본초학표지

大韓本草學會誌제 28 권제 5 호 (213 년 9 월 ) Kor. J. Herbology 213;28(5):113-119 ISSN 1229-1765 http://dx.doi.org/1.6116/kjh.213.28.5.113. Lipopolysaccharide 로유발된마우스대식세포의염증매개성 Cytokine 생성증가에대한참당귀물추출물의효능연구 한효상 중부대학교보건행정학과 Anti-inflammatory Effect of Angelicae Gigantis Radix Water Extract on LPS-stimulated Mouse Macrophages Hyo-Sang Han Department of Health Administration, College of Social Sciences, Joongbu University, Geumsan 312-72, Korea ABSTRACT Objectives : The purpose of this study was to investigate the effects of Angelicae Gigantis Radix Water Extract(AG) on the production of proinflammatory mediators in RAW 264.7 cells stimulated with lipopolysaccharide(lps). Method : RAW 264.7 cells were cotreated with AG( and ug/ml) and lipopolysaccharide(lps; 1 ug/ml) for 24 hours. After 24 hour treatment, using Bead-based multiplex cytokine assay, concentrations of various cytokines such as interleukin(il)-6, IL-1β, IL-1, tumor necrosis factor-alpha(tnf-α), granulocyte colony-stimulating factor(g-csf), granulocyte macrophage colony-stimulating factor(gm-csf), interferon inducible protein-1(ip-1), leukemia inhibitory factor(lif), lipopolysaccharide-induced chemokine(lix), monocyte chemoattractant protein-1(mcp-1), macrophage colony-stimulating factor(m-csf), macrophage inflammatory protein(mip)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted(RANTES) and vascular endothelial growth factor(vegf) were measured. Result : AG significantly inhibited LPS-induced production of TNF-α, MIP-1α, G-CSF, RANTES, IL-1, and M-CSF from LPS-stimulated RAW 264.7 cells at the concentrations of and ug/ml. AG significantly inhibited LPS-induced production of MIP-1β, MIP-2, GM-CSF, and IL-6 from LPS-stimulated RAW 264.7 cells at the concentrations of ug/ml. AG significantly inhibited LPS-induced production of VEGF from LPS-stimulated RAW 264.7 cells at the concentrations of ug/ml. But AG did not show any significant effect on the production of MCP-1, LIF, LIX, IP-1 and IL-1β from LPS-induced RAW 264.7 cells. Conclusion : These results suggest that AG has anti-inflammatory effect related with its inhibition of proinflammatory mediators such as TNF-α, MIP-1α, G-CSF, RANTES, IL-1, MIP-1β, MIP-2, GM-CSF, IL-6, VEGF and M-CSF in LPS-induced macrophages. Key words : Angelica gigantis Radix, Water Extract, macrophage, lipopolysaccharide, anti-inflammation, cytokine 서론 1) 최근면역매개물질인사이토카인 (cytokine) 의염증및면역반응조절기능에관한연구가활발히진행되고있고한약을대상으로한항염연구에대한보고도많이이루어지고있다. 여러가지사이토카인중패혈증과관련되는것은인터루킨 -6, 종양괴사인자등이며, 폐렴이나천식등은케모카인이관여되며, 염증성혈관신생은성장인자들과관련된다. 한의학에서많이사용되고있는참당귀또한염증질환치료에도많이응용되고있으므로, 대식세포 (macrophage) 에서분 교신저자 : 한효상, 충남금산군추부면대학로 21. 중부대학교보건행정학과 Tel : 41-7-6292 E-mail : hanhs@joongbu.ac.kr 접수 :213 년 8 월 15 일 수정 :213 년 9 월 15 일 채택 :213 년 9 월 17 일

