Original Article J Sasang Constitut Med 2012;24(4):51-61 http://dx.doi.org/10.7730/jscm.2012.24.4.51 사상체질처방의항동맥경화효능비교연구 - 열다한소탕, 양격산화탕, 청심연자탕, 태음조위탕 - 김온순, 김예지, 신현규 한국한의학연구원한약기초연구그룹 Abstract Anti-atherosclerotic Effects of Herbal Formulas for Sasang Constitutional Medicine Ohn-Soon Kim, Ye-Ji Kim, Hyeun-Kyoo Shin Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine. Objectives The purpose of this study was to investigate the anti-atherosclerotic effects of 4 herbal formulas for Sasang constitutional medicine (Yeoldahanso-tang: YDHST, Yanggyeoksanhwa-tang: YGSHT, Cheongsimyeonja-tang: CSYJT and Taeeumjowitang: TEJWT). Methods The antioxidant activities of herbal formulas were studied by measuring free radical scavenging activities on ABTS and DPPH. The inhibitory effects on LDL oxidation was evaluated by the formation of TBARS, REM and fragmentation of apolipoprotein B-100 (ApoB). Effects of herbal formulas on macrophage lipid accumulation were determined in native LDL and LPS co-incubated macrophages using Oil Red O staining. Results The scavenging activities on ABTS and DPPH of herbal formulas were increased in dose-dependent manner (YDHST>YGSHT>CSYJT>TEJWT). Herbal formulas reduced the oxidation properties of LDL induced by CuSO 4. YDHST, YGSHT and CSYJT showed strong suppressive effect on LDL oxidation than TEJWT. In addition, YDHST, YGSHT and CSYJT significantly inhibited foam cell formation in LDL/LPS stimulated RAW 264.7 cells. Conclusions These results demonstrate that YDHST, YGSHT and CSYJT have potentials on anti-atherosclerosis by antioxidative effect and suppressive effect on LDL oxidation. Key Words : Yeoldahanso-tang, Yanggyeoksanhwa-tang, Cheongsimyeonja-tang, Taeeumjowi-tang, Anti-atherosclerotic effect Received October 8, 2012 Revised November 9, 2012 Accepted November 28, 2012 Corresponding Author Hyeun-Kyoo Shin Basic Herbal Medicine Research Group, Korea Institute of Oriental Medicine, Yuseongdae-ro 1672, Yuseong-gu, Daejeon, 305-811, Korea. Tel: +82-42-868-9464 Fax: +82-42-864-2120 E-mail: hkshin@kiom.re.kr C The Society of Sasang Constitutional Medicine. All rights reserved. This is an open acess article distributed under the terms of the Creative Commons attribution Non-commercial License (http:// creativecommons.org/licenses/by-nc/3.0/)
