J Korean Soc Food Sci Nutr 한국식품영양과학회지 43(2), 224~230(2014) http://dx.doi.org/10.3746/jkfn.2014.43.2.224 제주연안갈조류추출물의신경세포보호효과 신동범 한은혜 박성수 제주대학교식품영양학과 Cytoprotective Effects of Phaeophyta Extracts from the Coast of Jeju Island in HT-22 Mouse Neuronal Cells Dong-Bum Shin, Eun-Hye Han, and Sung-Soo Park Dept. of Food Science and Nutrition, Jeju National University, Jeju 690-756, Korea ABSTRACT Marine algae have long been recognized as a health and beauty food, based on its anti-tumor, anti-inflammatory and anti-obesity activities. In this study, methanol extracts were prepared from 10 different phaeophyta, after which DPPH radical scavenging and cytoprotective activities of HT-22 cells against β-amyloid protein (Aβ), which has neurotoxic effects, were investigated. In DPPH experiments, Ecklonia cava and Ishige okamurai showed strong ROS scavenging activities, whereas eight other phaeophyta including Petalonia binghamiae (P. bin) showed weak ROS scavenging activities. To validate the cytoprotective effects of 10 different phaeophyta in Aβ-induced HT-22 cells, protein expression levels of APP, BACE1, inos, phosphorylated ERK1/2, phosphorylated p38 and phosphorylated JNK1/2 were determined along with MTT assay. In the MTT assay, P. bin showed the best effective cytoprotective activity at a concentrations of 25 μg/ml, whereas Sargassum confusum, Colpomenia sinuosa, Myelophycus simplex, and Sargassum hemiphyllum showed potential. Determination of protein expression levels related to Aβ-induced neurotoxicity in the five selected phaeophyta showed that P. bin inhibited BACE1 and inos expression in Aβ-induced HT-22 cells. These results indicate that the cytoprotective effects of P. bin are mediated by suppressing the pathways involving Aβ-induced ERK and p38 activation. Key words: edible phaeophyta, cytoprotection, β-amyloid protein (Aβ), mitogen-activated protein kinase (MAPK), Petalonia binghamiae 서 제주도연안에서자생하는해조류는 520여종이넘는것으로보고되었고, 이는한반도전체의약 70% 를차지하는수준이다 (1). 제주도연안은우리나라에서가장수온이높고다른지역에서볼수없는아열대성의남방계해조류를많이볼수있으며, 특히톳과모자반을이용한전통음식이많이전해내려오고있다 (2). 식용으로쓰이는해조류는소화흡수율이낮고영양가가낮아서예전에는영양학적측면에서관심을끌지못하였으나, 무기질, 비타민및식이섬유소가풍부하고최근다양한생리활성물질들을가지고있음이보고되고있어해조류에서기능성생리활성물질탐색에대한관심이지속적으로높아지고있다 (3). 