114 大韓本草學會誌 Vol. 28 No. 5, 213 비되는염증매개인자들 (proinflammatory mediators) 에대한참당귀의항염효과를충분히검토할필요가있다고사료된다. 當歸는 神農本草經 1) 中品에 味甘溫. 主咳逆上氣, 溫瘧寒熱洗洗在皮膚中, 婦人漏下絶子, 諸惡瘡瘍, 金瘡, 煮飮之. 라고처음收載된이후, 補血和血, 調經止痛, 潤燥滑腸의효능이있어月經不調, 經閉腹痛, 癥瘕結聚, 崩漏, 血虛頭痛, 眩暈, 痿痺, 腸燥便難, 癰疽瘡瘍, 跌打損傷을치료하는약물로사용되어왔다 2). 현재한국, 중국, 일본 3국에서사용하는당귀는그기원식물이각각달라한국에서는참당귀 Angelica gigas Nakai( 산형과 Umbelliferae), 중국에서는 A. sinensis (Oliv.) Diels를, 일본에서는大和當歸 A. acutiloba Kitag. 의根을자국의약전에규정하고있는실정이며당귀의기원에따른성분과약리적효과는상이한것으로알려져있다 2). 當歸의성분으로 Coumarin 유도체로서 decursinol 이참당귀의주성분으로분리되어있고, 기타 7-de-methyl-suberosine, umbelliferone, isoimperatorin, xanthyletin, nodakenin 등이알려져있다 3). 이외에도참당귀정유는 β-eudesmol 이 9.49%, α-pinene이 9.47%, limonene이 3.16%, elemol 이 1.47% 의조성을이루고있다 4). 참당귀의약리작용에대해서박등 5) 은당뇨개선효과를, 함등 6) 은항암및혈압조절효능을, 허등 7) 은항산화효과를, 이등 8) 은항미생물효과를보고하였다. 또한안등 9) 은면역증강효능을, 박등 1) 은미백효과를, 김등 11) 은심혈관질환의개선과치료효과를, 신등 12) 은간효소억제효과를, 김등 13) 은퇴행성신경질환에서신경보호효능을보고하였다. 이에저자는참당귀의항염효과에대하여알아보기위하여참당귀를열수추출하여얻은시료 (AG) 를대상으로 lipopolysaccharide (LPS; 지질다당체 ) 로유발된 RAW 264.7 cells의인터루킨 (interleukin; IL), 종양괴사인자알파 (tumor necrosis factor-alpha; TNF-α), 성장인자 (growth factor), 케모카인 (chemokine) 등의사이토카인 (cytokine) 생성증가에대한영향을조사하여유의한결과를얻었기에이에보고하는바이다. 1. 재료 재료및방법 1) 약재실험에사용된참당귀 (Angelica gigantis Radix; Angelica gigas의뿌리 ) 는한국대구의옴니허브주식회사로부터 212 년 5월에구입 (NO; 212-7) 하여기원의진위와품질의우열을가천대학교한의과대학본초학교실에서감정하였고, 약재는초음파세척기 (Branson, USA) 를이용하여불순물을제거하고실험에사용하였다. 2) Cell line 실험에사용된대식세포는마우스복강대식세포 (mouse macrophage line) 인 Raw 264.7 cells로서한국세포주은행 (KCLB, Korea) 으로부터구입하여사용하였다. 3) 시약및기기 본실험에사용된시약으로는 Dulbecco's modied essential medium(dmem, Sigma, USA), penicillin(sigma, USA), streptomycin(sigma, USA), FBS(Sigma, USA), Multiplex cytokine assay kit(panomics, USA) 등이사용되었다. 각시약의품질은분석용등급이상의것으로하여사용하였다. 본실험에사용된기기는 deep freezer(ilshin Lab Co, Korea), vortex mixer(vision Scientific Co, Korea), water bath(in-tron biotech., Korea), ice-maker(vision Scientific Co, Korea), 원심분리기 (Hanil, Korea), 세포배양기 (NUAIRE, USA), rotary vacuum evaporator(eyela, Japan), Bio-Plex 2(Bio-Rad, USA), 생물현미경 (Olympus, Japan), fume hood(hanil, Korea), 무균작업대 (Jeio thec, Korea), ultrasonic cleaner(branson, USA) 등이다. 2. 