J Korean Soc Food Sci Nutr 한국식품영양과학회지 39(4), 494~499(2010) DOI: 10.3746/jkfn.2010.39.4.494 큰잎모자반효소적추출물의항산화활성 고석천 1 강성명 1 안긴내 1 양현필 2 김길남 3 전유진 1 1 제주대학교해양생명과학과 2 ( 주 ) 키토라이프기술연구소 3 제주하이테크산업진흥원제주생물종다양성연구소 Antioxidant Activity of Enzymatic Extracts from Sargassum coreanum Seok-Chun Ko 1, Sung-Myung Kang 1, Ginnae Ahn 1, Hyun-Pil Yang 2, Kil-Nam Kim 3, and You-Jin Jeon 1 1 Dept. of Marine Life Science, Jeju National University, Jeju 690-756, Korea 2 Technology Research Center, Kitto Life Co., Ltd., Gyeonggi 459-0, Koera 3 Jeju Biodiversity Research Institute (JBRI) and Jeju Hi-Tech Industry Development Institute (HiDI), Jeju 697-943, Korea Abstract In this study, Sargassum coreanum was enzymatically hydrolyzed to prepare water-soluble extracts by using five carbohydrates (Viscozyme, Celluclast, AMG, Termamyl and Ultraflo) and five proteases (Protamex, Kojizyme, Neutrase, Flavozyme and Alcalase) and their potential antioxidant activity were evaluated. The Celluclast and Neutrase extracts of Sargassum coreanum exhibited better DPPH radical scavenging activities (92.42% and 92.78%, respectively) and hydrogen peroxide (H 2O 2) scavenging activities (58.28% and 57.97%, respectively) compared to those of other enzymatic extracts. These results suggest that Sargassum coreanum would be a good raw materials for antioxidant and enzymatic hydrolysis would be a good strategy to prepare antioxidant extracts from seaweeds. Key words: Sargassum coreanum, antioxidant, enzymatic extract, radical scavenging 서론산소는인체내소화및에너지생성등여러대사과정에관여하고생물의생존에가장필수적인물질이지만, 반응성이매우큰활성산소로전환되면생체에큰영향을미친다고알려져있다. 활성산소종 (reactive oxygen species; ROS) 의종류로는일반적으로 superoxide anion radical( O - 2 ), hydroxyl radical( OH), hydrogen peroxide(h 2O 2), singlet oxygen( 1 O 2) 등이있고, 이들 ROS는단백질, 불포화지방산등과결합하여과산화지질을생성하고, DNA나 RNA 등에손상을일으켜, 생체막의손상, 면역능력의약화와함께성인병및각종질병과노화더나아가서는암을유발시키게된다 (1-3). 이러한활성산소종생성의생체내적요인으로는세포대사작용, 산화효소, 박테리아작용이있으며, 세포외적요인으로는오염된공기, 대사율증가, 흡연, 특정항생제, 자외선등이있고, 체내에서는각종항산화효소와항산화물질로이를제어하고있다 (4). 따라서이러한활성산소를효과적으로억제하는안전하고경제적인천연물의생리활성 물질을이용한연구가활발히이루어지고있다. 제주도는지리적인조건과난류와한류, 연안수등담수계가교류하는이점을가지고있어해조류가풍부하게서식하고있다 (5). 