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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 46(4), 401~408(2017) 배암차즈기에탄올추출물의 3T3-L1 지방전구세포분화억제및지방축적저해효과 김성옥 1 김미려 2 황경아 3 박노진 4 정지숙 4 1 경성대학교식품응용공학부식품영양전공, 2 대구한의대학교한의과대학약리학교실 3 농촌진흥청국립농업과학원농식품자원부, 4 구례군농업기술센터구례야생화연구소 Inhibition of Differentiation and Anti-Adipogenetic Effect of the Salvia plebeia R. Br. Ethanol Extract in Murine Adipocytes, 3T3-L1 Cells Sung-Ok Kim 1, Mi-Ryeo Kim 2, Kyung-A Hwang 3, No-Jin Park 4, and Ji-Suk Jeong 4 1 Department of Food Science & Biotechnology (Nutrition), Kyungsung University 2 Department of Herbal Pharmacology, College of Korean Medicine, Daegu Hanny University 3 Department of Agrofood Resources, National Academy of Agricultural Sciences, Rural Development Administration 4 Wild Flower Institute of Gurye-gun Agricultural Center ABSTRACT Salvia plebeia R. Br. (Lamiaceae) has been used in folk medicines in Asian countries, including Korea and China, to treat inflammatory diseases. The focus of our research was on the anti-adipogenic activity of ethanol extract from Salvia plebeia R. Br. (SPE) in 3T3-L1 adipocytes. This study investigated inhibition of differentiation and lipogenesis upon SPE treatment in 3T3-L1 cells. The results reveal that SPE at non-cytotoxic concentration significantly suppressed triglyceride accumulation and reduced expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein-alpha, and sterol regulatory element-binding protein as adipogenic transcription factors in 3T3-L1 adipocytes compared to non-treated control cells. Inducible phosphorylation of AMP-activated protein kinase, acetyl CoA carboxylase, and hormone-sensitive lipase as well as carnitine palmitoyltransferase-1 mrna expression increased upon SPE treatment, which suppressed expression of fatty acid synthase. In conclusion, these results demonstrate that SPE can inhibit expression of adipogenic genes in 3T3-L1 adipocytes. Our study suggests that SPE has potential anti-obesity effects and is a novel therapeutic functional agent with anti-adipogenic activity via reduction of lipogenesis. Key words: anti-adipogenic activity, Salvia plebeia R. Br., adipocytes, inhibition differentiation, lipogenesis 서 에너지소비량보다영양소를과다섭취할경우에너지불균형에의해비만이유발되며, 최근서구화된식습관과영양분의과잉섭취로인해비만인구가증가하고있다 (1). 세계보건기구 (World Health Organization, WHO) 발표에의하면비만유병률이 1980년에비해 2008년전세계적으로거의두배증가하였다 (2). 