Korean J. Plant Res. 27(5):439-446(2014) http://dx.doi.org/10.7732/kjpr.2014.27.5.439 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article Murine Macrophage RAW 264.7 세포에서구실잣밤나무추출물의항염증효과 고영종 1,2, 송상목 1,3, 현우철 1, 양수경 1, 송창길 2, 이동선 3, 윤원종 1 * 1 ( 재 ) 제주테크노파크생물종다양성연구소, 2 제주대학교생명자원과학대학식물자원환경전공, 3 제주대학교생명자원과학대학응용생명공학전공 Anti-inflammatory Effect of Castanopsis cuspidata Extracts in Murine Macrophage RAW 264.7 Cells Yeong-Jong Ko 1,2, Sang Mok Song 1,3, Woo-Chol Hyun 1, Soo-Kyung Yang 1, Chang-Khil Song 2, Dong-Sun Lee 3 and Weon-Jong Yoon 1 * 1 Biodiversity Research Institute, Jeju TECHNOPARK, Seogwipo-si 699-943, Korea 2 Faculty of Biotechnology, Jeju National University, Jeju 690-756, Korea 3 Major of Plant Resource & Environment, Jeju National University, Jeju 690-756, Korea Abstract - This study describes a preliminary evaluation of the anti-inflammatory activity of Castanopsis cuspidata extracts. C. cuspidata was extracted using 80% ethanol and then fractionated sequentially with n-hexane, dichloromethane, ethylacetate, and butanol. To screen for anti-inflammatory agents effectively, we first examined the inhibitory effect of the C. cuspidata extracts on the production of pro-inflammatory factors and cytokines stimulated with lipopolysaccharide. In addition, we examined the inhibitory effect of C. cuspidata extracts on pro-inflammatory mediators (NO, inos, COX-2) in murine macrophage RAW 264.7 cells. The amounts of protein levels were determined by immunoblotting. Of the sequential solvent fractions of C. cuspidata, the n hexane, dichloromethane and ethylacetate fractions inhibited the mrna expression of pro-inflammatory cytokines (IL-1β and IL-6), production of NO, and the protein level of inos and COX-2. These results suggest that C. cuspidata may have significant effects on inflammatory factors and may be provided as a possible anti-inflammatory therapeutic plant. Key words - Castanopsis cuspidata, Pro-inflammatory factors, Cytokines, Lipopolysaccharide, RAW 264.7 cells 서언 염증반응은생체나조직에물리적작용이나화학적물질, 세균감염등의어떠한기질적변화를가져오는침습이가해질때그손상부위를수복재생하려는기전이며, 일단자극이가해지면국소적으로 histamine, serotonine, bradykinin, prostaglandins (PGs), hydroxyeicosatetraenoic acid (HETE), leukotriene 과같은혈관활성물질이유리되어혈관투과성이증대되면서염증을유발한다 (Tizard et al., 1986; Lee et al., 2006). 그러나 * 교신저자 (E-mail) : yyjkl@jejutp.or.kr These authors contributed equally to work. 지속적인염증반응은도리어점막손상을촉진하고, 그결과일부에서는암발생등의질환을이끈다 (Yoon et al., 2010; Nam et al, 2013). 