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1 J Korean oc Food ci Nutr 한국식품영양과학회지 43(10), 1491~1499(2014) 고콜레스테롤식이투여마우스에서복분자미숙과추출물과홍삼농축액복합투여의콜레스테롤개선효과 이민정 1 이수정 1 최혜란 1 이정현 1 정종태 1 최경민 2 차정단 2 황승미 2 박종혁 3 이주희 3 이태범 1 1 고창복분자연구소 2 진안홍삼연구소 3 임실치즈과학연구소 Cholesterol Improvement Effects of Co-treatment with Black Rasperry and Red Ginseng Extracts in Mice Fed a High Cholesterol Diet Min Jung Lee 1, u Jung Lee 1, Hye Ran Choi 1, Jung-Hyun Lee 1, Jong Tae Jeong 1, Kyung-Min Choi 2, Jeong-Dan Cha 2, eung-mi Hwang 2, Jong Hyuk Park 3, Joo Hee Lee 3, and Tae Bum Lee 1 1 Gochang Black Rasperry Research Institute 2 Institute of JinAn Red Ginseng 3 Imsil Research Institute of Cheese cience ABTRACT We examined the effects of unripe lack rasperry (UBR) and red ginseng (RG) extracts on cholesterol improvement in C57BL/6J mice fed a HCD (high cholesterol diet) for 12 weeks. Hepatic total lipid and total cholesterol contents were significantly induced in hyperlipidemic mice. However, supplementation with UBR, RG and simvastatin effectively reduced these lipid profiles. Further, UBR and co-treatment with UBR and RG increased expression of LDL receptor, REBP2, and R-B1 mrna compared with HCD. The ApoB/ApoA1 ratio was reduced y co-treatment with UBR and RG compared to treatment with UBR. In addition, histopathologic evaluation showed that co-treatment with UBR and RG suppressed lipid accumulation as well as FA and leptin expression in plasma. These results indicate that co-treatment with UBR and RG may e useful for the prevention of hypercholesterolemia. Key words: unripe lack rasperry, red ginseng, imvastatin, high cholesterol diet, hypercholesterolemia 서 식습관, 비만, 음주, 운동부족등으로인해발생하는건강문제, 즉잘못된생활습관으로인해발생하는생활습관병중에최근대사성질환이급증하고있으며, 특히심혈관계질환의발병률은수십년간지속적으로증가하여주요사망원인중하나가되어의학및영양학적측면에서사회적인문제가되고있다 (1,2). 대사증후군은복부비만, 고혈압, 이상지질혈증, 공복혈당장애가한사람에서군집적으로나타나는것을말하는데주요요인중의하나인고지혈증은저밀도지단백 (low density lipoprotein, LDL) 농도의이상증가, 중성지방의증가, 고밀도지단백 (high density lipoprotein, HDL) 의감소, 리포단백의증가, 지질분해감소와같은지방의생화학대사이상에의해발생하게된다 (3). 이러한질병의치료약물들 Received 13 June 2014; Accepted 5 August 2014 Corresponding auteror: Tae-Bum Lee, Gochang Black Rasperry Research Institute, Jeonuk , Korea tlee01@gri.re.kr, Phone: 론 은장기간복용으로인한부작용발생의문제점이있어치료보다는예방이필요한질환으로여겨지고있으며, 최근에는천연물의건강기능성에대한연구가활발히이루어지고있고이에대한관심과수요도증가하고있다 (4-6). 전국적으로재배되고있는복분자 (Ruus occidentalis) 는폴리페놀과플라보노이드, 안토시아닌, 트리테르페노이드등다양한생리활성물질을함유하고있어항암활성 (7), 면역개선 (8), 항염증 (9), 항산화및항균효과 (10) 등다양한생리활성에대한효능이밝혀진바있다. 최근연구에의하면복분자완숙과추출물보다복분자미숙과추출물이시험관시험에서 LDL을억제시키고체내에서는콜레스테롤생합성을억제하였으며, CD 36, scavenger receptor type A(R-A) 수용체를억제시킴으로써세포내의 ox- LDL의흡수를차단시키고세포안에서는 macrophage에있는 PPAR-γ를억제시킴으로인해 LDL 산화가억제되었으며, adipophilin의활성이억제됨에따라세포안에있는콜레스테롤방출을촉진시켜동맥경화를완화시킬수있다고보고한바있다 (11,12). 또한동물실험에서도 LDL을억제함으로써고지혈증및지질대사에효능이있음을확인하

