KOREAN J. FOOD SCI. TECHNOL. Vol. 49, No. 2, pp. 203~208 (2017) https://doi.org/10.9721/kjfst.2017.49.2.203 The Korean Society of Food Science and Technology 고지방식사로유도된신장산화스트레스를개선하는가압볶음무말랭이열수추출물효과 전연희 김미정 한성경 송영복 1 송영옥 * 부산대학교식품영양학과및김치연구소, 1 ( 주 ) 세전식품연구소 Effects of hot water extracts of roasted radish against renal oxidative stress induced by high-fat diet Yeonhui Jeon, Mijeong Kim, Seongkyung Han, Yeong-Bok Song 1, and Yeong Ok Song* Department of Food Science and Nutrition, Kimchi Research Institute, Pusan National University 1 Sejeon Co., Ltd., Jincheon Abstract The antioxidant and anti-inflammatory effects of roasted dried radish (RDR) against renal oxidative stress were examined in high-fat diet (HFD)-fed mice. The HFD was prepared by adding lard to chow diet to provide 50% of the calories from fat. Hot water extracts of dried radish (DR) or RDR were administered orally to mice at 237 mg/kg bw/day, whereas distilled water was administered as a vehicle for 12 weeks. Compared to the control group, renal reactive oxygen species, peroxynitrite, and thiobarbituric acid reactive substance level in the DR or RDR group were significantly decreased, whereas the glutathione level was increased (p<0.05). Protein expressions of antioxidant factors such as nuclear factor erythroid 2-related factor-2, heme oxygenase-1, glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase were significantly increased in the DR and RDR groups; however, nuclear factor-kappa B expression was suppressed (p<0.05). These antioxidant and anti-inflammatory effects of RDR were found to be significantly greater than those of DR. Keywords: radish, roasting, kidney, antioxidant effect, anti-inflammatory effect 서 고지방식사는비만, 고지혈증, 지방간, 동맥경화등과같은퇴행성질환을일으키는식사요인으로알려져있다 (1). 최근고지방식사는사구체신염, 사구체경화와같은만성신장질환의원인으로도보고되고있는데, 쥐에게 16 주간고지방식사를섭취시켰을때사구체섬유화가진행되었고이는고지방식사가염증반응에참여하는유도산화질소 (II) 합성효소 (inducible nitric oxide synthase, inos) 의발현을증가시켰기때문이었다고설명하였다 (2). 뿐만아니라고지방식사는마우스의신장에서지방산합성은촉진한반면지방산산화를억제하여신장에지방질축적을유도하였는데이는지방산합성을조절하는전사인자인 sterol regulatory element-binding protein-1 과효소인 fatty acid synthase 의발현을증가시킨반면지방산산화에관여하는 carnitine palmitoyltransferase-1 의발현은감소시켰기때문이라고보고하였다 (3). 그결과신장에지방질이유의적으로축적되었고, 지방질과산화물 (thiobarbituric acid reactive substance) 의농도가증가하였다고보고하였다 (4). 