J Korean oc Food ci Nutr 한국식품영양과학회지 2(), 199~1907(2013) http://dx.doi.org/.376/jkfn.2013.2..199 복분자미숙과물추출물의콜레스테롤개선효과 최혜란 이수정 이정현 권지웅 이희권 정종태 이태범 ( 재 ) 고창복분자연구소 Cholesterol-lowering Effects of Unripe Black Raspberry Water Extract Hye Ran Choi, u Jung Lee, Jung-Hyun Lee, Ji Wung Kwon, Hee Kwon Lee, Jong Tae Jeong, and Tae-Bum Lee Gochang Black Raspberry Research Institute, Jeonbuk 55-93, Korea ABTRACT We investigated the effects of unripe black raspberry water extract (UBR-W) and oxidation-ldl treatment on cholesterol levels. Experiments using an established human hepatocellular carcinoma cell line (HepG2) showed a time-dependent increase in expression of LDL receptor after UBR-W treatment. Expression of LDL receptor-related genes, such as REBP1 and 2, increased upon UBR-W treatment. However, expression of HDL-related genes was unaffected by UBR-W. HMG-CoA reductase activity was reduced by UBR-W treatment, whereas HMG-CoA mrna expression significantly increased. In addition, the ApoB/ApoA1 mrna level, which is a predictor of cardiovascular risk, was reduced in a time-dependent manner by UBR-W treatment. Macrophage-like cells (RAW 26.7) showed increased expression of ox-ldl-related genes, such as CD36, scavenger receptor-a, adipophilin, and PPAR-gamma, upon ox-ldl treatment compared to untreated control cells, and quantitative lipid analysis indicated a dramatic increase in lipid accumulation. However, UBR-W treatment significantly reduced expression of ox-ldl-related genes and largely prevented lipid accumulation. The results indicate that UBR-W mediates a cholesterol-lowering effect via inhibition of cholesterol synthesis and induction of LDL uptake through REBP. Key words: unripe black raspberry, cholesterol, LDL receptor, HMG-CoA reductase, ox-ldl 서 2007년통계청자료에의하면각종암과순환기계질환인뇌혈관질환과심장질환에의한사망자가한국인전체사망자수의 7.6% 를차지하였다. 이는식생활의다양화및서구화로인해고칼로리및동물성식품의섭취가증가됨에따라당뇨병, 고혈압, 고지혈증과같은각종성인병발병이늘어나고있으며, 특히심혈관계질환의발병율은지난수십년간계속증가하여주요사망원인중하나가되었다 (1-3). 특히지방질의과잉섭취로인해유발되는고지혈증및심혈관계질환은치료보다예방이필요한질환으로여겨져, 최근에는콜레스테롤합성을저해하는생리활성물질을천연물로부터찾아내려는연구가활발하게진행되고있다. 최대생산지인고창을비롯한전국으로재배되고있는복분자 (Rubus occidentalis)() 는유리당, 무기질의인, 철및칼륨이많이함유되어있고특히유기산과비타민 C가많이포함되어있으며, 항암활성 (5) 및항염증 (6), 항산화및항균효과 (7) 등다양한생리활성에대한효능이밝혀진바있다. 