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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 43(11), 1627~1634(2014) 고콜레스테롤혈증 ApoE Knockout 마우스에서기능성수정과의지질과산화및산화적 DNA 손상억제효과 박은주 1 백아란 2 김미정 2 이선우 1 이은지 1 최미주 1 이지현 2 송영옥 2 1 경남대학교식품영양학과 2 부산대학교식품영양학과및김치연구소 Inhibitory Effects of Functional Sujeonggwa (Cinnamon Drink) on Lipid Peroxidation and DNA Damage in DietInduced Hypercholesterolemic ApoE Knockout Mice Eunju Park 1, Aran Baek 2, Mijeong Kim 2, Seon Woo Lee 1, Eunji Lee 1, MiJoo Choi 1, Jeehyun Lee 2, and Yeong Ok Song 2 1 Department of Food and Nutrition, Kyungnam University 2 Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University ABSTRACT The recipe for sujeonggwa, a Korean traditional sweet drink containing cinnamon, ginger, sugar, or honey, was modified by replacing sugar with alternative sweeteners [stevia or shortchain frutooligosaccharide (scfos)] in order to improve the health functionality of sujeonggwa. The aim of this study was to evaluate the effects of modified sujeonggwa on lipid peroxidation and oxidized DNA damage in dietinduced hypercholesterolemic ApoE knockout mice. Hypercholesterolemia was induced in 6weekold male mice by administration of a high cholesterol diet (1.25% cholesterol, 0.5% cholic acid, and 10% coconut oil) for 4 weeks, after which mice were divided into five groups: sucrose solutionfed control group, sujeonggwa containing sucrose group, sucrose+stevia group, sucrose+stevia+scfos group, and commercially available sujeonggwa group as a positive control. After 6 weeks, sujeonggwa supplementation resulted in reduced hepatic thiobarbituric acid reactive substances (TBARS), regardless of sweetener type. However, reduction of hepatic TBARS by commercially available sujeonggwa was insignificant. Both endogenous and H 2O 2induced DNA damage in hepatocytes and splenocytes were significantly reduced only in the sujeonggwa containing stevia group compared to the sucrosefed control group. There were no significant effects of sujeonggwa supplementation on total radical trapping potential, lipid peroxidation, or DNA damage in blood. These results suggest that sujeonggwa has protective effects against hepatic lipid peroxidation and DNA damage in hepatocytes or splenocytes from dietinduced hypercholesterolemic ApoE knockout mice, and the type of sweetener should be modified to improve the health benefits of sujeonggwa. Key words: sujeonggwa, stevia, shortchain fructooligosaccharide, oxidized DNA damage, ApoE knockout mice 서 고콜레스테롤혈증은활성산소종 (reactive oxygen species, ROS) 의발생을촉진하여산화적스트레스상태를유발함으로써동맥경화를촉진하는것으로알려져있다. 