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Journal of Korean Medicine Rehabilitation Vol. 26 No. 3, July 2016 pissn 1229-1854 eissn 2288-4114 http://dx.doi.org/10.18325/jkmr.2016.26.3.51 Original Article 靑蛾地黃湯의 RAW264.7 Cell 에서항산화및항염증효능연구 윤정원 김순중 박동수세명대학교한의과대학한방재활의학과교실 Anti-Oxidative and Anti-Inflammatory Effects of Cheongajihwang-Tang Extract on RAW264.7 Cells Jeong-won Yoon, K.M.D., Soon-Joong Kim, K.M.D., Dong-Su Park, K.M.D. Department of Korean Rehabilitation Medicine, College of Korean Medicine, Se-Myung University RECEIVED June 23, 2016 ACCEPTED July 6, 2016 CORRESPONDING TO Soon-Joong Kim, College of Korean Medicine, Se-Myung University, 65 Semyung-ro, Jecheon 27136, Korea TEL (043) 649-1920 FAX (043) 649-1872 E-mail kimsj@semyung.ac.kr Objectives This study was designed to investigate whether the Cheongajihwang-Tang (CT) has an inhibitory effect association with oxidation or inflammation in RAW264.7 cells. Methods Cytotoxic activity of CT extract on RAW264.7 cells was evaluated by using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) solution. Nitric oxide production was measured using Griess reagent system. The total phenolic contents and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was measured to evaluate the anti-oxidative effects of CT. Dichlorofluorescin diacetate (DCFH-DA) has been used as a substrate for measuring intracellular oxidant production. Results Cheongajihwang-Tang does not impair the cell viability in tested concentration. CT showed anti-oxidative effects in vitro by decreasing electron donating ability, and also showed anti-inflammatory effects suppressing NO and ROS expression in LPS induced RAW264.7 activation. CT inhibited the generation of intracellular ROS production as dose dependant manner. Conclusions CT has anti-oxidative effects and anti-inflammatory activities. These results indicate that CT extract has an anti-inflammatory activities via anti-oxidative effects. (J Korean Med Rehabil 2016;26(3):51-58) Copyright 2016 The Society of Korean Medicine Rehabilitation Key words Cheongajihwang-Tang, Anti-oxidation, Anti-inflammatory, RAW264.7 서론»»» 염증은병원균침입이나조직손상에의해일어나는선천적면역반응으로다양한면역세포에의해진행되는복합적인과정이다 1). 염증성반응은크게급성반응과만성반응으로나눌수있는데, 장기간지속되는만성염증의경우노화로인한파킨슨질환, 노인성치매, 류마티스관절염, 동맥경화와같은다양한질환들의원인이된다 2). 노화는시간이경과함에따라생체내여러가지생리 적기능이저하되는현상을말하며아울러외부로부터오는 stress를감당하지못하게되는상태를의미한다 3). 노화의기전과관련하여많은이론들이대두되고있는데이중 free radical 과관련된학설이최근많은지지를받고있다 4). 인체내에는안정한상태의산소가환경적및생화학적요인등에의하여반응성이큰활성산소로전환된다. 이들활성산소는강한산화력으로세포막지방질을과산화시키고세포막투과성의변화를초래하고이것이축적된결과가노화와만성퇴행성질병의근본적인원인 www.e-jkmr.org 51