론 Received 2 eptember 2013; Accepted November 2013 Corresponding author. E-mail: tblee01@gbri.re.kr, Phone: 2-63-560-5170 최근연구에의하면복분자미숙과추출물이완숙과추출물보다총폴리페놀함량이 2배이상높게나타났으며, 시험관시험에서 LDL(low density lipoprotein) 의산화활성을억제하고 () 동물시험에서복분자미숙과물추출물이 LDL을억제함으로써고지혈증및지질대사에효능이있음을확인하였으며, 혈관질환의원인이되는염증성사이토카인인 TNF-α, IL-6, PGF2, 일산화질소등의생성을억제한다고보고한바있다 (9). 예비실험에서복분자완숙과와미숙과물추출물에의한복분자의콜레스테롤억제효과를알아본결과완숙과물추출물보다미숙과물추출물에서보다높은콜레스테롤억제효과를확인하였다 (data not shown). 따라서본연구에서는복분자미숙과물추출물을가지고실험을진행하였다. Apoliprotein B(ApoB) 는말초조직으로지질을전달하는 system 즉 forward transport system인 lipoprotein의중요성분인 apolipoprotein 구성성분이다 (). 간에서조직으로전달되는 lipoprotein complex(vldl, IDL 및 LDL) 를흔히 bad cholesterol이라고부르기도한다 (11). 간에서형성된 ApoB는각조직으로이동하여 LDL receptor의 ligand와같은역할을하며, 조직으로 cholesterol을이동시키는중요한역할을수행한다. LDL receptor는세포막에존재하는단백질로혈중의 LDL-cholesterol의유전적결
1900 최혜란 이수정 이정현 권지웅 이희권 정종태 이태범 함때문에혈중콜레스테롤이제거되지않아나이와상관없이고콜레스테롤혈증이유발된다고보고되었다 (). 이러한 LDL receptor 유전자의발현은세포내의 sterol 함량에의해조절되는데이는 sterol regulatory element binding protein(rebp) 이라는전사인자의활성화와밀접하게연관되어있다. 반면, 간이외의세포에과잉으로존재하는 cholesterol 은간으로이동하여담즙으로대사가되거나재활용하게된다. 즉이러한말초조직으로부터여분의 cholesterol을간으로이동시키는 reverse cholesterol transport(rct) 에관여하는 high density lipoprotein(hdl) complex를 good cholesterol이라한다 (13). Apoliprotein A-1(ApoA-1) 은 HDL의주된단백질구성성분으로서 RCT에결정적인역할을한다. ApoA-1은말초조직의세포막에존재하는 ATPbinding cassette transporter A1(ABCA1) 에결합하여콜레스테롤을 HDL로전달하게하며, 이렇게형성된 HDL은간으로이동되어대사된다 (1,15). 동맥경화발생가능성을높이는 ApoB 함유입자에는 ApoA-1이포함되어있지않기때문에 ApoA-1은동맥경화예방에관여한다고할수있다. 심혈관질환과이들 lipoprotein의구성은밀접한관련이있으므로심혈관질환의지표로사용한다. 한편, 혈중콜레스테롤의농도는크게생체내에서의생합성과분해의두과정에의하여조절되는데그중생합성과정이더크게영향을미치는것으로알려져있다. 콜레스테롤합성에있어서가장중요한단계는콜레스테롤의생합성의효소인 3-hydroxy-3-methylglutaryl(HMG)-CoA reductase 반응으로잘알려져있다. 이효소활성의조절은체내총콜레스테롤과혈청콜레스테롤수준에큰영향을미친다. 천연물질이나합성물질을이용하여고지혈증치료및동맥경화예방효과여부를조사할때이 HMG-CoA reductase 활성에미치는영향이많이연구되고있다. 또한동맥경화의주요발생과정은혈중콜레스테롤의주요전달체인저밀도지단백 (LDL) 이동맥벽에혈중농도의존적으로전달되어동맥벽의내피하에서지방성분의산화가시작되고, 이들이순환중인 monocyte를동맥벽으로유도하게됨으로써시작이된다. 동맥벽에침윤한 monocyte 는산화된 LDL에자극받은혈관내피세포가생산한 monocyte-colony stimulating factor(m-cf) 에의해 macrophage로분화하고, 이들세포의강력한산화능에의해 LDL 은완전한산화상태 (ox-ldl) 가되어 macrophage의표면수용체인 LDL receptor에결합하는능력을상실하고대신에 scavenger receptor(r) 를통하여 macrophage에수용됨으로써이들세포내의 cholesterol 농도를조절하게된다 (16). 결과적으로 macrophage에고농도로축적된 cholesteol은소위 foamy cells이되어동맥경화의초기병변인지방선조 (fatty streak) 를형성한다. 