특히갈조류는항암과항염증생리활성을갖는 fucoidan과 laminarin이라는각각의성분이알려져있다 (3,4). 그리고제주도연안에서서식하는갈조류를이용한생리활성물질을탐색하기위한활발한연구가이루어지고 론 Received 8 October 2013; Accepted 21 October 2013 Corresponding author. E-mail: foodpark@jejunu.ac.kr, Phone: +82-64-754-3552 있는데고리매추출물이인간혈액암세포주인 HL-60 세포의성장을억제하는효과가나타났고 (5), 미역쇠추출물은생쥐의 3T3-L1 세포와 B16/F10 세포에서각각항비만및미백활성이밝혀져미용기능성식품으로의가능성도제기되고있다 (6,7). 또한갈조류의효소적가수분해방법을이용한추출물에관한연구에서는과산화수소로부터의활성산소소거능이 90% 로높게나타났고 50% 의 DNA 손상을보호하는효과를나타냈으며 (8), 감태의분획물에서는암세포의증식억제효과가있었고항산화효과도합성항산화제인 BHA, BHT보다매우높게나타났다 (9). 비틀대모자반으로부터얻은 farnesylacetone 유도체는아세틸콜린의작용을저하시키는 cholinesterase의두가지형태인 acetylcholinesterase와 butyrylcholinesterase를억제시켜치매예방효과가기대되고있다 (10). 일반적으로치매에는알츠하이머형치매와혈관성치매가대표적인데, 혈관성치매는뇌출혈, 뇌졸중등뇌혈관에장애가생겨발생하는치매를말하며알츠하이머형치매는뇌신경세포의퇴행으로인하여발생하는치매로노인성치매환자의 60~70% 를차지하며나이가들수록이환율이높은것으로알려져있다 (11).
제주연안갈조류추출물의신경세포보호효과 225 알츠하이머병의병리학적특징으로는신경세포외부에축적되는노인반점 (senile plaques) 과신경세포의세포체내에엉켜진신경섬유덩어리 (neurofibrilary tangles) 를들수있다. 이중노인반점의생성은아밀로이드전구체단백질 (amyloid precursor protein; APP) 의용해과정에서유래된아밀로이드베타단백질 (β-amyloid protein; Aβ) 의축적에의하며축적된 Aβ가신경세포에독성으로작용하여신경세포에서염증반응이촉진되고결국신경세포의사멸을일으키게된다. 따라서알츠하이머병의주요병인은 Aβ 로생각되어지고있다 (12-14). 알츠하이머병의병인인 Aβ는다양한경로를통하여신경세포의사멸을일으키는것으로알려져있는데 Aβ의축적은신경세포에독성작용을나타내며이로인해세포에염증반응을일으키고, 염증반응시세포내에증가하는것으로알려진 inos의발현이증가함에따라활성산소의일종인일산화질소 (nitric oxide; NO) 가다량생성되고 NO의자극에의해세포신호전달체제인 mitogen activated protein (MAP) kinase pathway가작동되는것으로알려져있다 (15,16). Aβ는 APP의대사과정에서생성되는일부분으로서 39~43개내외의아미노산으로이뤄진펩타이드이다 (17). 아세틸콜린의저하가알츠하이머의발병원인이라고생각해왔던아세틸콜린가설이후에등장한아밀로이드베타가설은 Aβ가알츠하이머의발병원인으로작용할것이라는실험적증거들에의하여가장오래도록일반적으로받아들여지고있다 (18). Aβ의전구단백질 APP는 770개의아미노산으로구성되어진펩타이드로 amyloidogenic pathway와 non-amyloidogenic pathway의두가지로그대사과정이진행된다. Amyloidogenic pathway는 Aβ가형성되는경로로 β- secretase와 γ-secretase라는단백질분해효소에의하여 APP가대사되는데먼저 β-secretase에의해 APP가 sapp β와 C-terminal fragment-β(ctfβ) 로분해되고 CTFβ가다시 γ-secretase에의해 Aβ와 CTFγ로분해되는경로를거친다. Amyloidogenic pathway의대사산물인 Aβ가신경세포주변에축적되어신경세포독성을일으켜신경세포의손상과염증반응을촉진시키고결국신경세포의사멸을가져와알츠하이머병을일으킨다. Non-amyloidogenic pathway는 α-secretase라고알려진단백질분해효소가활성화되어 APP를분해하는것으로 α-secretase는 Aβ의 16~17 번째부분에작용하여 sappα와 CTFα가생성되며, CTFα 는다시 γ-secretase 작용에의해 short peptide(p3) 와 CTFγ로대사된다. Non-amyloidogenic pathway는 Aβ의일부분이잘려나가게되어 Aβ의축적이방해되며대사산물인 sappα 는세포성장및보호작용을보인다 (12,13,19-21). Aβ의형성에관여하는효소인 β-secretase는 aspartic protease의일종으로 BACE1과 BACE2가동정되어지고있으며 BACE1(β-site APP cleaving enzyme 1) 은이자, 뇌등에서발현이되며, 세포내의소포체와골지체와같은 APP가발현되는곳과같은위치에서발현된다. 