52 Anti-atherosclerotic Effects of Herbal Formulas for Sasang Constitutional Medicine I. 緖論 산소를이용해생명을유지하는모든생명체들은세포내항산화방어계와활성산소종간의恒常性을유지하며생존해나간다. 이러한恒常性이유지되지못하고불균형이초래될때산화적스트레스 (oxidative stress) 가유도되는데이는세포내다양한구성성분에손상을초래하여많은生理 病理學적현상에서중요한역할을하는것으로알려져있다 1. 동맥경화 (atherosclerosis) 는산화적스트레스에기인한질환중하나로, 특히동맥경화발병원인으로산화 LDL (low density lipoprotein) 이중요한역할을하는것으로알려져있다. 혈액내 LDL 은산화적스트레스에의해산화된 LDL (oxidized LDL) 로변화되어혈관내막내로유입되게된다. 유입된산화 LDL은대식세포의탐식작용에의해세포내로유입되어거품세포를형성하게된다. 거품세포는대식세포의염증반응및평활근세포의분화 증식및혈관내피세포의변화를유도하여플라크를생성함으로써동맥경화를유발하게된다 2,3. 식생활이서구화되어가는우리나라에서도동맥경화로인한순환기계질환이날로증가하고있는추세로효과적인치료와예방이절실히필요한실정이다. 四象醫學은東武李濟馬의저서인 東醫壽世保元 에서비롯된體質醫學으로四象人, 곧太陽人 太陰人 少陽人 少陰人으로나누어生理, 病理, 治療및豫防을달리접근한다 4. 四象體質에따른각四象處方들의生理活性에대한몇몇연구가진행되어왔으나, 동맥경화와관련된연구는미흡한실정이다. 본연구에서는少陽人處方인凉膈散火湯과太陰人處方熱多寒少湯, 淸心蓮子湯, 太陰調胃湯 4종의處方을선정하여항산화효과, LDL 산화억제효과및거품세포형성억제효과를비교분석함으로써산화적스트레스에의한동맥경화와같은순환기계질환의예방및치료의적용가능성을검토하였다. Ⅱ. 材料및方法 1. 재료 1) 처방추출본실험에사용된열다한소탕, 양격산화탕, 청심연자탕, 태음조위탕의처방구성약재들은 ( 주 ) 옴니허브 (Yeongcheon, Korea) 와 ( 주 )HMAX (Jecheon, Korea) 에서각각구입하였다. 각각의처방에따라약재를배합하고 10배의물을가한후 100 C에서 2시간동안무압환류추출법 ( 추출기 : 경서메디텍 COSMOS660) 을이용하여추출하였다. 추출액은약전표준체를사용하여거르고동결건조기 ( 일신동결건조기, PVTFD100R) 를사용하여처방추출물분말을얻었으며수득율은 Table 1과같다. 2. 실험방법 1) 항산화능측정가. ABTS 라디칼소거능측정 3-ethyl-benzothiazoline-6-sulfonic acid (ABTS, Sigma Chemical Co. St. Louis, MO) 라디칼을이용한항산화능측정은 ABTS + cation decol orization assay방법을 96 well plate에맞게수정하여실시하였다 5. 7 mm ABTS와 2.45 mm potassium perulfate 를최종농도로혼합하여실온인암소에서 24시간동안방치하여 ABTS + 를형성시킨후 743 nm에서 0.7의흡광도값을갖도록 phosphate buffer saline (PBS, ph 7.4) 로희석하였다. 96 well plate에 ABTS + 용액과시료를혼합하여실온에서 30 분간반응시킨후, microplate reader (Benchmark Plus, Bio-Rad. USA) 를사용하여 743 nm에서흡광도를측정하였다. 각시료추출물의라디칼소거활성은시료를첨가하지않은대조구의흡광도를 1/2로환원시키는데필요한시료의농도인 RC 50 값으로나타내었다. 이때활성비교를위하여 Vitamin C를사용하였다.
OS Kim et al. 53 Table 1. Combinations of Crude Drugs in Herbal Formulas Componented crude drugs (g) YDHST a) YGSHT b) CSYJT c) TEJWT d) 갈근 Puerariae Radix 15.0 황금 Scutellariae Radix 7.5 3.75 고본 Angelicae Tenuissimae Radix 7.5 나복자 Raphani Semen 3.75 3.75 7.5 길경 Platycodonis Radix 3.75 3.75 승마 Cimicifugae Rhizoma 3.75 백지 Angelicae Dahuricae Radix 3.75 생지황 Rehmanniae Radix 7.5 인동등 Lonicerae Flos 7.5 연교 Forsythiae Frucus 7.5 산치자 Gardeniae Fructus 