따라서본연구에서는복분자미숙과물추출물 (unripe black raspberry water extract: UBR-W) 의세포내콜레스테롤관련기전을확인하고, ApoB-1/ApoA-1 ratio를확인하여심혈관질환에미치는효능을살펴보며, LDL의산화억제기전을연구하여고지혈증개선에미치는효과를검증하고자하였다. 재료및방법 Phenolic compounds 함량분석복분자 ( 전북고창산 ) 추출물의 phenolic compounds 함량을분석하기위하여 11개표준품 (igma-aldrich Co., t. Louis, MO, UA) 을구입하여 HPLC(Agilent, Palo Alto, CA, UA) 를이용 Table 1, 2의조건으로함량분석을하였다. 실험재료간은지방대사의핵심인장기로서, 지방산과콜레스테롤의합성및대사, 분비를관장하는데혈중지질의구성성분인 HDL과 LDL의생성을알아보고자인간간암세포주 (human hepatocellular carcinoma cells: HepG2) 를사용하였고, 산화된 LDL에의한대식세포들의변화를알아보고자마우스대식세포주 (mouse macrophage-like cell line: RAW 26.7) 를사용하였다. 이들세포주에각각미치는효능을통하여콜레스테롤및다양한지방대사관련유전자들의발현에복분자미숙과물추출물 (UBR-W) 이어떠한영향 Table 1. HPLC condition for compounds analysis HPLC model Column Detector Column temperature Flow rate Injection Wavelength Mobile phase Agilent 10 series OP C1 (250.6 mm) UV/Vis 25 C 1.0 ml/min μl 360 nm 320 nm 20 nm 260 nm rutin, myricetin, luteolin, quercetin, kaempferol caffeic acid, p-coumaric acid, ferulic acid, resveratrol gallic acid ellagic acid A: 0.2 M ortho-phosphoric acid, ph 1.57 B: 20% 50 mm ammonium dihydrogen phosphate, ph 2.6 in 0% acetonitrile
복분자미숙과물추출물의콜레스테롤개선효과 1901 Table 2. HPLC solvent condition Gradient step Time (min) A (%) B (%) 1 2 3 5 6 7 0.0.0 15.0 20.0 25.0 30.0 32.0 0.0 95.0 0.0 70.0 60.0.0.0 95.0 95.0 을미치는지알아보고자실험을수행하였다. 5.0 20.0 30.0 0.0 90.0 90.0 5.0 5.0 시료추출복분자미숙과 1 kg에물 L를넣고환류냉각장치를부착한히팅맨틀을이용하여 2시간동안가열추출하였고, 이것을 2회반복하였다. 추출물은여과지 (ADVANTEC No. 2, Adventec Toyo Kaisha, Tokyo, Japan) 를이용여과하여감압농축후동결건조하였다. 세포배양 HepG2 세포, RAW 26.7 세포는한국세포주은행에서분양받았으며 Dulbecco's modified Eagle medium(dmem, Welgen, Daegu, Korea) 에 56 C에서 30분간열처리된 fetal bovine serum(paa Laboratories Inc., Etobicoke, ON, Canada) % 와항생제 (penicillin/streptomycin, Lonza, Walkersville, MD, UA) 1% 를첨가하여 37 C, 습도 90%, 5% CO 2 배양기에서배양하였다. 세포독성실험 (MTT) 배양이끝난세포의생존률은 Mosmann(17) 의방법에따라 MTT[3-(,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] 환원방법을이용하여측정하였다. MTT assay는대사가왕성한세포의농도를측정하는방법으로미토콘드리아의탈수소효소작용에의해 MTT tetrazolium이 MTT formazan[3-(,5-dimethylthiazole-2- yl)-2,5-diphenyl-tetrazolium bromide] 환원방법을이용하여 50 nm의파장에서흡광도가최대가되어살아있는세포수를반영한다. HepG2 세포는 96 well에 1 /ml 농도로 90 μl 분주하고, 복분자미숙과물추출물을처리한뒤 72시간배양후에 microplate reader(ynergy HT, Bio-Tek, Winooski, VT, UA) 를이용하여 50 nm에서흡광도를측정하여세포독성여부를확인하였다. 