뇌에서는 hippocampus, cortex, cerebellum에서그발현량이높다 (20-24). MAP kinase는크게 ERK(the extracellular signalactivated kinases), JNK(the c-jun N-terminal kinase), p38 등이있다. ERK(ERK1/2) 는성장호르몬의신호전달에주로관여하며세포의증식및분화에중추적인역할을담당한다. 반면에 stress activated protein kinase로분류되는 p38과 JNK는이름그대로세포외부의스트레스성자극에의해활성화되며염증반응, 세포사멸등에연관되어있다 (25-27). 이들 MAP kinase와뇌신경세포사멸과의연관성에대해최근연구가활발히진행중인데알츠하이머병과다운증후군환자의뇌에서 MAP kinase의활성이증가되었고 (28) Aβ가자극원이되어 p38과 ERK를활성화시키면서염증반응을유도하여신경세포의사멸이일어나게된다는보고가있다 (29). 이와같은결과들로보아알츠하이머발병의주요원인인 Aβ 생성에따르는세포내스트레스반응에 MAP kinase pathway가연관되어있음을알수있다. 많은연구들에서알츠하이머병의치료제로 Aβ의축적을억제하는물질이나 Aβ를생성시키는 APP 분해효소인 BACE1의저해제, 항산화제, 항염증제개발이진행되고있다 (30-33). 그러므로본연구에서는제주연안에서자생하는해조류중갈조류 10종의메탄올추출물을이용하여활성산소소거능과아밀로이드베타단백질 (Aβ) 이라는신경독성유발물질에대한 HT-22 신경세포보호효과를관찰해보았다. 재료및방법갈조류추출물제조본실험에서는제주도연안에서식하고있는갈조류 10종의추출물을제주테크노파크산하제주생물자원지원센터제주유용생물자원추출물은행으로부터채집해보관해놓았던것을분양받아사용하였다 (Table 1). 분양받은추출물은 80% 메탄올을용매로추출하여감압농축된것으로농도는 1 mg/ml가되게 dimethylsulfoxide(dmso, JT Baker, Phillipsburg, NJ, USA) 에녹여서실험에사용하였다. 세포배양본연구에서는흰쥐의 HT-22(hippocampal neuron cell line) 세포를사용하였다. HT-22 세포는 10% fetal bovine serum(fbs), 100 unit/ml penicillin-streptomycin이함유된 Dulbecco's modification of Eagle's medium(dmem, Gibco-BRL, Grand Island, NY, USA) 을사용하여 37 C, 5% CO 2 조건으로배양기 (MCO-17AI, SANYO Electric Biomedical Co., Ltd, Osaka, Japan) 에서배양하였다. 계대배양은 trypsin-edta(sigma-aldrich Co., St. Louis, MO, USA) 를사용하여 2~3일에한번씩시행하였다.
226 신동범 한은혜 박성수 Table 1. List of 10 phaeophyta Binomial name Korean name Sampling date Location Sargassum confusum (S. con) Colpomenia sinuosa (C. sin) Myelophycus simplex (M. sim) Petalonia binghamiae (P. bin) Ishige sinicola (I. sin) Dictyota dichotoma (D. dic) Dictyota coriacea (D. cor) Sargassum hemiphyllum (S. hem) Ishige okamurai (I. oka) Ecklonia cava (E. cav) 알쏭이모자반불레기말바위수염미역쇠넓패참그물바탕말참가죽그물바탕말짝잎모자반패감태 2005. 03. 09 2005. 03. 11 2005. 04. 07 2005.04.07 2005. 04. 22 2005. 04. 26 2005. 04. 26 2005. 05. 23 2005. 07. 19 2005. 10. 14 Oedo Sinchon Bukchon Bukchon Samyang Sasudong Sasudong Sindo Gimnyeong Wimi 전자공여능 (DPPH) 측정전자공여능의측정은 Blois의방법 (34) 을변형하여사용하였다. 