생활수준의향상에따라식이섭취패턴이총지방섭취량에서동물성지방으로부터섭취하는에너지비율이증가하면서체질량지수 (body mass index, BMI) 가 25(kg/m 2 ) 이상인비만인구가꾸준히증가하고있으며, 이로인한각종생활습관병이국민의건강을위 Received 11 January 2017; Accepted 30 March 2017 Corresponding author: Ji-Suk Jeong, Gurye Wild Flower Institute of Gurye-gun Agricultural Center, Jeonnam 57660, Korea herojisuk@hanmail.net, Phone: 론 협하는문제가되고있다. 비만은체내에지방조직이과다하게축적된상태를의미하는것으로혈장으로부터지방세포로유입된지방산과포도당이에스테르화하여주로중성지방의형태로축적되며, 단순히체중이증가하는것이외에당뇨, 고혈압, 지방간, 고콜레스테롤혈증, 심혈관질환, 호흡기질환, 종양, 불임, 우울증및사회부적응등을유발하여이차적으로심각한건강장애를초래할수있으므로비만을예방, 개선또는치료하는것이신체를건강하게유지하는데중요하다고할수있다 (3). 본연구에사용한배암차즈기 (Salvia plebeia R. Br.) 는꿀풀과 (Labiatae) 의두해살이풀로잎의모양이곰보같다고하여곰보배추, 뱀비늘처럼생겼다고뱀배추등으로불린다. 한방에서는향기로운가지가달린풀이라하여여지초 ( 荔枝草 ) 라고도하며, 설견초, 나인초, 수양이, 과동청이라고도한다 (4). 배암차즈기는쓴맛과떫은맛이강하고매운맛이나는특징이있으며, 민간에서는주로기침, 천식, 염증등에효과

2 402 김성옥 김미려 황경아 박노진 정지숙 가있는것으로알려져있다. 한의학적으로성미가고신, 량 ( 凉 ) 하며, 청혈해옥, 양혈산어, 이수소종등의효능이있어, 감모발열, 인후종통, 패열해수, 토혈, 뇨혈, 치창출혈, 신염누종, 백탁, 치질, 옹종창독, 습진소양, 질타손상등을치료한다고하였다 (5). 배암차즈기의주요성분으로는플라보노이드와페놀성물질, 사포닌, 강심배당체, 불포화스테롤, 정유등이있으며, 종자에는지방유를함유하고있다. 배암차즈기경엽을메탄올로추출하여추출액의에틸아세테이트분획을 1H NMR, 13C NMR 등으로구조를분석한결과, rosmarinic acid, luteolin 7-O-β-glucopyranoside, hispidulin 7-O-glucopyranoside 등이동정되었다 (6). 배암차즈기의연구는 2008년도처음 SCI 논문에서성분연구 (7) 가발표되고그후항산화성분이알려지면서아토피및염증관련질환등의연구 (8-11) 가다수발표되었으며, 최근에는비만과관련된연구 (12,13) 가일부보고되고있다. 본연구는배암차즈기에탄올추출물이지방의축적억제및지방합성관련유전자의 mrna, DNA 발현을조절하여비만예방, 개선또는치료용천연물을발굴하기위해 3T3- L1 전지방세포에서지방세포로성숙되는과정에서지방세포의분화와성숙단계에서분화를조절하는유전자 CCAAT/ enhancer-binding protein-alpha(c/ebpα), peroxisome proliferator-activated receptor gamma(pparγ), sterol regulatory element-binding protein(srebp-1c) 과지방세포의성숙단계에서지방합성및억제에관여하는유전자 phosphorylation of AMP-activated protein kinase (pampk), phosphorylation of acetyl CoA carboxylase (pacc), carnitine palmitoyltransferase-1 activity(cpt- 1), hormone-sensitive lipase(hsl), fatty acid synthase (FAS) 의전사유전자발현과단백질발현수준에미치는영향을조사하였다. 농축 (H-1000VW, NVC-2100, SB-1000, Eyela, Tokyo, Japan) 하여농축된시료를 -80 C 에서동결건조 (FDU-2100, Eyela) 하여배암차즈기에탄올추출물 (SPE) 을제조하였다. 세포배양 3T3-L1 마우스유래배아섬유아세포 (mouse embryo fibroblast) 는미국세포주은행 (ATCC, Rockville, MD, USA) 에서구입하였다. 먼저 10% bovine calf serum(bcs, Sigma- Aldrich Co., St. Louis, MO, USA) 을함유하는 Dulbecco s modified Eagle s medium(dmem, WELGENE Inc., Daegu, Korea) 으로 37 C, 5% CO 2 incubator에서배양하였다 (day 0). 8일동안배양한다음에전지방세포 (preadipocyte) 를분화유도배양액인 10% fetal bovine serum(fbs, Sigma- Aldrich Co.) 을함유한 DMEM 배지 (2 μg/ml insulin, 0.5 mm isobutylmethylxanthine, 2 μm dexamethasone 첨가 ) 로옮겨 SPE를처리하여 48시간배양하였다. 