3.75 박하 Menthae Herba 3.75 지모 Anemarrhenae Rhizoma 3.75 석고 Gypsum 3.75 방풍 Saposhnikoviae Radix 3.75 형개 Schizonepetae Spica 3.75 연자육 Nelumbinis Semen 7.5 산약 Dioscoreae Radix 7.5 천문동 Asparagi Radix 3.75 맥문동 Liriopis Tuber 3.75 3.75 원지 Polygalae Radix 3.75 석창포 Acori Graminei Rhizoma 3.75 3.75 산조인 Zizyphi Spinosi Semen 3.75 용안육 Longanae Arillus 3.75 백자인 Thujae Semen 3.75 감국화 Chrysanthemi Flos 3.75 의이인 Cocicis Semen 11.25 건율 Castaneae Semen 11.25 오미자 Schizandrae Fructus 3.75 마황 Ephedrae Herba 3.75 Total (g) 45 45 52.5 48.75 Yield (%) 29.0 16.2 20.5 19.9 a) b) c) d) Yeoldahanso-tang, Yanggyeoksanhwa-tang, Cheongsimyeonja-tang and Taeeumjowi-tang 나. DPPH 라디칼소거능측정 1-1-diphenyl-2-picrylhydrazyl (DPPH, Sigma Chemical Co.) 라디칼을이용한항산화능측정은 96 well plate을이용하여실시하였다 6. 96 well plate에 0.15 mm의 DPPH 용액과시료를혼합하여실온에서 30분간반응시킨후, 517 nm에서흡광도를측정하였다. 각처방추출물의항산화능은시료를녹인용매인 DMSO를대조군으로하여대조군에대한라디칼소거능을백분율로나타내었다. 활성비교를위하여 Vitamin C를사용하였다. DPPH radical scavenging activity=(1 A sample/a control) 100 2) LDL 산화억제활성측정가. Cu 2+ 에의한 LDL의산화반응은 LDL (Biomedical Technologies, Stoughton, MA) 100 μg과시료를혼합한후 25 μm CuSO 4 를첨가함으로써시작하였고, 37 에서 6시간동안반응시켰다. 반응후 thiobarbituric acid reacting substance (TBARS) 의형성 7, relative electrophoretic mobility (REM) 8, apob-100의 fragmentation 9 을측정하였다.
54 Anti-atherosclerotic Effects of Herbal Formulas for Sasang Constitutional Medicine 나. TBARS 측정산화반응이끝난 LDL 50 μg을취하여 TBARS Assay Kit (BioAssay Systems, Hayward, CA) 를이용하여제조사의 protocol에따라측정하였다. 산화된 LDL과의비교를통해상대적인비율을계산하였다. 다. REM 측정산화반응이끝난 LDL 10 μg을 0.8% agarose gel에 loading하여 TAE buffer (40 mm Tris, 40 mm acetic acid, and 1 mm EDTA) 에서 100 V로 30분동안전기영동한후, Coomassie brilliant blue R-250로염색하여이동거리를측정하였다. 라. ApoB-100 분절화측정산화반응이끝난 LDL 20 μg을 6% SDS-PAGE 에 loading하여 TAE buffer (40 mm Tris, 40 mm acetic acid, and 1 mm EDTA) 에서 100 V로 6시간동안전기영동한후, Coomassie brilliant blue R-250로염색하여관찰하였다. 3) 거품세포형성억제관찰가. RAW 264.7 세포주배양생쥐대식세포주인 RAW 264.7 세포는 American Type Culture Collection (ATCC, Manassas, VA) 에서분양받아사용하였다. RAW 264.7 세포는 5.5% fetal bovine serum (FBS), 1% penicillin-streptomycin 이첨가된 Dulbecco s modified eagle s medium (DMEM) 배지로 37, 5% CO 2 에서배양하였다. 세포배양에사용된모든시약은 Gibico Laboratories (Grand Island, NY) 제품을사용하였다. 