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1 Korean J. Plant Res. 28(5): (2015) Print ISSN Online ISSN Original Research Article 비타민나무잎에탄올추출물의 AMPK 활성화를통한 3T3-L1 지방전구세포의 adipogenesis 억제효과 정현주 1, 박주희 1, 김명조 2 * 1 삼성생약 ( 주 ) 바이오생명공학연구소, 2 강원대학교식물자원응용과학과 Ethanol Extract of Hippophae Rhamnoides L. Leaves Inhibits Adipogenesis through AMP-activated protein kinase (AMPK) Activation in 3T3-L1 Preadipocytes Hyeon Ju Jeong 1, Ju Hee Park 1 and Myong-Jo Kim 2 * 1 Biotechnology institute, Samsung Herb Medicine Agricutural Co., Chuncheon 24341, Korea 2 Department of Applied Plant Sciences, Kangwon National University, Chuncheon 24341, Korea Abstract - In the present study, we investigated the effect of 70% EtOH extract from Hippophae Rhamnoides L. leaves (HRL) on the anti-obesity effect in 3T3-L1 cells. The effects of HRL on lipid accumulation in 3T3-L1 cells were examined using Oil Red O staining. In addition, we examined the gene expression levels by using RT-PCR and western blot. The results of this analysis showed that 100 μg / ml HRL significantly increased the inhibition of lipid accumulation by 82.25%; significantly decreased the mrna expression of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and fatty acid synthase (FAS) in 3T3-L1 cells as well as the stimulated protein expression of AMP-activated protein kinase (AMPK); and suppressed the expression level of PPARγ. These results suggest that HRL can prevent adipogenesis through activation of AMPKα and inhibition of adipogenesis transcription factors. Key words - Hippophae Rhamnoides L. leaves, HRL, Adipogenesis, 3T3-L1, AMPK 서언 최근서구화된식습관과생활환경의변화로전세계적으로비만이심각한질환으로대두되고있다 (No, 2012). 비만은신체에너지의섭취와소비의불균형으로생기는질환으로고혈압, 고지혈증, 당뇨및심혈관질환과같은성인병발생가능성을증가시키는요소로밝혀지고있다 (Spiegelman and Flier, 2001). 그로인해현재다양한비만관련치료제가출시되고있지만, 이들은지방변, 두통, 오심, 우울증, 불안및인지관련증상등의부작용이보고되고있다 (Ballinger and Peikin, 2002; Lee, 2013). 따라서이러한부작용이적으면서우수한효과를기대할 * 교신저자 : kimmjo@kangwon.ac.kr Tel 수있는천연물을이용한항비만식이개발이요구되는실정이다 (Kim et al., 2010; Jeon et al., 2014; Choi et al., 2013; Shon et al., 2013). 지방세포내의지방구 (lipid droplet) 는지질의대사와조절등지방대사에중요한역할을하고, 지방구에축적되는지질은중성지방의분해와합성을조절하는것으로알려져있다. 따라서비만예방및관리에있어지방전구세포로부터분화된지방세포의형성억제와지방구내에존재하는중성지방의분해로인한 glycerol 의유출과정은중요한기전이며 (Frayn et al., 2003), 이때다양한전사인자와호르몬이관여한다. AMPactivated protein kinase (AMPK) 는지방산의합성과분해를매개함으로써체내에너지항상성유지의중요한역할을한다 (Assifi et al., 2005). 