의효능이있으며, 류마티스성관절염, 중기허약, 기혈쇠미, 사디산언, 근골불리, 타박상, 경폐, 산후유즙결핍을치료등에이용되어져왔으나 (Ahn, 1998), 생리활성보고가이루어지지않아본연구에서는좁은잎천선과나무알코올추출물의항산화및항염증효과를탐색함으로써향후식의약품소재등천연물소재로의활용가능성에대해알아보고자하였다. 재료및방법시료의추출본연구에사용한좁은잎천선과나무는서귀포시남원읍에서채취하였으며, 제주생물종다양성연구소의송관필박사의식물학적동정을거쳤으며, 실험에사용한시료의확증표본 ( 표본번호 JBRI ) 은제주생물종다양성연구소표본실에보관하고있다. 좁은잎천선과나무의잎은수세후열풍건조한다음마쇄기로갈아분말화하였다. 분말시료 300 g을 70% 발효주정 (EtOH) 으로 1회교반추출후여과하여감압농축하여용매를증발시켰다. 여기에서얻은 EtOH 추출물 (90 g) 을계통적추출방법에의하여 n-hexane (Hex) 분획 (2.73 g), dichloromethane (CH 2 Cl 2 ) 분획 (1.24 g), ethylacetate (EtOAc) 분획 (0.49 g), n-buthanol (BuOH) 분획 (7.32 g) 및 water 분획 (70.01 g) 을얻어실험시료로사용하였다. DPPH radical 소거활성에의한항산화활성검색항산화활성은 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma) 를이용하여시료의라디칼소거효과 (radical scavenging effect) 를측정하는 Blois 법을활용하였다 (Blois, 1958). DPPH 약 2 mg을 EtOH 15 ml에녹여 DPPH용액을제조하였다. 이용액 12 ml에 DMSO 6.25 ml를첨가한후, 517 nm의파장에서대조군의흡광도가 0.94~0.97이되도록 EtOH 로희석하여 10초간진탕시켰다. 그리고용매 1 ml에분말로추출된시료 1 mg을섞은후충분히녹이고, 준비된 DPPH 450 μl에시료용액 50 μl를넣어섞은후실온에서 10분간방치하였다가 517 nm에서흡광도를측정하였다. Xanthin oxide 억제및 superoxide 소거활성검색 Xantin/xanthine oxidase 에의한 uric acid 생성은 290 nm에서증가된흡광도에의해측정하였고, superoxide 의양은 nitroblue tetrazolium (NBT) 환원방법에의해측정하였다 (Fridovich, 1970; Nishikimi et al., 1972). 반응액으로는각농도별시료 (15.63, 31.25, 62.5, 125, 250, 500, 1000 μg / ml ) 와 0.5 mm xanthin 과 1 mm EDTA 를 200 mm phosphate buffer (ph 7.5) 100 μl에서준비하였고, 50 mu/ ml xanthin oxidase 를첨가하여 uric acid 의생성을유도하였다. Superoxide 소거활성은위반응액에 0.5 mm NBT 를첨가하여반응시켰다. Xanthine oxidase 억제및 superoxide 소거활성은각각생성된 -304-

3 uric acid 와 superoxide 의흡광도가 50% 감소할때나타나는시료의농도 (IC 50 ) 로표시하였으며, 각시료는 3회반복하여실험을실시하여평균값을구하였다. 세포배양 Murine macrophage cell line RAW 세포를 KCLB (Korean Cell Line Bank) 로부터분양받아 100 units/ ml penicilin-streptomycin 과 10% fetal bovine serum (FBS) 이함유된 DMEM 배지를사용하여 37, 5% CO 2 항온기에서배양하였으며, 3 4 일에한번씩계대배양을시행하였다. Lipopolysaccharide (LPS, E. coli serotype 0111:B4) 를 Sigma 로부터구입하여사용하였다. Nitric oxide (NO) assay RAW 세포 ( cells/ ml ) 를 DMEM 배지를이용하여 24 well plate에접종하고, 시험물질과 LPS (1 μg / ml ) 를함유한새로운배지를동시에처리하여 24시간배양하였다. 생성된 NO의양을 Griess 시약을이용하여세포배양액중에존재하는 NO - 2 의형태로측정하였다. 세포배양상등액 100 μl와 Griess 시약 [1% (w/v) sulfani-lamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid] 100 μl를혼합하여 96 well plate 에서 10분동안반응시킨후 ELISA reader 를이용하여 540 nm에서흡광도를측정하였다. 표준농도곡선은 sodium nitrite (NaNO 2 ) 를 serial dilution( 연속희석 ) 하여얻었다. 