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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 45(1), 27~34(2016) 저온숙성마늘의라디칼소거활성을통한산화스트레스억제효과 황경아 김가람 황유진 황인국 송진 농촌진흥청국립농업과학원농식품자원부기능성식품과 Oxidative Stress Inhibitory Effects of Low Temperature-Aged Garlic (Allium sativum L.) Extracts through Free Radical Scavenging Activity Kyung-A Hwang, Ga Ram Kim, Yu-Jin Hwang, In-Guk Hwang, and Jin Song Department of Agrofood Resources, National Academy of Agricultural Science, RDA ABSTRACT Garlic has drawn attention as a food material for its anti-oxidative and anti-inflammatory properties as well as for prevention and treatment of cancer. In order to increase efficiency, various aging methods for garlic have been attempted. In particular, thermally processed garlic is known to have higher biological activities due to its various chemical changes during heat treatment. Therefore, in this study, we investigated the anti-oxidative effects of garlic extracts aged at low temperature (60 70 C). In the results, 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis (3-ethylbenzo-thiazoline-6-sulfonate) radical scavenging activities and ferric reducing ability of low temperature-aged garlic (LTAG) were similar to those of raw garlic. LTAG also showed decreased lipopolysaccharide (LPS)-induced production of reactive oxygen species, although there were not significant differences among samples. In addition, xanthine oxidase activity was inhibited by LTAG; the 15 days and 60 C extract showed outstanding inhibition compared with the others. To understand the molecular mechanisms behind the anti-oxidative activity of LTAG, we performed quantitative real-time PCR analysis. The 30 days and 70 C extract upregulated mrna expression of antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD), Mn-SOD, glutathione peroxidase, and catalase in LPS-stimulated RAW cells. This result indicates that LTAG can be a functional food as a nature antioxidant and antioxidant substance. Key words: low temperature aged garlic, radical scavenging activity, reactive oxidative stress, RAW cell 서 생체내에서는활성산소및활성질소를포함하는자유라디칼이생성되면이를제거하기위해항산화방어시스템이작동되며, 정상적상태에서는이시스템을통해외부의자극으로부터신체를보호한다. 그러나항산화방어시스템의작동에이상이생기면신체는활성산소를제거하지못하는산화스트레스상태로빠지게되며, 세포와조직을구성하는단백질, 지질, 세포막과 DNA 등의손상을초래하여이로인한돌연변이및염증, 발암등각종질병을유발시킨다 (1-3). 