방법 1) 참당귀열수추출물제조참당귀 g을정확하게중량을측정한뒤, 환류추출기에 1차증류수 2, ml 와함께넣은뒤탕액이끓는시점으로부터 2 시간동안가열하여추출한다음추출액을 filter paper(advantec No.2, Japan) 로감압여과한여과액을 rotary vacuum evaporator 를이용하여농축액을얻었으며, 이농축액을동결건조기를이용하여건조한분말을시료로사용하였다. 동결건조추출물은 21.2 g을얻었으며, 수율은 42.4 % 였다. 2) 세포배양 RAW 264.7 cells는 37, 5% CO 2 조건에서 1% FBS, penicillin( U/mL, streptomycin( ug/ml) 이첨가된 DMEM 배지로세포배양기에서배양되었다. Raw 264.7 cells를 cm 2 flask(falcon, USA) 에서충분히증식된후배양 3 일간격으로배양세포표면을 phosphate buffered saline(pbs) 용액으로씻어준후 ml flask 당 1 ml의.% trypsin-edta 용액을넣고실온에서 1 분간처리한다음 trypsin 용액을버리고 37 에서 5 분간보관하여세포를탈착하여계대배양하였다. 탈착된세포는 1% FBS가첨가된 DMEM 배양액 1 ml에부유시킨다음새로운배양용기 ( ml culture flask) 에옮겨 1 : 2의 split ratio로세포배양기에서배양하였다. 3) 사이토카인 (Cytokine) 분비측정사이토카인분비와관련된시료의영향을알아보기위해 Politch 등 14) 을참조하여 Bio-Plex Cytokine Assay를다음과같이실험을시행하였다. 96 well plate에 1 1 5 cells/ml 의 cell을 ul씩넣고세포배양기에서 24 시간동안배양한후배지를버리고배양세포표면을 1 PBS 용액으로씻어준뒤각 well에 LPS 1 ug/ml를단독혹은다양한농도의시료와함께배지에담아처리하고 24 시간동안배양하였다. 배양이끝나면상등액 (cell culture supernatant) 을채취하여 filter plate(96 well type) 에미리준비되어있던 antibody-conjugated capture beads와결합시켰다. 결합된 capture beads가담긴 filter plate의각 well을 1 ul의 wash buffer로세척하였다. 세척이끝난뒤각 well에

Lipopolysaccharide 로유발된마우스대식세포의염증매개성 Cytokine 생성증가에대한참당귀물추출물의효능연구 115 detection antibody 를추가한후 3 분간배양하였고, 배양이끝난뒤 wash buffer로 3 회세척하고각 well에 streptavidin-pe를분주한뒤상온에서 3~ rpm의조건으로 3 분간진동배양 (shaking) 하였다. 배양이끝나면 wash buffer로 3 회세척한뒤각 well에 12 ul의 reading buffer를분주하고상온에서 3~ rpm의조건으로 5 분간진동배양 (shaking) 한후 Bio-Plex array reader(bio-plex 2) 을이용, 측정하고자하는사이토카인 (cytokine) 의양을조사, 비교하였다. 조사된사이토카인 (cytokine) 들은 IL-1β, IL-6, IL-1, TNF-α, granulocyte colony-stimulating factor(g-csf), granulocyte macrophage colony-stimulating factor(gm-csf), interferon inducible protein-1(ip-1), leukemia inhibitory factor(lif), lipopolysaccharide-induced chemokine(lix), monocyte chemoattractant protein-1(mcp-1), macrophage colony-stimulating factor(m-csf), macrophage inflammatory protein(mip)-1α, MIP-1β, MIP-2, Regulated on Activation, Normal T cell Expressed and Secreted(RANTES), vascular endothelial growth factor(vegf) 등이다. 3. 통계처리본실험에서얻은결과에대해서는 mean±sd 로나타내었으며, 대조군과각실험군과의평균의차이는 student's t-test 로분석하여 P-value값이.5 미만일때통계적으로유의한차이가있는것으로판정하였다. 결과 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells에처리한결과, ug/ml 의농도에서모두 LPS에의한 RANTES 생성증가를유의하게억제하였다 (Fig. 1). 5. M-CSF의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells에처리한결과, ug/ml 의농도에서모두 LPS에의한 M-CSF 생성증가를유의하게억제하였다 (Fig. 1). 