Antioxidant Effect of Sargassum hemiphyllum Extracts

짝잎모자반(Sargassum hemiphyllum) 추출물의 항산화 효과

박지혜¹,박선희¹, 김민지¹, 김꽃봉우리¹, 최정수², 안동현¹*

1부경대학교식품공학과/식품연구소

2경남정보대학교호텔외식조리계열

Received: September 6, 2016 / Revised: December 14, 2016 / Accepted: December 19, 2016

서 론

체내에서영양소의산화과정중산소의약 ^95% 이상은물

로환원되지만일부산소가불완전하게전자를흡수하는과 정에서 불안정한산소 환원체가되는데 이를 활성산소종 (reactive oxygen species, ROS)이라고한다^[1]. 활성산소는 superoxide radical (O₂⁻), hydroxyl radical (· OH), peroxyl radical (ROO·)과 같은 산소 중심의 ^radical과 ^singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroperoxide

(ROOH)와같은비라디칼종들을포함한다^. 활성산소는정

상적인대사과정뿐만아니라^, 자외선^, 스트레스^, 대기오염및 흡연과같은다양한환경적요인에의해서도생성된다^. 활성

산소가과잉생성될경우생체내에서는지질과산화반응 으로인한과산화물질의생성^, 단백질및 ^DNA의산화로인 한염증유발^, 암^, 동맥경화^, 고혈압^, 류마티스관절염과같 은각종질병과노화를촉진시킨다^{[2, 3].} 활성산소와질병과 의관련성을보고한연구가활발해짐에따라 free radical의 발생을억제시키고산화작용으로부터생체를보호할수있 는다양한항산화제가개발되고있다^{. BHA, BHT}와같은합 성항산화제의경우탁월한효과로폭넓게사용되고있으나 다량으로섭취하거나체내에축적이될경우생체효소및지 방의변화로피부자극^, 각종장애^, 성호르몬감소^, 신경계 통의이상등을일으킬수있으며^, 독성및발암성을나타내

는것으로보고되고있다[1, 4, 5]. 또한천연항산화제로널

리알려진 tocopherol은안전하기는하나단독으로는산화연

쇄반응저지능력이낮고가격이비싼단점이있다^[6]. 이러

한이유로천연물로부터항산화능력이우수하면서도독성 이없는천연항산화제에관한연구가절실히요구되고있 Antioxidant Effect of Sargassum hemiphyllum Extracts

Ji-Hye Park¹, Sun-Hee Park¹, Min-Ji Kim¹, Koth-Bong-Woo-Ri Kim¹, Jung-Su Choi², and Dong-Hyun Ahn¹*

1Department of Food Science & Technology/Institute of Food Science, Pukyong National University, Busan 48513, Republic of Korea

2Subdivision of Culinary Arts, Kyungnam College of Information and Technology, Busan 47011, Republic of Korea

This study was conducted to determine the antioxidant activities of ethanol and water extracts of Sargassum hemiphyllum. Antioxidant activities were evaluated by assessing total phenolic contents, 2,2-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging activity, chelating effect, reducing power, and using the ranci- mat method. Total phenolic contents in the ethanol and water extracts were 17.91 and 13.44 mg gallic acid equivalents/g, respectively. Ethanol extract showed higher DPPH radical scavenging activity than water extract and similar activity to that of BHT. The reducing power of ethanol and water extracts increased in a concentration-dependent manner. Particularly, ethanol extract was more effective than water extract.

Water extract showed a higher chelating effect compared to ethanol extract. The antioxidant index mea- sured by rancimat was lower than those in BHT, but the ethanol extract showed a higher value than the water extract. The ethanol extract showed higher antioxidant activity than the water extract, except for the chelating effect. These results suggest that the ethanol extract of Sargassum hemiphyllum has more potent antioxidant activity and may be used as a source of natural antioxidants.