최근건강수명연장에관심이높은현대인들의산화스트레스와질병에효과적인기능성소재를의약품보다식품을섭취하고자하는욕구가증대되어기능성식품시장확대및활성화를불러왔고, 또한관련연구도활발히진행되고있다 (4,5). 그중마늘이대표적으로생리활성이높은기능성소재로알려져있다. 마늘은 Allium 속식물로백합 Received 24 September 2015; Accepted 25 November 2015 Corresponding author: Kyung-A Hwang, Department of Agrofood Resources, National Academy of Agricultural Science, RDA, Jeonbuk 55365, Korea kah366@korea.kr, Phone: 론 과의다년초식물이며항균, 항암, 항노화, 항산화활성 (6), 혈압강화 (7) 와면역증강등의다양한생리활성이알려지면서기능성식품및의약품의원료로다방면에이용되고있다 (8,9). 마늘의주요한생리활성기능은플라보노이드와황화합물의영향에의한것이지만 (10), 생마늘특유의강한향과매운맛으로인해섭취시거부감을준다. 이러한요인을극복하기위해마늘이지니고있는생리활성기능은유지하면서강한향과매운맛을보완시키기위한연구방법이개발되고있는데, 그대표적인방법으로마늘에열처리를하거나숙성기간의변화를주는것이다. 이과정을통해가공된제품이흑마늘과홍마늘로이들은일정한온도에서숙성을거쳐원하는색을띠게되며, 가공정도에따라화학적성분변화및풍미, 생리활성기능의차이를나타낸다 (11). 숙성마늘은생마늘을 40~90 C의온도에서수십일간숙성시킴으로써 allicin 감소및폴리페놀과플라보노이드증가등의성분변화가일어난다. 이로인해마늘의강한냄새를감소시키고, 높아진과당함량으로인한단맛증가와더불어식감도한결부드러워진다 (12,13). Cho 등 (14) 에따르면생마늘을온도별로숙성시켰을때마늘의탄수화물이과당으로변환되고탈수반응을거쳐 5-hydroxymethyl-2-furaldehyde

2 28 황경아 김가람 황유진 황인국 송진 (HMF) 라는물질이생성되었고, 이는 75 C, 7일간의고온숙성과정을거치며급격히증가하기시작하였으나, 60 C의저온숙성에의해서는 HMF의생성이거의이루어지지않았음을보고하였다. 최근연구에의하면 HMF는 tyrosinase 활성을억제시켜피부미백효능을가지고있으며 (15), 적혈구의세포겸상화를억제하는기능이있다는것이보고되고있지만 (16), 돌연변이성물질인 5-sulfoxymethylfurfural 로활성화되면독성또는돌연변이성원및발암성을나타낼수도있음이보고되고있다 (17). 따라서최근마늘의기능성을증진시키되 HMF 함량을낮추는저온숙성마늘의개발이이루어지고있으나, 항산화등의생리활성에관한연구는미비한실정이다. 따라서본연구에서는생마늘을저온에서기간별숙성을통해제조한저온숙성마늘추출물의항산화활성을평가하고, 향후이들소재를활용한기능성식품개발및이를통한농산물부가가치창출에기여하고자한다. 재료및방법재료및시약실험에사용한마늘은 2015년경상북도의성군에서재배된통마늘을구입하여사용하였다. 2,2-Diphenyl-1-picrylhydrazyl(DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS), potassium peroxodisulfate, hydrochloric acid(hcl), 2,4,6-tris(2-pyridyl)-s-triazine) (TPTZ), sodium acetate anhydrate, acetic acid, iron(Ⅲ) chloride hexahydrate(fecl 3 6H 2O), iron(Ⅱ) sulfate heptahydrate(feso 4 7H 2O), lipopolysaccharide(lps), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(mtt), dimethyl sulfoxide(dmso) 는 Sigma- Aldrich Co.(St. Louis, MO, USA) 에서구입하였고, Dulbecco's modified Eagle medium(dmem), fetal bovine serum(fbs), penicillin-streptomycin(p/s), phosphate buffed saline(pbs) 은 Gibco(Waltham, MA USA) 제품을사용하였고, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) 는 Molecular probe(waltham, MA USA) 제품을구입하여사용하였다. 이외에분석을위한시약은필요에따라특급또는 1급을구입하여사용하였다. 