2 1492 이민정 이수정 최혜란 이정현 정종태 최경민 차정단 황승미 박종혁 이주희 이태범 였다 (13). 홍삼 (red ginseng) 은인삼 (Panax ginseng C.A. Meyer) 을껍질을벗기지않은상태로장시간증기로쪄서건조한것을말하는데, 이러한가공과정을거치면서인삼의생리활성물질인사포닌 (saponin) 의함량이증가하고인삼에없는새로운생리활성성분인 ginsenoside-rg2, Rg3, Rh1, Rh2 가생성된다 (14). 특히 R2는혈중콜레스테롤수치저하와배설촉진, 고콜레스테롤혈증으로야기되는동맥경화의개선효과가보고된바있다 (15). 이러한홍삼내에존재하는 ginsenoside는지방이나콜레스테롤의흡수, 이동속도를촉진시키고, 혈청콜레스테롤증가를억제한다는연구결과 (16,17) 와고지방식이를한실험동물에서 LDL 수용체의합성억제를완화시켜콜레스테롤을저하시켰다는보고가있다 (18). 이전시험관시험연구에서복분자미숙과와홍삼물추출물을이용하여인간간암세포주 (HepG2) 에서콜레스테롤합성억제효과를보였으며, 위장관세포주 (Caco-2) 에서는 HMG-CoA reductase 활성억제, LDL 억제및 HDL 증가, 콜레스테롤배출증가에관련된유전자를조사하였고, 단독투여시보다복합투여시부가적인효과를보였다. 하지만복분자미숙과 (100 μg/ml) 에비해홍삼 (800 μg/ml) 은높은농도에서효과를보여 (11) 이에본연구는복분자미숙과추출물과홍삼농축액을복합투여하여저농도에서고콜레스테롤식이를급여한실험동물에있어지질농도, 지질대사관련유전자발현등에미치는영향을조사하여기능성제품을개발하는기초자료로활용하고자하였다. 재료및방법시료추출고창에서재배된복분자미숙과의 10배용량의물을첨가한후 2시간동안농축추출기 (Cosmo 660, Kyungseo Machine, Incheon, Korea) 를이용하여 2회가열추출하였다. 추출물은여과하여농축한후동결건조 (PVTFD10R, Ilsinioase, Dounducheon, Korea) 하여사용하였다. 진안에서재배된인삼을이용한홍삼농축액은에탄올로 3회추출하여원심분리기를이용하여전분질을여과하였다. 여과된추출액을 C에서감압농축하여농축액을얻은후 C에서 3일간숙성하여 85 C에서 2시간살균하여사용하였다. 실험동물의사육환경및식이투여실험동물은 5주령된 C57BL/6J종수컷마우스를 ( 주 ) 중앙실험동물 (eoul, Korea) 로부터구입하여 1주일간일반사료로적응시켰다. 그런다음각실험군마다평균체중이약 g이되도록 Z 자식으로각군당 9마리씩 5개군으로나누어 stainless steel ottomed cage에 3마리씩분리사육하였다. 실험동물사육실의온도는 22±2 C, 상대습도 는 50±5% 로유지하였으며명암은 12시간주기로조절하였다. 본동물실험에서실험군의분류는정상대조군은 AIN- 76A 식이 (Normal) 를하였고, 고콜레스테롤대조군은 highcholesterol diet 식이 (CTL), 복분자미숙과추출물투여군은 high-cholesterol diet 사료에복분자미숙과물추출물 250 mg/kg/day(ubr) 를투여하였고, 복분자와홍삼복합투여군은 high-cholesterol diet 사료에복분자미숙과물추출물 125 mg/kg과홍삼농축액 125 mg/kg/day(ubr+ RG) 를복합투여하였고, 양성대조군은 high-cholesterol diet 사료에 imvastatin 10 mg/kg/day(imvastatin) 군으로나누어경구용주사기를이용하여 12주간매일일정한시간대 ( 오전 10시 ) 에경구투여를하였으며, 정상대조군과고콜레스테롤대조군에는같은양의증류수를투여하였다. 사료는실험동물용고지질이함유된고형사료와필터및자외선살균기로여과살균된정제수를자유로이섭취하도록하였으며매주식이를공급하였고, 음수, 식이, 체중은일주일에한번일정한시간대에디지털계량기를사용하여측정하였다. 식이섭취효율은실험전기간동안의체중증가량을같은기간동안에섭취한식이섭취량으로나누어산출하였다. 실험에사용한 imvastatin은 statin계의기본약물로간에서콜레스테롤의생성을조절하며, HMG-CoA reductase inhiitor로알려져있는동맥경화치료제로사용되고있는약이다 (19-22). 본동물실험은고창복분자연구소동물실험윤리위원회승인 (GBRI IACUC 14001) 하에진행되었다. 혈액및조직적출희생전 12시간절식시킨후 diethyl ether로마취시켜개복한즉시복대정맥으로부터혈액을채취하였다. 혈액은헤파린튜브에수집후, 3,000 rpm에서 15분간원심분리하여획득한혈장 (plasma) 을분석에사용하였다. 채취한간조직의일부는 10% 중성포르말린에고정하여병리조직제작과정을거쳐 hematoxylin&eosin(h&e) stain 후간조직의지질침착정도를확인하였고, 나머지조직은 70 C 에냉동보관한후분석에사용하였다. 