활성화된 AMPK 는 sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator- c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 비타민나무잎에탄올추출물의 AMPK 활성화를통한 3T3-L1 지방전구세포의 adipogenesis 억제효과 activated receptor γ (PPARγ) 및 fatty acid synthase (FAS) 와같은지방세포화인자의발현을억제함으로써지방합성을억제시키고 acetyl-coa carboxylase (ACC) 의비활성화와 carnitine palmitoyltransferase-1 (CPT-1) 의활성화를통해베타- 산화를촉진시켜미토콘드리아로지방산이동을증가시킨다 (Hardie, 2003; Foretz et al., 1998; Park et al., 2015). 분화초기에는 CCAAT/enhancer-binding protein β (C/EBPβ), C/EBPδ 와 SREBP-1c 가발현되고, 이들전사인자는후기전사인자인 PPARγ 와 C/EBPα 의발현을유도하여지방전구세포의분화를촉진하며섭취된에너지원으로부터중성지방의합성을촉진하여지방과간조직에저장한다 (Cao et al., 1991; Naowaboot et al., 2012). 또한, SREBP-1c 는 FAS, ACC 및 acetyl-coa synthase (ACS) 등의발현을조절하여지방산과중성지방의합성을조절하게된다 (Kolehmainen et al., 2001). 따라서항비만기능성소재개발연구는지방세포의분화과정을억제하거나지방분해를촉진하는소재를탐색하는연구가진행되어야한다. 본연구에서사용한비타민나무 (Hippophae rhamnoides L.) 는보리수나무과 (Elaeagnaceae) 의낙엽성관목으로북아시아와유럽이원산지이고, 산자나무, 갈매보리수나무등으로불리며 (Rousi, 1971), 항산화, 항미생물, 항염증, 항암, 위궤양치료, 간세포보호, 면역조절및피부보호효과등이알려져있다 (Chauhan et al., 2007; Zu et al., 2006; Ganju et al., 2005; Xing et al., 2002; Suleyman et al., 2001). 화학성분은폴리페놀류, flavonoid, flavonoid 배당체, pomolic acid, vomifoliol, β-sitosterol, protocatechuic acid 및 ursolic acid 등이알려져있다 (Yang et al., 2013; Maheshwari et al., 2011). 비타민나무잎추출물에존재하는 flavonoid는주로 quercetin, kaempferol 및 isorhamnetin 으로, 이성분들은지방세포분화및지방축적억제효과가있다고알려져있다 (Yang et al., 2011; Jang and Jeong, 2010; Bae et al., 2014). 비타민나무잎의항비만연구로는덖음차추출물의간장지질관련효소활성 (phosphatidate phosphohydrolase (PAP), fatty acid β-oxidation (β-oxidation), CPT, malic enzyme 등 ) 을측정하여내장비만의억제효과를확인하였으며 (Lee et al., 2011), 에탄올추출물을대상으로 PPARα, CPT-1, ACC의 mrna 발현을확인하여지방산산화로인한항비만효과가나타났음을 in vivo 연구로확인하였다 (Pichiah et al., 2012). 또한비타민나무잎의주성분인 flavonoid, tannin 등을대상으로지방축적억제효과를확인하였고 (Yang et al., 2013), polymethoxylated flavonoid 인 pentamethylquercetin 을대상으로 aiponectin 및 PPARγ 등의 mrna 와단백질발현 을확인하여혈당강하효과를확인하였다 (Chen et al., 2011). 하지만비타민나무잎추출물의지방생성및축적저해에의한항비만작용기전연구는미비한실정이다. 따라서본연구에서는비타민나무잎추출물을이용하여지방전구세포의분화억제능과세포내중성지방의생성및축적과관련된작용기전을분석함으로써체지방감소에도움이되는기능성식품소재개발을위한가능성을규명하고자하였다. 재료및방법재료본실험에시료로사용된비타민나무잎 (Hippophae rhamnoides L.) 은강원비타민나무영농조합 (Chuncheon, Korea) 에서제공받아사용하였고, 강원대학교식물자원응용공학과약용식물분류학실험실에서동정받았으며증거표본은강원대학교식물자원응용공학과식물표본실에보관하였다. 본실험에사용된 mouse embryo 유래의 3T3-L1 지방전구세포는한국세포주은행 (KCLB, Korea) 에서분양받았고, 세포배양에사용한 Dulbeco s modified Eagle s medium (DMEM), fetal bovine serum (FBS), bovine calf serum (BCS), penicilin/streptomycin solution (PS), phosphate buffered saline (PBS), trypsin-edta는 HyClone 사 (Logan, UT, USA) 로부터구입하여사용하였다. Insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX) 및 Oil Red O, 3-(4, 5-dimethyl thiazol-2-yl)-2, 5- diphenyhl tetrazolium bromide (MTT) 는 Sigma 사 (St. Louis, MO, USA) 에서구입하여사용하였다. 