세포독성평가 (LDH assay) RAW 세포 ( cells/ ml를 DMEM 배지에시험약물과 LPS (1 μg / ml ) 를동시에처리하여 24시간배양한후배양배지를얻어 3,000 rpm 에서 5분간원심분리하였다. LDH (lactate dehydrogenase) 활성을 non-radioactive cytotoxicity assay kit (Progma, USA) 를이용하여측정했으며, 96 well plate 에원심분리하여얻은배양배지 50 μl와 reconstituted substrate mix 를 50 μl를넣고, 실온에서 30분반응시킨후 50 μl의 stop solution 을넣어 490 nm에서흡광도를측정하였다. 각시료군에대한평균흡광도값을구하였으며, 대조군 (LDH control, 1:5000) 의흡광도값과비교하여세포독성을평가하였다. prostaglandin E 2 (PGE 2 ) 생성억제효능평가 RAW 세포를 DMEM 배지를이용하여 cells/ ml로조절한후 24 well plate 에접종하고, 5% CO 2 항온기에서 18 시간전배양하였다. 이후배지를제거하고 10배농도 (1 mg / ml ) 로조제된시험물질 50 μl와 450 μl의 LPS 최종농도 (1 μg / ml ) 를함유한새로운배지를동시에처리하여전배양과동일한조건에서배양하였다. 24시간후 Prostaglandin E 2 (PGE 2 ) 를측정하기위해배양배지를 12,000 rpm 에서 3 min 간원심분리하여상층액을얻었다. PGE 2 의정량은 PGE 2 ELISA kit (R&D System, Inc, USA) 를이용하여정량하였으며 standard에대한표준곡선의 r 2 값은 0.99 이상이었다. 염증성사이토카인 (TNF-α, IL-6, IL-1β) 생성억제효능평가 RAW 세포를 DMEM 배지를이용하여 cells/ ml로조절한후 24 well plate에접종하고, 5% CO 2 항온기에서 18 시간전배양하였다. 이후배지를제거하고 RAW 세포는 10배농도 (5, 10, 20 μg / ml ) 로조제된추출물시료 50 μl와 450 μl의 LPS (1 μg / ml ) 를함유한새로운배지를동시에처리하였고전배양과동일한조건에서배양하였다. 24 시간후배양배지를원심분리 (12,000 rpm, 3 min) 하여얻어진상층액의 pro-inflammatory cytokines 생성함량을측정하였다. 모든시료는정량전까지 20 이하에보관하였다. pro-inflammatory cytokines 정량은 mouse enzyme-linked immnunosorbent assay (ELISA) kit (R&D System Inc., Minneapolis, MN, USA) 를이용하여정량하였으며 standard 에대한표준곡선의 r 2 값은 0.99 이상이었다. Western blot analysis RAW 세포 ( cells/ ml ) 를 18시간전배양을하고, LPS (1 μg / ml ) 로자극을주고시료 (50 μg / ml ) 를동시에처리하여전배양과동일조건에서 24시간동안배양하였다. 배양이끝난후, 세포를 2~3 회 PBS (Phosphate Buffered Saline) 로세척후 300 μl의 lysis buffer 를첨가, 30분 ~1시간동안 lysis 시킨후원심분리 (15,000 rpm, 15 min) 하여세포막성분등을제거하였다. 단백질농도는 BSA (bovine serum albumin) 을표준화하여 Bio-Rad Protein Assay Kit 를사용하여정량하였다. 20~30 μg의 lysate를 8% mini gell SDS-PAGE (poly acrylamide gel electrophoresis) 로변성분리하여, 이를 PVDF membrane (BIORAD) 에 200 ma로 2시간동안 transfer 하였다

4 Korean J. Plant Res. 31(4) : 303~311(2018) 그리고 membrane 의 blocking 은 5% skin milk가함유된 TTBS (TBS+0.1% Tween 20) 용액에서상온에서 2시간동안실시하였다. inos 와 COX-2 의발현양을측정하기위해 1차항체로서 anti-mouse inos (Calbiochem) 와 anti-goat COX-2 (BD Biosciences) 를 TTBS용액에서희석 (1:1000) 하여상온에서 2시간반응시킨후 TTBS로 3회세정하였다. 2차항체로는 HRP (horse radish peroxidase) 가결합된 anti-mouse IgG와 antigoat IgG (Amersham Co.) 를 1:5000 으로희석하여상온에서 30 분간반응시킨후, TTBS 로 4회세정하여 ECL 기질 (Amersham Co.) 과 1분간반응후 X-ray 필름에감광하였다. 통계분석실험결과는 SAS Program (statistical analysis system) 을이용하여분석하였고, 평균치와표준편차로나타냈다. 그룹간의유의적인통계차를분석하기위하여 p<0.05의유의수준에서 Duncan's multiple range test 를실시하였다. 