대식세포는선천면역뿐만아니라획득면역등다양한숙주반응에관여하여항상성유지에관여하는것으로알려져있으며, 염증반응시에는 nitric oxide (NO) 와 cytokine 을생산하여감염초기에생체방어에중요한역할을한다 (Ding et al., 2009; Jeong et al, 2012). 내독소로잘알려진 lipopolysaccharide (LPS) 는그람- 음성균의세포외막에존재하며, RAW 264.7 세포와같은 macrophage 또는 monocyte 에서 tumor necrosis factor-α, interleukin-6 (IL-6), interleukin-1β (IL-1 β) 와같은 pro-inflammatory cytokine 들을증가시키는것으로알려져있다 (Lee et al., 2006; Kim and Moudgil, 2008). 또 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons -439- Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
韓資植誌 Korean J. Plant Res. 27(5) : 439~446(2014) 한이러한염증매개물질의형성은 phospholipase A2 의활성으로인해 arachidonic acid가 prostaglandin으로바뀌는과정및 NO형성과정으로이어지게된다 (McDaniel et al., 1996; Park et al, 2014). 체내염증과정에서는과량의 NO 및 prostaglandin E 2 (PGE 2 ) 등의염증인자가 inducible nitric oxide synthase (inos) 및 cyclooxygenase-2 (COX-2) 에의해형성된다. 이중 NO는체내방어기능, 신호전달기능, 신경독성, 혈관확장등의다양한생리기능을가지고있다. 일반적인 NO의형성은세균을죽이거나종양을제거시키는중요한역할을하지만, 염증상태에서 inos 에의해과잉생성된 NO는혈관투과성, 부종등의염증반응을촉진시킬뿐만아니라염증매개체의생합성을촉진하여염증을심화시키는것으로알려져있다 (Mu et al., 2001; Ryu et al., 2003; Kim et al., 2004). 참나무과식물인구실잣밤나무 (Castanopsis cuspidata (C. cuspidata)) 는상록활엽교목으로지리적으로일본, 중국, 타이완등에분포하며, 우리나라에서는제주도, 완도등남해안이남지역에분포한다 (Kang et al., 2002; Lee et al., 2010). 주로제주에서는하천을따라많이분포하며열매는날로먹거나구워서먹고, 떡이나묵에넣어서먹기도한다. 최근에는열매의영양성분분석이나식의약품소재의활용도에연구들이이루어졌으나잎과줄기등을이용한여러연구들은미흡한실정이다 (Kang et al., 2002; Lee et al., 2010). 본연구에서는자원식물로서활용가치가높은구실잣밤나무잎추출물을대상으로염증성질환의예방및치료제개발의기초자료로사용될수있는지탐색하기위해기능성을밝히고자극성에따라순차적으로용매분획하여추출물및분획물을가지고 LPS 로자극된 RAW 264.7 세포에서염증성매개인자인 NO의생성억제효과및 inos 그리고 COX-2 생성억제효능및염증성 cytokine 인 IL-1β 와 IL-6 의억제효과를조사하였다. 재료및방법식물재료제주도하천일대에자생하고있는구실잣밤나무 (Castanopsis cuspidata) 잎을 2013 년 9월경에채집하였으며, 채집된시료는증류수로 2~3 회수세한뒤물기를제거한뒤 2주간음건한후마쇄기로분쇄하여추출용시료로사용하였다. 용매계통분획구실잣밤나무잎에탄올추출물및순차적분획물의제조는 80% 에탄올 (EtOH) 및분획용헥산 (n-hexane), 디클로로메탄 (CH 2 Cl 2 ), 에틸아세테이트 (EtOAc) 그리고부탄올 (BuOH) 을사용하여용매의극성을이용한순차적추출법을사용하였다. 즉분말건조된시료 500 g에 80% 에탄올로 2회반복추출한후여과하여얻어진추출액을감압농축하여동결건조기로완전히건조시켰다. 건조된에탄올추출물 20 g에 10배량의증류수와동량의헥산을첨가하여분획한후감압농축하여헥산분획물을얻었다. 동일한방법으로디클로로메탄, 에틸아세테이트, 부탄올그리고물층을분획하여각각의순차분획물을얻었으며, 모든과정은 2회반복실시하였다. 세포배양대식세포계열 (Murine macrophage cell line) 인 RAW 264.7 세포는 American Type Culture Collection (ATCC, Manassas, VA, USA) 에서구입하여 10% fetal bovine serum (FBS) 와 penicillin-streptomycin 100 units/100 μg / ml이함유된 DMEM 배지 (GIBCO, Grand Island, NY, USA) 를사용하여 37, 5% CO 2 항온기에서배양하였으며, 3일에한번씩계대배양을시행하였다. Nitric oxide (NO) assay RAW 264.7 세포를 10% FBS 가첨가된 DMEM 배지를이용하여 1.5 10 5 cells/ ml로조절한후 24 well plate 에접종하고, 추출물시료와 LPS (1 μg / ml ) 를동시에처리하여 24시간배양하였다. 