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pissn 1229-1153 / eissn 2465-9223 J. Food Hyg. Saf. Vol. 30, No. 4, pp. 383~389 (2015) http://dx.doi.org/10.13103/jfhs.2015.30.4.383 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 비만흰쥐에서발효서목태의항산화효과 배귀정 하배진 * 신라대학교의생명과학대학제약공학과 Antioxidative Effect of Fermented Rhynchosia nulubilis in Obese Rats Gui-jeong Bae and Bae-jin Ha* Department of Pharmaceutical Engineering and College of Medical Life Science, Silla University, 1-1 San, Gwaebup-dong, Sasang-gu, Busan 46958, Korea (Received November 2, 2015/Revised November 29, 2015/Accepted December 17, 2015) ABSTRACT - This study was to examine the antioxidative activity of fermented Rhynchosia nulubilis (FRN) in obese rats. Oxidative stress due to reactive oxygen species (ROS) can cause oxidative damage to cells. Mitochondria are especially important in the oxidative stress as ROS have been found to be constantly generated as an endogen threat. Mitochondrial defense depends mainly on superoxide dismutase whereas microsomal defense depends on catalase, which is an enzyme abundant in microsomes. Seven weeks-aged female Sprague-Dawley rats were divided into four groups and fed high fat diets for 44 days. Also fermented Rhynchosia nulubilis was administered orally for 44 days at 7.5 ml/kg of body weight of rats. The antioxidative activities of fermented Rhynchosia nulubilis were measured by the superoxide dismutase, catalase, malondialdehyde levels in liver homogenate. The levels of malondialdehyde in FRN-treated groups were lower than those in obese groups. Superoxide dismutase and catalase levels were significantly increased. These results demonstrated that fermented Rhynchosia nulubilis had the inhibitive effects of oxidative stress in obese rats, suggesting that fermented Rhynchosia nulubilis would be used as an ingredient of the useful functional products. Key words : Obese, Rhynchosia nulubilis, Anti-oxidation, Fermentation 생활습관으로인한질병이사회적문제가됨에따라과실, 채소류, 두류, 곡류가가지고있는여러가지생체기능성물질에관심이높아지게되었다 14). 그중콩류는식이섬유소, isoflavone, steroids, saponin, carotene 등이함유되어있어항암작용및콜레스테롤농도저하, 심장질환예방등의효과가있는것으로보고되었다. 특히 Isoflavone 이다량함유된서목태 (Rhynchosia nulubilis) 는쥐눈이콩, 약콩이라고도한다. 서목태는어느식품보다해독성이탁월하여청혈작용이있고, 신체기능을강화하는약제로신약본초에보고되었으며, 골질환예방및치료에도사용되어왔다. 또한고혈압과당뇨병을예방하고노화방지와노인성치매예방및신장에좋다고알려져있다 16,26). 비만에대한연구들을살펴보면, 비만이지질과산화물형성을촉진시켜체내항산화시스템이불균형에이르는 *Correspondence to: Bae-jin Ha, Department of Pharmaceutical Engineering and College of Medical Life Science, Silla University, 1-1 San, Gwaebup-dong, Sasang-gu, Busan 46958, Korea Tel: 82-51-999-5466, Fax: +82-51-999-5636 E-mail: bjha@silla.ac.kr 과정과밀접한관련이있다고보고되어있다 18). 