결과및고찰 DPPH radical 소거활성에의한항산화활성검색항산화물질의가장특징적인기작은유리기와반응하는것으로유리기소거작용은활성라디칼 (free radical) 에전자를공여하여식물중의항산화효과나인체에서노화를억제하는척도로사용된다. DPPH는안정한유리기로 cysteine, glutathione 과같은황함유아미노산과 ascorbic acid, aromatic amine (ρphenylenediamine, ρ-aminophenol) 등에의해환원되어탈색되므로항산화물질의항산화능측정에많이이용되고있다 (Chang et al., 1998). 좁은잎천선과잎의조추출물과각각의유기용매분획물의실험결과높은라디칼소거활성을보이지는않았으나, EtOAc 분획물의라디칼소거활성에대한 IC 50 값은 μg / ml로나타났다 (Table 1). Xanthine oxidase 억제 Xanthine oxidase 는산화적환경에서 xanthine dehydrogenase 로부터생성된다. Xanthine oxidase 는 hypoxanthine을산화시켜최종적으로 uric acid 와산소를생성하며산소유리기와수소과산화기가이산소로부터발생하게된다. Uric acid 의축적은고요산혈증과통풍을유발시키며 uric acid 형성의억제제가이들질환을위한치료물질로서유용할것이다. 게다가 xanthine oxidase에의해생성된산소유리기는세포의손상을초래한다 (Chang et al., 1998). 좁은잎천선과잎조추출물과각각의유기용매분획물의 xanthin oxidase 활성억제에대한결과는일반적으로항산화제로활용되는타식물보다는낮은활성을보이고있다. 좁은잎천선과나무 EtOAc 분획물에서 xanthin oxidase 활성억제는 IC 50 값이 μg / ml로가장높게나타났다 (Table 1). superoxide 소거활성정상적인산화적인산화의과정동안소모되는전체산소의 0.4-4% 정도는 free radical superoxide ( O - 2 ) 로전환되며생성된 O - 2 는다른 reactive oxygen species (ROS) 로전환되어직접적또는간접적으로세포손상을유발하는것으로알려져있다. 정상적으로는 O - 2 는내인성항산화방어기전에의해 superoxide dismutase (SOD) 에의해빠르게과산화수소로전 Table 1. Comparison of antioxidant potential on 70% EtOH extract and solvent fractions of FES Treatment DPPH radical scavenging activity IC 50 ( μg / ml ) z Xanthine oxidase inhibitory activity Superoxide radical scavenging activity 70% EtOH ±0.92 * > ±2.50 * n-hexane fr. >1000 >1000 >1000 CH 2 Cl 2 fr ±0.03 ** > ±2.57 * EtOAc fr ±0.02 ** ±10.09 * 85.72±1.23 ** n-buoh fr ±0.59 ** > ±16.63 * H 2 O fr ±10.02 >1000 >1000 z IC 50 values were calculated from regression lines using five different concentrations in triplicate experiments. Values are the mean ± SEM of triplicate experiments. *, P<0.05; **, P<

5 환된다. 그러나이내인성항산화방어체계가세포내산화- 환원균형을유지하는데문제가생길경우결과적으로산화스트레스가일어나게되며이산화스트레스는직접적으로세포내거대분자의손상을일으키거나세포손상을일으키는데중요한역할을한다 (Korycka et al., 1979). 따라서산소유리기의자유기를소거할수있는물질또한산화적손상의예방에유용할것으로사료된다. 좁은잎천선과나무잎조추출물과분획물에대한 superoxide 소거활성의대한결과는 DPPH radical 소거활성및 xanthine oxidase 억제활성의결과보다는높은억제활성을보였다. 특히, CH 2 Cl 2 분획물과 EtOAc 분획물에서비교적높은 superoxide 소거활성을나타냈으며 IC 50 값은각각 98.84, μg / ml로나타났다 (Table 1). 세포독성에미치는영향 LDH 는모든세포의세포질안에존재하는효소로서 pyruvic acid 와 lactic acid 간의가역적전환에관여하여촉매작용을하며, LDH 를내포한조직이파괴될때혈액중으로흘러나와혈중 LDH 가상승한다. RAW 세포 ( cells/ ml ) 에시험약물과 LPS (1 μg / ml ) 를동시처리하여 24시간배양한후, LDH assay 방법을이용하여세포독성을확인한결과, 좁은잎천선과나무의조추출물및각각의유기용매분획물에서독성이나타나지않았음을확인할수있었다 (Fig. 1). Nitric oxide 생성억제효과활성산소중하나이며, 최근염증유발에중요한역할을하는것으로알려진 nitric oxide (NO) 생성에대한좁은잎천선과나 무잎조추출물과분획물의효과를알아보았다. 