해조류는종류와시기에따라성분에서다소차이를가지고있으며, 대부분이 40~65% 의탄수화물을가지고육상식물과는다른구조형태가많고, 해조류내의탄수화물이장관운동활성화 (6) 및혈관내콜레스테롤침착방지 (7), 중금속배출촉진 (8), anti-plasmin 억제활성 (9), 고지혈증개선 (10) 항돌연변이 (11), 항균 (12), 항바이러스활성 (13) 등식용해조류로부터생리활성물질들이확인되면서기능성식품으로서의개발에관심이모아지고있다. 그중에서도갈조류는 xanthopophyll, pigment, fucoidan, phycocolloids, phlorotannins 그리고 fucoxanthin과같은다양한생리활성물질을함유한다고알려져있고 (14), 이러한생리활성물질은많은연구자들에의해항산화, 항응고, 항고혈압, 항균그리고항암활성을가진다는것이보고된바있다 (12,15-18). 그러나이러한갈조류에속하는큰잎모자반에대한생리활성효과에대한연구는찾아보기힘들고, 비 Corresponding author. E-mail: youjinj@jejunu.ac.kr Phone: 82-64-754-3475, Fax: 82-64-756-3493
큰잎모자반효소적추출물의항산화활성 495 수용성물질추출, 낮은수율, 고비용, 환경오염과독성이부각되고있는유기용매추출법이주를이루고있어새로운추출방법의대안이요구되어지고있다. 이전연구에서유기용매의단점을보완하기위해다양한효소를이용하는효소적가수분해방법을시도하여추출물을얻은후활성산소에대한높은소거능과암세포에대한높은증식억제활성이보고된바있다 (19,20). 이에본연구에서는수용성추출물을높은수율로얻을수있고, 독성없이친환경적인추출방법인당분해효소와단백질분해효소를이용하는효소적가수분해방법을이용하여갈조류에한종류인큰잎모자반으로부터수용성성분을추출한후각효소추출물의항산화효과를확인하고자하였다. 재료및방법시약단백질분해효소 (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase) 와당분해효소 (Viscozyme, Celluclast, AMG, Termamyl, Ultraflo) 는 Novo사 (Novozymes Nordisk, Bagsvaerd, Denmark) 에서구입하여사용하였고, 1,1-diphenyl-2-pricrylhydrazyl(DPPH), 2- deoxyribose, peroxidase, ABTS(2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid) 는 Sigma 회사 (St. Louis, MO, USA) 에서구입하여사용하였다. 그외모든시약은분석용특급시약을사용하였다. 큰잎모자반본실험에사용된큰잎모자반 (Sargassum coreanum) 은제주도연안에서식하고있는갈조류로제주도연안에서채집한후흐르는물로수세하여염분과협잡물을제거하고동결건조하여사용하였다. 일반성분분석큰잎모자반의일반성분은 AOAC법 (21) 에따라수분함량은 105 o C 상압건조법, 조지방은 Soxhlet 추출법, 조단백질 은 Kjeldahl법, 조회분은 5 o C 건식회화법으로분석하였으며, 탄수화물함량은고형분의총량에서수분, 회분, 조단백질그리고조지방의함량을뺀값으로나타내었다. 효소적추출물의조제효소적추출은 Heo 등 (22) 의방법으로 Table 1의조건에따라가수분해하였다. 동결건조후곱게간큰잎모자반 1 g을각각 100 ml의증류수와혼합한후 100 μl의단백질분해효소와당분해효소를첨가하였다. 효소적가수분해반응물은 12시간동안최적의조건에서효소반응을통해추출한후각추출물을 3,000 g에서 20분간원심분리하여잔사를제거한상층액을취한후여과하여농도를 2 mg/ml로조절한후실험시료로사용하였다. 추출물의수율측정수율은큰잎모자반으로부터유효성분을제조방법에따라획득한다음양을 % 로계산하였다. 총 polyphenol 함량측정큰잎모자반의총 polyphenol 함량측정은 Shetty 등 (23) 에준하여수행하였다. 각추출물 1 ml에 95% 에탄올용액 1 ml와증류수 5 ml를넣어혼합한후 % Folin- Ciocalteu reagent(sigma Chemical, St. Louis, MO, USA) 0.5 ml를넣고 5분간반응시켰다. 여기에 5% Na 2CO 3 용액 1 ml를가한후어두운상태에서 1시간동안반응시킨후 725 nm에서흡광도를측정하였다. 이때표준검량곡선은표준물질로 gallic acid를사용하여동일한방법으로작성된표준곡선으로부터총 polyphenol 함량으로환산하였다. DPPH radical 소거활성측정각추출물의 free radical 소거활성은 Blois(24) 의방법을변형하여측정하였다. 