Journal of Nutrition and Health (J Nutr Health) 2015; 48(1): 1 ~ 8 http://dx.doi.org/10.4163/jnh.2015.48.1.1 pissn 2288-3886 / eissn 2288-3959 Research Article 고지방 / 고콜레스테롤식이를섭취한마우스에서자색고구마열수추출물보충이지방간저항성에미치는영향 * 이유진 1 양윤경 2 김유진 1 권오란 1 이화여자대학교식품영양학과, 1 숭의여자대학교식품영양과 2 Effects of an aqueous extract of purple sweet potato on nonalcoholic fatty liver in high fat/cholesterol-fed mice* Lee, You Jin 1 Yang, Yoon Kyoung 2 Kim, You Jin 1 Kwon, Oran 1 1 Department of Nutritional Science and Food Management, Ewha Womans University, Seoul 120-750, Korea 2 Department of Nutritional Science and Food Management, Soongeui Women s College, Seoul 100-751, Korea ABSTRACT Purpose: Anthocyanins from purple sweet potato (PSP) have been investigated in vitro and in animals and found to have a protective effect against oxidative hepatic damage. In this study, we investigated that aqueous extract of PSP can ameliorate the dysfunction of lipid metabolism in mice fed a high fat/cholesterol diet. Methods: Forty C57BL/6J mice were randomly divided into 5 groups (n = 8) and fed one of the following diets for 8 weeks; normal fat (NF) diet; high fat/ cholesterol (HFC) diet; HFC with 1.25% PSP (HFPL) diet; HFC with 2.5% PSP (HFPM) diet; HFC with 5% PSP (HFPH) diet. Results: Non-alcoholic fatty liver was manifested in the HFC group by showing increased levels in plasma alanine aminotransferase (ALT) activity, total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), increased level of TC and presence of many large lipid droplets in the liver, and increased fat cell size in the HFC group compared with the NF group. However, administration of HFC induced a significant decrease in food intake, resulting in decrease in fat mass. Coadministration of PSP did not lead to reversal of body weight changes, ALT activity, and lipid levels in plasma and the liver, but suppressed excess enlargement of the fat cell size through increasing carnitine palmitoyltransferase-1 (CPT-1) gene expression in the liver. Accordingly, the number of fat droplets in the liver was reduced in PSP administered groups. Conclusion: Taken together, these results suggest that PSP may have a protective effect on the dysfunction of lipid metabolism. Conduct of further studies on the coordinated regulation of PSP for lipid metabolic homeostasis at the liveradipose tissue axis is needed. KEY WORDS: purple sweet potato, aqueous extract, high fat/cholesterol diet, non-alcoholic fatty liver 서론 지방간은비정상적으로많은지방 ( > 5%) 이간에축적된상태로, 과도한알코올섭취, 당뇨병, 고지혈증및비만이주요위험요인이나알코올섭취없이도동물성지방을과다하게섭취하고운동량이감소한경우비알코올성지방간이유도될수있다. 