J Korean Soc Food Sci Nutr 한국식품영양과학회지 45(9), 1239~1248(2016) 호박즙, 옥수수수염차, 팥차및혼합물이식이유도비만동물모델에서체중과항산화활성에미치

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1 J Koren So Food Si Nutr 한국식품영양과학회지 45(9), 1239~1248(216) 호박즙, 옥수수수염차, 팥차및혼합물이식이유도비만동물모델에서체중과항산화활성에미치는영향 박재희 이은지 박은주 경남대학교식품영양생명학과 Effet of Pumpkin, Corn Silk, Adzuki Ben, nd Their Mixture on Weight Control nd Antioxidnt Ativities in High Ft Diet-Indued Oesity Rts Je-Hee Prk, Eunji Lee, nd Eunju Prk Deprtment of Food, Nutrition nd Biotehnology, Kyungnm University ABSTRACT Pumpkin juie (PJ), orn silk te (CT), nd dzuki en te (AT) hve long een used for tretment of oesity in Kore. This study investigted the effiy of PJ, CT, AT, nd their mixture (PCA) on ltertion of ody weight nd ntioxidnt metolism in high-ft diet (HFD)-indued oese rts. After eing fed HFD for 4 weeks, SD rts were divided into six groups fed norml diet (ND), HFD, HFD+PJ [25 mg/kg ody weight (BW)], HFD+CT (25 mg/kg BW), HFD+AT (25 mg/kg BW), nd HFD+PCA (PJ : CT : AT=1:1:1, 25 mg/kg BW) for nother 9 weeks. HFD onsumption resulted in totl lipid, triglyeride, nd totl holesterol umultion in dipose tissue, whih ws redued y dministrtion of PJ, CT, AT, or PCA. The plsm oxygen rdil sorne pity vlue nd hepti glutthione peroxidse tivity signifintly inresed ompred to the HFD group. The liver thiorituri id retive sustnes ws signifintly lower in the PCA group thn the HFD group. HFD-indued DNA dmge in heptoytes, s mesured y omet ssy, deresed in the PJ, AT, nd PCA-supplemented groups. The PCA group exerted superior ntigenotoxi effet ompred to other tretments. PCA reovered the onentrtion of plsm diponetin, whih ws redued y HFD. Adipoyte surfe re (%) ws signifintly higher in the HFD group thn the ND group, signifintly lower in the PJ nd PCA groups thn the HFD group, nd not signifintly different ompred with the ND group. Bsed on the results, supplementtion of PJ, CT, AT, nd PCA exhiited lipid-lowering effets in dipoytes of HFD-indued oese rts. Furthermore, the PCA group exhiited superior ntioxidnt tivity in ll treted groups. This study suggests tht mixed everge onsisting of PJ, CT, nd AT my e signifint soure of nturl ntioxidnts, whih might e helpful in preventing oesity nd progress of vrious oxidtive stresses indued y HFD. Key words: diet-indued oese, pumpkin, orn silk, dzuki en, physiologil tivity 서 비만은현재전세계적으로심각한건강문제로떠오르고있으며, 우리나라또한국민건강영양조사결과비만유병률이 1998년 26.% 에서 214년 31.5% 로크게증가한것으로나타났다 (1). 비만은생활습관상의원인또는유전적원인에의한체내지방축적이과잉된상태로높은산화적스트레스와항산화대사의불균형은만성적인산화적스트레스의상태를나타내게되며 (2,3), 이는심혈관계질환이나당뇨, 호흡기질환, 골관절염같은질병을유발하게한다. 따라서비만을치료하려는방법에많은관심이모여지고있 Reeived 23 My 216; Aepted 2 June 216 Corresponding uthor: Eunju Prk, Deprtment of Food, Nutrition nd Biotehnology, Kyungnm University, Chngwon, Gyeongnm 51767, Kore E-mil: pej@kyungnm..kr, Phone: 론 으나 (4), 비만을치료하려는방법들중약물요법들에서부작용이상당수보고되고있어, 최근에는식품및천연물에서체중조절에효과적인기능성소재들을찾아내고이들의작용기전을밝히는연구가활발히진행되고있다 (5-8). 민간요법은예로부터민간에서전해져오는질병치료를목적으로시행되어온방법들로, 한의학이나현대의학의근본이생활속에서시작되고있다는점에서의료행위의원조라할수있다 (9). 전통적으로체중감소를위해행해지는민간요법으로는호박즙이가장많이음용되고있으며그다음으로는옥수수수염차, 팥삶은물이음용되고있다. 늙은호박 (Cuurit mosht Duh.) 은민간요법에서부종에약효가좋다고하여해독용으로널리사용되고있으며 (1), 옥수수수염 (orn silk) 은벼과에속하는옥수수 (Ze mys Linne) 의수염으로고혈압, 혈당강하, 이뇨작용등에효능이있다고보고되고있다 (11,12). 옥수수수염이포함된생약복합물은 3T3-L1의지방축적억제율을농도의존적으로