6. VEGF의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells 에처리한결과 ug/ml의농도에서 LPS에의한 VEGF 생성증가를유의하게억제하였다 (Fig. 1). 7. MIP-1β 의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells 에처리한결과 ug/ml 의농도에서 LPS에의한 MIP-1β 생성증가를유의하게억제하였다 (Fig. 1). 8. MIP-2의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells 에처리한결과 ug/ml의농도에서 LPS에의한 MIP-2 생성증가를유의하게억제하였다 (Fig. 1). 1. TNF-α 생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells에처리한결과, ug/ml 의농도에서모두 LPS에의한 TNF-α 생성증가를유의하게억제하였다 (Fig. 1). G-CSF production TNF-α production N C C+AG C+AG RANTES production MIP-1α production N C C+AG C+AG 2. MIP-1α 의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells에처리한결과, ug/ml 의농도에서모두 LPS에의한 MIP-1α 생성증가를유의하 M-CSF production N C C+AG C+AG N C C+AG C+AG VEGF production N C C+AG C+AG N C C+AG C+AG 게억제하였다 (Fig. 1). MIP-1β production MIP-2 production 3. G-CSF의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells에처리한결과, ug/ml 의농도에서모두 LPS에의한 G-CSF 생성증가를유의하게억제하였다 (Fig 1). 4. RANTES의생성증가에미치는영향 N C C+AG C+AG N C C+AG C+AG Fig. 1. Effects of AG on cytokine production (TNF-α, MIP-1α, G-CSF, RANTES, M-CSF, VEGF, MIP-1β, MIP-2) in LPS-stimulated RAW 264.7 cells measured by Multiplex bead-based cytokine assay after 24 h incubation. Normal group (N) was treated with media only. Control group (C) was treated with LPS (1 ug/ml) only. C+AG group was cotreated with LPS and ug/ml of Angelicae Gigantis Radix water extract. C+AG group was cotreated with LPS and ug/ml of Angelicae Gigantis Radix water extract. Values are the mean ± SD of more than three independent experiments. P <.5 vs. Normal. P <.5 vs. Control. P <.1 vs. Control.

116 大韓本草學會誌 Vol. 28 No. 5, 213 9. GM-CSF 의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells 에처리한결과 ug/ml의농도에서 LPS에의한 GM-CSF 생성증가를유의하게억제하였다 (Fig. 2). 1. IL-6 의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells 에처리한결과 ug/ml의농도에서 LPS에의한 IL-6 생성증가를유의하게억제하였다 (Fig. 2). 11. IL-1 의생성증가에미치는영향 LPS(1 ug/ml) 와함께 AG( and ug/ml) 를 24 시간동안 RAW 264.7 cells에처리한결과, ug/ml 의농도에서 LPS에의한 IL-1 생성증가를유의하게억제하였다 (Fig. 2, ). 