생성된 NO의양은 Griess 시약 [1%(w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid] 을이용하여세포배양액중에존재하는 NO - 2 의형태로측정하였다. 세포배양상등액 100 μl와 Griess 시약 100 μl를혼합하여 96 well plates 에서 10분동안반응시킨후 microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA) 를사용하여 540 nm에서흡광도를측정하였다. 생성된 NO의양은 sodium nitrite (NaNO 2 ) 를 standard로비교하였다. RNA 분리및 RT-PCR RAW 264.7 세포로부터의 total RNA 추출은 TRI-reagent (MRC, Cincinnati, OH, USA) 를이용하였으며, RNase-free한조건하에서실시하였다. cdna 합성은 cdna 합성 kit (Promega, madison, WI, USA) 를가지고 1 μg의 total RNA 를 oligo(dt) 18 primer, dntp (0.5 μm), 1 unit RNase inhibitor 그리고 M-MuLV reverse transcriptase (2U) 로 70 5분, 25 5분, 37 60분, -440-
Murine Macrophage RAW 264.7 세포에서구실잣밤나무추출물의항염증효과 그리고 70 에서 10분 heating 시킴으로서반응을중지시켰다. Polymerase chain reaction (PCR) 은합성된 cdna 로부터유전자를증폭시키기위하여 2 μl cdna, 4 μm의 5 과 3 primer, 10X buffer (10 mm Tris-HCl, ph 8.3, 50 mm KCl, 0.1% Triton X-100), 250 μm dntp, 25 mm MgCl 2, 1 unit Taq polymerase (Promega, Madison, WI, USA) 를섞고 distilled water 로전체를 25 μl로맞춘다음 Perkin-Elmer Thermal Cycler (Perkin- Elmer Co., Norwalk, CT, USA) 를이용하여 PCR 을실시하였다. 이때 PCR cycle은 inos 와 COX-2 는 94 /45 초, 56 /45 초, 72 /60초, 30회이고 β-actin 는 94 /45 초, 58 /45 초, 72 /60 초, 30 회이며, PCR 에의하여생성된산물은 1.5% agarose gel 에서전기영동을실시하고 ethidium bromide 로염색하여특정밴드를확인하였다 (Table 1). Immunoblotting 배양이끝난세포를수집하여 2~3 회 PBS (phosphate buffered saline) 로세척한후 1 ml 의 RIPA buffer (sigma, St. Louis, MO, USA) 을첨가하여 30분동안 lysis 시킨후 12,000 rpm 에서 20분간원심분리하여세포막성분등을제거하였다. 단백질농도는 BSA (bovine serum albumin) 를표준화하여 Bio-Rad Protein Assay Kit 를사용하여정량하였다. 30 μg의 lysate 를 8~12% mini gel SDS-PAGE 로변성분리하여, 이를 PVDF (polyvinylidene difluoride) membrane (BIO-RAD, Richmond, CA, USA) 에 200 ma로 2시간동안 transfer하였다. 그리고 membrane 의 blocking 은 5% skim milk 가함유된 TTBS (0.1% Tween 20 + TBS (ph 7.4, 1l 기준 8 g NaCl, 0.2 g KCl, 3 g Tris)) 용액에서상온에서 2시간동안실시하였다. inos 의발현양을검토하기위한항체로는 anti-mouse inos (1:1000, Calbiochem, La Jolla, CA, USA) 를 COX-2 의발현양을검토하기위한항체로는 anti-mouse COX-2 (1:1000, BD Biosciences Pharmingen, San Jose, CA, USA) 을 TTBS 용액에서희석하여상온에서 2시간반응시킨후 TTBS로 3회세정하였다. 2차항체로는 HRP (horse radish peroxidase) 가결합된 anti-mouse IgG (Amersham Pharmacia Biotech, Little Chalfont, UK) 를 1:5000 으로희석하여상온에서 30분간반응시킨후, TTBS 로 3회세정하여 ECL 기질 (Amersham Biosciences, Piscataway, NJ, USA) 과 1~3 분간반응후 X-ray 필름에감광하였다. 