2 124 박재희 이은지 박은주 증가시켰으며, 지방축적에관여하는효소 glyerol-3-phosphte dehydrogense(gpdh) 의활성을억제시켰다. 또한, 고지방식이로유도한비만동물모델에서옥수수수염함유생약복합물의체중과식이효율감소에의한항비만효과가보고되었으며 (13), in vitro 수준에서옥수수수염의항산화활성이보고되었다 (14,15). 팥 (Vign ngulris vr. nipponensis) 에는사포닌이풍부하게함유되어있고, 사포닌은소변을원활하게배출하는이뇨효과가있으며부기를빼주는데도움이되는것으로보고되고있다 (16). 최근에는단일소재보다는천연소재혼합섭취로항비만효과를확인한연구 (13,17) 들이보고되는추세로, in vivo 수준에서체중감소에도움이되는것으로보고된호박즙, 옥수수수염차, 팥차추출물혼합시그효과를살펴보는것이의미가있을것으로생각된다. 비만및대사증후군발병기전연구및치료제개발을위한동물모델로는 spontneous mutnt, trnsgeni mutnt, knokout( 또는 knok in) 등의유전자변이동물모델이주이다. 그동안비만연구에서주로사용되어온 o/o 또는 d/d 마우스는 C57 Bl/6J 마우스를대상으로렙틴또는렙틴수용체변이를유발시킨종으로서 leptin resistne 및제2형당뇨병연구에적합한모델이나, 이러한단일유전자변이에의한비만이매우드물게나타난다 ( 식욕억제에관여하는 MC4-R 유전자변이의경우전체비만의약 4%) 는점을감안하면인체비만연구적용에있어한계가있다. 그러므로비만이사회적관심과문제로대두하고비만에의해유발되는질병의치료에관심이집중됨에따라동물을이용한다양한식이성비만모델 (dietry oesity models) 개발이진행되고있다 (18). 그리고여러연구를통해고지방식이 (high-ft diet, HFD) 를통한비만모델은체중조절이나비만기전연구에적합한모델로알려져있다 (19). 따라서본연구에서는 SD rt에 4주동안고지방식이를급여하여식이유도비만동물모델로만든후민간요법으로체중감량을위해음용되어온호박즙, 옥수수수염차, 팥차를 9주동안음료의형태로공급하여이추출물들의항산화활성과체중감소에어떠한영향을미치는지비교해보고자하였으며, 이들세가지음료를혼합섭취시그상승효과를살펴보고자하였다. 재료및방법시료준비본실험에사용된호박, 옥수수수염, 팥추출물들은일반적으로음용하는형태와같은조건으로제조하여실험에사용하였다. 호박추출물은 212년가을에수확된늙은호박 (4~5 kg/ 개 ) 을구입하여꼭지를제거하고수세정선한후일정한크기로절단하여오쿠 (OC-83R, Gyeonggi, Kore) 를사용하여원액으로제조하였다. 옥수수수염은강원 도에서 212년에생산되어건조된것을구입하여사용하였다. 옥수수수염차는옥수수수염 2 g에 1 L의물을가하고 3분동안침출하여옥수수수염을건져낸후추출물만을시료로사용하였다. 팥은창원시농협에서 211년에생산된것을구입하여사용하였다. 팥차는건조된팥 5 g에 1 L의물을가하고 3분동안열탕처리하여팥을건져낸후추출물만을시료로사용하였다. 각추출물은동결건조후분말화하여사용하였다. 이때추출수율은호박즙 1.5%, 옥수수수염차 2.4%, 팥차 1% 로계산되었다. 실험동물사육 5주령의 Sprgue-Dwley 수컷흰쥐 42마리를 1주일간동물사육실환경에적응시킨후고지방식이로비만을 4주간유도한뒤 6그룹으로나누어 9주간사육하였다. AIN-76 기본식이를기준으로정상대조군 (norml diet, ND) 은기본식이에물을함께섭취하였고, 고지방식이섭취군 (high-ft diet, HFD) 은 lrd 15% 를첨가한고지방식이에물을함께섭취하였다. 고지방식이 (HFD)+ 호박즙 (pumpkin juie, PJ) 섭취군은호박즙분말 (25 mg/kg BW) 이첨가된물을함께섭취하였고, 고지방식이 (HFD)+ 옥수수수염차 (orn silk te, CT) 섭취군은옥수수수염차분말 (25 mg/kg BW) 이첨가된물을함께섭취하였다. 고지방식이 (HFD)+ 팥차 (dzuki en te, AT) 섭취군은팥차분말 (25 mg/kg BW) 이첨가된물을함께섭취하였고, 고지방식이 (HFD)+ 혼합음료 (PCA) 섭취군은호박즙분말 : 옥수수수염차분말 : 팥차분말을동량의비율로혼합하여 25 mg/kg BW의농도로섭취하였다. 이때각분말은실험동물들의하루평균물섭취량인 2 ml에혼합하여공급하였고, 각추출물이혼합된음료를모두섭취한것을확인한후증류수를자유식 (d liitum) 으로공급하였다. 실험동물은경남대학교동물사육실에서 stinless steel wire ge에한마리씩분리하여사육하였고, 해당식이를증류수또는음료와함께자유로이섭취하도록하였다. 사육기간동안사육실의온도는 25±5 C, 습도는 5% 를유지하였으며명암은 12시간 (7:~19:) 을주기로자동조절하였다. 식이섭취량과음료섭취량은매일기록하였고, 일주일간격으로체중을측정하였다. 본동물실험의연구계획서는경남대학교동물실험윤리위원회의심의및승인을거쳐수행되었다 (KUIAC-13-8). 혈액분리및장기채취 13주간의사육이끝난후 12시간공복을유도한흰쥐를가볍게마취시킨다음대동맥에서전혈을취하여 lithiumheprini polystyrene tue에담아 3, rpm에서 3분간원심분리한후혈장을취하였다. 혈액을취한후간과지방조직을적출하여차가운.9% 생리식염수로세척하여표면의혈액을제거하고여과지로물기를닦아액체질소에급속냉동시켜분석전까지 -8 C에보관하였다.

