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대한한의학회지제 32 권제 1 호 (211 년 1 월 ) J Korean Oriental Med 211;32(1):175-184 Original Article 독활물추출물이대식세포면역활성에미치는영향 이종한, 김윤상, 임은미 경원대학교한의과대학부인과학교실 Effects of Angelicae Pubescentis Radix Water Extract on Immune Property in RAW 264.7 Macrophages Jong-Han Lee, Yoon-Sang Kim, Eun-Mee Lim Dept. of Gynecology, College of Oriental Medicine, Kyungwon University Objectives: The purpose of this study was to investigate the effects of Angelicae pubescentis Radix water extract (ACE) on immune properties in macrophage cells. Methods: The cells were divided into two groups: As a control, the first was not treated with ACE, and the other was treated with ACE. Together with the cell viability, productions of nitric oxide (NO) and cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α by treating of ACE were monitored. Results: 1. There was no decrease of the cell viability after 24 hr incubation, but a significant decrease after 48 hr incubation with all four concentrations (25, 1, 2, and 4 μg / ml ) of ACE. 2. A significant increase in the production of NO was observed in the concentrations above 5 μg / ml of ACE after 24 hr incubation. 3. Further, after 48 hr incubation, the critical concentration of ACE for the increase was reduced to 25 μg / ml. 4. The production of IL-1β significantly increased with the ACE concentrations of 1 and 2 μg / ml after 24 hr incubation. 5. The production of IL-6 significantly increased with the ACE concentration of 2 μg / ml after 24 hr incubation. 6. A significant increase in the production of TNF-α was detected with ACE concentrations of 5, 1, and 2 μg / ml after 24 hr incubation. Conclusions: These show that ACE increases mouse macrophage NO production at concentrations above 5 μg / ml, and the cytokines (IL-1β, IL-6, and TNF-α) at concentrations above 2 μg / ml. These results suggest that ACE improves macrophage immune property. Key Words : Angelicae pubescentis radix, cytokine, immune property, macrophage, nitric oxide 서론부인과에서면역력의저하는산후감염, 자궁경부암등의종양질환및각종생식기감염질환등에서중요한문제이다 1). 한의학에서는 正氣在內邪不可干, 邪氣所湊其氣必虛 2) 라고하여, 發病機制를 邪正鬪爭 으로보아正氣는면역기능에대한조절로해석할수있다 3). 독활은辛溫發散, 祛風勝濕하는效能이있어부인 Received:2 December 21 Revised:3 December 21 Accepted:3 December 21 Correspondence to: 김윤상 (Yoon-Sang Kim) 인천광역시남동구구월동 12-1 번지경원대학교부속길한방병원한방부인과 Tel:+82-32-468-33, Fax:+82-32-468-433, E-mail:komy@kyungwon.ac.kr 175