에탄올에용해시킨 4 10-4 M DPPH 용액 2.9 ml에각추출물 0.1 ml를넣고혼합하여 30분간반응시킨후 516 nm에서흡광도를측정하였다. DPPH free radical 소거활성은다음과같이계산하였다. DPPH free radical 소거활성 (%)=[1-( 대조구흡광도- 시료흡광도 )/ 대조구흡광도 ] 100 Table 1. Optimum hydrolytic conditions of particular enzymes Enzyme Viscozyme Celluclast AMG Termamyl Ultraflo Protamex Kojizyme Neutrase Flavourzyme Alcalase Optimum conditions ph Temperature ( o C) 4.5 4.5 4.5 7.0 7.0 8.0 60 60 60 40 40 Enzyme characteristics Arabanase, cellulase, β-glucanase, hemi-cellulase and xylanase Catalyzing the breakdown of cellulose into glucose, cellobiose and higher glucose polymer An exo-1,4-α-d-glucosidase A heat-stable α-amylase A heat-stable multi-active β-glucanase Hydrolysis of food proteins Boosting of the soya sauce fermentation An endoprotease Containing both endoprotease and exopeptidase activities A endo protease
496 고석천 강성명 안긴내 양현필 김길남 전유진 Hydrogen peroxide 소거활성측정각추출물의 hydrogen peroxide 소거활성은 Müller의방법 (25) 에따라수행하였다. 0.1 M phosphate buffer(ph 5.0) 와각추출물 100 μl을각각 96 microwell plate에넣어혼합시키고, 여기에다시 20 μl의 hydrogen peroxide를첨가시키고 37 o C에서 5분간반응시켰다. 반응이끝난후 1.25 mm ABTS와 peroxidase(1 unit/ml) 를각각 30 μl 첨가하여최종적으로 37 o C에서 10분간반응시킨후 405 nm에서흡광도를측정하였다. Hydrogen peroxide 소거활성은다음과같이계산하였다. Hydrogen peroxide 소거활성 (%)=[1-( 대조구흡광도- 시료흡광도 )/ 대조구흡광도 ] 100 Metal chelating 활성측정 Metal chelating 활성은 Decker와 Welch의방법 (26) 에따라측정하였다. 시료 0.1 ml에증류수 0.1 ml와 0.5 mm FeCl 2 0.025 ml를혼합한후 5 nm에서흡광도 ( 시료흡광도 1) 를측정하고이혼합물에 2.5 mm ferrozine 0.025 ml를첨가한후실온에서 20분간방치한다음 5 nm에서흡광도 ( 시료흡광도 2) 를측정하였다. Metal chelating 활성은다음과같이계산하였다. Metal chelating 활성 (%)=[1-( 시료흡광도 2- 시료흡광도 1)/( 대조구흡광도 2-대조구흡광도 1)] 100 positive control: EDTA 통계처리본실험의결과자료는 3회반복측정하여얻은결과를평균 ± 표준편차로나타내었으며, 통계처리는 ANOVA (one-way analysis of variance) 를이용하여다중범위검정 (Duncan's multiple range test) 을실시하여차이를검정하였다. 결과및고찰큰잎모자반의일반성분본실험에사용한큰잎모자반의일반성분조성은 Table 2와같다. 탄수화물의함량이전체의약 67% 를차지하였다. 여러해조류들중에갈조류는다당류를비롯한탄수화물성분을다량으로함유하고있다고알려져있는데, 본실험에서또한가장많은함량을차지하고있는것을확인하였다. 또한각종미네랄및비타민들을다량으로함유하고있어서회분함량도 12.77% 를나타내었고, 단백질의함량이 14.4% 를차지하였으며, 수분이약 4.3%, 지방의함량이약 1.33% 내외의함량을나타내었다. 추출수율각효소적추출물의유효성분즉, 수율은 Table 3에나타내었다. 모든추출물에서 40% 이상의비교적높은수율을보였다. 단백질분해효소추출물에서는 Neutrase 추출물이 47.91%, 당분해효소추출물에서는 Celluclast 추출물이 51.91% 의가장높은수율을보였다. 