3 호박즙, 옥수수수염차, 팥차및그혼합물의체중감소와항산화효과 1241 혈장, 간및지방조직의지질농도측정혈장총콜레스테롤과 HDL-콜레스테롤농도는 Allin 등 (2) 의방법에기초를둔진단 kit(choongwe Phrm Corp., Seoul, Kore) 을이용하여비색법으로측정하였다. 중성지방농도는 lipse-glyerol phosphte oxidse 방법 (21) 에기초를둔진단 kit(choongwe Phrm Corp.) 을이용하여비색법으로측정하였다. LDL-콜레스테롤은 Friedewld 등 (22) 에의한계산법 [ 총콜레스테롤-(HDL-콜레스테롤- 중성지방 /5)] 으로산출하였다. 간과지방조직의총지질함량은 Folh 등 (23) 의방법으로분석하였다. 지방조직의총콜레스테롤과중성지방농도는진단 kit(choongwe Phrm Corp.) 을이용하여비색법으로측정하였다. 지방조직의형태학적관찰부고환지방의조직을 1% neutrl uffered formlin을사용하여고정후탈수및포매과정을거쳐파라핀블럭을제작하였다. 그리고두께 4 μm의관상절편으로제작한후 xylene으로파라핀을제거하고, hemtoxylin과 eosin으로염색하여광학현미경 (Nikon, Tokyo, Jpn) 으로관찰하였다. 부고환지방의표면적의측정은 Imge J softwre(ntionl Institute of Mentl Helth, Bethesd, MD, USA) 를사용하여측정하였다. 아디포넥틴분석혈장아디포넥틴은 rt diponetin ELISA kit(assy Pro, St. Chrles, MO, USA) 을이용하여측정하였다. 혈장 oxygen rdil sorne pity(orac) 측정 2,2'-Azois(2-midinopropne) dihydrohloride(aaph) 를 peroxyl rdil genertor로사용하여최종농도는 2 nm이되도록시료에처리하였으며, Ou 등 (24) 의방법에따라형광표준용액 fluoresein의최종농도는 4 nm이되도록처리하였다. Control stndrd로 1 μm의 Trolox를사용하였으며, 각시료는 FLUOstr OPTIMA miro-plte reder(bmg Lteh, Ortenerg, Germny) 에의해 exittion wvelength 485 nm, emission wvelength 535 nm에서 15 yle 동안측정되었다. ORAC vlue는각시료의형광값감소곡선아래부분의총면적을산출하여 1 μm Trolox equivlent(te) 로나타내었고, 최소 3번반복하여실험하였다. 적혈구, 간항산화효소활성측정 Glutthione-peroxidse(GSH-Px) 의활성은간조직을 25 mm potssium phosphte uffer(ph 7.) 1 ml와함께균질화한후, 1, g에서 2분간원심분리하였다. 사이토졸층을포함하는 25 μl의상층액을 1 mm EDTA, 1 mm NN 3, 1 mm GSH, 2 mm NADPH 그리고 1 unit 의 glutthione redutse와함께실온에서 5분동안방치한후, 이혼합물에 2.5 mm H 2O 2 25 μl를더해반응을측정한것으로 H 2O 2 감소율은 spetrophotometer를이용하여 34 nm에서 7초간측정되었으며조직의단백질은 BCA protein ssy로정량하였다. 간조직의 tlse 활성은간을균질화한다음 6 g에서 1분간원심분리하였다. 이때얻은상층액을 1, g 에서 2분동안원심분리하여얻은 pellet에 1 RBC를처리하여 1분동안아이스에방치한후다시 1, g에서 2분간원심분리하여 pellet을 5 mm N-K phosphte uffer(ph 7.) 로세척한다음실험에사용하였다. 실험방법은 N-K phosphte uffer 6 μl와시료 5 μl 를섞어 3 mm hydrogen peroxide 3 μl를첨가한후 UV/VIS spetrophotometer로 24 nm에서 3초동안 hydrogen peroxide의감소량을측정하였다. 조직의단백질정량은 BCA protein ssy(piere Biotehnology, Rokford, IL, USA) 를이용하였으며, 활성도는단백질 1 mg이시간당생성시킨 hydrogen peroxide를 nmole로나타내었다. 간지질과산화물측정간.1 g을 1.15% KCl 용액에넣고균질화한시료및 stndrd 4 μl와 TBARS 용액 [.8% TBA : 2% eti id(ph 3.5) : 8.1% SDS=15:15:2(v/v)] 1.6 ml를혼합한후, oil th를이용하여 95 C에서 1시간동안반응시켰다. 반응이끝난후차가운 ie wter에 1분간냉각시키고, 증류수 1 ml와 n-utnol : pyridine solution=15:1 혼합용액 5 ml를첨가하여충분히혼합한다음 4, rpm으로 15분간원심분리하였다. 원심분리후상층액 2 μl를취해 54 nm에서흡광도를측정하였다. 간의 protein 함량을 BCA protein ssy로측정한후최종값은 μm/mg protein으로나타내었다. 간세포 DNA 손상측정간세포의 DNA 손상도는해부당일 ollgense(15 units/1 g liver) 가함유된 1 ml의 Hnk's Buffered Slt Solution(HBSS) 용액이담긴 flsk에옮겨 37 C shking inutor에서 1분간보관한후 4 g에서 5분간원심분리하였다. 상층액만취해 7 g에서다시한번 1분간원심분리한후바닥에모인세포를취해 15 μl의 1% LMA와섞은다음, 1% norml melting grose gel(nma) 을미리입혀둔슬라이드위에현탁액이골고루분산되도록분주하여 over glss로덮어 4 C 냉장고에보관하였다. 4 C 냉장고에서젤이굳으면 over glss를벗기고그위에다시 1% LMA 용액 75 μl를분주하여다시냉장고에보관하였다. 슬라이드의산화적스트레스처리는 2 μm H 2O 2 용액을 5분간처리한후 PBS로세척하였다. Cell lysis를위해미리차갑게준비해둔 lkli lysis uffer(2.5 M NCl,

