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1 J Korean Med Obes Res 2015;15(1): pissn , eissn JKOMOR Original Article 백출, 어성초, 금은화, 황금, 황련과 Metformin 의병용투여시항산화효과및전지방세포분화억제효과 장세주ㆍ한경선ㆍ왕경화ㆍ채희성 1 ㆍ최영희 1 ㆍ진영원 1 ㆍ최한석 2 ㆍ김호준 동국대학교한의과대학한방재활의학과교실, 1 동국대학교약학대학약학교실, 2 동국대학교의과대학내분비내과교실 Effect of Atractylodes Rhizoma Alba, Houttuyniae Herba, Lonicerae Flos, Scutellariae Radix and Coptidis Rhizoma Extracts Combined with Metformin on the Antioxidant and Adipocyte Differentiate Inhibition Seju Chang, Kyungsun Han, Jing-Hua Wang, Hee-Sung Chae 1, Yong Hee Choi 1, Young-Won Chin 1, Han-Seok Choi 2, Hojun Kim Department of Oriental Rehabilitation Medicine, College of Korean Medicine, Dongguk University, 1 Department of Pharmacy, College of Pharmacy, Dongguk University, 2 Department of Endocrinology Medicine, College of Medicine, Dongguk University Received: May 18, 2015 Revised: May 31, 2015 Accepted: June 1, 2015 Correspondence to: Hojun Kim Department of Oriental Rehabilitation Medicine, Dongguk University Ilsan Oriental Hospital, College of Korean Medicine, Dongguk University, 27 Dongguk-ro, Ilsandong-gu, Goyang , Korea Tel: Fax: kimklar@dongguk.ac.kr Copyright 2015 by The Society of Korean Medicine for Obesity Research Objectives: This study was to investigate the antioxiative capacity, antiobesity effects of Atractylodes Rhizoma Alba, Houttuyniae Herba, Lonicerae Flos, Scutellariae Radix, and Coptidis Rhizoma on Raw and 3T3-L1 cell lines. Methods: Three different types of herb extracts (A. Rhizoma Alba, H. Herba, L. Flos, S. Radix, and C. Rhizoma; water 100%, ethanol 30%, ethanol 100%) were used in this study. Total polyphenol compound, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, reactive oxygen species (ROS) activity, NO production and cell proliferation were measured. Results: Total polyphenol compound measurement of L. Flos, A. Rhizogenes, and C. Rhizoma extracts were higher than A. Rhizoma Alba, H. Herba. DPPH radical scavenging activity, ROS activity and NO production of S. Radix, C. Rhizoma extracts were lower than L. Flos, A. Rhizoma, and H. Herba. Conclusions: Metformin and S. Radix, C. Rhizoma, A. Rhizoma Alba, and L. Flos extracts combination groups showed synergistic effect on adipocyte differentiation inhibition and antioxidative activity. Key Words: Metformin, Herbal medicine, Combination, Antioxidative, Adipocyte differentiation 서론 비만은섭취한에너지가신체활동과성장에소비한에너지량보다많아체내지방이과도하게축적되는현상으로 1) 고지혈증, 고혈압, 당뇨병및동맥경화증등각종질환의발병률을증가시키는요소이며 2) 비만으로인한지방세포의크기와양적증가는조직에산화스트레스를증가시킨다. 이 러한산화스트레스는많은지방세포에아디포카인 (adipokines) 을발현시키고, 활성산소 (reactive oxygen species, ROS) 를증가시킨다 3). ROS는산화스트레스에의해생성되며당뇨병, 신부전, 암등여러가지다양한질환의병인으로인정되고있다 4). ROS는산소를포함하는화학적반응성이높은분자로서 superoxide anion (O 2-), hydrogen peroxide (H 2O 2), 24