세포독성평가 (LDH assay) RAW 264.7 세포 (1.5 10 5 cells/ ml ) 를 DMEM 배지에추출물시료와 LPS (1 μg / ml ) 를동시처리하여 24시간배양한후배양배지를얻어 3,000 rpm 에서 5분간원심분리하였다. LDH (lactate dehydrogenase) assay 는 non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA) 를이용하여측정했으며, 96 well plate 에원심분리하여얻은배양배지 50 μl와 reconstituted substrate mix 를 50 μl를넣고, 실온에서 30분반응시킨후 50 μl의 stop solution 을넣은후 microplate reader (Bio-TEK Instruments Inc., Vermont, WI, USA) 를사용하여 490 nm에서흡광도를측정하였다. 각시료군에대한평균흡광도값을구하였으며, 대조군 (LDH control, 1:5000) 의흡광도값과비교하여세포독성을평가하였다. Table 1. Sequences of primer and fragment sizes of the investigated genes in RT-PCR analysis Gene Primer sequences Fragment size (bp) inos z F 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' 496 R 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3' R 5'-CACTACATCCTGACCCACTT-3' COX-2 y 696 R 5'-ATGCTCCTGCTTGAGTATGT-3' F 5'-CAGGATGAGGACATGAGCACC-3' IL-1β x 447 R 5'-CTCTGCAGACTCAAACTCCAC-3' IL-6 w F 5'-GTACTCCAGAAGACCAGAGG-3' 308 R 5'-TGCTGGTGACAACCACGGCC-3' F 5'-GTGGGCCGCCCTAGGCACCAG-3' β-actin 603 R 5'-GGAGGAAGAGGATGCGGCAGT-3' inos : inducible nitric oxide synthase, y COX-2 : cyclooxygenase-2, x IL-1β : interleukin-1β, w IL-6 : interleukin-6. -441-
韓資植誌 Korean J. Plant Res. 27(5) : 439~446(2014) 통계분석모든실험은 3회이상반복으로이루어졌으며, 실험결과는각항목에따라평균치 ± 표준편차 (SD) 를구하여신뢰수준 95%(p<0.05) 에서통계적유의차를평가하였다. 결과및고찰 Nitric oxide 생성억제효과활성산소중하나이며, 염증유발에중요한역할을하는것으로알려진 NO 생성에대한구실잣밤나무추출물과분획물의효과를확인하였다. 생성된 NO 양을 Griess 시약을이용하여세포배양액중에존재하는 NO - 2 의형태로측정하였다. 그결과 80% 에탄올추출물, 헥산과디클로로메탄분획물에서대조군 에탄올추출물및분획물의수율구실잣밤나무잎시료 (500 g) 를 80% 에탄올로추출한후여과하여얻어진추출액을감압농축하여조추출물 128 g (25.69%) 을얻었다. 그리고여기서얻어진에탄올추출물 (20 g) 을 10배량의증류수로현탁시킨후에헥산 (n-hexane), 디클로로메탄 (CH 2 Cl 2 ), 에틸아세테이트 (EtOAc) 그리고부탄올 (BuOH) 등을순차적으로분획하여헥산층에서 1.25 g (6.28%), 디클로로메탄층에서 1.28 g (6.44%), 에틸아세테이트층에서 4.05 g (20.28%), 부탄올층에서 5.06 g (25.33%) 및잔사인물층에서 8.15 g (40.78%) 의분획물을얻었다. 각순차적분획물의수율은 Table 2에나타내었으며, 추출에사용한구실잣밤나무 80% 에탄올추출물의수율은약 18.25% 였다. 에탄올추출물에대한각순차분획물중헥산분획물수율이 6.28% 로가장낮았고, 물분획물이 40.78% 로가장높은수율을보였다 (Scheme 1). Table 2. Effect of NO production on treated concentration of 80% EtOH extract and solvent fractions of C. cuspidata in RAW 264.7 cells Samples Inhibition (%) 100 ug/ ml 50 ug/ ml 80% EtOH 47.43 30.54 n-hexane 37.73 14.01 CH 2 CI 2 91.99 81.57 EtOAc 72.22 z 58.57 ByOH N/D N/D H 2O N/D N/D The production of nitric oxide was assayed from culture medium of cells co-treated with LPS (1 μg / ml ) in the presence of 80% EtOH extract and solvent fractions of C. cuspidata (100 and 50 μg / ml ). z cytotoxicity. Scheme 1. Systematic purification using solvent partitioning from C. cuspidata. -442-