항산화는생체내에서 Free radical 반응을야기시키는활성산소 (Reactive oxygen species, ROS) 들이계속생성되어세포가손상되는산화적스트레스에대한작용이다 22,27). 생체내에는이러한활성산소로인한질병과노화를대항하기위해서생체내에서 SOD (Superoxide dismutase), CAT (Catalase), GPX (Glutathione peroxidase) 등과같은효소적방어체계의항산화제를생성하며, 외부에서체내로공급이가능한비효소적방어체계로는 Vitamin C와 Vitamin E, Uric acid, Flavonoid 계열및 Polyphenol류등의천연항산화제와합성항산화제인 Butylated hyroxyanisole (BHA) 와 Butylated hydroxytoluene (BHT) 등이있다 7,17,27). 이러한항산화물질은질환의발병과관련된불안정한분자들인활성산소를효과적으로제거하여항산화및항염증효과를나타낸다고보고된다 6,7,20). 본연구에서는발효서목태엑기스및음료추출물의비만에의한항산화활성을비교및분석하여기능성식품에서비만에대한항산화효과에기초자료를제공하고자한다. 383

384 Gui-jeong Bae and Bae-jin Ha Materials and Methods DPPH radical 소거활성측정항산화활성의대표적인지표인 radical 소거활성 (radical scavenging activity) 은 Blois 4) 의방법을변형하여발효서목태엑기스에대한 1,1-diphenyl-2-picrylhydrazyl (DPPH) 의전자공여능으로측정하였다. 즉, 추출물의농도별희석액 50 µl에 0.15 mm DPPH 용액혹은에탄올 150 µl을첨가하여총액의부피가 200 µl가되도록하였다. 이반응액을실온 (25 o C) 에서 30 분간방치한후 96 well plate reader 기기 (AT/Infinite M200, Tecan) 를사용하여 517 nm에서흡광도를측정한후, 아래와같은식으로 DPPH radical 소거율을계산하였다. 양성대조군으로 3,5-di-tert-4-butylhydroxytoluene (BHT, Sigma, USA) 를사용하였다. DPPH radical scavenging activity(%) = {1 (S SB)/(C CB)} 100 S: Absorbance of sample SB: Absorbance of sample blank C: Absorbance of ethanol CB: Absorbance of ethanol blank 총폴리페놀함량측정총폴리페놀함량은 Folin-Denis법 9) 을응용하였다. 각각 10,000, 1,000, 500, 100 µg/ml이되도록희석한후희석액 800 µl에 1 N Folin-ciocalteu's phenol reagent (Sigma, USA) 100 µl을첨가하여잘혼합하고실온 (25 o C) 에서 3 분간방치하였다. 200 µl의 10% sodium carbonate(10% Na 2 CO 3 ) 를혼합하여실온에서 1 시간동안암실상태로방치한후 Table 1. Experimental design of rats Experimental group NOR(6) CON(6) FRN-E(6) day 11-54 day 11-54 food dose of sample 42 g/day of Normal diet 42 g/day of High-fat diet 42 g/day of High-fat diet 7.5 ml/kg of water-fed 7.5 ml/kg of water-fed 7.5 ml/kg of FRN-fed 42 g/day of High-fat diet FRN-B(6) 7.5 ml/kg of FRN-fed NOR: Normal control group CON: High fat diet treated group FRN-E: Fermented Rhynchosia nulubilis extracts (100%) and high fat dietary group FRN-B: fermented Rhynchosia nulubilis extracts (8%) and high fat dietary group 96 well plate reader 기기 (AT/Infinite M200, Tecan) 를이용하여 725 nm에서흡광도를측정하였다. 총폴리페놀함량은에탄올에녹인 Gallic acid를최종농도가 0, 50, 100, 150, 200, 250 µg/ml이되도록하여 725 nm에서흡광도를측정하여표준검량곡선을작성하였으며, 총폴리페놀함량은 µg gallic acid equivalents(gae)/ml로나타내었다. 실험재료실험에사용된발효서목태엑기스는 ( 주 ) 할머니청국장에서제공받았다. 발효서목태는전통발효방법을사용하였으며발효과정은다음과같다. 먼저서목태를수돗물로세척하고 5 시간동안수돗물에서방치했다. 팽윤시킨서목태는물과혼합 (3:1, by w/v) 하여볏짚과함께 70 o C에 Fig. 1. Comparison of body weight of rats for 44 days. NOR: Normal control group, CON: High fat diet treated group, FRN-E: Fermented Rhynchosia nulubilis extracts (100%) and high fat dietary group, FRN-B: Fermented Rhynchosia nulubilis extracts (8%) and high fat dietary group. The number of experiment aminals is given in parenthesis.