이와같은결과로보아단백질분해효소추출물에서는 Neutrase 추출물이, 당분해효소추출물에서는 Celluclast 추출물이다른효소추출물보다많은생리활성물질이포함되어있을것이라사료된다. Lee 등 (2) 의연구에서도추출수율이높은효소추출물에서가장우수한항산화활성이나타났다고보고되었다. 총 polyphenol 함량페놀성물질은식물계에널리분포되어있는 2차대사산물의하나로서다양한구조와분자량을가진다. 이들은 phenol hydroxyl기를가지기때문에단백질및기타거대분자들과결합하는성질을가지며, 항산화효과등의생리활성기능도가진다 (27). 일반적으로페놀성화합물이항산화작용을하는대표적인물질로보고되어있어큰잎모자반의효 Table 2. Chemical compositions of Sargassum coreanum (%) Scientific name Moisture Ash Protein Carbohydrate Lipid Sargassum coreanum 4.30 1) 12.77 14.40 67.21 1.33 1) The values are averages of triplicate determinations. Table 3. Yields and polyphenolic contents in enzymatic extracts of Sargassum coreanum Carbohydrases Proteases Enzyme extract Yield (%) Polyphenolic content (μg/ml) Viscozyme Celluclast AMG Termamyl Ultraflo Protamex Kojizyme Neutrase Flavourzyme Alcalase 47.32±0.17 c1) 51.91±0.24 a 41.25±0.13 g 41.85±0.28 f 43.17±0.23 e 47.12±0.32 c 45.71±0.15 d 47.94±0.12 b 43.57±0.27 e 43.43±0.15 e 1) Means in the same column bearing different superscripts are significantly different (p<0.05). 108.53±0.32 g 115.15±0.31 d 91.65±0.23 j 94.63±0.42 i 99.17±0.45 h 120.63±0.42 b 116.45±0.37 c 124.25±0.33 a 113.77±0.45 e 112.03±0.48 f
큰잎모자반효소적추출물의항산화활성 497 소적추출물에함유된페놀성화합물의함량을측정하였다. 각효소적추출물의페놀성화합물함량을측정한결과는 Table 3과같다. 단백질분해효소추출물에서는 Neutrase 추출물이 0.1242 mg/ml, 당분해효소추출물에서는 Celluclast 추출물이 0.1151 mg/ml로가장많은페놀성화합물을함유하였다. DPPH radical 소거활성 Free radical은생물학적손상의주요요인으로알려져있고, DPPH는천연항산화제의 free radical 소거활성을평가하는데일반적으로사용된다 (28). 따라서큰잎모자반의각효소적추출물로 DPPH free radical 소거능을측정한결과를 Fig. 1에나타내었다. 각효소적추출물은 90% 전후의우수한소거활성을보였으며, 단백질분해효소추출물에서는 Neutrase 추출물이 92.78%, 당분해효소추출물에서는 Celluclast 추출물이 92.42% 로서가장높은소거활성을보였다. 이러한소거활성은이전의연구에서보고된상용되어지고있는항산화제 (α-tocopherol, BHA 및 BHT) 와유사한수준이거나보다높은소거활성을보여우수한항산화력을가지고있는것으로보인다 (29). 이러한결과는갈조류인톳, 감태, 잘록이고리매를이용하여효소적추출을통해얻은효소추출물들의우수한 DPPH radical 소거활성이보고된연구결과와유사하였다 (30-32). 또한 DPPH radical에대한많은연구들이페놀성화합물과의긍정적상호관계를보고된바있는데 (33,34), 이러한연구결과와비교하여볼때효소적추출물이유기용매추출물보다페놀성화합물함량이낮음에도불구하고 DPPH radical 소거활성이높게나타났는데, 이러한결과는큰잎모자반효소적추출물의유효성을입증하는것이라사료된다. Hydrogen peroxide 소거활성 Hydrogen peroxide는체내각기관들의 DNA에심각한손상을초래해각종질병과노화를유발한다고알려져있다 (35). 이러한 hydrogen peroxide 소거활성을측정한결과를 Fig. 2에나타내었다. 모든효소적추출물에서약 40~60% 의소거활성을보였다. 