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1 Journal of Korean Medicine Rehabilitation Vol. 24 No. 3, July 2014 pissn eissn Original Article 細辛酒錠抽出物이 LPS 로유발된 RAW Cell 의염증및항산화반응에미치는영향 이옥진 오민석대전대학교한의과대학한방재활의학과교실 Anti-oxidation and Anti-inflammatory Effect of Asiasari Radix in RAW Cells Yu-Chen Lee, K.M.D., Min-Seok Oh, K.M.D. Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Dae-Jeon University RECEIVED June 19, 2014 REVISED July 21, 2014 ACCEPTED July 22, 2014 CORRESPONDING TO Min-Seok Oh, Department of Rehabilitation Medicine of Korean Medicine, College of Korean Medicine, Dae-Jeon University, 1136, Dunsan-dong, Seo-gu, Daejeon , Korea TEL (042) FAX (042) Objectives The purpose of this study was to investigate the Anti-oxidation and anti-inflammatory effects of ethanol extract from asiasari radix (AR) on lipopolysaccharide (LPS)-induced in RAW Cells Methods Anti-oxidative effects of AR were measured by scavenging activities of 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and production of reactive oxygen species (ROS) in RAW cells. Anti-inflammatory effects of AR were measured by mediators including nitric oxide(no), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necosis factors-α (TNF-α) and inos, IL-1β, IL-6, TNF-α mrna expression in RAW cells. Results Total phenolic content was expressed 28.77±1.67. DPPH radical Scavenging was increased depend on AR ethanol extract. ABAT radical Scavenging was increased depend on AR ethanol extract. Production of ROS was significantly decreased by AR ethanol extract on concentration of 100 (μg/ml). Production of NO was significantly decreased by AR ethanol extract on concentration of 100 (μg/ml). Production of IL-1β, interleukin-6 and TNF-α were increased depend on AR ethanol extract. And Production of interleukin-6, TNF-α were significantly decreased AR ethanol extract. inos, IL-1β, IL-6, TNFα mrna expression of RAW cells was increased depend on AR ethanol extract. Conclusions According to this study, AR ethanol extract has anti-oxidative and anti-inflammatoy effects. (J Korean Med Rehab 2014;24(3):99-110) Copyright 2014 The Society of Korean Medicine Rehabilitation Key words Asiasari radix (AR), Anti-oxidation, Anti-inflammatory, RAW cell 서론»»» 염증반응이란대식세포를포함한면역세포들이항원의침입이나조직손상과같은자극에의해손상부위로이동하여항원을제거하고, 손상의결과로생성된물질을제거하는것을말한다 1,2). 대식세포는감염초기에생체방어 에중요한역할을하는세포로, NO, cytokine 등과같은염증매개물질을생산하여염증반응에관여하게되는데, 염증매개물질이과량생산되면염증성질환을유발함으로써숙주에치명적인결과를초래할수있다 3-6). 염증반응의억제는염증질환을치료하는데있어중요한목표가되는데, 기존항염제에비해한약물을이용한 99