고콜레스테롤혈증으로인한산화적스트레스는동맥내벽의부착인자의발현을촉진하고, 동맥내피세포의투과성을높여대식세포및 LDL 콜레스테롤의이동을촉진하여지방병변을일으킴으로써병의진행을촉진한다 (1,2). 또한동맥경화의발병은나이가들수록증가하는데, 이는노화에따라활 Received 14 July 2014; Accepted 25 July 2014 Corresponding author: Yeong Ok Song, Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University, Busan , Korea yosong@pusan.ac.kr, Phone: 론 성산소종에의한생체분자 ( 단백질, 지질및 DNA 등 ) 의산화를억제하는항산화체계및 repair system의활성이저하되기때문이라고보고되었다 (3,4). 수정과제조에사용하는계피, 생강및후추는방향, 건위혈액순환촉진, 식욕증진, 소화촉진등의약리효과가알려져있다. 계피 (Cinnamomum cassia) 는계수나무껍질로활성성분인 cinnamaldehyde와 cinnamic acid의지질저하효과 (5,6) 를포함한기능성연구가널리진행되어왔다. 생강 (Zingiber officinale) 은아시아에서널리사용되어온약용식물로관절염, 근육통, 후두염, 변비, 구토, 고혈압, 및감염성질환등의다양한질병치유에사용되어왔다 (7). 생강의유효성분인 gingerol, zingerone 그리고 shogal의항산화효과는많은연구를통해잘알려져있으며, 특히 6 shogaol의유리기소거효과가높은것으로알려져있다 (8). 최근에는항염증, 항궤양및항암, 항당뇨및콜레스테롤저

2 1628 박은주 백아란 김미정 이선우 이은지 최미주 이지현 송영옥 하효과등이보고되고있다 (911). 비만은만성질환의원인으로알려져있으며현대인의경우당함량이높은음료의섭취가비만의원인으로지적되고있어최근세계음료시장은단맛은느껴지나저칼로리인음료그리고카페인이함유되어있지않은기능성음료형태로시장이확대되고있다. 수작의궤 ( 受爵儀軌 ) (1765) 및 조선요리법 ( 朝鮮料理法 ) (1936) 에기록된전통수정과제조법에의하면수정과의단맛을내는급원으로꿀또는설탕을사용하고있어수정과섭취시상당량의칼로리가얻어지고이는콜라나사이다등의청량음료섭취로부터얻어지는칼로리와유사한것으로조사되었다 ( 시판수정과 44 kcal/ 100 ml, 코카콜라 44 kcal/100 ml). 설탕이함유된콜라등과같은청량음료의섭취는최근청소년비만의가장큰원인으로지적되고있어세계음료시장은단맛은유지하면서칼로리는낮은음료를개발하기위해설탕대체재를사용하고있다. 천연감미료인 stevia는설탕의 50~300배의감미를지니고있지만 (12) 칼로리는거의없어상업용으로사용되고있으나쓴맛이제품에남아기호를떨어뜨린다고지적되고있다 (13). Stevia는간에서지방산의 βoxidation에관여하는효소인 acylcoa oxidase(aco), acylcoa synthetase(acs), carnitine palmitoyltransferase(cpt)Ⅰ의발현을증가시켜혈중지질농도를낮추는효과가보고되고있다 (12). 또한 stevia는 TNFα, IL6, IL1β의농도를감소시키는항염증효과가있음이보고되었다 (14). 새로운감미료로소개되고있는 short chain fructooligosaccharide(scfos) 는 fructose 가 3분자정도결합하고있는비소화성탄수화물로단맛은설탕의 25% 정도로약하나 prebiotics로써역할이보고되고있다 (15). scfos는 bifidobacteria의증식을초래하며 (16) 단쇄지방산의생성을촉진하여혈중지질을저하하는효과가보고되고있는데, 이는지방산합성효소 (17) 나콜레스테롤합성효소의발현을억제하기 (18) 때문이라고보고되고있다. 본연구에서는전통음청류인수정과의세계화일환으로대체당을사용하여관능성이탁월한기능성수정과음료를개발한후, 개발된수정과음료의항산화및산화적 DNA 손상억제효능을살펴보고자하였다. 재료및방법수정과시료제조고문헌에의거하여전통수정과제조법을재현하고꿀을설탕으로대체하는일반수정과레시피를개발하였다. 이후수정과의단맛을일정하게유지하면서칼로리를낮추기위하여설탕의일부를 stevia 또는 scfos로대체한기능성수정과음료 (sujeonggwa drink) 레시피를개발하였다. 곶감은수정과의기호에영향을미치지않는다는선호도조사결과를바탕으로 (19) 본연구에서는곶감을수정과레시피 에서제외하였다. 통계피를 3 cm 길이로자른후 17배 (w/v) 의물로, 그리고생강 (3 mm 두께 ) 은 10배 (w/v) 의물을넣고전기레인지 (Swiss Military Electric Range, Gyeonggi, Korea) 화력 2,000(550 C) 에서 10분간끓인후화력 1,600(400 C) 에서 50분간끓여열수추출하였다. 추출물은여과지로걸러잔사를제거한후 1:1(v/v) 로혼합하여수정과음료의기본 base로사용하였고감미를맞추었다. 기능성수정과제조를위해사용한설탕대체원은 stevia( 그린비아스위트, 전원식품, Gyeonggi, Korea) 와 scfos(#71920, Allergy Research Group, Alameda, CA, USA) 이다. 