본초학표지

大韓本草學會誌제 29 권제 4 호 (2014 년 7 월 ) Kor. J. Herbology 2014;29(4):53-59 ISSN 1229-1765(Print), ISSN 2288-7199(Online) http://dx.doi.org/10.6116/kjh.2014.29.4.53. 川芎茶調散加味方주정추출물이산화적손상및염증에미치는영향 박지원 1#, 심부용 1, 이해진 2, 김동희 1* 1 : 대전대학교한의과대학병리학교실, 2 : 대전대학교미용의학 Effect of Cheongungdajosan-gamibang on oxidative damage and inflammation Ji Won Bak 1#, Boo-Yong Sim 1, Hae jin Lee 2, Dong Hee Kim 1* 1 : Department of Pathology, College of Oriental Medicine, Daejeon University, 2 : Department of College of Beauty & Health Medicine, Daejeon University ABSTRACT Objectives : The purpose of this study was verification of the anti-inflammation and anti-oxidant effect of Cheongungdajosan-gamibang extract (CG) in mouse macrophage, RAW 264.7 cells. Methods : We have basically using LPS-stimulated RAW 264.7 cells. The cell toxicity was determined by MTT assay. To evaluate the anti-inflammatory effect of Cheongungdajosan -gamibang, amount of nitric oxide(no) was measured using the NO detection kit and the IL-1β, IL-6 and TNF-αexpression was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Also, free radical scavenging assay has tested for DPPH and ABTS radical activity as well as the contents of total polyphenol. Results : In this study, 96.6% or higher cell viability was observed in all tested groups from 1, 10, 100 μg / ml in RAW 264.7 cells. The RAW 264.7 cells were induced by lipopolysaccharide (LPS) and CG 1, 10, 100 μg / ml. The CG decreased nitric oxide (NO) production activity dose dependently, especially at 100 μg / ml of 55%. The production of IL-1β, IL-6 and TNF-αwere decreased by 51%, 78% and 35% in CG treated 100 μg / ml. CG showed dose-dependent suppression activity of reactive oxygen species (ROS) production, especially at 100 μg / ml of 37%. DPPH radical scavenging activity and ABTS cation decolorization were activated over 86% and 88% in CG at 1,000 μg / ml concentration. Conclusions : According to the results, we thought that CG showed anti-inflammatory and antioxidant activities on the RAW 264.7 cells in mouse macrophage. Therefore, this research is expected to provide the fundamental data about the natural material analysis of relating to the anti-inflammation and antioxidant. Key words : Cheongungdajosan-gamibang, Anti-oxidant, Anti-inflammatory, Free radical, Cyotokine 서론 1) 인체에는산화촉진물질과산화억제물질이균형을이루고있는데여러가지요인에의해그균형이무너지게되면산화적스트레스를일으키게되며점차세포손상과병리적질환을야기키시게된다. 