시료제조비타민나무잎추출물 (HRL) 은파쇄된시료에 10배수의 70% 에탄올을첨가하여상온에서 12시간씩 2회반복하여추출하였다. 추출액은 filter하여불순물을제거한다음, 감압농축하여농축물을얻었으며, 이를동결건조하여사용하였다. 3T3-L1 세포배양및분화 (diferentiation) 유도 3T3-L1 지방전구세포는 10% BCS 와 1% PS 가함유된 DMEM 배지를이용하여 37, 5% CO 2 의조건에서배양하였다. 세포의분화는 cells/ ml의농도로 6 well plate 에세포를분주한후, 100% confluent 한상태가되도록배양하였다. 2일후 10% FBS, 1% PS 및분화유도물질 MDI (0.5 mm IBMX, 1 μm DEX, 10 μg / ml insulin) 가첨가된 DMEM 배지를처리하고, 그이후 -583-

3 韓資植誌 Korean J. Plant Res. 28(5) : 582~590(2015) 10% FBS 와 10 μg / ml insulin만을포함한 DMEM 배지로교체하여지방세포분화를유도하였다. 시료의처리는분화유도배지첨가시점부터같이처리하였다. 세포독성 HRL 의세포독성은 Ishiyanma et al. (1996) 의 MTT assay 로실험하였다. 세포를 cells/well의농도로 96 well plate 에 100 μg씩분주한후 37, 5% CO 2 incubator 에서 24시간배양하여일정농도로희석된추출물을첨가한후다시 24시간배양하였다. 배양완료후 5 μg / ml농도의 MTT 시약을 100 μg씩분주한다음 37, 5% CO 2 incubator 에서 4시간배양하였다. 배지를제거하고 DMSO 100 μg를가하여생성된 formazan을녹인후 ELISA microplate reader (Model 680, Biorad Laboratories Inc., Hercules, CA, USA) 를이용하여 540 nm에서흡광도를측정하였다. 세포생존율은다음의식에따라계산하였다. Cell viability (%) = ABS sample /ABS control 100 ABS sample: Absorbance of the experimental sample ABS control : Absorbance of the control Oil Red O 염색을이용한세포내중성지방측정 HRL 에의해지방세포분화시나타나는중성지방축적저해를확인하기위하여 3T3-L1 지방전구세포의분화유도시추출물을농도별 (0, 10, 25, 50, 100 μg / ml ) 로처리하였다. 배지를제거한후, 10% formalin (Intron, Seongnam, Korea) 을처리하여상온에서 1시간고정시켰다. 고정후증류수와 60% isopropanol 로세척한후세포들을완전히건조시키고, Oil Red O working solution 을 20분간처리하여세포안에축적된지방구들을염색하였다. 현미경을사용하여염색된세포를관찰한후, 100% isopropanol 을이용하여세포내염색되어있는 Oil Red O를용출시켜 510 nm에서흡광도를측정하여지방축적을확인하였다. RT-PCR 을이용한 mrna 분석지방세포분화가완료된세포를 PBS로세척하여 harvest 한후 Total RNA Extraction Kit (Intron) 를이용하여 RNA 를분리하였다. 추출된 RNA 를이용하여 cdna 를합성한후 template 로사용하여지방세포분화발현인자인 SREBP-1c, PPARγ, C/EBPα 를확인하고, 지방축적, 합성및저장에관련된 FAS 의 primer (Genotech, Daejeon, Korea) 를이용하여 RT-PCR 을 실시하였다. Primer 염기서열은 Table 1과같고, PCR 조건은초기변성 94 5분, 변성은 94 30초, annealing 은 56 (PPARγ), 58 (SREBP-1c, FAS), 60 (C/EBPα) 60 초, 신장반응은 72 60초로하여 35cycle 을진행하였다. 데이터는 UVIband Software (UVItec, Cambridge, UK) 로분석하였으며유전자의발현량은 differentiation media 만처리한대조군을 1.0 으로간주하여상대적인값을측정하였다. Western blot 을통한단백질발현분석분화된 3T3-L1 세포를 harvest 하여 lysis buffer (Cell Signaling Technology, Danvers, MA, USA) 를넣고 30분간용출시킨후 13,000 g에서 10분간원심분리하여상층액을얻었다. 정량한단백질은 4-20% SDS-polyacrylamide gel (Biorad Laboratories Inc.) 