2 이옥진 오민석 항염제의경우부작용이적고안전성이높아, 현재한약물제제를통해염증성매개물질로알려진 NO와염증유발 cytokine 등을억제시킬수있는물질을찾기위한연구가활발히진행되고있다 7-11). 細辛 (Asarum sieboldii Miq.) 은리풀 (Asarum sieboldii) 이나민족두리풀 (Asarum sieboldii var. seoulensis) 의뿌리및전초로 12) 性味가辛ㆍ溫하며發散解表, 袪風止痛, 溫經通絡 13-16) 효능이있다. 이 17), 지 18) 는 microglial cell을이용하여細辛의항염증효과에대한보고하였으며, 정 19) 은대식세포를이용하여細辛추출물의항염증효과를보고하였다. 그간細辛이염증기전과항산화기전에미치는영향에대한연구가이루어지지않았던것으로 RAW cell을이용하여細辛이항염및항산화에미치는영향을찾아보고이러한細辛의효능이향후관절염의치료에응용될수있는지를알아보고자하였다. 또한酒錠추출물이가수분해추출보다수득율이우수하다는보고 20,21) 되고있으나. 細辛을酒錠추출하여 RAW cell 을이용한항염항산화연구는접하지못하였기에, 저자는 Lipoplysaccharide (LPS) 로유도된 RAW cell에細辛酒錠抽出物을처리하여항염및항산화반응등을관찰한결과유의한성적을얻었기에보고하는바이다. 재료및방법»»» 1. 재료 1) 세포세포는한국세포주은행 ( 서울, 한국 ) 에서 RAW 세포를구입하여사용하였다. 2) 약재細辛 (Asiasari Radix 이하 AR로표기 ) 은 옴니허브 ( 대구, 한국 ) 에서구입하였고, 대전대학교지역혁신센터난치성면역질환의동서생명의학연구센터 (Traditional and biomedical research center, TBRC) 에서정선후사용하였다. 3) 시약 Co., U.S.A.), dulbecco's phosphate buffered saline (D-PBS: Welgene, Korea), ether (Sigma Co., U.S.A.), dulbecco's modified eagle's medium (DMEM: Gibco BRL Co., U.S.A.), 우태아혈청 (fetal bovine serum: FBS, Invitrogen Co., U.S.A.), lipopolysaccharide (LPS: Sigma Co., U.S.A.), cell viability assay kit (Daeillab sevice, Korea), nitric oxide detection kit (Intron Biotechnology, Korea), dimethyl sulfoxide (DMSO: Sigma Co., U.S.A.), 1,1 diphenyl-2-picryl-hydrazyl (DPPH: Sigma Co., U.S.A.), penicillin (Hyclone, Co., U.S.A.), streptomycin (Hyclone Co., U.S.A.), agarose (FMC Co., U.S.A.), 2',7'-dichlorodihydrofluorescin diacettate (DCFH-DA: Sigma Co., U.S.A.), anti-biotic (Gibco BRL Co., U.S.A.), trypan blue (Sigma Co., U.S.A.), ethanol (Merck Co., Germany), mouse cytokine milliplex map immunoassay kit (Millipore Co., U.S.A.), total RNA RNA purification kit는 GeneAll 사 ( 서울, 한국 ), 酒錠은 주정판매월드 ( 전주, 한국 ), HNO 3 ( 덕산, 한국 ), As, Pb, Hg, Cd standard solution (SCP Science, Canada), acetonitrile ( 덕산, 한국 ) 을사용하였다. 4) 기기기기는 rotary vacuum evaporator (Büchi B-480 Co., Switzerland), freeze dryer (EYELA FDU-540 Co., Japan), CO 2 incubator (Forma scientific Co., U.S.A.), clean bench (Vision scientific Co., Korea), autoclave (Sanyo Co., Japan), vortex mixer (Vision scientific Co., Korea), spectrophotometer (Shimadzu Co., Japan), centrifuge (Sigma Co., U.S.A.), deep-freezer (Sanyo Co., Japan), thermocycler system (MWG Biotech Co., Germany), ice-maker (Vision scientific Co., Korea), plate shaker (Lab-Line Co., U.S.A.), ELISA reader (Molecular Devices Co., U.S.A.), ICP (Shimadzu, Co., Japan), 수은분석기 (Teledyne Leeman Labs, U.S.A.), high performance liquid chromatography (HPLC: Shimadzu, Co., Japan), 유세포분석기 (Flow cytometer, Becton Dickinson, Co., U.S.A.), Light Microscope (Carl Zeiss, Co., Germany) 등을사용하였다. 시약은 isopropanol (Sigma Co., U.S.A.), gelred (Sigma 100 J Korean Med Rehab 2014;24(3):99-110