0.2 mm 1,1-diphenyl-2-picrylhydrazyl(DPPH) 을 180 μl, 1 mg/ml 농도의갈조류추출물 20 μl를섞어총량이 200 μl가되게 96 well plate에분주하고암실에서 30분반응후 VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA) 를사용하여 517 nm에서흡광도를측정하였다. 대조군으로는널리알려져있는합성항산화제인 butyl hydroxyanisole(bha) 을사용하였으며전자공여능은다음과같은방법으로계산하였다. 전자공여능 (%)=(1- 시료의흡광도 ) 100 Blank 의흡광도 세포생장률측정 갈조류추출물이아밀로이드베타에의한독성으로부터신경세포의생존및성장에미치는영향을관찰하고자 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(mtt) 분석법을수행하였다. MTT 분석법은세포의생장률을측정하는방법으로서살아있는세포의미토콘드리아내의 dehydrogenase가황색수용성물질인 MTT (Gibco-BRL) 에의해 dark blue formazan을생성하는원리로이환원되는정도를흡광도로측정함으로써알아보는방법이다 (35). HT-22 세포를 96 well plate에세포수가 5 10 4 /well이되게분주하여 24시간배양후배양액을제거하고 fetal bovine serum이포함되지않은배양액을분주한후 200 µm 아밀로이드베타단백질 (Aβ 1-42) 1 μl와갈조류추출물 25, 50 μg/ml를처리하여 37 C, 5% CO 2 조건으로배양기에서배양하였다. 48시간후 5 mg/ml 농도의 MTT reagent 10 μl를분주하여 4시간배양하였고상층액을제거한후 100 μl의 DMSO를첨가하여 5분정도 incubation 시켜생성된 blue formazan을완전히녹여준후 VersaMax tunable microplate reader로 540 nm에서흡광도를측정하였다. Aβ 1-42 와갈조류추출물처리군과 Aβ 1-42 처리군의세포생존율은 Aβ 1-42 와갈조류추출물을처리하지않은 HT-22 세포의세포생존율을 100% 로정하고 이에대해상대적인세포생존율로구하였다. 단백질발현양상분석아밀로이드베타단백질 (Aβ) 의신경독성에대한신경세포보호효과를가진갈조류추출물이신경세포사멸경로중작용되는부분을알아보고자 Western blot을통해 APP, BACE1, inos, MAP kinase의단백질발현양상을살펴보았다. HT-22 세포 (5 10 5 ) 에 200 µm 아밀로이드베타단백질 (Aβ 1-42) 10 μl와 25 μg/ml의갈조류추출물을처리하여 37 C, 5% CO 2 배양기에서배양하였다. 48시간후배양한세포를수확하여 PBS(phosphate buffered saline) 로 2 회세척후, 500 μl의 lysis buffer를첨가하여초음파분쇄기로분쇄한후 10,000 rpm에서 15분간원심분리하여그상층액을취한후단백질농도는 2D quant kit(amersham Biosciences, Piscataway, NJ, USA) 를이용하여정량하였다. 30 μg의 lysate를 12% sodium dodecyl sulfate polyacrylamaide gel에서전기영동을이용하여단백질을분리하였다 (36). 분리된단백질을 PVDF(polyvinylidene fluoride) membrane(millipore, Bedford, MA, USA) 에 200 ma로 2시간동안 transfer 한후 5% skim milk가함유된 TTBS(Tris buffered saline containing 0.1% Tween 20) 에넣어상온에서 1시간정도 blocking 시킨후 TTBS로 10분간 3회세척하였다. APP(1:5,000, Sigma-Aldrich Co.), BACE1(1:2,000, Sigma-Aldrich Co.), inos(1: 10,000, Abcam, Inc., Cambridge, MA, USA), ERK1/2(1: 1,000, Sigma-Aldrich Co.), phospho ERK1/2(1:1,000, Sigma-Aldrich Co.), p38(1:1,000, Sigma-Aldrich Co.), phospho p38(1:1,000, Sigma-Aldrich Co.), JNK(1:1,000, Sigma-Aldrich Co.), phospho JNK(1:1,000, Sigma- Aldrich Co.) 로희석한 1차항체를처리하여상온에서 1시간반응시킨후 TTBS로 10분간 3회세척한다음 2차항체로는 HRP(horseradish peroxidase) 가결합된 anti-rabbit IgG(Bio-Rad, Hercules, CA, USA) 와 anti-mouse IgG (Bio-Rad) 를 1:5,000으로희석하여상온에서 1시간반응시켰다. 그후 membrane을 TTBS로 10분간 3회세척한다음 ECL(enhanced chemiluminescence, Amersham