축적된지방질과산화물은체내대사과정중 *Corresponding author: Yeong Ok Song, Department of Food Science and Nutrition, Pusan National University, Busan 46241, Korea Tel: +82-51-510-2847 Fax: +82-51-583-3648 E-mail: yosong@pusan.ac.kr Received September 24, 2016; revised November 16, 2016; accepted December 12, 2016 론 생성된활성산소종과결합하여산화반응및염증반응을촉진시킴으로써산화스트레스 (oxidative stress) 를유발한다고알려져있다 (5). 산화스트레스란체내에서활성산소종, 활성질소종및지방질과산화물등의유리기를지니고있는물질의생성이높아이를제거하는산화방지시스템의균형이깨어진상태를일컫는것으로암, 동맥경화, 당뇨병, 그리고심혈관질환등과같은퇴행성질환의발병과연관이있다 (6,7). 산화스트레스가발생하면 DNA, RNA 및효소등과같은중요한기능을수행하는단백질뿐아니라세포및조직에도손상을초래하여기능장애를일으킨다 (8). 산화스트레스는 nuclear factor-kappa B (NF-κB) 를활성화하여염증관련사이토카인 (cytokine) 및케모카인 (chemokine) 의생성을촉진한다 (9). 체내산화방지방어체계에는토코페롤 (tocopherol), 아스코브산 (ascorbic acid) 및글루타싸이온 (glutathione) 과같은산화방지물질과초과산화물제거효소 (superoxide dismutase, SOD), 카탈레이스 (catalase, CAT), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1) 및 glutathione S-transferase (GST) 와같은산화방지효소가존재한다 (10). 무 (radish, Raphanus sativus L.) 는십자화과 (Cruciferae) 채소로민간에서감기등의예방및치료목적으로상용되어왔다 (11). 문헌에따르면무는내복근이라하여소화촉진과어패류또는면류의중독해소에효과가있고, 무종자는내복자라하여기담, 혈담, 천식, 늑간신경통등에사용하였다고한다 (12). 무의기능성성분으로글루코시놀레이트 (glucosinolate) (13), 설포라판 (sulforaphane)(14), 과산화효소 (peroxidases)(15) 및 L- 트립토판 (tryp- 203
204 한국식품과학회지제 49 권제 2 호 (2017) tophan) (16) 등이알려져있으며, 이들의산화방지효과 (16), 항균효과 (17), 항암효과 (18) 및항염증효과 (19) 는잘알려져있다. 무에함유되어있는 allyl-isothiocyanate 와설포라판과같은황함화합물은산화방지효소의전사인자인 nuclear factor erythroid 2- related factor-2 (Nrf2) 의발현을통해 GST, HO-1 등과같은산화방지효소합성을촉진한다고알려져있다 (20,21). 본연구팀은무말랭이차를개발하기위하여무말랭이를가압볶음한후열수추출물을제조하여 2, 2-diphenyl-1-picrylhydrazyl hydrate (DPPH) 라디칼소거능및산화스트레스를유발한 LLC-PK1 cell 의세포생존율에대한연구를수행하였을때가압볶음무말랭이는무말랭이보다라디칼소거능과산화스트레스개선효과가높았는데이는가압볶음중메일라드 (maillard) 반응에의해산화방지기능성이증가하였기때문이라고하였다. 무말랭이에는존재하지않았던 5- 하이드록시메틸푸르푸랄 (hydroxymethylfurfural) 이가압볶음무말랭이에서확인되었다 (22). 식품가공법에서볶음공정은향미및산화방지기능성을증진시키기위해차제조에흔히사용되고있으며, 율무 (23), 수수 (24), 결명자 (25) 및흰민들레 (26) 등을볶음하였을때 DPPH 라디칼소거활성이증가하였다고보고하였다. 본연구는가압볶음무말랭이의산화스트레스억제효과를고지방식사를섭취한마우스의신장에서확인하고자하였다. 재료및방법 재료및시약고랭지무 (Unduryeong, Gangwon-do, Korea) 를농수산물시장 (Busan, Korea) 에서구입하여사용하였다. 본실험에사용한시약인 7H 2 O-에틸렌다이아민테트라아세트산 (ethylenediaminetetraacetic acid), 싸이오바비투르산 (thiobarbituric acid) 는 Bio Basic Inc. (Markham, ON, Canada) 에서구입하여사용하였고, 소혈청알부민 (bovine serum albumin), 2',7'-dichlorofluorescein-diacetate (DCF- DA), dihydrorhodamine 123, 5,5'-dithiobis (2-nitrobenzoicacid), protease inhibitor cocktail은 Sigma Chemical Co. (St. Louis, MO, USA) 에서구입하여사용하였다. Bio-Rad protein assay kit와 Laemmli sample buffer는 Bio-Rad Laboratories (Hercules, CA, USA) 에서구입하였고웨스톤블롯분석 (western blot assay) 에사용된 CAT (F-17, sc-34285), cycloxygenase (COX)-2 (29, sc- 19999), GPx (B-6, sc-133160), GST (B-14, sc-138), HO-1 (H- 105, sc-1078), NF-κB (A, sc-109), Nrf2 (H-300, sc-13032), SOD (FL-154, sc-11407) 항체는 Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) 에서구입하여사용하였고, inos (PA1-036) 은 Thermo Fisher Scientific Co. (Waltham, MA, USA) 에서구입하여사용하였다. 