3 細辛酒錠抽出物이 LPS 로유발된 RAW Cell 의염증및항산화반응에미치는영향 2. 방법 1) 검액의조제細辛 30 g을 80% 酒錠 500 ml에넣어 3시간동안환류추출후여과액을얻어 rotary vacuum evaporator 에서감압농축하였다. 농축된용액을 freeze dryer로동결건조하여 23.9 g의분말을얻었다. 얻어진분말은냉동고에서보관하며필요한농도로 3차증류수에희석하여사용하였다. 2) 안전성검사 (1) 중금속검사납, 비소, 카드뮴분석의경우細辛酒錠抽出物 0.5 g 을극초단파시료전처리장치전용용기에넣고질산 10 ml을넣은후, 용기를후드안에정치시켜발생가스를제거하고극초단파시료전처리장치를사용하여분해하였다. 분해가끝난다음분해액을여과지로여과하여용량플라스크에넣고물을넣어적절하게표준액의농도범위로희석하여검액으로하였다. 따로질산 10 ml를극초단파시료전처리장치전용용기에넣어검액조제와같은방법으로조작하여공시험액으로사용하였다. 준비된검액, 표준액및공시험액을가지고유도결합플라즈마분광계 (inductively coupled plasma, ICP) 를이용하여검량선을작성하고공시험액으로보정하여검액을측정하였다. 수은분석의경우細辛酒錠抽出物 50 mg을정확하게달아특별한전처리과정없이수은분석기를이용하여측정하였다. (2) 세포독성측정 Raw 세포는 96 well plate에 cells/well로분주하여 24시간동안배양하였다. 실험을하기전에새로운배양액으로교체하였고, 細辛을각각 1, 10, 100 (μg/ml) 의농도로처리하여다시 24시간동안배양하였다. 배양후 10 μl의 WST solution 을첨가하여세포배양기 (37 o C, 5% CO 2 ) 에서 30분간반응시켰다. 반응후 450 nm에서흡광도의변화를측정하여대조군에대한세포생존율을백분율로표시하였다. (3) HPLC 분석細辛酒錠抽出物 30 mg을 80% 酒錠 1 ml에녹여 0.45 μm membrane filter로여과후 10 μl를 HPLC 시료로사용하였다. HPLC는 Shimadzu (Japan) 사의 system controller (CBM-20A), pump (LC-20AD), column oven (CTO-20A), diode array detector (SPD-M20A) 를사용하였으며, column 은 ZORBAX Eclipse Plus C18 ( mm, 5 μm) 을사용하였다. 이동상은 water(a) 와 acetonitrile(b) 로 gradient elution system 을적용시켜 0~5분 (0% B), 5~40분 (30% B), 40~60분 (80% B), 60~80분 (100% B) 로설정하였다. 유속은 1.0 ml/min이었으며 column 온도는 40 o C를유지하였고, ultraviolet rays of wavelength (UV wavelength) 는 210 nm로설정하여분석하였다. 3) 항산화효능측정 (1) Total polyphenol 함량총폴리페놀함량은 Folin-Ciocalteu 시약을이용하는방법으로측정하였다. 추출시료용액 1 ml에 50% Foiln- Ciocalteu's phenol reagent 0.5 ml를가하여실온에서 3 분간반응시켰다. 반응용액에 Na 2 CO 3 포화용액 1 ml와 7.5 ml 증류수를차례로혼합하여 30분간정치시킨뒤, 12,000 rpm에서 10분간원심분리한후상등액을취해 760 nm에서흡광도를측정하였다. 총폴리페놀함량은 gallic acid를표준물질로이용하여작성한검량선에따라함량을구하였으며측정단위로는 GAE (Gallic acid equivalent)/g을사용하였다. (2) DPPH 소거능 Radical소거활성시험은안정한 radical DPPH를사용하는방법이다. 細辛酒錠抽出物은최종농도가 1, 10, 100, 1,000 (μg/ml) 의농도로될수있게희석시켰으며, 에탄올에용해시킨 0.2 mm의 DPPH 용액 150 μl와細辛酒錠抽出物을각각 100 μl씩혼합하여 37 o C에서 30 분간반응시켰다. 반응후 517 nm에서흡광도를측정하였다. 대조군은시료액대신증류수를넣었으며 DPPH 용액의대신酒錠을넣어보정값을얻었다. DPPH radical 소거율은아래의식에따라계산하였다. 소거율 (%)=( 대조군의흡광도 - 시료첨가군의흡광도 ) 100 대조군의흡광도 (3) ABTS 소거능 ABTS assay 방법 22) 을응용하여측정하였다. 기존에보고된방법을 96 well plate에맞게수정하여실시하였다

4 이옥진 오민석 細辛酒錠抽出物은최종농도가 1, 10, 100, 1,000 (μg/ml) 의농도로될수있게희석시켰으며, ABTS 용액은 7.4 mm ABTS (2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) 와 2.6 mm potassium persulphate 를제조한후, 암소에하루동안방치하여양이온 (ABTS ㆍ+) 을형성시킨다음 734 nm에서흡광도를측정하여흡광도값이 1.5 이하가나오도록희석하고, 희석된 ABTSㆍ + 용액 150 μl 와 AR 抽出物을각각 5 μl 혼합하고, 실온에서 10분간반응시킨후, 734 nm에서흡광도를측정하였다. 항산화능은증류수를대조군으로하여대조군에대한 ABTS radical 소거능을백분율로나타내었다. ( 시료첨가군의흡광도 ) 소거율 (%)=(1- ) 100 대조군의흡광도 (4) 세포내 ROS 생성 Raw 세포에서 reactive oxygen species (ROS) 를측정하기위하여 2',7'-dichlorofluorescin diacetate (DCF-DA) 를이용하였다. 12 well plate에 Raw 세포를 cells/well 이되게분주하여 24시간동안배양하였다. 배양후새로운배양액으로교체하였으며, 細辛酒錠抽出物을 1, 10, 100 (μg/ml) 의농도로처리하고, LPS 1 μg/ml 의농도로처리한후, 다시 24시간동안 37 o C, 5% CO 2 배양기에서배양하였다. 배양후, 1,200 rpm 에서 5분간원심분리하여모은세포를차가운 PBS로 2회세척하고, DCF-DA 10 μm이되도록첨가하여 15분동안암소, 상온에두었다. 염색후차가운 PBS를넣어 1,200 rpm에서 5분간원심분리한다음상청액을제거하고다시 PBS 400 μl를부유시켜유세포분석기 (Flow cytometer, Becton Dickinson, Franklin Lakes, NJ U.S.A.) 