산화적스트레스의직접적인원인이되는활성산소종과활성질소종은인체가활동하기위한에너지공급대사과정에서이루어지는생화학적산화반응중에발생이된다 1). 일반적으로노화, 발암, 심장병및순환계등의문 제를억제하기위해 BHT (butylated hydroxy toluene), BHA (butylated hydroxy anisole), PG(propylgallate), TBHQ (tertiaty butylhydroquinone) 등이경제적이고, 우수한항산화제로써널리사용되고있으나, 복용기간이길어지면간, 폐, 신장, 위장및순환계에심각한부작용을초래한다 2,3). 염증은체내에서외부로부터의물리적, 화학적자극이나세균의감염으로인해손상된조직을수복하거나재생하려는생체방어기전의하나이기도하지만, 각종질환을일으키는 * 교신저자 : 김동희. 대전광역시동구대학로 62 대전대학교한의과대학 Tel : 042-280-2623 FAX : 042-280-2624 E-mail : dhkim@dju.kr # 제 1 저자 : 박지원. 대전광역시동구대학로 62 대전대학교한의과대학 Tel : 042-280-2828 FAX : 042-280-2624 E-mail : jiwon3240@naver.com 접수 :2014 년 6 월 27 일 수정 :2014 년 7 월 18 일 채택 :2014 년 7 월 23 일

54 大韓本草學會誌 Vol. 29 No. 4, 2014 원인이되기도한다. 여러자극에의해과량생산된염증매개인자는 tumor necrosis factor-α(tnf-α), interleukin 1β(IL-1β) 등과같은사이토카인을생산하여다양한염증반응을일으킨다 4). 염증반응이만성적으로일어나면염증매개물질이과도하게분비되어암세포의성장촉진, 인슐린의저항성증가, 동맥경화의악화등다양한병리학적기전에관여한다고알려져있다 5). 川芎茶調散加味方의기본방인川芎茶調散은宋代陳 6) 의 太平惠民和劑局方 에최초로수록된처방이며, 元代危 7) 의 世醫得效方 에는茶調散으로기재되어있다. 그처방은川芎, 荊芥, 甘草, 防風, 白芝, 薄荷, 細辛, 羌活 8종의약재로구성되어있고, 外感風邪로인한頭痛, 傷風壯熱, 目眩鼻塞, 鼻塞聲重등의증상에많이사용되는처방이며, 임상에서는증상의경증, 중증도에따라원방에다양한한약재를가감하여활용하고있다 6,7). 기존의川芎茶調散에대한실험적연구로는전 8) 의 ' 高血壓및高脂血症에대한川芎茶調散의實驗的硏究 ', 주 9) 의 ' 川芎茶調散의鎭痛效果및作用機轉에관한硏究 ' 등이있으나, 川芎茶調散加味方의항산화와항염증효과에대한연구는보고된바없다. 항산화활성을지닌천연원료들은염증성질환에도효과를보인다고알려져있어 10), 이에저자는원방인川芎茶調散에홍 11) 의연구에서항산화효능이입증된화피를가미하여川芎茶調散加味方의항산화효능과항염증효능을검증하고자하였다. 1. 약재 재료및방법 본실험에사용한천궁다조산가미방 (Cheongungdajosan-gamibang 이하 CG로표기 ) 의구성약재들은 옴니허브에서구입하였고, 대전대학교지역혁신센터난치성면역질환의동서생명의학연구센터 (TBRC) 에서정선후사용하였다. 그내용과분량 (1첩) 은다음과같다 (Table 1). Table 1. The Prescription of CG Herbal medicine name Pharmacognostic name Weight(g) 川芎 Cnidium officinale Makino 12 荊芥 Schizonepeta tenuifolia var. japonica 12 甘草 Glycyrrhiza uralensis 6 防風 Ledebouriella seseloides 6 白芷 Angelica dahurica 6 樺皮 Betula platyphylla Suk. var. japonica Hara 6 辛夷 Magnolia denudata Desrousseaux 4 薄荷 Mentha arvensis var. piperascens 3 細辛 Asiasarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa 3 Total amount 58 2. 세포배양 실험에사용된 RAW 264.7 세포는한국세포주은행에서구입하였고, 10% fetal bovine serum (FBS) 와 1% penicillin 으로조성된 DMEM 배지 1 ml을넣어부유시켜세포배양기 (37, 5% CO 2) 에서배양하였다. 계대배양횟수는 5회이상으로하였고, 시료들을처리하기전에 24시간을적응시켰다. 3. 시약및기기사용된시약은 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), penicillin (Hyclone, Co., U.S.A.), 1,1-diphenyl-2-picryl-hydrazyl (DPPH) 는 Sigma-Aldrich 사 (st. Louis, MO, USA), Folin-Ciocalteu's phenol reagent 는 Merck사 (Darmstadl, Germany), mouse cytokine milliplex map immunoassay kit (Millipore Co., U.S.A.) 