1,2 2012년식품의약품안전처조사연구에따 르면우리나라성인의비알코올성지방간유병률은 2004 년 11.5% 에서 2010 년 23.6% 로가파른증가세를보이고있다. 3 다양한과일, 곡류, 채소에널리함유되어있는안토시아닌은수용성플라보노이드계화합물로강한항산화능을바탕으로항염증, 산화스트레스감소와세포사멸에대한보호효과를가지고있다고알려져있다. 4-7 최근기능성식품소재로많은관심이집중되고있는자색고구마는비타 Received: November 12, 2014 / Revised: December 14, 2014 / Accepted: January 28, 2015 *This study was performed with the support of the Food Functionality Evaluation Program funded by the Ministry of Agriculture, Food and Rural Affairs through Korea Food Research Institute and Cooperative Research Program for Agriculture Science & Technology Development (Project title: Research on New Functional Food Materials with Domestic Agricultural Products, Project No. PJ00845002) Rural Development Administration, Republic of Korea. To whom correspondence should be addressed. tel: +82-2-3277-6860, e-mail: orank@ewha.ac.kr 2015 The Korean Nutrition Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
2 / 자색고구마열수추출물의지방간저항성 민, 무기질, 식이섬유, 베타카로틴뿐아니라안토시아닌함량이높은것으로알려져있다. 또한자색고구마에함유된안토시아닌은동일한함량의딸기, 적양배추, 들깨유래안토시아닌에비해안전성이높다는보고도있다. 8 Han 등 9 및 Sakatani 등 10 은자색고구마를메탄올로추출하여안토시아닌의함량을 200 mg/kg bw까지높인후항산화능을밝힌바있다. Hwang 등 11,12 은자색고구마의간손상억제기능을보고하였는데, 이들도역시 C-18 Sep-Pak을사용하여분리한안토시아닌분획을시험물질로사용하였다. 작용기전으로는 nuclear factor erythroid 2-related factor 2 (Nrf-2) 발현증가와동시에 cyclooxygenase 2 (COX-2) 와 inducible nitric oxide synthase (inos) 발현억제를제안하였다. 이들은동일한시험물질을 HepG2 세포와비만동물에적용하여자색고구마의간지질축적억제기능을보고하였으며, 이때에는 adenosine monophosphate-activated protein kinase (AMPK) signaling pathway 활성화를기전으로제안하였다. 이러한사전연구결과는자색고구마에함유된안토시아닌의항산화, 간보호기능을뒷받침할수있지만, 실제로이러한방법으로제조된안토시아닌은생산율이 2.5% 이하로매우낮고용매잔류등의문제가있으므로기능성식품으로사용에제한을준다. 따라서본연구에서는생산율을높일뿐아니라안토시아닌이외에자색고구마에함유된 chlorogenic acid, caffeic acid 등다양한 phytochemical 15 을함유한기능성식품원료를개발하기위한목적으로자색고구마의열수추출물의간손상보호효과를시험하였다. 이를위해고지방 / 콜레스테롤식이로비알콜성지방간을유도한동물모델을사용하여간조직의지방축적및대사개선효과를알아보았다. 연구방법 시험물질시험물질인자색고구마열수추출물 (PSP) 은재단법인전라남도생물산업진흥재단에서공급받았다. 자색고구마는세척하고조각내어 95~100 o C 끓는물에서 90분간추출하고, 15~30 o C에서냉각하여여과하였다. 여과물은 15,000 g에서원심분리하여, 20 Brix로농축하여진공데시케이터에서건조하였다. PSP의영양성분은 100 g당탄수화물 83.93 g, 조회분 4.50 g, 조단백 3.78 g, 조지방 0.01 g, 수분 6.45 g이며, 총안토시아닌함량은1.32 g로표준화하였다. 실험동물및식이 5 주령의수컷 C57BL/6 마우스 (Orient Bio, Daejeon, Korea) 를공급받아 7일간물과표준식이 (5L79 Purina rat & mouse 18% chow, Charles River Laboratories Inc. Wilmington, MA) 를자유롭게공급하며적응시켰다. 적응기간후난괴법으로 8마리씩 5군으로분류하였다. 정상대조군 (normal fat; NF) 은 AIN 73A diet을공급하였고, 고지방 / 콜레스테롤시험군은각각자색고구마함량에따라실험대조군 (high fat/cholesterol control, HFC), 저용량군 (HFC + 1.25% PSP, HFPL), 중용량군 (HFC + 2.5% PSP, HFPM) 및고용량군 (HFC + 5% PSP, HFPH) 으로나누어서 8주간공급하였다. 실험식이조성은 Table 1과같다. 실험실의사육조건은온도 22~24 o C, 습도 45 ± 5%, 조명 12 시간명 / 암 (6:00~18:00/18:00~6:00) 로항상유지시켰다. 