이틀후 30 μg SPE/2 μg 인슐린 /ml 10% FBS DMEM 배지에서 48시간더배양한다음에 10% FBS DMEM 배지에서 96시간동안배양하여성숙한지방세포 (adipocytes) 에서 RNA 와단백질을분리하여유전자의발현변화를측정하였다. 세포생존율측정 3T3-L1 세포를 96-well plate에 cells/ml로분주하여안정화한후 SPE를농도별로처리하여 24시간배양한다음배지를제거하고 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide(mtt) 시약을 0.5 mg/ml 농도로첨가하여 37 C, 5% CO 2 항온기에서 2시간반응시킨후배지는제거하고 DMSO로불용성결정을용해시킨다음 ELISA reader(tecan Austria GmbH, Grödig, Austria) 로 570 nm 파장에서흡광도를측정하였다. 재료및방법배암차즈기에탄올추출물제조본실험에사용된배암차즈기 (Salvia plebeia R. Br.) 는전라남도구례군농업기술센터친환경시험장 ( 위도 , 경도 ) 노지에서재배된것으로 3월중순에길이 10~20 cm의것을채취하여지상부를사용하였다. 지상부는이물질제거및수세후열풍건조기 (SH-FDO 150, Samheung, Sejong, Korea) 로 45 C에서 12시간동안열풍건조하였다. 열풍건조한시료는분쇄기 (SMX-4OOODY, Shinil, Seoul, Korea) 로분쇄하여 -80 C deep freezer (MDF-U53V, Sanyo, Osaka, Japan) 에동결보관하면서추출물제조에사용하였다. 분쇄한시료를각각 20 g씩정량하고 400 ml의 70% 에탄올로 heating mentles(whm 12295, Daihan Scientific Co., Ltd., Wonju, Korea) 을이용하여에탄올추출물을제조하였다. 추출은 70 C에서 3시간동안 1시간씩 3회반복추출하여, filter paper로여과한후감압 Oil Red O 염색세포분화중 30 μg/ml SPE로처리된세포의배지를제거하고 D-PBS로세포를세척한후, Cacodylate buffer (ph 7.4) 로 2시간고정한다음 Oil Red O(Sigma-Aldrich Co.) 로염색하였다. 세포염색이끝난후 40% isopropyl alcohol(2-propanol, ) 로 3번세척한다음건조하여세포내염색된지방구의크기를광학현미경 (Motic AE 31, Viking Way Richmond, British Columbia, Canada) 으로관찰하였으며, 염색된지방세포의지방함량을측정하기위하여 100% isopropyl alcohol로지방을추출하여분광광도계 (Lambda 45, Perkin Elmer UV/VIS, Shelton, CT, USA) 로 510 nm 파장에서흡광도를측정하였다. RNA 분리및역전사중합효소연쇄반응 (RT-PCR) SPE 30 μg/ml 농도로처리된 3T3-L1 분화세포를 D-PBS(DPBS, SH ) 로세척한후 mrna 양을측정하기위해 RNeasy(Qiagen Sciences Inc., Gaithers-

3 배암차즈기에탄올추출물의항비만효능 403 Table 1. Oligonucleotides sequence used in PCR Genes 1) AMPK ACC SREBP1c C/EBPα Sequence (5' 3') (F) 5'-CCA GGT CAT CAG TAC ACC AT-3' (R) 5'-CTG CCA AAG GAT CCT GGT GA-3' (F) 5'-GGACCACTGCATGGAATGTTAA-3' (R) 5'-TGAGTGACTGCCGAAACATCTC-3' (F) 5'-ATCCGCTTCTTACAGCACAG-3' (R) 5'-CCAATTAGAGCCATCTCTGC 3' (F) 5'-GCA ACG CCG CCT TTG GCT TT-3' (R) 5'-AGT GCG CGA TCT GGA ACT GC-3' PPARγ (F) 5'-ATT CTG GCC CAC CAA CTT CGG-3' (R) 5'-TGG AAG CCT GAT GCT TTA TCC CCA-3' CPT-1 (F) 5'-CTCAGTGGGAGCGACTCTTCA-3' (R) 5'-GGCCTCTGTGGTACACGACAA-3' FAS (F) 5'-CCCTGAAATCCCAGCACTTC-3' (R) 5'-GGCATGGCTGCTGTAGGGGT-3' HSL (F) 5'-GGTGACACTCGCAGAAGACAATA-3' (R) 5'-GCCGCCGTGCTGTCTCT-3' GAPDH (F) 5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' (R) 5'-AGC CTT CTC CAT GGT GGT GAA GAC-3' 1) AMP-activated protein kinase (AMPK), acetyl CoA carboxylase (ACC), sterol