을사용하였으며, 기기는 rotary vacuum evaporator (Büchi B-480 Co., Switzerland), freeze dryer (EYELA FDU-540 Co., Japan), spectrophotometer (Shimadzu Co., Japan), ELISA reader (Molecular Devices Co., U.S.A.), Flow cytometry system (BD Biosciences immunocytometry systems, U.S.A.), 주정추출에사용된주정은 주정판매월드 (Jeonju, Korea) 에서구입하여사용하였다. 4. 시료추출 CG 2첩에 80% 주정 (C 2H 5OH) 1000 ml을넣고 3시간동안환류추출후여과액을얻어 rotary vacuum evaporator 에서감압농축하였다. 농축된용액을 freeze dryer로동결건조하여분말 9.4 g을얻었으며, 얻어진분말은초저온냉동고 (-80 ) 에서보관하면서실험에따라필요한농도로증류수에희석하여사용하였다. 5. 세포독성측정 Raw 264.7 세포는 96 well plate에 2 10 4 cells/well로분주하여 24시간동안배양하였다. 새로운배양액으로교체하였고, CG를각각 1, 10, 100 ( μg / ml ) 의농도로처리하여다시 24시간동안배양하였다. 배양후 10 μl의 WST solution 을첨가하여세포배양기 (37, 5% CO 2) 에서 30분간반응시켰다. 반응후 450 nm에서 ELISA reader기를이용해흡광도의변화를측정한후대조군에대한세포생존율을백분율로표시하였다. 6. 항산화효능측정 1) 총폴리페놀함량측정총폴리페놀함량은 Folin-Ciocalteu 시약을이용하는방법으로측정하였다. 추출시료용액 1 ml에 50% Foiln-Ciocalteu's phenol reagent 0.5 ml를가하여실온에서 3분간반응시켰다. 반응용액에 Na 2CO 3 포화용액 1 ml와 7.5 ml증류수를차례로혼합하여 30분간정치시킨뒤, 12,000 rpm에서 10 분간원심분리한후상등액을취해 760 nm에서흡광도를측정하였다. 총폴리페놀함량은 gallic acid를표준물질로이용

川芎茶調散加味方주정추출물이산화적손상및염증에미치는영향 55 하여작성한검량선에따라함량을구하였으며측정단위로는 GAE(Gallic acid equivalent)/g 을사용하였다. 2) 1,1-diphenyl-2-picryl hydrazyl(dpph) radical 소거능측정자유라디칼소거활성실험은안정한자유라디칼 DPPH를사용하는방법으로주정에용해시킨 0.2 mm의 DPPH 용액 150 μl와 CG 1, 10, 100, 1,000 ( μg / ml ) 의농도가되게 100 μl씩각각혼합하고, 37 에서 30분간반응시킨후, 517 nm에서흡광도를측정하였다. 대조군은시료액대신증류수을넣었으며, DPPH 용액대신주정을넣어보정값을얻었다. 자유라디칼소거율은아래의식에따라계산하였다. 다시 24시간동안배양하였다. N1 buffer와 N2 buffer를각각 10분간반응후 540 nm에서흡광도를측정하였다. Nitrite standard 의농도별표준곡선을이용하여배양액의 NO 농도를결정하였다. 2) Cytokine 생성량측정 Raw 264.7 cells을 12 well plates에 2 10 5 cells/ ml이되도록분주하고, 24시간동안배양한후, CG를 1, 10, 100 ( μg / ml ) 의농도로처리하고, LPS 1 μg / ml을처리하였다. 24 시간동안배양한후세포배양액을수거하여배양액에함유된 IL-1β, IL-6, TNF-α 를 custom-made 4-plex cytokine Milliplex panel을이용하여측정하였다. 소거율 (%) = ( 대조군의흡광도 - 시료첨가군의흡광도 ) 100 대조군의흡광도 3) 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(abts) radical 소거능측정 ABTS assay 방법은기존에보고된방법을 96 well plate 에맞게수정하여실시하였다. CG를최종농도가 1, 10, 100, 1,000 ( μg / ml ) 의농도로될수있게희석시켰으며, ABTS 용액은 7.4 mm ABTS (2,2 azino bis- (3-ethylbenzothiazoline-6-sμlfonic acid)) 와 2.6 mm potassium persulphate 를제조한후, 암소에하루동안방치하여양이온 (ABTS +) 을형성시킨다음 734 nm에서흡광도를측정하여흡광도값이 0.7±0.03 이되게희석하였다. 희석된 ABTS + 용액 150 μl와 CG 추출물을각각 5 μl혼합하고, 실온에서 10분간반응시킨후, 734 nm에서흡광도를측정하였다. 