혈장에서 ApoB/ApoA1 ratio는 Enzyme-Linked Immunosorent Assay(ELIA) Kit for Apolipoprotein A1과 ApoB(Cloud-Clone Corp, Houston, TX, UA) 를각각측정하여 ratio 값을구하였고, 렙틴 (leptin) 의농도는 Quantikine Human Leptin Immunoassay(R&D ystem, Inc., Minneapolis, MN, UA) kit로측정하였으며, FA(fatty acid synthase) 는 MOUE Fatty Acid ynthase ELIA kit(cusaio, Wuhan, China) 를사용하였다. 간조직중의지질분석간의총지질은간조직을 Folch법으로추출한후정량하였으며, 추출한간지질을시료로하여간의총콜레스테롤 (Biovision Cholesterol Quantitation kit, Bio-Vision Inc.,

3 복분자와홍삼의콜레스테롤개선효과 1493 Mountain View, CA, UA) 과중성지방 (Triglyceride Fluorometric Assay Kit, Cayman, Ann Aror, MI, UA) 양을측정용 kit를사용하여측정하였다. RNA 분리간조직으로부터 RNA를추출하기위하여실험동물의간 20 mg을 RNA 추출시약 (RNAiso PLU, Takara, Otsu, Japan) 1 ml에넣고, homogenizer로균질화한후상온에 5분동안방치하였다. 이후 chloroform 200 µl를넣어 20 초간 vortex 하였고 15,000 rpm으로 10분동안원심분리한후상층액을새로운튜브에옮겼다. 상층액과동량으로 isopropanol을첨가한다음 10분동안방치한후이를다시 15,000 rpm에서 10분동안원심분리하여 RNA 침전물을획득하였다. RNA 침전물을 0.1% diethyl pyrocaronate (DEPC) 와혼합한 75% 에탄올을넣어 washing 하여 15,000 rpm에서 5분동안원심분리한후에탄올을제거하여실온에서침전물을건조시켜 0.1% DEPC water에녹였다. RNA 농도 (1 OD=40 µg/ml) 는 spectrophotometer (DUR 730 spectrophotometer, Beckman, Fullerton, CA, UA) 를이용하여 260 nm에서측정하였다. YBR Green Ⅰ kit(roche, Mannheim, Germany) 를이용하여 Light Cycler 2.0(Roche) 에서증폭하였다. 실험에사용된 primer의염기서열과 PCR 조건은 Tale 1과같으며유전자의정량분석은 Light Cycler oftware 4.0(Roche) 을이용하였다. 간의병리조직학적분석간조직내지방의축적을확인하기위해서적출된간조직의일부를 10% 중성포르말린에고정하였다. 병리조직학적검사를위한통상적인방법을사용하여파라핀포매한다음 4 µm의두께로절편을만들고슬라이드제작한후 H&E 염색을실시하고 Nikon Eclipse E200(Nikon, Kanagawa, Japan) 현미경을이용하여 NAH(nonalcoholic steatohepatitis) score를측정하였다. NAH score란비알코올성지방간염수치로거대수포성지방증, 간세포손상과혼합염증반응이라는 3가지병변이주로 3구역, 중심소엽에분포하는것을특징으로한다. NAH score 측정은 H&E 염색을한슬라이드를광학현미경으로조사하여지방증, 풍선변성, 소엽염증정도의점수를합산하여 5점이상이면 NAH로, 2점이하이면 NAH가아닌것으로판정하였다 (23). 실시간역전사중합효소연쇄반응 (real-time PCR quantification) First strand cdna를 50 mm Tris-HCl(pH 8.3), 75 mm KCl, 3 mm MgCl 2, 10 mm DTT(Invitrogen, Carlsad, CA, UA), 1 U/µL RNasin(Invitrogen), 1 mm each dntp, oligo(dt) ng과 MMLV reverse transcriptase(invitrogen) 200 U가함유된 20 µl의용액에서총 RNA 2 µg으로부터합성하였다. cdna를합성한뒤 HMG-CoA reductase, LDL 생성관련인자인 LDL receptor(ldlr) 와전사인자인 sterol regulatory element inding protein-2(rebp-2) mrna 발현정도와 HDL 관련인자인 scavenger receptor B1(R-B1) 및콜레스테롤배출관련 transporter인 ATP inding cassette transporter A1(ABCA1) mrna의발현정도를확인하였다. 실시간역전사중합효소연쇄반응은 Fasttart DNA Master 통계분석모든분석자료는평균 ± 표준오차 (mean±e) 로나타내었으며실험결과는 P 12.0K(P Inc., Chicago, IL, UA) 를이용하여 Duncan's multiple range test에의해 P<0.05 수준에서통계처리하였다. 결과및고찰체중증가량, 음수섭취량, 식이섭취량 C57BL/6J 5주령마우스에고콜레스테롤식이를급여하여복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군인 imvastatin을 12주간경구투여하여각군의체중증가량, 음수섭취량, 식이섭취량을 Tale 2에나타내었다. 