나. Oil Red O 염색 24 well plate에분주한 RAW 274.7 세포 (5 x 10 4 cell/ml) 에 LPS (100 ng/ml) 와 LDL (50 μg/ml) 를처리하고각각의처방추출물을농도별로처리한후 24시간동안배양하였다. 배양이끝난세포는 10% formalin을이용하여고정한후, 0.6% Oil Red O (Sigma Chemical Co.) 용액으로 3분간염색하여광학현미경으로관찰하였다. 이미지분석을위하여 MetaMorph Software (Molecular Decives) 를이용하였다. 4) 통계처리실험값은 Mean ± S.E.M. 으로표시하였다. 실험결과에대한통계학적유의성검정은 ANOVA 검정을적용하였으며, Dunnet s multiple comparison test를이용하여 p-value가 0.05 미만일경우유의한것으로판정하였다. Ⅲ. 結果 1. 항산화능비교 4종처방의항산화능을비교하고자추출물을농도별로조제한후 ABTS와 DPPH 라디칼소거활성을측정하였다. ABTS 라디칼의소거활성을비교한결과 4종처방의 RC 50 값은열다한소탕 ; 23.07±1.35 μg/ml, 양격산화탕 ; 37.74±0.94 μg/ml, 청심연자탕 ; 117.28± 1.48 μg/ml, 태음조위탕 ; 204.84±2.15 μg/ml, 양성대조군인 Vitamin C는 3.03±1.56 μg/ml(table 2) 으로측정되었다 (Table 2). DPPH 라디칼의소거활성을비교한결과는 Fig. 1와같다. 추출물의농도가증가함에따라점차적으로 DPPH 라디칼의소거활성이증가하는경향을보였으며, 추출물의농도 200 μg/ml에서열다한소탕 ; 43.64%, 양격산화탕 ; 44.13%, 청심연자탕 ; 34.77%, 태음조위탕 ;12.87% 의라디칼소거활성을보였다. 2. LDL 산화억제활성비교 4종처방의 LDL 산화억제효과를검색하고자 Cu 2+ 에의해 LDL 산화를유도하고, 처방추출물을처리하여 LDL 산화정도를비교분석하였다. LDL 산화정도는 3가지방법으로확인하였다. 먼저 TBARS assay 를수행한결과 Fig. 2에나타난바와같이 4종처방모두농도
OS Kim et al. 55 Table 2. Scavenging Effects of Herbal Formulas on ABTS radical. Herbal formulas Concentration(μg/mL) Scavenging effect(%) RC 50 1) (μg/ml) 12.5 YDHST a) 25 50 12.5 YGSHT b) 50 100 50 CSYJT c) 200 400 50 TEJWT d) 200 400 2.5 Vitamin C 10 35.46±2.45 58.48±0.78 91.22±1.31 28.63±0.66 73.14±0.37 97.24±1.37 30.83±1.32 74.71±1.96 98.38±0.87 23.31±0.82 55.75±1.18 81.72±2.54 42.94±2.32 96.82±1.05 1) Concentration required for 50% reduction of ABTS + at 30 min reaction. a) Yeoldahanso-tang, b) Yanggyeoksanhwa-tang, c) Cheongsimyeonja-tang and d) Taeeumjowi-tang 23.07±1.35 37.74±0.94 117.28±1.48 204.84±2.15 3.03±1.56 Fig. 1. DPPH radical scavenging activity of herbal formulas. YDHST: Yeoldahanso-tang, YGSHT: Yanggyeoksanhwa-tang, CSYJT: Cheongsimyeonja-tang and TEJWT: Taeeumjowi-tang. Each value is the mean ± S.E.M. of triplicate determinations. Fig. 2. Effects of herbal formulas on Cu 2+ -induced lipid peroxidation in LDLs. Indicated concentrations of herbal formulas or Vitamin C (50 μg/ml) and LDLs were incubated with CuSO 4 for 6 h at 37. The level of LDLs was assessed by TBARS. YDHST: Yeoldahanso-tang, YGSHT: Yanggyeoksanhwa-tang, CSYJT: Cheongsimyeonja-tang and TEJWT: Taeeumjowi-tang. Each value is the mean ± S.E.M. of triplicate determinations.