4 1242 박재희 이은지 박은주 1 mm EDTA, 1 mm Tris) 에 1% Triton X-1을섞은후슬라이드를담가 4 C의암실조건에 1시간동안침지시켰다. Lysis가끝난슬라이드는전기영동수조에배열하여차가운 eletrophoresis uffer(3 mm NOH, 1 mm N 2EDTA) 를채워 2분간 unwinding 후, 25 V/3±3 ma 의전압을걸어 2분간전기영동을실시하였다. 전기영동이끝나고.4 M Tris uffer(ph 7.5) 로충분히세척하고 ethidium romide로핵을염색하여형광현미경 (LEICA DMLB, Wetzlr, Germny) 의 CCD mer(nikon) 를통해보내진세포핵이미지를 omet imge nlyzing system(komet, version 5., Kineti Imging, Liverpool, UK) 이설치된컴퓨터로분석하였다. 통계처리모든데이터의통계처리는 SPSS/Windows 18.(IBM, Chigo, IL, USA) 을이용하여분석하였고, 결과는평균 ± 표준오차로나타내었다. 각항목은일원배치분산분석 (onewy ANOVA) 을시행하여, Dunn's multiple rnge test 로신뢰수준은 P<.5에서평균값들에대해그룹간의유의성차이를검증하였다. 결과및고찰체중증가량, 식이섭취량및식이섭취효율초기체중, 최종체중, 체중증가량, 식이섭취량및식이섭취효율은 Tle 1에나타내었다. 초기체중에서는그룹간유의성이나타나지않았으나, 최종체중에서는 ND군보다 HFD군의체중이 19.2% 유의적증가를보여고지방식이섭취로인한비만유도를확인할수있었다. 고지방식이섭취군들간의최종체중비교시 HFD군에비해음료섭취그룹이 6~9% 유의적으로감소하였다. 이는 HFD군보다음료섭취군들 (HFD+CT, HFD+AT, HFD+PCA) 의식이섭취효율이유의적으로낮은데서기인한것으로생각한다. 지방조직무게의변화실험동물의지방조직무게의변화를 Tle 2에나타내었다. 부고환지방조직무게는 HFD군이 ND군보다유의적으로증가하였고, HFD군에비해 HFD+PCA군은유의적으로감소하였으며, 호박즙, 옥수수수염차, 팥차개별섭취군은감소하는경향만나타내었다. 다른부위의지방조직무게는그룹간유의적인차이를나타내지않았다. 전체지방조직무게또한 HFD군이 ND군보다유의적으로높았으며, HFD +PCA군은유의적으로감소하는결과를나타내었는데이는부고환지방조직무게가반영된결과로생각된다. 호박즙, Tle 1. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on the ody weight hnge, energy intke, food intke, nd food effiieny rtio in diet-indued oese rts ND 1) HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Initil ody weight (g) Body weight fter 4 weeks (g) Finl ody weight (g) Body weight gin (g) Food intke (g/d) FER 5) (%) 195.9±2.2 NS2)3) 293.3±3.1 4) 382.1± ± ±. 9.5± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.3 1) ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. 2) Vlues re the men±se for 7 nimls in eh group. 3) NS: no signifint differene. 4) Vlues with different letters in the sme row re signifintly different t P<.5 y Dunn's multiple rnge test. 5) FER: food effiieny rtio. Tle 2. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on dipose tissue weight in diet-indued oese rts (g/kg BW) Suutneous Epididyml Retro-peritonel Adominl Brown dipose tissue Totl dipose tissue ND 1) HFD HFD+PJ HFD+CT HFD+AT HFD+PCA 8.1±.4 NS2)3) 11.9±.6 4) 2.7±.3 NS 4.7±.4 NS.8±.1 NS 28.1± ±1. 17.± ±.3 5.8±.5.8± ± ± ±.9 3.1±.5 6.1±.6.9±.1 36.± ± ±1. 3.7±.2 6.5±.7.8± ± ± ±.8 3.5±.4 6.6±.7.9± ± ± ± ±.4 5.7±.7 1.± ±2.9 1) ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. 2) Vlues re the men±se for 7 nimls in eh group. 3) NS: no signifint differene. 4) Vlues with different letters in the sme row re signifintly different t P<.5 y Dunn's multiple rnge test.