생성된 NO 양을 Griess 시약을이용하여세포배양액중에존재하는 NO - 2 의형태로측정하였다. 실험결과 n-buoh 분획물과 H 2 O 분획물을제외하고좋은 NO 생성억제효과를보였으며, 그중 CH 2 Cl 2 분획물과 EtOAc 분획물에서대조군인 LPS 단독처리군에비해강력한 NO 생성억제효과를관찰할수있었으며, 항산화활성이높게나타나는 EtOAc 분획물에서농도의존적으로 NO 생성이억제하는것을확인할수있었다 (Fig. 1, Fig. 2). Pro-inflammatory cytokine 생성억제효과 Pro-inflammatory cytokine 들은정상조직에서발현될뿐만아니라병변과정에서그발현정도가증가되며, 암촉진과정에서일어나는피부염증에중요한역할을한다. TNF-α, IL-1 β, 그리고 IL-6 등과같은 cytokine 들은인간의염증성피부질환과관련이있음은이미많이보고되어왔다. 또한여러염증질환과알러지현상에 cytokine 들에대한항체를처리하였을때증상이완화되었다 (Kim and Moudgil, 2008; Kim et al., 2009). 염증단계에중추적역할을하고있는 cytokine 인 IL-1 β와 IL-6 의발현을저해시키거나 COX-2 활성저해에기인하는 PGE 2 의생성억제를통해 pro-inflammatory fator 의증가를수반하는병변과정을조절할수있을가능성이높다 (Ren and Torres, 2009; Heo et al., 2010). 대식세포인 RAW 세포로부터 Pro-inflammatory cytokine 의생성억제정도를확인한결과, 좁은잎천선과나무잎의 CH 2 Cl 2 분획물과 EtOAc 분획물에서 TNF-α, IL-6 와 IL-1β 의생성이조추출물과타분획물보다상대적으로높게억제되었으며항산화활성이높게나타나는 EtOAc 분획물에서농도의존적으로억제하는것을확인할수있었다 (Fig. 3). Fig. 1. The effects of 70% EtOH and solvent fractions of FES on cell viability and nitric oxide production in RAW cells. The production of nitric oxide was assayed in the culture medium of cells stimulated with LPS (1 μg / ml ) for 24 h in the presence of the 70% EtOH extract and the solvent fractions of FES (100 μg / ml ). Cytotoxicity was determined using the LDH method. Values are the mean ± SEM of triplicate experiments. *, P<0.05; **, P<0.01. Fig. 2. Inhibitory effect of EtOAc fraction of FES on nitric oxide production in RAW cells. The production of nitric oxide was assayed in the culture medium of cells stimulated with LPS (1 μg / ml ) for 24 h in the presence of the EtOAc fraction of FES (25, 50 and 100 μg / ml ). Values are the mean ± SEM of triplicate experiments. *, P<0.05; **, P<

$Korean J. Plant Res. 31(4) : 303~311(2018) (A) (B) (C) Fig. 3. Inhibitory effect of 70% EtOH extract and solvent fractions (above) and EtOAc fraction (below) of FES on cytokines production in RAW 264.$

6 Korean J. Plant Res. 31(4) : 303~311(2018) (A) (B) (C) Fig. 3. Inhibitory effect of 70% EtOH extract and solvent fractions (above) and EtOAc fraction (below) of FES on cytokines production in RAW cells. Cells ( cells/ ml ) were stimulated by LPS (1 μg / ml ) for 24 h in the presence of 70% EtOH extract and solvent fractions from FES (100 μg / ml ). Supernatants were collected, and the cytokines concentration in the supernatants was determined by ELISA. Values are the