regulatory element binding protein (SREBP-1c), CCAAT/enhancer-binding protein-alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), carnitine palmitoyltransferase-1 activity (CPT-1), fatty acid synthase (FAS), and hormone sensitivity lipase (HSL). burg, MD, USA) 시약으로총 RNA를추출하고정량하여 SuperScript TM First-Stand Synthesis system(invitrogen Life Technologies, Rockville, MD, USA) 을이용하여 2 μg RNA로역전사 (reverse transcription, RT) 를수행하였다. 합성된 RT product(template cdna) 에 2.5 mm dntp, 10 buffer, DEPC water, premixed primer(geno- Tech, Daejeon, Korea) 및 Taq DNA polymerase를넣고 Mastercycler gradient(eppendorf, Hamburg, Germany) 를이용하여 PCR 방법으로증폭하였다. 사용한 primer의서열은 Table 1에나타내었다. PCR 반응은 94 C에서 1분간 1 cycle 반응후 94 C 45초, 58 C 45초간, 72 C 1분간 40회반복반응시켰으며, 72 C에서 10분간 extension을시행한후반응을완료하여증폭된산물은 1.5% agarose gel에전기영동하고 ethidium bromide(et-br, Sigma-Aldrich Co.) 로염색한후, Gel Doc(Vilber Lourmet, Marnela-Vallaee Cedex, France) 을이용하여 DNA band를확인하였다. mrna 발현량은 L Process(Version 2.01, Fujifilm, Stamford, CT, USA) 와 Multi Gaugesoftware(Version 2.01, Fujifilm) 를이용하여정량하였다. Western blots을이용한단백질발현분석 SPE 처리 3T3-L1 세포에서단백질을분리하여지방세포분화및지방생성관련단백질의발현변화를 western blot으로분석하기위해시료처리가끝난배양세포에차가운단백질 lysis RIPA buffer를첨가하여균질화시켜 4 C에서 30분간반응시킨후원심분리하여그상층액의단백질 농도를 Bio-Rad 단백질정량시약 (Bio-Rad, Hercules, CA, USA) 으로정량하여단백질시료를만든다음, 8~10% SDS-PAGE를분리한후 PVDF membrane으로 transfer 시켰다. Membrane을상온에서 5%(w/v) nonfat dry milk 를함유한 TBS-T(0.1% Tween 20 in TBS) 에서 1시간 incubation 하여비특이적인단백질들에대한 blocking을실시하고 TBS-T로 15분세척하였다. 준비된막에 1차대상단백질 [pampk, pacc, SREBP-1c, C/EBPα, PPARγ, CPT-1, FAS, HSL(, Dallas, TX, USA), Table 2] 의 1차항체와 horseradish peroxidase가부착된 2차항체로각각처리한다음 membrane 수세후 ECL(Amersham Life Science Corp., Arlington Heights, IL, USA) detection 시약으로각각단백질발현량은 L Process(Version 2.01, Fujifilm) 와 Multi Gauge software(version 2.01, Fujifilm) 를이용하여정량분석하였다. 이때양성대조물질로라스베라톨 (Sigma-Aldrich Co.) Table 2. List of antibodies used in western blot Primary antibody Antibody information Dilution pampk pacc SREBPc1 CPT-1 C/EBPα PPAR-γ FAS HSL β-actin 1:20,000

4 404 김성옥 김미려 황경아 박노진 정지숙 을같은조건으로처리하여비교하였다. 통계처리 실험결과는평균 (mean)± 표준편차 (standard deviation, SD) 로나타내었고, 통계적유의성은 GraphPad Prism 5 program(graphpad Software, Inc., La Jolla, CA, USA) 의 Student s t-test를이용하여 P 값이 0.05 미만 (P<0.05) 인경우에유의한것으로인정하였으며, 대조군과의통계학적유의성 (*) 을각각표기하였다. 본실험은독립적으로 3번이상실시하였다. Cell viability (% of control) c a 결과및고찰 배암차즈기에탄올추출물의추출수율 SPE는 19.5% 의수율을나타내었다 (data not shown). 