윤정원 김순중 박동수 이된다 5). 또한산화적스트레스는세포구조를빠른속도로붕괴시킴으로써염증반응에관여하게된다 6). 또한노화과정에서염증반응이지속적으로일어남으로써활성산소의생성증가와 inos 유도에따른 NO의대량생성이세포및조직손상을가져와노화과정을촉진한다고정 7) 등이말하였다. 이렇게산화적스트레스와염증은상호관계를통하여노화를진행시킨다. 그러므로각종만성퇴행성질환을치료또는개선하기위해서생체의항산화기능및항염기능을증가시킬수있는물질의공급이필요하다고사료된다. 최근천연물, 한약재를포함한다양한약용식물들에서생리활성물질들을찾는연구가활발하게진행되고있으며특히전세계적으로식물자원에서항암, 항산화및항염등에효과가있는기능성물질을다량함유하는자원을선발하는연구가다양한각도에서진행되고있다 8). 靑蛾地黃湯은 晴崗醫鑑 9) 에수록되어있는처방으로靑蛾丸 10) 에六味地黃湯 11) 을合方加味한것으로腎虛腰痛에쓰이는처방이다. 靑蛾丸은胡桃, 補骨脂, 杜冲, 六味地黃湯은熟地黃, 山藥, 山茱萸, 澤瀉, 牡丹皮, 白茯苓의약물로구성되어있으며모두腎虛에사용되는처방이다. 腎虛는동양의학에서노화의중요원인으로보고있다 12). 靑蛾丸과六味地黃湯의항산화작용과항염작용에대한관련연구로는문 13), 정 14), 서 15), 이 16) 등의연구가있으며, 이들을합방한靑蛾地黃湯에대한연구로는골다공증에미치는영향 17) 외에는없었다. 이에저자는靑蛾地黃湯도靑蛾丸과六味地黃湯같이항산화및항염효과가있을것이라고기대하여이를밝히고자 LPS로염증이유도된 RAW264.7 mouse macrophage cell line을이용하여靑蛾地黃湯추출물이나타내는염증억제반응을관찰하였으며, 유효한결과가있었기에보고하는바이다. 재료및방법»»» 1. 재료 1) 약재본실험에사용한靑蛾地黃湯 ( 이하 CT라한다 ) 은에이 치맥스 ( 제천, 대한민국 ) 으로부터구입하여사용하였다. 처방의구성과용량은다음과같다 (Table I). 실험에사용하기위해靑蛾地黃湯 5첩분량 ( 약 430 g) 을 10배중량의증류수를넣고 80 o C에서 3시간동안추출한후감압농축기 (EYELA, Japan) 및동결건조기 (Labcono, USA) 를이용하여추출물 (74.64 g, 회수율 19.07%) 을만들었으며, 이를냉동실에보관하였다가실험직전에필요한농도로만들어사용하였다. 세포실험을수행하는경우에는물추출물중부유물및잔사제거를위해추출물을 70% 에탄올에녹인후용해된부분만다시감압농축및동결건조를수행한후 dimethyl sulfoxide (DMSO) 에실험에필요한농도로용해한후이를증류수로희석하여세포실험을진행하였다. 2) 시약 실험에사용한시약은다음과같다. DMSO (Junsei chemical Co., Japan) 와 30% hydrogen peroxide (Junsei chemical Co., Japan), 추출에사용한 Methanol (SK chemicals Co., Korea), 세포배양액인 Dulbecco's Modifide Eagle Medium (Welgene, Korea), Fetal bovine serum (Gibco BRL, USA) 과 Penicillin -streptomycin (Gibco BRL, USA), Folin & Ciocalteu's phenol reagent (Sigma-Aldrich Co., USA) 와 Sodium carbonate (Sigma-Aldrich Co., USA), Diethylene glycol (Sigma-Aldrich Co., USA), 2,2-Diphenyl-1-pikryl-hydrazyl (Sigma-Aldrich Co., USA) 등을사용하였다. Table I. The Compositions of Cheongajihwang-Tang Extracts Herb name Pharmacognostic name Amount (g) 熟地黃 Rehmanniae Radix Preparata 16 山藥 Discoreae Rhizoma 8 山茱萸 Corni Fructus 8 杜冲 Eucommiae Cortex 6 補骨脂 Psoraleae Fructus 6 白茯笭 Poria 6 牧丹皮 Moutan Cortex 6 澤瀉 Alismatis Rhizoma 6 五味子 Schizandrae Fructus 2 (7 粒 ) 胡桃肉 Juglandis Semen 22 (5 枚 ) Total 86 52 J Korean Med Rehabil 2016;26(3):51-58