12. MCP-1, LIF, LIX, IP-1, IL-1β 의생 성증가에미치는영향 LPS(1 ug/ml) 에의해서유발된 RAW 264.7 cells의 MCP-1, LIF, LIX, IP-1, IL-1β의생성증가에대해서 AG는별다른변화를나타내지않았다 (Fig. 2). GM-CSF production IL-1 production LIF production IP-1 production N C C+AG C+AG N C C+AG C+AG N C C+AG C+AG N C C+AG C+AG IL-6 production MCP-1 production LIX production IL-1β production N C C+AG C+AG N C C+AG C+AG N C C+AG C+AG N C C+AG C+AG Fig. 2. Effects of AG on cytokine production (GM-CSF, IL-6, IL-1, MCP-1, LIF, LIX, IP-1, IL-1β) in LPS-stimulated RAW 264.7 cells measured by Multiplex bead-based cytokine assay after 24 h incubation. Normal group (N) was treated with media only. Control group (C) was treated with LPS (1 ug/ml) only. C+AG group was cotreated with LPS and ug/ml of Angelicae Gigantis Radix water extract. C+AG group was cotreated with LPS and ug/ml of Angelicae Gigantis Radix water extract. Values are the mean ± SD of more than three independent experiments. P <.5 vs. Normal. P <.5 vs. Control. P <.1 vs. Control. 고찰 補血藥에속하는當歸는 (Angelicae Gigantis Radix) 의기원식물은 대한약전 15) 에 참당귀 Angelica gigas Nakai ( 산형과 Umbelliferae) 의뿌리 라고收載되었으며, 李時珍 16) 은當歸라는이름은 古人娶妻爲嗣續也. 當歸調血爲婦人要葯, 有思夫之意, 故有當歸之名. 이라고하였다. 性은溫하고, 味는甘辛하다. 참당귀의함유성분으로는 coumarin 계의 decursin, decursinol angelate 와 nodakentin, umbelliferon, β -sitosterol 등이알려져있다 17). 약리작용으로는최근에파골세포형성저해효과 18), 베타아밀로이드에의한기억력장애에대한보호효과 19), 뇌경색보호효과 2) 등이보고되었다. 염증의반응은그원인과반응조직의차이에관계없이아주유사한변화를보이는데이런현상은조직손상직후에일어나는변화로손상에의하여생체내국소부위에유리되는공통적인물질요인의존재를추정하게하며, 염증의반응을중개하는물질인화학적매개체로는프로스타글란딘 (prostaglandin, PG), 산화질소 (nitric oxide, NO), 염증을유발하는여러가지사이토카인 (cytokine) 등이있다 21). 또한염증반응을유도하는주요한화학전달매개물질로는크게즉시형혈관투과성항진에관여하는 amine류 (serotonin, histamine 등 ) 와 kinin류 (bradykinin 등 ), 지연형반응에주로작용하는사이토카인 (cytokine) 류등으로분류할수있다 22). LPS는그람음성박테리아에서유래하는내독소 (endotoxin) 의일종으로대식세포 (macrophage) 등의면역세포를자극하여일산화질소 (nitric oxide, NO) 와사이토카인 (cytokine) 등각종의전염증성인자들 (proinflammatory mediators) 의생성증가를유도하며 23), 면역세포특히대식세포 (macrophage) 로하여금염증촉발물질의급격한생성증가를유발한다 24). 본연구에서는참당귀의항염효과를알아보기위해, 참당귀를열수추출하여제조한시료 (AG) 를대상으로 LPS로유발된 Raw 264.7 cells의 interleukin(il)-6, IL-1β, IL-1, tumor necrosis factor-alpha(tnf-α), granulocyte colony-stimulating factor(g-csf), granulocyte macrophage colony-stimulating factor(gm-csf), interferon inducible protein-1(ip-1) leukemia inhibitory factor(lif), lipopolysaccharide-induced chemokine(lix), monocyte chemoattractant protein-1(mcp-1), macrophage colony-stimulating factor(m-csf), macrophage inflammatory protein(mip)-1α, MIP-1β, MIP-2, Regulated on Activation Normal T cell Expressed and Secreted(RANTES), vascular endothelial growth factor(vegf) 등의사이토카인 (cytokine) 생성증가를유발하고, 이에대한참당귀물추출물 (AG) 의영향을알아보기위해실험을수행하였다. 본연구진은 AG가 ug/ml의농도까지마우스대식세포에대하여세포독성을나타내지않음을확인하고다음실험을진행하였다. TNF-α 는 LPS에의해활성화된대식세포 (macrophage), 단핵백혈구 (monocyte), 비만세포 (mast cell), 호중구 (neutrophil), 성상세포 (astrocyte), 섬유아세포 (fibroblast) 등의많은세포에서분비되는사이토카인 (cytokine) 으로세포성면역반응이나세포성장, 분화등에관련되어있다 ). AG가 LPS로유발된