저온숙성마늘추출물제조밀폐용기에통마늘을각각 20통씩밀봉하여 15일 60 C (1560), 15일 70 C(1570), 30일 60 C(3060), 30일 70 C (3070), 60일 60 C(6060) 의조건으로배양기에서숙성시킨후, 동결건조 (Lyoph-pride Series Freeze Dryer, ilshinbiobase, Dongducheon, Korea) 하여믹서기 (HR 2860, Phillips Co., Eindhoven, Netherlands) 로 10분간분쇄하였다. 분쇄한저온숙성마늘 5 g에 70% 주정 200 ml 를넣어 30분간초음파추출하고, 상온에서 1시간방치한후상등액을회수하였다. 동일한과정을 3회반복하여여과 지 (Whatman No. 2, GE Healthcare Ltd., Little Chalfont, UK) 로여과한후감압농축 (EYELA CCA-1110, Tokyo Rikakikai Co.) 한다음, 동결건조기를이용하여분말화해시료로사용하였다. 이렇게얻어진저온숙성마늘에탄올추출물분말은 -70 C deep freezer에보관하였으며, 분말시료는 DMSO에녹여각각 50, 100, 250 μg/ml 농도로 PBS에희석하여실험에사용하였다. DPPH 라디칼소거능측정 DPPH 라디칼소거능은 Mensor(18) 의방법에따라측정하였다. 즉 0.3 mm DPPH 용액에 2.5배의저온숙성마늘추출물을혼합한후실온에서 30분간반응시킨다음, 분광광도계 (SpectraMax M5, Molecular Devices, LLC., Sunnyvale, CA, USA) 를이용하여 518 nm에서흡광도를측정하였다. 음성대조군은시료대신 PBS로실험하였고, 라디칼소거능은백분율 (%) 로나타내었다. DPPH radical scavenging =(1- ABSsample activity (%) ABS ) 100 control ABTS 라디칼소거능측정 ABTS 라디칼소거능측정은 Re(19) 의방법에따라수행하였으며, 7 mm ABTS 용액과 2.45 mm potassium persulfate를 1:1로혼합하고실온의어두운곳에서 12~16시간보관하여 ABTS 라디칼을생성시켰다. ABTS 라디칼은 732 nm에서흡광도가 0.7±0.02가되도록조정하여사용하였다. 농도를조정한 ABTS 용액 1.8 ml에저온숙성마늘농도별추출물 0.2 ml를가하여실온에서 30분간반응시킨후분광광도계 (Molecular Devices, LLC.) 를이용하여 732 nm에서흡광도를측정하였다. 음성대조군은시료대신 PBS 로실험하였고, 라디칼소거능은백분율 (%) 로나타내었다. ABTS radical scavenging =(1- ABSsample activity (%) ABS ) 100 control 환원력측정 환원력측정은 FRAP법 (20) 을이용하여수행하였다. 반응액은 300 mm acetate buffer(ph 3.6), 40 mm HCl에녹인 10 mm TPTZ와 20 mm FeCl 3 6H 2O를각각 10:1:1 (v:v:v) 의비율로실험직전에만들어사용하였다. 저온숙성마늘농도별추출물 10 μl에반응액 300 μl를혼합하여 4분간실온에서반응시킨후 593 nm에서흡광도를측정하였다. 추출물의환원력은 FeSO 4 를표준물질로하여얻은검량선으로계산하여나타내었다. 세포배양 RAW 마우스대식세포는한국세포주은행 (Seoul, Korea) 에서분양받아사용하였다. RAW 세포는 10% FBS와 1% P/S를포함한 DMEM 배지를사용하여 37 C, 5% CO 2 배양기에서배양하였다. 배양된세포는 2일에한

3 저온숙성마늘의산화스트레스억제활성 29 번계대배양하면서실험에사용하였다. 세포독성측정 RAW 세포를 cell/well로 96 well plate에분주하여 2시간배양한후시료를농도별 (50, 100, 250 μg/ ml) 로처리하여 24시간배양하였다. 배양완료후 5 mg/ ml의 MTT 용액을각 well에첨가하여 37 C에서 4시간동안재배양한후생성된 formazan을 DMSO에녹여 540 nm에서흡광도를측정하였다. Intracellular reactive oxygen species(ros) 억제활성측정 RAW 세포를 cell/well로 6 well plate에분주하여 2시간동안배양한후저온숙성마늘추출물을처리하였으며, 산화스트레스유발을위해추출물처리 1시간뒤에 LPS를각 well에처리하여 24시간배양하였다. 배양완료후 no phenol-dmem 배지에 10 µm DCFH-DA를첨가하여빛을차단한상태에서 37 C에서 30분반응후 PBS에 0.1% BSA를첨가하여제조한 buffer로 2회세척한후 flow cytometry(bd FACSCalibur, Becton Dickinson, San Jose, CA, USA) 를이용하여측정하였다. Xanthine oxidase 저해활성측정 Xanthine oxidase 저해활성은 xanthine oxidase assay kit(abcam plc., Cambridge, UK) 을이용하여측정하였다. 