Antioxidative Effect of Fermented Rhynchosia nulubilis in Obese Rats 385 서 5 시간동안쪘다. 찐서목태열수추출물은 30 o C에서 48 시간동안발효한후, 120 o C에서멸균하였다. 발효물은미세모슬린천을이용하여여과한후여과된추출액을실험에사용하였다 (yield: 2% w/w, dry weight). 실험동물및식이실험동물은 8 주령이고몸무게가 175 g Sprague-dawley (SD) 계암컷을 ( 주 ) 셈타코코리아로부터구입하여사용하였다. 각실험에쓰이는 RAT 은매일일정한시간에시료를투여하였고 (Table 1), 또한체중을매일일정한시각에측정하였다 (Fig. 1). 쥐의활동성을주기위해 12 시간낮과밤의주기로 Auto control system기기 (SS-2200, SYSTRONICS, KOREA) 를사용하여온도 24 ± 2 o C와습도 60 ± 5% 로유지하여 54 일간사육하였다. 쥐들은난괴법 (randomized complete block design) 에의해 4 군으로각군당 6 마리로나누어실험하였다. 각각의그룹은정상군 (NOR: 생수투여 + 정상식이 ), 대조군 (CON: 생수투여 + 60% high fat diet), (FRN-E: 100% 발효서목태엑기스투여 + 60% high fat diet), (FRN- B: 8% 발효서목태엑기스투여 + 60% high fat diet) 으로표기하여실험하였다 (Table 1). 10 일간적응기간을가진후, 44 일간급식하고 55 일째되는날 C 로마취하고개복하여장기의상태를확인하였고, 하대정맥에서혈액을채취하였으며, 4 엽의간을모두적출한뒤식염수로세척하여여분의혈액을제거하였다. 그리고혈액은윈심분리한후혈청을획득하여실험에사용하기전에 74 o C에보관하였고, 간또한 74 o C에보관하여실험에사용하였다. 고지방식이투여 ( 주 ) 셈타코에서고지방식이를구입하였으며식이의조성은 Table 2와같다. 간균질물의총단백질측정단백질의정량은 Lowry법 21) 으로 750 nm에서흡광도를측정하였고, 표준단백질시료로 Bovine Serum Albumin (BSA) 을사용하였다. 간조직중 superoxide dismutase (SOD) 활성측정 SOD 효소활성의측정은 Beauchamp와 Fridovich법 3) 의변형된방법에따라 0.2 M K-phosphate buffer (ph 7.4) 를 6720 µl, 1 mm xanthine 100 µl, 1% sodium deoxychlorate 300 µl, 1.5 mm KCN 300 µl, 0.2 mm cytochrome C 1500 µl를넣은혼합액에 sample 80 µl를넣고, xanthine oxidase 원액 10 µl를넣은후 Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 를이용하여 550 nm에서의흡광도변화를 2 분동안측정하였다. 효소의활성도는 superoxide dismutase standard (Sigma Co., USA) Table 2. Formulation of high-fat diet PRODUCT mg % kcal % Protein 26.2 20 Carbohydrate 26.3 20 Fat 34.9 60 Total 100 kcal/gm 55.24 Ingredient mg kcal Caseun, 80 Mesh 200 800 L-Cystine 3 12 Corn Starch 0 0 Maltodextrin 10 125 500 Sucrose 68.8 275.2 Cellulose, BW200 50 0 Soybean Oil 25 225 Lard* 245 2205 Mineral Mix S10026 10 0 10 0 DjCalcium Phosphate 13 0 Calcium Carbonate 5.5 0 Potassium Citrate, 1 H 2 O 16.5 0 Vitamin Mix V10001 10 40 Choline Bitartrate 2 0 Total 773.85 4057 Formulated by E.A, Ulman, Ph.D. Research Diet, Inc. Typical analysis of cholesterol in lard = 0.95 mg/g Cholesterol(mg)/4057 = 232.8 Cholesterol(mg)/kg = 300.8 를표준액으로사용하여비교측정하였다. 간조직중 catalase (CAT) 의활성측정 CAT 효소활성측정은 Aebi법 1) 으로측정하였다. 측정하기에앞서효소의효율에따라결과가달라지므로희석배율을정하여선측정후일정희석배율에서가장좋은효과가보이는희석배율을선택하여측정한다. 1.9 ml의 PBS (0.05 M, ph 7.0) 와 0.1 ml의간균질화물을첨가하여잘혼합한다. 혼합한뒤 H 2 용액 1ml를첨가한뒤 Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 를이용하여 240 nm에서 1 분 30 초간측정한다. CAT 활성도의표기는 U/mg protein으로하였다. 간조직중 malondialdehyde (MDA) 의정량간의 MDA 수치는 Ha의방법 13) 에따라 1ml의 sodium dodecyl sulfate(7% SDS) 와균질화한간시료 0.5 ml과혼합한다. 37 o C, 30 분간반응시킨뒤 0.67% thiobarbituricacid 시약을 2ml 첨가하여한시간동안끓는물에서가열하였다. 가열후즉시냉각시킨뒤부탄올 5ml을첨가하고 3000 rpm에서 10 분간원심분리한다. 원심분리

