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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 46, No. 4, pp. 511~515 (2014) The Korean Society of Food Science and Technology 고량강으로부터분리된 galangin 의 RAW 세포주에서 LPS 로유도된 nitric oxide 생성저해활성 이화진 * 세명대학교자연약재과학과 Inhibitory Effect of Galangin from Alpinia officinarum on Lipopolysaccharideinduced Nitric Oxide Synthesis in RAW macrophages Hwa Jin Lee* Department of Natural Medicine Resources, Semyung University Abstract In a screen for plant-derived inhibitors of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW macrophage cells, a flavonol isolated from the chloroform extract of Alpinia officinarum was isolated. The structure of the flavonol was found to be 3,5,7-trihydroxy-2-phenylchromen-4-one (galangin, GLG) by using spectroscopy. GLG exhibited an inhibitory effect (IC 50 value: 26.8 µm) on NO production in LPS-stimulated RAW murine macrophage cells. Moreover, GLG suppressed expressions of inducible nitric oxide synthase (inos) protein and mrna in a dose-dependent manner. Keywords: Alpinia officinarum; Zingiberaceae; nitric oxide; inducible nitric oxide synthase; flavonol 서 Nitric oxide (NO) 는혈압조절, 신경전달, 혈액응고, 면역기능등의역할을하는것으로알려져있으며, 여러조직과세포에서 L- arginine으로부터 Nitric Oxide Synthase (NOS) 에의해생성된다 (1). NO를생성하는 NOS는세포질내에항상미량으로존재하는 constitutive form (cnos) 와면역학적변화에의해유도되는 inducible form (inos) 으로나눌수있으며, cnos는 Ca ++ /calmodulin 의존적이며단시간동안소량의 NO를생성하여신경전달물질로서작용하거나혈관근육세포의이완을통해혈압강하작용을하는등정상적인생리기능을담당하며, 신경세포에존재하는 neuronal NOS (nnos) 와내피세포에존재하는 endothelial NOS (enos) 로구분할수있다 (2). 반면 inos는 Ca ++ /calmodulin 비의존적이며혈관내피세포, 근육세포를포함한다양한조직에서 lipopolysaccharide (LPS), interleukine 1 (IL-1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) 등에의해유도되어장시간동안다량의 NO를생성하여병리학적혈관확장, 세포독성, 조직손상등유해작용을나타내고, 패혈증환자에게서 septic shock 을일으키는것으로보고되고있다 (3). 또한 NO가혈관투과성증가, 부종등염증반응을야기하거나 cyclooxygenase (COX) 의활성을촉진시켜 prostaglandin 등의생합성을촉진하여염증반응 *Corresponding author: Hwa Jin Lee, Department of Natural Medicine Resources, Semyung University, Jecheon, Chungbuk , Korea Tel: Fax: hwalee@semyung.ac.kr Received May 26, 2013; revised June 3, 2014; accepted June 4, 2014 론 을심화시키는것으로나타난다 (3-6). 고량강 ( 高良薑, Alpinia officinarum Hance) 은생강과 (Zingiberaceae) 에속하는다년생초본의뿌리줄기로서양강 ( 良薑 ), 신강 ( 身薑 ), 소신강 ( 小身薑 ), 고신강 ( 膏身薑 ) 이라고도일컫고, 주요성분으로는 1,8-cineol, methyl cinnamate, α-cadinene 등의정유성분과 galangol 등의신미성분이알려져있다 (7). 예부터방향성건위, 진통, 진토약으로서소화불량, 구토, 복통, 하리에응용하고, 중국에서는전통적으로비장과위관련질환에대해탁월한치료효과를나타내는것으로알려져있다 (8). German Commission E Monograph 에는식욕감퇴와소화불량에사용되는것으로기재되어있으며, 미국의 FDA 에서는 generally regarded as safe (21CFR section , ) 로규정하고있다 (9). 한편, 고량강의약리활성에대한연구는항산화, 항암, 혈관이완및항염증작용등이보고되어있다 (10-13). 본연구에서는고량강추출물과고량강으로부터분리한단일화합물이 LPS 로활성화된 RAW 대식세포주에서의 NO 생성에미치는효과와, 고량강으로부터분리된 NO 생성저해제가 inos 의발현에미치는영향에대해보고하고자한다. 재료및방법 식물재료및활성성분의분리고량강 (4.5 kg) 을환류냉각하면서메탄올로 3 번추출한것을실험재료로사용하였다. 고량강메탄올추출물로부터극성에따른용매분획을얻기위하여메탄올추출물 (400 g) 을물에분산하고헥산 (47.5 g), 클로로포름 (25.5 g), 에틸아세테이트 (18.1 g) 순서로각각의가용분획을얻었다. 클로로포름가용분획 (2.24 g) 을 hexane:ethylacetate (30:1 10:1) 의조건으로실리카겔컬럼크로마토그래피를행하여용출물을크게 6 개의분획으로나누고각분 511