2차항체 α-tubulin antibody (EP1332Y) (ab52866), rabbit anti-goat IgG H&L (HRP) (ab6741), donkey anti-rabbit IgG H&L (ab6802), rabbit anti-mouse IgG H&L (HRP) (ab6728) 는 Abcam Inc. (Cambridge, UK) 에서구입하여사용하였다. 그외의기타시약은특급시약을구입하여사용하였다. 가압볶음무말랭이제조무는껍질을제거하고 3 3 0.5 cm 크기로자른다음, 상온에서 12 시간예비건조한후 50 o C 로설정된건조기 (USD-6533F, Kyung Dong Navien, Seoul, Korea) 에서 24 시간건조하였다. 가압볶음무말랭이제조는재래시장에서사용하는튀밥용제조기를사용하였다. 압력 4.5 kg/cm 2 에서 2 분간가압볶음하여본실험에사용하였다. 시료의추출및제조무말랭이와가압볶음무말랭이에각각 20 배 (w/v) 의물을넣고 100 o C 에서 1 시간추출하였다. 열수추출물을여과 (No 2, Whatman, Springfield Mill, UK) 한후, 감압농축기 (Rotavator R-200, Bchi, Switzerland) 에서 1 차농축하고, 동결건조하여실험에사용하였다. 무말랭이와가압볶음무말랭이의열수추출물수율은 36% ( 동결건조물량 100/ 가압볶음무말랭이양 ) 으로한약재인당귀 (27) 및감초의열수추출물수율 (28) 과유사하였다. 당귀의수율은 38-40% 그리고감초의열수추출물수율은 31.6% 로보고되었다. 실험동물의사육및식사 4주령수컷 C57BL/6을구입하여 (DooYeol Biotech, Seoul, Korea) 1 주일사육실에적응시킨후, 각군의평균체중이유사하도록 8마리씩 4군으로나누었다. 고지방식사는 chow diet (2018S Teklad global 18% protein rodent diet, Harlan Teklad, Madison, WI, USA) 에라드를섞어 (20%, w/w) 총열량의 50% 를지방에서공급할수있도록제조하였다. 정상식사군 (normal diet group, NOR group) 은 chow diet를공급하고, 존대 (sonde) 를사용하여증류수를경구투여하였고, 실험군은고지방식사를공급하면서증류수를공급하는대조군 (control group, CON group), 무말랭이열수추출물을투여한무말랭이추출물투여군 (DR group), 그리고가압볶음무말랭이열수추출물을투여하는가압볶음무말랭이추출물투여군 (RDR group) 이었다. 쥐에공급한 DR 및 RDR 열수추출물의농도는다음과같이산출하였다. 성인 (60 kg) 이하루 2 잔의차 (2 g 건조차 / 팩 ) 를마신다고가정하였을때가압볶음무말랭이 4g이필요하고추출수율 36% 와및사람과쥐의대사속도차 (10배) 를감안하여산출하였을때쥐체중 kg 당하루공급량은 237 mg이었다. 사육실의온도는 22±1 C, 습도는 55±5% o 를유지하였으며, 명암주기는 12시간으로조절하였다. 동물사육기간은 12주이었으며, 실험중식사와물은자유롭게섭취하도록공급하였다. 동물실험과정은부산대학교의동물실험윤리위원회의승인후수행하였다 (PNU-IACUC, approval No. PNU- 2015-0824). 해부및시료채취 12 주사육후 12 시간동안절식시킨후 zoletil (30 mg/kg bw, Virbac Laboratories, Carros, France) 과 xylazine (10 mg/kg bw, Bayer Korea, Seoul, Korea) 으로마취시켜개복하였다. 인산완충소금물 (phosphate buffered saline) 으로하대정맥을통해관류한뒤해당장기를적출하여인산완충소금물로헹군후여과지로물기를제거하고, 무게를측정한후액체질소에서바로동결한후 80 oc 에서보관하였다. 신장균질액및 post-mitochondrial fraction 제조신장조직에 9 배의인산완충소금물을첨가한후균질기 (PT-MR 3100, Kinematica Inc,. Lucerne, Switzerland) 를이용하여조직을균질화하여지방질과산화물및글루타싸이온실험시료로사용하였다. post-mitochondrial fraction 은조직균질액을 4 o C 에서 2,012 g 로 15 분간원심분리하여상층액을얻은후, 이를 4 o C 에서 18,627 g 로 15 분간원심분리한후상층액을얻어서 reactive oxygen species (ROS) 및 peroxynitrite (ONOO ) 실험에사용하였다. 산화스트레스관련인자측정지방질과산화물농도는 Ohkawa 등 (29) 의방법에의해말론알