을사용하여전기영동후 PVDF membrane (Biorad Laboratories Inc.) 에 transfer 하였다. 5% Skim milk (0.1% Tween 20 containing PBS, PBST) 용액에서 1시간동안 nonspecific binding site 를 blocking한뒤 1차항체 [anti- PPARγ, anti-ampk, anti-β-actin (1:1000), Cell Signaling Technology] 로 4 에서 overnight 하고 2차항체 [anti-rabbit IgG or anti-mouse IgG linked with horseradish peroxidase, Santa Cruz Biotechnology, Inc. USA] 로상온에서 1시간 incubation 하였다. ECL solution (YoungInFrontier Co., Gasandong, Seoul, Korea) 을이용하여 antibody-bound protein 을 detection 하였다. 통계처리모든결과는 SAS (ver. 9.2, SAS Institute Inc., NC, USA) 통계프로그램을이용하여분석하였고평균 ± 표준편차로표시하였으며, 실험군간평균의차이는 one-way ANOVA 로유의성을확인하였다. 대조구인분화 control 에대한시료처리구의통계적유의성은 Tukey s multiple comparison test 로검정하였고 p < 0.05 이상일때만통계적유의성이있는것으로판단하였다. 결과및고찰세포독성 HRL 이 3T3-L1 세포에미치는영향을알아보기위하여 MTT assay를통해세포독성을측정하였다 (Fig. 1). 추출물을처리하지않은대조군의세포증식율을 100% 하였을때, 100 μg / ml의농도에서 ± 1.35% 로 80% 이상의세포생존율을나타내었다. 식물추출물에의한세포생존율에관한연구를보면칡잎추출 -584-

Each bar represents the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 compared to control cells. 물의경우에는 100 μg / ml농도에서 89.3% 의생존율을나타내었으며 (Lee et al.

4 비타민나무잎에탄올추출물의 AMPK 활성화를통한 3T3-L1 지방전구세포의 adipogenesis 억제효과 Fig. 1. Effects of HRL (extract of Hippophae rhamnoides L. leaves) on cell viability. 3T3-L1 cells were treated with different concentrations (0~100 μg / ml ) of HRL for 24 h. Cell viability was measured by MTT assay. The viability of untreated control cells was defined as 100%. Each bar represents the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 compared to control cells. 물의경우에는 100 μg / ml농도에서 89.3% 의생존율을나타내었으며 (Lee et al., 2014), 두메부추추출물의 100 μg / ml농도에서독성을나타내지않아 (Choi and Kim, 2014a) 항비만제및원료개발에있어서유효한물질의가능성을나타내었다. 따라서본연구는세포증식에크게영향을미치지않는 100 μg / ml이하의농도 10, 25, 50, 100 μg / ml로 3T3-L1 세포에처리하여지방세포분화억제능을확인하였다. 중성지방축적억제효과 Oil Red O 염색시약은중성지질, 콜레스테롤만을염색하고, 세포내축적된지방구의중성지방을염색하여세포의붉은색정도를통해분화정도를확인할수있다 (Choi et al., 2013). 따라서 3T3-L1 세포분화과정에서 HRL 이지방구생성을억제하는지확인하기위해 Oil Red O 염색법을이용하였다. 분화처리군 (Con) 의경우, 세포질내지방구의형성이활발하게유도되는것을확인하였고, HRL 을 10, 25, 50, 100 μg / ml로처리하였을때농도의존적으로붉은색이적게관찰되어지질축적이감소함을확인하였다 (Fig. 2). 이를정량분석한결과, 분화처리군 (Con) 에비해 HRL 25, 50, 100 μg / ml처리군이각각 20.76, 및 82.25% 로중성지방축적을억제하는것으로나타났다 (Fig. 2). 이는 Yoon et al. (2010) 이보고한목향추출물의경우 100 μg / ml에서약 10% 축적억제를나타낸연구결과와비교하였을때 HRL 의우수한지방분화능억제효과를관찰할수있었다. 