항산화능은증류수를대조군으로하여대조군에대한 ABTS 라디칼소거능을백분율로나타내었다. 8. 통계처리실험결과는실험결과는 SPSS 11.0의 unpaired student's T-test 및 ANOVA test를사용하여통계처리하였으며 p<0.05, p<0.01 및 p<0.001 수준에서유의성을검정하였다. 결과 1. 세포독성 RAW 264.7 세포의세포생존율을측정한결과, 대조군을 100.0±7.4% 로나타냈을때, CG 투여군 1, 10, 100 ( μg / ml ) 농도에서 100.6±5.3%, 99.4±7.8%, 96.6±5.0% 의세포생존율을나타내었다 (Fig. 1). 소거율 (%) = (1- 시료첨가군의흡광도 ) 100 대조군의흡광도 4) 세포내 ROS 측정 Raw 264.7 세포에서 reactive oxygen species (ROS) 를측정하기위하여 12 well plate에 Raw 264.7 세포를 2 10 5 cells/well 이되게분주하여 24시간동안배양하였다. 배양후새로운배양액으로교체하였고 CG를각각 1, 10, 100 ( μg / ml ) 의농도와 LPS 1 μg / ml의농도를처리하여다시 24시간동안배양하였다. 배양후, 세포를 DCF-DA로염색한후유세포분석기를이용하여형광강도의세기에따른변화를분석하였다. 7. 항염증효능측정 1) Nitric oxide(no) 측정 NO의농도는배양액내의 nitric oxide 농도를 griess reagent system을이용하여측정하였다. Raw 264.7 세포를 96 well plate에 2 10 4 cells/well 로분주하여 24시간동안배양한후새로운배양액으로교체하였고 CG를각각 1, 10, 100 ( μg / ml ) 의농도와 LPS 1 μg / ml의농도를처리하여 Fig. 1. Cell viability of CG extract in Raw 264.7 cells. Cells were treated with 1, 10, 100 ( μg / ml ) of CG for 24 hours. Cell viability was determined using the WST assay. The results were expressed as mean ± S.D. 2. 항산화효능에미치는영향 1) 총폴리페놀함량 CG 주정추출물에존재하는총폴리페놀함량을 gallic acid 를표준물질로하여측정한결과, Table Ⅱ와같이총폴리페놀함량은 39.9 ± 1.0 mg GAE (Gallic acid equivalent)/g 의수치를나타내었다 (Table 2). Table 2. Total phenolic contents of ethanol extract from CG. Samples Total phenolics ( mg GAE 1) /g ext.) CG-ethanol ext. 39.9 ± 1.0

56 大韓本草學會誌 Vol. 29 No. 4, 2014 1) Total phenolic contents was expressed as milligram of gallic acid equivalent (GAE) per gram of extract. 2) 1,1-diphenyl-2-picryl hydrazyl(dpph) radical 소거능에미치는영향 CG 주정추출물의 DPPH 소거율은 1, 10, 100, 1000 ( μg / ml ) 농도에서 5.6 ± 0.4%, 11.8 ± 2.4%, 43.6 ± 4.9%, 86.0 ± 3.8% 로나타나, 농도의존적으로증가함을나타내었다 (Fig. 2). Fig. 4. Effects of CG extract on the ROS production in Raw 264.7 cells. The Raw 264.7 cells were stimulated with LPS and treated with medium, CG (1, 10, 100 μg / ml ) for 24 hours. The ROS production was analysed following incubation with DCFH-DA by flow cytometry. The results are expressed as mean ± S.D (Significance of results, *** p < 0.001). 3. 항염증효능에미치는영향 Fig. 2. DPPH free radical scavenging activity of CG extract at various concentration. Extracts were incubated with DPPH solution at 37 for 30 mins. Activities were determined by measurement of absorbance at 517 nm. The results were expressed as mean ± S.D. 1) Total Nitric oxide(no) 생성에미치는영향 CG 주정추출물의 NO 생성량은대조군을 100±5.2% 로나타냈을때, 정상군은 5.8 ± 2.2%, CG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 87.0 ± 7.2%, 87.8 ± 6.1%, 45.2 ± 4.5% 로나타나, 대조군에비해 100 μg / ml농도에서유의성있는 ( *** p < 0.001) 감소를나타내었다 (Fig. 5). 