가압볶음무말랭이의신장산화스트레스개선효과 205 데하이드 (malonaldehyde, MDA) 표준곡선을이용하여계산하고 nmol 말론알데하이드로나타내었다. 글루타싸이온농도는 disulfide reagent {0.1 M sodium phosphate buffer (ph 8), 0.01 M 5,5'- dithiobis} 을이용하여 Ellman(30) 의방법으로측정하였다. ROS 및 ONOO 농도는 post-mitochondrial fraction를사용하여 ROS 농도는 DCF-DA를이용한 Ali 등 (31) 의방법으로 ONOO 농도는 dihydrorhodamine 123 buffer를이용한 Kooy 등 (32) 의방법으로측정하였다. ROS와 ONOO 는분당형광 (fluorescence) 강도로표시하였다. Western blot assay 웨스톤블롯분석시료로 whole cell 추출물을사용하였다. 즉, 신장조직에 nonidet-p40 lysis buffer {50 mm Tris (ph 8.0), 5 mm ethylenediaminetetraacetic acid (ph 8.0), 150 mm NaCl, 1% NP- 40, 1 mm D,L-dithiothreitol, 1 mm phenylmethylsulfonyl fluoride} 와 protease inhibitor cocktail을혼합하여첨가후균질화하였다 (PT- MR 3100, Kinematica Inc.). 균질액을 1시간동안얼음에보관한후 4C에서 18,627 g로 20분간원심분리하여상층액을얻었다. o 시료의단백질농도를측정한후일정량의단백질이함유된 whole cell 추출물에 Laemmli sample buffer와 β-mercaptomethanol을첨가하여혼합하고이를 sodium dodecyl sulphate-polyacrylamide gel에 loading 한후 90 V 전압으로 2시간동안전기영동 (Power Pac300. Bio-Rad) 하여단백질을분리하였다. 분리된단백질은니트로셀룰로스막 (nitrocellulose membrane) (0.45 μm pore size, Whatman) 으로이동시킨후 5% 탈지우유 (skim milk) 에서 1 시간동안 blocking 하였다. 니트로셀룰로스막을 1차항체용액에충분히잠기게하여 4C에서밤새반응시켰다. 이후세척하여 o 1차항체를제거하고 2차항체와반응시켰다. 2차항체와의반응은상온에서 1시간동안지속하였다. 반응후니트로셀룰로스막을세척하여부착되지않은항체를제거하고, enhanced chemiluminescence 용액으로발색시켜 CAS-400SM (Davinch-K, Seoul, Korea) 으로촬영하였다. 단백질발현은 image J software (Image J, National Institutes of Health, Bethesda, MD, USA, http:// www.rsb.info.nih.gov/ij) 사용하여측정하였고, 단백질의발현정도는 α-tubulin에대한비율로보정한뒤발현정도는정상식사군에대한비율로표시하였다. 통계처리 SPSS/PC package 20 (IBM, New York, NY, USA) 을사용하여, 평균과표준편차를구하였으며, one-way ANOVA 를실시한후, Duncan s multiple range test 로사후검정을실시하여 p<0.05 수준에서유의차를검증하였다. 결과및고찰 가압볶음무말랭이추출물의신장산화스트레스개선효과고지방식사를섭취한 CON group 의 ROS, ONOO 및지방질과산화물농도는 NOR group 에비해각각 135.0%, 202.0% 및 137.4% 증가되었다 (Table 1, p<0.05). 본결과는고지방식사섭취가신장의 ROS(33) 및지방질과산화물의농도 (34) 를높인다는보고와일치하였다. 이러한 CON group 의 ROS, ONOO 및지방질과산화물농도는무말랭이열수추출물투여에의해감소되었는데, RDR group 의 ONOO 와지방질과산화물농도는 DR group 에비해각각 52.2% 및 27.2% 감소한것으로보아유리기제거효과는 RDR group 이높은것을확인할수있었다. 체내산화방지물질인글루타싸이온농도는 NOR group 에비해 CON group 에서 70.3% 유의적으로감소하였고 (p<0.05), DR 및 RDR group 의글루타싸이온농도는 CON group 에비해각각 262.2% 및 370.7% 유의적으로증가하였다 (p<0.05). 특히 RDR group 의글루타싸이온농도는 DR group 에비해 141.4% 유의적으로증가하였다 (p<0.05). 이상의결과에의하면고지방식사는신장에서 ROS 생성및지방질과산화를촉진시켜산화스트레스를유발하였고, 무말랭이열수추출물섭취에의해신장의산화스트레스를개선하는효과가확인되었다. 