대체당의첨가량은제조사의성분을바탕으로산술적으로계산한다음첨가하여기능성수정과를제조하고관능검사로평가한후 Table 1에나타난바와같이최종으로첨가량을확정하였다. 실험식이및동물사육고콜레스테롤식이를제조하기위하여분말 chow diet (2018S Teklad global 18% protein rodent diet, provided by Harlan Teklad, Madison, WI, USA) 를구입하여여기에 cholesterol 1.25%, cholic acid 0.5% 그리고 coconut oil 10%(w/w) 를중량비로첨가하였다. 실험기간중사료와물은제한없이공급하였으며매일식이섭취량을기록하였고체중은 7일단위로측정하였다. 본연구에서는고콜레스테롤혈증이유발된쥐에다양한수정과를경구로투여하여수정과및기능성수정과의항유전독성효과를확인하고자하였다. 6주령의수컷 apolipoprotein E knockout(apoe KO) 마우스 (SLC Inc., Hamamatsu, Japan) 35마리를구입하여 1주일간적응시킨후 1마리씩개별케이지에넣어사육하였다. 사육실의온도는 22±1 C, 습도는 55±5% 로유지하였고, 조명은 12시간주기로조절하였다. 콜레스테롤식이를 4주간섭취시켰을때혈장콜레스테롤농도가 1,000 mg/dl 이상으로모든동물에서고콜레스테롤혈증이유발되었음 (20) 을꼬리채혈을통해확인하였다. 수정과의효과를확인하기위하여각군의콜레스테롤농도가동일하게 5군의실험군으로나누었다. 본연구에사용된실험군은대조군인설탕물섭취군 (sucrose solution fed group, Control), 설탕첨가수정과음료섭취군 (sujeonggwa containing sucrose, Sucrose), 스티비아첨가수정과음료섭취군 (sujeonggwa containing sucrose+stevia, Stevia), scfos 첨가수정과음료섭취군 (sujeonggwa containing sucrose+stevia+scfos, scsfo), 그리고양성대조군으로시판수정과를섭취시킨군 (Vilac sujeonggwa drinks, Positive control) 이다. 매일동일한시간에수정과와설탕물을존대를사용하여 6주간경구투여하였다. 식이는콜레스테롤식이를계속공급하였다. 수정과투여량은성인 1일음료섭취량 (21) 을기준으로산출하였다. 사람과동물의차이를보정하여 ( 보정계수 10) 산출하였을때마우스의하루수정과섭취량은약

3 기능성수정과의지질과산화및산화적 DNA 손상억제효과 1629 µl/25 g이었다. 경구로투여할수있는최대용량인 100 µl로만들기위하여진공농축기 (Rotavapor R200, Büchi, Flawil, Switzerland) 에서수정과를농축하였다. 시료의당도를측정하여 ( 당도계, AR 200, Reichert, Inc., Depew, NY, USA) 농축한수정과의당의함량에변화가없음을확인하였다. 대조군에투여할설탕물은일반수정과의설탕농도와동일하게제조하였다. 긍정대조군은시판되고있는캔수정과 (Vilac sujeonggwa, Paldo, Naju, Korea) 를사용하였다 (Table 1). 동물실험은부산대학교동물실험윤리위원회 (Pusan National UniversityInstitutional Animal Care and Use Committee, PNUIACUC, Approval Number PNU ) 의승인을받은후수행하였다. 해부및시료채취 10주간사육후마우스를 12시간동안절식시킨다음마취하고해부하였다. 마취제는 zoletile 50(zolazepan과 tiletamine 혼합물, 30 mg/kg bw, Virbac Laboratories, Carros, France) 과 xylazine(10 mg/kg bw, Bayer Korea, Seoul, Korea) 을혼합한용액을사용하였고복강으로주사하였다. 혈액은하대정맥에서채취한후 comet assay를위해 20 μl를이용하고남은혈액을 3,000 rpm, 4 C에서 20 분간원심분리 (COMBI514R, Hanil Science Industry, Incheon, Korea) 하여혈장을얻어 80 C에보관하면서실험에사용하였다. 채혈후냉각시킨 phosphate buffered saline(cold PBS) 을하대정맥을통해관류하여남은혈액을제거한뒤장기를적출하여여러차례씻은후여과지로수분을완전히제거하고무게를측정하였다. 무게를측정한장기는간 1 g과비장전체를 comet assay를위해사용하였고, 나머지장기는액체질소로급속냉동시켜분석시까지 80 C에보관하였다. 혈장총항산화능 (TRAP) 측정총항산화능은 RiceEvans와 Miller(22) 의방법을변형하여실험하였다. 각시료는 ABTS[2,2azinobis(3ethylbenzothiazoline6sulfonate), 150 µm] 와 metmyoglobin(2.5 µm) 을 H 2O 2(75 µm) 로활성화시켜형성된 ferryl myoglobin 라디칼종의상호작용에의해생성된 ABTS radical cation의흡광도를측정하는것으로흡광도의억제정도는혈장에들어있는 antioxidant capacity에비례하게된다. 유리시험관에 PBS buffer, ABTS, metmyoglobin을넣고혼합한후, plasma를넣고 H 2O 2 로반응시켜 734 nm의파장에서 UV/VIS spectrophotometer(shimadzu UV 1601, Kyoto, Japan) 를이용하여흡광도를측정하였다. 