를이용하여형광강도의세기에따른변화를분석하였다. 4) 항염증효능측정 (1) NO NO (Nitric oxide) 농도는 griess reagent system을이용하여측정하였다. Raw 세포는 96 well plate에 cells/well 로분주하여 24시간동안배양후새로운배양액으로교체하였고, 細辛酒錠抽出物을 1, 10, 100 (μg/ml) 의농도로처리하고, LPS 1 μg/ml의농도로처리하여다시 24시간동안세포배양기 (37 o C, 5% CO 2 ) 에서배양하였다. N1 buffer 50 μl를각 well에처리하여 10분간상온에서반응한후, N2 buffer 50 μl를각 well에처리하고 10분간반응시켰다. 반응후 540 nm 에서흡광도를측정하였다. Nitrite standard의농도별표준곡선을이용하여배양액의 NO 농도를결정하였다. (2) Cytokine 세포내에서염증성사이토카인을측정하기위하여 luminex를사용하였다. 12 well plate에 Raw 세포를 cells/well 이되게분주하여 24시간동안배양후새로운배양액으로교체하였고, 細辛酒錠抽出物을 1, 10, 100 (μg/ml) 의농도로처리하고, LPS 1 μg/ml의농도로처리하여다시 24시간동안세포배양기 (37 o C, 5% CO 2 ) 에서배양하였다. 원심분리후상청액으로 interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necosis factors-α (TNF-α) 를측정하였다. 5) 유전자발현 (1) Ribonucleic acid (RNA) 추출셀에 RNAzolB 1,000 μl를넣고 chloroform (CHCl 3 ) 200 μl를첨가한후 15초간다시혼합하였다. 이를얼음에 2분간방치하였고, 13,000 rpm에서원심분리한후약 400 μl의상층액을회수하여 isopropanol 400 μl와동량혼합후천천히흔들고얼음에서 15분간방치하였다. 이를다시 13,000 rpm에서원심분리한후 80% EtOH로수세하고 1분간 vacuum pump 에서건조하여 RNA를추출하였다. (2) 역전사중합효소연쇄반응역전사 (reverse transcription) 반응은 RT premix kit의 mixture (reaction buffer, dntps mixture, RNase inhibitor, stabilizer, oligo dt15 primer) 를사용하여 total RNA 1 μg이되게 diethyl pyrocarbonate (DEPC) 처리된증류수에최종부피가 20 μl가되도록하여첨가하였다. 이 20 μl의반응혼합액을잘섞은뒤 2,000 rpm에서 5초간원심침강하여 45 o C heating block에서 60분동안반응시켜 first-strand complementary DNA (cdna) 를합성하였다. 이를다시 95 o C에서 5분동안방치하여 M- MLV RT를불활성화시킨후합성이완료된 cdna 를 polymerase chain reaction (PCR) 에사용하였다. (3) RT-PCR RT-PCR은 DNA polymerase 1 U/tube에 250 mm 102 J Korean Med Rehab 2014;24(3):99-110

5 細辛酒錠抽出物이 LPS 로유발된 RAW Cell 의염증및항산화반응에미치는영향 Table I. The Sequences of Primers in Used This Study Primer F/R Sequences inos F ATT GAT GGA GAG GGT CCA GC R ACC TGG AGG CAA GAG CTG AT IL-1β F GTG TCT TTC CCG TGG ACC TT R TCG TTG CTT GGT TCT CCT TG IL-6 F CCT TCC TAC CCC AAT TTC CA R CGC ACT AGG TTT GCC GAG TA TNF-α F AGC ACA GCC AGC ATG ATC CG R GTT TGC TAC GAC GTG GGC TA β-actin F GAG GTT CGA TGA TGC AGT GG R CCC AGG ATA GGA CTC AGG GA Table II. Content of Pb, As, Cd and Hg in 80% Ethanol Extract of AR Pb As Cd Hg Permissive density (mg/kg) AR N.D. N.D. N.D.: not detected. AR: asiasari radix. Fig. 1. Cell viability of AR extract in Raw cells. Raw cell was treated with 1, 10 and 100 (μg/ml) of AR extract for 24 hr. Cell viability was measured using an MTT assay. The results were expressed as mean±s.d. from three independent experiments. AR: asiasari radix. dntps mix, RT buffer (10 mm Tris-HCl, ph 9.0, 30 mm KCl, 1.5 mm MHCl 2 ) 를포함한 mixture 에각샘플과 primer를넣고 PCR을시행하였다. PCR 조건은 denaturation을위해 94 o C에서 10초, annealing 을위해 inos는 57 o C에서 10초, IL-1β는 62 o C에서 10초, IL-6는 59 o C에서 10초, TNF-α는 64 o C에서 10초, b-actin은 61 o C에서 10초수행하였으며, extension 을위해 72 o C에서 30초로 35 cycles 에수행하였고, 사용된 primer 는아래와같다 (Table I). 