(176) 대한한의학회지제 32 권제 1 호 (211 년 1 월 ) 과에서風濕痺痛을치료하고, 熟地黃, 當歸, 川芎, 續斷, 杜沖, 白芍藥및桑寄生등을配合하여産後身痛, 下肢痛에사용하며 4), 강활과비교하여羌之氣淸行氣而發散營衛之邪, 獨之氣濁行血而溫養營衛之氣, 羌有發表之功, 獨有助表之力 5) 이라하여독활의효능을인체의질병에대한邪正鬪爭에서正氣를도와서邪氣를내친다 6,7) 는개념으로보았다. 부인과에서면역에대한기존연구는艾葉, 淫羊藿등개별한약재와처방으로구성된연구가있었지만 8-2) 독활의면역활성동에대한보고는없었다. 이에본연구에서는독활이대식세포면역활동에어떤영향을미치는지알아보기위하여열수추출하여얻은추출물 ( 이하 ACE) 로마우스대식세포에전처리후배양하여 cell viability와 NO 생성을관찰하였고, IL-1β, IL-6 및 TNF-α 등의 cytokine 생성에미치는영향을조사하여유의한결과를얻었기에이에보고하는바이다. 실험 1. 재료 1) 약재실험에사용된獨活 (Angelicae Pubescentis Radix; root of Aralia continentalis KITAGAWA, 옴니허브, Korea) 은 28년 11월에구입 (NO: 28-11-9) 하였고, 약재는사용전에초음파세척기 (ultrasonic cleaner) 를이용하여불순물을제거하고실험에사용하였다. 2) Cell line 실험에사용된 mouse 대식세포는 Raw 264.7 cell line( 한국세포주은행, Korea) 을사용하였다. 3) 시약및기기 (1) 시약본실험을위해서 ethyl alcohol(samchun Chemical, Korea), methyl alcohol(samchun Chemical, Korea), DMSO(Sigma, USA), DMEM(Sigma, USA), 1 phosphate buffered saline(pbs, Sigma, USA), EDTA(Sigma, USA), acetic acid(sigma, USA), isopropanol(sigma, USA), trypsin-edta(sigma, USA), NO assay kit (Oxford Biomedical Research, USA) 및 Bio-Plex cytokine assay kit(panomics, USA) 등이사용되었다. (2) 기기본실험에사용된기기는 CO 2 incubator (NUAIRE, USA), pulverizer(rong tsong, Taiwan), rotary vacuum evaporator(eyela, Japan), air compressor(tamiya, Japan), homogenizer(omni, USA), research microscope (Becton dickinson, USA), centrifuge(hanil, Korea), fume hood(hanil, Korea), clean bench(jeio thec, Korea), ultrasonic cleaner(branson, USA), microplate reader(bio-rad, USA), thermo aluminum bath(fine PCR, USA), vortex mixer(vision Scientific Co, Korea), water bath(intron biotech, Korea), ice-maker (Vision Scientific Co, Korea) 및 Bioplex-2(Bio-rad, USA) 등이다. 2. 방법 1) ACE의제조獨活 5 g을정확하게측정하고, 환류추출기에 1 차증류수 2, ml와함께넣어끓는시점으로부터 2시간동안가열추출한다음추출액을 filter paper (Advantec No.2, Japan) 로감압여과하고이여과액을 rotary vacuum evaporator를이용하여농축액을얻었다. 이농축액을동결건조기를이용하여건조한분말을시료로사용하였고, 동결건조추출물은 11.46 g을얻었으며, 수율은 22.91% 이었다. 2) 세포배양 Raw 264.7 cells은 37, 5% CO 2 조건에서 1% fetal bovine serum(fbs), penicillin(1 U/ ml ) 및 streptomycin(1 μg / ml ) 등이첨가된 DMEM 배지로배양되었다. Cells은 75 cm2 flask(falcon, USA) 에서충분히증식된후배양 3일간격으로배양세포표면을 PBS 용액으로씻어준뒤 5 ml flask 당 1 176