제주연안갈조류추출물의신경세포보호효과 227 Biosciences) 기질과 5~10분간반응후 X-ray 필름 (Eastman Kodak, Rochester, NY, USA) 에감광하였다. 통계처리모든실험은 3회반복으로이루어졌으며, 그결과는 SPSS software(ver. 12.0K, SPSS Inc., Chicago, IL, USA) 를사용하여처리하였다. 각측정군의평균과표준편차를산출하고분산분석후유의차가있는경우 Duncan의다중범위검정으로 P<0.05 수준에서유의성을검정하였다. 결과및고찰제주연안갈조류추출물이아밀로이드베타단백질의신경독성에대한신경세포보호효과를탐색하기위해항산화활성 (DPPH 라디칼소거능 ), 세포생존에미치는영향 (MTT assay), 신경독성에관여하는 HT-22 세포내단백질의발현정도를조사하였다. 전자공여능측정분석본연구에서는갈조류 10종의메탄올추출물과일반적으로잘알려진합성항산화제인 BHA를대조군으로하여 DPPH 라디칼소거능을측정해보았다 (Fig. 1). 그결과대조군은 83.68% 의 DPPH 라디칼소거능을보였고갈조류중가장높은활성을보인것은감태 (Ecklonia cava) 로 78.24% 로대조군과비슷한결과를나타내었다. 그리고갈조류중패 (Ishige okamurai) 는 59.69% 의소거능을나타내서감태와더불어천연항산화제로서가능성을나타내었으며나머지갈조류는 50% 미만의라디칼소거능을나타내서라디칼소거능이외의생리활성능력을탐색해보는것이바람직할것으로보였다. 갈조류추출물은많은연구에서항산화활성의우수성을나타내었는데특히 fucan과같은황화다당은 polyphenol 화합물과더불어항산화활성을갖는갈조류의주요성분으로알려져있다 (37). 톳 (Hizikia fusiformis) 자숙액에탄올추출물을이용한 DPPH 라디칼소거능에서는 1,000 μg/ml Electron Donating Activity (% of control) 100 80 60 40 20 0 Con BHA S. con C. sin M. sim P. bin I. I. sin D. sin dic D. cor S. hem I. oka E. cav Fig. 1. Electron donating activity of methanol extracts from 10 phaeophyta in the concentrations of 1 mg/ml. All data were obtained in triplicate and are presented as means±sd. Significant differences between treatments with the Student's t-test in an one-way analysis of variance. BHA: positive control group. P< 0.05 compared to control. 일때 85%, 100 μg/ml일때 77% 의소거능을나타내어갈조류가천연항산화제로서의개발가능성이있음을밝혔다 (38). 또한갈조류인셀만모자반 (Sargassum kjellmanianum) 으로부터분리한 phlorotannins 성분이우수한지질과산화억제효과를보인다고밝혀졌는데 (39) 최근노화를비롯한여러가지성인병의원인이 free radical의산화에의한것임이밝혀지고있어 phlorotannins가풍부한갈조류의섭취는노화를비롯한만성성인병예방에큰도움이될것이다 (40). 세포생장률측정을통한세포보호효과분석아밀로이드베타단백질의독성에의한신경세포생존율을측정함으로써갈조류의 HT-22 세포보호효과를살펴보았다 (Table 2). 배양한세포에아밀로이드베타단백질 (Aβ 1-42) 을처리하고갈조류추출물을 25 μg/ml 처리하였을때 Aβ 1-42 만을처리한군에서는 85.88% 의신경세포생존율을보였으며미역쇠 (Petalonia binghamiae) 에서는 120.34%, 바위수염 (Myelophycus simplex) 은 116.49%, 불레기말 (Colpomenia sinuosa) 은 108.47%, 짝잎모자반 (Sargas- Table 2. Cytoprotective effects of methanol extract of phaeophyta in Aβ-treated HT-22 neuronal cell line Phaeophyta name Amyloid-beta (con) Sargassum confusum (S. con) Colpomenia sinuosa (C. sin) Myelophycus simplex (M. sim) Petalonia binghamiae (P. bin) Ishige sinicola (I. sin) Dictyota dichotoma (D. dic) Dictyota coriacea (D. cor) Sargassum hemiphyllum (S. hem) Ishige okamurai (I. oka) Ecklonia cava (E. cav) Mean±SD. The mean difference is significant at P=0.05. Cell viability (% of control) 25 μg/ml 50 μg/ml 85.88±14.31 98.12±7.08 108.47±6.93 116.49±9.13 120.34±10.49 33.97±7.79 75.51±5.6 52.51±4.88 102.17±9.55 45.00±2.7 93.39±9.88 82.81±4.03 114.54±13.54 105.77±7.21 63.19±4.23 113.02±13.69 13.26±1.56 32.40±7.61 22.21±4.56 102.53±7.47 12.73±1.15 54.02±12.65
228 신동범 한은혜 박성수 sum hemiphyllum) 102.17% 로 Aβ 1-42 만을처리한군에비해유의적으로높은신경세포생존율을보였다. 알쏭이모자반 (Sargassum confusum) 과감태는각각 98.12%, 93.39% 로 Aβ 1-42 만을처리한군에비하여높은신경세포생존율을보였으나유의적이지는않았다. 그외갈조류에서는생존율이 80% 미만으로세포보호효과를나타내지못하였다. 갈조류추출물을 50 μg/ml 처리하였을때 Aβ 1-42 만을처리한군에서는 82.81% 의신경세포생존율을보였고알쏭이모자반은 114.54%, 미역쇠는 113.02%, 불레기말은 105.77 %, 짝잎모자반은 102.53% 의생존율을보여 Aβ 1-42 만을처리한군에비해유의적으로높은신경세포생존율을보였다. 나머지 6종의갈조류에서는 70% 미만의세포생존율을나타내 Aβ의독성에대한세포보호효과를나타내지못하였다. 선행되었던연구중원추리추출물을이용하여 Aβ에의하여유발된 PC12 세포에서의독성을억제하는효능을연구한결과에서는애기원추리, 죽대및각시원추리추출물은 200 μg/ml까지의농도에서생존율이 100~120% 를유지함을확인하였다 (41). 해조류추출물을대상으로 bromobenzene으로간독성을유발한흰쥐의 in vitro 실험에서는톳, 꽈베기모자반 (Sargassum siliquastrum), 지충이 (Sargassum thunbergii) 추출물이과산화지질생성을억제한다는연구결과가있었으며 (42) 감태에서유래된 eckol이산화적스트레스에의한 U79-4 세포손상을억제하는것으로나타났다 (43). 세포생장률측정분석결과알쏭이모자반, 불레기말, 바위수염, 미역쇠, 짝잎모자반이 Aβ의신경독성에대해서세포를효과적으로보호하는것으로보였고대조군에비해높은신경세포생존율을나타내고있어신경세포생존율의증가뿐만아니라신경세포의증식에도영향을미친다는것을알수있었다. 신경세포보호효과에연관된단백질발현양상분석세포생장률측정분석을통해아밀로이드베타단백질 (A β) 의신경독성에대한신경세포생존률이유의적으로높았던알쏭이모자반, 불레기말, 바위수염, 미역쇠, 짝잎모자반추출물을이용하여신경세포사멸과관련된단백질발현양상을살펴보았다. 알츠하이머병의병인으로알려진 Aβ의전구단백질이 APP와 APP로부터아밀로이드베타를생성시키는단백질분해효소인 BACE1(β-site APP cleaving enzyme 1) 의단백질발현양상을살펴본결과, APP는대조군에비해알쏭이모자반과, 불레기말, 바위수염을처리하였을때높거나비슷한발현양상을나타냈고, 미역쇠와짝잎모자반을처리하였을때는대조군보다낮은발현양상을나타내었다. BACE1 의경우에는다섯가지갈조류모두에서대조군에비해낮은발현양상을나타내었고바위수염에서가장낮은발현양상을나타내었다. 따라서미역쇠추출물이아밀로이드베타 (A) Con (B) S. con C. sin M. sim P. bin S. hem APP BACE1 inos Con S. con C. sin M. sim P. bin S. hem Fig. 2. Cytoprotective effects of selected 5 phaeophyta on APP and BACE1 protein expression associated with Aβ accumulation (A) and inos protein expression associated with NO generation (B) in Aβ-induced HT-22 neuron cell line. 