혈장의 TRAP 농도는 trolox의 calibration curve를이용하여계산한후 TEAC(Trolox equivalent antioxidant capacity, mm) 로나타냈다. 혈장및간과산화지질 (TBARS) 측정혈장및간의지질과산화물농도를 thiobarbituric acid related substances(tbars) 의함량으로나타내었다. 혈장 50 µl에 0.05 N HCl µl, 0.67% TBA 용액 167 µl를넣고 95 C의 water bath에서 30분간반응시킨후얼음물에서냉각시켰다. 여기에 15% methanol이함유된 n butanol 0.7 ml를첨가한후 2, rpm에서 10분간원심분리시켜얻은상층액의흡광도를 540 nm에서측정하였다. 간의지질과산화물농도를측정하기위해선간조직균질액 0.1 ml에 1% phosphoric acid 0.6 ml와 0.67% TBA 용액 0.2 ml를각각넣고충분히혼합한뒤 95 C의 water bath에서 45분간반응시킨후얼음물에서냉각시켰다. 반응종결을위해 0.8 ml의 nbutanol을첨가한후 3,000 rpm 에서 10분간원심분리시켜얻은상층액의흡광도를 540 nm에서측정하였다. 지질과산화물농도는 malondialdehyde(mda) 표준곡선을이용하여 nmol MDA로나타내었 Table 1. Recipe for the various sujeonggwa drinks Ingredient Cinnamon extracts 1) (ml) Ginger extracts 2) (ml) Sugar (g) Stevia 3) (g) scfos 4) (g) Control Sujeonggwa containing Sucrose Sucrose+Stevia Sucrose+Stevia+scFOS Positive control 5) kcal/100 ml ) Cinnamon sticks and water (1:17, w/v) were boiling for 10 min at 2,000 (equivalent to 550 C) of electric range (Swiss Military Electric Range, Gyeonggi, Korea) followed by boiling for 50 min at 1,400 (equivalent to 400 C). 2) Sliced ginger and water (1:10, w/v) were heated for 10 min at 2,000 (equivalent to 550 C) of electric range (Swiss Military Electric Range, Gyeonggi, Korea) followed by heating for 50 min at 1,400 (equivalent to 400 C). 3) Stevia product (Steviasweet, Jeonwonfood, Gyeonggi, Korea) used in this study was composed of 25% stevia and 75% glucose which is commercially available on the market. 4) scfos: shortchain fructooligosaccharide (#71920, Allergy Research Group, Alameda, CA, USA). 5) Commercially available sujeonggwa (Vilac sujeonggwa, Vilac, Korea) supplemented group. 6) NA: not available. NA 6) NA NA

4 1630 박은주 백아란 김미정 이선우 이은지 최미주 이지현 송영옥 다 (23). 산화적 DNA 손상측정을위한간및비장시료준비간 1 g을잘게자른후 240 unit collagenase(ec , Worthing Biochemical Corp., Freehold, NJ, USA) 가포함된 Hank's balanced salt solution(hbss) 완충용액에담가 37 C에서 30분간처리하였다. 효소처리후 427 rpm에서 5분간원심분리하여얻은상층액을모아다시 1,785 rpm에서 10분간원심분리한후하층의 cell pellet을 comet assay에이용하였다. 적출한비장전체를 RPMI 1640 배양액 (Gibco, Grand Island, NY, USA) 으로씻은다음멸균유리봉으로분쇄하여세포를유리시킨후, 세포현탁액을 100 µm nylon cell strainer(bd Biosciences, San Jose, CA, USA) 에통과시켜배양액으로 2번세척하고 3,000 rpm에서 10분간원심분리하였다. 적혈구를제거하기위해 RBC lysis buffer 10 ml를넣고 5분간실온방치후 954 rpm에서 10분간원심분리하여얻은 cell pellet을 comet assay에이용하였다. 백혈구, 간, 비장의산화적 DNA 손상측정 DNA 손상을측정하기위해 Singh 등 (24) 의방법을변형하여 comet assay를실시하였다. 