mean ± SEM of triplicate experiments. *, P<0.05; **, P<0.01. Inhibitory effect of 70% EtOH extract and solvent fractions (above) and EtOAc fraction (below) of FES on IL-1β(A), IL-6 (B) and TNF-α(C) production in RAW cells, respectively. Fig. 4. Inhibitory effects of the 70% EtOH extract and solvent fractions of FES on PGE 2 production in RAW cells. Cells ( cells/ ml ) were stimulated by LPS (1 μg / ml ) for 24 h in the presence of the 70% EtOH extract and the solvent fractions from FES (100 μg / ml ). Supernatants were collected, and the PGE 2 concentration in the supernatants was determined by ELISA. Values are the mean ± SEM of triplicate experiments. *, P<0.05; **, P<0.01. Prostagladin E 2 (PGE 2 ) 생성에미치는영향 Macrophage RAW 세포에서염증성인자인 PGE 2 억제효과를 ELISA kit 를이용하여정량하였다. 그결과 PGE 2 생성억제는 n-hexane 분획물, n-buoh 분획물과 H 2 O 분획물을제외한유기용매분획물에서높은억제효과를보여주고있다. CH 2 Cl 2 분획물은 92%, EtOAc 분획물은 84% 의억제효과를보였으며 (Fig. 4), 항산화활성이높게나타나는 EtOAc 분획물에서 PGE 2 생성이농도의존적으로억제되고있다 (Fig. 5). 이러한 Fig. 5. Inhibitory effects of EtOAc fraction of FES on PGE 2 production in RAW cells. Cells ( cells/ ml ) were stimulated by LPS (1 μg / ml ) for 24 h in the presence of EtOAc fraction of FES (25, 50 and 100 μg / ml ). Supernatants were collected, and the PGE 2 concentration in the supernatants was determined by ELISA. Values are the mean ± SEM of triplicate experiments. *, P<0.05; **, P<0.01. 결과 LPS에의해발현되는 PGE 2 억제에영향을준다는것을확인할수있었다. inos 생성에미치는영향 inos 는평소에는세포내에존재하지않으나일단유도되면장시간동안다량의 NO를생성하며, 생성된 NO는병리적인혈관확장, 세포독성, 조직손상등과같은생체에유해한작용을나타낸다. 그리고, 염증상태에서 inos 에의해생성된 NO는혈관투과성, 부종등의염증반응을촉진시킬뿐만아니라염증매 -308-

7 Fig. 6. Inhibitory effect of 70% EtOH extract and solvent fractions of FES on the protein level of inos and COX-2 in RAW264.7 cells. RAW cells ( cells/ ml ) were pre-incubated for 18 hr, and the cells were stimulated with LPS (1 μg / ml ) in the presence of 70% EtOH extract and solvent fractions of FES (100 μg / ml ) for 24 hr. inos and COX-2 protein levels were determined using immunoblotting method. Fig. 7. Inhibitory effect of EtOAc fraction of FES on the protein level of inos and COX-2 in RAW264.7 cells. RAW cells ( cells/ ml ) were pre-incubated for 18 hr, and the cells were stimulated with LPS ( μg / ml ) in the presence of FES fraction (EtOAc - 25, 50, 100 μg / ml ) for 24 hr. 개체의생합성을촉진하여염증을심화시키는것으로알려져있다 (Tesuka et al., 2001; Santos-Gomes et al., 2003). RAW 세포에 LPS (1 μg / ml ) 를사용하여 inos 의생성을유도한후좁은잎천선과나무잎조추출물과분획물을처리하여단백질생성에대한억제정도를 western blotting을통해알아보았다. 