386 Gui-jeong Bae and Bae-jin Ha 후상층액을취하여 535 nm의파장에서흡광도를측정 (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 하였다. 표준시약으로는 1,1,4,4-tetra-ethoxypropane를사용하였다. 통계적분석본실험에대한모든실험결과는평균치와표준편차로나타내었고, 통계적유의성은 IBM SPSS Statistics Ver. 20 (IBM, NY, USA) 를이용한 one-way ANOVA로검정하였으며, 사후검증으로 Duncan s post-hoc test과 t-test를실시하였고유의성은 p < 0.01과 p < 0.001로하였다. Results and Discussion DPPH radical 소거효과측정발효서목태의 DPPH radical 소거효과결과는 Fig. 2와같다. 발효서목태엑기스의 DPPH radical 소거효과는각각 22.18 ± 0.63%, 10.65 ± 2.47%, 4.93 ± 0.63%, 3.56 ± 0.17% 이며, 발효서목태음료에서는각각 6.37 ± 0.54%, 3.77 ± 0.73%, 2.87 ± 0.15%, 1.80 ± 1.20% 로발효서목태엑기스가발효서목태음료보다 DPPH radical 소거효과가높았으며, 농도의존적으로감소하였다. 홍등 14) 의연구에따르면, 대두와서목태열수추출물의 DPPH에대한전자공여능은농도가증가함에따라증가하였고, 서목태추출물은 73.05%, 대두추출물은 57.70% 로본연구에서도사용된서목태가대두보다높은전자공여능을나타냈다. Dordevic 등 8) 의연구에서메밀, 보리, 밀및호밀의 DPPH radical 소거능을연구한결과, 메밀이가장높은소거능을보였으며고농도로갈수록높은소거능을나타냈다. 또한 L. rhamnosus와 S. cerevisiae로각각발효를했을때, 비슷한소거능을보였지만비발효일때보다는높은소거능을나타냈다. 이에따라본연구에서는발효한서목태를사용하였다. 총폴리페놀함량측정발효서목태의총폴리페놀함량을측정하는결과는 Fig. 3과같다. 발효서목태엑기스의총폴리페놀함량은각각 229.08 ± 0.73 µg/ml, 36.84 ± 0.57 µg/ml, 18.74 ± 0.05 µg/ ml, 12.11 ± 0.37 µg/ml이며, 발효서목태음료에서는각각 77.01 ± 0.60 µg/ml, 35.76 ± 0.41 µg/ml, 12.74 ± 0.08 µg/ml, 10.02 ± 0.06 µg/ml로발효서목태엑기스가발효서목태음료보다폴리페놀의함량이더높았으며, 농도가높을수록폴리페놀함량이높았다. 박등 24) 의연구에의하면, 된장의총폴리페놀함량이 4.17 ± 0.11 mm gallic acid equivalent로가장높게나타났으며, 그외대두식품의경우메주 (2.310 ± 0.09 mm gallic acid equivalent), 청국장 (1.785 ± 0.07 mm gallic acid equivalent) 및콩 (0.585 ± 0.03 mm gallic acid equivalent) 순으로폴리페놀함량이높게나타났다. Arellano-gonzalez 등 2) 의연구에따르면, 발효를하지않은커피는 335 ± 71 w/(g/kg) 이고, 발효를한커피는 322.4 ± 30.1 w/(g/kg) 로써발효를한커피가더높은함량을나타냈다. 이에따라본연구에서는발효한서목태를사용하였다. 간에서의 CAT 활성변화 CAT 는 H 2 를무독성의 H 2 O로환원시키거나 methanol, ethanol, formic acid 및 phenol과같은 hydrogen donor의산화에관여하는효소로서지방산화에의해생성된유리기를제거하는것으로알려져있으며활성산소종을소거시키는작용을한다 15,23). Fig. 2. Scavenging activity of fermented Rhynchosia nulubilis and BHT on free radical. FRN-B: Fermented Rhynchosia nulubilis extracts (8%), FRN-E: Fermented Rhynchosia nulubilis extracts (100%), BHT: positive control. All the values were expressed as means ± S.D. (n = 6), a,b,c,d,e are different (P < 0.01) group