3) 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(abts) 소거능에미치는영향 CG 주정추출물의 ABTS 소거율은 1, 10, 100, 1000 ( μg / ml ) 농도에서 0.2 ± 2.3%, 1.9 ± 2.4%, 31.8 ± 1.5%, 87.7 ± 3.7% 로나타나, 농도의존적으로라디칼소거능이증가함을나타내었다 (Fig. 3). Fig. 5. Effects of CG extract on NO production in LPS-stimulated Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of CG for 24 hours. The results are expressed as mean ± S.D (Significance of results, *** p < 0.001). Fig. 3. ABTS free radical scavenging activity of CG extract at various concentration. Extracts were 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 4) ROS 생성저해활성에미치는영향 CG 주정추출물의 ROS 생성저해활성은대조군을 100.0 ± 0.3% 로나타냈을때, 정상군은 42.1 ± 3.7%, CG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 74.2 ± 3.9%, 72.4 ± 2.2%, 63.1 ± 5.2% 로, 대조군에비해농도의존적이며유의성있는 ( *** p < 0.001) 감소를나타내었다 (Fig. 4). 2) IL-1β생성에미치는영향 CG 주정추출물의 IL-1β생성량을측정한결과, 대조군을 100.0 ± 6.9% 로나타냈을때, 정상군이 10.3 ± 11.0%, CG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 94.8 ± 7.2%, 82.7 ± 7.8%, 48.8 ± 0.2% 로대조군에비해농도의존적인감소를나타내었고, 100 μg / ml농도에서유의성 ( *** : p <0.001) 있는감소를나타내었다 (Fig. 6). Fig. 6. Effects of CG extract on LPS-stimulated IL-1βproduction in Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of CG extract for 24 hours. The results are expressed as mean ± S.D (Significance of results, *** p < 0.001).

川芎茶調散加味方주정추출물이산화적손상및염증에미치는영향 57 3) IL-6 생성에미치는영향 CG 주정추출물의 IL-6 생성량을측정한결과, 대조군을 100.0 ± 6.0% 로나타냈을때, 정상군이 3.4 ± 0.8%, CG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 36.6 ± 3.0%, 30.1 ± 2.1%, 22.3 ± 4.9% 로대조군에비해농도의존적이며유의성있는 ( *** : p <0.001) 있는감소를나타내었다 (Fig. 7). Fig. 7. Effects of CG extract on LPS-stimulated IL-6 production in Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of CG extract for 24 hours.. The results are expressed as mean ± S.D(Significance of results, *** p < 0.001). 4) TNF-α 생성에미치는영향 CG 주정추출물의 TNF-α생성량을측정한결과, 대조군을 100.0 ± 1.0% 로나타냈을때, 정상군이 16.3 ± 0.3%, CG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 91.2 ± 1.0%, 79.8 ± 2.0%, 65.2 ± 8.4% 로대조군에비해농도의존적인감소를나타내었고, 100 μg / ml농도에서유의성 ( * : p <0.05) 있는감소를나타내었다 (Fig. 8). Fig. 8. Effects of CG extract on LPS-stimulated TNF-αproduction in Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of CG extract for 24 hours.. The results are expressed as mean ± S.D (Significance of results, * p < 0.05). 