5 호박즙, 옥수수수염차, 팥차및그혼합물의체중감소와항산화효과 1243 옥수수수염차, 팥차개별섭취군역시부고환지방조직무게의영향으로전체지방조직무게가감소하는경향만보였다. 비만은체지방의증가, 특히복강내에위치한지방조직의증가가건강상의위해요인으로작용한다고보고하여 (25) 호박즙, 옥수수수염차, 팥차혼합음료가체중뿐만아니라체지방감소에도영향을미치는것으로생각된다. Do 등 (26) 의연구에서늙은호박물추출물 (.5~5 mg/ml) 을 3T3- L1 세포에처리하여지방축적이억제되는것을농도의존적으로확인하였지만, 그유효성분에대해서는보고하고있지않았다. 사포닌은리파아제활성을낮추어섭취된지방이소화관에서흡수되는것을저해함으로써지방조직의무게가증가하는것을억제하는것으로보고되고있는데 (16), 팥에서는 dzuki sponin Ⅰ~Ⅷ 종이보고되고있으며 (27), 옥수수수염에도사포닌이함유된것으로보고되었다 (11, 12). 따라서본연구에서는고지방식이에의한지방조직무게증가에대해각각의시료유효성분의개별효과보다는이들성분의상승작용이부고환지방과총지방무게감소에효과적이었던것으로생각된다. 지방조직의형태학적분석과지방세포의표면적비만은에너지섭취와소비간의불균형으로인해과도하게체지방이축적되는현상으로지방세포의수와크기가증가하게된다. 따라서지방세포크기의측정은항비만효능을입증할수있는효과적인방법으로잘알려져있다 (28). 지방조직에서그룹간의유의적차이를보였던부고환지방세포표면적을비교해보면 HFD군은 ND군보다유의적으로지방세포표면적이증가하였으며, HFD+PJ 군 (-78%) 과 HFD +PCA군 (-65%) 의지방세포표면적은 HFD군에비해유의적으로감소하였으며, ND군과유의적차이를나타내지않 았다 (Fig. 1). HFD+PJ군에서지방세포표면적감소는지방세포에서중성지방농도가현저하게감소에의한것으로생각한다. Rhee 등 (29) 의연구에서도고지방식이로유도된비만쥐에녹차카테킨공급시부고환지방의크기가감소하였는데, 이는중성지방의합성이감소하여체지방축적이감소하므로인해지방세포의 hypertrophy를억제한것으로보고하였다. Med 등 (3) 은아디포넥틴농도가내장지방과음의상관관계가있음을보고하여, HFD+PCA군의부고환지방조직세포표면적감소는증가한아디포넥틴에의한것으로생각된다. 부고환지방조직의형태학적분석은 Fig. 2 에나타내었다. 아디포넥틴에미치는영향지방세포에서분비되는아디포넥틴은체내지방축적및식욕을조절하고염증을감소시키며인슐린민감도를촉진하고, 비만인에서혈청아디포넥틴의수치가정상인의혈청아디포넥틴보다감소하는것으로보고되고있다 (31,32). 아디포넥틴은 ND군에비해 HFD군에서유의적으로감소하였고, HFD군에비해 HFD+CT군과 HFD+PCA군이유의적으로증가하였다 (Fig. 3). 항산화제는산화적스트레스를낮추어아디포넥틴발현을조절하는것으로보고되고있으며 Adipoyte surfe re (% of ND) ND HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Fig. 1. Epididyml dipoytes surfe re of oese rts fed the experimentl diets for 9 weeks. ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+ CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. Men surfe re for suutneous dipoytes ws mesured using Imge J softwre. Eh r represents the men±se (n=7 per group). Brs with different letters re signifintly different t P<.5 y Dunn's multiple rnge test. Fig. 2. Histologil nlysis of epididyml dipose tissue of rts fed the experimentl diets for 9 weeks. ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. All setions were stined with hemtoxylin nd eosin; mgnifition, 1. Mgnifition r=1 μm.

6 1244 박재희 이은지 박은주 Adiponetin (μg/ml) ND HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Fig. 3. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on dipoytokine levels in plsm. ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+ CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. Eh r represents the men±se (n=7 per group). Brs with different letters re signifintly different t P<.5 y Dunn's multiple rnge test. (33,34) 포도씨 proynidins(35), tehins(36), γ-oryznol(37) 등의항산화제들이아디포넥틴발현을증가시킨연구결과들이보고되었다. 따라서 HFD+PCA군에서호박즙, 옥수수수염차, 팥차의항산화성분들에의해고지방식이에의한산화적스트레스가감소되어아디포넥틴농도가증가한것으로생각된다. 액, 간, 분변의지질농도가고지방식이섭취에의해서는유의하게증가하였으나함초분말섭취에따른차이는나타나지않아, 이는함초동결건조분말섭취가장내지방의흡수나혈중지질농도에영향을미치지않는것으로보고하였다. 그러나함초분말섭취에의해서부고환지방조직의지방축적억제효과는확인하였다. 반면부고환지방조직에서총지질함량, 중성지방, 총콜레스테롤농도는 HFD군이 ND군보다유의적으로높았으며, 음료섭취군은 HFD군에비해유의적으로낮게나타났다 (Fig. 4). 이는늙은호박물추출물 (5 mg/kg) 이고지방식이 ( 콜레스테롤 15%, sodium holte 1%, 옥수수유 84%) 로유도된동물모델에서혈중콜레스테롤과중성지질을낮추는효과를보고한 Lim과 Choi(1) 의연구결과와유사하였다. Be 등 (39) 은인삼의사포닌이간세포내콜레스테롤과지방산의생합성및분해를촉진시키는한편콜레스테롤대사를항진시키는것으로보고하고있으며, 또한콜레스테롤장내흡수억제효과로인해체중, 부고환지방조직무게감소, 지방성분축적이억제된것으로보고하였다. 따라서본연구결과에서도늙은호박내유효성분과옥수수수염과팥에들어있는사포닌의작용으로지방조직내지질농도가감소한것으로생각한다. 그리고지방조직의무게와지질농도에유의적효과를보인늙은호박의생리활성성분과그작용기전에관하여는추후체계적인연구가필요할것으로생각된다. 혈장, 간조직, 부고환지방조직지질분석혈장과간조직의지질분석시모든그룹에서유의적인차이를나타내지않았다 (Tle 3). Kim 등 (17) 의결과에서도고지방식이군 (AIN-76 식이 +2.5% lrd 첨가 ) 의혈액총콜레스테롤, 중성지질, HDL-콜레스테롤은정상식이군과유의적차이가나타나지않았는데그이유에대해서는언급하고있지않았다. 고지방식이로유도한비만흰쥐에함초분말의항비만효과를탐색한 Kim 등 (38) 의연구에서도혈 혈장 ORAC 값에미치는영향혈장 ORAC ssy는혈장에서 peroxy rdil 소거능을측정하는방법으로, 그결과는 Fig. 5에나타내었다. ORAC 활성은정상식이군보다고지방식이섭취에의해유의적으로감소하지는않았지만, 고지방식이와호박즙, 옥수수수염차, 팥차혼합물섭취시고지방식이섭취군보다 ORAC 활성이유의적으로증가하였다. HFD+PCA군을제외한나머지음료섭취군은 HFD군에비해증가하는경향만보였다. 고지 Tle 3. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on plsm nd hepti tissue lipid profiles ND 1) HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Plsm lipid profile (mg/dl) Triglyeride Totl holesterol HDL-holesterol LDL-holesterol Hepti tissue lipid profile (mg/g) Totl lipid Triglyeride Totl holesterol 68.8±4.6 NS2)3) 157.9±3.6 NS 37.1±1.6 NS 134.5±3.7 NS 19.3±1.5 4).56±.3 NS.62±.4 NS 67.4± ± ± ±4. 18.±1.3.6±.4.68± ± ± ± ± ±.9.58±.2.71± ± ± ± ± ±1.3.57±.3.63± ± ± ± ± ±.9.54±.1.58± ± ± ± ± ±.8.55±.1.58±.2 1) ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. 2) Vlues re the men±se for 7 nimls in eh group. 3) NS: no signifint differene. 4) Vlues with different letters in the sme row re signifintly different t P<.5 y Dunn's multiple rnge test.