그결과단백질수준에서도 n-buoh 분획물과 H 2 O 분획물을제외한조추출물과유기용매분획물에서좋은억제효과를보였으며, 그중 CH 2 Cl 2 분획물과 EtOAc 분획물에서대조군인 LPS 단독처리군에비해강한억제효과를나타내었고 (Fig. 6) 항산화활성이높게나타나는 EtOAc 분획물에서 PGE 2 생성이농도의존적으로억제되고있음도확인했다 (Fig. 7). 이러한결과 NO의생성억제가 inos 발현억제를통한것으로여겨진다. COX-2 발현에미치는영향다수의염증억제약물들의작용기전은 prostagladin 합성억제를나타내며이는 COX-2의생성및활성저해에의한것이다. COX 는 COX-1 과 COX-2로나뉘어지는데다양한세포에서각각다른발현경향을나타낸다. COX-1 은위및신장기능의유지, 혈소판의형성에필요한 prostagladin 의합성에작용하 며, 상대적으로 COX-2는동물이나인간의염증반응부위에서발현된다 (Masferrer et al., 1994; Hyun et al., 2004). 따라서, COX-2에의한 prostagladin의합성은염증반응을매개하는것으로여겨진다. RAW 세포에 LPS (1 μg / ml ) 로자극을주고좁은잎천선과나무잎조추출물과분획물을처리하여확인한결과, 조추출물, n-buoh 분획물과 H 2 O 분획물을제외한유기용매분획물에서억제효과를나타났으며 (Fig. 6) 항산화활성이높게나타나는 EtOAc 분획물에서 COX-2 발현이농도의존적으로억제됨을확인하였다 (Fig. 7). 따라서, 본연구결과에서는좁은잎천선과나무잎의알코올추출물을순차적으로분획한 CH 2 Cl 2 분획물과 EtOAc 분획물이천연물유래항염증소재로서산업화이용가능성을확인하였다. 특히, 항산화활성과항염증활성을보이고있는 EtOAc 분획물을활용한추가연구를통해식의약품소재등천연물소재로서의활용가능성이높을것으로사료된다. 적요본연구는좁은잎천선과나무잎추출물을식의약품소재등천연물소재로활용하기위하여항산화및항염증활성에대한 -309-

8 Korean J. Plant Res. 31(4) : 303~311(2018) 예비평가를기술하였다. 좁은잎천선과나무는 70% 에탄올을사용하여추출한다음헥산, 디클로로메탄, 에틸아세테이트및부탄올을사용하여순차적으로분획하였다. 항산화및항염증제효과를효과적으로스크리닝하기위해좁은잎천선과나무잎추출물이산화스트레스 (DPPH, xanthine oxidase and superoxide) 생성에미치는억제효과를확인하였다. 또한, LPS 로활성화된대식세포 RAW 세포에서염증성인자 (NO, inos, COX-2, PGE 2, IL-6 and IL-β) 의생성에대한좁은잎천선과나무잎추출물의억제효과를확인하였다. 좁은잎천선과나무잎추출물의용매분획물중디클로로메탄과에틸아세테이트분획물은산화스트레스 (DPPH, xanthine oxidase and superoxide) 의생성감소가있었고, 좁은잎천선과나무잎추출물의헥산과디클로로메탄분획물은염증유발인자 (NO, inos, COX-2, PGE 2, IL-6, and IL-β) 의생성을억제하였다. 또한, 디클로로메탄분획물은염증성사이토카인 (TNF-α, IL-6, and IL-1β) 의생성을억제하였다. 이러한결과는좁은잎천선과나무잎추출물이산화스트레스및염증유발인자에유의한영향을미치고있어산화방지제및항염증제와같은천연물소재로활용될수있을것으로사료된다. 사사본연구는농림축산식품부의재원으로농림식품기술기획평가원의농생명산업기술개발사업의지원을받아연구되었음 ( ). References Ahn, D.K Illustrated Book of Korean Medicinal Herbs. Kyohaksa Publishing Co., Seoul, Korea. p. 855 (in Korean). Bae, S.J The effects of anticarcinogenic activity of Solanum thberosum peel fractions. J. Korean Soc. Food Sci. Nutr. 31: Blois, M.S Antioxidant determinations by the use of a stable free radical. Nature 181: Branen, A.L Toxicological and biochemistry by the use of a stable free radical. Nature 181: Carocho, M. and I.C. Ferreira A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methologies and future perspectives. Food Chem. Toxicol. 