56 Anti-atherosclerotic Effects of Herbal Formulas for Sasang Constitutional Medicine Fig. 3. Effects of herbal formulas on electrophoretic mobility of Cu 2+ -induced oxidized LDLs. Indicated concentrations of herbal formulas or Vitamin C (50 μg/ml) and LDLs were incubated with CuSO 4 for 6 h at 37. The electrophoretic mobility of LDLs were detected by agarose gel electrophoresis. (A) The stained gel is representative of two independent experiments. (B) Relative electrophoretic mobility (REM) indicated the distances moved from the origin were calculated and presented. YDHST: Yeoldahanso-tang, YGSHT: Yanggyeoksanhwa-tang, CSYJT: Cheongsimyeonja-tang and TEJWT: Taeeumjowi-tang. 의존적으로저해효과를나타냈으며, IC 50 (the half maximal inhibitory concentration) 값은열다한소탕 ; 33.54 μ g/ml, 양격산화탕 ; 31.23 μg/ml, 청심연자탕 ; 115.38 μg/ml 으로계산되었다. 하지만태음조위탕의경우실험최고농도인 1000 μg/ml 에서도 47% 의저해효과만을나타냈다. REM assay 결과 4종처방추출물모두산화반응에의한전기적이동차를농도의존적으로감소시키는것을확인하였다 (Fig. 3). 추출물의농도 125 μg/ml에서열다한소탕 ; 84.89%, 양격산화탕 ; 93.02%, 청심연자탕 ; 67.62%, 태음조위탕 ; 66.66% 의이동억제효과를나타냈다. LDL 산화정도를나타내는지표인 apob-100의분절화를측정한결과, Cu 2+ 에의해산화반응을유도한 LDL에서는 apob-100이거의분절되어온전한단백질 band를관찰할수없었지만, 처방추출물과함께산화반응을유도한 LDL의 apob-100은농도의존적으로분절화가회복되는것을관찰할수있었다 (Fig. 4). 3. 거품세포형성억제활성비교 LPS와 LDL을처리하여배양한대식세포는대조군에서거의관찰되지않는 Oil Red O 염색양성반응이다수관찰되었다. Oil Red O 염색은주로거품세포에존재하는세포질내의지방입자에서나타나게되는데, 4종처방을처리한거품세포에서이러한 Oil Red O 염색을비교함으로써거품세포형성억제활성을비교분석하였다. 태음조위탕을제외한열다한소탕, 양격산화탕, 청심연자탕을처리한대식세포에서거품세포형성이억제되는것을확인하였다 (Fig. 5). Ⅳ. 考察 한국인의사망원인통계에의하면동맥경화로인한심근경색, 뇌졸중을비롯한순환기계질환은사망원인의 2위로악성종양다음으로높은순위를차지하
OS Kim et al. 57 Fig. 4. Effects of herbal formulas on Cu 2+ -mediated ApoB fragmentation in LDLs. Indicated concentrations of herbal formulas or Vitamin C (50 μg/ml) and LDLs were incubated with CuSO 4 for 6 h at 37. LDLs were loaded onto 6% SDS-PAGE for electrophoresis. The stained gel is representative of two independent experiments. M, standard molecular weight markers. YDHST: Yeoldahanso-tang, YGSHT: Yanggyeoksanhwa-tang, CSYJT: Cheongsimyeonja-tang and TEJWT: Taeeumjowi-tang. Fig. 5. Inhibitory effects of herbal formulas on the foam cell formation. RAW264.7 cells were co-incubated with LPS (100 ng/ml) and LDL (50 μg/ml) for 24 h in the presence or absence of herbal formulas. The lipid laden macrophages (foam cell) were stained for neutral lipids with oil red O. (B) The quantitative data were presented means ± S.E.M. *p < 0.01, as compared to cells cultured with LPS and LDL. YDHST: Yeoldahanso-tang, YGSHT: Yanggyeoksanhwa-tang, CSYJT: Cheongsimyeonja-tang and TEJWT: Taeeumjowi-tang. Each value is the mean ± S.E.M. of triplicate determinations.