Keywords: Antioxidant effect, DPPH radical scavenging, Sargassum hemiphyllum

*Corresponding author

Tel: +82-51-629-5831, Fax: +82-51-629-5824 E-mail: [email protected]

© 2017, The Korean Society for Microbiology and Biotechnology

다^. 최근천연해양식물중에함유된다양한생리활성물질에 관한연구가성행하여^[7] 항균^{[8, 9],} 항암^[10], 항산화^{[8, 11],}

항아토피^[12] 물질에관한연구가이루어져기능성식품및

약품개발소재로서주목받고있다^.

짝잎모자반(Sargassum hemiphyllum)은모자반과의갈조 류로손가락모양의뿌리에서나온줄기는외가닥으로길어 지거나두갈래로갈라져윗부분에서가지를낸다^. 잎은얇 고주걱모양이며기포는알모양으로끝에가시모양의잎을 가진다^. 상부의잎이좌우대칭이아니기때문에짝잎모자반 이라고불리며우리나라동해안과제주지역및일본해역 에분포한다^. 짝잎모자반에대한연구를보면항알러지^[13], 항돌연변이및암세포증식억제^[14] 효과등이보고되고있 으며세포내지질과산화물생성억제에관한연구^[15]가 보고되고있다^. 본연구에서는짝잎모자반의에탄올및물 추출물을대상으로항산화능을알아보고이를바탕으로식 품산업에항산화물질로서의적용가능성을검토하고자하 였다^.

재료 및 방법

실험재료

본실험에사용한짝잎모자반(S. hemiphyllum)은부산인 근해안에서채취하여담수로깨끗이씻은다음세절하고동 결건조하여잘게분쇄한후−²⁰℃에서동결저장하면서사 용하였다^.

추출

동결건조된분말을 ^95% 에탄올로추출한다음잔사에물

을이용하여추출하였다^. 먼저 ^95% 에탄올을시료의 ¹⁰배량 가하여실온에서 Shaker (Dongwon Science Co., Korea)를

이용하여 ^{180 rpm}으로교반하면서 ²⁴시간추출한후원심

분리기(UNION 32R, Hanil Co., Korea)로 ^{1,977 ×g}에서

10분간원심분리하였다^. 상층액을취한후남은잔사에동

일한용매를가하여같은방법으로 ²회반복추출하였다^. 에 탄올추출후남은잔사는 ³⁷℃에서건조하여물추출에사 용하였다^. 물추출물은건조된잔사에 ¹⁰배량의증류수를가 하여에탄올과동일하게 ³회반복추출하여취하였다^. 상층 액은 여과지로 여과한 후 ³⁷℃ water bath에서 ^rotary evaporator (RE200, Yamato Co., Japan)를이용하여감압 하에농축하고 ³⁷℃에서건조하였다^. 건조된시료는−²⁰℃ 에보관하면서실험에사용하였다^.

총 페놀 화합물 함량 측정

총페놀화합물함량은 Folin-Denis법^[16]을변형하여측 정하였으며 Folin-Ciocalteu's 시약이페놀성화합물에의해

환원되어몰리브덴청색으로발현되는원리를이용하였다^. 초순수 ^{6.5 ml}에 각각의 시료 ^{0.5 ml}을 가한 후 ^Folin- Ciocalteu’s 용액 ^{0.5 ml}을혼합하여상온에서 ³분간정치시 켰다^. 다음무수탄산나트륨포화용액 ^{1 ml}을첨가하고전 체가 ^{10 ml}이되도록초순수 ^{4.4 ml}을혼합하여상온에서 ¹시 간방치시킨후 UV/visible spectrophotometer (GENESYS 10 UV, USA)로 ^{765 nm}에서흡광도를측정하였다^. 표준물

질은 gallic acid를사용하였으며동일한방법으로측정하여

얻은표준곡선으로부터총페놀화합물함량을정량하였다^. 총페놀화합물함량은 mg gallic acid equivalents/g 단위로 나타내었다^.

DPPH radical 소거능 측정

DPPH radical 소거능은 Blois [17]의방법을변형하여측 정하였다^. 시료 ^{0.5 ml}에 0.2 mM DPPH (2,2-diphenyl-2- picrylhydrazyl) 용액을 ^{0.5 ml} 가하고혼합하여 ³⁰분간방 치시킨후 UV/visible spectrophotometer로 ^{517 nm}에서흡 광도를측정하였다^. 대조구는시료대신용매를가하여동일 한방법으로측정하였으며시료자체의색에대한흡광도값 을보정해주기위해 0.2 mM DPPH 대신메탄올을넣어같 은방법으로흡광도를측정하였다. DPPH radical 소거능은 다음과같이계산하였다^.