2% agarose gel에전기영동하여, 유전자발현여부를측정하였다. UV로촬영하여각그룹별로 band를확인하였고, RNA 발현을수치로나타내었다. 3. 통계처리실험결과는 statistical package for the social sciences (SPSS) 11.0의 unpaired student's T-test를사용하여통계처리하였으며 p<0.05, p<0.01 및 p<0.001 수준에서유의성을검정하였다. Fig. 2. HPLC chromatogram of 80% ethanol extract of AR. Column; ZORBAX Eclipse Plus C18 ( mm, 5μm), sample injection volume; 10μl, mobile phase; H 2O(A)/ACN(B) gradient elution: 0~5 min (0% B), 5~40 min (30% B), 40~60 min (80% B), 60~80 min (100% B), flow rate; 1.0 ml/min, detector; PDA detector. AR: asiasari radix. 결과»»» 1. 안전성검사 1) 중금속함량 細辛酒錠抽出物의중금속함량을측정한결과, 납과비소의경우기준치이하로검출되었고나머지중금속의경우검출되지않았다 (Table II). 2) 세포독성 Raw 세포주에서細辛의세포생존율을측정한결과, 대조군을 100.0±7.0% 로나타냈을때細辛처리군은각각 1, 10, 100 (μg/ml) 농도에서 105.4±17.0%, 86.0± 8.3%, 94.2±11.2% 의세포생존율을나타내었다 (Fig. 1)

6 이옥진 오민석 Table III. Total Phenolic Contents of 80% Ethanol Extract of AR Samples Total phenolics (mg GAE 1) /g ext.) Asiasari radix 28.77±1.67 1) Total phenolic contents was expressed as milligram of gallic acid equivalent (GAE) per gram of extract. AR: asiasari radix. Fig. 4. ABTS free radical scavenging activity of AR extract at various concentration. Extract was incubated with ABTS solution at RT for 10 mins. Activities were determined by measurement of absorbance at 517 nm. The results were expressed as mean±s.d. from three independent experiments. ABTS: 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid). AR: asiasari radix. Fig. 3. DPPH free radical scavenging activity of AR extract at various concentration. Extract was incubated with DPPH solution at 37 o C for 30 mins. Activities were determined by measurement of absorbance at 517 nm. The results were expressed as mean±s.d. from three independent experiments. DPPH: 1,1 diphenyl-2-picryl-hydrazyl. AR: asiasari radix. 3) HPLC pattern 분석 細辛酒錠抽出物의경우 210 nm에서 HPLC를이용하여 pattern 분석한결과, 50.87분, 53.61분, 56.80분, 분대에 peak를확인할수있었다 (Fig. 2). 2. 항산화효능에미치는영향 1) 총폴리페놀함량총폴리페놀함량을 gallic acid를표준물질로하여측정한결과, 폴리페놀함량이 28.77±1.67 mg/g으로나타났다 (Table III). 2) DPPH 소거능 DPPH 소거율은 1 μg/ml 농도에서 0.0±1.0%, 10 μg/ ml 농도에서 1.2±1.6%, 100 μg/ml 농도에서 15.1± 2.3%, 1,000 μg/ml 농도에서 55.2±3.9% 로나타나농도의존적으로증가하였다 (Fig. 3). Fig. 5. Effect of AR extract on ROS production in Raw cells. Raw cell was treated with 1, 10 and 100 (μg/ml) of AR extract and LPS (1 μg/ml) for 24 hr. The ROS production was analyzed by flow cytometry. The results were expressed as mean±s.d. from three independent experiments. Statistically significant value was calculated by compared with control group by student's t-test (*p<0.05). ROS: reacive oxygen species. LPS: lipopolysaccharide. AR: asiasari radix. Norma: normal group. Con: LPS- induced group. 3) ABTS 소거능 ABTS 소거율은 1 μg/ml 농도에서 0.3±0.7%, 10 μg/ ml 농도에서 1.6±0.8%, 100 μg/ml 농도에서 9.3± 0.6%, 1,000 μg/ml 농도에서 85.6±1.2% 로나타나농도의존적으로증가하였다 (Fig. 4). 4) ROS 의생성 ROS 생성저해활성은대조군을 100.0±1.0% 로나타 104 J Korean Med Rehab 2014;24(3):99-110