고찰 활성산소종 (reactive oxygen species, ROS) 은초과산화이온 (superoxide anion radical : O - 2 ), 과산화수소 (hydrogen peroxide : H 2O 2), 수산화라디칼 (hydroxyl radical : OH) 등이있고, 불안정하며반응성이높아주위의화합물과전자를잃거나얻으려는특성이있다. 또한체내여러생체물질들과쉽게반응하여세포생체막을구성하는성분인불포화지방산에영향을주는데, 이때과산화반응이일어나면서체내에과산화지질을축적하게된다. 이로인해생체기능이저하되어암, 동맥경화, 염증, 당뇨, 노화및순환기장애등과같은여러질환을일으키는원인이된다 12,13). 활성질소종 (reactive nitrogen species, RNS) 은일산화 질소 (nitric oxide : NO), 이산화질소 (nitrogen dioxide : NO 2), 아질산 (nitrous acid : HNO 2), 퍼옥시니트리트 (peroxynitrite : ONOO-) 과같은물질이며, 대식세포, 호중구및다른면역세포들의면역반응으로다량생성되고, 또한활성산소도같이생성된다 14). 산화적스트레스는세포가활성산소에노출되었을때 superoxide dismutase (SOD), catalase 및 glutathionine peroxidase 와같은항산화효소를활성화시켜세포내산화적스트레스를제거하려는방어적역할을하기도한다 15), 그러나과도하게생성된활성산소로부터의손상을막기위해서는항산화제의도움이필요하다. 이에최근천연물질을이용한항산화제에대한연구와개발이활발이이루어지고있고, 항산화효능을가진물질들이뛰어난함염증작용을수반하고있다고보고된바가있으며 16), 한의학적인측면에서도많은연구발표가이루어지고있다 17). 본실험에앞서 CG의 Raw 264.7 세포에대한생존율을관찰한결과, 모든농도에서 96% 이상의세포생존율을나타내어안전성이검증되었다. 페놀성화합물들은연쇄반응과정에서 alkyl radical 이나 alkylperoxy radical 에수소를공여하여 radical 을제거시킴으로써산화를억제하는것으로알려져있다 18). 즉, 폴리페놀의주성분인 flavonoid, tannins 등의총량인총페놀함량은항산화활성에서는중요한인자로작용하며, 총페놀함량이증가함에따라산화억제작용이활발히이루어진다는것을알수있다. 본실험에서 gallic acid를표준물질로하여 CG 주정추출물의총폴리페놀함량을측정한결과, 39.87 mg /g 으로나타났다. DPPH는강한산화력을가진유리라디칼계시약으로, DPPH에의한항산화활성은화합물이 DPPH radical 에전자를공여함으로써전자공여체인항산화제와만나전자를얻어환원이되면서자유기를소거하는활성이다 19). ABTS radical 을이용한항산화능의측정은 potassium persulfate 와의반응에의해생성된 ABTS free radical 이추출물내의항산화물질에의해제거되어 radical 특유의색인청록색이탈색되는것을이용한방법으로 ph에민감하게반응하는 DPPH 실험에비해비교적넓은범위의 ph에적용될수있는항산화측정방법이다 19,20). 본실험에서 DPPH radical 과 ABTS radical 소거효능은모두농도의존적으로증가함을나타내유의적인항산화능을확인할수있었다. 활성산소 (reactive oxygen species, ROS) 는원자나분자가안정적인상태로유지하기하기위해주위에맴도는전자와짝을이루고, 짝을이루지못한전자는다른세포를파괴하여안정적인상태로환원을하려는활성상태가발생하게되는데, 이런활성이강한상태에있는물질들인유리기중하나이다. 유리기가생성이되는원인들로는음식물을섭취하여에너지로전환시키기위해산화와환원이이루러지는과정, 운동으로인한산소섭취량의증가, 고강도운동또는무산소운동으로인한산소공급이적은경우, 운동중에받은충격이나생화학적인반응으로야기되는근육의손상으로인해생기는염증반응, 대사증가와호르몬분비증가등으로알려져있다 21,22). 본실험에서 ROS 생성저해능을측정한결과, 모든농도에서유의적인결과를나타내었다. 선행연구에서라디칼을환원시키거나상쇄시키는능력이클수록높은항산화활성을나타내고, 폴리페놀을함유한천연물은질병과노화의원