본연구는이화여자대학교동물실험윤리위원회의승인을받아수행하였다 (IACUC No. 2013-01-082). 체중, 식이섭취량, 혈액및장기채취체중은일주일에 2번일정한시간에측정하였고, 식이섭취량은매일일정한시간에측정하여평균섭취량을계산하 Table 1. Composition of experimental diets 1) (Unit: g/kg diet) Group High-fat/cholesterol diet NF Ingredients HFC HFPL HFPM HFPH Cornstarch 150 150 150 150 150 Sucrose 500 337 324.5 312 287 Casein 200 200 200 200 200 Corn oil 50 30 30 30 30 Lard 0 170 170 170 170 Cholesterol 0 10 10 10 10 Sodium cholate 0 3 3 3 3 Cellulose 50 50 50 50 50 Mineral mix#200000 2) 35 35 35 35 35 Vitamin mix#300050 3) 10 10 10 10 10 DL-Methionine 3 3 3 3 3 Choline Bitrate 2 2 2 2 2 Purple sweet potato 0 0 12.5 25 50 Total 1,000 1,000 1,000 1,000 1,000 Total calorie (kcal) 3,920 4,708 4,708 4,708 4,708 Carbohydrates (% as kcal) 67.5 42 42 42 42 Protein (% as kcal) 20.8 17 17 17 17 Lipid (% as kcal) 11.7 41 41 41 41 1) NF, normal fat control (AIN 73A diet); HFC, high fat control; HFPL, HFC + PSP 1.25%; HFPM, HFC + PSP 2.5%; HFPH, HFC + PSP 5% 2) Mineral mixture (g/kg mix): CaHPO 4 500; NaCl 74; K 2 H 6 O 7 H 2 O 220; K 2 SO 4 52; MgO 24; MnCO 3 3.50; Fe(C 6 H 5 O 7 ) 6H 2 O 6 ; ZnCO 3 1.6; CuCO 3 0.3; KIO 3 0.01; Na 2 SeO 3 5H 2 O 0.01; CrK(SO 4 ) 2 0.55; sucrose, finely powdered 118 3) Vitamin mixture (g/kg mix): thiamin HCl 0.6; riboflavin 0.6; pyridoxin HCl 0.7; niacin 3.0; calcium pantothenate 1.6; folic acid 0.2; biotin 0.02; vitamin B12 (0.1%) 1.0; vitamin A palmitate (500,000 IU/g) 0.8; vitamin D3 (400,000 IU/g) 0.25; vitamin E acetate (500 IU/g) 10.0; menadione sodium bisulfate 0.08; sucrose finely powdered 981.15
Journal of Nutrition and Health (J Nutr Health) 2015; 48(1): 1 ~ 8 / 3 였다. 실험동물을 12시간이상절식시킨후, CO 2 가스와에테르로보정하여호흡마취하였다. 혈액은복대동맥에서채혈하여 EDTA 가처리된튜브에넣어 4 o C, 3,500 rpm, 10분동안원심분리한후혈장을 -80 o C에분석전까지보관하였다. 간과지방조직은적출하여생리식염수로세척후수분을제거하고, 액체질소로급속동결하여 -80 o C에보관하여분석에사용하였다. 적출한간과지방조직일부는 10% 중성포르말린용액에고정하여병리학적분석에사용하였다. 혈장의생화학적지표성분분석혈장의 asparate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglycerides (TG) 와 high density lipoprotein cholesterol (HDL-C) 농도는측정용 kit (Asan Pharmaceutical, Seoul, Korea) 를이용해측정하였다. 혈장 low density lipoprotein cholesterol (LDL-C) 수준은 Freidewald 등 12 의방법에따라 LDL cholesterol = total cholesterol HDL cholesterol (triglyceride/ 5) 식으로계산하였다. 간조직의지질수준분석간조직의지방은 Folch 등 13 의방법에따라추출하였다. 추출된간조직내지방의 TC, TG는측정용 kit (Asan Pharmaceutical, Seoul, Korea) 를이용한효소법으로측정하였다. 유전자발현분석 TRIzol (Ambion, Austin, TX, USA) 을이용하여간조직에서 total RNA를추출하였다. 추출된 RNA는 cdna reverse transcription kit (Applied Biosystems, Foster City, CA, USA) 를사용하여 cdna로합성하였다. 합성된 cdna 는 TaqMan real-time PCR systems (Applied Biosystems) 을사용하여 mrna 측정에사용하였다. 