이종한외 2 인 : 독활물추출물이대식세포면역활성에미치는영향 (177) ml의.25% trypsin-edta용액을넣고실온에서 1 분간처리한다음 trypsin용액을버리고 37 에서 5 분간보관하여세포를탈착하여계대배양하였다. 탈착된세포는 1% FBS가첨가된 DMEM 배양액 1 ml에부유시킨다음새로운배양용기 (5 ml culture flask) 에옮겨 1:2의 split ratio로 CO 2 배양기 (37, 5% CO 2) 에서배양하였다. 3) 세포독성검사 (MTT assay) Raw 264.7 cells에나타내는세포독성유발효과를알아보기위하여 Mosmann 등 21) 의방법을응용하여 MTT assay를실시하였다. 96 well plate에 1 1 4 cells/well의 cell을 1 μl씩넣고 37, 5% CO 2 incubator에서 24시간동안배양한후배지를버리고배양세포표면을 1 PBS 용액으로씻어주었다. 같은양의배지와 PBS에녹인, 25, 5, 1, 2 및 4 μg / ml등의시료를각 well에처리하고 24 혹은 48시간동안배양하였다. 배양이끝난후 PBS에녹인 1 μg / ml MTT(Sigma, USA) 를 1 μl씩각 well에처리하여알루미늄호일로차광시킨뒤 2 시간동안같은조건에서배양하였다. 배양액을모두제거한후 DMSO를 1 μl처리하고 37 에서 2시간방치다음 microplate reader를이용하여 49 nm에서흡광도를측정하였다. cell viability는다음공식으로계산되었다. Viability(%) = 1 AT / AC AC - absorbance of control. AT - absorbance of tested extract solution. 4) NO 생성측정세포로부터생성되는 NO의양은 Weissman 등 22-24) 의방법을응용, 세포배양액중에존재하는 NO를 Griess 시약을이용하여측정하였고, NO의농도를추정하기위해 microplate reader를이용, 54 nm에서흡광도를측정하여 NO의생성정도를비교하였다., 25, 5, 1, 2 및 4 μg / ml등의다양한시료를배지에담아각 well에처리하고 24 혹은 48 시간동안 37, 5% CO 2 incubator에서배양한후세포배양상등액 6 μl을채취하여여기에 Griess 시약 1 μl을혼합하여 15분동안반응시킨뒤 microplate reader를이용하여 54nm에서흡광도를측정하였다. 세포의 NO 생성은다음공식으로계산되었다. Productions of NO(%) = 1 AT / AC AC - absorbance of control. AT - absorbance of tested extract solution. 5) Cytokine 생성측정면역단백질분비와관련된시료의영향을알아보기위해 Politch 등 25) 의방법을응용하여다음과같이실험을시행하였다. 96 well plate에 1 15 cells/ ml의 cell을 1 μl씩넣고 37, 5% CO 2 incubator 에서 24 시간동안배양한후배지를버리고배양세포표면을 1 PBS 용액으로씻어준뒤각 well에, 5, 1 및 2 μg / ml등의다양한농도의시료와함께배지에담아처리하고 24시간동안배양하였다. 배양이끝나면상등액을채취하여 Bio-Plex Suspension Assay System 을이용, Quantitative Multiplexed Cytokine/Chemokine Assay를실시하여 IL-1β, IL-6 및 TNF-α 등의마우스대식세포 cytokine 생성에대한시료의영향을계산, 비교하였다. 3. 통계처리본실험에서얻은결과에대해서는 mean ± SD로나타냈으며, 대조군과각실험군과의평균의차이는 Student s t-test로분석하여 p-value가.5 미만일때통계적으로유의한차이가있는것으로판정하였다. 결과 1. 세포생존율의변화 ACE가마우스대식세포생존율에미치는영향을비교한결과 24시간처리시생존율의변화는없었다 (Table 1, fig. 1). 177

(178) 대한한의학회지제 32 권제 1 호 (211 년 1 월 ) Table 1. Effects of ACE on Cell Viability in RAW 264.7 Cells for 24 hr Incubation. Table 2. Effects of ACE on Cell Viability in RAW 264.7 Cells for 48 hr Incubation. ACE Concentration( μg / ml ) Cell Viability(% of normal) ACE Concentration( μg / ml ) Cell Viability(% of normal) Normal 1. ± 15.47 25 99.58 ± 11.43 5 91.82 ± 1.8 1 97.7 ± 11.5 2 97.56 ± 1.78 4 14.3 ± 13.96 Normal : Not treated with ACE. Normal 1. ± 8.88 25 89.42 ± 8.72 5 95.25 ± 9.92 1 83.85 ± 8.77 2 75.96 ± 9.21 4 72.17 ± 7.26 p <.5 compared to normal. Cell Viability (% of normal) 14 12 1 8 6 4 2 Normal 25 5 1 2 4 Fig. 1. Effects of ACE on cell viability in RAW 264.7 cells for 24 hr incubation. 