58 大韓本草學會誌 Vol. 29 No. 4, 2014 인이되는 ROS와 RNS의감소와연관이있다고알려져있고 23), ROS는생성후적절하게제거되지않아체내에축적이되면단백질이나지질등의손상을야기하고, 활성산소에의한지질과산화물생성으로인해세포가파괴되어각종기능장애를일으킨다. 이러한활성산조종이정상적소멸하지않고잔존하면자유라디칼에의해산화적스트레스를받게됨으로각종질병의원인이된다고알려져있다 24). 이에본연구결과에서도 CG 투여군에서자유라디칼감소능과 ROS 생성억제가서로부합되어나타나, 항산화에대한효능이있음을뒷받침할수있다. 염증반응에있어서항산화작용을연구는중요하다. 과생산되어조절이불가능한 reactive oxygem species(ros) 는정상적인세포에산화적스트레스로작용한다. 이러한 ROS는일차적으로산화적스트레스뿐만아니라염증과관련된 nitric oxide(no), interleukin-1β(il-1β), interleukin-6 (IL-6), tumor necrosis factor-α(tnf-α) 와같은염증성 cytokine 을자극하여과발현한다고알려져있어, 항산화와항염증은상호연관되어있다고할수있다 25). 그람-음성균의세포외막에존재하는 lipopolysaccharide(lps) 는대표적인내독소중의하나로서, Raw 264.7과같은 macrophage와 monocyte에서 interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α(tnf-α) 와같은염증성 cytokine 의분비를촉진하며 inos의발현을유도하여 NO를생성한다고알려져있다. NO는인체에서다양한생리활성을나타내는인자이기도하지만과다생산되면활성산소와결합하여, 과산화질소등의다른독성유리기물질을만들어내어염증질환을유발시키거나악화시키는인자로작용한다 26,27). 이에 NO 소거능을관찰한결과, 100 μg / ml농도에서만유의적인감소를나타내었다. IL-1β는외부항원을감지한대식세포에서분비하는비특이염증반응을개시하는초기면역반응물질로써 LPS나 TNF-α와같은다른사이토카인에의해유도가되며, 대식세포, 호중구등에의해생산이되는염증유발사이토카인이다. 이런 IL-1β가과도하게생성되면감염성질환이나자가면역질환등을유발한다 28). 본실험에서 IL-1β분비량억제능은 100 μg / ml농도에서만유의적인감소를나타내었다. IL-6는염증반응시분비되는친염증성사이토카인으로단핵구나대식세포에서분비되며림프구를활성화시켜항체생산을증가시키는것으로, 염증성병변에서항상증가하는것으로보고되고있다. 본실험에서 IL-6 분비량억제능은모든농도에서유의적인감소를나타내었다. TNF-α는 T cell과대식세포를활성화하고다른 pro-inflammatory cytokine 들을증가시켜서염증반응을일으키는데중요한역할을한다. 주로활성화된대식세포에서대량생산되지만, 림포이드계의세포 (lymphoid cells), 비만세포 (mast cells), 내피세포 (endothelial cells) 을비롯하여생체에존재하는다양한세포에서도생산된다. 또한, 면역세포들의집합과면역매개물질들의생산을자극하는데중요한역할을담당하며, 인체내의미생물들을포식하는대식세포의능력을증진시키고육아종 (granuloma) 을형성하는등, 숙주의방어기전에있어서도핵심적인역할을수행한다 29). 본실험에서 TNF-α 분비량억제능은 100 μg / ml농도에서만유의적인감소를나타내었다. 선행연구에서는염증반응이시작 이되면산화적스트레스, lymphotoxin, LPS 등의자극을받아 NF-κB 염증인자가활성화되어 IL-1β, IL-6, TNFα를포함하는주요염증촉진단백질의유전자발현을조절하며, COX-2, inos의활성화를조절하여염증반응전반에걸쳐중요한역할을한다고보고된바있고 30), 이에본연구에서 CG 투여군이사이토카인생성억제와 NO의생성저해활성에미치는영향을관찰하였을때, 상호부합되는결과가나타나, 항염증에대한효능이실험적으로일부입증이되었다고판단된다. 결론 본연구는 CG의산화적손상및염증에미치는효능을객관적으로검증하기위해실험을통하여다음과같은결론을얻었다. 1. CG의세포독성평가에서는 97% 이상의세포생존율을나타내었다. 2. CG의총폴리페놀함량이 39.87 mg /g 로나타났고, DPPH radical 소거능, ABTS radical 소거능과 ROS 생성억제능을통한항산화효능평가에서농도의존적인감소와더불어유의적인결과가도출되었다. 3. CG의항염증효능은 NO 생성저해능과 IL-1β, IL-6, TNF-α소거능에서모두농도의존적인감소와유의적인결과가도출되었다. 이상의실험에서 CG의항산화, 항염증효능이농도의존적이고유의한결과가도출되어, CG가산화적손상및염증에대한효과가있음을실험적으로입증되었다. 향후개별약물간또는원방인천궁다조산과의효능비교연구에서 inos 발현또는 peroxynitrite 생성에대한영향등의실험을추가하여심도있는연구가이루어져야할것으로사료된다. 감사의글 본연구는지식경제부지정대전대학교난치성면역질환의동서생명의학연구지역혁신센터의지원에의한것입니다. References 1. Kim HH. The Study on Verification of Cosmeceutical Activities from Aster glehni Fr. Schm. and Application of oleosome on Advanced formulation. Daegu Haany University. 2010. 2. Fubini B, Hubbard A. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic Biol Med. 2003 ; 34(12) : 1507-16.

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