먼저 RAW 세포를 cell/well로 12 well plate 에분주하여 2시간동안배양한후저온숙성마늘추출물을처리하였으며, 산화스트레스유발을위해추출물처리 1시간뒤에 LPS를각 well에처리하여 24시간배양하였다. 배양한세포를 PBS로 2회세척한다음, PBS를이용하여세포를분리하였다. 분리한세포에 assay buffer를첨가하여 16,000 rpm에서 10분간원심분리하고상등액만취하였다. 상등액 50 μl와반응액 50 μl를혼합하여 37 C에서 1시간반응시킨후 570 nm에서흡광도를측정하였다. 항산화효소의유전자발현 RAW 세포를 cell/well로 12 well plate에분주하여 2시간동안배양한후저온숙성마늘추출물을처리하였으며, 산화스트레스유발을위해추출물처리 1시간뒤에 LPS를각 well에처리하여 24시간배양하였다. 배양한세포의 total RNA는 RNeasy mini kit(qiagen, Hilden, Germany) 으로추출하였고, reverse transcription system kit(promega, Madison, WI, USA) 을사용하여 cdna 를합성한다음, SYBR Green mix(qiagen) 를사용하여 mrna 발현정도를 Real-Time PCR Detection System (CFX96 TM, Bio-Rad, Hercules, CA, USA) 으로분석하였다. Endogenous control은 GAPDH를사용하였으며, 유전자발현에이용된 Cu/Zn-superoxide dismutase(sod), Mn-SOD, glutathione peroxidase(gpx), catalase의 primer는 Table 1에나타내었다. 통계분석모든실험은 3회이상실시하여얻은결과들을 mean± standard error of mean(sem) 으로나타내었고, 각실험결과로부터 Student's t-test를구한후 P<0.05인경우유의성이있는것으로판단하였다 (SPSS 12.0, IBM Co., Armonk, NY, USA). 결과및고찰저온숙성마늘의라디칼소거능 DPPH, ABTS, FRAP 방법은가장널리이용되는항산화활성측정방법중하나이다. 그중 DPPH 와 ABTS는라디칼의일종으로 DPPH는자유라디칼이지만 ABTS는양이온라디칼이라는것에서차이를보이며, 항산화물질의종류에따라라디칼에대한결합도가각기다르므로이를통해항산화능의차이를판단할수있다 (21). 저온숙성마늘추출물의 DPPH 라디칼소거능을비교한결과는 Fig. 1A와같으며, 처리농도가증가할수록라디칼소거능은증가하였다. 생마늘추출물의경우 250 μg/ml 농도에서약 20% 의소거능을나타내었으나, 대부분의저온숙성마늘추출물은 250 μg/ Table 1. Primers used for semi-quantitative RT-PCR of the antioxidant enzymes Target gene Primer Sequence (5' 3') Mn-SOD Cu/Zn-SOD Catalase Glutathione peroxidase GAPDH GGGTTGGCTTGGTTTCAATAAGGAA AGGTAGTAAGCGTGCTCCCACACAT CAGCATGGGTTCCACGTCCA CACATTGGCCACACCGTCCT AAGACAATGTCACTCAGGTGCGGA GGCAATGTTCTCACACAGGCGTTT CTCGGTTTCCCGTGCAATCAG GTGCAGCCAGTAATCACCAAG GAGCCAAAAGGGTCATCATC TAAGCAGTTGGTGGTGCAGG

30 황경아 김가람 황유진 황인국 송진 A B C Fig. 1. DPPH (A) and ABTS (B) radical scavenging activities and FRAP (C) assay of low temperature-aged garlic extract.

Values are the mean±sem of three replications (n=3). * P<0.05 compared to raw garlic (0000). ml 농도에서생마늘추출물보다더높은소거능을보였다. 특히 60일 60 C 추출물은약 30% 의소거능을보였으며, 30일 70 C 추출물도우수한소거능을보였다.

또한 Moreno 등 (23) 의연구에서는 14일동안 50 C에서숙성시킨마늘추출물의항산화활성은 amadori 화합물에의한것이고, 이화합물은 maillard 반응의첫단계에서주로생성되며이것이라디칼소거능에기여하는것으로보고되고있다.