2 장세주외 : 한약재와 Metformin 병용투여시항산화및전지방세포분화억제효과 hydroxyl radical (OH), peroxinitrite (NO 3-), hypochlorite ion (OCl-) 등을포괄하여이르는용어인데 5) 반응시간이매우짧은반면에반응력이커서염증과세포자멸사를유발시킨다 6). 특히당뇨병환자에서는 ROS의생성과산화의방어기전사이의불균형으로인해산화적손상이잘발생되고, 이런 ROS는인슐린을생성하고분비하는 -cell을파괴시킨다 7). 이에체내산화스트레스를조절하는것은비만관리및당뇨치료에있어필수적이며추후발생할수있는여러합병증의예후에도중요한영향을미치게될것으로생각된다. 여러선행연구에서비만에천연항산화물질을처치후항산화효소를분석한바있고 3), 경제적인천연항산화제발굴을위해과일이나채소의부산물을이용한항산화연구를진행하고있다 8). 그외에도참마와 selenium을병용투여하여지방대사물과지질과산화의작용을통한항산화작용상승효과와 9) streptozotocin 으로당뇨유발한쥐에택사와 chromium 의병용투여를통해혈당과조직내 glycogen 및지질대사에미치는영향에대한연구 10) 등을통해한약재와다른약물과의병용투여시항산화효과와혈당및지질대사에미치는영향에대한연구를하였다. Metformin 은현재제2형당뇨병의환자의 1차약물요법으로사용되고있으며저혈당증상을유발하지않으며혈당을낮추어주어세계적으로 1,200 만명정도가복용하는것으로알려져있다 11). Metformin 은혈당강하작용외에도혈관내피세포에서의염증반응을억제하는작용이있으며 12) 제2형당뇨병의유발인자인비만과인슐린저항성이염증반응과밀접한연관을갖고있음에따라 metformin 을염증반응제어를위한연구에도활용이가능할것으로생각된다. 대표적으로쥐에게 metformin 과대황을병용투여하여항산화효과가증가한연구가있었으며 13) 이를통해 metformin 과다른한약물을병용투여시항산화효과및항염증작용에대해규명하며나아가비만치료에활용할연구가필요할것으로생각된다. 본연구에서는백출, 어성초, 금은화, 황금, 황련을세가지추출방법을퉁해얻은각각의추출물을 metformin 과함께 Raw 세포와 3T3-L1 세포에처리하여총페놀함량분석, 1-diphenyl-2-picrylhydrazyl (DPPH) 라디컬소거능, ROS 생성량, NO 생성량, 세포생존율을측정하였다. 또한 Oil Red O staining을통해지방세포의지방축적억제효과를관찰하였다. 상기실험을통해각각의군에서한 약의추출법에따른효과차이및 metformin 과병용투약시약효상승효과를검증하고자하였다. 재료및방법 1. 실험재료 Macrophage cell line인 RAW 세포는한국세포주은행 (KCLB, Seoul, Korea) 에서분주받아사용하였다. 세포배양은 Dulbecco s modified Eagle s medium (DMEM) 에 10% fetal bovine serum (FBS) 과 2 mml-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin이함유된배지를사용하였다. CO 2 incubator 37 o C, 5% CO 2-95% O 2 조건하에서배양하였다. 실험에사용된백출, 어성초, 금은화, 황금, 황련추출물 (water 100%, 에탄올 30%, 에탄올 100%) 은동국대학교약학대학에서제공받아사용하였다. 각각의세포를정상군 ( 이하 normal, N) 황련열수추출물 (water 100%) 처리군 ( 이하 HLW), 황련 30% 에탄올추출물처리군 ( 이하 HL30), 황련 100% 에탄올추출물처리군 ( 이하 HL100), 황금열수추출물처리군 ( 이하 HGW), 황금 30% 에탄올추출물처리군 ( 이하 HG30), 황금 100% 에탄올추출물처리군 ( 이하 HG100), 백출열수추출물처리군 ( 이하 BCW), 백출 30% 에탄올추출물처리군 ( 이하 BC30), 백출 100% 에탄올추출물처리군 ( 이하 BC100), 금은화열수추출물처리군 ( 이하 GMHW), 금은화 30% 에탄올추출물처리군 ( 이하 GMH30), 금은화 100% 에탄올추출물처리군 ( 이하 GMH100), 어성초열수추출물처리군 ( 이하 OSCW), 어성초 30% 에탄올추출물처리군 ( 이하 OSC30), 어성초 100% 에탄올추출물처리군 ( 이하 OSC100) 등총 15개의군으로만들었다. 2. 세포배양 RAW cell line과 3T3-L1 cell line은 DMEM에 10% FBS와 2 ml glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin 이함유된배양액을사용하여 37 o C, 5% CO 2, 90% humidity의조건하에서배양되었다. 배양된세포는 2 3일에한번씩배양액을바꾸어주면서배양하여세포분화가최대에도달하였을때 phosphate buffered saline (PBS) 으로세포를세척한후 trypsin-ethylenedia- 25

3 minetetraacetic acid 용액으로부착된세포를분리한후원심분리하여세포를모은다음세포와배지를잘혼합하여계대배양하여사용하였다. 3. Total phenolic compound 함량측정각각의추출물 40 l와 Folin-ciocalteu s reagent 200 l, water 1,160 l를혼합하여 3분간 incubator 에서반응시키고, 20% Na 2CO l를첨가한다. 2시간반응시킨후에원심분리하여상층액을 96 well plate에 200 l/well 이되도록옮기고, microplate spectrophotometer를이용하여 765 nm에서흡광도를측정하였다. 총페놀함량은 gallic acid를표준용액으로검량선을구하고계산하였다. 4. DPPH 라디컬소거능분석 DPPH 라디컬소거능측정은각각의추출물 40 l에 300 M DPPH/EtOH 용액을 760 l 첨가하고 37 o C에서 30분동안반응시킨후, microplate reader에서 515 nm 파장으로흡광도를측정하였다. 