by one-way ANOVA followed by Duncan s post hoc test. Fig. 3. Contents of fermented Rhynchosia nulubilis on total polyphenol. FRN-B: Fermented Rhynchosia nulubilis extracts (8%), FRN-E: Fermented Rhynchosia nulubilis extracts (100%). All the values were expressed as means ± S.D. (n = 6), a,b,c,d,e are different (P < 0.01) group by one-way ANOVA followed by Duncan s post hoc test.

Antioxidative Effect of Fermented Rhynchosia nulubilis in Obese Rats 387 Fig. 4. Effect of fermented Rhynchosia nulubilis on CAT levels in liver homogenate. NOR: Normal control group, CON: High fat diet treated group, FRN-E: Fermented Rhynchosia nulubilis extracts (100%) and high fat dietary group, FRN-B: Fermented Rhynchosia nulubilis extracts (8%) and high fat dietary group. All the values were expressed as means ± S.D. (n = 6), ***means each group was significantly different (P < 0.001) for the value of CON in student s test. Fig. 5. Effect of fermented Rhynchosia nulubilis on SOD levels in liver homogenate. NOR: Normal control group, CON: High fat diet treated group, FRN-E: Fermented Rhynchosia nulubilis extracts (100%) and high fat dietary group, FRN-B: Fermented Rhynchosia nulubilis extracts (8%) and high fat dietary group. All the values were expressed as means ± S.D. (n = 6), ***means each group was significantly different (P < 0.001) for the value of CON in student s test. 비만을유도한 rat의간균질액에서의 CAT 의활성변화는 Fig. 4와같다. CAT 는정상군이 0.57 ± 0.06 nmol/mg protein, 대조군은 0.38 ± 0.01 nmol/mg protein, 발효서목태엑기스섭취군은 0.57 ± 0.08 nmol/mg protein, 발효서목태음료섭취군은 0.56 ± 0.02 nmol/mg protein로, 발효서목태엑기스섭취군과발효서목태음료섭취군이대조군에비해 94.7~100% 증가하여정상군으로의회복을나타냈다. Carillon 등 5) 의연구에서는정상군과비만을유도시킨대조군의 CAT 활성보다 SODB( 멜론 ) 을섭취한군이현저하게 CAT 활성이증가되었으며내인성발현또한 50% 증가한것으로나타났다. 이를통해 CAT 가증가된것은활성산소를제거하여생체내대사과정에서생성된과산화물로부터생체조직을보호하여손상된간조직의기능을회복시킨것이기때문에발효서목태가 CAT 활성에유의적인효과가있는것으로사료된다. 간에서의 SOD 활성변화체내에축적된과량의활성산소는암을발생시키거나노화를촉진하는등유해한영향을미친다. 이러한활성산소는과식, 과도한스트레스및흡연, 지나친운동으로야기되는과호흡등에의해그양이증가하는데이러한활성산소를체내에서가장먼저분해시키는역할을하는효소가바로 SOD이다. SOD라는효소는 25세까지는체내에서자연적으로생성되므로별문제가없으나, 25세이후부터는 SOD 발생량이줄어들어서신체는점점노화되어간다, SOD는생체내의항산화방어기구중에서효소적방어계의하나로 superoxide radical을환원시켜 H 2 로전환시킴으로써산소독으로부터생체를보호한다 10). 비만을유도한 rat의간균질액에서 SOD의활성변화 는 Fig. 5와같다. 정상군이 86.33 ± 0.58 nmol/mg protein 로나타났고대조군은 70.33 ± 0.58 nmol/mg protein, 발효서목태엑기스섭취군은 79.20 ± 7.26 nmol/mg protein, 발효서목태음료섭취군은 84.22 ± 9.40 nmol/mg protein로발효서목태섭취군의 SOD 수치가대조군에비해 55.4~ 86.8% 증가하여정상군으로의회복을보였다. 