같은방법으로 RAW 26.7 세포는 96 well에 /ml 농도로 90 μl 분주한뒤 2시간동안배양한후 50 nm에서흡광도를측정하여세포독성여부를확인하였다. HMG-CoA reductase activity assay 복분자미숙과물추출물의 HMG-CoA reductase activity 억제를확인하기위해 HMG-CoA reductase activity assay kit(igma-aldrich Co.) 를사용하였다. RNA 분리및역전사-실시간중합효소연쇄반응 (real-time RT-PCR) HepG2 세포에서복분자미숙과물추출물처리시간에따른 LDL과 HDL 관련유전자발현변화를확인하고자 UBR-W를 2시간, 시간, 72시간동안처리하였고, 이후 RNA 추출시약 (RNAiso PLU, TAKARA, Otsu, Japan) 을사용하여세포를분리한뒤 RNA를분리하였다. 분리된 total RNA 2 μg을사용하여 cdna를합성한뒤, LDL 관련인자인 LDL receptor와전사인자인 REBP1 및 REBP2 mrna의발현정도와 HDL 관련인자인 ABCA1, R-B1 mrna의발현정도및동맥경화지표로알려져있는 Apo B1/ApoA1 ratio를 real-time PCR을통해확인하였다. RAW 26.7 세포역시 6 well에 5 5 개로분주하고, LDL의산화를일으키기위해 ox-ldl(hi-tbar ox-ldl) 을농도별로처리하였다. 복분자물추출물은 ox-ldl 처리 30분전에전처리하였다. 이후복분자미숙과물추출물처리후 2시간뒤에 Trizol 시약을사용하여세포를분리하여 RNA를획득하였고, total RNA 2 μg을사용하여 cdna를합성하였다. 이후지방대사에관련된유전자인 CD36, PPARγ, R-A, adipophilin mrna의발현정도를 real-time PCR을통해확인하였다. Real-time PCR 수행은 Light Cycler 2.0 Instrument (Roche, Mannheim, Germany) 를사용하였고, 표준대조군 (internal housekeeping gene) 으로 β-actin을사용하였다. PCR 조건은 95 C에서 분동안 pre-running 후, 95 C에서 15초, 60 C에서 초, 72 C에서 초, 60 C에서 60초의간격으로 50회반복하여증폭하였다. 각각의 primer의염기서열은 Table 3과같다. 통계처리실험결과는평균 ± 표준편차형태로나타내며두그룹간의통계적인분석은 tudent's t-test 방법을이용하였다. P<0.05인경우통계적인유의성이있는것으로간주하였다. 결과및고찰복분자미숙과물추출물의세포독성검사 (MTT) MTT assay를통해세포독성을측정하였는데복분자미숙과물추출물에대한세포독성을알아보기위해활성화된 HepG2 세포에농도의존적으로처리하여 72시간후세포생존율을측정하여 Fig. 1의결과를얻었다. 그결과 3.5 0 μg/ml 모든농도에서세포독성이없는것을확인할수있었다. 따라서복분자미숙과물추출물을이용한콜레스테롤관련유전자및 HMG-CoA reductase 활성측정시세포생존율에영향을주지않는 0 μg/ml 농도로설정하
1902 최혜란 이수정 이정현 권지웅 이희권 정종태 이태범 Table 3. equence of primers for PCR Gene &A Nucleotide sequences LDL receptor REBP1 REBP2 ABCA1 R-B1 ApoB1 ApoA1 β-actin CD36 Adiponectin R-A PPAR-γ β-actin A A A A A A A A A A A A A GAG TAC ACC AGC CTC ATC C GCT GAT GAC GGT GTC ATA G CTC AGA TAC CAC CAG CG CTC ACC GTA GAC AAA GAG AAG TAG ACC GCT CAC GGA TT AGG CAT CAT CCA GTC AAA C AAT GTC AAG GTG TGG TTC AAT A CTG CTG CTT GGT GAG AT AGC TCA ACA ACT CCG AC GCT GTA GAA CTC CAG CGA GTG CAA CGA CCA ACT TGA GCA CTT CCC AGT GAT AGC CCA GTT TGA AGG CTC CG CTC AGG CCC TCT GTC TC CAC TGT GCC CAT CTA CG CTT AAT GTC ACG CAC GAT TTC GAG CCA TCT TTG AGC CTT TCA GAT CCG AAC ACA GCG CCA AGG ATT CTG TAG CCA GCA ACA GTG GGA CTC ATC GGT GTC TGA ATG TCA GAG TCC GTG AAT CTA GTC TGA GGT CGT TGG TG CCG AGA ACC ATC CAG TTG A CGG GAA GGA CTT TAT GTA TGA AGA GAA GCT GTG CTA TGT T CAC AGG ATT CCA TAC CCA AG Length of PCR products 159 bp 163 bp bp 162 bp 200 bp 175 bp 156 bp 157 bp 05 bp 523 bp 177 bp 277 bp 179 bp 여진행하였다. RAW 26.7 세포역시농도의존적으로처리하여 2시간후세포생존율을측정하였다. 