靑蛾地黃湯의 RAW264.7 Cell 에서항산화및항염증효능연구 3) 세포실험에사용된세포는 mouse macrophage RAW 264.7 cell line으로한국세포주은행 (KLCB, Korea) 에서구입하였다. 2. 방법 1) 총폴리페놀함량총 polyphenol 함량은 Folin-Denis 의방법 18) 을참고하여사용하였으며, Folin-Ciocalteu 시약을이용하는방법으로측정하였다. 추출시료용액 1 ml에 50% Foiln-Ciocalteu's phenol reagent 0.5 ml를가하여실온에서 3분간반응시켰다. 반응용액에 Na 2 CO 3 포화용액 1 ml와 7.5 ml 증류수를차례로혼합하여 30분간정치시킨뒤 12000 rpm에서 10분간원심분리한후상등액을취해 ELISA reader (BioTek, USA) 로 760 nm에서흡광도를측정하였다. 총 polyphenol 함량은 gallic acid를표준물질로이용하여작성한검량선에따라함량을구하였으며측정단위로는 GAE (Gallic acid equivalent)/g 을사용하였다. 2) 항산화효과각농도별추출물을 0.2 ml, 1.5 10-4 M 2,2-Diphenyl- 1-pikryl-hydrazyl (DPPH) 용액 1.8 ml을혼합하여암실에서 30분간반응시켜 ELISA reader 를이용해 510 nm에서흡광도를측정하였다. 위실험은 Blois의방법 19) 을응용하였다. 전자공여능은시료첨가한샘플과첨가하지않은샘플차이를백분율로구하였다. 양성대조군으로서아스코르브산 (ascorbic acid) 을이용하였다. 3) 세포배양마우스유래대식세포주인 RAW264.7 세포는 10% fetal bovine serum (FBS) 과 1% penicillin-streptomycin을포함하는 DMEM 배지를사용하였다. 세포는 37 o C, 5% CO 2 조건에서배양하였다. 4) RAW264.7 세포독성시험추출물에대한세포생존률을측정하기위해세포개수를세어 well 당 2 10 5 cells 가되도록분주하여 37 o C, 5% CO 2 조건의 incubator 에서 18시간배양하였다. 이후부착 된각세포들에추출물을농도별로처리하고, 1시간뒤 5 μg/ml 의농도인 LPS를처리하여 24시간처리하였고 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay 방법으로분석하였다. 이는 Carmichaeal 등의방법 20) 을응용하였다. 세포가부착되어있는 plate에 MTT solution (1 mg/ml) 을동량반응시켜 2시간동안 incubator 에서반응시키고상층액을제거한후남아있는 formazan 을용해시켜 fluorescence reader 를이용해 570 nm에서흡광도를측정하였다. 각데이터들은다음과같이계산하였다. Percent values (%) =[1-(Control Abs-Sample Abs)/Control Abs)] 100 5) NO 생성억제효과측정약물처리후 NO를측정하기위해각농도별추출물을다른 plate에옮겨 Griess reagent [1% (w/v) sulfanilamide, 1% (w/v) naphtylethylendiaminde in 30% acetic acid] 와동량반응시켜 ELISA reader 를이용해 540 nm 에서흡광도를측정하였다. 6) DCFH-DA에의한 intracellular ROS 측정각추출물에대한산화적손상을보호하는효과를확인하기위해세포를 2 10 5 cells가되도록각 well에분주하여 37 o C, 5% CO 2 조건의 incubator 에서 18시간배양하였다. 이후부착된각세포들에추출물을농도별로처리하고, 1시간뒤 5 μg/ml 의농도인 LPS를처리하여 24 시간처리하였다. 약물처리후배지를제거한다음, Phosphate Buffer Saline (PBS) 로희석된 10 um 2' 7-dichlorofluorescein diacetate (DCFH-DA) 를가하고, 30분간배양하였다. PBS로 2회세척한후에 ELISA reader 를이용해 excitation 485 nm, emission 530 nm에서형광강도를측정하였다. DCF 형광광도의증가율은대조군과비교하여퍼센트 (%) 로계산하였다. 7) 통계분석모든실험은 3번반복하고, 실험결과는평균 ± 표준편차 (mean±s.d.) 로표현하였다. 대조군및실험군간의통계적유의성은일원분산분석 (one-way ANOVA with www.e-jkmr.org 53