타겟유전자의 primer는 sterol regulatory element binding protein 1c [SREBP-1c; Mm00550338_m1], fatty acid synthase [FAS; Mm01204974_m1], carnitine palmitoyltransferase-1 [CPT1; Mm01231183_m1] 및 β-actin [Actb; Mm00607939_s1] 을사용하였으며, mrna 발현량은 CT method을이용하여 β-actin값에대한상대적인양으로계산하였다. 병리조직학적관찰적출된간및지방조직은즉시 10% phosphate-buffered formalin에넣어고정한후흐르는물에서과잉의고정액을제거하고, ethyl alcohol을이용하여조직속의수분을제거하였다. Xylene을이용하여조직내의 alcohol을제거 한후 paraffin 처리를하여조직공간을채워 5 μm으로박절하여슬라이드에부착하였다. Hematoxylin-eosin (H&E) 으로염색한후 200 에서조직상태를관찰하였다. 통계분석모든실험결과는 Statistical Analysis Systems package version 9.3 (SAS Institute, Cary, NY, USA) 을이용하여분석하였으며, 평균 ± 표준오차 (S.E.) 로나타내었다. 군간분석항목별차이는일원배치분산분석 (one-way analysis of variance) 를수행한후, Duncan s multiple range test를이용하여 p < 0.05 수준에서유의성을검증하였다. 결 식이섭취량및체중식이섭취량은 NF군에비해 HFC군에서유의적으로감소하여총에너지섭취량은모든군에서유의적인차이를보이지않았다 (Fig. 1A, 1B). 주간체중증가를관찰한결과 NF군은지속적으로체중이증가한반면 HFC군은 5주이후부터체중증가가둔화되었다. 그결과 8주간총체중증가량은 NF군이 HFC군에비해유의하게높은것으로나타났다 (Fig. 1C, 1D). PSP 보충은식이섭취량및에너지섭취량에영향을미치지않았다. 그러나체중증가에는영향을미쳐, 중용량처리된 HFPM군은 2~4주에 HFC군에비해유의하게체중증가가억제된것으로나타났으나, 이후 5주부터는다른군들과같은수준으로증가되었다. 그결과 8주간총체중증가량이가장낮은것으로나타났으나, HFC군에비해통계적으로유의한차이는나타내지않았다. 한편고용량처리된 HFPH군은저용량및중용량처리군에비해 NF군에가장유사한체중증가패턴을나타냈다 (Fig. 1C, 1D). 혈장 ALT, AST 활성도및간조직의변화혈장 AST 수준은모든군간유의적인차이를보이지않았다 (Fig. 2A). 혈장 ALT 수준은 NF군에비해 HFC군에서유의적으로높았으며, 이는 PSP 보충으로극복되지못하였다 (Fig. 2B). 고지방 / 콜레스테롤식이와 PSP 보충에의한간의병리조직학적변화를관찰한결과는 Fig 2C와같다. NF군에비해 HFC군에서지방구 (lipid droplet) 의수가유의적으로증가하였다. PSP 처리군에서지방구의수가줄어드는경향을보였으며, 특별히 HFPH군은지방구의수가유의하게감소하는것으로관찰되었다. 혈중지질및지단백질수준혈장 TG 수준은 NF 군에비해 HFC 군에서유의적으로 과
4 / 자색고구마열수추출물의지방간저항성 Fig. 1. Effects of purple sweet potato extract on food intake and body weight in high fat/cholesterol diet-fed C57BL/6 mice: (A) food intake, (B) energy intake, (C) total body weight gain and (D) body weight changes by week. NF, normal fat control; HFC, high fat control; HFPL, HFC + PSP 1.25%; HFPM; HFC + PSP 2.5%; HFPH, HFC + PSP 5%. Values are expressed as Mean ± SE (n = 8 for each group). Means with different letters on the bar are significantly different from each other at p < 0.05 by Duncan s multiple range test. Fig. 2. Effects of purple sweet potato extract on liver enzymes in plasma and liver histology in high fat/cholesterol-fed C57BL/6 mice: (A) AST, (B) ALT and (C) liver histology. NF, normal fat control; HFC, high fat control; HFPL, HFC + PSP 1.25%; HFPM; HFC + PSP 2.5%; HFPH, HFC + PSP 5%. Values are expressed as Mean ± SE (n = 8 for each group). Means with different letters on the bar are significantly different from each other at p < 0.05 by Duncan s multiple range test. Histology bar represents 50 μm.