단백질분해효소추출물에서는 Neutrase 추출물이 57.97%, 당분해효소추출물에서는 Celluclast 추출물이 58.28% 의소거활성으로가장높은소거활성을보였는데, Heo 등 (22) 의보고에서보다는비교적낮은활성을나타내었다. Kim 등 (36) 은항산화성의정도는식물의종류및이들에함유되어있는항산화유효성분의종류와추출방법에따라현저한차이가있음이보고된바있다. Metal chelating 활성 Peroxyl과 alkoxyl 라디칼은분자재구성을통해여러형태의과산화물로전환되고, 그중지질과산화물은그자체로는상당히안정적이나철이온과같은전이금속에의해분해가촉진되어생성된지질과산화분해산물들은다양한유해작용을한다고알려져있다 (37,38). 이와관련한 metal chelating 활성을측정한결과를 Fig. 3에나타내었다. 단백질분해효소추출물에서는 Neutrase 추출물이 59.51%, 당분해효소추출물에서는 Celluclast 추출물이 48.88% 의소거활성으로가장높은소거활성을보였다. Lee 등 (39) 은해수미세 Fig. 2. Hydrogen peroxide scavenging activity of enzymatic extracts from Sargassum coreanum (2 mg/ml). Abbreviations are the same as in Fig. 1. Means with different letters are significantly different (p<0.05). NS Fig. 1. DPPH free radical scavenging activity of enzymatic extracts from Sargassum coreanum (2 mg/ml). V: Viscozyme ext, C: Celluclast ext, AMG: AMG ext, T: Termamyl ext, U: Ultraflo ext, P: Protamex ext, K: Kojizyme ext, N: Neutrase ext, F: Flavourzyme ext, A: Alcalase ext. NS: not significant. Fig. 3. Metal chelating activity of enzymatic extracts from Sargassum coreanum (2 mg/ml). Abbreviations are the same as in Fig. 1. Means with different letters are significantly different (p<0.05).
498 고석천 강성명 안긴내 양현필 김길남 전유진 조류를이용한효소적추출물에서우수한 metal chelating 활성을보고한바있고, 이는친수성성분들에의한다고하였다. 요 본연구에서는제주연안에서식하는갈조류중큰잎모자반의항산화활성을확인하기위하여큰잎모자반으로부터효소적가수분해를이용하여추출물을제조한후이들로부터활성산소종의소거활성을확인하였다. 여기서효소적가수분해를이용하여제조된큰잎모자반의효소적추출물은항산화활성이우수하였으며, 효소적추출물간의유의적차이는없었다. 또한단백질분해효소추출물에서는 Neutrase 추출물이항산화활성이우수하였으며, 당분해효소추출물에서는 Celluclast를이용하여제조된효소적추출물이다소우수한항산화효과를나타내었다. 이와같은결과는여러가지효소들이복합되어진효소가세포벽에있는섬유질이나당단백질혹은알긴산고분자물질등을분해시키는작용을유도하였기때문이라고사료된다. 따라서수용성의추출물은유기용매추출물의단점을보완할수있을뿐만아니라큰잎모자반효소적추출물의생리활성물질은식품을포함한다양한분야에응용시킬수있을것으로사료되며, 이상의결과로큰잎모자반은잠재적인기능성식품소재로서의가능이충분하다고판단된다. 문 1. Choi SH, Kim EK, Lee SJ, Jeon YJ, Moon SH, Lee CH, Jeon BT, Park IS, Park TK, Kim B, Park SH, Park PJ. 2008. ESR spectroscopy investigation of antioxidant activity and protective effect on hydroyl radical-induced DNA damage of enzymatic extracts from Picrorrhiza kurroa. J Food Biochem 32: 708-724. 2. Lee SH, Kim KN, Cha SH, Ahn GN, Jeon YJ. 2006. 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