이러한결과는비타민나무에서존재하는 quercetin, kaempferol 및 isorhamnetin 등의 flavonoid 성분의영향으로생각되며, Yang et al. (2013) 의보고에따르면비타민나무에서분리된 Fig. 2. Effect of the HRL (extract of Hippophae rhamnoides L. leaves) on the lipid accumulation in 3T3-L1 cells. Differentiation of confluent 3T3-L1 cells was initiated in DMEM containing MDI (0.5 mm IBMX, 1 μm DEX and 10 μg / ml insulin). Following 10-day differentiation, differentiated adipocytes were fixed and stained with oil-red O in order visualize lipid droplets. Pre, preadipocyte; Con, differentiated adipocyte. Each bar represents the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 compared to differentiated adipocyte (Con). quercetin, kamepferol 및 isorhamnetin 등의성분이각각 45.6, 42.2 및 44.3% (30 μm) 의중성지방축적억제효과를나타내는것을확인하였다. 이와같은결과로 HRL 처리가지방구의생성을저해시켜지방축적을억제하는효과가있음을확인하였다. RT-PCR을이용한 mrna 발현량측정 Adipogenesis는지방전구세포가지방세포로분화되는과정으로, 이를직간접적으로조절하는유전자및단백질들이최근다양한연구로인해잘알려져있으며 (White and Stephens, 2010), SREBP-1c, C/EBP family, PPARγ 등이있다. SREBP-1c 는 지방산이나콜레스테롤합성에필수전사인자이고 (Park, 2005), 지방세포에서 PPARγ 와 C/EBPα 의발현을유도하여지방생성을촉진시키며, 지방산생성에관여하는효소인 FAS 와 ACC 등의발현을조절한다 (Fajas et al., 1999). PPARγ 는 adipogenesis 과정에서 ap2 promoter 위치에결합하여 adipogenesis 를조절하고, C/EBPα 는 PPARγ 와의강한상승작용을통해지방전구세포의분화후기과정을촉진한다 (Hauser et al., 2000; Darlington et al., 1998). 따라서본연구에서는 HRL 에의한지방전구세포의분화억제효과를확인하기위하여 adipogenic transcription -585-

5 韓資植誌 Korean J. Plant Res. 28(5) : 582~590(2015) factor 및관련유전자들의발현을 RT-PCR 을이용하여확인하였다 (Fig. 3). 그결과분화를유도한대조군그룹 (Con) 에서 SREBP-1c, PPARγ, C/EBPα 및 FAS 의 mrna 발현이증가하였으나, 분화과정에서 HRL 을처리하여분화를유도한경우 SREBP-1c, PPARγ, C/EBPα 및 FAS 의 mrna 발현이모두농도의존적으로감소하였음을확인하였다 (Fig. 3). 특히, HRL 100 μg / ml의농도에서 SREBP-1c, PPARγ 및 C/EBPα 는분화처리군 (Con) 대비, 각각 0.87, 0.91 및 0.35 배감소하였으며 (Fig. 3), 이들전사인자들의하위인자인 FAS 의 mrna 발현도 0.49 배감소하는것을확인하였다 (Fig. 3D). 이전의연구는비타민나무잎에탄올추출물이 PPARα 및 CPT-1 의발현증가에영향을미쳐지방산베타 -산화를촉진시킴으로써항비만효과를나타낸다고보고된바있고 (Pichiah et al., 2012), 덖음차추출물식이그룹에서 PAP, β-oxidation 및 CTP 의활성을저해시켜 fatty acid esterification pathway 를통한혈당강하효과를나타낸다고보고된바있으나 (Lee et al., 2011), 본연구에서는비타민나무잎에탄올추출물이 SREBP-1c, PPARγ, C/EBPα 및 FAS 의발현을감소시켜지방합성이억제되는것을확인할수 Fig. 3. Effects of HRL (extract of Hippophae rhamnoides L. leaves) on adipogenic genes expression in 3T3-L1 cells. Differentiation of confluent 3T3-L1 cells was initiated in DMEM containing MDI (0.5 mm IBMX, 1 μm DEX and 10 μg / ml insulin). Total RNA was extracted and cdna was prepared. Equivalent amounts of cdna were amplified using primers specific for SREBP-1c (A), PPAR-γ (B), C/EBP-α (C), FAS (D), and GAPDH. Pre, preadipocyte; Con, differentiated adipocyte. Each bar represents the mean ± SEM (n = 3) * p < 0.05, ** p < 0.01 compared to differentiated adipocyte (Con)