본연구결과는무추출물은 zearalenone 로산화스트레스를유발한쥐의신장에서지방질과산화물의농도를낮추었다는보고 (35) 와일치하였다. 무의산화방지물질로는글루코시놀레이트 (13), 설포라판 (14), 과산화효소 (15) 및 L- 트립토판 (16) 등이알려져있다. 이러한무말랭이의산화스트레스개선효과는가압볶음무말랭이군에서더욱증가하였는데, 이는볶음과정중메일라드반응에의해산화방지물질의생성이증대하였기때문이라고사료된다. 본연구팀의선행연구에서무말랭이를가압볶음하였을때, 총폴리페놀함량및메일라드반응중간생성물인 5- 하이드록시메틸푸르푸랄의농도가증가하였으며, 가압볶음무말랭이열수추출물의 DPPH 라디칼소거능및산화스트레스가유발된 LLC-PK1 cell 의세포생존율이무말랭이군에비해높았다 (22). 율무 (23), 수수 (24), 결명자 (25), 흰민들레 (26) 등을볶음하였을때 DPPH 라디칼소거능이증가하였다는보고는본연구결과와유사하였다. 가압볶음무말랭이추출물의산화방지효과고지방식사를섭취한 CON group 의 Nrf2 및이에의해조절되는산화방지효소인 HO-1, GST, 초과산화물제거효소, 그리고 GPx 의단백질발현은 NOR group 에비해유의적으로낮았다 (Fig. 1, p<0.05). 이러한현상은무말랭이추출물을섭취시킨 DR 및 RDR group 에서증가하여무는산화를방지하는효과가있음을확인하 Table 1. Protective effect of hot water extracts of roasted radish against renal oxidative stress induced by high-fat diet ROS ONOO TBARS Group 1) (Flu/min/mg protein) (nmol MDA/g tissue) GSH (mmol/g tissue) NOR b2391±921 ab 3096±856 b 18.28±4.86 b 3.83±0.67 a CON 3227±755 a 6254±751 a 25.12±2.41 a 1.14±0.69 c DR b2173±841 ab 2394±984 b 20.90±2.85 b 2.98±0.64 b RDR 1432±442 b 1144±377 c 15.22±1.05 c 4.22±0.32 a Data are mean±sd (n=8 each group). 1) Mice were fed high-fat diet for 12 weeks with oral administration of distilled water (DW) (CON group), hot water extracts of dried radish (DR group) or hot water extracts of roasted dried radish (RDR group), respectively. Normal group (NOR group) was fed chow diet with oral administration of DW. a-c Data with different letters in the column are significantly different with one-way ANOVA followed by Duncan s multiple range test at p<0.05.
206 한국식품과학회지제 49 권제 2 호 (2017) Fig. 1. Protein expression of Nrf2, HO-1, GST, SOD, CAT, and GPx of C57BL/6 mice fed high-fat diet for 12 weeks. Data are mean±sd (n=8 each group). Mice were fed high-fat diet for 12 weeks with oral administration of distilled water (DW) (CON group), hot water extracts of dried radish (DR group) or hot water extracts of roasted dried radish (RDR group), respectively. Normal group (NOR group) was fed chow diet with oral administration of DW. a-d Data with different letters are significantly different with one-way ANOVA followed by Duncan s multiple range test at p<0.05. 였다. 특히가압볶음무말랭이추출물군인 RDR 군의효과는 DR 군에비해유의적으로높았는데, Nrf2 의발현은 123.9% 증가하였고, HO-1, 카탈레이스및 GPx 의발현은각각 123.7%, 143.1% 및 140.6% 유의적으로증가하였다 (p<0.05). 본연구결과는무의성분중함황물질인설포라판은산화방지효소계를촉진시켜산화스트레스를억제한다는보고와일치하였다. 쥐에설포라판을정맥주사한후신장에산화스트레스를유발하였을때지방질과산화물의생성이감소하였는데이는설포라판이 Nrf2 를활성화시켜 HO-1 과 GPx 와같은산화방지효소의발현이증가하였기때문이라고보고하였다 (36). 