7 호박즙, 옥수수수염차, 팥차및그혼합물의체중감소와항산화효과 1245 Totl lipid (mg/g) d Triglyeride (mg/g) d Totl holesterol (mg/g) ND HFD HFD PJ HFD CT HFD AT HFD PCA +PJ +CT +AT +PCA ND ND HFD HFD PJ HFD CT HFD AT PCA HFD +PJ +CT +AT +PCA ND ND HFD HFD PJ HFD CT HFD AT HFD PCA +PJ +CT +AT +PCA Fig. 4. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on epididyml dipose tissue lipid profiles. ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. Eh r represents the men±se (n=7 per group). Brs with different letters re signifintly different t P<.5 y Dunn's multiple rnge test. 방식이에의해라디칼생성이증가한것을확인할수있었으며, 호박즙, 옥수수수염차, 팥차를각각섭취하는것보다세가지를혼합하여섭취하였을때라디칼소거효과가증가하는것을확인할수있었다. 늙은호박의경우카로틴색소는비타민 A의전구체로서뿐만아니라활성산소의소거제 (4) 로작용함으로써항산화작용을발휘하는기능성성분으로보고되고있으며, lutein, α-rotene, β-rotene이 3% 정도함유되어있다 (41). 옥수수수염유래플라보노이드로는 mysin, pimysin, methoxymysin 등이있으며이중 mysin은옥수수수염에가장많이함유된대표적인기능성물질로종양세포주에대한세포독성효과및라디칼소거활성등이보고되어있다 (42,43). 팥의색소는 nthoynin계의 ynidin으로알려져있으며 (44), 이들색소는항산화 (45) 및항종양효과 (46) 를나타내는것으로보고되고있다. TE (1 μm Trolox equivlent) ND HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Fig. 5. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on oxygen rdil sorne pity (ORAC) nd totl rdil trpping pity (TRAP) in plsm. ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. Eh r represents the men±se (n=7 per group). Brs with different letters re signifintly different t P<.5 y Dunn's multiple rnge test. 간지질과산화물형성에미치는영향지질과산화물은활성산소종등산화적스트레스에의해체내각종지질성분으로부터생성된다. 생성된지질과산화물은세포막의투과성을변형시켜 DNA, 탄수화물, 단백질, 지질의손상을유발하여노화, 고혈압, 동맥경화, 심장병등각종질병을일으킬수있다 (47). 간에서의지질과산화물을분석한결과를살펴보면 HFD군이 ND군보다증가하는경향을보였고, HFD+PCA군은 HFD군보다유의적으로감소하였다 (Fig. 6). 홍삼성분이지질과산화에미치는효과를살펴본 Sung 등 (48) 은홍삼에함유된사포닌을비롯하여정유성분, 폴리아세틸렌, 페놀성분, 배당체및산성펩타이드등 (49) 의생리활성성분이지질과산화억제효과가있다고보고있다. 따라서 HFD+PCA군의지질과산화정도가 HFD군에비해억제된이유는옥수수수염과팥에함유된사포닌과플라보노이드, 안토시아닌과늙은호박의카로틴간의상승작용으로인한것으로생각한다. TBARS (μm/mg protein) ND HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Fig. 6. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on TBARS in liver. ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. Eh r represents the men±se (n=7 per group). Brs with different letters re signifintly different t P<.5 y Dunn's multiple rnge test.