류등 12) 의연구에따르면대조군의 SOD 활성이 9.84 ± 2.50 U/mg protein이고, 발효대두를 200 mg/kg 첨가군에서 16.47 ± 3.06 U/mg protein으로 40% 증가했다. 본연구에서의비만유도흰쥐의 SOD 활성이더높았으므로대두보다서목태가간에서의 SOD 활성에상승효과가있을것으로예측된다. 이를통해발효서목태가간에서의 SOD 활성증가에도움을주는것으로사료된다. 간에서의 MDA 변화과산화지질은 oxygen radicals에의해불포화지방산에서일어나는연쇄반응으로 oxygen radicals의직접적인작용보다는철이온존재하에 superoxide와 H 2 의상호작용에의해형성되는 OH 0 에의해간접적으로일어나며, 이의주된손상장소가 DNA나세포막이다 11). MDA는발암의가능성이있고, 돌연변이를일으킬수있으며, 고도로반응성이높은 free radical들은여러가지세포의중요한구성성분들, 즉지방세포막, DNA, 단백질등을변형시키는것으로알려져있다 12). 비만을유도한 rat의간균질액에서 MDA의활성변화는 Fig. 6과같다. 정상군이 22.32 ± 1.13 nmol/mg protein 로나타났고대조군은 35.05 ± 1.23 nmol/mg protein, 발효서목태엑기스섭취군은 23.57 ± 1.12 nmol/mg protein, 발효서목태음료섭취군은 23.77 ± 1.14 nmol/mg protein로

388 Gui-jeong Bae and Bae-jin Ha Fig. 6. Effect of fermented Rhynchosia nulubilis on MDA levels in liver homogenate. NOR: Normal control group, CON: High fat diet treated group, FRN-E: Fermented Rhynchosia nulubilis extracts (100%) and high fat dietary group, FRN-B: Fermented Rhynchosia nulubilis extracts (8%) and high fat dietary group. All the values were expressed as means ± S.D. (n = 6), ***means each group was significantly different (P < 0.001) for the value of CON in student s test. 발효서목태섭취군의 MDA 수치가대조군에비해 88.6~ 90.1% 감소하여정상군에가까운수치를보였다. 성등 28) 의연구에서는 MDA량이물추출물투여군은 2.28%, 알코올추출물및지용성추출물투여군은 18.53%, 30.1% 씩감소함을보였으며, 총사포닌투여군은 13.25% 로물추출물을제외한모든추출물에서유의성있게감소함을보였다. 본연구에서의비만유도흰쥐의 MDA량이더감소하였으므로사포닌보다서목태가지질과산화억제에더효과가있을것으로예측된다. 이를통해발효서목태가지질과산화를억제하는것으로사료된다. 국문요약 본연구에서는유도비만쥐에서발효서목태의항산화효과를연구하기위하여체중 175 g의 SD계암컷흰쥐 24 마리를대상으로정상군, 고지방식이군, 발효서목태엑기스급여군, 발효서목태음료급여군으로분류하여 54 일간사육하였다. in vitro 항산화활성에서는발효서목태엑기스가높은활성및함량을나타냈다. in vivo 항산화활성에서는발효서목태섭취군이고지방식이대조군에비해 CAT, SOD 활성은높은활성을보였으며, MDA량은감소현상을보였다. 이와같은결과를통해발효서목태가항산화효소의증가로활성산소의제거능을향상시킬뿐만아니라, 생체내대사과정에서생성된과산화물로부터생체조직을보호하여손상된간조직의기능을회복시킨것으로사료된다. References 1. Aebi, H.: Catalase in vitro, In: Packer L, ed methods in Enzymology. N. Y. Academic, 105, 121-126 (1984). 2. Arellano-gonzalez, M. A., Ramirez-coronel, M. A., Torresmancera, M. T., Perez-morales, G. G. and Saucedo-castaneda, G.: Antioxidant Activity of Fermented and Nonfermented Coffee (Coffea arabica) Pulp Extracts. Food Technol. Biotechnol, 49, 374-378 (2011). 3. Beauchamp, C. and Fridovich, I.: Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical biochemistry, 44, 276-287 (1971). 4. Blois, M.S.: Antioxidant determination by the use of a stable free radical. Nature, 26, 1199-1200 (1958). 