Journal of Nutrition and Health (J Nutr Health) 2015; 48(1): 1 ~ 8 / 5 Fig. 3. Effects of purple sweet potato extract on plasma lipid levels in high fat/cholesterol-fed C57BL/6 mice: (A) TG, (B) TC, and (C) LDL- C. NF, normal fat control; HFC, high fat control; HFPL, HFC + PSP 1.25%; HFPM; HFC + PSP 2.5%; HFPH, HFC + PSP 5%. Values are expressed as Mean ± SE (n = 8 for each group). Means with different letters on the bar are significantly different from each other at p < 0.05 by Duncan s multiple range test. 낮았으나 (Fig. 3A), TC 수준은 NF군에비해 HFC군에서유의적으로높았다 (Fig. 3B). LDL-C 수준도 TC와같은패턴을나타냈다 (Fig. 3C). PSP 보충은고지방 / 콜레스테롤식이의영향을변화시키지못하였다. 간조직의지질및관련유전자발현간조직에서 TC 수준은 NF군에비해 HFC군에서유의적으로높았으며 (Fig. 4A), TG 수준은모든군간유의적인차이를보이지않았다 (Fig. 4B). 지방합성전사인자인 SREBP-1c 와지방산합성에직접관여하는 FAS 유전자는 NF군과 HFC군에서차이가없는것으로나타났다. 그러나지방산산화에관여하는 CPT1 유전자는 NF군에비해 HFC군에서유의하게낮게발현되는것으로나타났다 (Fig. 4C). PSP 보충은간조직의 TC 및 TG 수준에영향을미치지못하였으나, 유전자발현에대해서는저용량군인 HFPL 군에서 SREBP-1c 및 FAS 유전자의발현이유의적으로증가하였으며, CPT1 발현도증가하는경향을나타냈다. 지방조직의무게및조직의변화지방조직의무게및조직의형태를비교한결과는 Fig. 5 에제시되었다. 지방조직의무게는 NF군에비해 HFC군에서유의하게감소하였으나 (Fig. 5A), 병리조직학적분석결과지방세포의크기는 NF군에비해 HFC군에서비대해진것으로나타났다 (Fig. 5B). PSP 보충은지방조직의무게에영향을미치지못하였으나, 지방세포의비대를억제하는것으로나타났다. 고찰 본연구는고지방 ( 열량의 41%)/ 콜레스테롤 ( 식이의 1%) 식이로지방간을유도한실험동물에서자색고구마열수추출물의간손상에보호효과를시험하는것을목적으로수행되었다. 5주령 C57BL/6 마우스에 8주간의고지방 / 콜레스테롤식이를섭취시킨결과, 간과지방조직의변화그리고유의하게증가된혈장 ALT, TC, LDL-C 및간의 TC 함량을근거로비알코올성지방간이유도되었음을확인할수있었다. 그러나식이섭취량은 NF군에비해 HFC군에서유의하게감소하여총에너지섭취량이유사하였으며, 체중증가량및지방조직의무게는오히려 HFC군에서더낮은것으로관찰되었다. 그럼에도불구하고 HFC군의간
6 / 자색고구마열수추출물의지방간저항성 Fig. 4. Effects of purple sweet potato extract on lipid and mrna levels in the liver of high fat/cholesterol-fed C57BL/6 mice: (A) TC, (B) TG, and (C) mrna levels. NF, normal fat control; HFC, high fat control; HFPL, HFC + PSP 1.25%; HFPM; HFC + PSP 2.5%; HFPH, HFC + PSP 5%; SREBP-1c, sterol regulatory element-binding protein-1c; FAS, fatty acid synthase; CPT-1, carnitine palmitoyltransferase-1. Values are expressed as Mean ± SE (n = 8 for each group). Means with different letters on the bar are significantly different from each other at p < 0.05 by Duncan s multiple range test. Fig. 5. Effects of purple sweet potato extract on fat mass and epididymal fat tissue histology in high fat/cholesterol-fed mice: (A) adipose tissue weight and (B) adipose tissue histology. NF, normal fat control; HFC, high fat control; HFPL, HFC + PSP 1.25%; HFPM; HFC + PSP 2.5%; HFPH, HFC + PSP 5%. Values are expressed as Mean ± SE (n = 8 for each group). Means with different letters on the bar are significantly different from each other at p < 0.05 by Duncan s multiple range test. Histology bar represents 50 μm.