5. Intercellular ROS activity assay 3T3-L1 세포를 6 well-plate 에 cells/well로 seeding 하고 18시간이지난후, 각각의추출물을 200 g/ ml를첨가하여 6시간동안반응시킨다. 이후 Dulbecco s phosphate buffered saline (DPBS) 로 washing하고 0.25% TE 100 l/well을첨가한후, 1,000 rpm으로 5분간원심분리한다. 상등액을제거한후 DPBS 1 ml를첨가한후 fluorescence activated cell sorter (FACS) tube로옮긴다. 1,000 rpm으로 5분간원심분리한후상등액을제거한다. 500 l/tube dihydrohodamine (1:200 PBS dilution; 5 10 M) 을첨가한후 37 o C에서 30분간 incubation 하고 DPBS 용액으로 3번 washing 한다. 다시 DPBS 1 ml를첨가하여 37 o C에서 10분간 incubation 한후 FACS detection 하였다. 6. NO assay 24 well-plate 에 Raw 세포를 cells/well로 seeding하고 24시간후각각의추출물을 200 g/ml를첨가하여 24시간동안반응시켰다. Lipopolysaccharide (LPS) 0.1 g/ml를각각의 well에처리하고 24시간동안 37 o C 5% CO 2 incubator에서배양후세포배양상등액 100 l를채취하여여기에그리스시약 100 l를혼합하여 15분동안반응시킨후 microplate reader를이용하여 540 nm에서흡광도를측정하였다. 7. Cell viability assay 96 well-plate에 3T3-L1 세포를 cells/well로 seeding하고 24시간후에각각의추출물들을 well당 100 l 씩분주하여 24시간반응시키고, EZ-Cytox WST regent 10 l씩각 well에가하고 2시간동안배양한후 microplate reader를이용해 450 nm에서흡광도를측정하였다. 8. Oil red O staining assay 마우스지방세포인 3T3-L1 cell line을 6 well plate에서 cells/well로 seeding한후세포가 full confluence가될때까지배양한다. 세포가 full confluence 가될때까지배양한이후에배지를 1 um dexamethasone, 0.5 mm isobutylmethylxanthine, 10 g/ml insulin을포함한 DMEM 배지 (differentiation media) 로바꾼후 48시간동안배양한다. 이후 10 g/ml의 insulin을포함하는 DMEM 배지 (maturation media) 를처리한후분화를유도한다. 분화가유도된세포는 lipid droplet의크기와개수가증가하며, 이러한분화지방세포를다양한농도의샘플과양성대조군을처리한후 Oil O Red staining을통해서지방축적을억제하는효과를분석한다. 9. 통계방법실험결과는평균값 ± 표준편차 (mean±standard deviation) 로표시하였다. DPPH 라디컬소거능, 세포생존율분석은총 3회의실험을수행하였다. Independent t-test를통해군간비교, paired t-test를통해전후비교를하였으며, 통계적유의성은 P값이 0.05 이하인경우유의한것으로간주하였다. 결과 1. 한약재 5종의추출방법에따른항산화활성비교 황련, 황금, 백출, 금은화, 어성초의추출방법에따른총 26

4 장세주외 : 한약재와 Metformin 병용투여시항산화및전지방세포분화억제효과 Table 1. Total Phenolic Compound and DPPH Radical Scavenging Activity of Five Herbs Herb Extraction procedure Total phenolic compound (mg/g) DPPH scavenging IC50 ( g/ml) Coptidis Rhizoma Water 100% Ethanol 30% Ethanol 100% Scutellariae Radix Water 100% Ethanol 30% Ethanol 100% Atractylodes Water 100% 0.12 Rhizoma Alba Ethanol 30% 0.06 Ethanol 100% 2.53 Lonicerae Flos Water 100% Ethanol 30% Ethanol 100% Houttuyniae Herba Water 100% Ethanol 30% Ethanol 100% DPPH: 1-diphenyl-2-picrylhydrazyl. 페놀함량과 DPPH 라디컬소거능을비교한결과는 Table 1 에나타냈다. 총폴리페놀함량및 DPPH 소거능모두백출군에서가장낮은결과를보였다. 특히 DPPH 라디컬소거능의결과값은측정되지않았다. 황련군에서는총페놀함량이 30% 에탄올추출물에서가장높게측정된반면황금은 100% 열수추출물, 금은화와어성초는에탄올 100% 추출물에서가장높은폴리페놀함량을나타냈다. DPPH 라디컬소거능은황련에서가장우수한결과는나타냈고그중열수추출물이가장뛰어난효과를보였다. 황금과어성초는에탄올 100% 군에서가장좋은 DPPH 소거능을보였으며금은화의경우 30% 에탄올군에서높은 DPPH 소거능을보였다. 