8 1246 박재희 이은지 박은주 Tle 4. Effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on erythroyte nd hepti ntioxidnt enzyme tivities ND 1) HFD HFD+PJ HFD+CT HFD+AT HFD+PCA GSH-Px (μm/mg protein) Ctlse (mm/mg protein) 286.6±35.9 2)3) 237.1± ±.2 NS 1.± ±16.3.9± ±78. 1.± ± ± ± ±.1 1) ND, norml diet; HFD, high-ft diet; HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. 2) Vlues re the men±se for 7 nimls in eh group. 3) Vlues with different letters in the sme row re signifintly different t P<.5 y Dunn's multiple rnge test. 4) NS: no signifint differene. 간조직항산화효소활성에미치는영향비만은지질과산화물의형성을촉진해체내항산화시스템의불균형을일으키는과정과관련이있다 (5,51). 지방이축적될경우몸전체에서산화스트레스가증가하는양상을보이는데, 비만쥐의경우지방조직에서활성산소종의생산이선택적으로많이이루어진다. 생체내에는이러한산화적손상을예방하거나복구하는체계로 superoxide dismutse(sod), glutthione(gsh), glutthione peroxidse(gsh-px), glutthione S-trnsferse(GST), tlse(cat) 등이있다 (52). 비만으로인한산화스트레스는항산화효소활성을감소시켜항산화방어시스템을저하시킨다고알려진바활성산소종과항산화효소와의항상성의불균형은 DNA, 단백질및지질의산화변형을통하여노화를촉진하며심혈관질환, 염증성질환및암과같은질환을유발하게된다 (53,54). 간항산화효소활성을분석한결과는 Tle 4에나타내었다. 간조직 GSH-Px는 HFD군과 ND군간의유의적차이는없었으나고콜레스테롤식이섭취군에서감소하는경향을나타내었고, 옥수수수염차섭취군과호박즙, 옥수수수염차, 팥차혼합물섭취군은고콜레스테롤식이섭취군보다 GSH-Px 활성이유의적으로증가했으며, 이들내함유된항산화성분의상승작용에의한결과로생각된다. GSH-Px는 GSH와과산화수소로부터 GSSG와물, 알코올을생성하는반응을촉매하여생체내과산화수소나지질과산화물을제거하는작용을한다 (55). 간조직 tlse는모든그룹에서유의적인결과를나타내지못하였다. 간세포 DNA 손상에미치는영향 Comet ssy는소형전기영동겔방법으로인체에유해한독성물질에의한 DNA 손상정도를간단하고빠르게측정하여유전독성의발생여부를알아내는좋은도구로이용되고있다 (56). 간세포 DNA 손상은알칼리환경의전기영동을이용한 omet ssy를수행한후 DNA 손상정도를세포의파괴된파편의길이로측정하였다. 고지방식이로비만을유도한동물의간세포에 H 2O 2 2 μm 농도를처리하여내재적인 DNA 손상정도를관찰하였고, 이에따른간세포의 DNA 손상에대한음료섭취효과는 Fig. 7에제시하였다. 본실험에서 ND군에비해 HFD군은유의적으로 DNA 손상이증가하였다. 이는고지방식이에의한활성산소의증 Til DNA (%) d ND HFD HFD+PJ HFD+CT HFD+AT HFD+PCA Fig. 7. Antigenotoxi effet of pumpkin juie (PJ), orn silk te (CT), dzuki en te (AT), nd the mixture of PJ, CT, nd AT (PCA) on high-ft diet indued DNA dmge in heptoytes. ND, norml diet; HFD, HFD+PJ, HFD supplemented with pumpkin juie; HFD+CT, HFD supplemented with orn silk te; HFD+AT, HFD supplemented with dzuki en; HFD+PCA, HFD supplemented with the mixture of PJ, CT, nd AT. Eh r represents the men±se (n=7 per group). Brs with different letters re signifintly different t P<.5 y Dunn's multiple rnge test. 가로체내지질과산화물이과다생성되면서 DNA 손상이많이유발되는것으로생각한다. HFD군에비해 HFD+PJ 군, HFD+AT군, HFD+PCA군은 DNA 손상이유의적으로감소하였으며, 그효과는 HFD+PCA군에서가장높게나타났다. 이결과로호박즙, 팥차각각의항산화성분들이고지방식이로유도된 DNA 손상회복효능을가진다는것을확인할수있었으며, 그효과는호박즙, 옥수수수염차, 팥차의혼합물에함유된항산화성분들의상승작용에의해증가한것으로생각된다. 요 4주간식이로유도된비만동물에서호박즙 (25 mg/kg BW), 옥수수수염차 (25 mg/kg BW), 팥차 (25 mg/kg BW) 와이들혼합음료 ( 호박즙분말 : 옥수수수염차분말 : 팥차분말, 1:1:1, 25 mg/kg BW) 를 9주간투여하여그효능을알아본결과호박즙, 옥수수수염차, 팥차개별섭취시체중감소효과는보였으나그기전은본연구에서규명하지못하였다. 그러나혼합음료섭취군에서는지방조직무게의유의적감소로인한체중저하효능을확인할수있었다. 부고환지방 약 d

9 호박즙, 옥수수수염차, 팥차및그혼합물의체중감소와항산화효과 1247 조직의표면적은고지방식이섭취군 (HFD) 군에비해고지방식이 + 혼합음료섭취군 (HFD+PCA) 군에서유의적으로감소하였으며, HFD+PCA군의아디포넥틴은 HFD군에비해유의적으로증가하였다. 음료섭취군들중고지방식이 + 혼합음료섭취군 (HFD+PCA) 은고지방식이섭취군 (HFD) 보다 ORAC vlue가증가하였고, GSH-Px의간항산화효소에서도 HFD군보다 HFD+PCA군이유의적으로증가하였다. 또한, HFD+PCA군은고지방식이에의한지질과산화물생성을유의적으로감소시켰으며, 비만으로인한산화적스트레스에따른 DNA 손상을현저히낮추었다. 따라서산화적스트레스에의해유도되는비만치료에호박즙, 옥수수수염차, 팥차를각각섭취하기보다는이들세가지를동일한비율로혼합섭취시각각의항산화효능의상승작용에의한항비만효과가현저하게나타낼것으로생각한다. 감사의글 이논문은 211년도정부 ( 미래창조과학부 ) 의재원으로한국연구재단의지원을받아수행된기초연구사업임 (No. NRF ). REFERENCES 1. Koren Sttistil Informtion Servie. Oesity. kr/sttistislist/sttistislist_1list.jsp?vwd=mt_ztitle &prentid=d#sucont (essed Jn 216). 