6 비타민나무잎에탄올추출물의 AMPK 활성화를통한 3T3-L1 지방전구세포의 adipogenesis 억제효과 있었다. Park et al. (2013) 에의하면지방세포분화는 adipogenic transcription factor와지방형성관련효소의상호작용으로조절되고 SREBP-1c, PPARγ 및 C/EBPα 의발현억제로지방세포의분화를억제한다고보고된바있으며이외에도본연구결과는 adipogenic transcription factor 의발현억제를통한지방세포분화억제를제시한 Kim et al. (2014), Hwang et al. (2014) 및 Choi and Kim (2014b) 의보고와일치한다. 따라서 HRL 은 3T3-L1 지방전구세포에서 adipogenic transcription factor인 SREBP-1c 의발현을억제시킴으로써 SREBP-1c 의타겟유전자이며지방형성과정에주요인자인 PPARγ 와 C/EBPα 의발현을억제하고지질의합성수송, 저장에관여하는 FAS 의발현에영향을미쳐지방분화를억제를이끌어세포내중성지방의축적이감소되는것으로사료된다. Western blot을이용한단백질발현량측정 HRL 의지방생성및분해와관련된단백질발현과의연관성을확인하기위해 western blot 을이용하여 AMPKα, PPARγ 의발현량을측정하였다 (Fig. 4). AMPK 는세포내의에너지항상성유지역할을하는효소로지방의대사조절에중요한역할을한다. AMPK 는 AMP 가증가되면인산화를통해활성화되고지방산산화를증가시키며지방합성을억제한다. AMPK 는활성화되어 SREBP-1c, PPARγ 및 FAS 와같은지방세포화인자의발현을억제함으로써지방합성을억제시키고 ACC 의비활성화와 CPT-1 의활성화를통해베타- 산화를촉진시켜미토콘드리아로지방산이동을증가시킨다 (Hardie, 2003; Foretz et al., 1998; Fryer, 2002). HRL 을농도별로처리한결과, AMPK 및 PPARγ 의발현이유의성있는변화를나타냈고, P-AMPK 의발현은 100 μg / ml의농도에서 3.5배증가되는것을확인하였으며 Fig. 4. Effects of HRL (extract of Hippophae rhamnoides L. leaves) on adipogenic protein expression in 3T3-L1 cells. Differentiation of confluent 3T3-L1 cells was initiated in DMEM containing MDI (0.5 mm IBMX, 1 μm DEX and 10 μg / ml insulin). Western blot was performed using P-AMPKα (A), AMPKα (B), PPARγ (C), and β-actin. Pre, preadipocyte; Con, differentiated adipocyte. Each bar represents the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 compared to differentiated adipocyte (Con)

7 韓資植誌 Korean J. Plant Res. 28(5) : 582~590(2015) PPARγ 의발현은 0.1 배감소되는것을확인하였다. 이러한결과는비타민나무잎에존재하는 quercetin 의영향으로생각된다. Ahn et al. (2008) 의연구에따르면 quercetin 이 AMPK 의발현을증가시켜 adipogenesis 를억제시키고, PPARγ, C/EBPα, SREBP-1 및 FAS 의발현감소로지방전구세포에서지방세포로의분화를억제시킨다고보고된바있다. 따라서 HRL 은활성화된 AMPK 의발현을증가시킴으로써, 지방형성과정에주요인자인 PPARγ 의발현에영향을준것으로생각되며, 이는 3T3-L1 지방전구세포에서지방세포로의분화억제를이끌어세포내중성지방의축적이감소되는것으로판단된다. 적요본연구는 HRL 의 3T3-L1 지방전구세포의분화과정중에 HRL 이지방의축적에미치는영향을확인하였다. MTT assay 를이용하여세포독성을측정한결과 100 μg / ml의농도에서도세포증식에영향을미치지않는것을확인하였고, 이와같은결과를토대로 Oil Red O 염색법을이용하여지방세포분화억제능을측정하였다. 그결과, HRL 의경우 100 μg / ml의농도에서 82.25% 지방축적억제능을나타내었다. 지방생성에영향을미치는유전자발현량을측정하기위해 RT-PCR 법과 western blot 법을시행하였다. HRL 은 SREBP-1c, PPARγ 와 C/EBPα 의 mrna 발현을억제시켰고, 지방생성에영향을미치는효소인 FAS 의생성을조절하는것으로나타났다. 또한, HRL 처리로 AMPKα 의단백질발현이증가하였으며, PPARγ 의발현량이감소하는것을확인하였다. 이상의결과들로부터 HRL 은 AMPKα 의활성화를통한지방합성을억제를보유하고있는바, 향후항비만기능성소재로활용될수있을것으로생각한다. 