Journal of Nutrition and Health (J Nutr Health) 2015; 48(1): 1 ~ 8 / 7 조직에서생화학적, 병리학적차이를나타낸것은총에너지섭취량보다는급여된지방의종류가간건강에중요함을시사한다. 14 NF군은총에너지섭취량의 12% 를불포화지방의형태로섭취하였으나, HFC군은총에너지섭취량의 41% 를불포화지방및포화지방 3:17의비율로섭취하였을뿐아니라별도로 1% 수준의콜레스테롤도제공되었다. 총에너지섭취량은동일하나, 에너지원이다른 NF군과 HFC군은체중증가패턴도서로다르게관찰되었다. NF군은 8주간의실험기간중지속적으로체중이증가하였으나, 포화지방급원의지방에서제공하는에너지 % 가높은 HFC 군은 5주까지체중증가를나타내다가그이후부터는체중증가가둔화되는것으로관찰되었다. 이는과도한포화지방의섭취로지방세포변형이나타난것으로추정된다. 즉, NF군에서는과잉의열량이지방세포로축적되는 건강한비만 (healthy obesity) 을유지한반면, HFC군에서는지방세포가더이상과잉열량을축적하는역할을담당하지못하는 나쁜비만 (bad obesity) 이유도되었다고추정할수있다. 15 포화지방의과다섭취는지방세포를과다하게비대화하여 NF-κB경로에따른염증반응이나타나는것을동물실험결과로보고된바있다. 16 그러나불포화지방산이함유된식이를과량섭취하였을때체중이서서히증가하는반면, 과도한지방침착은억제하는경로에대해서는아직그기전이불확실하다. 17 이러한지방조직의대사이상은다시간조직과혈액의지질대사에영향을미친것으로판단된다. 지방세포의크기가최대로증가하면, 지방조직은더이상지방을축적하지못하므로, 과잉의지방은간으로유입되어중성지방, 인지질, 콜레스테롤에스테르로재합성된후 very low density lipoprotein (VLDL) 의형태로혈액으로방출된다. 그러나 TC 및 LDL-C의축적으로간조직이손상되어 TG는간에축적되고반면, 혈액으로유출되는 TG 농도가감소된것으로추정된다. 18 이결과는간조직에서 SREBP-1c 및 FAS 유전자발현의변화없이 CPT-1 유전자발현이감소된것과일치한다. SREBP-1c는간조직에서강하게발현되며 TG 및지방산합성에관여하는전사활성유전자이며, 19 acetyl-coa carboxylase (ACC) 와 FAS 등을조절한다. 20 CPT-1은지방산을미토콘드리아내로유입시키는데관여하는지방산산화의속도제한효소이다. 21 또한이결과는간조직의 TG 수준으로는확인되지못하였으나, 간조직에서지방구가증가한것과도일치한다. PSP 보충은 HFC군의혈액및간손상지표를개선시키지못하였다. 그러나특별히고용량의 PSP 보충은저용량및중용량보충군에비해 NF군에가장가까운체중증가패턴을나타냈다. 또한지방조직및간조직에도영향을미 쳐지방조직의무게가증가하고크기는작아지는경향을나타냈으며간조직의지방구의수도감소하는결과를나타냈다. 한편 PSP 보충은지방조직의대사이상을완화시켜간으로지질유입을억제하는것으로추정될수있으나, 통계적인유의성을나타낼만큼충분한효과를나타내지못하였다. 따라서 PSP 섭취군에서도간으로의지질유입은계속진행되는것으로보이며, 간조직에서 SREBP-1c 및 FAS 유전자발현이 HFC군과 PSP 보충군에서차이가없었다는것으로뒷받침할수있다. 또한 PSP 저용량군에서중용량및고용량군에비해 SREBP-1c 및 FAS 유전자발현이유의하게높았던것도중용량이상에서보충의효과가나타남을뒷받침한다. 이상의결과는고지방식이 ( 열량의 45%) 를 4주간섭취시킨 ICR mice에자색고구마의 anthocyanin 분획을경구투여하였을때, 간의지질축적이억제되는효과를관찰한황등 6 의결과와상이한것으로이는시험물질의제조방법의차이 ( 열수추출 vs. anthocyanin 분획 ) 에기인하는것으로추정할수있다. 황등의연구에서사용된시험물질은순도와함량을높인분획물로 anthocyanin 함량은 200 mg/kg bw 이었다. 그러나본연구에사용된시험물질은 anthocyanin 함량이각각 32.5, 65, 130 mg/kg bw으로낮았다. 본연구의결과를요약하면, C57BL/6 마우스에게 8 주간고지방 / 콜레스테롤식이를섭취시킨결과, 지방세포의비대가유도되었으며간조직에서지방구의증가, 혈액및간의 TC 증가, 혈액의 ALT 활성증가를특징으로하는지질대사이상이초래되었다. 동일한기간동안, 고지방 / 콜레스테롤식이와 PSP를보충시킨군에서는지방세포의크기가비교적작게유지되었으며, 간조직의지방구도적어진것으로관찰되었다. 혈액지표는유의적으로개선하지못하였으나, 이결과만으로도 PSP는지방세포를건강하게유지하는방법으로간보호효과가있는것으로사료된다. 따라서향후에는 PSP가지방세포와간조직의상호대사축에서어떤작용기전이있는지에대한실험이필요할것으로생각된다. 요약 자색고구마열수추출물의간보호기능을확인하기위해 C57BL/6 마우스를사용하여시험하였다. 지방간유도를위해 8주간고지방 / 콜레스테롤식이를급여하였으며, 자색고구마열수추출물은 1.25, 2.5, 5% 의수준으로식이에함께넣어같은기간동안제공하였다. 간조직의병리학적분석, 혈장 ALT 활성도, 간및혈장의 TC 수준을바탕으로비알콜성지방간모델이형성되었음을확인하였다.