본연구팀의선행연구 (14) 에의하면배암차즈기물추출물의수율 22.5% 보다 70% 에탄올추출물의수율이낮았다. Choi 등 (12) 은 30, 50, 70, 95% 주정용매로추출한경엽조추출물이 16.5~18.6%, 전초조추출물은 19.5~23.9% 의수율을나타내었으며, 주정함유량에따른수율차이는보이지않았다. Park 등 (15) 의연구에의하면배암차즈기는수분 %, 조지방 8.89%, 조회분 1.24%, 조단백 8.03% 로다른잎채소보다높은조지방, 조단백함량을나타내고있으며, 클로로필 a는 11.61%, 클로로필 b는 5.28% 로총클로로필함량이 16.88% 로상당히높은편이었다. 세포독성배암차즈기에탄올추출물의세포독성을확인하기위하여 24시간동안 SPE를각각농도별 (0.1, 1, 10, 25, 50, 100, 200, 500 μg/ml) 로처리한후 MTT assay를실시하여세포생존도변화를측정하였다. 그결과 Fig. 1과같이대조군 (Control) 보다 SPE 처리농도에의존적으로지방세 a b Control c d SPE (μg/ml) Fig. 1. Effect of the Salvia plebeia R. Br. ethanol extract (SPE) on viability of 3T3-L1 cells. MTT assay. Results were expressed as the mean±sd of three independent experiments. Different letters (a-f) above the bars indicate significant differences at P<0.05. Student s t-test was performed using GraphPad Prism 5 program. * P<0.05, control vs. SPE. e * f f 포의생존율이감소하였으며, 50 μg/ml 이상의농도에서는통계적으로유의한세포독성이나타나는것을확인하였다. 따라서세포생존율 91% 로세포독성을유발하지않는농도인 30 μg/ml의농도에서이후실험을수행하였다. Jeong 등 (16) 은배암차즈기물추출물을대식세포 RAW264.7에 25, 50, 100 μg/ml 농도로처리하여 25 μg/ml 이상농도에서농도의존적으로생존도가억제된것으로보고하여비슷한수준이었으나, Jeong 등 (17) 은배암차즈기물추출물이대식세포 RAW264.7에 50, 100, 250, 500 μg/ml 농도로처리한모든농도에서 100% 이상의생존율을보여 RAW264.7 세포에서독성을나타내지않는것으로보고하였다. 이때 Jeong 등 (16) 은 100 C에서 3시간, Jeong 등 (17) 은 100 C에서 12시간을추출한추출물을사용하였다. Choi 등 (12) 은 30, 50, 70, 95% 주정으로상온에서 24시간동안침지추출한경엽지상부와전초추출물을비만세포 3T3-L1에처리한결과, 경엽 95% 주정추출물은세포독성을나타내었고, IC 50 농도는 μg/ml였다. 경엽 30, 50, 70% 와전초의모든추출물 200 μg/ml 처리농도에서세포독성을나타내지않는것으로보고되어, 추출부위와추출온도, 추출시간등에따라세포독성을나타내는추출물의농도가달라지는것을확인할수있었다. 중성지방축적억제효과비대지방세포로유도된 3T3-L1에 SPE가지방축적에미치는효과를 Oil Red O 용액으로염색하여지방세포의염색정도로확인하였으며, 그결과는 Fig. 2에나타내었다. Oil Red O 염색은지방세포내축적된지방구의중성지방을붉은색으로염색하여세포의붉은색정도를통해분화정도를시각화할수있다. 현미경을통한관찰을통해대조군의세포에비해서 SPE 처리군에서 30% 로유의적으로세포분화억제와지방축적감소효과를확인할수있으며, 양성대조군인 35 μm fenofibrate 처리세포 35% 와도비슷한수준을나타내었다. Isopropyl alcohol로염색된지방구의중성지방량측정결과에서도대조군에비해 SPE 처리군에서지방축적감소가유의적으로감소한것을확인하였다. 따라서 SPE는지방세포의분화억제와지방세포의중성지방축적을감소시키는효과가뛰어남을알수있었다. 구절초메탄올추출물 (18) 을 50~500 μg/ml의시료처리범위에서농도의존적으로지방세포형성을감소하였으며, 300, 400, 500 μg/ml의처리군에서각각 35, 61, 68% 의억제능을, 비타민나무잎 (19) 은 25, 50, 100 μg/ml 처리군이각각 20.76, 45.29, 82.25% 로중성지방축적을억제하는것으로나타나 SPE가구절초메탄올추출물과비타민나무잎보다지방생성억제능이높은것을확인할수있었다. 배암차즈기추출물이지방세포의전사유전자발현및단백질발현에미치는영향체내지방축적은조직내지방세포의비대 (hypertro-

배암차즈기에탄올추출물의항비만효능 405 A B Control 35 μm Fenofibrate 30 μg/ml SPE (positive control) Cont 35 μm SPE Fenofibrate (30 μg/ml) Fig. 2. Effect of SPE (Salvia plebeia R. Br.