그결과복분자미숙과물추출물을 6.25 00 μg/ml 농도별로처리했을때 0% 세포생존율을보여세포독성이나타나지않음을확인할수있었다. 또한복분자미숙과와완숙과추출용매로서물과 25%, 50%, 75% 에탄올을사용하여추출한뒤각각의추출물을이용하여간암세포에서콜레스테롤생성억제효과를스크리닝한결과, 복분자미숙과물추출물이콜레스테롤생성억제효과가가장뛰어남을확인하였고, 향후실험에서는복분자미숙과물추출물을선택하여실험을진행하였다. Fig. 1. Effect of UBR-W on the survival of HepG2 cells. The cytotoxicity was determined using the MTT assay after 72 hr treatment with UBR-W. UBR-W: unripe black raspberry water extract. 복분자미숙과물추출물의 LDL receptor, REBP1 및 REBP2 mrna 발현에미치는영향 HepG2세포에서 LDL receptor, REBP1 및 REBP2 mrna 발현에미치는영향을확인해본결과 (Fig. 2), 복분자미숙과물추출물을 0 μg/ml 처리하였을때 2시간때부터 2.배증가되었으나그이상의시간 ( 및 72시간 ) 에서는증가경향을보이지않았다. 적포도주유래 polyphenolic compounds(1) 처럼복분자미숙과에함유되어있는다량의폴리페놀류가 LDL receptor 유전자의발현을증가시켜콜레스테롤의세포내유입증가를통한세포내떨어진콜레스테롤양회복및혈중의콜레스테롤양을낮추는데기인한다고할수있다. 복분자미숙과는완숙과에비해훨씬더많은총폴리페놀및총플라보노이드를함유하고있을뿐만아니라 (), 복분자완숙과와미숙과물추출물의폴리페놀함량을조사한결과완숙과에비해미숙과가
복분자미숙과물추출물의콜레스테롤개선효과 1903 ellagic acid 함량은 5.3배, garlic acid는 53배더높은함량을가지고있다 (Table ). LDL receptor의발현을증가시키는전사인자로알려져있는 REBP1과 REBP2 역시복분자미숙과물추출물의처리시간이증가할수록그발현량이증가하였고, 특히 REBP2의발현이현저하게증가되었다 (Fig. 2). 전사인자의하나인 REBPs는 REBP1a, 1c와 REBP2의 3종류의 isoform으로존재하며 (1), REBP1a와 REBP1c는같은유전자에서다른 promoter 부위를사용하여만들어지고 REBP2는다른유전자에서만들어진다. REBP1은지질대사관련중성지방과인지질의합성에관여하고 REBP2 는콜레스테롤대사에작용한다 (19). 따라서복분자미숙과물추출물에의한 LDL receptor mrna의증가는 LDL receptor의발현을조절할수있는 REBPs mrna의발현증가와관련이있음을확인하였다. Fig. 2. Effect of UBR-W extract (0 μg/ml) on expression of LDL receptor, REBP1 and REBP2 mrna in HepG2 cell line. The level of mrna expression was determined by real-time RT-PCR and normalized by that of mrna β-actin, which was used as a control for RNA. Values are mean±e of triplicate determinations. * tatistically significant from the control (P< 0.05). 복분자미숙과물추출물의 ABCA1 및 R-B1 mrna 발현에미치는영향 HepG2 세포에서 HDL관련유전자인 ABCA1 및 R-B1 mrna 발현에미치는영향을확인해본결과 (Fig. 3), 복분자미숙과물추출물은 ABCA1 및 R-B1 mrna 발현에영향을미치지않음을확인할수있었다. HDL은좋은콜레스테롤로불리며혈액속 HDL의비율이높을수록동맥경화의위험이낮다고판단한다. ATP-binding cassette transporter A1(ABCA1) 은 HDL 콜레스테롤운송에관여한다고보고되어있다 (20,21). 콜레스테롤유출에관여하는 ABCA1은콜레스테롤을세포외로분비시켜분비된콜레스테롤이간에서담즙산으로바뀌어체외로배출하는기능을담당한다 (22). 