2 512 한국식품과학회지제 46 권제 4 호 (2014) 획에대해 NO 생성저해활성을검정하였다. NO 생성저해활성이좋은 6 번분획 (786 mg) 에대해실리카겔컬럼크로마토그래피 (chloroform:methanol, 7:1 2:1) 와역상 (C 18 ) 컬럼크로마토그래피 (55% acetonitrile 80% acetonitrile) 등을행하여강한활성을나타내는단일화합물 (7.5 mg) 을분리하였으며, 순도는역상컬럼을이용한 high performance liquid chromatography (HPLC, Shimadzu HPLC system with UV monitor, Kyoto, Japan) 로확인하였다. 세포주배양과시료의처리 RAW 대식세포주 (ATCC, Rockville, MD, USA) 를 10% FBS (fetal bovine serum), 2 mm L-arginine 과 100 µg/ml 의 penicillin, streptomycin (Life technologies, Frederick, MD, USA) 이포함된 DMEM 배지로 37 o C, 5% CO 2 incubator 에서배양하였다. RAW 세포를 cells/ml 하여 48 well plate 에분주하여 12 시간동안부착시키고새로운배지로교환한다음 LPS 를 1 µg/ml 이되도록가하였다. 시료는 dimethylsulfoxide (DMSO) 에용해시켜보관하고, 세포에처리시최종농도 0.1% DMSO 용액이되도록 LPS 와동시에배양액에가한다음 37 o C, 5% CO 2 incubator 에서일정시간배양하여각 well 의배양액으로유리된 NO 생성정도를측정하였다. 시료의세포에대한독성은 MTT 시험법으로측정하고세포에대해독성이없는농도에서활성을검정하였다. MTT 시험법을이용한시료의세포독성평가 RAW 세포를 cells/ml 로하여 96 well plate 에 100 µl 씩분주하여 12 시간동안부착시키고새로운배지로교환한다음 LPS 를 1µg/mL 이되도록가하였다. 시료는최종농도 0.1% DMSO 용액이되도록 LPS 와동시에배양액에가한다음 37 o C, 5% CO 2 incubator 에서 24 시간배양하였다. 각 well 의배양액을제거후 PBS 로한번세척한후 methylthiazolydiphenyltetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) 용액을가하였다. 37 o C, 5% CO 2 incubator 에서 4 시간배양한후 solubilization 용액을가하고, 570 nm 에서흡광도를측정하였다. Griess reagent를이용한배양액중의 NO 생성량의측정 NO는 2-3초의짧은 life time을가진불안정한물질로대부분 NO 2 형태로전환되므로생성된 NO의양은 NO 2 의형태로정량하였다. 세포배양상등액 100 µl와 Griess 시약 (0.1% N-(1- naphthyl)ethylenediamin 2HCl, 1% sulfanilamide in 5% conc. H 3 PO 4 in H 2 O) 150 µl를혼합하여 10분동안반응시키고발색정도를 microplate reader (Molecular Devices, Sunnyvale, CA, USA) 를이용하여 570 nm에서흡광도를측정하였으며검량선작성을위해 NaNO 2 를표준품으로사용하였다. Westernblot analysis RAW 세포를대조군, LPS 처리군, LPS+ 시료처리군으로나누어처리한후 20 시간배양하였다. 배양액을제거하고세포를 lysis buffer (Cell Signaling Technologies, Beverly, MA, USA) 를이용하여 lysis 시키고, Bradford 방법을사용하여단백질을정량한후, 8% SDS-PAGE 를이용하여전기영동한후, PVDF membrane 에 transfer 하였다. inos 항체 (BD Biosciences, Franklin Lakes, NJ, USA) 를결합시키고 horseradish peroxidase linked type 의 2 차항체를결합시킨후, ECL detection reagents 를이용한형광항체법으로 inos 단백질의발현정도를관찰하였다. Reverse transcription-polymerase chain reaction (RT-PCR) 법 RAW 세포를대조군, LPS처리군, LPS+ 시료처리군으로나누어처리한후 6시간배양하였다. 배양액을제거하고 PBS로세척후신속히 Trizol (Life technologies) 을가하여세포를 lysis 시킨뒤, 제품설명서에따라 RNA를분리하였다. 