5 배암차즈기에탄올추출물의항비만효능 405 A B Control 35 μm Fenofibrate 30 μg/ml SPE (positive control) Cont 35 μm SPE Fenofibrate (30 μg/ml) Fig. 2. Effect of SPE (Salvia plebeia R. Br. ethanol extract) on Oil Red O staining and triglyceride contents in differentiated 3T3-L1 adipocytes. A, captured with a microscope; B, quantified by the lipid accumulation using spectrophotometer. Results were expressed as the mean±sd of three independent experiments. Student s t-test was performed using GraphPad Prism 5 program. * P<0.05, control vs. SPE. Fenofibrate was a positive control. phy) 와과형성 (hyperplasia) 으로진행되며, 지방세포과형성은지방세포 (preadipocytes) 의활발한증식과활성화된지방세포분화과정으로유도되기때문에지방세포의분화 (adipogenesis) 는지방세포의과형성뿐만아니라분화된세포내의활성화된지방질생합성 (lipogenesis) 작용에의하여생성된지방구축적이유도된다 (20). 지방세포의과형성과지방전구세포의증식과분화에의해조절되며, 지방세포의비대는지방의합성과지방분해의균형에의해조절된다 (21). 비대지방세포로유도된 3T3-L1 세포에서 PPARγ, C/EBPα, SREBPc1, ACC, AMPK, CPT-1, HSL 및 FAS의전사유전자발현및단백질발현에미치는 SPE의효과를조사한결과, Fig. 3과같다. 지방세포에서초기지방세포분화에중요한역할을하는 PPARγ와 C/EBPα는 adipogenesis 과정을조절하는전사인자로서두인자의발현은 adipogenic gene의발현을조절함으로써지방조직에서지방산합성을증가시키고중성지방을생산하므로 adipogenesis 조절에 PPARγ와 C/EBPα 발현을억제한다 (22). 또한, 인슐린신호전달과관련된유전자의발현과성숙한지방세포에서포도당과지질대사를조절하여지방세포의분화를더욱촉진시켜분화과정을완성시킨다 (23,24). 따라서지방세포분화후기에발현되며여러호르몬인 adipocytokine과 adipogenic protein 발현에중요한역할을하는전사조절인자이다. SPE의 PPARγ, C/EBPα 유전자전사량과단백질발현억제활성을확인한결과, SPE 처리세포에서대조세포에비해 PPARγ, C/EBPα 발현이유의적으로감소를나타내어 SPE가지방세포에서지방분화및지방축적을억제하는것으로보인다. 또한, PPARγ, C/EBPα의타깃유전자는 SREBPc1, FAS의활성을유도하여지방합성을한다 (25). 따라서 SPE 처리시 SREBPc1, FAS의유의적감소는 PPARγ, C/EBPα 발현이유의적으로감소로기인한것을알수있다. 반대로호르몬자극지방분해효소 (HSL) 유전자발현과활성은 SPE의처리세포에서유의적증가를보여지방분해효과를나타내었다. AMPK와 ACC 유전자전사량과인산화단백질활성은대조세포와비교해서유의적으로증가를나타내었다. AMPK는세포내항상성유지 역할을하는효소 (26) 로이것의활성화는포도당및지방산산화증가와간에서지방합성과포도당신생억제, 췌장에서인슐린분비조절을하는중요한매개인자로작용한다. ACC 는지방합성과콜레스테롤합성을조절하는효소단백질인 ACC의인산화증가로효소활성이불활성화하므로지방세포에서지방합성의억제를나타낸다 (27). ACC 인산화증가는지방산베타산화를활성화시키는 CPT-1의발현을증가시키므로포화지방산의미토콘트리아안으로유입이증가하고일련의지방산산화가일어나므로지방축적은감소하고인슐린민감도는증가한다 (28,29). 따라서 SPE 처리시 CPT-1의유의적증가는 SPE가지방세포에서의지방합성억제효과가있음을알수있었다. 식방풍추출물처리시 C/EBPα, FABP4, FAS 등모든유전자에서농도의존적으로발현이감소하였으며, PPARγ는 250 μg/ml 농도에서부터유의적으로유전자발현의억제효능을보였다 (30). 이에반해 SPE는 30 μg/ml에서유전자발현억제효능이양성대조군라즈베라톨과비슷한효능을보여식방풍추출물보다낮은농도에서효과를나타내었다. 비타민나무잎에탄올추출물을처리한결과, SREBP-1c, PPARγ, C/EBPα 및 FAS 발현을감소시켰으며, 100 μg/ml의농도에서 p-ampk 의발현은 3.5배증가하고 PPARγ의발현은 0.1배감소하여지방합성이억제된다고보고하였다 (19). Yang 등 (21) 과 Jeong 등 (19) 은 quercetin 등플라보노이드계열의성분이지방세포분화에영향을준것으로시사하였다. Jeong 등 (14) 에의하면열풍건조배암차즈기물추출물의총폴리페놀과플라보노이드함량은각각 mg/gae/g과 mg QUE/g 수준이었다. 이상의결과를종합하면 3T3-L1 지방세포에서 SPE 처리시지방분화초기전사인자인 PPARγ, C/EBPα의발현을조절하여이들전사인자의타깃유전자로알려진 SREBPc1, FAS의발현을조절하여지방세포의분화억제와지방합성을억제할뿐만아니라 AMPK 인산화단백질발현증가로지방세포에서의지방합성관련효소단백질인 ACC 불활성을증가시키고, 증가된 ACC 불활성화는미토콘트리아내지방산산화를활성화하는 CPT-1을활성화시키므로지방

6 406 김성옥 김미려 황경아 박노진 정지숙 A B C D E F G H Fig. 3. Effect of Salvia plebeia R. Br. extract on mrna (black) and protein (white) expressions in the 3T3-L1 cells. GAPDH and β-actin was used as a loading control in RT-PCR and western blot. Densitometry was performed using L Process and Multi Gauge software (Version 2.01, Fujifilm), and represent the average densitometric analyses as compared with β-actin. The data were expressed as the mean±sd (n=3) from three in* dependent experiments. P<0.05, significant between the vehicle control (-), Resveratrol as positive control (+) and SPE treated cells. SPE; Salvia plebeia R. Br. ethanol extracts.