따라서 ABCA1의발현이증가하게되면혈중 HDL의농도가증가하게되고최종적으로콜레스테롤을체외로배출하여체내의콜레스테롤을낮추게된다. 또한 HDL 대사에있어매우중요한유전자로는 R-B1 이있다. R-B1은 HDL의선택적흡수에관여하며간과 Table. Phenolic and flavonoid compound profiles of Rubus occidentalis Gene Caffeic acid Ellagic acid Ferulic acid Gallic acid Kaempferol Luteolin Myricetin p-coumaric acid Quercetin Resveratrol Rutin Unripe black raspberry (μg/g) Ripe black raspberry (μg/g) Extraction condition (% ethanol) Extraction condition (% ethanol) 0 25 50 75 0 0 25 50 75 0 172,502 3,62 1 2 632 72 6,57 7 69 50 116 1 2 6,352 0 2 22 11 160 2 6,16 2 32 36 1 0 222 7,76 17 2 22 6 1 15 262 1,600 0 6 1 7 13 72 2 1,52 136 0 99 1,32 6 6 6 956 1,15 6 6 2 92 1,0 6 50 20 76
190 최혜란 이수정 이정현 권지웅 이희권 정종태 이태범 Fig. 3. Effect of UBR-W extract (0 μg/ml) on expression of ABCA1, R-B1 mrna in HepG2 cell line. There is no statistically difference between control and treatment groups. Fig.. Effect of UBR-W extract (0 μg/ml) on HMG-CoA reductase activity and mrna expression in HepG2 cell line. * tatistically significant from the control (P<0.05). 지방대사조직에가장많이분포하고콜레스테롤을간을통해체외로배출하는역할을담당한다 (23,2). 위의실험결과를통하여간세포에서복분자미숙과물추출물은 LDL receptor와 REBPs mrna의발현을증가시켜 LDL의농도를낮추지만, HDL 유전자의발현에는영향을미치지않음을확인할수있었다. 복분자미숙과물추출물의 HMG-CoA reductase 발현과활성에미치는영향콜레스테롤합성은많은연구들에의하면최종산물에의한피드백으로조절된다고알려져있다. 이의조절에가장중요한인자로 LDL receptor와 HMG-CoA reductase가있다. HMG-CoA reductase는콜레스테롤생합성단계에서작용하는 rate-limiting enzyme으로써스테롤이나이소프레노이드계화합물의생합성경로의중간단계인 mevalonic acid의합성을매개하는역할을한다 (25). 여러역학연구 (26-2) 에서혈중콜레스테롤치나혈중 LDL-콜레스테롤수치가높을수록관상동맥성심장질환에걸릴위험성이높아진다고보고해왔다. 동맥경화치료에사용되는대부분의 statin 계열의약은 HMG-CoA reductase의저해제로작용하는데, 값이너무고가이며장기복용시일부부작용이있다고알려져있다. 따라서독성이없고안전한식품소재를대상으로 HMG-CoA reductase 활성저해제조성물 을개발한다면그의의가크다고할수있겠다. 본연구에서는 HepG2 세포에서복분자미숙과물추출물이 HMG-CoA reductase의발현과활성에어떠한영향을미치는지확인하였다 (Fig. ). 복분자미숙과물추출물을처리한시간이증가할수록 HMG-CoA reductase mrna의발현이각각 73.6%, 73.5%, 1.5% 유의성있게증가되었다. 그러나복분자미숙과물추출물을처리하였을경우농도에의존적으로 HMG-CoA reductase의활성이감소하였다. 이러한결과는 statin을처리시 HMG-CoA reductase mrna 발현증가와함께 HMG-CoA reductase의활성감소와같은동일한결과를보여주었다. 적포도주와녹차를이용한동맥경화와관련된선행연구에서도같은결과를보여주고있다 (1,29). 즉, 복분자미숙과물추출물이 HMG-CoA reductase를저해함으로써활성을감소시키고, 이것이원인이되어결과적으로간에공급되는콜레스테롤의양이감소하는현상을보상하기위해 HMG-CoA reductase mrna의발현이증가되는것으로생각된다 (30). 이러한결과는콜레스테롤및중성지방합성에관여하는전사인자인 REBPs mrna를활성화하여 LDL receptor의증가와동시에 HMG-CoA reductase mrna 양을증가시켜콜레스테롤항상성을유지한다고볼수있다.