DPPH radical scavenging effect (%) =

(1 − 시료첨가구의흡광도^/무첨가구의흡광도^{) × 100}

환원력 측정

환원력은 Oyaizu [18]의방법을변형하여측정하였다^. 시 료 ^{0.5 ml}에 0.2 M sodium phosphate buffer (pH 6.6) 2.5 ml를첨가한후 potassium ferricyanide 용액 ^{2.5 ml}를 가해충분히혼합하여 ⁵⁰℃에서 ²⁰분간반응시킨다^. 반응이 끝나면 10% trichloroacetic acid (TCA) 용액 ^{2.5 ml}를첨가 한다음 ^{1977 ×g}에서 ¹⁰분간원심분리하였다^. 상층액 ^{2 ml} 에초순수 2 ml과 0.1% iron(III) chloride 용액 0.4 ml를가

하여혼합한후초순수 ^{4.4 ml}를가하여 ^{700 nm}에서흡광

도를측정하였다^. 대조구는 ascorbic acid를사용하였으며^, 환원력은다음과같이계산하였다^.

Reducing power (Abs) =

시료첨가구의흡광도 − 공시험의흡광도 금속봉쇄력 측정

금속봉쇄력은 ^Shimada 등^[19]의방법에따라 측정하였 다^. 시료 ^{0.2 ml}에 초순수 ^{0.74 ml}를 혼합한 후^{, 2 mM} FeCl₂용액 ^{0.02 ml}와 ferrozine 용액 ^{0.04 ml}를첨가하여

실온에서 ²⁰분간방치한후 ^{562 nm}에서흡광도를측정하였 다^. 대조구는시료대신용매를가하여동일한방법으로측 정하였다^. 또한시료자체의색에대한흡광도값을보정해 주기위해시료대신에동량의증류수를가해흡광도를측 정하였다^.

Chelating effect (%) =

(1 −시료첨가구의흡광도^/무첨가구의흡광도^{) × 100}

유지 산화 안정도 측정

산화 안정도 실험은 rancimat (743 Metrohm Co.,

Switzerland)을이용하여 ^lard의산패유도기간을측정하였다^. Reaction vessel에 ^{lard oil}을 ^{3.0 g} 취하고최종농도가 ^{5, 1, 0.5}

mg/ml이 되도록 시료를 첨가한 다음 ^vortex하여 혼합한다^.

Rancimat 기계에 reaction vessel을주입한다음 ¹⁰⁰℃에서시

간당 ^{20 L}의여과된공기를주입하여산화시켰다^. 이때발생하

는휘발성산화생성물이 ^{65 ml}의초순수가들어있는 absorption

vessel로이행될때나타나는전기전도도의변화에따라자동적

으로산출된유도기간으로부터산화안정도를측정하였다^. 추출 물의항산화정도를측정하고동시에추출물을첨가하지않은

것을대조구로하여항산화 정도를 비교하여 antioxidant

index (AI)로나타내었다^{. AI}는각항산화제를첨가한실험구 의유도기간을대조구의유도기간으로나눈값으로^, 유지산 화안정도수치가큰것일수록항산화활성이높음을의미한 다^{[20, 21].}

통계 처리

본 실험결과에 대한 유의차 검정은 SAS program (Statistical analytical system V8.2, SAS Institute Inc.,

USA)을이용하여평균값을분산분석하였으며^{, Duncan}의

다중검정법을통해 p < 0.05 수준에서각처리구간유의성 을검정하였다^.