Murine Macrophage RAW 264.7 세포에서구실잣밤나무추출물의항염증효과 인 LPS 단독처리군에비해높은 NO 생성억제효과를관찰할수있었다 (Table 2, Fig. 1). 특히디클로로메탄분획물인경우 50 μg / ml의농도에서도세포독성효과도보이지않으면서우수한 NO 생성억제효과를보였다. inos 및 COX-2 생성억제효과 RAW 264.7 세포에 LPS (1 μg / ml ) 를사용하여 inos 와 COX-2 의생성을유도한후구실잣밤나무잎추출물과분획물을 100 μg / ml농도로처리하여 inos 와 COX-2 의 mrna 발현억제정 Fig. 1. Effect of 80% EtOH extract and solvent fractions from C. cuspidata extracts on the production of nitric oxide in LPS-stimulated RAW 264.7 cells. The production of nitric oxide (NO) was assayed in the culture medium of cells co-treated with LPS (1 μg / ml ) for 24 h in the presence of 80% EtOH extract and solvent fractions from C. cuspidata extracts (100 μg / ml ). Values are the mean ± standard deviation (SD) of triplicate experiments. *, P < 0.05; **, P < 0.01. Fig. 2. Inhibitory effects of 80% EtOH extract and solvent fractions of C. cuspidate on the mrna expression of pro-inflammatory mediators in RAW 264.7 cells. RAW 264.7 cells (1.0 10 6 cells/ ml ) were pre-incubated for 18h, and the mrna expressions of inos and COX-2 were determined from 24 h cultures of cells co-treated lipopolysaccharide (1 μg / ml ) in the presence of 80% EtOH extract and solvent fractions of C. cuspidate (100 μlμg / ml ). -443-
韓資植誌 Korean J. Plant Res. 27(5) : 439~446(2014) 도를확인하였다. 그결과, inos 와 COX-2 의단백질발현양상과마찬가지로헥산, 디클로로메탄분획물에서대조군인 LPS 단독처리군에비해 mrna 발현억제효과를나타내었다 (Fig. 2). RAW 264.7 세포에 LPS (1 μg / ml ) 를사용하여 inos 의생성을유도한후큰비쑥추출물과분획물을 50 μg / ml농도로처리하여 inos 발현을 immunoblotting를통해확인하였다. 그결과 immunoblotting에의한 inos 와 COX-2 의단백질생성은헥산과디클로로메탄분획물에서대조군인 LPS 단독처리군에비해강한억제효과를나타내었다 (Fig. 3). 본연구결과, NO 생성억제효과와초기- 염증성인자생성억제효과가가장우수한디클로로메탄분획물이 inos 의발현또한강하게억제시키는걸로보아 NO의생성억제기전은 inos 발현억제를통해이루어진것으로여겨진다. 세포독성에미치는영향 LDH 는모든세포의세포질안에존재하는효소로서 pyruvic acid와 lactic acid 간의가역적전환에관여하여촉매작용을하며, LDH 를내포한조직이파괴될때혈액중으로흘러나와혈중 LDH 가상승한다. RAW 264.7 세포 (1.5 10 5 cells/ ml ) 에시험약물과 LPS (1 μg / ml ) 를동시처리하여 24 h 배양한후, LDH assay 방법을이용하여세포독성을확인한결과, 에틸아세테이트분획물인경우 100 μg / ml이상의농도에서다소세포독성이나타났으나다른분획물에서는세포독성이나타나지않았고 50 μg / ml농도에서는모든추출물및분회물에서세포독성이나타나지않았다 (Table 2, Fig. 1). Pro-inflammatory cytokine 생성억제효과 Pro-inflammatory cytokine 들은정상조직에서발현될뿐만아니라병변과정에서그발현정도가증가되며, 암촉진과정에서일어나는피부염증에중요한역할을한다. TNF-α, IL-1β, 그리고 IL-6 등과같은 cytokine 들은인간의염증성피부질환과관련이있는것으로보고되어왔다 (Kim et al., 2008). 또한여러염증질환과알러지현상에 cytokine 들에대한항체를처리하였을때증상이완화된것으로보고되었다 (Kim et al., 2008; Kim et al., 2009). 