48시간처리시 25, 1, 2 및 4 μg / ml등의농도군에서유의한감소를보였다 (Table 2, fig. 2). 2. NO 생성량의변화 ACE가마우스대식세포의 NO 생성에미치는영향을비교한결과 24시간처리시 5, 1, 2 및 4 μg / ml등의농도군에서유의한증가를보였다 (Table 3, Fig. 3). 48시간처리시모든농도군에서유의한증가를보였다 (Table 4, Fig. 4). Cell Viability (% of normal) 12 1 8 6 4 2 Normal 25 5 1 2 4 Fig. 2. Effects of ACE on cell viability in RAW 264.7 cells for 48 hr incubation. p <.5 compared to normal. 3. IL-1β 생성량의변화 ACE가마우스대식세포의 IL-1β 생성에미치는영향을비교한결과 24시간처리시 1, 2 μg / ml등의농도군에서유의한증가를보였다 (Table 5, Fig. 5). 4. IL-6 생성량의변화 ACE가마우스대식세포의 IL-6 생성에미치는영향을비교한결과 24시간처리시 2 μg / ml의농도군에서유의한증가를보였다 (Table 6, Fig. 6). 178

이종한외 2 인 : 독활물추출물이대식세포면역활성에미치는영향 (179) Table 3. Effects of ACE on NO Production of RAW 264.7 Cells for 24 hr Incubation. Table 4. Effects of ACE on NO Production of RAW 264.7 Cells for 48 hr Incubation. ACE Concentration( μg / ml ) NO production(% of normal) ACE Concentration( μg / ml ) NO production(% of normal) Normal 1. ± 1.74 25 99.56 ± 1.96 5 111.8 ± 2.55 1 127.89 ± 2.42 2 136.5 ± 4.22 4 142.1 ± 3.4 p <.5 compared to normal. Normal 1. ± 4.73 25 16.96 ± 5.53 5 142.83 ± 9.13 1 211.85 ± 19.9 2 292.61 ± 16.2 4 34.43 ± 18.55 p <.5 compared to normal. NO production (% of normal) 16 14 12 1 8 6 4 2 Normal 25 5 1 2 4 NO production (% of normal) 4 35 3 25 2 15 1 5 Normal 25 5 1 2 4 Fig. 3. Effects of ACE on NO production of RAW 264.7 cells for 24 hr incubation. p <.5 compared to normal. Fig. 4. Effects of ACE on NO production of RAW 264.7 cells for 48 hr incubation. p <.5 compared to normal. 5. TNF-α 생성량의변화 ACE가마우스대식세포의 TNF-α 생성에미치는영향을비교한결과 24시간처리시모든농도군에서유의한증가를보였다 (Table 7, Fig. 7). 고찰인체가자신의조직을손상시키려는유기물이나독소에대해방어하는능력을면역이라하고, 최근 부인과에서는종양의발생기전, 산후질환및갱년기질염치료등과관련하여면역에대한관심이증가하는추세이며 1), 특히한약이생체활성조절물질로서면역과관련된치료제로활용여부에대한관심도많다. 韓醫學에서는 正氣在內邪不可干, 邪氣所湊其氣必虛 2) 라고하여, 發病機制를 邪正鬪爭 으로보았고, 正氣와邪氣의투쟁과정에서正氣가虛하여적응력과항병력이감소하면邪氣의침입이용이해져질병이발생한다고보았다 26). 이와더불어한의 179