결과 및 고찰

총 페놀 화합물 함량

천연물에서 얻어지는페놀 화합물은 phenolic hydroxyl

(OH)기를가지기때문에단백질및기타거대분자들과쉽

게결합하여항산화및항암등의다양한생리활성을가지

는것으로알려져있다^[22]. 페놀화합물의항산화작용은수

산기를통한수소공여로라디칼들과쉽게공명으로안정화 될수있는구조를가지고있어서항산화활성을가지는것

으로보고되고있다^[23]. 따라서천연물의항산화연구중에

서페놀화합물의함량과항산화활성의연관성에대한연

구가활발히이루어지고있다^. 짝잎모자반에탄올및물추

출물의총페놀화합물함량을측정한결과(Table 1), 에탄

올추출물의경우 17.91 mg gallic acid equivalents/g이었으 며물추출물의경우 13.44 mg gallic acid equivalents/g으 로나타나에탄올추출물이물추출물보다더높은함량을 나타내었다^. 용매에따라추출되는페놀화합물의양이다른 것은용매의극성에따라추출되는물질이매우다르게나 타나기때문이다^{[11]. Lee} 등^[24]의연구에서와송의잎^, 줄 기및뿌리각각의총페놀화합물함량을측정한결과세처 리구모두에탄올추출물이물추출물보다페놀화합물함 량이더높았으며 ^Kim 등^[25]의연구에서해조류 ⁶종의페 놀화합물함량을측정한결과시험된 ⁶종모두에탄올추 출물이물추출물보다페놀화합물함량이더높아본연구 결과와일치하였다^.

DPPH radical 소거능

항산화물질은 free radical에전자나수소를공여하여복

합체를 만든다. DPPH radical 소거능 측정에 사용되는

DPPH는짙은자주색을나타내는질소중심의 ^radical로서

radical 전자의비편재화에의해비교적안정한상태로존

재한다^{[26]. DPPH}는다른 free radical들과결합하여안정 한복합체를만들어항산화활성이있는물질과만나게되

면 ^radical이소거되어탈색되는것을비색정량하여항산화

활성을측정한다^. 짝잎모자반추출물의 DPPH radical 소거

능을 측정한 결과(Table 2), 에탄올 추출물에서는 ¹ 및

0.5 mg/ml의농도에서 ^96% 이상의높은라디칼소거능을보

였으며 0.1, 0.05 및 0.005 mg/ml의농도에서각각 ^{62, 28} 및 ^5%의라디칼소거능을보여대조구인 ^BHT와유의적인 차이가없었다^. 또한물추출물에서는 ^{1, 0.5} 및 ^{0.1 mg/ml} 의농도에서각각 ^{92, 90} 및 ^44%로대조구인 ascorbic acid

와비교하여물추출물이 ^{0.1 mg/ml}의농도에서활성이현

저히감소하는것으로나타났다^. 이는꽈배기모자반 ^absolute

에탄올추출물의 ^{0.5 mg/ml}의농도에서 ^95%의라디칼소거

능을보인 ^Cho 등^[6]의보고와유사하였다^. 총페놀함량은

DPPH 라디칼소거능과높은상관관계가있으며폴리페놀

이식물의항산화성에서주요역할을하고있으며^[27], 추출 Table 1. Total phenolic contents (TPC) of Sargassum hemi- phyllum extracts.

TPC (mg/g of dry sample)

Ethanol 17.91 ± 0.27^a

Water 13.44 ± 0.08^a

aMeans with different superscripts in the same column are sig- nificantly different (p < 0.05).

물이함유하고있는페놀성화합물이항산화활성에주된역 할을하여총페놀화합물의함량이증가하면항산화활성

도증가하는것으로알려져있다^[28].

환원력

환원력의측정은시료에존재하는 reductones이수소원

자를제공함으로써 free radical 연쇄를변환시키며, reductones 는또한과산화의일정한전구물질과반응하여과산화의형 성을방해하는것으로항산화활성과직접적인연관이있는

것으로알려져있다^{[29, 30].} 시료중에항산화제와같이환

원력을 가진 성분이 존재하게 되면 ^Fe3+/ferricyanide

complex를 ^Fe2+ 상태로환원시키면서청색을띠게되는데환 원력에서의흡광도수치는그자체가시료의환원력을나타 내어높은환원력을가지는물질은흡광도수치가높게나