4 30 황경아 김가람 황유진 황인국 송진 A B C Fig. 1. DPPH (A) and ABTS (B) radical scavenging activities and FRAP (C) assay of low temperature-aged garlic extract. 0000, raw garlic; 1560, 15 days and 60 C aged; 1570, 15 days and 70 C aged; 3060, 30 days and 60 C aged; 3070, 30 days and 70 C aged; 6060, 60 days and 60 C aged. Values are the mean±sem of three replications (n=3). * P<0.05 compared to raw garlic (0000). ml 농도에서생마늘추출물보다더높은소거능을보였다. 특히 60일 60 C 추출물은약 30% 의소거능을보였으며, 30일 70 C 추출물도우수한소거능을보였다. Shin 등 (22) 의연구에따르면온도를다르게하여숙성한마늘추출물의 DPPH 라디칼소거능은숙성온도가높을수록그활성이우수하였다고보고하였으며, 이는본실험의결과와유사한것을확인할수있었다. 또한 Moreno 등 (23) 의연구에서는 14일동안 50 C에서숙성시킨마늘추출물의항산화활성은 amadori 화합물에의한것이고, 이화합물은 maillard 반응의첫단계에서주로생성되며이것이라디칼소거능에기여하는것으로보고되고있다. 본연구의저온숙성마늘도숙성과정중 maillard 반응으로인해 amadori 화합물이생성될것으로생각되며, 이화합물이항산화활성에영향을줄것으로생각된다. ABTS 라디칼소거능은항산화물질이 ABTS 양이온과반응하여양이온이소거되면서특유의청록색이탈색되며이의흡광도를측정하여항산화능을측정하는방법이다 (18). Fig. 1B에나타낸것과저온숙성마늘추출물의 ABTS 라디칼소거능은 DPPH 라디칼소거능결과와유사하게추출물의농도에의존적으로증가하였다. 100 μg/ml 농도에서생마늘추출물과저온숙성마늘추출물은유사한 ABTS 라디칼소거능을보였으나, 250 μg/ml 농도에서는모든저온숙성마늘추출물이생마늘추출물보다더높은소거능을나타내는것을확인하였다. 특히 30일 70 C 추출물은약 40% 의소거능을보였으며, 60일 60 C 추출물도우수한소거능을보였다. Lee 등 (24) 은생마늘에비해열처리한마늘의항산화능이우수하였고, ABTS 라디칼소거능의경우숙 성기간과정도에따라차이를보였으며, 갈변물질이많이생성될수록소거능이더증가함을보고한바있다. 본연구결과저온숙성마늘추출물의 ABTS 라디칼소거능이 DPPH 라디칼소거능보다더높은결과가나타났는데, 그이유는 ABTS의경우수소공여항산화제와연쇄절단형항산화제로서의능력을모두측정할수있고, 수용성, 지용성물질모두에쓰이는방법이기때문에 ABTS 라디칼소거능이더높게나타난것으로판단된다. 환원력측정법은 ferric ion이 ferrous로전환되는과정을분석하여추출물이 Fe 3+ 을 Fe 2+ 로환원시킬때 Fe 2+ 이나타내는흡광도를측정하여환원력을계산하는방법으로라디칼소거능의항산화측정법과다른메커니즘의측정법이다 (25). 생마늘추출물과저온숙성마늘추출물의환원력을측정한결과는 Fig. 1C와같다. 모든추출물이농도의존적으로환원력이증가하였고 250 μg/ml 농도에서생마늘추출물과 30일 70 C 추출물이비슷한환원력을보였으며, 60일 60 C 추출물은더높은환원력을나타냈다. Hwang 등 (26) 에따르면총페놀및총플라보노이드의함량이높은추출물이라디칼소거능과환원력도높게나타난다는결과가보고되어있다. 생마늘을숙성시켰을때총페놀화합물및플라보노이드의함량이증가된다는결과 (24) 로봤을때본연구의저온숙성마늘추출물의항산화활성은페놀화합물과많은연관이있는것으로판단된다. 세포독성평가저온숙성마늘추출물이 RAW 세포에대한독성유무를확인하기위하여저온숙성마늘추출물을농도별로처

저온숙성마늘의산화스트레스억제활성 31 거하는항산화방어계가항상균형을이루고있으며 (28), 항산화연구에서 ROS 소거능은중요한지표로사용된다. 실험에사용한 LPS는그람음성세균의세포외벽물질로산화스트레스를유발시키는인자로잘알려져있다 (29). Fig. 3에나타낸바와같이 LPS를처리하였을때대조군에비해 ROS가많이생성되는것을확인할수있었다.