염증단계에중추적역할을하고있는 cytokine 인 IL-1β 와 IL-6 의발현을저해시키거나 COX-2 활성저해에기인하는 PGE 2 의생성억제를통해 pro-inflammatory factor 의증가를수반하는병변과정을조절할수있을가능성이높다고보고되었다 (Heo et al., 2010; Ren et al., 2009). 대식세포인 RAW 264.7 세포로부터 pro- Fig. 3. Inhibitory effects of 80% EtOH extract and solvent fractions of C. cuspidate on the protein level of inos and COX-2 in RAW 264.7 cells. RAW 264.7 cells (1.0 10 6 cells/ ml ) were pre-incubated for 18 h, and the cells were co-treated with lipopolysaccharide (1 μg / ml ) in the presence of 80% EtOH extract and solvent fractions of C. cuspidate (100 μg / ml ) for 24 h. inos and COX-2 protein levels were determined using immunoblotting method. -444-
Murine Macrophage RAW 264.7 세포에서구실잣밤나무추출물의항염증효과 Fig. 4. Inhibitory effects of 80% EtOH extract and solvent fractions of C. cuspidate on the mrna expression of pro-inflammatory cytokines in RAW 264.7 cells. RAW 264.7 cells (1.0 10 6 cells/ ml )were pre-incubated for 18 h, and the mrna expressions of pro-inflammatory cytokines were determined from 24 h cultures of cells co-treated with lipopolysaccharide (1 μg / ml ) in the presence of 80% EtOH extract and solvent fractions of C. cuspidate (100 μg / ml ). inflammatory cytokine 의 mrna 발현정도를 RT-PCR를통해확인한결과, LPS 자극과함께처리하여 IL-6 와 IL-1β 생성억제에대한구실잣밤나무추출물및용매분획물을 100 μg / ml농도로처리하였을때, IL-6 생성억제는 80% 에탄올추출물, 디클로로메탄과에틸아세테이트분획물에서억제효과가나타났으며, IL-1β 생성억제는 80% 에탄올추출물과디클로로메탄분획물에서억제효과를나타내었다 (Fig. 4). 적요본연구는구실잣밤나무 (C. cuspidate) 잎추출물의항염증활성이염증성매개인자생성저해및염증성사이토카인의억제와관련이있을것으로예상되어짐에따라, 구실잣밤나무를대상으로 80% 에탄올를가지고추출한후추출물을극성에따라순차적으로용매분획을실시하여, 구실잣밤나무 80% 에탄올추출물및용매분획물들이염증반응의주체가되는대식세포계열인 RAW 264.7 세포에서 LPS 로유도된 NO의생성억제효과, 그리고 inos 와 COX-2 의 mrna 와단백질발현억제효과및 IL-6 와 IL-1β 와같은염증성사이토카인생성억제효과등을알아보았다. 대식세포계열인 RAW 264.7 세포에 LPS 로자극을주고구실잣밤나무 80% 에탄올추출물및분획물을처 리하여확인해본결과, 헥산, 디클로로메탄및에틸아세테이트분획물에서 NO의생성억제효과가강하게나타났으며, 헥산과디클로로메탄분획물에서는 inos 와 COX-2 생성억제효과가다른분획물에비해강하게나타났으며염증성사이토카인생성억제효능도 80% 에탄올추출물및분획물들이다소차이는있었지만헥산과디클로로메탄분획물에서 IL-6 와 IL-1β 에서생성억제효과가나타났다. 이러한결과는구실잣밤나무잎에서유효성분추출을통한항염증물질의연구또는예방하거나치료할수있는염증억제성분의분리및그작용기전연구에중요한기초자료가될것이라사료된다. 또한구실잣밤나무추출물로부터염증억제성분을도출하고자활성분획인디클로로메탄분획물에대하여활성성분의분리가진행중이다. References Ding, C., F. Cicuttini, J. Li and G. Jones, 2009. Targeting IL-6 in the treatment of inflammatory and autoimmune diseases. Expert Opin Investig Drugs. 18:1457-1466. Heo, S.J., W.J. Yoon, K.N. Kim, G.N. Ahn, S.M. Kang, D.H. Kang, A. Affan, C. Oh, W.K. Jung and Y.J. Jeon. 2010. Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW -445-
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