58 Anti-atherosclerotic Effects of Herbal Formulas for Sasang Constitutional Medicine 고있다. 동맥경화는노화와더불어산화적스트레스가주요발병원인인질환으로그기전은다음과같다. 혈중 LDL 이산화적스트레스에의해산화 LDL 로변환되면이산화된 LDL 은거품세포형성, 염증반응, 평활근세포및혈관내피세포의분화와증식등의다양한반응을촉진하여혈관내벽에플라크를생성함으로써동맥경화를유발하게된다. 따라서초기의산화반응을조절하는것이동맥경화의예방과치료에중요할것으로여겨진다. 본연구에서는 4종四象處方의동맥경화에대한효능을알아보고자하였다. 東醫壽世保元 에따르면凉膈散火湯은少陽人의胃受熱裏熱病의하나인上消證에활용되는처방이며, 熱多寒少湯과淸心蓮子湯은太陰人의肝受熱裏熱病의하나인燥熱證에활용되는처방이며, 太陰調胃湯은太陰人胃完受寒表寒病의하나인胃脘寒證에활용되는처방이다. 4 이러한 4종處方의기존의실험적연구결과들을살펴보면凉膈散火湯은주로허혈에의한뇌손상의보호효과 10, 당뇨와비만과같은대사질환개선효과 11,12, 항염증 13 및면역활성효과 14 등이보고되었으며, 熱多寒少湯은주로뇌질환과관련하여보호효과 15 및산화적손상에대해방어효과 16 가보고되었다. 淸心蓮子湯은항산화효과 17 와더불어항노화효과 18 및항알레르기효과 19 가보고되었으며, 太陰調胃湯은항산화효능을통한간기능보호효과 20 및비만을억제하며항고지혈증효과 21 가있다고알려져있다. 각각의處方에대한단편적인항산화효능에대한연구는일부이루어져있지만동맥경화와관련하여체계적인연구는전무하다시피하였다. 동맥경화에대한효능을규명하기위한일환으로먼저처방들의항산화능을측정하였다. 측정결과 ABTS 소거활성과 DPPH 소거활성모두열다한소탕, 양격산화탕, 청심연자탕, 태음조위탕의순서로관찰되었다. 이는다양한항산화물질의항산화능을검색한결과 ABTS 소거활성과 DPPH 소거활성간의높은상관관계가존재한다는보고 22 와일관된결과로생각된다. ABTS 라디칼소거활성법은 in vitro 뿐만아니라 in vivo에서도항산화능을측정하기위해널리이용되고있는방법으로수소공여항산화제 (hydrogen-donating antioxidant) 와연쇄절단형항산화제 (chain-breaking antioxidant) 모두를측정할수있고, 수용상 (aqueous phase) 과유기상 (organic phase) 모두에적용이가능한방법이기때문에본연구에서도소거능이상대적으로높게측정되었다 23,24. DPPH 라디칼소거활성법은실제항산화활성과연관성이높은방법으로시료내항산화활성물질이활성라디칼에전자를공여하여탈색을유도하는원리를이용한방법으로이러한전자공여작용은지방질산화를억제시키는척도나동맥경화를억제하는척도로사용되고있다 25. DPPH 소거활성법을이용한태음조위탕의기존연구결과 20 에서도우리의결과 (12.87%, 19.38%) 와유사하게추출물의농도 200 μg/ml과 400 μg/ml에서각각 15.58%, 28.26% 의라디칼소거활성을관찰하였다. LDL은혈장단백질 (plasma protein) 중밀도가 1.025 에서 1.058에해당하는지단백 (lipoprotein) 으로중심부는소수성인중성지방 (triglyceride) 과 cholesterol ester (CE) 로이루어져있으며극성인인지질과콜레스테롤에의해둘러싸여있으며, apob-100 단백질을포함하고있다 26. ApoB-100 단백질은 LDL-수용체에결합하는리간드로작용하게되는데, 산화된 LDL은수용체에결합하지못하고대식세포에의해탐식되어동맥경화의주요한발병원인인거품세포를형성하게된다. 라디칼소거활성을통해항산화효과를확인한처방들이실제로 LDL 의산화를억제하는효능을나타내는지 Cu 2+ 에의한 LDL 산화모델을이용하여관찰하였다 27. LDL 산화정도는지질과산화의증가 (TBARS assay) 와 apob-100의음성전하의증가 (REM assay), apob-100의분절화유도를평가함으로써측정하였다. 4종처방모두농도의존적으로 LDL의산화를억제하는것을확인하였다. 태음조위탕추출물의경우 LDL 을구성하는성분중 apob-100의산화를억제하는효능은다른처방추출물과비교하여크게차이가없었으나, 지질과산화를억제하는효능은현저하게낮은것으로관찰되었다. 즉열다한소탕, 양격산화탕, 청심