5 저온숙성마늘의산화스트레스억제활성 31 거하는항산화방어계가항상균형을이루고있으며 (28), 항산화연구에서 ROS 소거능은중요한지표로사용된다. 실험에사용한 LPS는그람음성세균의세포외벽물질로산화스트레스를유발시키는인자로잘알려져있다 (29). Fig. 3에나타낸바와같이 LPS를처리하였을때대조군에비해 ROS가많이생성되는것을확인할수있었다. ROS가생성된세포에생마늘추출물과저온숙성마늘추출물을처리하였을때모든시료에서높은 ROS 생성억제효과를나타내었으나추출물간의유의적인차이는보이지않았다. Fig. 2. Effects of low temperature-aged garlic extracts on cell viability in RAW cells. 0000, raw garlic; 1560, 15 days and 60 C aged; 1570, 15 days and 70 C aged; 3060, 30 days and 60 C aged; 3070, 30 days and 70 C aged; 6060, 60 days and 60 C aged. Values are the mean±sem of three replications (n=3). * P<0.05 compared to CTRL. 리하여 24시간후에세포생존율을측정한결과대조군의세포생존율을기준으로 50, 100, 250 μg/ml 농도에따른유의적인차이를나타내었다 (Fig. 2). 생마늘추출물은 250 μg/ml 농도에서약 30% 정도의세포독성을나타내었으나, 저온숙성마늘추출물은농도에상관없이세포독성을보이지않았다. 따라서 RAW 세포에대한추출물의항산화활성연구를위한적정농도를 100 μg/ml 농도로설정하고실험에사용하였다. ROS 억제활성평가산화스트레스의주요인자인 ROS는생체내조직에손상을주고변형을유발하여여러대사과정및생화학적변화를일으키는원인물질로 (27) 생체내에서는이러한 ROS를제 Xanthine oxidase 저해활성측정생마늘추출물과저온숙성마늘추출물의 xanthine oxidase 저해활성을측정결과는 Fig. 4와같다. 생마늘추출물의 xanthine oxidase 활성은약 55% 가저해되었으며, 대부분의저온숙성마늘추출물도 xanthine oxidase 활성을저해시키는것을확인하였다. 특히 15일 60 C 추출물이약 69% 의높은저해효과를보였으며, 15일 70 C 추출물도 47% 의저해효과를보였다. 마늘의숙성기간이길어지고, 온도가높아짐에따라 xanthine oxidase 활성이감소함을확인할수있었다. 여러연구에의하여플라보노이드가 xanthine oxidase의활성을저해하는물질로잘알려져있으며 (30), Kang 등 (31) 의연구에서황마늘에탄올추출물에서플라보노이드의함량이증가한다고보고하였다. 또한 xanthine oxidase 는 hydroxy기가 methyl화되면저해효과가감소된다고보고되고있고 (32), 이는마늘의숙성기간이길어지고고온에서처리할경우 HMF의생성량이증가한다는연구결과에따라 (33) xanthine oxidase의저해활성능은 HMF의영향 Fig. 3. Effect of low temperature-aged garlic extracts on the production of reactive oxygen species in RAW cells. 0000, raw garlic; 1560, 15 days and 60 C aged; 1570, 15 days and 70 C aged; 3060, 30 days and 60 C aged; 3070, 30 days and 70 C aged; 6060, 60 days and 60 C aged.

32 황경아 김가람 황유진 황인국 송진 Fig. 4. Effect of xanthine oxidase inhibition by low temperatureaged garlic extracts in RAW 264.7 cells.

Values are the mean±sem of three replications (n=3). * P<0.05 compared to CTRL. 을받는것으로생각된다. 항산화효소의유전자발현 LPS로유도된산화스트레스를억제하기위한기전을확인하기위해 RAW 264.