2. Ferretti G, Bhetti T, Moroni C, Svino S, Liuzzi A, Blzol F, Bihieg V. 25. Proxonse tivity in highdensity lipoproteins: omprison etween helthy nd oese femles. J Clin Endorinol Met 9: Higdon JV, Frei B. 23. Oesity nd oxidtive stress: diret link to CVD?. Arteriosler Throm Vs Biol 23: Antiptis VJ, Gill TP. 21. Oesity s glol prolem. In Interntionl Text Book of Oesity. Björntorp P, ed. John Wiley & Sons In., Chihester, West Sussex, UK. p Seo YH. 25. Ptent trend of nti-oesity supplementry food. Food World 8: Hn LK, Kimur Y, Okud H. 25. Anti-oesity effets of nturl produts. Stud Nt Prod Chem 3: Moro CO, Bsile G. 2. Oesity nd mediinl plnts. Fitoterpi 71: S73-S Rylm S, Dell-Fer MA, Bile CA. 28. Phytohemils nd regultion of the dipoyte life yle. J Nutr Biohem 19: Ku BH Kore folk mediine. Prkhul Press In., Seoul, Kore. p Lim JP, Choi H. 21. Effets of the wter extrt from Cuurit mxim Duhesne on inflmmtion nd hyperlipidemi in rts. Koren J Med Crop Si 9: Velzquez DV, Xvier HS, Btist JE, de Cstro-Chves C. 25. Ze mys L. extrts modify glomerulr funtion nd potssium urinry exretion in onsious rts. Phytomediine 12: Ru O, Wurglis M, Dingermnn T, Adel-Twn M, Shuert-Zsilvez M. 26. Sreening of herl extrts for tivtion of the humn peroxisome prolifertor-tivted reeptor. Phrmzie 61: Chin HS, Pk KJ, Pk SH, Kim JK. 29. The effets of herl extrt mixture on nti-oesity. J Koren So Food Si Nutr 38: Mksimović Z, Mlenić D, Kovević N. 25. Polyphenol ontents nd ntioxidnt tivity of Mydis stigm extrts. Bioresour Tehnol 96: Mksimović ZA, Kovević N. 23. Preliminry ssy on the ntioxidnt tivity of Mydis stigm extrts. Fitoterpi 74: Hn LK, Kimur Y, Okud H. 23. Anti-oesity effets of te sponins. 7th Interntionl Symposium on Green Te, Seoul, Kore. p Kim HS, Kim TW, Kim DJ, Hwng HJ, Lee HJ, Choe M. 27. Effets of nturl plnts supplementtion on dipoyte size of the epididyml ft pds in rts. J Koren So Food Si Nutr 36: Vsselli JR, Weindruh R, Heymsfield SB, Pi-Sunyer FX, Boozer CN, Yi N, Wng C, Pietroelli A, Allison DB. 25. Intentionl weight loss redues mortlity rte in rodent model of dietry oesity. Oes Res 13: Cho JH, Lee NJ, Hong SH, Kim DK, Shin S, Prk JH, Kng JK, Kim YB, Hwng SY. 25. Effet of Hwlgidn SJ-21 on oesity indued y high-ft diet in Zuker rts. L Anim Res 21: Allin CC, Poon LS, Chn CS, Rihmond W, Fu PC Enzymti determintion of totl serum holesterol. Clin Chem 2: MGown MW, Artiss JD, Strndergh DR, Zk B A peroxidse-oupled method for the olorimetri determintion of serum triglyerides. Clin Chem 29: Friedewld WT, Levy RI, Fredrikson DS Estimtion of the onentrtion of low-density lipoprotein holesterol in plsm, without use of the preprtive ultrentrifuge. Clin Chem 18: Folh J, Lees M, Slone Stnley GH A simple method for the isoltion nd purifition of totl lipides from niml tissues. J Biol Chem 226: Ou B, Hmpsh-Woodill M, Prior RL. 21. Development nd vlidtion of n improved oxygen rdil sorne pity ssy using fluoresein s the fluoresent proe. J Agri Food Chem 49: Björntorp P The ssoitions etween oesity, dipose tissue distriution nd disese. At Med Snd Suppl 723: Do GP, Lee HJ, Do JR, Kim HK Antioesity effet of the Cuuit mosht Duh extrts in 3T3-L1 dipoyets. Koren J Food Preserv 19: H TJ, Lee BW, Prk KH, Jeong SH, Kim HT, Ko JM, Bek IY, Lee JH Rpid hrteristion nd omprison of sponin profiles in the seeds of Koren Leguminous speies using ultr performne liquid hromtogrphy with photodiode rry detetor nd eletrospry ionistion/mss spetrometry (UPLC-PDA-ESI/MS) nlysis. Food Chem 146: Moon GA, Choi SM, Kim SH, Kim SS, Kng JY, Yoon Y. 23. Humn nd niml study on the nturl food for oesity nd metoli syndrome risk ftors. J Koren So Food Si Nutr 32: Rhee SJ, Kim KR, Kim HT, Hong JH. 27. Effets of tehin on lipid omposition nd dipose tissue in oese rts fed high ft diet. J Koren So Food Si Nutr 36: Med N, Tkhshi M, Funhshi T, Kihr S, Nishizw H, Kishid K, Ngretni H, Mtsud M, Komuro R, Ouhi

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