윤정원 김순중 박동수 Dunnett's post hoc test) 을통해확인하였다. 모든그래프에서 p 값은 0.05이하일때통계적으로유의한것으로간주하였다. 결과»»» 1. 총폴리페놀함량 CT 추출물에존재하는총 polyphenol 함량을 gallic acid를표준물질로하여측정한결과, polyphenol 함량이 39.76±1.63 mg/g으로나타났다 (Table II). 2. DPPH free radical 소거능 산화적 free radical 에전자를공여하여산화를억제시키게하는방법으로서항산화효과를보는것을목적으로하였다. 항산화능은페놀화합물의양과상관관계가있는 Table II. Total Phenolic Contents of Cheongajihwang-Tang Extract Sample Total phenolics (mg*) CT 39.76±1.63 The values represent mean±s.d. of triplicate independent experiments. *Total phenolic contents was expressed as milligram of gallic acid equivalent (GAE) per gram of extract. CT: Cheongajihwang-Tang. 것으로보고되고있으며 21), 본연구에서 CT과양성대조군의전자공여능을농도별로측정하여비교한결과 10 mg/ml 농도에서 33.4±3.7% 의소거능을보여, 양성대조군으로사용한 ascorbic acid 30.3±1.9% 의소거능과비교하여통계적으로유의한높은효과를나타냈다 (Fig. 1). 3. 세포독성측정 CT에의한대식세포의독성을 MTT assay에의해확인한결과농도별약물을주입하였을때각농도에서대식세포에대한독성을일으키지않은것으로나타났다 (Fig. 2). 4. Nitric oxide 측정 RAW264.7 cell의 NO 생성억제효능을측정하기위해 CT을농도별로처리한결과, 1 mg/ml, 2 mg/ml, 및 4 mg/ml 투여군에서각각 23.3±0.4 μm, 19.1±0.7 μm, 및 12.3±1.0 μm의농도의존적경향으로 NO의생성을억제하였고, 이는대조군 CT0군의 10.8±0.3 μm의 NO 생성량과비교하여통계적으로유의한 NO 생성억제효과를나타내었다 (Fig. 3). 5. Intracellular ROS 생성저해 RAW264.7 cell의 ROS 생성억제효능을측정하기위 Fig. 1. DPPH free radical scavenging activity of ascorbic acid and Cheongajihwang-Tang (CT) extract. The results are expressed as mean±s.d. of three independent experiments. Significantly different from ascorbic acid (*p<0.05). Significantly different from control group ( p<0.05). Fig. 2. Effect of Cheongajihwang-Tang (CT) extract on the cell viability of RAW264.7 cells. The values represent mean±s.d. of three independent experiments. Cell viability expressed as a percentage of the control (CT0). 54 J Korean Med Rehabil 2016;26(3):51-58

靑蛾地黃湯의 RAW264.7 Cell 에서항산화및항염증효능연구 Fig. 3. Effects of Cheongajihwang-Tang (CT) extract on NO production in LPS-stimulated RAW 264.7 cells. The values represent mean±s.d. of three independent experiments. Asterisk indicates a significant difference at *p<0.05 level. 해 CT을농도별로처리한결과, 2 mg/ml 및 4 mg/ml 투여군에서각각 81.9±6.2%, 및 65.3±1.8% 로대조군 (CT0) 과비교하여통계적으로유의한그리고농도의존적으로 ROS 생성을억제하였음을관찰할수있었다 (Fig. 4). 고찰»»» 활성산소는산소의화학적특성으로인해생성되는산소프리라디칼 (oxygen free radical) 과이들로부터유래되는산소화합물을말한다 22). 인체내에는산화억제기구가활성화되어있어서산화촉진물질과산화억제물질이균형을이루고있지만여러가지요인들에의하여이균형상태가깨지면산화적스트레스가유발된다 23). 과잉으로생성된활성산소는세포를구성하는지질, 단백질, 탄수화물및 DNA와반응하여산화적손상및효소활성을변화시켜세포손상을가져와 24) 세포사를일으키며, 고혈압, 동맥경화, 심부전, 류마티스관절염, 알레르기, 암및노화등에깊이관련되어있다 25). 또한, 활성산소의과도한생성및그로인한 NO의축적은대식세포의지나친활성을유도하여생체내필수방어기전인염증의과활성화를일으켜염증관련질환을유발하기도한다 26). 