(18) 대한한의학회지제 32 권제 1 호 (211 년 1 월 ) Table 5. Effects of ACE on IL-1β Production of RAW 264.7 Cells. ACE Concentration( μg / ml ) IL-1β production(pg/ ml ) Normal 4.3 ±.5 5 4.8 ±.5 1 7. ±.82 2 15.4 ± 2.69 p <.5 compared to normal. Table 6. Effects of ACE on IL-6 Production of RAW Cells. ACE Concentration( μg / ml ) IL-6 production(pg/ ml ) Normal 395.8 ± 74.47 5 418.9 ± 56.87 1 469.9 ± 11.47 2 1237.5 ± 215.88 p <.5 compared to normal. IL-1 production ( g/ml) 2 18 16 14 12 1 8 6 4 2 Normal 5 1 2 IL-6 production ( g/ml) 16 14 12 1 8 6 4 2 Normal 5 1 2 Fig. 5. Effects of ACE on IL-1β production of RAW 264.7 cells. p <.5 compared to normal. 학적치료법중 正虛者扶正, 邪實者祛邪 라는치료원칙은면역학적입장에서正氣와邪氣의상황을동시에고려한좋은치료방편이었음을입증하는연구가많이보고되었다 3,27). 扶正法은인체의안정성을증강시키기위한항병력조절과면역효능을높이는치료방법으로볼수있는반면, 祛邪法은면역효능감소의원인을제거하는치료법으로볼수있는데, 正氣虛弱과면역기능저하의상관성, 祛邪法를통한면역균형의도모및扶正과祛邪의비율조절을통한면역저하와면역과민의치료법선택등의연구들이기존에보고된바있다 26,28). 또다른연구들을살펴보면扶正의방법은면역반응을촉진시켜益衛氣 Fig. 6. Effects of ACE on IL-6 production of RAW cells. p <.5 compared to normal. 補元氣養血氣益肺健脾補腎機能을포괄하고, 祛邪의방법은면역반응을억제하며, 祛散風邪淸熱解毒活血化瘀滌痰化濁등을포괄한다 29) 고도보는한편약물분류중補氣補血補陰補陽하는약물들은대부분면역강화작용이있으며, 實證에사용하는祛風除濕淸熱解毒活血化瘀毒性攻堅의효능이있는약물은대부분면역억제작용이있다는보고도있었다 28). 獨活은오가과 ( 五加科두릅나무과 Araliaceae) 에속한독활 Aralia continentalis KITAGAWA(Aralia cordata THUNB.) 의뿌리를건조하여사용하며 3), 정유성분 1~2%, 스테아린산.7%, 수지, 살리실산과, 디테르펜산, 구리, 망간및니켈등이함유되 18

이종한외 2 인 : 독활물추출물이대식세포면역활성에미치는영향 (181) Table 7. Effects of ACE on TNF-α Production of RAW 264.7 Cells. ACE Concentration( μg / ml ) TNF-α production(pg/ ml ) Normal 14.3 ± 3.4 5 263. ± 47.45 1 97.5 ± 125.75 2 2216.3 ± 411.7 p <.5 compared to normal. TIIF- production ( g/ml) 3 25 2 15 1 5 Normal 5 1 2 Fig. 7. Effects of ACE on TNF-α production of RAW 264.7 cells. p <.5 compared to normal. 어있다 31). 性은溫無毒하고, 味는辛苦하며 3), 辛溫發散, 祛風勝濕하는效能으로熟地黃, 當歸, 川芎, 續斷, 杜沖, 白芍藥및桑寄生등을配合하여부인과임상에서는産後身痛과下肢痛의치료에사용해왔다 4). 강활 ( 羌活 ) 과비교하여羌之氣淸行氣而發散營衛之邪, 獨之氣濁行血而溫養營衛之氣, 羌有發表之功, 獨有助表之力 7) 이라하여, 독활에대한기존연구는독활의항염증작용 32), 항균활성에대한연구 33) 외다수 34-46) 의연구는있었지만독활의면역활성에대한연구는없었다. 위에서살펴본正氣와邪氣의투쟁관점은오늘날면역이론과유사한개념으로보이며, 한의학의 正氣와면역조절작용에대한연구도있었다 47-49). 또한한의학에서正氣는면역기능에대한조절로해석할수있다고했다 3). 그래서저자는독활의辛溫發散, 祛風勝濕의작용을인체의질병에대한邪正鬪爭에서正氣를도와서邪氣를내치는효능으로생각하고, 독활이대식세포와관련된효소에어떤영향을미치는지관심을가지게되었다. 본연구에서는獨活을물추출하여얻은시료 ACE로마우스대식세포를이용하여 cell viability, NO 생성, IL-1β, IL-6 및 TNF-α 등의 cytokine 생성에미치는영향을조사하였다. ACE의마우스대식세포의생존율에미치는영향을확인하기위하여 MTT assay를수행한결과 24 시간동안배양한경우에는유의한변화가나타나지않았으나, 48시간의배양에서는 25, 1, 2 및 4 μg / ml등의농도군에서유의한감소를나타내었다. NO는일종의세포신호전달물질로 NO 합성효소 (nitric oxide synthase; NOS) 에의해체내에서생산되고, NOS에의해만들어진 NO는혈압을감소시키는혈관확장과혈소판활동을조절하며, 면역반응에도기여함으로써신경전달조절과세포분화에관여하고있으며새로운염증매개물질로주목하고있다 