5. Carillon, J., Romain, C., Bardy, G., Fouret, G., Feilletcoudray, C., Gaillet, S., Lacan, D., Cristol, J. and Rouanet, J.: Cafeteria diet induces obesity and insulin resistance associated with oxidative stress but not with inflammation: improvement by dietary supplementation with a melon superoxide dismutase. Free Radical Biology and Medicine, 65, 254-261 (2013). 6. Choi, S. H.: Pharmacological and Molecular studies of Atracylodes japonica Koidzumi on anti-oxidant activity, M. S. Thesis, KyungHee University, 1-29 (2011). 7. Doh, E.S., Chang, J.P., Kil, K.J., Choi, M.S., Yang, J.K., Yun, C.W., Jeong, S.M., Jung, Y.H., Lee, G.H.: Antioxidative Activity and Cytotoxicity of Fermented Allium victorialis L. Extract. Korean J. Plant Res., 24., 30-39 (2011). 8. Dordevic, T. M., Siler-Marinkovic, S. S. and Dimitrifevicbrankovic, S. I.: Effect of fermentation on antioxidant properties of some cereals and pseudo cereals. Food Chemistry, 119, 957-963 (2010). 9. Folin, A.D. and Denis, W.: A colorimetric method for the determination of phenols (and phenolderivatives) in urine. J. Biol. Chem., 22, 305-308 (1915). 10. Ha, B.J. and KIm, M.H.: Effect of Chondroitin Sulfate on Collagen Maturity and Aging. J. Food Hygiene and Safety, 14, 45-54 (1999). 11. Ha, B.J., Kim, H.J., Lee, S.H., Ha, J.M., Lee, S.H., Lee, J.H., Lee, D.G., Park, E.K. and Nam, C.S.: The Hepatoprotective Effects of Epimedii Herba through the Antioxidation. J. of Life Sci., 15, 572-577 (2005). 12. Ha, B.J.: Effects of Rhynchosia molubilis Saponin on Hepatotoxicity and Pathology. J. of Life Sci., 16, 186-191 (2006). 13. Ha. B. J.: Studies on the Antilipidperoxidation effect of Brazilin and Hematoxylin. Ph. D. Dissertation. Seoul university, Seoul, Korea (1985). 14. Hong, J.Y., Shin, S.R., Kong, H.J., Choi, E.M., Woo, S.C., Lee, M.H. and Yang, K. M.: Antioxidant activity of extracts from soybean and small black bean. Korean J. Food Preserv., 21, 404-411 (2014). 15. Kang, M.H., Lee, J.H., Lee, J.S., Kim, J.H., Chung, H.K.: Effects of Acorn Supplementation on Lipod Profiles and Antioxidant Enzyme Acticities in High Fat Diet-Induced Obese Rats. Korean J. of nutrition, 37, 169-175 (2004). 16. Kang, S.A. and Han, J.A.: Acetycholinesterase Inhibiting Effect and Free Radical Scavenging Effect of Soybean(Glycine max) and Yak-Kong(Rhychosia nolubilis)*. J. East

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