7 細辛酒錠抽出物이 LPS 로유발된 RAW Cell 의염증및항산화반응에미치는영향 냈을때정상군은 74.6±3.8%, 細辛투여군은 1 μg/ml 농도에서 100.6±3.6%, 10 μg/ml 농도에서 90.3±8.7%, 100 μg/ml 농도에서 83.3±8.2% 로나타나며특히 100 μg/ ml 농도에서유의성있게감소하였다 (Fig. 5). 3. 항염증효능에미치는영향 1) NO의생성 NO 생성량은대조군을 100.0±9.8% 로나타냈을때정상군은 42.9±10.4%, 細辛투여군은 1 μg/ml 농도에서 92.2±5.9%, 10 μg/ml 농도에서 88.8±9.4%, 100 μg/ ml 농도에서 51.2±3.8% 로나타나며특히 100 μg/ml 농도에서유의성있게감소하였다 (Fig. 6). 이 10.0±3.0 pg/ml, 細辛투여군은 1 μg/ml 농도에서 ±261.7 pg/ml, 10 μg/ml 농도에서 ± pg/ml, 100μg/ml 농도에서 899.8±215.3 pg/ml으로모든농도에서농도의존적으로감소하였고, 모든농도에서유의성있게감소하였다 (Fig. 8). (3) TNF-α의생성 TNF-α 생성량은대조군이 ± pg/ml, 정상군이 191.3±92.1 pg/ml, 細辛투여군은 1 μg/ml 2) Cytokine 에미치는영향 (1) IL-1β의생성 IL-1β 생성량은대조군이 17.9±1.0 pg/ml, 정상군이 4.1±1.5 pg/ml, 細辛투여군은 1 μg/ml 농도에서 17.0±1.3 pg/ml, 10 μg/ml 농도에서 10.3±2.9 pg/ml, 100 μg/ml 농도에서 5.8±4.1 pg/ml 로모든농도에서농도의존적으로감소하였으며, 특히 10 μg/ml 농도에서유의성있게감소하였다 (Fig. 7). (2) IL-6의생성 IL-6 생성량은대조군이 ±135.9 pg/ml, 정상군 Fig. 7. Effect of AR extract on LPS-induced IL-1β production in Raw cells. Raw cells were treated with 1, 10 and 100 (μg/ml) of AR extract in the presence of LPS (1 μg/ ml) for 24 hr. The results were expressed as mean±s.d. from three independent experiments. Statistically significant value was calculated by compared with control group by student's t-test (*p<0.05). interleukin-1β (IL-1β). Fig. 6. Effect of AR extract on LPS-induced nitric oxide (NO) production in Raw cells. Raw cell was treated with 1, 10 and 100 (μg/ml) of AR extract and LPS (1 μg/ml) for 24 hr. The amount of nitric oxide in supernatant was measured using Griess reagent. The results were expressed as mean±s.d. from three independent experiments. Statistically significant value was calculated by compared with control group by student's t-test (***p<0.001). NO: nitric oxide. Fig. 8. Effect of AR extract on LPS-induced IL-6 production in Raw cells. Raw cells were treated with 1, 10 and 100 (μg/ml) of AR extract in the presence of LPS (1 μg/ml) for 24 hr. The results were expressed as mean±s.d. from three independent experiments Statistically significant value was calculated by compared with control group by student's t-test (***p<0.001). IL-6: interleukin

8 이옥진 오민석 농도에서 ± pg/ml, 10 μg/ml 농도에서 ± pg/ml, 100 μg/ml 농도에서 ± pg/ml 으로모든농도에서농도의존적으로감소하였고, 모든농도에서유의성있게감소하였다 (Fig. 9). 4. 유전자발현에미치는영향 mrna 발현에미치는영향을보기위해 1, 10, 100 μg/ ml 농도로처리한후, LPS 처리된대조군의 mrna 발현량을 100% 로보았다. inos, IL-1β, IL-6, TNF-α는농도 Fig. 9. Effect of AR extract on LPS-induced TNF-α production in Raw cells. Raw cells were treated with 1, 10 and 100 (μg/ml) of AR extract in the presence of LPS (1 μg/ ml) for 24 hr. The results were expressed as mean±s.d. from three independent experiments. Statistically significant value was calculated by compared with Control group by student's t-test (*p<0.05, **p<0.01). TNF-α: tumor necosis factors-α. 