복분자미숙과물추출물의콜레스테롤개선효과 1905 복분자미숙과물추출물의 ApoB1/ApoA1 ratio에미치는영향 HepG2 세포에서 ApoB1/ApoA1 ratio 는 ApoB1 과 ApoA1 의 mrna의발현을통해분석하였다. ApoB1은시간에따라발현이감소되는경향을보였고, ApoA1은 2시간, 시간에서는뚜렷한증가를나타내지않았으나 72시간에서 51% 유의성있게증가됨을확인하였다 (Table 5). 그러나 ApoB1/ApoA1 ratio는복분자물추출물의처리시간이증가함에따라시간의존적으로현저한감소를나타내었고, 72시간에는 60% 의감소를나타내었다 (Fig. 5). Apolipoprotein B1/Apolipoprotein A1 ratio(apob1/ ApoA1 ratio) 는이상지질혈증, 비만, 대사성질환, 심혈관질환의위험도를예측하는데유용한임상적지표로알려져있다 (31). ApoA1은고밀도지질단백의가장큰구성요소이며, ApoB는 chylomicron에존재하고중성지방이풍부한초저밀도지질단백과저밀도지질단백의체내양을반영한다. 간으로의비에스테르화지방산 (nonesterified fatty acid) 유입이증가하는경우간에서의 ApoB 분비가증가되고이화작용 (catabolism) 에장애가생겨 ApoB의상승을일으킨다. 최근연구결과에의하면 LDL-cholesterol보다는 apoprotein B가더중요하며 LDL-C/HDL-C보다 ApoB1/ ApoA1이더중요하다는연구들이발표되었다 (31-33). 그러므로앞으로는이상동맥경화의평가와치료기준을 Apo B1/ApoA1 ratio로해야한다는주장이설득력을얻고있 Table 5. Change of ApoB1 and ApoA1 ratio treated with UBR- W (0 μg/ml) in HepG2 cell line ample % ApoB1 ratio % ApoA1 ratio CONT 2 hr hr 72 hr 0±0..63±1.92 * 73.36±.23 * 90±2. * 0±1. 2.35±1.5 * 7.21±5.07 * 206.61±6.55 * CONT, control; UBR-W, unripe black raspberry water extract. * tatistically significant from the control (P<0.05). 다. 따라서복분자미숙과물추출물이 ApoB1/ApoA1 ratio 를현저히감소시킨결과로미루어고지혈증개선에효과가있는것으로사료된다. 복분자미숙과물추출물의 ox-ldl 농도별처리에따른 mrna 발현에미치는영향혈액내에이동하는 LDL이내피세포안으로침투하게되면 ox-ldl 형태로변형이된다. 변형된 LDL은 macrophage에있는 CD36, scavenger receptor(r-a) 수용체에의해흡수되고많은 LDL이축적된 macrophage는거품세포로변형이되는데, peroxisome proliferator activated receptor gamma(ppar-γ) 또한연관성이있음이알려져있다. 이거품세포가염증물질을분비하고동맥경화및다양한질병을유발한다고알려져있다 (3,35). Adipophilin이활성화되면세포내에있는콜레스테롤의방출을촉진시킴으로인하여세포내에항상성을유지시키고, 최근논문에의하면 LDL 산화와도관련이있다고보고되었다 (36). ox-ldl은생체내에서일어나는 LDL의산화반응을나타내기위해사용하였다. ox-ldl은.5, 25, 50 μg/ml 농도로 2시간동안처리하였다. 그결과 ox-ldl의농도가증가함에따라 CD36, R-A, adipophilin, PPAR-γ mrna 의발현이농도의존적으로증가하였다. ox-ldl 50 μg/ml 농도에서 CD36은 7.5배, R-A는 2.배, adipophilin은 11 배, PPAR-γ는 1.6배정도증가함으로써유의성있는결과를나타내었다 (Fig. 6A). 