3T3-L1 세포에각한약추출물을처리한후세포내 ROS 활성도를측정한결과대조군은 29.5%, metformin 2 mm군은 25.1% 가측정되었다. OSCW+Met 1 mm군은 28.3%, OSC30+Met 1 mm군은 27.8%, OSC100+ Met 1 Fig. 1. The level of ROS activity in 3T3-L1 cells. HL: Coptidis Rhizoma water 100% extract, HL30: C. Rhizoma ethanol 30% extract, HL100: C. Rhizoma ethanol 100% extract, HG: Scutellariae Radix water 100% extract, HG30: S. Radix ethanol 30% extract, HG100: S. Radix ethanol 100% extract, BC: Atractylodes Rhizoma Alba water 100% extract, BC30: A. Rhizoma Alba ethanol 30% extract, BC100: A. Rhizoma Alba ethanol 100% extract, GMH: Lonicerae Flos water 100% extract, GMH30: L. Flos ethanol 30% extract, GMH100: L. Flos ethanol 100% extract, OSC: Houttuyniae Herba water 100% extract, OSC30: H. Herba ethanol 30% extract, OSC100: H. Herba ethanol 100% extract. 27

mm군은 24.1%, HLW+Met 1 mm군은 28.3%, HL30+ Met 1 mm군은 36.7%, HL100+Met 1 mm군은 41.0%, HGW+Met 1 mm군은 17.3%, HG30+Met 1 mm군은 19.6%, HG100+Met 1 mm군은 22.0%, BCW+Met 1 mm군은 31.5%, BC30+Met 1 mm군은 29.

5 mm군은 24.1%, HLW+Met 1 mm군은 28.3%, HL30+ Met 1 mm군은 36.7%, HL100+Met 1 mm군은 41.0%, HGW+Met 1 mm군은 17.3%, HG30+Met 1 mm군은 19.6%, HG100+Met 1 mm군은 22.0%, BCW+Met 1 mm군은 31.5%, BC30+Met 1 mm군은 29.5%, BC100+ Met 1 mm군은 34.6%, GMHW+Met 1 mm군은 26.8%, GMH30+Met 1 mm군은 27.0%, GMH100+Met 1 mm 군은 20.9% 가측정되었다 (Fig. 1). 2. Metformin과 5종의한약재병용투여시염증억제능및세포생존율비교백출, 금은화, 황금, 황련, 어성초의단독추출물에 metformin 1 mm을첨가하여 3T3-L1 세포에서세포생존율을측정한결과는 Fig. 2에나타내었다. GMHW군 200 g/ml 에서 0.459, GMH30군 200 g/ml에서 0.45, GMH 100군 200 g/ml에서 0.455, HLW군 200 g/ml에서 0.485, OSCW군 200 g/ml에서 0.459가측정되어, 대조군의측정값 0.597에비해통계적으로유의하게감소하였음을관찰할수있었다 (P<0.05). 이외의군들에서도경미한증가 및감소가보였지만통계적으로유의성은보이지않았다. Metformin과다섯가지한약의병용투여및한약의단독투여시염증생성억제능력을평가하기위해 NO 생성을측정한결과, 대조군은 2.08, LPS를처리한군은 8.05로측정되었고 LPS군이 N군에비해통계적으로유의하게증가한것을확인하였다 (P=0.017). GMH100군은 5.83, HGW 군은 3.20, HG30군은 3.15, HG100군은 4.10, HLW군은 3.45, HL30군은 2.79, HL100군은 2.64, OSC30군 5.69로측정되어 LPS군과비교시통계적으로유의하게감소하였다. HL30군은 2.79로통계적으로가장유의한것으로나타났다 (P=0.0019). 이외의군들에서도경미한감소가보였지만통계적으로유의성은보이지않았다. Metformin 1 mm 을병행투여한군에서는 BC100군은 4.93, GMH30군은 5.25, GMH100군은 5.82, HGW군은 2.92, HG30군은 2.89, HG100군은 3.64, HLW군는 2.97, HL30군은 2.61, HL100군은 2.36, OSCW군은 4.57, OSC30군은 4.87, OSC100군은 4.15로측정되어 LPS군과비교시통계적으로유의하게감소하였다 (P<0.05) HG100군은 3.74로통계적으로가장유의한것으로나타났다 (P=0.0036). 이외의 Fig. 2. The level of cell proferation in 3T3-L1 cells. BC: Atractylodes Rhizoma Alba water 100% extract, BC30: A. Rhizoma Alba ethanol 30% extract, BC100: A. Rhizoma Alba ethanol 100% extract, GMH: Lonicerae Flos water 100% extract, GMH30: L. Flos ethanol 30% extract, GMH100: L. Flos ethanol 100% extract, HG: Scutellariae Radix water 100% extract, HG30: S. Radix ethanol 30% extract, HG100: S. Radix ethanol 100% extract, HL: Coptidis Rhizoma water 100% extract, HL30: C. Rhizoma ethanol 30% extract, HL100: C. Rhizoma ethanol 100% extract, OSC: Houttuyniae Herba water 100% extract, OSC30: H. Herba ethanol 30% extract, OSC100: H. Herba ethanol 100% extract. *P-value<0.05 as compared to 24 hours group. P-value<0.05 as compared to control. 28