체중증가량은정상대조군에비해고콜레스테롤식이군에서체중증가를보였으나, Tale 1. PCR primers and condition for real-time PCR Gene & A Nucleotide sequences HMG-CoA reductase LDL receptor REBP2 ABCA1 β-actin A A A A A CCTGACACTGAACTGAAGCG TCTTTCCAGAACACAGCACG AAGACGTGCTCCCAGGATGACTTC GTGCTCCTCATCTGACTTGTCCTTG ATATCATTGAAAAGCGCTAC ATTTTCAAGTCCACATCACT AGTCACAGCTCTGTGCTCTGG GTTTGTCTCCTTCGAAATGTCA AGAGAAGCTGTGCTATGTT CACAGGATTCCATACCCAAG Length of PCR products (p) Annealing ( C)

4 1494 이민정 이수정 최혜란 이정현 정종태 최경민 차정단 황승미 박종혁 이주희 이태범 Tale 2. Effects of extracts of unripe lack rasperry and red ginseng supplementation on ody weight gain, water intake, and food intake in C57BL/6J mice fed a HCD for 12 weeks Group Normal CTL UBR250 UBR125+RG125 imvastatin Initial ody weight (g) 20.92±0.44 N 20.66± ± ± ±0.34 Final ody weight (g) 28.76±0.72 N 30.07± ± ± ±0.75 Body weight gain (g/90 days) 0.65±0.04 N 0.78± ± ± ±0.04 Water intake (g) 27.08±0.64 a 25.76±0.41 a 23.67± ± ±0.69 a Food intake (g) 18.8±0.24 N 20.5± ± ± ±1.42 1) Normal: normal group, CTL: high cholesterol diet, UBR250: high cholesterol diet+unripe lack rasperry water extract 250 mg/kg, UBR125+RG125: high cholesterol diet+unripe lack rasperry water extract 125 mg/kg+red ginseng 125 mg/kg, imvastatin: high cholesterol diet+imvastatin 10 mg/kg. Values are expressed as mean±e (n=9). N: not significant. Different superscript letters (a,) in the same column indicate significant differences etween groups at P<0.05 y Duncan's multiple range test. 고콜레스테롤식이군과비교하였을때복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군간의체중증감, 식이섭취량에서는큰차이를보이지않았다. 간조직중의지질분석 C57BL/6J 5주령마우스에복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군인 imvastatin을 12주간실험식이를급여한후간조직의지질함량에미치는영향은 Tale 3과같다. 간조직중에정상대조군과비교했을때고콜레스테롤식이군에서 total lipid와 total cholesterol의양이유의성있게증가되었다. 증가된고콜레스테롤식이군에비해 total lipid와 total cholesterol은양성대조군을처리한그룹에서각각 26.6%, 22.8% 의유의성있는감소결과를보여주었다. 고콜레스테롤식이군과비교하였을때 total cholesterol은복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군에서각각 26.2%, 31.7% 의감소를나타내었으며, 이는복분자와홍삼복합투여군의부가적효과 (additive effect) 를나타내는것으로확인하였다. Triglyceride는투여군간의차이를나타내지않았다. Tale 3. Effects of extracts of unripe lack rasperry and red ginseng supplementation on hepatic total lipid, total cholesterol and triglyceride in C57BL/6J mice fed a HCD for 12 weeks Group Normal CTL UBR250 UBR125+RG125 imvastatin Total lipid (mg/g tissue) 0.06±0.01 c 0.15±0.02 a 0.11± ± ±0.04 Total cholesterol (mg/g tissue) 1.64±0.07 c 3.50±0.22 a 2.58± ± ±0.13 Triglyceride (mg/g tissue) 329.5± ± ± ±17.53 a 244.3±13.6 c Values are expressed as mean±e (n=9). Different superscript letters (a-c) in the same column indicate significant differences etween groups at P<0.05 y Duncan's multiple range test. 