얻어진 RNA에 reverse transcriptase (Life technologies) 와 hexamer (Cosmo, Seoul, Korea) 를가한후 42 o C에서 1시간반응시켜 cdna로전환하였다. 얻어진 cdna의일정량과 primer (inos; sense 5'-ATGTC- CGAAGCAAACATACA-3', antisense 5'-TAAGGTCCAGGAAG- TAGGTG-3', β-actin; sense 5'-TGTGATGGTGGGAATGGGTCAG- 3', antisense 5'-TTTGATGTCACGCACGATTTCC-3'), polymerase (Promega, Madison, WI, USA) 를가하여 inos mrna의발현정도를관찰하였다. 자료의통계적처리실험결과에서얻은모든값은 3 회이상반복실험한결과의평균 ± 표준편차로표시하였다. 각실험군간의측정치비교는 oneway analysis of vaiance (ANOVA) 와 student s t-test 를이용하여 p<0.05 인값에대해유의적인것으로처리하였다. 결과및고찰 고량강추출물의 NO 생성저해활성고량강 (Alpinia officinarum Hance, Zingiberaceae) 메탄올추출물에대해극성에따른각용매분획을얻고, 활성을가진용매분획으로부터활성검정을병행한각종칼럼크로마토그래피법으로활성성분을추적하였다. 활성검정은 LPS 를처리한 RAW 세포에서 NO 생성저해정도로평가하였다. 메탄올추출물및용매분획에의한 NO 생성저해활성실험결과, LPS 단독처리군에비해메탄올추출물은 16%, 클로로포름가용분획은 70% 의 NO 생성저해활성을나타내었다. 추출물및용매분획의세포처리농도는 10 µg/ml 이었고, 이때 RAW 세포에대한세포독성은나타나지않았다 (Fig. 1). 따라서, 클로로포름층에대해활성성분추적을위한컬럼크로마토그래피를실시하였다. 고량강으로부터활성성분의분리와단일활성성분의 NO 생성저해활성클로로포름층에대해재료및방법에서기술한것과같이실리카겔및역상컬럼크로마토그래피를행하여강한활성을나타내는단일화합물을분리하였으며, 순도는역상컬럼을이용한 HPLC 로확인하였다. 분리된화합물은분자량 의황갈색결정상으로실리카겔 TLC 상에서 10% 황산에의해주황색으로발색되었으며, 분광학적분석자료를통해그구조를 3,5,7-trihydroxy-2-phenylchromen-4-one (galangin, GLG, Fig. 2A) 으로규명하였고, 보고된문헌의자료와도일치하였다 (14). 강력한 NO 생성저해활성을나타내는클로로포름층으로부터분리한단일화합물 GLG 에대해 LPS 로활성화한 RAW 세포로부터유도된 NO 생성저해능력을평가하였다 (Fig. 2B). 그결과, LPS 단독처리군에서 NO 가과량생산되는것을확인할수있었으나, 화합물 GLG 처리군에서는농도의존적으로 NO 생성을저해하는것을확인하였으며, NO 생성저해활성에대한 IC 50 값은 26.8 µm 이었다. 또한, GLG 의 NO 생성량의변화가세포독성에의한영향과는무관함을 MTT assay 결과로확인하였다 (Fig. 2B). GLG 는항암 (15), 혈압강하 (16), 항알러지 (17), 항비만 (18), 항염증 (19) 등의다양한약리활성을보이는것으로보고된바있으

$고량강의 NO 생성저해활성 513 Fig. 1. Effect of solvent fractions of Alpinia officinarum on LPSinduced NO production. RAW 264.7 cells were treated with LPS (1 µg/ml) and/or solvent fractions of A.$

3 고량강의 NO 생성저해활성 513 Fig. 1. Effect of solvent fractions of Alpinia officinarum on LPSinduced NO production. RAW cells were treated with LPS (1 µg/ml) and/or solvent fractions of A. officinarum (10 µg/ml) for 20 h, and the amount of nitrite in supernatant was measured using Griess reagent (bar). Cell viability assay was performed with MTT colorimetric assay ( ). AOM: methanol extract, AOHx: hexane layer, AOC: CHCl 3 layer, AOEA: EtOAc layer. 며, 또한합성품 GLG 혹은천연물로부터유래한 GLG 가 J774A.1 대식세포주, 복막대식세포모델에서염증억제효능을나타내는것으로도알려져있어, 본연구의결과와도일치하는것으로생각된다 (20-21). 고량강추출물로부터분리한단일화합물의 inos 발현저해활성 inos 의발현을저해하여과도한 NO 생성을저해하는것으로알려져있는천연 inos 저해제로는 lueolin(22), kazinol B(23), quercetin(24), kaempferol(25) 과같은 flavonoid, liganan(26,27), Fig. 2. The structure of GLG from Alpinia officinarum (A) and the effect of GLG on LPS-induced NO production (B). RAW cells were treated with LPS (1 µg/ml) and/or various concentrations of GLG for 20 h, and the amount of nitrite in supernatant was measured using Griess reagent (bar). Cell viability was performed using MTT colorimetic assay ( ). The values are expressed as the means±sd of three individual experiments. *p<0.05. terpenes(28,29) 및 alkaloids(30,31) 등다양한구조의화합물들이보고되어있다. 