장세주외 : 한약재와 Metformin 병용투여시항산화및전지방세포분화억제효과 Fig. 3. The comparison of NO production between herbal treatment and metformin and herb combination treatment in Raw 264.7 cells.

Rhizoma Alba ethanol 100% extract, GMH: Lonicerae Flos water 100% extract, GMH30: L. Flos ethanol 30% extract, GMH100: L. Flos ethanol 100% extract, HG: Scutellariae Radix water 100% extract, HG30: S.

6 장세주외 : 한약재와 Metformin 병용투여시항산화및전지방세포분화억제효과 Fig. 3. The comparison of NO production between herbal treatment and metformin and herb combination treatment in Raw cells. LPS: lipopolysaccharide, BC: Atractylodes Rhizoma Alba water 100% extract, BC30: A. Rhizoma Alba ethanol 30% extract, BC100: A. Rhizoma Alba ethanol 100% extract, GMH: Lonicerae Flos water 100% extract, GMH30: L. Flos ethanol 30% extract, GMH100: L. Flos ethanol 100% extract, HG: Scutellariae Radix water 100% extract, HG30: S. Radix ethanol 30% extract, HG100: S. Radix ethanol 100% extract, HL: Coptidis Rhizoma water 100% extract, HL30: C. Rhizoma ethanol 30% extract, HL100: C. Rhizoma ethanol 100% extract, OSC: Houttuyniae Herba water 100% extract, OSC30: H. Herba ethanol 30% extract, OSC100: H. Herba ethanol 100% extract. *P-value<0.05 as compared to LPS group. P-value< 0.05 as compared to control. 군들에서도경미한감소가보였지만통계적으로유의성은보이지않았다 (Fig. 3). 3. 한약과메트포민병용투여시지방분화억제능력평가대조군은 0.417이측정되었다. BC30군과 1 mm metformin을병용투여한군은 0.197, GMH30군과 1 mm metformin을병용투여한군은 0.224로측정되었으며통계적으로유의한것으로나타났다 (P<0.05). BC30 군이 GMH30 군에비해측정값이낮게측정되었으며통계적으로가장유의한것으로나타났다 (P=0.016). 이외의군들에서도경미한감소가보였지만통계적으로유의성은보이지않았다 (Fig. 4). 고찰 Fig. 4. Oil Red O staing 3T3-L1 cells treated with 5 types of herbs combination with metformin. CON: control, HL: Coptidis Rhizoma water 100% extract, HL30: C. Rhizoma ethanol 30% extract, HL100: C. Rhizoma ethanol 100% extract, HG: Scutellariae Radix water 100% extract, HG30: S. Radix ethanol 30% extract, HG100: S. Radix ethanol 100% extract, BC: Atractylodes Rhizoma Alba water 100% extract, BC30: A. Rhizoma Alba ethanol 30% extract, BC100: A. Rhizoma Alba ethanol 100% extract, GMH: Lonicerae Flos water 100% extract, GMH30: L. Flos ethanol 30% extract, GMH100: L. Flos ethanol 100% extract, OSC: Houttuyniae Herba water 100% extract, OSC30: H. Herba ethanol 30% extract, OSC100: H. Herba ethanol 100% extract. *P-value<0.05 compared to metformin alone. 백출에관한대표적인약리적인연구로는항산화효과가잘알려져있으며 14), 백출열수추출액이 Th1 type 사이토카 인유도및대식세포에서 NO 조절등에관여하여면역반응에관여한다고보고된바가있다 15). 또한백출이 phospho- 29