백혈구, 간, 비장의 DNA 손상정도를확인하기위해전혈, 간세포및비장세포현탁액을각각 0.7% low melting agarose(lma) 와섞은후, 1% normal melting agarose gel(nma) 이 precoating 된슬라이드위에고르게분주한후커버글래스로덮어 4 C 냉장고에보관하였다. 각샘플당두개의슬라이드를제작하여한개의슬라이드는산화적스트레스무처리용 (PBS 처리 ) 으로사용하고, 다른한개의슬라이드는 DNA에산화적스트레스를가하기위해백혈구는 400 µm, 간과비장세포는 200 µm H 2O 2 용액에슬라이드를담가 5분간처리한뒤 PBS로세척하였다. 4 C 냉장고에서젤이굳으면커버글래스를벗기고그위에다시 0.7% LMA 용액 100 µl로한겹더덮었다. Cell lysis를위해미리준비해둔차가운 alkali lysis buffer(2.5 M NaCl, 100 mm EDTA, 10 mm Tris) 에사용직전 1% Triton X100을섞은후슬라이드를담가 4 C, 암실조건에서 1시간동안침지시켰다. Lysis가끝난슬라이드를전기영동수조에배열하고 4 C 차가운 electrophoresis buffer(300 mm NaOH, 10 mm Na 2EDTA) 를채워 20분간 unwinding 시킨후 25 V/300±3 ma의전압을걸어 20분간전기영동을실시하였다. 전기영동이끝나고 0.4 M Tris 완충용액 (ph 7.5) 으로충분히세척하고 ethidium bromide로핵을염색하여형광현미경 (Leica, Wetzlar, Germany) 에서관찰하고 CCD camera(nikon, Tokyo, Japan) 를통해보내진각각의세포핵이미지는 comet image analyzing system(kinetic Imaging, Komet 4.0, Liverpool, UK) 이설치된컴퓨터상에서분석하였다. 각세포의내재적 DNA 손상도와 H 2O 2 에의한산화적 DNA 손상 도는핵으로부터이동한 DNA 손상정도 (% Tail DNA) 를정량하여측정하였다. 통계처리모든자료의처리는 SPSSPC+ 통계 package Ver. 21 (IBM, Chicago, IL, USA) 을사용하여처리하였다. 각항목에따라평균치 ± 표준편차 (SD) 를구하고, 군별유의성검증을위해서는 oneway 분산분석 (ANOVA) 을시행하여 F 값을구한뒤, Duncan's multiple range test를이용하여각군간의유의성차이 (P<0.05) 를검증하였다. 또한각항목에대하여 Pearson 상관분석을실시하였다. 결과및고찰저칼로리기능성수정과레시피개발관능평가에의하면계피, 생강, 통후추, 곶감, 꿀을사용해제조한전통수정과는감미가너무높았고, 특히서양인과중국인은너무맵다고표현하여후추의매운맛에대한거부감이높았다. 곶감의첨가유무에대한차이는 Seo 등 (19) 의연구와마찬가지로나타나지않았다 (data not shown). 기능성수정과의단맛은일반수정과와비슷하나칼로리는일반수정과에비해 stevia 첨가수정과는약 33%, 그리고 scfos 첨가수정과는 85% 의칼로리를내었다. 순수한 stevia는칼로리가거의없는장점이있는반면뒷맛이쓰게느껴지는단점이보고되고있으며 (13), 본연구에서도설탕의일부만 stevia로대체하였음에도불구하고약간의쓴맛이느껴졌다. 한편 scfos는장운동을촉진하는기능이있어첨가할수있는양이제한되어있으므로수정과의단맛을유지하기위해설탕, stevia 그리고 scfos를복합적으로첨가하였다. scfos를첨가한기능성수정과는설탕또는 stevia로제조한수정과와유사한단맛을내었으며내국인뿐만아니라외국인을대상으로한관능평가에서좋은점수를받았다 (data not shown). 체중증가량, 식이섭취량, 식이효율콜레스테롤식이를 4주간섭취로고콜레스테롤혈증 (>1,000 mg/dl) 이유발된마우스에다양한종류의수정과를 6주동안경구투여하였을때설탕물을섭취한 control group, 시판수정과를섭취한 positive group, 그리고설탕만사용하여제조한일반수정과를섭취한 sucrose group 에서체중증가량이다소높았으나전실험군간유의적인차이가없었다. 식이섭취량및식이효율역시군간유의적인차이가없었다 (data not shown). 수정과음료의항산화및지질산화억제효과 TBARS는 free radical에의해체내막불포화지방산이산화되어생성된지질과산화물이며 (25), 노화및심혈관질환, 암등각종질병의원인이되기도한다 (26). 고콜레스테

5 기능성수정과의지질과산화및산화적 DNA 손상억제효과 1631 A B C Fig. 1. Correlation between hepatic TBARS and H 2O 2 induced DNA damage of leukocyte (A), hepatocyte (B), and splenocyte (C) in hypercholesterolemic ApoE KO mice. The correlation coefficients (r) and the correspondent significance values (P) are indicated. 롤식이에의해유도된고콜레스테롤혈증은간, 혈장, 적혈구, 심장등의조직에서지질과산화물 TBARS의생성을증가시키며 (27), 지질과산화과정의여러단계에서 DNA의손상을일으킨다 (28). 본연구결과에서도간의 TBARS 함량이높을수록백혈구, 간, 비장세포의산화적 DNA 손상도유의적으로증가하는것으로나타났다 (Fig. 1). 