7 배암차즈기에탄올추출물의항비만효능 407 축적을억제시킬것으로생각된다. 현재배암차즈기의항비만활성물질에대한보고가없어추가적으로물질규명및분자수준의대사적검정을위해동물실험이필요할것으로본다. 요 본연구는배암차즈기에탄올추출물 (SPE) 을유효성분으로함유하는지방생성및축적저해효능을조사하였다. 배암차즈기에탄올추출물은마우스배아섬유아세포 (mouse embryo fibroblast) 유래지방세포인 3T3-L1에서지방세포분화를억제하는효능을보유하고있었으며, 지방세포내중성지방의농도를감소시키는효능을보유하고있었다. 또한, PPARγ, C/EBPα, SREBP-1c, pacc, pampk, CPT- 1, 지방산합성효소 (FAS, fatty acid synthase) 발현억제, 호르몬자극지방분해효소 (HSL, hormone sensitivity lipase) 활성화등지방합성관련인자들의발현을조절하는효능을보유하고있는것으로확인되었다. 이상의결과로 SPE 가지방세포분화및지방대사에관련된인자들의발현을조절함으로써지방생성및지방축적을저해하는효능을보유하기때문에배암차즈기를활용한비만개선을위한소재로서의활용이가능할것으로보인다. 약 감사의글 이논문은산업통상자원부지역특화기술융복합연구지원사업 (R ) 의항염증효능식물탐색후속연구로구례군의구례야생화연구소운영연구개발비에의해수행되었습니다. REFERENCES 1. Sim MO, Lee HI, Ham JR, Seo KI, Lee MK Longterm supplementation of esculetin ameliorates hepatosteatosis and insulin resistance partly by activating AdipoR2-AMPK pathway in diet-induced obese mice. J Funct Foods 15: WHO/Europe approaches to obesity. int/en/health-topics/noncommunicable-diseases/obesity/ obesity (accessed Jan 2017). 3. Noh SK Functional action of flavonoids for treatment of obesity. Food Industry and Nutrition 7(2): Salvia plebeia R. Br.. selectplantpilbkdtl.do?plantpilbkno=33589 (accessed Jan 2017). 5. Jo SY, Lee U, Kim EY, Lee SJ, Her JW, Yoon TJ A study on the anti-inflammatory and anti-allergic effect of Salvia plebeia R. extracts. Korean J Pharmacogn 41: Cho KJ, Bae SC, Kim JB, Kim SS, Kang SA, Kim MB Development of functional medicinal food materials and separation of physiological active substances from native plants. Ministry of Agriculture, Food and Rural Affairs (GOVP ), Sejong, Korea. p Jin XF, Lu YH, Wei DZ, Wang ZT Chemical fingerprint and quantitative analysis of Salvia plebeia R.Br. by high-performance liquid chromatography. J Pharm Biomed Anal 48: Choi JK, Oh HM, Lee S, Kwon TK, Shin TY, Rho MC, Kim SH Salvia plebeia suppresses atopic dermatitislike skin lesions. Am J Chin Med 42: Wu F, Wang H, Li J, Liang J, Ma S Homoplantaginin modulates insulin sensitivity in endothelial cells by inhibiting inflammation. Biol Pharm Bull 35: Choi JK, Oh HM, Park JH, Choi JH, Sa KH, Kang YM, Park PH, Shin TY, Rho MC, Kim SH Salvia plebeia extract inhibits the inflammatory response in human rheumatoid synovial fibroblasts and a murine model of arthritis. Phytomedicine 22: Gao H, Liu Y, Li K, Wu T, Peng J, Jing F Hispidulin induces mitochondrial apoptosis in acute myeloid leukemia cells by targeting extracellular matrix metalloproteinase inducer. Am J Transl Res 8: Choi SI, Kwak H, Kim JY, Choi JG, Lee JH Antiadipogenic effects of Salvia plebeia R. Br. extracts by extraction conditions in 3T3-L1 preadipocytes. Korean J Med Crop Sci 23: Won HR Effect of Salvia plebeia water extract on antioxidant activity and lipid composition of rats fed a high fat-high cholesterol diet. Korean J Community Living Sci 27: Jeong JS, Kim YJ, Choi BR, Go GB, Son BG, Gang SW, Moon SM Antioxidant and physicochemical