사사본연구는산업통상자원부와한국산업기술진흥원의지역특화산업육성사업 ( 과제번호 R ) 과농촌진흥청공동연구어젠다사업 ( 과제번호 PJ009859) 의지원으로수행된연구결과입니다. References Ahn, J.Y., H.J. Lee, S.N. Kim, J.H. Park and T.Y. Ha The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways. Biochem. Biophys. Res. Commun. 373(4): Assifi, M.M., G. Suchankova, S. Constant, M. Prentki, A.K. Saha and N.B. Ruderman AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/ carbohydrate-refed rats. AM. J. PHYSIOL. ENDOC. M. 289(5):E Bae, C.R., Y.K. Park and Y.S. Cha Quercetin-rich onion peel extract suppresses adipogenesis by down-regulating adipogenic transcription factors and gene expression in 3T3-L1 adipocytes. J. Sci. Food Agric. 94(13): Ballinger, A. and S.R. Peikin Orlistat: its current status as an anti-obesity drug. Eur. J. Pharmacol. 440(2-3): Cao, Z., R.M Umek and S.L. McKnight Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Gene. Dev. 5(9): Chauhan, A.S., P.S. Negi and R.S. Ramteke Antioxidant and antibacterial activities of aqueous extract of Seabuckthorn (Hippophae rhamnoides) seeds. Fitoterapia 78(7-8): Chen, L., T. He, Y. Han, J.Z. Sheng, S. Jin and M.W. Jin Pentamethylquercetin improves adiponectin expression in differentiated 3T3-L1 cells via a mechanism that implicates PPARγ together with TNF-α and IL-6. Molecules 16(7): Choi, H.Y. and G.H. Kim. 2014a. Inhibitory effects of Allium senescens L. methanol extracts on reactive oxygen species production and lipid accumulation during differentiation in 3T3-L1 cells. Korean J. Food Sci. Technol. 46(4): (in Korean). Choi, H.Y. and G.H. Kim. 2014b. Inhibitory effects of Allium sacculiferum Max. methanol extracts on ROS production and lipid accumulation during differentiation of 3T3-L1 cells. J. Korean Soc. Food Sci. Nutr. 43(6): (in Korean). Choi, J.H., Y.H. Park, I.S. Lee, S.P. Lee and M.H. Yu Antioxidant activity and inhibitory effect of Aster scaber Thunb. extract on adipocyte differentiation in 3T3-L1 cells. Korean J. Food Sci. Technol. 45(3): (in Korean). Darlington, G.J., S.E. Ross and O.A. MacDougald The role of C/EBP genes in adipocyte differentiation. J. Biol. Chem. 273(46): Fajas, L., K. Schoonjans, L. Gelman, J.B. Kim, J. Najib, G. Martin, J.C. Fruchart, M. Briggs, B.M. Spiegelman and J. Auwerx Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and -588-

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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,