전구체물질의감소효과와더불어신경독성물질인 Aβ 생성기전중단백질분해효소인 BACE1의활성을저해함으로써 Aβ 생성을감소시키는효과를보였다 (Fig. 2A). 녹차에서분리된 catechin계화합물이 BACE1을저해한다는보고가있었다 (44). 세포에서염증반응이일어날때발현되어세포의사멸에관여하는 inos는대조군에비해갈조류추출물을처리한군에서는낮은발현양상을나타내었다 (Fig. 2B). 이결과로보아갈조류추출물이세포손상과염증을유도시키는 NO의생성을유발하는 inos 단백질의발현을저해하는효과가밝혀졌다. 청각 (Codium fragile) 추출물의항염증효과를살펴본연구에서도 RAW264.7 세포에치주질환균에서유도된 LPS와청각추출물을처리한결과 NO 활성을현저히감소시켜높은항염증효과를나타내었으며 inos 활성도저해시킨다는보고도있다 (45). 신경세포의사멸과관련된 MAP kinase의활성화된상태의발현양상을살펴보았다 (Fig. 3). PC12세포에서 Aβ의처리로 ERK와 p38을통해 inos의발현이증가한다는연구결과가있었고 (16) MAP kinases pathway가알츠하이머병의병인인 Aβ와밀접한관련이있다는많은보고가있었다 (28,29). 본연구에서는 Aβ의신경독성으로인해신경세포에서활성화된 MAP kinase의저해양상을분석하였다. 그결과 HT-22 세포에서 ERK와 p38의 Aβ에의한활성화를저해한해조류는미역쇠와바위수염이었으며, 이중미역쇠는식용으로쓰이는갈조류로알려져있다 (Fig. 3). 실험동물의 ischemia model에서 ERK 활성화정도는 ischemia가일어난중심부위 (ischemic core) 에서는감소하지만주변부위 (penumbra) 에서는활성화됨이알려져있다. 다른실험결과에서는 ischemic core에서도 ERK 활성화가증가함이보고된바있다. 또한 p38이신경세포의사멸에관여함이알려진것은 in vitro 실험에서신경성장호르몬인 NGF 결핍이나흥분상태시에유발된신경세포사멸에 p38 활성화가관련되어있으며, 최근뇌졸중실험동물모델에서의연구결과를
제주연안갈조류추출물의신경세포보호효과 229 P-ERK1/2 ERK1/2 P-JNK 1/2 JNK 1/2 P-p38 p38 Fig. 3. Cytoprotective effects of selected 5 phaeophyta on P- ERK1/2 and P-p38 MAPK protein expression associated with NO generation and P-JNK SAPK protein expression in Aβ-induced HT-22 neuron cell line. P represents phosphorylated form of MAPK. 보면 p38이뇌의 microglia에서활성화되며 p38 저해제들이우수한신경세포보호작용을나타낸다는보고가있다 (46-48). 본연구결과들을종합해볼때, 미역쇠추출물은 DPPH 소거를통한항산화활성은낮지만 Aβ의축적을야기하는 BACE1 단백질의발현을저해함과동시에 Aβ의신경독성으로인한 MAP kinase 활성화중 inos 활성화와연관된 ERK와 p38의활성화를저해함으로써 HT-22 신경세포에염증반응이감소되고결국신경세포사멸이줄어들게된다. 그러므로미역쇠추출물의 Aβ의축적감소및 Aβ의신경독성에대한신경세포보호효과가밝혀짐에따라식용갈조류인미역쇠를활용하여알츠하이머형치매예방에도움이되는건강기능성식품소재의개발이가능하다고사료된다. 요 항암, 항염증및비만억제등의생리활성을지닌해조류는최근건강기능식품, 기능성화장품그리고의약품산업분야에서미용과건강식품소재로각광받고있다. 본연구에서는 10종의갈조류메탄올추출물을이용하여 1,1-diphenyl- 2-picrylhydrazyl(DPPH) 라디칼소거능과아밀로이드베타단백질 (Aβ) 의신경독성에대한 HT-22 신경세포보호효과를조사함으로써천연물로부터안전하고새로운신경세포보호소재를개발해내고자한다. DPPH 라디칼소거능의경우미역쇠를포함한 8종의갈조류에서는비교적낮은활성산소소거능을보인반면, 감태와패에서강력한활성산소소거능이나왔다. Aβ의신경독성에대해 10종의갈조류추출물이갖는 HT-22 신경세포보호효과를검증하기위해 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(mtt) 분석과 APP, BACE1, inos 단백질의발현양상및 ERK1/2, p38, JNK1/2 단백질의활성화양상을분석했다. MTT 분석결과, Aβ의신경독성으로부터미역쇠가 25 μg/ml의농도에서가장효과적으로세포를 약 보호하고있는것으로나타났고, 알쏭이모자반, 불레기말, 바위수염, 짝잎모자반도세포보호효과가있는것으로나타났다. 세포보호효과가있는것으로밝혀진 5종의갈조류를가지고수행한단백질발현분석결과, 미역쇠는 Aβ의신경독성에의해 HT-22 신경세포에서발현되는단백질인 BACE1과 inos의발현을저해하였다. 