8 / 자색고구마열수추출물의지방간저항성 고지방 / 콜레스테롤식이의급여는식이섭취량을감소시켜총에너지섭취량은시험군간차이가없었으나, 포화지방을급원으로하였을때지방세포의비대와혈장 TC, 간 TC, 간의지방구를증가시키는것으로관찰되었다. 한편자색고구마열수추출물을고지방 / 콜레스테롤식이와함께섭취시킨결과, 고지방 / 콜레스테롤식이로인한지질대사이상을유의하게변화시키지못해혈액및간손상지표를개선시키지못하였으나, 지방조직의크기는작게유지하고간의지방구형성은억제하는것으로관찰되었다. 이상의결과로자색고구마열수추출물은지질대사개선을통해간보호효과를갖음을알수있었다. 향후에는자색고구마열수추출물이지방세포-간의상호지질대사에미치는영향을추가적으로연구해야할것으로사료된다. 감사의글 본연구에사용된자색고구마열수추출물을제공해주신전라남도생물산업진흥원에감사드립니다. References 1. Samuel VT, Liu ZX, Qu X, Elder BD, Bilz S, Befroy D, Romanelli AJ, Shulman GI. Mechanism of hepatic insulin resistance in nonalcoholic fatty liver disease. J Biol Chem 2004; 279(31): 32345-32353. 2. Lee E, Kim WJ, Lee YJ, Lee MK, Kim PG, Park YJ, Kim SK. Effects of natural complex food on specific enzymes of serum and liver and liver microstructure of rats fed a high fat diet. J Korean Soc Food Sci Nutr 2003; 32(2): 256-262. 3. Ministry of Food and Drug Safety. Influence of dietary intake on non-alcoholic fatty liver disease in Korean. Cheongwon: Ministry of Food and Drug Safety; 2012. 4. Hna KH, Lee JC, Kim JH, Lee JS. Manufacture and physiological functionality of Korean traditional liquor by using purple-fleshed sweet potato. Korean J Food Sci Technol 2002; 34(4): 673-677. 5. Rossi A, Serraino I, Dugo P, Di Paola R, Mondello L, Genovese T, Morabito D, Dugo G, Sautebin L, Caputi AP, Cuzzocrea S. Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation. Free Radic Res 2003; 37(8): 891-900. 6. Hwang YP, Choi JH, Han EH, Kim HG, Wee JH, Jung KO, Jung KH, Kwon KI, Jeong TC, Chung YC, Jeong HG. Purple sweet potato anthocyanins attenuate hepatic lipid accumulation through activating adenosine monophosphate-activated protein kinase in human HepG2 cells and obese mice. Nutr Res 2011; 31(12): 896-906. 7. Ramirez-Tortosa C, Andersen ØM, Cabrita L, Gardner PT, Morrice PC, Wood SG, Duthie SJ, Collins AR, Duthie GG. Anthocyanin-rich extract decreases indices of lipid peroxidation and DNA damage in vitamin E-depleted rats. Free Radic Biol Med 2001; 31(9): 1033-1037. 8. Hwang YP, Choi JH, Yun HJ, Han EH, Kim HG, Kim JY, Park BH, Khanal T, Choi JM, Chung YC, Jeong HG. Anthocyanins from purple sweet potato attenuate dimethylnitrosamine-induced liver injury in rats by inducing Nrf2-mediated antioxidant enzymes and reducing COX-2 and inos expression. Food Chem Toxicol 2011; 49(1): 93-99. 