타난다^{[31, 32].} 짝잎모자반추출물의환원력을측정한결과

(Table 3), 에탄올추출물의경우 1, 0.5, 0.1 mg/ml의농도에 서각각 0.35, 0.20, 0.06의값을보였으며물추출물의경우 에탄올추출물과같은농도에서각각 0.20, 0.11, 0.04의값 을보여에탄올추출물이물추출물보다높은환원력을가 지는것으로나타났다^. 에탄올과물추출물모두대조구인 ascorbic acid보다는낮은환원력을보였다^. 이는 ^Kim 등^[11]

의연구에서패추출물의환원력을측정한결과총페놀화 합물함량이비교적높은발효주정추출물의환원력은높고 총페놀화합물함량이낮은물추출물은환원력이낮다고 보고되어본연구와유사한결과를나타내었다^.

금속봉쇄력

Fe, Cu, Co, Ni, Sn과같은금속이온인자는산화환원이 용이한금속이지만이들의금속염은지질산화과정에서촉 매로작용할수있는금속이다^. 철의축적으로인한과잉현 상은 철과 생체 내 존재하는 ^H2O₂와의 Fenton reaction (Fe²⁺+H2O2 → ^{Fe3++OH−H+OH·)}에의해세포노화및세포 손상을 야기하는강력한 hydroxy radical이나 superoxide

radical 등의생성을촉진하여세포내지질및단백질의산

화를촉진하고^, 식품의가공및저장중에는지방질의산화

를촉진한다^[33]. 짝잎모자반추출물의금속봉쇄력은 ^Table

4와같다^. 짝잎모자반에탄올추출물의경우 ^{1 mg/ml}의농 도에서 ^9%의낮은활성을나타내었으며물추출물의경우 1 및 ^{0.5 mg/ml}의농도에서각각 ^28% 및 ^19%의활성을나 타내어에탄올추출물보다물추출물의활성이더높게나 타났다^. 이에대조구인 ^EDTA의경우 ^{1, 0.5} 및 ^{0.1 mg/ml}의

농도에서모두 ^100%에가까운값을보여에탄올과물추출

물모두 ^EDTA보다낮은금속봉쇄력을보였다^. 이는 ^Lee 등 [24]의연구결과에서와송의에탄올추출물은 DPPH radical 소거능과환원력에서높은활성을나타내었고^, 물추출물은 금속봉쇄력에서더높은활성을보인것으로보고되었으며^, Lee 등^[34]의연구결과에서노랑느타리버섯균사체에탄올

추출물은 ^{5 mg/ml}의농도에서 ^60% 이상의항산화능을가지

나금속봉쇄력은 ^10% 미만으로나타나본연구와비슷한결 과를보였다^. 따라서항산화능의주된기작이금속봉쇄력에기 인한것이아닌것으로사료되었으며이들의결과와꽈배기모

자반추출물이유사한경향을보이는것으로나타났다^[35].

Table 2. DPPH radical scavenging effect of Sargassum hemiphyllum extracts.

(unit : %)

1 mg/ml 0.5 mg/ml 0.1 mg/ml 0.05 mg/ml 0.005 mg/ml

Ethanol 96.29 ± 0.06^a 96.34 ± 0.00 62.03 ± 0.31 28.21 ± 0.66 5.56 ± 1.49

Water 92.24 ± 0.13 90.63 ± 0.07 44.18 ± 0.33 =^b =

BHT 94.63 ± 0.15 94.40 ± 0.13 79.61 ± 0.71 56.06 ± 3.03 10.82 ± 0.57

Ascorbic acid 95.08 ± 0.19 95.17 ± 0.05 95.60 ± 0.11 95.73 ± 0.05 18.67 ± 1.10

aMeans with different superscripts in the same row and column are significantly different (p < 0.05).

bNot done.

Table 3. Chelating effect of Sargassum hemiphyllum extracts.

(unit : %)

1 mg/ml 0.5 mg/ml 0.1 mg/ml

Ethanol 009.03 ± 0.80^a -^b -

Water 028.02 ± 1.56 019.51 ± 0.37 - EDTA 100.04 ± 0.08 100.00 ± 0.00 99.65 ± 0.60

aMeans with different superscripts in the same row and column are significantly different (p < 0.05).

bLess than 5%.