의존적으로억제되었고, β-actin 으로정규화를한결과, inos의경우, 1, 10, 100 μg/ml 의농도에서 17.4%, 23.6%, 66.1%, IL-1β의경우, 1, 10, 100 μg/ml의농도에서 61.0%, 72.9%, 74.0%, IL-6의경우, 1, 10, 100 μg/ ml의농도에서 7.4%, 10.9%, 56.1%, TNF-α의경우, 1, 10, 100 μg/ml 의농도에서 22.3%, 30.2%, 56.4% 가감소하였다 (Fig. 10). 고찰»»» 염증반응은어떤자극에대한생체조직의비특이적면역반응으로써조직의변성, 순환장애와삼출, 조직증식세가지를유발하는복잡한병변이다 23,24). 대식세포는초기염증반응에관여하는대표적인면역세포로서동물체내모근조직에존재하며외부이물질을탐지하고포식하여죽은세포를제거하는포식작용과더불어외부자극을통해염증사이토카인, ROS, NO, PGE 2 등의다양한염증물질분비를통해초기염증반응을유도하게된다 25-28). 염증매개물질이과량생산되면기관지염, 관절염등의염증성질환을유발함으로써질환을악화시키는데기여하게된다. 따라서각종염증질환을효과적으로감소시킬수있는항염증제제및치료보조제개발에대한연구가많이이루어지고있으며, 최근한약재로부터유래되는생리활성물질이나단일화합물이치료제개발의표적물질로주목받고있다 29). 최근에골관절염의진행에중요한역학을하는 tumor Fig. 10. Effects of AR on lipopolysaccharide(lps)-induced mrna production in RAW cells. Cells treated with 1, 10 or 100 (μg/ml) of AR in the presence of 1 (μg/ml) LPS or with LPS alone for 24 hrs. Total RNA was isolated and the mrna expression levels of inos, IL-1β, IL-6 and TNF-α were detected by RT-PCR. Bar graph showed band intensity of PCR products. 106 J Korean Med Rehab 2014;24(3):99-110

9 細辛酒錠抽出物이 LPS 로유발된 RAW Cell 의염증및항산화반응에미치는영향 necrosis factor-α (TNF-α), interleukin-1β (IL-1β) 등의 cytokine 을억제하는면역학적관점의약물에대한가진행되고있다 30-32). 또한체내에서생성되는산화스트레스는염증반응을유발하는원인중하나가되므로, 다량의항산화성분을섭취하여산화스트레스를억제함으로써염증반응의유발을감소시켜만성질환을예방, 치료하고자하는연구가활발히진행되고있다 33). 細辛은족두리풀 (Asarum sieboldii) 이나민족두리풀 (Asarum sieboldii var.seoulensis) 의뿌리및전초를한약재로사용하며 12) 性味는辛 溫 無毒하고, 歸經은肺 腎에속하며, 效能은發散解表, 祛風止痛, 溫肺化痰, 溫經通絡, 行水通竅등으로알려져있다 13-16). 細辛에대한연구로는 LPS로활성화된복강대식세포에서細辛抽出物의항염증효과에대한연구 19), 細辛정유추출물의피부진균및기회감염진균에대한항진균효과의연구 12), 빈용한약재의진통및소염활성에대한연구 34) 등이있으나, 염증기전에관계된 cytokine 과항산화에대한연구는미비한것으로사료되었기에 RAW cell을이용하여細辛의항염및항산화에미치는영향을알아보고자하였다. 본연구에서는細辛의 RAW cell에서세포독성, 항산화효과및 NO 생성, 염증성지표로서 IL-1β, IL-6, TNF-α의염증 cytokine, 유전자발현에미치는영향에대해분석하였다. 먼저약재의안정성검증을위해중금속함량, 세포독성검사, HPLC pattern 분석을실시하였다. 細辛酒錠抽出物의중금속함량을측정한결과, 납과비소의경우기준치이하로검출되었고나머지중금속의경우검출되지않았으며 (Table II), 세포생존율은대조군을 100.0±7.0% 로나타냈을때細辛酒錠抽出物은각각 1, 10, 100 (μg/ml) 농도에서 105.4±17.0%, 86.0±8.3%, 94.2±11.2% 의세포생존율을나타내어 (Fig. 1) 비교적안전한약물임을알수있었다. 細辛酒錠抽出物을 210 nm에서 HPLC를이용하여 pattern 분석한결과, 50.87분, 53.61분, 56.80분, 분대에 peak를나타내었다 (Fig. 2). Free radical 은정상적인신체대사과정중에생성된한개이상의비공유전자를가진불안정한상태의이온으로 DNA 손상을일으키고단백질의과산화물을생성하며지질과산화를유발하는등여러염증반응과연관되어노 화및조직손상과밀접한관련이있다 35). 폴리페놀은식물에함유되어있으며, 대사과정중에발생하는유해한과산화물질을제거하는작용 (free radical scavenging) 이나항산화작용을나타낸다. 또폴리페놀화합물은만성질환의원인의하나로생각되어지고있는 oxidative stress나다른여러가지생리적요인을제거하여결과적으로는세포나조직을보호하고, 파괴를방지하는역할을한다 36). DPPH는짙은자주색을나타내는질소중심의 radical 로항산화활성을가진시료와반응하게되면노란색으로탈색되어감소하는데이의감소정도를측정하여 DPPH radical 소거활성을분석하였다 37). ABTS 소거능은 ABTS radical 이항산화물질과반응하여 ABTS radical 특유의청록색이탈색되어감소하는정도를측정하는방법이다 37). ROS는모든생명체에서일반적인세포사과정및여러요인에의해지속적으로활성화되는데 38), ROS가일정농도이상이지속되면 39) 영양분파괴로인한불균형이초래되며, 이에따라더욱산화적스트레스가증가하고 ROS에의한유리기생성이촉진되어생체막지질을파괴하게되다 40). 