노화란생명체에서진행되는일련의퇴행성변화를일으키는과정으로형태적, 기능적퇴축, 예비력과적응력의저 Fig. 4. Effect of Cheongajihwang-Tang (CT) extract on the intracellular reactive oxygen species (ROS) generation of RAW264.7 cells. The values represent mean±s.d. of three independent experiments. ROS generation expressed as a percentage of the control (CT0). Asterisk indicates a significant difference at *p<0.05 level. 하로사망에귀착되는보편적인생리현상을말한다 27). 최근생활수준이향상되고인간의수명이증가함에따라노화와관련된각종퇴행성질환에관심이커지고있으며, 그원인이활성산소에기인된것이라는산소유해설이점차인정받고있다 28). 이러한관점에서볼때생체내항산화방어시스템을증가시키거나 reactive oxygen species (ROS) 를조절할수있는항산화제개발연구의필요성이강조되고있고이에대한탐색이활발히진행되고있다 29). 활성산소를조절하는항산화제는합성항산화제와천연항산화제로구분되며합성항산화제의경우, 다량섭취시여러가지독성을나타낼수있는것으로알려져있어안전한천연항산화제연구에대한관심이높아지고있다. 식물속에함유된 phytochemical 을비롯한천연생리활성물질들은체내에들어오면항산화작용이나세포손상을억제하는작용을통해항암, 항균, 항돌연변이등의예방기능을가지고있는것으로알려져왔다 30). 본연구에서사용한靑蛾地黃湯은 晴崗醫鑑 9) 에수록되어있는처방으로靑蛾丸에六味地黃湯을合方加味한것이다. 靑蛾丸은 太平惠民和劑局方 10) 에최초로기재된처방으로胡桃, 補骨脂, 杜冲의약물로구성되어있어溫腎强腰의효능이있으며, 腎陽虛의만성요통에사용된다 31). www.e-jkmr.org 55

윤정원 김순중 박동수 六味地黃湯은 小兒藥證直訣 11) 에최초로수록된처방으로熟地黃, 山藥, 山茱萸, 澤瀉, 牡丹皮, 白茯苓의약물로구성되어있다. 滋陰補腎, 瀉火의효능이있어肝腎陰虛, 虛火上炎으로인한증상에적용한다 31). 素問ㆍ上古天眞論 32) 에 女子七歲腎氣盛 八八則齒髮去腎者主水受五臟六腑之精而藏之, 天壽過度氣脈常通而腎氣有餘也 라하여長壽하는것은腎氣의盛衰與否에의하여결정된다고하였으니腎氣虛衰는노화의중요원인이다 12). 항산화및항염작용과관련하여靑蛾丸은김 12), 정 14) 등의연구가있으며六味地黃湯은문 13), 서 15), 이 16) 등의연구가있다. 그러나이들을합방한靑蛾地黃湯에대한항산화및항염관련연구는아직없다. 이에저자는靑蛾地黃湯항산화효과및항염증효과를밝히고자하였다. 본연구에서는靑蛾地黃湯의항산화및항염증효능에미치는영향을알아보기위하여총 polyphenol 함량, DPPH free radical 소거능, NO 생성억제효과, ROS 생성억제효과를평가하였다. Lipopolysaccharide (LPS) 는병원균내독소로서인지질, 다당류및소량의단백질로구성되며 RAW264.7 cell 과같은대식세포에서 tumor necrosis factor-alpha (TNFα inflammatory cytokine) 을증가시키며염증을유발한다 33). 또한 reactive oxygen species (ROS) 생성을촉진하고 nitric oxide (NO) 를과도하게활성화시켜염증을유발시켜조직의손상을초래한다 34). 따라서 LPS는염증반응을연구하는실험모델로많이사용되고있다 35). Polyphenol 화합물은 flavonoids, anthocyanins, tannins, catechins, isoflavones, lignanas, resveratrols 등을총칭하며, 식물계에널리분포되어있고과일및엽채류에다량함유되어있다 36). 페놀성항산화제들은연쇄반응에서 alkyl radical이나 alkylperoxy radical에수소를공여하여그 radical 을제거함으로써산화를억제하는작용을가진물질로알려져있다 37). DPPH (1,1-Diphenyl-2-picryl-hydrazyl) 라디컬소거능은항산화제가안정한자유라디칼인 DPPH와반응하면서 DPPH를 DPPHH로환원시켜, 흡광도를감소시키는원리를이용하여항산화활성을측정하는방법으로가장널리사용되고있다 38). Nitric oxide (NO) 는동맥이나다른혈관의이완및신경계의신호전달물질로작용하면서외부에서침입한미 생물에대한방어분자로알려져있다 39). 그러나과도한 NO의형성은혈관투과성부종등의염증반응을촉진시킬뿐만아니라염증매개체의생합성을촉진하여염증을심화시키는것으로알려져있다 40). 