6 32 황경아 김가람 황유진 황인국 송진 Fig. 4. Effect of xanthine oxidase inhibition by low temperatureaged garlic extracts in RAW cells. 0000, raw garlic; 1560, 15 days and 60 C aged; 1570, 15 days and 70 C aged; 3060, 30 days and 60 C aged; 3070, 30 days and 70 C aged; 6060, 60 days and 60 C aged. Values are the mean±sem of three replications (n=3). * P<0.05 compared to CTRL. 을받는것으로생각된다. 항산화효소의유전자발현 LPS로유도된산화스트레스를억제하기위한기전을확인하기위해 RAW 세포에저온숙성마늘추출물을처리한후 LPS로자극하여항산화효소의 mrna를 RT-PCR 로측정한결과 (Fig. 5), Cu/Zn-SOD와 Mn-SOD의 mrna 발현은 LPS로자극한세포에비해 30일 70 C 추출물의유전자발현이유의적으로증가하는것을확인하였다. GPx와 catalase 유전자발현도 30일 70 C 추출물과 60일 60 C 추출물에서역시증가하는것을확인하였다. Superoxide dismutase(sod), GPx, catalase, glutathione reductase 등은세포내의효소성항산화제로이들의활성을증가시키기위한많은연구가진행되고있다 (34). SOD는세포내외로부터발생하는다량의 superoxide(o - 2 ) 를과산화수소 (H 2O 2) 로전환시켜 superoxide(o - 2 ) 를제거하는중요한역할을하는효소로알려져있다 (35). 정상적인상황에서는세포내에존재하는 Cu/Zn-SOD와 Mn-SOD로 O - 2 를비활성화시킬수있으나 (36), 염증, 질병등과같은특수상황에서는다량의 O - 2 가존재하게되므로 SOD 활성이잘이루어지지않아조직손상을초래할수있다 (37). 또한 GPx는 glutathione과 SOD에의해생성된 H 2O 2 를이용하여물분자로전환시키는효소이며 (38) catalase는 dismutation 반응을통해 H 2O 2 를물과산소로분해시키는효소로, 두효소모두생체내에서활성산소로부터생체를보호하는역할을한다 (39). 따라서 30일 70 C 숙성마늘추출물은항산화효소의활성을증가시킴으로써산화스트레스를감소시켜세포손상을낮출수있을것으로생각되며, Kim 등 (40) 의논문에서약용식물에의해 SOD, catalase, GPx의활성이증가함으로써자유라디칼생성억제및활성산소에의한산화적손상으로부터생체를보호하였다는결과와유사한것을확인하였다. A B C D Fig. 5. Effect of low temperature-aged garlic extracts on expression of antioxidant mrna in RAW cells. 0000, raw garlic; 1560, 15 days and 60 C aged; 1570, 15 days and 70 C aged; 3060, 30 days and 60 C aged; 3070, 30 days and 70 C aged; 6060, 60 days and 60 C aged. Values are the mean±sem of three replications (n=3). * P<0.05 compared to LPS-stimulated.

7 저온숙성마늘의산화스트레스억제활성 33 요 본연구에서는마늘의숙성기간 (15일, 30일, 60일 ) 과온도 (60 C, 70 C) 를달리한저온숙성마늘과생마늘의항산화효과를비교 분석하였다. DPPH와 ABTS의라디칼소거능과 FRAP법에의한환원력을측정한결과 250 μg/ml에서생마늘추출물보다 30일 70 C 추출물과 60일 60 C 추출물의항산화활성이우수하였다. 세포내활성산소생성은 15 일 60 C 추출물과 30일 70 C 추출물에서높은억제효과를보였으며, xanthine oxidase에대한활성저해효과역시 15일 60 C 추출물에서우수하였다. 항산화효소의유전자발현은 LPS를처리한군과생마늘추출물보다 30일 70 C 추출물에서높은효과를보였다. 본연구결과를통해저온숙성마늘이생마늘보다항산화활성이우수하다는것을확인함으로써차후항산화건강기능식품소재로서의활용이가능할것으로판단되나, 저온숙성마늘추출물의체내생리활성메커니즘규명을위해동물실험등의추가적인연구를계속적으로수행할예정이다. 약 감사의글 본성과물은농촌진흥청연구사업 ( 세부과제명 : 저온숙성마늘의피로개선효능평가및작용기전구명, 세부과제번호 : PJ ) 의지원에의해이루어진것임. REFERENCES 1. Halliwell B, Gutteridge JM, Cross CE Free radicals, antioxidants, and human disease: where are we now?. J Lab Clin Med 119: Jiang WY Therapeutic wisdom in traditional Chinese medicine: a perspective from modern science. Trends Pharmacol Sci 26: Park DJ, Kim SG, Yoon MK, Park SY, Kim YH, Lee SJ, Choi YW Effects of extracts from raw and black garlic on anticancer, antioxidant, antiinflammatory and whitening. Korean J Hort Sci Technol 28: Kim JH, Ahn MS, Kim GH, Kang MH Antioxidant and antimicrobial activities of Pleurotus eryngii extracts prepared from different aerial part. Korean J Food Sci Techonol 38: Atoui AK, Mansouri A, Boskou G, Kefalas P Tea and herbal infusions: Their antioxidant activity and phenolic propolis. Food Chem 89: Kang MJ, Yoon HS, Shin JH Chemical properties and biological activity of garlic (Allium sativum L.) shoots. J Agric Life Sci 46: Chun HJ, Park JE Effect of heat treatment of