이는미역쇠의세포보호효과가 Aβ의신경독성으로부터일어난 ERK와 p38의활성화에연관된세포신호전달경로를억제하는것으로보인다. 그러므로특히식용갈조류인미역쇠는 Aβ에의해유도된신경독성에대해서신경세포보호효과를갖는건강기능식품소재로서의가치가충분한것으로사료된다. 감사의글 이논문은 2013학년도제주대학교학술진흥연구비지원사업에의하여연구되었으며이에감사드립니다. REFERENCES 1. Lee YP, Kang SY. 2001. A catalogue of the seaweeds in Korea. Jeju National University Press, Jeju, Korea. p 477. 2. Cha SH, Ahn GN, Heo SJ, Kim KN, Lee KW, Song CB, Cho SK, Jeon YJ. 2006. Screening of extracts from marine green and brown algae in Jeju for potential marine angiotensin-1 converting enzyme (ACE) inhibitory activity. J Korean Soc Food Sci Nutr 35: 307-314. 3. Noda H, Amano H, Arshima K, Hashimoto S, Nisizawa W. 1989. Studies on the antitumor activity of marine algae. Bull Japan Soc Sci Fish 55: 1259-1264. 4. Schwartsmann G, Brondani da Rocha A, Berlinck RG, Jimeno J. 2001. Marine organisms as a source of new anticancer agents. Lancet Oncol 2: 221-225. 5. Kim SC, Park SY, Hyoun JH, Cho H, Kang JH, Lee YK, Park DB, Yoo ES, Kang HK. 2004. The cytotoxicity of Scytosiphon lomentaria against HL-60 promyelocytic leukemia cells. Cancer Biother Radiopharm 19: 641-648. 6. Kang SI, Kim MH, Shin HS, Kim HM, Hong YS, Park JG, Ko HC, Lee NH, Chung WS, Kim SJ. 2010. A water-soluble extract of Petalonia binghamiae inhibits the expression of adipogenic regulators in 3T3-L1 preadipocytes and reduces adiposity and weight gain in rats fed a high-fat diet. J Nutr Biochem 21: 1251-1257. 7. Yoon HS, Koh WB, Oh YS, Kim IJ. 2009. The anti-melanogenic effects of Petalonia binghamiae extracts in α-melanocyte stimulating hormone-induced B16F10 murine melanoma cells. J Korean Soc Appl Biol Chem 52: 564-567. 8. Heo SJ, Park EJ, Lee KW, Jeon YJ. 2005. Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresour Technol 96: 1613-1623. 9. Athukorala Y, Kim KN, Jeon YJ. 2006. Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava. Food Chem Toxicol 44: 1065-1074. 10. Ryu G, Park SH, Kim ES, Choi BW, Ryu SY, Lee BH. 2003. Cholinesterase inhibitory activity of two farnesylacetone derivatives from the brown alga Sargassum sagamianum. Arch Pharm Res 26: 796-799. 11. Panayi AE, Spyrou NM, Iversen BS, White MA, Part P.
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