9. Han KH, Matsumoto A, Shimada K, Sekikawa M, Fukushima M. Effects of anthocyanin-rich purple potato flakes on antioxidant status in F344 rats fed a cholesterol-rich diet. Br J Nutr 2007; 98(5): 914-921. 10. Sakatani M, Suda I, Oki T, Kobayashi S, Kobayashi S, Takahashi M. Effects of purple sweet potato anthocyanins on development and intracellular redox status of bovine preimplantation embryos exposed to heat shock. J Reprod Dev 2007; 53(3): 605-614. 11. Hwang YP, Choi JH, Choi JM, Chung YC, Jeong HG. Protective mechanisms of anthocyanins from purple sweet potato against tertbutyl hydroperoxide-induced hepatotoxicity. Food Chem Toxicol 2011; 49(9): 2081-2089. 12. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18(6): 499-502. 13. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957; 226(1): 497-509. 14. Lovejoy JC. The influence of dietary fat on insulin resistance. Curr Diab Rep 2002; 2(5): 435-440. 15. Wildman RP. Healthy obesity. Curr Opin Clin Nutr Metab Care 2009; 12(4): 438-443. 16. Siriwardhana N, Kalupahana NS, Cekanova M, LeMieux M, Greer B, Moustaid-Moussa N. Modulation of adipose tissue inflammation by bioactive food compounds. J Nutr Biochem 2013; 24(4): 613-623. 17. Posey KA, Clegg DJ, Printz RL, Byun J, Morton GJ, Vivekanandan-Giri A, Pennathur S, Baskin DG, Heinecke JW, Woods SC, Schwartz MW, Niswender KD. Hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet. Am J Physiol Endocrinol Metab 2009; 296(5): E1003-E1012. 18. Nam KS, Kim J, Noh SK, Park JH, Sung EG. Effect of sweet persimmon wine on alcoholic fatty livers in rats. J Korean Soc Food Sci Nutr 2011; 40(11): 1548-1555. 19. Yun TS, Min AK, Kim NK, Kim MK, Cho HC, Kim HS, Hwang JS, Ryu SY, Park KG, Lee IK. Effects of alpha-lipoic acid on SREBP-1c expression in HepG2 cells. J Korean Endocr Soc 2008; 23(1): 27-34. 20. Marceau P, Biron S, Hould FS, Marceau S, Simard S, Thung SN, Kral JG. Liver pathology and the metabolic syndrome X in severe obesity. J Clin Endocrinol Metab 1999; 84(5): 1513-1517. 21. Bruce CR, Hoy AJ, Turner N, Watt MJ, Allen TL, Carpenter K, Cooney GJ, Febbraio MA, Kraegen EW. Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is sufficient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance. Diabetes 2009; 58(3): 550-558.