garlic added diet on the blood of spontaneously hypertension rat. J Korean Soc Food Sci Nutr 26: Katsuzi N, Park M, Ha SD, Kim GH Effects of garlic extract for protecting the infection of influenza virus. J Korean Soc Food Nutr 29: Kim HK, Kwak HJ, Kim KH Physiological activity and antioxidative effect of garlic (Allium sativum L.) extract. Food Sci Biotechnol 11: Leelarungrayub N, Rattanapanone V, Chanarat N, Gebicki JM Quantitative evaluation of the antioxidant properties of garlic and shallot preparations. Nutrition 22: Choi DJ, Lee SJ, Kang MJ, Cho HS, Sung NJ, Shin JH Physicochemical characteristics of black garlic (Allium sativum L.). J Korean Soc Food Sci Nutr 37: Lawson LD, Wang ZJ Allicin and allicin-derived garlic compounds increase breath acetone through allyl methyl sulfide: use in measuring allicin bioavailability. J Agric Food Chem 53: Choi YH, Shim YS, Kim CT, Lee C, Shin DB Characteristics of thiosulfinates and volatile sulfur compounds from blanched garlic reacted with alliinase. Korean J Food Sci Technol 39: Cho KJ, Cha JY, Yim JH, Kim JH Effects of aging temperature and time on the conversion of garlic (Allium sativum L.) components. J Korean Soc Food Sci Nutr 40: Sharma VK, Choi J, Sharma N, Choi M, Seo SY In vitro anti-tyrosinase activity of 5-(hydroxymethyl)-2-furfural isolated from Dictyophora indusiata. Phytother Res 18: Abdulmalik O, Safo MK, Chen Q, Yang J, Brugnara C, Ohene-Frempong K, Abraham DJ, Asakura T Hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibits sickling of red blood cells. Br J Haematol 128: Monien BH, Frank H, Seidel A, Glatt H Conversion of the common food constituent 5-hydroxymethylfurfural into a mutagenic and carcinogenic sulfuric acid ester in the mouse in vivo. Chem Res Toxicol 22: Mensor LL, Menezes FS, Leitão GG, Reis AS, dos Santos TC, Coube CS, Leitão SG Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phytother Res 15: Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26: Benzie IF, Strain JJ The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 239: Wang M, Li J, Rangarajan M, Shao Y, LaVoie EJ, Huang TC, Ho CT Antioxidative phenolic compounds from sage (Salvia officinalis). J Agric Food Chem 46: Shin JH, Choi DJ, Chung MJ, Kang MJ, Sung NJ Changes of physicochemical components and antioxidant activity of aged garlic at different temperatures. J Korean Soc Food Sci Nutr 37: Moreno FJ, Corzo-martinez M, Casrillo MD, Villamiel M Changes in antioxidant activity of dehydrated onion and garlic during storage. Food Res Int 39: Lee SJ, Shin JH, Kang MJ, Jung WJ, Ryu JH, Kim RJ, Sung NJ Antioxidants activity of aged red garlic. J Life Sci 20: Yoo KM, Kim DO, Lee CY Evaluation of different methods of antioxidant measurement. Food Sci Biotechonol 16: Hwang CR, Kang MJ, Shim HJ, Sub HJ, Kwon OO, Shin JH Antioxidant and antiobesity activities of various color resources extracted from natural plants. J Korean Soc Food Sci Nutr 44: Kader KN, Coyle CH Reactive oxygen and nitrogen species: implications for cardiovascular device engineering.

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Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,