본연구에서는靑蛾地黃湯추출물에포함된폴리페놀함량을 gallic acid를표준물질로하여측정한결과, 폴리페놀함량이 39.76±1.63 mg/g으로나타났다. DPPH radical scavenging activity는 CT과양성대조군의전자공여능을농도별로측정하여비교한결과 10 mg/ml 농도에서 33.4±3.7% 의소거능을보여, 양성대조군으로사용한 ascorbic acid 30.3±1.9% 의소거능보다좋은활성을나타내었으며통계적으로유의하였다. NO 생성억제효능을측정한결과본연구에서 LPS 단독으로처리한경우 NO 생성이상승함을확인하였고 CT을농도별로처리한결과농도의존적경향으로 NO 생성을억제하였다. 또한대조군 CT0군의 NO 생성량과비교하였을때통계적으로유의한 NO 생성억제효과를나타내었다. ROS가정상적으로소거되지않았을때는잔존하는자유라디칼에의해산화적스트레스를일으키게된다 41). 세포내생성된 ROS는비형광을나타내는 DCF-DA 를산화시켜형광물질인 DCF로전환시키는데이렇게전환된형광물질의양이 ROS의양을나타낸다 42). 본실험에서역시 LPS에의해유도된 ROS 생성억제효능을측정하기위해 CT을농도별로처리한결과대조군 (CT0) 과비교하여통계적으로유의한그리고농도의존적으로 ROS 생성을억제하였다. 다만, 靑蛾丸과六味地黃湯을연구한논문에서는쥐의신장과뇌조직에서 peroxynitrite (ONOO - ) 의제거능, superoxide dismutase (SOD) 및 glutathione peroxidase (GSH) 와같은항산화효소의활성등을측정하여 12-16) 본실험에서의항산화및항염효과측정방법과달라靑蛾地黃湯과비교분석을할수없는한계점을보인다. 향후연구에서실험방법을통일한다면합방하기전과후의처방효과를비교해볼수있는좋은연구가될것이다. 이상의연구결과를종합하면, 靑蛾地黃湯은항산화효과가있으며이로인한유효한항염증효과를나타내, 염증을동반하여나타나는다양한만성퇴행성질병의연구에응용될수있으리라사료된다. 56 J Korean Med Rehabil 2016;26(3):51-58

靑蛾地黃湯의 RAW264.7 Cell 에서항산화및항염증효능연구 결론»»» 靑蛾地黃湯추출물의항산화활성이마우스대식세포에서의항염증, 세포내활성산소를억제하는연구를수행하여아래와같은결과를얻었다. 1. 靑蛾地黃湯추출물의폴리페놀함량은 39.76±1.63 mg/g이다. 2. 靑蛾地黃湯추출물의 DPPH radical 소거능은일부양성대조군보다통계적으로유의하게뛰어난항산화효과를관찰할수있었다. 3. 靑蛾地黃湯추출물은실험농도에서세포생존율에영향을미치지않았다. 4. 靑蛾地黃湯추출물의대식세포내 NO 생성을억제하는것을확인한결과, 농도의존적으로유의하게 NO 생성을억제하였다. 5. 靑蛾地黃湯추출물은대식세포내 ROS 생성을농도의존적으로유의하게억제하였다. 이상의실험결과靑蛾地黃湯은항산화과정에의한항염증효능이있음을관찰할수있었다. References»»» 1. Bae GC, Bae DY. The anti-inflammatory effects of ethanol extract of Allium Hookeri cultivated in South Korea. Kor.J.Herbology. 2012;27(6):55-61. 2. Greig FH, Kennedy S, Spickett CM. Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation. Free Radic Biol Med. 2012;52(2):266-80. 3. Lee DU. Oxidative stress and age-related changes in microsomal mixed function oxidase activity. Kor.J.Gerontol. 1991;1(2):187-201. 4. Volicer L, Crino PB. Involvement of free radicals in dementia of the Alzheimer s type, a hypothesis. Neurobiol Aging. 1990;11:567-71. 5. Kim HJ, Jin CB, Lee YS. Anti-oxidative activities of phenolic compound isolated from Inonotus obliquus. Kor.J.Pharmacognogy. 2007;38:1-16. 6. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of aging. Nature. 2000;408(6809):239-47. 7. Chung HY, Kim HJ, Kim JW. The inflammation hypothesis of aging : Molecular modulation by calorie restriction. Ann.N.Y Acad Sc. 2001;928:327-35. 