Table 4. Reducing power of Sargassum hemiphyllum extracts.

(unit : Absorbance at 700 nm) 1 mg/ml 0.5 mg/ml 0.1 mg/ml Ethanol 0.35 ± 0.00^a 0.20 ± 0.00 0.06 ± 0.00 Water 0.20 ± 0.00 0.11 ± 0.00 0.04 ± 0.00 Ascorbic acid 1.92 ± 0.01 0.94 ± 0.02 0.19 ± 0.00

aMeans with different superscripts in the same row and column are significantly different (p < 0.05).

유지 산화 안정도

Rancimat에의한항산화지수는시료를첨가한후유지를

일정한온도로가열하면서공기를주입하면유지가산화되 어 aldehyde, ketone 등의저분자량휘발성산화생성물이발 생하기시작한다^. 이러한휘발성산화생성물들이증류수의 전기전도도를증가시키면이차이를측정하여유도기간을 산출함으로써일정조건에서의유지산패의정도를측정하거 나항산화제의효율을분석할수있는것이다^[24]. 짝잎모자

반추출물의유지산화안정도를알아보기위해 ^rancimat에

의한항산화도를알아본결과(Table 5), 에탄올추출물및물 추출물은 5, 1, 0.5 mg/ml의농도에서각각 1.52, 1.49, 0.80 및 0.32, 0.25, 0.22의유지산화억제능을보여농도가증가 함에따라유의적인차이가없는것으로나타났으며두추 출물사이에도유의적인차이가거의없는것으로나타났다^. 대조구인 ^BHT와비교해보았을때 ^BHT의유지산화억제 능은 12.51, 8.17, 6.17로에탄올및물추출물보다활성이높 은것으로나타났다^. 이는 ^Kim 등^[36]의연구에서패 ^70% 발

효주정추출물을감마선조사한후 ^rancimat에의한항산화

도를측정한결과 ^{5 mg/ml}의농도에서 ^1.07의유지산화억 제능을보여짝잎모자반에탄올및물추출물의결과와큰 차이를보이지않았다^. 항산화물질과그상태에따라유도기 간이다르게나타나는것은각각의항산화물질은과산화물 의생성을제어할수도있고항산화물질내부에함유한금 속성분등에의해서과산화물의생성을촉진할수도있기 때문이다^[37].

요 약

본연구는짝잎모자반(S. hemiphyllum)을 ^95% 에탄올과 물로각각추출하여항산화활성을조사하였다^. 짝잎모자반 추출물의총페놀화합물함량을측정한결과^, 에탄올추출 물과물추출물각각 17.91 mg/g과 13.44 mg/g으로에탄올 추출물이물추출물보다더높은페놀화합물함량을나타내

었다. DPPH radical 소거능측정결과 1, 0.5, 0.1 mg/ml의 농도에서에탄올및물추출물은각각 96, 96, 62% 및 ^92, 90, 44%로에탄올추출물이더높은 ^radical 소거능을보였 으며^, 에탄올추출물은천연항산화제인 ^BHT와유사한소 거능을보였다^. 환원력의경우에탄올과물추출물모두대

조구인 ascorbic acid에비하여낮은값을보였으며에탄올

및물추출물간에는유의적인차이가나타나지않았다^. 금 속봉쇄력의경우 ^{1 mg/ml}의농도에서에탄올추출물은 ^9%

의활성을나타냈으며물추출물은 ^28%의활성을보여에탄 올추출물보다더높은활성을나타내었다^{. Rancimat}에의 한유지산화억제능의경우에탄올추출물이물추출물보 다더높은활성을보였지만대조구인 ^BHP에비해낮은항 산화력을나타내었다^. 짝잎모자반추출물은뛰어난 ^DPPH

radical 소거능으로높은항산화활성을가지며특히물추

출물보다에탄올추출물에서더높은활성을가지는것을알 수있었다. 따라서본연구결과, 짝잎모자반은천연항산화 소재로서의활용가능성이내재되어있다고사료된다^.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NO. 2012R1A6A1028677).

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