51: Chang, Z.J., S.C. Kuo, S.C. Chan, F.N. KO and C.M. Teng Antioxidant properties of butein isolated from Dalbergia odorifera. Biochem. Biophys. Acta 1392: Coleman, M.D., S. Fernandes and L.A. Khanderia A preliminary evaluation of a novel method to monitor a triple antioxidant combination (vitamin E, C and α-lipoic acid) in diabetic volunteers using in vitro methaemoglobin formation. Environ. Toxicol. Pharmacol. 14: Corl, M.M Antioxidant activity of tocopherol and ascorbylpalmitate and their mode of action. J. Am. Oil Chem. Soc. 51: Fridovich, I Quantitative aspects of the production of superoxide anion radical by milk xanthine oxidase. J. Biol. Chem. 245: Halliwell, B. and J.M.C. Gutteridge Free Radicals in Biology and Medicine, 2 nd ed. Glarendon Press, Oxford, UK. Heo, S.J., W.J. Yoon, K.N. Kim, G.N. Ahn, S.M. Song, D.H. Kang, A. Affan, C. Oh, W.K. Jung and Y.J. Jeon Evaluation of anti-inflammatory effect of fucoxanthin isolation from brown algae in lipopolysaccharide-stimulated RAW macrophages. Food Chem. Toxicol. 48: Hyun, E.A., H.J. Lee, W.J. Yoon, S.Y. Park, H.K. Kang, S.J. Kim and E.S. Yoo Inhibitory Effect of Salcia officinalis on the inflammatory cytokines and inducible nitric oxide synthesis in murine macrophage RAW YAKHAK HOEJI 48: Ismaki, P. and J. Punnonen Pro-and anti-inflammatory cytokine in rheumatoid arthritis. Ann. Med. 29: Jang, T.W., S.H. Nam and J.H. Park Antioxidant activity and inhibitory effect on oxidative DNA damage of ethyl acetate fractions extracted from cone of red pine (Pinus densiflora). Korean J. Plant Res. 29: (in Korean). Kim, E.Y. and K.D. Moudgil Regulation of autoimmune inflammation by pro-inflammatory cytokines. Immunol. Left. 120:1-5. Kim, J.H., R.A. Bachmann and J. Chen Interleukin-6 and insulin resistance. Vitam. Horm. 80: Korycka-Dahl, M., T. Richardson and C. Hicks Superoxide dismutase activity in bovine milk serum. J. Food Protection 42: Lee, T.B Illustrated Flora of Korea. Hyangmoonsa Publishing Co., Seoul, Korea. p. 990 (in Korea). Masferrer, J., B.S. Zweifel, P.T. Manning, S.D. Hauser, K.M. Leahy, W.G. Smith, P.C. Isakson and K. Seibert Selective inhibition of inducible cyclooxygenase 2 in vivo is anti-inflammatory and nonulcerogenic. Proc. Natl. Acad