Lipopolysaccharide 로유발된마우스대식세포의염증매개성 Cytokine 생성증가에대한참당귀물추출물의효능연구 117 마우스대식세포의 TNF-α 생성증가에미치는영향을비교하기위해 24시간동안처리한결과, ug/ml의농도에서 LPS에의한 TNF-α 생성증가를유의하게억제시켰다. MCP-1은염증반응을개시하는중요한요소로서섬유아세포, 내피세포, 혈관내평활근세포, 단핵구, T-세포등염증과관련된여러세포에서분비된다고알려져있다 26). AG가 LPS로유발된마우스대식세포의 MIP-1α 생성증가에미치는영향을비교하기위해 24시간동안처리한결과, ug/ml의농도에서 LPS에의한 MIP-1α 생성증가를유의하게억제시켰다. G-CSF는골수의과립백혈구전구세포와줄기세포의증식과분화를촉진하며 27), G-CSF-receptor 는골수의미성숙세포에존재하며, G-CSF에의해자극되면성숙과립구로증식ㆍ분화하기시작하며, 의학적으로 G-CSF는백혈구생성을촉진하여혈중조혈모세포의수를증가시키고, 암환자의 chemotherapy 이후호중구감소증을회복시키는데사용한다 28). AG가 LPS로유발된마우스대식세포의 G-CSF 생성증가에미치는영향을비교하기위해 24시간동안처리한결과, ug/ml의농도에서 LPS에의한 G-CSF 생성증가를유의하게억제시켰다. RANTES는호산구가혈중에서기도표면으로이동하는데관여하는케모카인들중하나로 IL-5와함께천식, 폐에서호산구에대한주요한화학주성인자이다 29). AG가 LPS로유발된마우스대식세포의 RANTES 생성증가에미치는영향을비교하기위해 24시간동안처리한결과, ug/ml의농도에서 LPS에의한 RANTES 생성증가를유의하게억제시켰다. 사람의 M-CSF는지방단백질 (lipoprotein) 의일종으로단핵구, 중성구및혈소판의생산을자극하는조혈인자로서의작용외에도혈중콜레스테롤 (cholesterol) 치를낮춰주고태반형성, 임신유지, 단핵구의항종양활성증폭등의작용을가지고있다 3). AG가 LPS로유발된마우스대식세포의 M-CSF 생성증가에미치는영향을비교하기위해 24시간동안처리한결과, ug/ml의농도에서 LPS에의한 M-CSF 생성증가를유의하게억제시켰다. VEGF는주요한성장인자로서 34-45 kd homodimeric glycoprotein 이며혈관내피세포에서발현되는매우특이한유사분열촉진인자로서 31) 배아발육 (embryonic development), 창상치유, 고형종양의성장및복수생성등의정상및비정상적인과정에주요한역할을한다 32). AG가 LPS로유발된마우스대식세포의 VEGF 생성증가에미치는영향을비교하기위해 24시간동안처리한결과 ug/ml의농도에서 LPS에의한 VEGF 생성증가를유의하게억제시켰다. MIP-1β는 CC chemokine 의하나로서면역반응과염증반응에있어서작용을하며, 특히단핵구, T림프구, 호산구등에대한화학주성작용을하여여러질환에서역할을한다 33). AG가 LPS로유발된마우스대식세포의 MIP-1β 생성증가에미치는영향을비교하기위해 24시간동안처리한결과 ug/ml의농도에서 LPS에의한 MIP-1β 생성증가를유의하게억제시켰다. MIP-2는호중구의모집과정에서중요한역할을하는 mouse C-X-C chemokine이며 MIP-2 유전자의전사가빠르게 LPS 대식세포계통의세포자극에의해유도된다 34). AG가 LPS로유발된마우스대식세포의 MIP-2 생성증가에 미치는영향을비교하기위해 24시간동안처리한결과 ug/ml의농도에서 LPS에의한 MIP-2 생성증가를유의하게억제시켰다. GM-CSF는국소에서작용하는사이토카인으로활성화된 T 세포와단핵식세포, 혈관내피세포, 섬유아세포그리고점막과결체조직의비반세포들이생성하는당단백이다. GM-CSF 는정상과립구와대식세포의전구세포분화에필요한성장인자일뿐만아니라과립구, 단핵구그리고호산구의전구세포에작용하여이들세포들에대한성장인자분비, 수명연장그리고기능항진등을나타낸다 35). AG가 LPS로유발된마우스대식세포의 GM-CSF 생성증가에미치는영향을비교하기위해 24시간동안처리한결과 ug/ml의농도에서 LPS에의한 GM-CSF 생성증가를유의하게억제시켰다. IL-6는항원, IL-4, IL-5에의해활성화되어증식하는 B 임파구의분화의최종시기에작용해서분비형의다량의항체를생성시킨다. 또한 T 임파구의증식과분화를유도해서혈액간세포와형질세포를증식시켜간세포의급성기단백생성과신경세포의분화를촉진하는등의다양한생물활성을나타낸다36). AG가 LPS로유발된마우스대식세포의 IL-6 생성증가에미치는영향을비교하기위해 24시간동안처리한결과 ug/ml의농도에서 LPS에의한 IL-6 생성증가를유의하게억제시켰다. 