반면항산화활성을가진비타민 E, C, ferulic acid 등은고콜레스테롤혈증에의해증가한 TBARS 농도를낮추는것으로보고되었다 (29,30). 본연구에서 ApoE KO 마우스에고콜레스테롤혈증유발후 6주간수정과음료를섭취시켜체내지질산화에미치는효과를살펴보았을때간 TBARS 농도는설탕물을섭취한대조군에비해수정과음료군인 sucrose군, stevia 첨가군, 그리고 scfos 첨가군의 TBARS 농도는각각 28%, 33.9%, 31.6% 로유의적으로감소하여수정과의지질과산화억제효과를확인할수있었다 (Table 2). 그러나지질과산화억제에대해서대체감미료사용의추가적인감소효과는없는것으로나타났다. 따라서수정과의간 TBARS 감소효과는주재료인계피와생강에의한것으로사료된다. Azab 등 (31) 은감마방사선에의해산화적손상이유발된쥐에서계피추출물섭취에의해간의 TBARS 농도가 32% 정도유의적으로감소되었다고보고하였다. 또한 54명의건강한성인을대상으로계피차를 10일간섭취시켰을때혈장 TBARS 농도가유의적으로감소된것으로보고하였다 (32). 생강의경우살충제 lindane에의해산화적스트레스가유발된흰쥐에서 4주간의생강섭취는혈장 TBARS를유의적으로감소시키는반면 glutathione peroxidase 등의항산화효소의활성을조절하는것으로보고되었다 (33). 시판수정과섭취군의경우간 TBARS 농도는대조군에비해 19% 정도감소하는경향을보였으나유의적이지않았다. 혈장 TBARS의경우설탕물을섭취한대조군에비해시판수정과를제외한수정과음료섭취군에서감소하는경향을보여주었으나유의성은없었으며, 혈장총유리기포집항산화능 (TRAP) 도실험군간의유의적인차이가없는것으로나타났다 (Table 2). 이상의결과로동일한설탕을섭취함에도불구하고이를수정과의형태로섭취할경우지질산화억제효과를나타냄 Table 2. Effect of functional sujeonggwa drink on total antioxidant capacity and lipid peroxidation in hypercholesterolemic ApoE KO mice 1) Group 2) Control Sucrose Stevia scfos Positive control Plasma TRAP (mm) 0.78±0.08 NS 0.72± ± ± ±0.04 Plasma TBARS (nmol MDA/mL) 34.7±3.0 NS 25.4± ± ± ±11.2 Hepatic TBARS (nmol MDA/g liver) 31.0±5.7 a 22.2±5.7 b 20.5±4.9 b 21.2±7.9 b 25.1±6.8 ab Data are means±sd (n=7). 1) Plasma cholesterol concentration of ApoE KO mice reached over 1,000 mg/dl after 4 weeks of 1.25% cholesterol diet consumption with mean value of 1,077.3±164.0 mg/dl (n=35). Mean value of plasma triglyceride concentration of ApoE KO mice (n=35) at 4th weeks was 163.0±39.9 mg/dl in the range of to mg/dl. 2) Control: sucrose solution fed control group, Sucrose: sujeonggwa containing sucrose supplemented group, Stevia: sujeonggwa containing sucrose+stevia supplemented group, scfos: sujeonggwa containing sucrose+stevia+scfos supplemented group, Positive control: commercially available sujeonggwa (Vilac sujeonggwa, Vilac, Korea) supplemented group. NS Data within each column are not significantly different. a,b Data with different letters within the column are significantly different with oneway ANOVA followed by Duncan's multiple range test at P<0.05 을확인할수있었으며, 감미료의종류를달리하여설탕의양을줄여제조한수정과도지질산화억제효과가증가함을확인할수있었다. 기능성수정과음료의백혈구, 간, 비장세포 DNA 손상에대한보호효과 Comet assay는세포수준의 DNA 손상정도를정량적으로측정가능한실험기법이며, Folkmann 등 (3) 과 Dalboni 등 (4) 의보고에의하면 comet assay를이용하여측정한 ApoE KO 마우스의간, 백혈구산화적 DNA 손상이고콜레스테롤혈증및노화에의해유의적으로증가한반면, 포도주스, 스피루리나같은항산화물질은고콜레스테롤에의해유도된 DNA 손상을유의적으로감소시키는것으로나타났다