따라서본연구에서도고량강으로부터분리한 Fig. 3. Effects of GLG from A. officinarum on the expression of LPS induced inos protein (A) and mrna (B). (A) RAW cells were treated with GLG (10, 30 and 50 µm) and/or LPS (1 µg/ml) for 20 h. Cell lysates were prepared and the inos, COX-2, and β-actin protein levels were determined by Western blot. (B) RAW cells were incubated in the presence of LPS (1 µg/ml) with or without GLG (10, 30 and 50 µm) for 6 h. The mrna levels for inos and β-actin were determined by RT-PCR from total RNA extracts. β-actin was used as an internal control. Images are the representative of three independent experiments that shows similar results. The values are expressed as the means±sd of three individual experiments. *p<0.05.

4 514 한국식품과학회지제 46 권제 4 호 (2014) flavonol 구조의화합물 GLG 가 inos 발현에저해활성을나타내는지확인하고자하였다. 화합물 GLG 가 inos 의발현에미치는영향을알아보기위하여, inos 항체를이용하는 Western blot 실험을수행한결과, 화합물 GLG 가농도의존적으로 inos 단백질발현을억제하는것을관찰하였다 (Fig. 3A). 또한화합물 GLG 의 inos 단백질발현억제가 mrna 발현저해에의한것인지를확인하기위해 inos primer 를이용하여 RT-PCR 실험을수행한결과, 화합물 GLG 에의해 inos mrna 의발현이농도의존적으로억제되는것을확인하였다 (Fig. 3B). 이로써고량강으로부터분리한 flavonol 구조의화합물 GLG 의 NO 생성저해활성은 inos mrna 의발현억제를통한 inos 효소단백질의생성저하에의한것임을확인할수있었다. 요 각종염증성질환및패혈증으로인한치명적인저혈압을예방치료하는약물개발을위한기초연구로서유도성 NOS (inducible nitric oxide synthase, inos) 에의한 NO 의과다생성을저해하는성분을천연물로부터찾아내고자본연구를수행하였다. NO 생성저해활성의검정은대식세포주인 RAW 세포를 LPS 로활성화한후, 유도되는 inos 에의해생성되는 NO 를 Griess 시약을이용해 NO 2 의형태로정량하였다. 또한 Western blot 실험및 RT-PCR 실험을시행하여 inos 의 mrna 의발현및단백합성에대한영향을조사하였다. 고량강 (Alpinia officinarum Hance, Zingiberaceae) 의메탄올추출물로부터극성에따른용매분획을시행하여활성성분을분리하고분광학적분석법을이용하여분리한단일성분이 flavonol 구조인 3,5,7-trihydroxy-2-phenylchromen- 4-one (galangin, GLG) 임을확인하였다. 작용기전을알아보기위해, Western blot 및 RT-PCR 실험결과, 분리한 flavonol 성분 (GLG) 의 NO 생성저해활성은 inos mrna 발현을저해하여 inos 효소단백질의생성이억제됨에기인하는것으로확인하였다. 따라서, 고량강추출물로부터분리한 flavonol 화합물 (GLG) 이 inos 발현의억제를통해다량의 NO 생산을저해함으로써, 고량강 (Alpinia officinarum) 의 NO 과량생성과관련된염증성질환에대한응용가능성이클것으로기대된다. 약 감사의글 이논문은 2013 학년도세명대학교교내학술연구비지원에의해수행된연구임. References 1. Garthwaite J. New insight into the functioning of nitric oxidereceptive guanylyl cyclase: physiological and pharmacological implications. Mol. Cell Biochem. 334: (2010) 2. Crane BR, Arvai AS, Ghosh DK, Wu C, Getzoff ED, Stuehr DJ, Tainer JA. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. 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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,