간의병리조직학적관찰각실험군들의간조직의병리조직학적변화는 Fig. 1과같다. 일반사료를먹은정상대조군은모든개체에서간에유의한병변과지방간이관찰되지않았다. 반면비만이유도된고콜레스테롤식이군에서는간세포내크고작은공포모양의지방구가침착되어간손상과지방간소견이관찰되었다 (Fig. 1). 병리조직학적검사를위하여통상적병리조직을제작한다음 H&E 염색을실시한후먼저 NAH score 를실시하였다 (Fig. 1). NAH score는비알콜성지방간염을말하며알코올성간질환에서보이는병리학적인특징을수치화한결과로서, 본실험그룹의 NAH score는정상대조군 0.2±0.14, 고콜레스테롤식이군 5.8±0.83, 복분자미숙과추출물투여군 2.6±0.50, 복분자미숙과물추출물과홍삼농축액복합투여군 1.4±0.53, 양성대조군은 3.1±0.60 으로, 복분자미숙과물추출물과홍삼농축액복합투여군이고콜레스테롤식이군에비교하여비알콜성지방간염의점수가유의적으로낮게나타났다. 복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군에서는고콜레스테롤식이군에비교하여간손상의정도가현저히감소되어있으며, 간세포내크고작은공포모양의지방구가현저히감소되었음을확인하였다. 고콜레스테롤식이로비만을유도하고대조약물로 imvastatin을투여한양성대조군에서발견된간세포내크고작은공포모양의지방구가현저히줄어들었다. 병리조직학적검사결과고콜레스테롤식이로비만이유도된군에서지방간병변이유의하게유발되어있는것을확인하였으며, 복분자미숙과추출물만투여한군과복분자미숙과물추출물과홍삼농축액복합투여군모두에서지방세포침윤과같은지방간병변의감소를확인하였다. 그중에서복분자미숙과물추출물과홍삼농축액복합투여군은고콜레스테롤식이군보다간세포내지방구가현저히줄어든것을확인할수있었고, 양성대조군보다지방세포침윤과같은지방간병변의감소가확인되었다. 간조직내 LDL과 HDL 관련유전자에미치는영향 C57BL/6J 5주령마우스에복분자미숙과추출물투여군,

5 복분자와홍삼의콜레스테롤개선효과 1495 Non-alcoholic steatohepatitis (NAH) score d a Normal CTL UBR250 UBR125+ simvastatin RG125 c Fig. 1. NAH score and histologic findings of liver tissue y the extracts of unripe lack rasperry and red ginseng treatment in hyperlipidemic mice fed the HCD for 12 weeks. NAH, non-alcoholic steatohepatitis. H&E stain, 400. cale ar=20 µm. Values are expressed as mean±e (n=9). Different letters (a-d) aove the ars indicate significant differences etween groups at P<0.05 y Duncan's multiple range test. 복분자와홍삼복합투여군과양성대조군인 imvastatin을 12주간경구투여한후에간조직내에서콜레스테롤관련유전자발현을조사하였다 (Fig. 2). HMG-CoA reductase는콜레스테롤생합성과정에중요하게작용하는효소이다. 현재동맥경화치료에사용되고있는 statin 계열의약물들은 HMG-CoA reductase의저해제로작용하고있으며, HMG-CoA reductase의활성은감소시키지만감소된콜레스테롤에대한항상성유지를위해 HMG-CoA reductase 유전자의발현은증가하게된다 (24). 따라서 HMG-CoA reductase의활성을조절하면혈중콜레스테롤의수치를조절할수있다는사실을바탕으로복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군에서고콜레스테롤식이군과비교하였을때각각 2.6배, 1.8배, 3.0배증가로양성대조군이가장많이증가하였지만복분자미숙과추출물도비슷한효과를보였다. 복분자미숙과추출물이 HMG-CoA reductase를저해함으로써활성을감소시키고, 이것이원인이되어결과적으로간에공급되는콜레스테롤의양이감소하는현상을보상하기위해 HMG-CoA reductase mrna 의발현이증가되는것으로생각된다 (25). uzuki 등 (26) 의연구결과에서도 C57BL/6 mice에녹차추출물을섭취시켰을때고지방및고당질 (sucrose) 식이에의해혈장콜레스테롤농도가상승하는것을억제시키는효과가관찰되었으며, 이는녹차가 HMG-CoA reductase 활성을억제하였기때문이라고설명하였다. 혈중지단백질은 LDL과 HDL로나뉘는데 LDL은혈관에쌓이는지질단백질로서혈중콜레스테롤의약 3/4를형성하고 (27) 간에서조직과세포로콜레스테롤을운반한다. 간에서의 LDLR은혈중의 LDL cholesterol과 IDL cholesterol 을간으로흡수하여다시담즙의형태로재흡수되도록한다. 또한혈중콜레스테롤을제거하고세포내콜레스테롤 양에따라발현이조절되어균형을유지하는데중요한역할을한다. LDLR의발현은콜레스테롤농도를낮출뿐만아니라세포내콜레스테롤항상성을유지하기위해 HMG-CoA reductase의발현을증가시킨다고알려져있다 (24). 