7 Akt가역할을담당하는세포성장억제를통해비만세포의성장을억제하며 16), 백출및발효백출의위장관계보호효과 17) 도보고된바있다. 어성초에관한연구로는항염증효능 18), 대동맥평활근세포이주및증식억제효과 19), 면역활성효과 20), 염증성매개물질에대한억제효과 21) 에관한실험적연구가보고되었다. 어성초를함유하는처방인가미패독산은항알레르기활성과항염증활성효과를나타내어알레르기와염증의치료제로서개발가능성이있다는보고가있다 22). 금은화는폴리페놀등다양한생리활성물질을함유하여항산화및항균력이있으며 23), 금은화및금은화전초가 Raw cell에서 LPS로유도된 NO의생성, inos, COX-2 및 cytokine 에영향을미쳐 gram-negative bacterial infection에의한염증반응에서염증관련지표들을억제함을보고한바있다 24). 황금의성분으로는 flavonoid계화합물로서, baicalein, biacalin, chrysin, oroxylin-a, oroxylin- -7-O-glucuronide, wogomin, wogonoside, skullcapflavone, dihydrobaicalin 등이있으며 25), 황금에관한국내연구로는항염및항과민, 항균및항바이러스, 항산화작용에관한보고되었다 26). 이밖에황금추출물의섭취는혈장과간의총콜레스테롤과중성지방의농도는낮추고, 혈장의 high density lipoprotein (HDL)-콜레스테롤농도, 총콜레스테롤에대한 HDL-콜레스테롤비율은높이고동맥경화지수를낮추어항고지혈증효과가있음을보고한바있다 27). 황련에관한연구로는위점막보호효과 28), 항균효과 29), 항암효과 30) 등에관한실험적연구가보고되었다. 황련의임상적유용의가능한분야로는심혈관계효과, 간보호효과, 항당뇨효과가보고한바있다. 또한황련의 berberrubine과 coptisine 성분의수산화라디칼제거능력은기존항산화제인 trolox보다강력한것으로보고되었다 31). 본연구는항염증, 항지질등의효과가있는백출, 어성초, 금은화, 황금, 황련추출물을 metformin 과병용하였을때항염증항산화효과를알아보기위하여 total polyphenol compound 함량, total phenolic compound 함량, DPPH 라디컬소거능, ROS, NO 생성량을측정하였으며, 세포독성여부에대해 cell proliferation 을측정하였다. Total phenolic compound 함량에서황금, 황련, 금은화추 출물이백출, 어성초추출물들에비해높았으며그중에서 HGW군과 HG30군이 mg/g, mg/g으로가장높게측정되었다. 황금의경우물을이용한추출법이 total phenolic compound를추출하는데용이할것으로생각된다. DPPH 라디컬소거능은황련추출물이가장높았고, 황금추출물, 금은화추출물은어성초추출물에비해낮았다. 백출추출물은 radical 소거능을측정할수없었으며, 이는추출된 total phenolic compound 함량이가장낮음에따라항산화효과가나타나지않는것으로생각된다. 어성초, 황련군에서 total phenolic compound의추출량이증가할수록라디컬소거능이증가되는것으로나타났으나황금, 금은화군의경우 total phenolic compound 함량의증가와라디컬소거능과의상관관계가나타나지않는것으로보아추후지속적인연구를통해추출방법에따른 total phenolic compound 구성성분차이및라디컬소거능과의규명도필요할것으로생각된다. ROS 측정시어성초, 황금, 금은화 1 mm metformin 처리군이 N군의 29.5% 보다낮게측정되었으며황금 1 mm metformin 처리군은 metformin 2 mm 단독처리군의 25.1% 보다낮게측정되었다. 이중 HGW 1 mm metformin 처리군은 17.3% 로가장낮게측정되었다. NO 측정시에는 HL30 단독투여군이 2.79로통계적으로가장유의한것으로나타났으며 metformin 병행투여시 HG100군은 3.74로통계적으로가장유의한것으로나타났다. 상기의결과들로보아황금과황련추출물들이 metformin과병용투여하였을때어성초, 금은화, 백출추출물들에비해항산화작용에효과적임을알수있었다. 황금의경우에탄올을이용한추출물이물을이용한추출물에비해항산화와관련있는성분추출에유의함을알수있었다. 세포독성에대해알기위해시행한 cell proliferation에서는 GMHW군 200 g/ml, GMH30군 200 g/ml, GMH100군 200 g/ml, HLW군 200 g/ml, OSCW군 200 g/ml 처리하였을때유의하게감소하였다. 전체적으로살펴봤을때경미한증가와감소가보였지만대조군의측정값과수치상큰차이가없어유해하지않다는것을알수있었다. 지방세포분화억제를위해시행한 Oil Red O staing 결과에서는 BC30군과 GMH30이유의하게감소하였다. 30

8 장세주외 : 한약재와 Metformin 병용투여시항산화및전지방세포분화억제효과 BC30군과 1 mm metaformin과병행투여시 0.224로측정되었으며통계적으로가장유의한것으로나타났다. 이번실험을종합하면황금과황련추출물들이 metformin과병용투여하였을때황산화작용에효과적이며지방세포분화억제에는백출과금은화추출물들이 metformin과병용투여하였을때유의성있는것으로관찰되었다. 그리고황금은에탄올 100%, 금은화는에탄올 30% 로추출하였을경우좋은결과를나타내었다. 한약물을다양한추출법을통한추출물을 metformin 과병용하여상호작용효과에대해알아보려하였다. 이를통해추출법에따라추출물의구성성분차이가다양하게날것으로생각되며이를다른약물과병용하였을때상호작용또한다양하게나타날것으로생각된다. 본연구를통해추후효과적인 metformin과한약물의병용을위한추출방법과제형에대한연구및 metformin 과한약물의상호작용규명을위한연구에필요한유효성분추출에관한연구가지속적으로이어져야될것으로생각된다. 결론 Metaformin과백출, 어성초, 금은화, 황금, 황련추출물 (water 100%, 에탄올 30%, 에탄올 100%) 을함께처리하여황산화작용과세포독성, 3T3-L1 지방세포분화억제에대해분석한결과다음과같은결론을얻었다. 1. 황금, 황련, 금은화추출물들은 total phenolic compound 함량측정시백출, 어성초추출물들에비해높게측정된다. 2. 황금, 황련추출물은 DDPH radical 소거능, ROS, NO 측정시, 금은화, 백출, 어성초추출물에비해낮게측정된다 (Fig. 3). 