changes in Salvia plebeia R. Br. after hot-air drying and blanching. J Korean Soc Food Sci Nutr 43: Park SI, Kim TS, Park CG, Kang MH Nutritional and sensory of green leafy vegetables cultivated from medicinal plant seed. J East Asian Soc Diet Life 22: Jeong JS, Kim YJ, Ahn EK, Shin JY, Go GB, Son BG Antioxidative activities and qualitative characteristics of substitute tea using Salvia plebeia R. Br.. Korean J Food Cook Sci 31: Jeong H, Sung M, Kim Y, Ham H, Choi Y, Lee J Anti-inflammatory activity of Salvia plebeia R. Br. leaf through heme oxygenase-1 induction in LPS-stimulated RAW264.7 macrophages. J Korean Soc Food Sci Nutr 41: Park JA, Jin KS, Kwon HJ, Kim BW Antiobesity activity of Chrysanthemum zawadskii methanol extract. J Life Sci 25: Jeong HJ, Park JH, Kim MJ Ethanol extract of Hippophae Rhamnoides L. leaves inhibits adipogenesis through AMP-activated protein kinase (AMPK) activation in 3T3-L1 preadipocytes. Korean J Plant Res 28: de Ferranti S, Mozaffarian D The perfect storm: obesity, adipocyte dysfunction, and metabolic consequences. Clin Chem 54: Yang JY, Della-Fera MA, Rayalam S, Ambati S, Hartzell DL, Park JH, Baile CA Enhanced inhibition of adipogenesis and induction of apoptosis in 3T3-L1 adipocytes with combinations of resveratrol and quercetin. Life Sci 82: Morrison RF, Farmer SR Hormonal signaling and transcriptional control of adipocyte differentiation. J Nutr 130: 3116S-3121S. 23. Ntambi JM, Kim YC Adipocyte differentiation and gene expression. J Nutr 130: 3122S-3126S. 24. Rangwala SM, Lazar MA Peroxisome proliferatoractivated receptor γ in diabetes and metabolism. Trends

8 408 김성옥 김미려 황경아 박노진 정지숙 Pharmacol Sci 25: Ambati S, Yang JY, Rayalam S, Park HJ, Della-Fera MA, Baile CA Ajoene exerts potent effects in 3T3-L1 adipocytes by inhibiting adipogenesis and inducing apoptosis. Phytother Res 23: Hardie DG The AMP-activated protein kinase pathway-new players upstream and downstream. J Cell Sci 117: Kahn BB, Alquier T, Carling D, Hardie DG AMP-activated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1: Zhang BB, Zhou G, Li C AMPK: An emerging drug target for diabetes and the metabolic syndrome. Cell Metab 9: González-Barroso MM, Anedda A, Gallardo-Vara E, Redondo- Horcajo M, Rodríguez-Sánchez L, Rial E Fatty acids revert the inhibition of respiration caused by the antidiabetic drug metformin to facilitate their mitochondrial β-oxidation. Biochim Biophys Acta 1817: Jung HK, Sim MO, Jang JH, Kim TM, An BK, Kim MS, Jung WS Anti-obesity effects of Peucedanum japonicum Thunberg L. on 3T3-L1 cells and high-fat diet-induced obese mice. Korean J Plant Res 29: 1-10.

농림수산식품부장관귀하 이보고서를 팥의대사성질환개선및기능성규명 에관한연구의최종보고서로제출 합니다 년 2 월 11 일 - 1 -

농림수산식품부장관귀하 이보고서를 팥의대사성질환개선및기능성규명 에관한연구의최종보고서로제출 합니다. 2013 년 2 월 11 일 - 1 - 연구기관명 : 부산대학교 연구책임자 : 정명호 연구원 : 손용해 연구원 : 장민경 - 2 - Ⅰ. 제목 팥의대사성질환개선및기능성규명 Ⅱ. 연구개발의목적및필요성 팥의대사성질환개선소재발굴과그기능성을과학적으로증명하기위해, 팥의당뇨,