포화지방과콜레스테롤의급여는혈중의 LDLR의활성을낮아지게하는데이와같이본실험에서도정상대조군에비해고콜레스테롤식이군에서 LDLR 활성이떨어졌다. 반면에복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군에서콜레스테롤식이군과비교하였을때각각 6.7배, 2.6배, 2.3배증가하여복분자미숙과추출물투여군이가장높은 mrna 발현을보였다. 이는 LDLR 발현을높여혈중의콜레스테롤수치를낮추는기작으로작용했을것으로추측된다. 복분자는생리활성물질의일종인폴리페놀과플라본 (flavone) 이풍부하게함유되어있는데, 폴리페놀중 ellagic acid 및 gallic acid 와홍삼의사포닌계열의 ginsenoside 중에서 Rg2, Rd, Rg3 등은혈중콜레스테롤의함량을낮춰고지혈증과동맥경화에예방효과가있는것으로알려져있다 (11,28,29). Bursill 등 (30) 의연구에서토끼에서카테킨을다량함유한녹차추출물의투여시 LDLR이증가하였다는보고와일치하는결과를얻었다. 혈중콜레스테롤이증가하면 LDLR 유전자발현을조절하는 REBPs의발현이증가하고, 증가된 REBPs 유전자는 LDLR의발현을증가시켜세포내로의콜레스테롤흡수를촉진하여혈중콜레스테롤농도를낮춘다 (24). REBPs 는 LDLR 외에도지방합성효소, HMG-CoA reductase 발현을조절한다 (31). REBPs 유전자는 REBP-1 과 REBP- 2로나눠지는데 REBP-1은지질대사관련중성지방과인지질의생합성에관련된유전자의조절에관여하며, REBP- 2는콜레스테롤의생합성과대사에관련된유전자를조절한다 (32). 실험결과고콜레스테롤식이군에비해복분자미숙

6 1496 이민정 이수정 최혜란 이정현 정종태 최경민 차정단 황승미 박종혁 이주희 이태범 A D B E C Fig. 2. Effects of extracts of unripe lack rasperry and red ginseng on hepatic mrna levels of HMG-CoA reductase, LDL receptor, REBP-2, ABCA1, and R-B1 genes in HCD fed mice for 12 weeks. The level of mrna expression was determined y real-time RT-PCR quantification and normalized y that of mrna β-actin, which was used as a control for RNA. Values are expressed as mean±e (n=9). Different letters (a-c) aove the ars indicate significant differences etween groups at P<0.05 y Duncan's multiple range test. 과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군모두유의적으로증가하였으며, 특히복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군에서는 4배이상의발현률을보여콜레스테롤대사에관여하여유전자를조절할것으로사료된다. 콜레스테롤유출 (cholesterol efflux) 에관여하는 transporter인 ABCA1 유전자의발현또한콜레스테롤대사에있어매우중요하며콜레스테롤을세포외로분비시켜분비된콜레스테롤이간에서담즙산으로바뀌어체외로배출하는역할을담당한다 (33). 따라서본연구에서는복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군에서 ABCA1 유전자의발현이높아졌으나유의적인차이는보이지않았다. Lee 등 (11) 의연구에의하면위장관세포주에복분자미숙과추출물을 100 µg/ml 처리한결과, ABCA1의발현이농도의존적으로증가됨을보고하였다. 본연구에서복분자미숙과 추출물이 ABCA1 유전자발현을 1.6배증가시키는것으로나타나혈중콜레스테롤을배출시키고, 혈중에서 free Apo A1에콜레스테롤을전달하는기전에중요한역할을할것으로생각된다. R-B1은체내콜레스테롤대사에있어서 HDL의선택적흡수에관여하며간과지방대사조직에가장많이분포한다 (34). 기존의연구자들에의해 R-B1 유전자는체내의콜레스테롤을간을통해체외로배출하는역할을담당하며이러한기능을통해뇌심혈관계질환의원인이되는동맥경화에대응하는기작을가지는걸로알려졌다 (35). 복분자미숙과추출물투여군과복분자미숙과물추출물과홍삼농축액복합투여군에서 R-B1 유전자의발현이 3.2 배증가하여혈중 HDL 농도를올리는데중요한역할을할것으로사료된다. 폴리페놀중 caffic acid와 ferulic acid가 ABCG1과 R-B1의발현을증가시켜 HDL 콜레스테롤을증가시킨다고보고하였고 (36), resvertrol이 ABCA1의발현을증가시켜콜레스테롤배출을촉진시킨다고보고된바