5). ACE는 24시간의배양에서는 5 μg / ml이상의농도군에서, 48시간의배양에서는 25 μg / ml이상의모든농도군에서대식세포의 NO의생성을유의하게증가시켰다. IL-1β 는 TNF-α와그기능이유사하며, 적절히발현되면생체방어기능으로서 B림프구와 T림프구의증식과분화를촉진시키며항염증반응, 항종양작용및조혈작용등을하나, 과잉또는장기적인생성이일어나게되면생체침습작용으로발열반응, 염증, 조직파괴, 쇼크등을일으킬수도있다 5). ACE 는 24시간의배양에서마우스대식세포의 IL-1β 생성을 1, 2 μg / ml의농도군에서유의하게증가시켰다. IL-6은 T림프구, B림프구, macrophage, 골수기질세포, 섬유아세포및내피세포등에서기원하며, 181

(182) 대한한의학회지제 32 권제 1 호 (211 년 1 월 ) T림프구와 B림프구기능을조절하여생체내염증의초기반응과조혈에작용하며, 면역반응과골흡수에도관여한다 5). ACE는 24시간의배양에서마우스대식세포의 IL-6생성을 2 μg / ml의농도군에서유의하게증가시켰다. TNF-α는활성화된 macrophage, fibroblast 등에의해분비되는 cytokine으로염증반응을매개하는데, neutrophils, endothelial cell 및 B세포등을자극하여 chemokines, prostaglandins, proteases 및 growth factors 등을분비하도록하며많은양의 cytokine이분비되면패혈성쇼크가발생하기도한다 5). ACE는 24시간의배양에서마우스대식세포의 TNF-α 생성을 5, 1 및 2 μg / ml등의농도군에서유의하게증가시켰다. 이러한결과는 ACE가대식세포의 NO, IL-1β, IL-12 및 IL-6 등의면역반응의매개체를증가시키며, 이러한면역매개인자를이용한침입성병원체및노화세포잔존물제거등의대식세포면역활성을증진시키는효능이있다고볼수있다. 이는한의학적인正氣와邪氣의개념으로볼때獨活은正氣를도와서邪氣를내쳐인체내면역기능의개선에도움이될수있을것으로보인다. 결론본연구에서는獨活을물추출하여얻은시료 ACE로마우스대식세포를 24시간, 48시간배양하여 cell viability와 NO생성을관찰하였고, 24시간배양하여 IL-1β, IL-6 및 TNF-α 등의 cytokine 생성에미치는영향을조사하여다음과같은결론을얻었다. 1. 세포생존율을측정한결과 24시간배양한군에서는유의한변화가없었고, 48시간배양한군에서는 25, 1, 2 및 4 μg / ml등의농도에서유의한감소를보였다. 2. 마우스대식세포의 NO 생성은 24시간배양한군에서는 5, 1, 2 및 4 μg / ml등의농도에서유의한증가를보였다. 3. 마우스대식세포의 NO 생성은 48시간배양한 군에서는모든농도에서유의한증가를보였다. 4. 마우스대식세포의 IL-1β 생성은 1, 2 μg / ml의농도군에서유의한증가를보였다. 5. 마우스대식세포의 IL-6 생성은 2 μg / ml의농도군에서유의한증가를보였다. 6. 마우스대식세포의 TNF-α 생성은 5, 1 및 2 μg / ml등의농도군에서유의한증가를보였다. 이러한결과는 ACE가대식세포의 NO, IL-1β, IL-6 및 TNF-α 등의면역매개인자의생성을촉진함으로써, 생성된면역매개인자를이용한침입성병원체및노화세포잔존물제거등의대식세포면역활성을증진시키는효능이있을것으로보인다. 감사의글 이연구는 21년도경원대학교지원에의한결과임. 참고문헌 1. 대한산부인과학회교과서편찬위원회. 부인과학. 2nd. Seoul: 칼빈서적. 1997;132. 2. 陳夢雷等. 醫部全錄. 北京 : 人民衛生出版社. 1982;1 冊 : 894, 32. 3. 문준전등. 동의병리학. 서울 : 고문사. 199;78-86. 4. 임은미. 여성본초학. 서울 : 전국의학사. 25;165-6. 5. 신풍문편집부. 신편중약대사전. 臺北 : 신풍문출판공사. 1982;2534-8. 6. 송병기. 한방부인과학. 서울 : 행림출판. 199;17-8. 7. 한의부인과학교재편찬위원회. 한의부인과학. 서울 : 정담. 22;3. 8. Lee EH, Kim TH. Effects of Boheo-tang and Bohe-tang plus Cervi Pantotrichum Cornu: on Immune Response in Postpartum mice. The journal of Oriental OB&GY. 29;22(3):25-36. 9. Ko JM, Choe CM, Cho HB, Kim SB. Antiarthritis Effect of Jeonsaenghwalhyeoltanggamibang through Immune Modulation. The journal of 182

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