6 1632 박은주 백아란 김미정 이선우 이은지 최미주 이지현 송영옥 A B C Fig. 2. Inhibitory effect of functional sujeonggwa drink on endogenous or H 2O 2 induced DNA damage in leukocyte (A), hepatocyte (B), and splenocyte (C) in hypercholesterolemic Apo E KO mice. Cells were untreated (light shading) or treated with H 2O 2 treated for 5 min on ice (dark shading). Data with different letters above the bars are significantly different with oneway ANOVA followed by Duncan's multiple range test at P<0.05. NS: Data are not significantly different. (34,35). 본연구에서 comet assay를이용하여다양한세포에대한기능성수정과의 DNA 손상억제효과를살펴본결과 (Fig. 2), stevia 첨가수정과섭취군이설탕물만을섭취한대조군에비해간, 비장세포의내재적 (endogenous) 또는 H 2O 2 로유도된산화적 DNA 손상에대한억제효과가있는것으로나타났다. 백혈구의 DNA 손상의경우 stevia군에서대조군에비해감소하는경향을보여주었으나통계적유의성은없었다. 시판수정과의경우간세포의내재적 DNA 손상만을억제하는효능이있는것으로나타났으며 (Fig. 1B), 설탕만으로제조한수정과나 scfos가첨가된수정과섭취 군의경우내재적또는 H 2O 2 로유도된산화적 DNA 손상이대조군에비해감소하는경향을보여주었으나유의성은없었다. 계피와생강은인체유래세포주 (HepG2, HCT116 cell) 의 DNA 손상에대해보호효과가있는것으로보고되었으나 (36,37), 본연구에서는계피와생강만의 DNA 손상억제효과는미미한것으로보인다. 반면설탕을줄이는대신천연감미료인 stevia를첨가한수정과의경우간과비장에서유의한 DNA 손상보호효과가관찰되었는데, 이는 stevia의항산화및산화적 DNA 손상억제효능 (38) 과관련이있는것으로사료된다. 체내면역을담당하는비장세포에 A B C D E Fig. 3. Comet image of hepatocyte in hypercholesterolemic ApoE KO mice. A: Control, B: Sucrose, C: Stevia, D: scfos, E: Positive control group.

7 기능성수정과의지질과산화및산화적 DNA 손상억제효과 1633 서 stevia 첨가기능성수정과의산화적 DNA 손상억제효능에대한 comet 이미지는 Fig. 3에제시하였다. 본연구결과 stevia 첨가수정과는고콜레스테롤혈증및 H 2O 2 에의해유도되는산화적 DNA 손상을효과적으로억제시킨다는것을알수있었다. 요약본연구는전통음청류인수정과의세계화일환으로대체당을사용하여관능성이탁월한기능성수정과음료를개발한후, 개발된수정과음료의항산화및산화적 DNA 손상억제효능을살펴보고자하였다. 고콜레스테롤식이공급으로고콜레스테롤혈증이유발된 10주령의 ApoE KO 마우스를대조군인설탕물섭취군 (Control), 설탕첨가수정과음료섭취군 (Sucrose), 스티비아첨가수정과음료섭취군 (Stevia), scfos 첨가수정과음료섭취군 (scsfo), 그리고양성대조군으로시판수정과를섭취시킨군 (Positive control) 으로나누어실험식이를 6주간공급한후, 혈장 TRAP, 혈장및간의 TBARS, 백혈구, 간세포및비장세포의내재적또는산화적스트레스로인한 DNA 손상정도를측정하였다. 모든군에서체중증가량, 식이섭취량, 조직무게는유의적차이가없었다. 간 TBARS 농도는설탕물을섭취한대조군에비해설탕의농도를달리하여제조한수정과음료군인 sucrose군, stevia 첨가군, 그리고 scfos 첨가군의 TBARS 농도가유의적으로감소하여수정과의지질과산화억제효과를확인할수있었다. 설탕을줄이는대신 stevia를첨가하여제조한기능성수정과섭취군에서설탕물만을섭취한대조군에비해간, 비장세포의내재적 (endogenous) 또는 H 2O 2 로유도된산화적 DNA 손상에대한억제효과가있는것으로나타났다. 백혈구의 DNA 손상의경우 stevia군에서대조군에비해감소하는경향을보여주었으나통계적유의성은없었다. 시판수정과의경우간세포의내재적 DNA 손상만을억제하는효능이있는것으로나타났으며, 설탕만으로제조한수정과나 scfos가첨가된수정과섭취군의경우내재적또는 H 2O 2 로유도된산화적 DNA 손상이대조군에비해감소하는경향을보여주었으나유의성은없었다. 이상본연구의결과, 고콜레스테롤혈증이유발된 ApoE KO 마우스에서수정과의보충섭취는간지질과산화를개선시키고 stevia 첨가수정과의경우간및비장에서 DNA 손상억제효과가있음을확인할수있었다. 본연구결과가수정과의세계화에기여할수있기를기대하며, 향후본연구를기반으로한분자생물학적수준의기전연구가필요하다고사료된다. 