볶음커피열수추출물은과산화수소로산화스트레스를유발한 NR8383 큰대식세포 (macrophages) 에서생커피의열수추출물보다 Nrf2 의발현이유의적으로증가했다고보고하였다 (37). 또한볶은보리추출물은혈액과간에서노화로인해생성되는지방질과산화물을감소시켰다고보고하였다. 이는볶음과정중생성된산화방지물질이산화방지효소인초과산화물제거효소및 GPx 의발현을증가시켰기때문이라고설명하여 (38) 볶은무말랭이추출물에의해산화방지효소의발현이증가한본연구의결과와유사하였다. 가압볶음무말랭이추출물의항염증효과산화스트레스가유발된쥐의신장조직에서염증조절전사인자인 NF-κB 와염증효소인 inos 및 COX-2 의발현정도를확인하였을때 NOR group 에비해 CON group 의 NF-κB 와 inos 단백질발현이유의적으로증가하였다 (Fig. 2, p<0.05). NF-κB 발현은 DR 및 RDR group 에서 CON group 에비해각각 40.4% 및 59.3% 유의적으로감소하였으며 inos 발현은 DR 및 RDR group 에서 CON group 에비해각각 13.9% 및 22.8% 유의적으로감소하였다. 특히, RDR group 의 NF-κB 단백질발현은 DR group 에비해 31.7% 유의적으로낮았다 (p<0.05). NF-κB 에의해전사가조절되는 COX-2 의발현은 NF-κB 와같은경향성을보였으나그차 Fig. 2. Protein expression of NF-κB, inos, and COX-2 of C57BL/6 mice fed high-fat diet for 12 weeks. Data are mean±sd (n=8 each group). Mice were fed high-fat diet for 12 weeks with oral administration of DW (CON group), hot water extracts of dried radish (DR group) or hot water extracts of roasted dried radish (RDR group), respectively. Normal group (NOR group) was fed chow diet with oral administration of DW. a-d Data with different letters are significantly different with one-way ANOVA followed by Duncan s multiple range test at p<0.05. 이가유의적이지않았다. 산화스트레스유발시생성된 ROS 는 NF-κB 를활성화하여 inos 및 COX-2 와같은염증성효소의생성을촉진시킴으로써염증반응을촉진한다 (9). 반면 Nrf2 와같은산화방지전사인자는 HO-1 등의산화방지효소의발현을증가시킴으로써 NF-κB 의활성을억제시켜염증을예방한다고보고되
가압볶음무말랭이의신장산화스트레스개선효과 207 고있다 (39). 무의성분중 allyl-isothiocyanate 와설포라판추출물은 lipopolysaccharide 를처리한대식세포에서 Nrf2 발현과 HO-1 의전사를증가시켰고 NF-κB 발현및 inos 의전사를억제하였다. 또한고지방식사와 allyl-isothiocyanate 추출물을경구투여했을때간에서 Nrf2 와 HO-1 의발현이증가하였고, inos 의발현은억제되었다고보고하였다 (40). 본연구에서관찰된가압볶음무말랭이의항염증효과는볶음과정중생성된산화방지물질에의해산화스트레스가억제된효과로생각된다. 가압볶음무말랭이추출물의섭취는체내산화방지물질인글루타싸이온을증가시키고 Nrf2 와같은산화방지전사인자와산화방지효소의발현을증가시킴으로써 NF-κB 가감소한것으로사료된다. 요 고지방식사를섭취한쥐의신장에서가압볶음무말랭이추출물의산화방지, 항염증효과를확인하였다. 실험군은 chow diet 와증류수를경구투여하는 NOR group, 고지방식사와증류수를경구투여하는 CON group, 고지방식사와 237 mg/kg bw/day 농도의 DR 과 RDR 를각각경구투여하는 DR group, RDR group 으로나누어 12 주간사육하였다. 신장의산화스트레스지표인 ROS, ONOO, 그리고지방질과산화물의농도를확인한결과 CON group 이 NOR group 에비해유의적으로증가하였다. 반면 DR group 과 RDR group 에서유의적으로감소하였다. 체내산화방지지표인글루타싸이온의농도와산화방지단백질의발현은 DR group 과 RDR group 모두 CON group 에비해증가하였다. NF-κB 발현은 CON group 이 NOR group 에비해증가하였으나 DR group 과 RDR group 은모두감소하였다. RDR group 은 DR group 에비해 ONOO 및지방질과산화물농도가감소하였고글루타싸이온농도와산화방지관련단백질발현중 Nrf2, HO-1, 카탈레이스, 그리고 GPx 가증가하였고염증반응전사인자인 NF-κB 의발현이낮아졌다. 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