이를바탕으로 LDL의산화를유도한뒤복분자미숙과물추출물을처리하여혈중지질개선효과를확인하였다. 복분자미숙과물추출물은 ox-ldl 처리 30분전에처리하였고농도는 50 μg/ml로시행하였다. 그결과 ox-ldl을처리한그룹에서지방산화에관련된유전자들의발현이현저하게증가하였고 (CD36은 16배, R-A는 2배, adipophilin은 1배, PPAR-γ는 1.3배 ), 복분자미숙과물추출물을처리한그룹에서는각각 60%, 32.6%, 1.7%, 50% 감소하였다. 특히 CD36, PPAR-γ에서유의성있게억제되는경향을확인할수있었다. 따라서복분자미숙과물추출물이 LDL 산화와관련된유전자를억제함으로써혈중지질개선에효과가있는것으로사료된다 (Fig. 6B). 요 약 Fig. 5. Inhibitory effect of UBR-W extract (0 μg/ml) on ApoB1/ ApoA1 mrna in HepG2 cell line. * tatistically significant from the control (P<0.05). 복분자미숙과물추출물이콜레스테롤개선에미치는영향을측정한결과는다음과같다. 간세포주 (HepG2 cells) 에서복분자미숙과물추출물은 REBPs를증가시킴으로써혈액내에 LDL을 LDL receptor를통해서세포안으로흡수시키고, 콜레스테롤합성에관여하는 HMG-CoA reductase 활성을억제하면서체내콜레스테롤을조절하였다. 그러나 HDL의생성에관여하는유전자 (ABCA1, R-B1) 는변화를보이지않았다. 결과적으로복분자미숙과물추출물이
1906 최혜란 이수정 이정현 권지웅 이희권 정종태 이태범 A B Fig. 6. Effect of various concentration of ox-ldl on expression CD36, R-A, adipophilin, and PPARγ mrna (A) and UBR-W extract on mrna expression of CD36, R-A, adipophillin, and PPARγ by ox-ldl (B) in RAW26.7 cells. * P< 0.05 vs. control; # P<0.05 vs. ox-ldl 50 μg/ml. LDL receptor를통해서 LDL을억제시키고체내에서는콜레스테롤생합성을억제하였으며, HDL의생성에는관여를하지않음을확인하였다. 또한 ApoB1/ApoA1 ratio 값을통해서동맥경화지표를확인해본결과유의성있게수치가감소함을확인하였고, 이는미숙과물추출물이콜레스테롤을개선하여동맥경화를예방할것으로기대한다. 또한대식세포 (RAW 26.7 cells) 에서복분자미숙과물추출물이 CD 36, R-A 수용체를억제시킴으로인해세포내의 ox-ldl 의흡수를차단시키고, 세포안에서는 macrophage에있는 PPAR-γ를억제시킴으로인해 LDL 산화가억제되었다. Adipophilin의활성이억제됨에따라세포안에있는콜레스테롤방출을촉진시킴으로인해동맥경화를완화시킬수있다고사료된다. 감사의글 본연구는고창군지역농식품선도클러스터육성사업과고창복분자연구소건립사업의일환으로수행되었으며이에깊이감사드립니다. REFERENCE 1. 2006 Annual report on the cause of death statistics. 2007. Korea National statistical Office. 2. Huh KB. 1990. The present status if nutrition-related diseased and its countermeasures. Korean J Nutr 23: 197-207. 3. Moon J. 1996. Nutritional problems of Korean. Korean J Nutr 29: 371-30.. Eu G, Chung BY, Bandopadhyay R, Yoo NH, Choi DG,
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