3. Oil red O staining 결과백출, 금은화추출물은다른추출물에비해지방세포분화억제하는경향이있다 (Fig. 4). 4. 단독투여시황련은에탄올 30% 추출물이, metaformin 병용투여시황금은에탄올 100% 로추출물이효과가가장좋은것으로관찰되었다. 5. Metformin 과황금, 황련, 백출, 금은화추출물의병용은 metformin 단독사용시보다염증조절및지방세포분화억제작용효과가유의하게나타났으며, 추후지속적인 연구를통해각각의한약물과 metformin 상호작용이해및약효증가시키는병용약물에대한개발이필요할것으로생각된다. 감사의글 본연구는한국보건산업진흥원을통해보건복지부 ( 양한방융합기반기술개발사업 ) 의재정지원을받아수행된연구임 (HI14C0558). References 1. Lee MA, Park JK, Um MH, Jeon JW, Lee JM, Park YK. Lipolytic effect of Sparassis crispa extracts in differentiated 3T3-L1 cells and high fat diet-induced obese mice. J Korean Soc Food Sci Nutr ; 41(12) : Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell ; 104(4) : Carriere A, Carmona MC, Fernandez Y. Mitochondrial reactive oxygen species control the transcription factor CHOP-10/ GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect. J Biol Chem ; 279(39) : Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in nor-mal physiological functions and human disease. Int J Biochem Cell Biol ; 39(1) : Lee SS. Role of reactive oxygen speciec in allergic rhinitis. Korean J Otorhinolaryngol-Head Neck Surg ;55(9) : Chung HY. Aging and carcinogenic mechanisms induced by free fadicals. Korean J Gerontol ; 2(1) : Kim HJ, Lee SG, Park SJ, Yu MH, Lee EJ, Lee SP, et al. Antioxidant Effects of extracts from fermented red ginseng added with medicinal herbs in STZ-induced diabetic rats. Korean J Food Sci Thchnol ; 44(3) : Kim MJ, Kim YG, Kim HS, Cheong C, Jang KH, Kang AS. Effect of antioxidant activities in ethanol extract of apple peel, grape peel and sweet potato peel ad natural antioxidant. J Korean Acad Ind Coop Soc ; 15(6) : Kim MH. Effects of H2O-fraction of Dioscorea japonica Thunb and selenium on lipid peroxidation in streptozotocin-induced diabetic rats. Korean J Food Cook Sci ; 17(4) : Lim SJ, Park JE. Effects of H 2O Fraction of alisma canaliculatum with chromium on blood glucose levels and lipid metabolism in streptozotocin-induced diabetic rats. Korean J Food Sci Technol ; 34(4) :

9 11. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F. Cellular and molecular mechanisms of metformin: an overview. Clin Sci ; 122(6) : Balavoine GGA, Geletii YV. Peroxynitrite scavenging by different antioxidants. Part 1: convenient assay, Nitri Oxide ; 3(1) : Patel RP, McAndrew J, Sellak H, White CR, Jo H, Freeman BA, et al. Biological aspects of reactive nitrogen species. Biochim Biophys Acta ; 1411(2-3) : Han JH, Kim JH, Kim SG, Jung SH, Kim DH, Kim GE, et al. anti-oxidative compounds from the aerial parts of Atractylodes macrocephala Koidzumi. Yakhak Hoeji ; 51(2) : Shin SW, Lee YS, Park JH. Kwon TK, Suh SI, Kwon YK. Comparison of immunomodualtory effects of water-extracted Ginseng Radix, Pilose Asia-bell, Astragali Radix, Astractylodes Rhizoma alba and Dioscoreae Rhizoma. Korean