7 복분자와홍삼의콜레스테롤개선효과 1497 Tale 4. Effects of extracts of unripe lack rasperry and red ginseng supplementation on ApoB/ApoA1 ratio in C57BL/6J mice fed a HCD for 12 weeks ample ApoB (ng/ml) ApoA1 (ng/ml) Normal CTL UBR250 UBR125+RG125 imvastatin 0.57±0.02 a 0.43± ± ± ± ±0.06 a 1.39± ±0.12 a 1.99±0.09 a 1.68±0.18 a Values are expressed as mean±e (n=9). Different superscript letters (a,) in the same column indicate significant differences etween groups at P<0.05 y Duncan's multiple range test. 있다 (37). 홍삼의주성분인진세노사이드중 R1이동맥경화유도세포주에서콜레스테롤배출을촉진한다는보고도있다 (38). ApoB는 LDL을구성하는지질단백질로 chylomicron에존재하며중성지방이풍부한초저밀도지질단백과저밀도지질단백의체내양을반영하고 ApoA1은 HDL의가장큰구성요소이다. Apolipoprotein B/Apolipoprotein A1 ratio(apob/apoa1 ratio) 는이상지질혈증, 비만, 대사성질환, 심혈관질환의위험도를예측하는데유용한임상적지표로알려져있다. 간으로의비에스테르화지방산 (nonesterified fatty acid) 유입이증가하는경우간에서의 ApoB 분비가증가되고이화작용 (cataolism) 에장애가생겨 ApoB의상승을일으킨다. 최근연구결과에의하면 LDLcholesterol보다는 ApoB가더중요하며 LDL-c/HDL-c보다 ApoB/ApoA1 ratio가더중요하다고발표하였다 (39). 복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군모두에서고콜레스테롤식이군과비교하여 ApoB/ApoA1 ratio가유의적으로감소하는경향을보였고, 특히복분자미숙과물추출물과홍삼농축액복합투여군에서가장높은감소율을나타내었다 (Tale 4). 이결과로미루어보아복분자와홍삼복합투여가콜레스테롤개선에효과가있는것으로사료된다. Leptin (pg/ml) c a 0 Normal CTL UBR250 UBR125+ simvastatin RG125 Fig. 3. Effects of extracts of unripe lack rasperry and red ginseng on plasma leptin level in mice fed with HCD. Values are expressed as mean±e (n=9). Different letters (a-c) aove the ars indicate significant differences etween groups at P<0.05 y Duncan's multiple range test. 하게증가하였으며, 홍삼과상엽복합투여군에서유의성있게감소하여 leptin의저항성이증가하였다는보고가있다 (41). 복분자미숙과추출물은 ellagic acid, ferulic acid, gallic acid, kaempferol, luteolin, quercetin, resveratrol, rutin 등다양한폴리페놀을함유하고있는데 (12), 이중 gallic acid가고지방식이동물모델에서콜레스테롤저하및혈청 leptin을억제한다고보고한바있다 (42). 또한홍삼은 Rg1, R1, R2 및 Rg3 등다양한진세노사이드를함유하고있는데, 이중 R1이비만동물모델에서염증성사이토카인을억제하고 p-tat3 전사인자에의해 leptin의함량을증진시킨다고보고하였다 (43). 지방산합성효소 (fatty acid synthase, FA) 는 ATP 등의 nucleoside triphosphate를요구하지않고 condensation 반응을촉매시키며간에서합성된지방산은중성지방으로전환되어초저밀도리포단백질 (very low density lipoprotein, VLDL) 의형태로혈관을순환하며지방조직에중 40 a c a 혈장의지질대사관련효소측정 Leptin은지방세포에서분비되어체지방을일정하게유지하기위한호르몬으로혈액순환을통해 adipocyte에서분비되며식욕조절인자로지방조직이많을수록혈중 leptin 의농도는증가하게되는데이는 leptin 작용에대한저항성때문이다 (40). Leptin의농도는고콜레스테롤식이군에서가장높이나타났으며, 투여군모두에서감소하였지만복분자미숙과물추출물과홍삼농축액복합투여군에서만유의적으로감소하였다 (Fig. 3). 본실험은복분자미숙과물추출물과홍삼농축액복합투여군의농도가가장낮게나타났으나체중과는상관관계를보이지않았다. 이와같은연구결과로홍삼과상엽을복합투여하였을때체중감소는나타나지않았으나혈중 leptin의농도는고과당식이에서현저 FA (pg/ml) a 0 Normal CTL UBR250 UBR125+ simvastatin RG125 Fig. 4. Effects of extracts of unripe lack rasperry and red ginseng on plasma fatty acid synthase (FA) level in mice fed with HCD. Values are expressed as mean±e (n=9). Different letters (a,) aove the ars indicate significant differences etween groups at P<0.05 y Duncan's multiple range test.