9 Sci. 91: McCartney-Francis, N., J.B. Allen, D.E Mizel, J.E. Albina, Q.W. Xie, C.F. Nathan and S.M. Wahl Suppression of arthritis by an inhibitor of nitric oxide synthase. J. Exp. Med. 178: Moncada, S., R.M. Palmer and E.A. Higgs Nitric oxide: Physiology pathophysiology, and pharmacology. Pharmacol. Rev. 43: Nathan, C Nitric ocide as a secretory product of mammalian cells. FASEB J. 6: Nishikimi, M., N.A. Roa and K. Yagi The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46: Ren, K. and R. Torres Role of interleukin-1 beta during pain and inflammation. Brain. Res. Rev. 60: Santos-Gomes, P.C., R.M. Seabra, P.B. Andrade and M. Fernandes- Ferreira Determination of phenolic antioxidant compounds produced by calli and cell suspensions of sage (Salvia officinalis L.). J. Plant Physiol. 160: Sim, M.O., H.J Lee, J.H Jang, H.E Lee, H.K Jung, T.M Kim, J.H No, J. Jung, D.E. Jung and H.W. Cho Antiinflammatory and antioxidant effects of Spiraea prunifolia Sieb. et Zucc. var. simpliciflora Nakai in RAW cells. Korean J. Plant Res. 30: (in Korean). Storck, M., M. Scgilling, K. Burkhardt, R. Prestel, D. Abendroth and C. Hammer Production of proinflammatory cytokines and adgesion molecules in ex-vivo xenogeneic kidney perfusion. Transpl. Int. 7:S647-S649. Stuehr, D.J., H.J. Cho, N.S. Kwon, M.F. Weise and C.F. Nathan Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: An FAD-and FMN-containing flavoprotein. Proc. Natl. Acad. Sci. USA 88: Talalay, P. and A.M. Benson Elevation of quinone reductase activity by anticarcinogenic antioxidants. Advances in Enzyme Regulation 20: Tesuka, Y., S. Irikawa, T. Kaneko, A.H. Banskota, T. Nagaoka, Q. Xionh, K. Hase and S. Kadota Screening of chinese herbal drug extracts for inhibitory activity on nitric oxide production and identification of an active compound of Zanthoxylum bugeanum. J. Ethnopharmacol. 77: Wefers, H., T. Komai, P. Talalay and H. Sies Protection against reactive oxygen species by NAD(P)H: quinone reductase induced by the dietary antioxidant butylated hydroxyanisole (BHA). Federation of European Biochemical Societies 169: Weisz, A., L. Cicatiello and H. Esumi Regulation of the mouse inducible type nitric oxid synthase gene promoter by interferon-γ, bacterial lipopolysaccharide, and NG-monomethyl- L-arginine. Biochem. J. 316: Williams, G.M., C.X. Wang and M.J. Iatropoulos Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. I. Chronic feeding studies. Food Chem. Toxicol. 28: Yun, H.Y., V.L. Dawson and T.M. Dawson Neurobiology of nitric oxide. Crit. Rev. Neurobiol. 10: (Received 14 April 2018 ; Revised 2 June 2018 ; Accepted 11 June 2018) -311-

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,