OS Kim et al. 59 연자탕이대략 100 μg/ml 의농도에서 50% 이상의지질과산화물의생성을억제하는데비해, 태음조위탕의경우 1000 μg/ml 에서도 50% 미만의억제효능을나타냈다. 마지막으로생쥐대식세포주인 RAW 264.7 세포를이용한거품세포모델을이용하여처방의항동맥경화효능을측정하였다 28. LPS와 LDL를동시에처리한대식세포는세포내 cholesterol ester의축적을유도해거품세포를형성하게되는데. 이러한거품세포내지방축적을 Oil Red O 염색을통해확인하였다. 거품세포형성을억제하는효능은항산화효능및 LDL 산화억제효능과일관되게열다한소탕, 양격산화탕, 청심연자탕에서강하게관찰되었다. 태음조위탕의경우실험최고농도인 200 μg/ml 농도까지유의적인거품세포억제효능을관찰할수없었다. 이는 TBARS assay 결과, 태음조위탕이다른처방보다현저하게낮은산화억제효능을갖는것과상관관계가있으며, LDL을구성하고있는지질과단백질중지질의산화를억제하는것이거품세포의형성을억제하는데더효과적일것으로여겨진다. 이상의결과를종합해보았을때, 4종의四象處方이항산화효능을통해 LDL 산화를억제함으로써동맥경화발병의주요원인이거품세포의형성을억제하는것을확인하였다. 특히열다한소탕, 양격산화탕, 청심연자탕의경우거품세포의형성을강력하게억제하는것을확인할수있었다. 본연구결과는四象處方이기존의적응증외에동맥경화와같은산화스트레스에의한질환의예방과치료에이용될수있는기초자료가될것으로기대된다. 1. ABTS 라디칼의소거활성을비교한결과, 추출물의농도가증가함에농도의존적으로라디칼의소거활성이증가하였으며, 4종처방의 RC 50 값은열다한소탕 ; 23.07±1.35 μg/ml, 양격산화탕 ; 37.74±0.94 μ g/ml, 청심연자탕 ; 117.28±1.48 μg/ml, 태음조위탕 ; 204.84±2.15 μg/ml으로측정되었다. 2. DPPH 라디탈의소거활성을비교한결과, 추출물의농도가증가함에농도의존적으로라디칼의소거활성이증가하였으며, 추출물의농도 200 μg/ml 에서열다한소탕 ; 43.64%, 양격산화탕 ; 44.13%, 청심연자탕 ; 34.77%, 태음조위탕 ; 12.87% 의라디칼소거활성을보였다. 3. TBARS assay 결과, 4종처방모두농도의존적으로 LDL 산화를억제하였으며, IC 50 값은열다한소탕 ; 33.54 μg/ml, 양격산화탕 ; 31.23 μg/ml, 청심연자탕 ; 115.38 μg/ml으로관찰되었다. 태음조위탕은실험최고농도인 1000 μg/ml에서 47% 의저해효과를나타냈다. 4. REM assay 결과, 4종처방모두농도의존적으로 LDL 산화를억제하였으며, 추출물의농도 125 μg/ml 에서열다한소탕 ; 84.89%, 양격산화탕 ; 93.02%, 청심연자탕 ; 67.62%, 태음조위탕 ; 66.66% 의억제효과를나타냈다. 5. ApoB-100 단백질의분절화를통해 LDL 산화를확인한결과, 4종처방모두농도의존적으로분절화가회복되는것을관찰할수있었다. 6. 열다한소탕, 양격산화탕, 청심연자탕의거품세포형성억제효과를 Oil Red O 염색을통해확인하였다. 이상의결과로열다한소탕, 양격산화탕, 청심연자탕은항동맥경화활성이있는것으로판단된다. Ⅴ. 結論 4종의四象處方인熱多寒少湯, 凉膈散火湯, 淸心蓮子湯, 太陰調胃湯의항동맥경화활성을연구한결론은다음과같다. Ⅵ. 感謝의말씀 본연구는한국한의학연구원표준한방처방 EBM 구축사업 (K12030) 의지원으로진행되었습니다.
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