8 1498 이민정 이수정 최혜란 이정현 정종태 최경민 차정단 황승미 박종혁 이주희 이태범 성지방을전달한다. FA의발현은 Fig. 4에나타내었다. 복분자미숙과추출물투여군, 복분자미숙과물추출물과홍삼농축액복합투여군과양성대조군모두고콜레스테롤식이군에비해유의적으로낮아졌다. 이와같이복분자미숙과추출물투여군과복분자미숙과물추출물과홍삼농축액복합투여군은지방산합성효소를저해함으로써고콜레스테롤식이를급여한마우스의체내지질저하에기여한것으로사료된다. Wang 등 (44) 은녹차 catechin의 galloyl moiety 가 FA의활성을억제한다고보고하였고, Yasui 등 (45) 또한 EGCG 를제외한녹차의수용성성분역시 Bal/c mice의간에서 FA, HMG-CoA reductase 및 acetyl-coenzyme A caroxylase alpha 등지질대사에관련하는효소의발현을낮추어혈중지질을낮추었다는연구결과를보고하였다. 요 본연구는복분자미숙과물추출물과홍삼농축액으로 C57BL/6J 수컷마우스를이용하여콜레스테롤억제효과및지질대사에미치는영향을살펴보고, 기능성식품으로의가능성을알아보았다. 실험동물은 C57BL/6J 수컷마우스를사용하였으며, 12주동안고콜레스테롤식이를급여하면서복분자미숙과물추출물과홍삼농축액을경구투여하였다. 그결과로고콜레스테롤식이군과비교하였을때복분자미숙과추출물과홍삼농축액식이군에서체중증가량, 식이섭취량에는변화가없었다. 콜레스테롤합성의가장중요한기작인 LDL receptor와 REBP2 발현에서는복분자미숙과추출물투여군과복분자와홍삼복합투여군모두유의적으로증가하여세포안으로의 LDL 흡수를촉진시킴으로써체내콜레스테롤을낮추는것으로판단된다. HDL 관련유전자발현에서도복분자미숙과추출물투여군과복분자와홍삼복합투여군에서 HDL uptake 유전자인 R-B1의발현을증가시켜콜레스테롤개선효과를보였다. 또한간의형태학적변화에서는고콜레스테롤식이군과비교하였을때복분자미숙과물추출물과홍삼농축액복합투여군에서지방형성이현저히줄어들었고, leptin과 FA의농도를측정한결과유의적으로감소하였다. 결과적으로복분자미숙과물추출물과홍삼농축액복합투여가콜레스테롤과고지혈증예방에긍정적인효과를보일것으로사료된다. 약 감사의글 본연구는산업통산자원부의지역특화기술융복합연구지원사업의 2013년도연구비지원으로이루어졌으며이에감사드립니다. REFERENCE 1. Lim, hin H, ong JH, Kwak H, Kang M, Yoon JW, Choi H, Cho I, Park K, Lee HK, Jang HC, Koh KK Increasing prevalence of metaolic syndrome in Korea. Diaetes Care 34: MHWK Yearook of Health and Welfare tatistics. Report of the Ministry of Health and Welfare, eoul, Korea. p Moon J Nutritional prolems of Korean. Korean J Nutr 29: Mckenny JM Lipid management: tools for getting to the goal. Am J Manag Care 7: Miettinen TA Cholesterol asorption inhiition: a strategy for cholesterol-lowering therapy. Int J Clin Pract 55: hin MK, Han H Effects of methanol extracts from amoo (Pseudosasa japonica Makino) leaves extracts on lipid metaolism in rats fed high fat and high cholesterol diet. Korean J Dietary Culture 17: Eu G, Chun BY, Bandopadhyay R, Yoo NH, Choi DG, Yun J Phylogenic relationships of Ruus species revealed y randomly amplified polymorphic DNA markers. J Crop ci Biotech 11: eeram NP, Adams L, Zhang Y, Lee R, and D, cheuller H, Heer D Blackerry, lack rasperry, lueerry, cranerry, red rasperry and strawerry extracts inhiit growth and stimulated apoptosis of human cancer cells in vitro. J Agric Food Chem 54: Chen T, Hwang HJ, Rose ME, Nines RG, toner GD Chemopreventive properties of lack rasperries in N-nitrosomethylenzylamine-induced rat esophageal tumorigenesis: down regulation of cyclooxygenase-2, inducile nitric oxide synthase and c-jun. Cancer Res 66: Wang Y, Jiao H cavenging capacity of erry crops on superoxide radicals, hydrogen peroxide, hydroxyl radicals and singlet oxygen. J Agric Food Chem 48: Lee J, Lee MJ, Ko YJ, Choi HR, Jeong JT, Choi KM, Cha JD, Hwang M, Jung HK, Park JH, Lee TB Effects of extracts of unripe lack rasperry and red ginseng on cholesterol synthesis. Korean J Food ci Technol 45: Choi HR, Lee J, Lee JH, Kwon JW, Lee HK, Jeong JT, Lee TB Cholesterol lowering effects of unripe lack rasperry water extract. J Korean oc Food ci Nutr 42: Yang HM, Oh M, Lim, hin HK, Oh Y, Kim JK Antiinflammatory activities of Ruus coreanus depend on the degree of fruit ripening. Phytother Res 22: Lee H, Park JH, Cho N, Yu HJ, You K, Cho CW, Kim DC, Kim YH, Kim KH ensory evaluation and ioavailaility of red ginseng extract (Rg1, R1) y complexation with γ-cyclodextrin. Korean J Food ci Technol 41: Yokozawa T, Koayashi T, Oura H, Kawashima Y tudies on the mechanism of the hypoglycemic activity of ginsenoside-r2 in streptozotocin-diaetic rats. Chem Pharm Bull (Tokyo) 33: Joo CN, Koo JH Biochemical study of some parmacological effects of Panax ginseng C. A. Meyer. Korean Biochem J 13: Yoon H, Joo CN tudy on the preventive effect of ginsenosides against hypercholesterolemia and its mechanism. Korean J Ginseng ci 17: Kang BH, Koo JH, Joo CN Effect of saponin fraction of Panax ginseng C. A. Meyer on lood serum lipoprotein distriution of cholesterol fed raits. Korean J Ginseng ci 10:

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