8. Kim HJ, Ahn MS, Kim GH, Kang MH. Antioxidant and antimicrobial activity of Pleurotus eryngii extracts prepared from different aerial part. Kor.J.Food Sci Technol. 2006;38:799-804. 9. Kim YH. Cheonggangeuigam. Seoul:Seongbosa. 2001:304. 10. Jin SM. Taepyeonghyeminhwajegukbang. Beijing:Ren min wei sheng Publishing INC. 1985:175. 11. Qian Yi. Soayakjeungjikgyeol. Seoul:Yeogang Publishing INC. 2002:11. 12. Kim SH, Jeong JC. Peroxynitrite scavenging activity and its mechanism of Cheonga-hwan. Kor.J.oriental medicine. 2002;23(4):55-63. 13. Moon SS, Kim BS, Kang JS. Study on antioxidant action of Yukmijihwang-tang. Kor.J.oriental physiology&pathology. 2003;17(2):436-442. 14. Jeong JC. Increased antioxidant enzyme activities and scavenging effects of oxygen free radicals by Cheongahwan. Journal of Korean Medicine. 1997;18(2):355-65. 15. Seo YE, Lee EA, Bae HS, Shin MK, Hong MC. Antioxidant effects of the Herbs Composing Yukmijihwang-tang on PC12 cell. Kor.J.Oriental Physiology&Pathology. 2003; 17(1):203-8. 16. Lee GH, Yoo DY. Evaluation of Anti-Inflammatory Effects of Yukmijihwangtang and Individual Drug Substances Based on the Extraction Methods. The Journal of oriental obstetrics&gynecology. 2012;25(2):89-107. 17. Kim MS, Seo BI, Kwak MA, Jee SY. Effect of Chungajihwangtang on osteoporosis in ovariectomized rats. Kor.J.Herbology. 2003;18(2):49-58. 18. Velioglu YS, Mazza G, Gao L, Oomah BD. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J Agric Food Chem. 1998;46(10):4113-7. 19. Kandaswami CE. Middleton E Jr. Free Radical Scavenging and Antioxidant Activity of Plant Flavonoids. Adv Exp Med Biol. 1994;366:351-76. 20. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity. Cancer Res. 1987;47(4):943-6. 21. Gheldof N, Engeseth NJ. Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples. J Agric Food Chem. 2002;50(10):3050-5. 22. Choi BK, Jeong SY, Park GS, Cho JH. Reactive Oxygen Substances and Diseases. Shinil Publishing INC. 2004;1:7-15. 23. Park SN. Skin aging and antioxidants. Kor.J.Soc Cosmetic Chem. 1997;23:75-132. 24. Stadtman ER, Berlett BS. Reactive oxygenmediated protein oxidation in aging and disease. Drug Metab Rev. 1998;30(2):325-43. 25. Virag L, Szabo E, Gergely P and Szabo C. Peroxynitrite induced cytotoxicity: mechanism and opportunities for www.e-jkmr.org 57

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