감사의글본연구는농림축산식품부한식세계화용역연구사업에의해이루어진것으로연구비지원에감사드립니다. REFERENCES 1. Stranahan AM, Cutler RG, Button C, Telljohann R, Mattson MP Dietinduced elevations in serum cholesterol are associated with alterations in hippocampal lipid metabolism and increased oxidative stress. J Neurochem 118: Sudhahar V, Kumar SA, Mythili Y, Varalakshmi P Remedial effect of lupeol and its ester derivative on hypercholesterolemiainduced oxidative and inflammatory stresses. Nutr Res 27: Folkmann JK, Loft S, Møller P Oxidatively damaged DNA in aging dyslipidemic ApoE / and wildtype mice. Mutagenesis 22: Dalboni SP, Campagnaro BP, Tonini CL, Vasquez EC, Meyrelles SS The concurrence of hypercholesterolemia and aging promotes DNA damage in apolipoprotein Edeficient mice. Open J Blood Dis 2: Kannappan S, Jayaraman T, Rajasekar P, Ravichandran MK, Anuradha CV Cinnamon bark extract improves glucose metabolism and lipid profile in the fructosefed rat. Singapore Med J 47: Huang B, Yuan HD, Kim DY, Quan HY, Chung SH Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferatoractivated receptorγ (PPARγ) and AMPactivated protein kinase (AMPK) pathways. J Agric Food Chem 59: Ali BH, Blunden G, Tanira MO, Nemmar A Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): a review of recent research. Food Chem Toxicol 46: Dugasani S, Pichika MR, Nadarajah VD, Balijepalli MK, Tandra S, Korlakunta JN Comparative antioxidant and antiinflammatory effects of [6]gingerol, [8]gingerol, [10]gingerol and [6]shogaol. J Ethnopharmacol 127: Oboh G, Akinyemi AJ, Ademiluyi AO, Adefegha SA Inhibitory effect of aqueous extract of two varieties of ginger on some key enzymes linked to type2 diabetes in vitro. J Food Nutr Res 49: Choudhury D, Das A, Bhattacharya A, Chakrabarti G Aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells. Food Chem Toxicol 48: Fuhrman B, Rosenblat M, Hayek T, Coleman R, Aviram M Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein Edeficient mice. J Nutr 130: Park JE, Cha YS Stevia rebaudiana Bertoni extract supplementation improves lipid and carnitine profiles in C57BL/6J mice fed a highfat diet. J Sci Food Agric 90: Choi SY, Jang EG, Hwang IK The sensory characteristics and estimation of shelflife by Q10 values with mixtures of highintensity sweeteners for beverage during storage. Korean J Food Cookery Sci 21: Cho BO, Ryu HW, So Y, Cho JK, Woo HS, Jin CH, Seo KI, Park JC, Jeong IY Antiinflammatory effect of austroinulin and 6Oacetylaustroinulin from Stevia rebaudiana in lipopolysaccharidestimulated RAW264.7 macrophages. Food Chem Toxicol 62: Bornet FR, Brouns F, Tashiro Y, Duvillier V Nutritional aspects of shortchain fructooligosaccharides: natural occurrence, chemistry, physiology and health implications.

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