본연구에서항산화활성에미치는영향을알아보고자총폴리페놀, DPPH 소거능, ABTS 소거능, ROS의생성에대해측정한결과細辛酒錠抽出物에존재하는총폴리페놀함량은 gallic acid를표준물질로하여 28.77±1.67 mg/g으로나타났다 (Table III). 細辛酒錠추출물의농도가상승함에따라 DPPH 소거효율과 ABTS 소거효율이증가하였다 (Fig. 3, 4). ROS는細辛 100 μg/ml 농도에서유의성있는감소를나타내었다 (Fig. 5). 이를통해細辛의항산화활성이유효함을알수있다. NO는높은반응성을가진생체생성분자로써 NO synthase (NOS) 에의해 L-arginine 으로부터생성된다 29,41). NO는신경전달, 혈관의이완및세포매개성면역반응에관여하는데특히대식세포를 LPS로자극하면 inducible NOS (inos) 가발현되어 NO을생성하게된다. 이렇게생성된 NO는염증반응을매개하는역할을하게된다 41-44). IL-1β는대식세포의탐식작용을도와주는등각종면역반응을조절하는것으로알려져있는중요한염증매개인자로, 대식세포, 호중구, 상피세포, 내피세포에의해생산되고, LPS와같은박테리아산물이나 TNF-α와같은 107

10 이옥진 오민석 다른 cytokine 에의해서유도되며, 과잉또는장기적인생성이일어나게되면생체침습작용으로발열, 염증, 조직파괴, 쇼크등을일으킬수있다 45-47). IL-6는대식세포와 T cell에의해생산되며, TNF-α와 IL-1과함께급성기단백반응의유도체로서 anti-inflammatory 와 pro-inflammatory cytokine 으로알려져있다 48). TNF-α는대식세포와비만세포등에서분비되며, 많은자가면역질환에서염증의개시및유지에핵심적역할을하는것으로알려져있고, 종양세포에서는세포독작용을염증세포에서는 IL-1과유사한염증유발작용을하여세포의증식과분화를조절한다 49-51). 본연구에서 RAW 세포에서의細辛의항염증효과에대한 NO 생성량과염증사이토카인에미치는영향을알아보고자 NO, IL-1β, IL-6, TNF-α의생성량을측정하여다. NO 항목에서는細辛투여군 100 μg/ml 농도에서유의성있는감소를나타내었다 (Fig. 6). IL-1β, IL-6, TNF-α 항목은모든농도에서농도의존적으로감소하였으며, IL-1β는특히 10 μg/ml 농도에서유의성있는감소를나타내었다 (Fig. 7). IL-6은모든농도에서유의성있는감소를나타내었다 (Fig. 8). TNFα는모든농도에서유의성있는감소를나타내었다 (Fig. 9). 이러한결과는細辛이 IL-1β, IL-6, TNF-α, NO의생성을억제시켜염증질환을효과적으로감소시킬수있는것으로사료된다. inos는염증반응시대량으로생성되는것으로 LPS, interferon-γ (TFN-γ), IL-1 및 TNF-α 등의자극에의해대식세포, 혈관평활근세포, 내피세포, 간세포및심근세포등에서장시간다량의 NO를생성하는것으로알려져있다 52-55). inos의지속적인발현에의해 NO가과도하게생산되면패혈증이나관절염등여러가지염증성질환또는자가면역질환을일으키며, 그발병과증상을악화시키는데깊게관여하고있다. 또한류마티스관절염이나변형성관절염환자의활막세포또는연골세포에서 inos mrna 나단백질의발현이보고되고있으며, 활막액중에 NO의농도가상승하여염증을악화시키는것으로알려져있다 56). 항염증성기전과관련된 inos, IL-1β, IL-6, TNF-α의유전자발현에미치는영향을 RAW cell에서 inos, IL-1β, IL-6, TNF-α mrna 발현을 RT-PCR로분석하였다. 그결과 LPS에細辛酒錠抽出物을처리한경우유전 자발현양이줄어드는것을확인할수있었다 (Fig. 10). 이는 LPS에의해유도된 inos, IL-1β, IL-6, TNF-α가細辛酒錠抽出物에의해유전자발현이억제됨으로써항염증효과가있다고생각된다. 본논문에서는細辛酒錠추출물이세포독성을나타내지않으면서 DPPH와 ABTS 소거능의증가와 ROS 생성량의감소를통해항산화효과가있음을알수있었고, NO 및염증사이토카인의생성량의감소, 유전적검사를통해항염증효과를확인하였다. 細辛을酒錠으로추출하여실험을하였는데酒錠抽出이가수분해추출보다수득율이우수하다고보고 20,21) 되고있으며, 총폴리페놀, ABTS radical 소거능, 세포내 ROS 생성측정 HPLC 분석를통하여細辛에대한정밀한검사를유도하였다. 細辛의주정추출물이 LPS로유발된각종염증매개물질등의생성증가를유의하게억제시키는것으로나타나, 細辛이염증매개물질의과다배출로인하여발생하는관절염등의질환의치료에영향을미칠수있는것으로볼수있으며, 향후이러한항염및항산화작용에의하여관절염의치료뿐만아니라예방등을응용될수있을것으로사료된다. 골관절염치료에대한기초연구의일환으로, LPS로유도된 RAW cell에細辛酒錠抽出物을처리하여항염및항산화반응등을관찰한결과유의한성적을얻었기에향후지속적인연구로임상적인활용이기대된다. 결론»»» LPS로유발된 RAW cell을이용한細辛酒錠抽出物의항산화활성, 항염증효능, 유전자발현을관찰한결과다음과같은결론을얻었다. 1. 항산화효과 1) 총폴리페놀은 28.77±1.67 mg/g으로나타났다 2) DPPH 소거능은농도의존적으로증가하였다. 3) ABTS 소거능은농도의존적으로증가하였다. 4) ROS의생성량은 100 (μg/ml) 농도에서유의성있게감소하였다. 108 J Korean Med Rehab 2014;24(3):99-110

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