J Orient Physiol Pathol ; 18(4) : Kim CK, Kim M, Oh SD, Lee SM, Sun B, Choi GS, et al. Effects of Atractylodes macrocephala Koidzumi rhizome on 3T3-L1 adipogenesis and an animal model of obesity. J Ethnopharmacol ; 137(1) : Han KS, Kim KC, Kim HJ. Effects of Atractylodes macrocephala Koidzumi rhizome on 3T3-L1 adipogenesis and an animal model of obesity. J Korean Med Obes Res ; 13(1) : Hwang IS, Kim YJ, Park YS, Kim HJ, Kim DH, Park W. Anti-inflammatory effect of Houttuyniae Herba water extract on LPS-induced RAW mouse macrophages. Korean J Herbol ; 29(4) : Han JH, Park SN, Yoon MS, Choi OB. Effects of Houttuynia cordata Thunb extract inhibits on the migration and proliferation of vascular smooth muscle cell. Korean J Pharmacogn ; 42(2) : Kim JH, Kim YS, Lim EM. Immune enhancing effect of Houttuyniae Herba on mouse macrophage. J Orient Obstet Gynecol ; 25(2) : Lee HJ, Kim KJ. The effects of Houttuyniae Herba on the mastcell-mediated inflammatory responses. J Korean Orient Med Ophthalmol Otolaryngol Dermatol ; 22(2) : Kim DN, Kim JY, Han EH, Oh KN, Kim SH, Jin MR, et al. Gamipaidok-san possesses antiallergic and anti-inflammatory activities. Korean J Orient Physiol Pathol ; 19(6) : Bae JH, Kim MS, Kang EH. Antimicrobial effect of Lonicerae Flos extracts on food-borne pathogens. Korean J Food Sci Technol ; 37(4) : Lee DE, Lee JR, Kim YW, Kwon YK, Byun SH, Shin SW, et al. Inhibition of lipopolysaccharide-inducible nitric oxide synthase, TNF-, IL-1 and COX-2 Expression by flower and whole plant of Lonicera japonica. Korean J Orient Physiol Pathol ; 19(2) : Kim HC. Hanyakyakri-hak. Seoul : Jipmoondang : Kim KS, Kim LH, Rhee YJ, Lee SH, Choi JH, Ko HN. Analysis on research trend of studies related with Scutellariae Radix in Korea. Korean J Orient Physiol Pathol 2011 ; 25(6) : Yoon HJ, Park YS. Effects of Scutellaria baicalensis water extract on lipid metabolism and antioxidant defense system in rats fed high fat diet. J Korean Soc Food Sci Nutr ; 39(2) : Mou JC, Lee SN, Kim MG, Sea IB, Leem KH. Effects of Coptidis Rhizoma herbal acupuncture extract on the acute gastric mucosal lesion progression induced by compound 48/80 in rats. Korean J Herbol ; 28(1) : Sea HS. An experimental study on the anti-bacterial effect of Coptidis Rhizoma extract compared with quntity of Pseudomonas aeruginosa. J Korean Inst Herb Acupunct ; 10(1) : Tang J, Feng Y, Tsao S, Wang N, Curtain R, Wang Y. Berberine and Coptidis Rhizoma as nov- el antineoplastic agents: a review of traditional use and biomedical investigations. J Ethnopharmacol ; 126(1) : Kim KG, Lee HT, Ku KH, Hong JW, Cho SI. Review of pharmacological effects of Coptidis Rhizoma and its bioactive compounds. J Korean Oriental Med ; 33(3) :

Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,