이밖에 LPS에의한 MCP-1, LIF, LIX, IP-1, IL-1β 의사이토카인 (cytokine) 생성증가는폐손상과같은호흡기염증질환에악화작용을하는것으로알려져있으나, 본연구에서는 AG가별다른유의성을나타내지않았다. Fig. 1에는주로인터루킨류의결과를표시하였고, Fig. 2에는성장인자들의결과를표시하였다. 이와같이 AG가 LPS에의해유발된마우스대식세포의다양한사이토카인 (cytokine) 생성증가를유의하게억제함은 AG가대식세포의염증매개인자과잉생성으로인한각종의염증성질환악화, 예를들면류마티스성관절염악화 37), 폐렴및급성호흡기질환의악화 38), 자궁내막증식증 (endometriosis) 유발촉진 39) 등을완화할수있는항염효능이있는것으로해석될수있다. 향후참당귀물추출물을이용한항염치료제개발을위하여분자수준의기전연구등더욱세심한연구가요구되어지는바이다. 결론 본연구에서참당귀를열수추출하여얻은시료 (AG) 를대상으로 LPS로유발된마우스대식세포의 IL-6, IL-1β, IL-1, TNF-α, G-CSF, GM-CSF, IP-1, LIF, LIX, MCP-1, M-CSF, MIP-1α, MIP-1β, MIP-2, RANTES, VEGF 등의사이토카인 (cytokine) 생성증가에대한영향을측정하여다음과같은결론을얻었다. 1. LPS(1 ug/ml) 와함께 AG( and ug/ml) 가포함된배지에 Raw 264.7 cells를 24 시간동안배양한결과, LPS에의해유발된세포의 TNF-α, MIP-1α, G-CSF, RANTES, M-CSF, IL-1의생성증가를, ug/ml의농도에서유의하게억제하였다.

118 大韓本草學會誌 Vol. 28 No. 5, 213 2. LPS(1 ug/ml) 와함께 AG( and ug/ml) 가포함된배지에 Raw 264.7 cells를 24 시간동안배양한결과, LPS에의해유발된세포의 VEGF의생성증가를 ug/ml의농도에서유의하게억제하였다. 3. LPS(1 ug/ml) 와함께 AG( and ug/ml) 가포함된배지에 Raw 264.7 cells를 24 시간동안배양한결과, LPS에의해유발된세포의 MIP-1β, MIP-2, GM-CSF, IL-6의생성증가를 ug/ml의농도에서유의하게억제하였다. 이상의실험결과는 AG가 LPS에의해유발된마우스대식세포의사이토카인 (cytokine), 케모카인 (chemokine), 성장인자 (growth factor) 등의염증매개물질생성증가를유의하게억제함을나타내는것이며, 이는 AG가염증매개물질의증가로인한종창 ( 腫脹 ), 통증 ( 痛症 ) 등을개선할수있는염증완화제로서의응용가능성이있음을나타내는것이며, 앞으로참당귀물추출물의항염효능에대한보다깊은연구가필요한것으로생각되는바이다. References 1. Wu B. Shennongbencaojing. Beijing : Renminweishengchubanshe. 1982 : 64. 2. Kim IR, Kim HC, Kuk YB, Park SJ, Park YK, Park JH, Seo BI, Seo YB, Shin MK, Lee YJ, LeeYC, Lee JH, Leem KH, Cho SI, Chung JK, Joo YS, Choi HY. Boncho-Hak. Seoul : Young-Lim Press. 27 : 629-31. 3. Kim DH, Kim HM, Ryu JH, Eom JY, Kim SC, Yang JH, Cho MK, Lim JP, Hong SH. Hanbangyakrihak. Seoul : Shinilbukseu. 27 : 337-43. 4. Chi HJ, Kim HS. Studies on Essential Oils of Plants of Angelica Genus in Korea (I) Essential Oils of Angelicae gigantis Radix. Kor J Pharmacogn. 1988 ; 19(4) : 239-47. 5. Park MJ, Kang SJ, Kim AJ. Hypoglycemic Effect of Angelica gigas Naki Extract in Streptozotocin-induced Diabetic Rats. Kor J Food Nutr. 29 ; 22(2) : 246-51. 6. Ham MS, Kim SS, Hong JS, Lee JH, Chung EK, Park YS, Lee HY. Screening and Comparison of Active Substances of Angelica gigas Nakai Produced in Kangwon and Angelica acutiloba Kitogawa Produced in Japan. Kor J Appl Microbiol Biotechnol. 1996 ; 24(5) : 624-9. 7. Heo SJ, Yang MO, Cho EJ. Analysis of Umbelliferaeceae Wild Plants and Antioxidative Activity of Pork Meat Products Added with Wild Plants. Kor J Soc Food Cookery Sci. 21 ; 17(5) : 456-63. 8. Lee S, Shin DS, Kim JS, Oh KB, Kang SS. Antibacterial coumarins from Angelica gigas roots. Arch Pharm Res. 23 ; 26(6) : 449-52. 9. Ahn KS, Sim WS, Kim HM, Han SB, Kim IH. Immunostimulating Components from the Root of Angelica gigas Nakai. Kor J Pharmacogn. 1986 ; 27(3) : 4-61. 1. Park SK, Hong SK, Kim HJ, Kim BY, Kim TG, Kang JS, Kim DU. Cosmetic Effect of Angelica gigas Nakai Root Extracts. Kor Chem Eng Res. 29 ; 47(5) : 553-7. 11. Kim GS, Park CG, Jeong TS, Cha SW, Baek NI, Song KS. ACAT(Acyl-CoA:cholesterol Acyltransferase) Inhibitory Effect and Quantification of Pyranocurmarin in Different Parts of Angelica gigas Nakai. J Appl Biol Chem. 29 ; 52(4) : 188-94. 12. Shin KH, Han JM, Lee IR. Effect of the Constituents of Angelicae gigantis Radix on Hepatic Drug Metabolizing Enzymes. Kor J Pharmacogn. 1996 ; 27(4) : 323-7. 13. Kim YO, Ha NN, Boo YM, Park SY, Park JY, Yu YB, Shin JS, An DK, Kim HC. Neuroprotective Effect of Angelica gigas Extracts on the Brain Ischemia Induced by Four-Vessel Occlusion in Rats. Kor J Herbology. 22 ; 17(2) : 151-7. 14. Politch JA, Tucker L, Bowman FP, Anderson DJ. Concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men. Hum Reprod. 27 ; 22(11) : 2928-35. 15. Korea Food and Drug Administration. The Korean Herbal Pharmacopoeia. Seoul : Korea Food and Drug Administration. 212 : 26-7. 16. Chen GT. Benchogangmutongshi. Beijing : Xueyuanchubanshe. 1992 : 615-21. 17. Ahn KS, Sim WS, Kim IH. Decursin a cytotoxic agent and protein kinase c activator from the root of angelica gigas. Planta medica. 1996 ; 62(1) : 7-9. 18. Kil JS, Kim MG, Choi HM, Lim JP, Boo Y, Kim EH, Kim JB, Kim HK, Leem KH. Inhibitory effects of Angelicae Gigantis Radix on osteoclast formation. Phytother Res. 28 ; 22(4) : 472-6. 19. An JJ, Kim DH, Moon YS, Jung JS, Ahn EM, Baek NI, Song DK. Protection against beta-amyloid peptideinduced memory impairment with long-term administration of extract of Angelica gigas or decursinol in mice. Prog Neuropsychopharmacol Biol Psychiatry. 24 ; 28(1) : -3. 2. Kang SY, Kim YC. Neuroprotective coumarins from the root of Angelica gigas Structure-activity relationships. Arch Pharm Res. 27 ; 3(11) : 1368-73. 21. Daehanbyeongrihakhoe. Byeongrihak. Seoul : Komoonsa. 1995 : 71-14.

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