01 (10-11-OK).hwp

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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 44(2), 165~172(2015) 천연식물로부터추출한색소성분의항산화및항비만활성 황초롱 1 강민정 1 심혜진 1 서화진 2 권오운 2 신정혜 1 1 ( 재 ) 남해마늘연구소 2 ( 재 ) 경북천연염색산업연구원 Antioxidant and Antiobesity Activities of Various Color Resources Extracted from Natural Plants Cho-Rong Hwang 1, Min Jung Kang 1, Hye Jin Shim 1, Hwa Jin Suh 2, Oh Oun Kwon 2, and Jung-Hye Shin 1 1 Namhae Garlic Research Institute 2 Gyeongbuk Natural Color Institute ABSTRACT The objective of this study was to investigate the antioxidant and antiobesity activities of various color resources extracted from natural plants such as, clove, persimmon, gall nut, amur cork, gardenia, safflower, and annatto. Total phenolic content was the highest in gall nut extract (2, mg/kg) followed by clove extract (1, mg/kg). DPPH, and ABTS radical scavenging activities and ferric reducing antioxidant power (FRAP) were also higher in gall nut extract. α-glucosidase inhibitory activity (IC 50) was highest in persimmon extract (22.83 μg/ml) followed by gall nut extract. α-amylase and lipase inhibitory activities were also higher in persimmon extract (49.45% and 61.01%, respectively). Lipid accumulation in 3T3-L1 cells was lower in persimmon, clove, and annatto extracts (81.54%, 83.36%, and 85.70% at 20 μg/ml, respectively). Triglyceride content in 3T3-L1 cells was lowest in clove extract (66.11%) followed by persimmon extract (88.88%). The results of this study suggest that gall nut extract has the highest antioxidant activity, whereas persimmon and clove extract show the antiobesity activities by inhibition of digestive enzymes and fat accumulation in 3T3-L1 cells. These extracts are useful materials for the development of antioxidant and antiobesity functional foods. Key words: natural plants, color resource, antioxidant, antiobesity 서 최근친환경에대한사람들의욕구가증대되면서복합기능을갖는다기능성천연색소소재에대한관심이나날이증가하고있다. 더욱이천연색소는합성색소에비해환경과인체에대한독성및발암성이적고항균, 항암, 항염등의생리적기능성이있음이알려짐에따라섬유염색에만한정되어이용되던것이근래에는식품, 화장품, 비누, 모발염색및페인트등다양한분야에소재로응용되고있다 (1). 천연색소는크게두가지로분류될수있는데첫째는보통식품으로사용하지않고착색목적으로사용하는아나토, 코치닐, 치자, 오배자및홍화황색소등이있고둘째는보통식품으로사용하는포도과피, 파프리카, 홍국, 비트및카카오색소등이있는데, 이러한색소는주로식물류에서그원료를추출하는것이대부분이나코치닐색소와같이선인장에기생하 Received 10 October 2014; Accepted 17 December 2014 Corresponding author. Jung-Hye Shin, Namhae Garlic Research Institute, Namhae, Gyeongnam , Korea whanbee@hanmail.net, Phone: 론 는연지벌레를원료로추출하는동물성색소도존재한다 (2). 식물체는다양한형태의항산화물질을함유하고있는데, 그중에서도페놀성화합물은대표적인식물유래의색소성분으로생체내의 DNA 손상, 암유발, 노화등다양한질병의원인이되는유리라디칼에의한손상을방지함으로써생체를보호하는중요한유용성분으로주목받고있다 (3,4). 그중안토시아닌은플라보노이드계색소로적자색식재료에많이함유되어있고항암, 항산화, 항바이러스및면역증강등에효과가있는것으로보고되어있으며 (5), 멍게껍질로부터추출한 alloxanthin, halocynthiaxanthin, halocynthiaxanthin과같은카로테노이드색소는항산화활성및염증억제효능이있는것으로보고되어있다 (6). 시금치나갓으로부터추출한클로로필색소또한강력한항산화효과를가지는것으로보고되고있다 (7,8). 본연구에서사용한천연색소소재로정향 (, Syzygium aromaticum) 은꽃봉오리를주로염료로이용하는데정향유의주성분인 eugenol은항균성, 항곰팡이성및항바이러스등에효과가있고, 정향추출물로염색할경우염색포의항균성과자외선차단효과가증가되는것으로보고되어

2 166 황초롱 강민정 심혜진 서화진 권오운 신정혜 있다 (9,10). 감 (, Diospyros kaki) 에서분리되는카로티노이드색소는주로탄닌성분으로산화 환원반응을기질로하여색상을나타내는특징을가지며 (11), 폴리페놀성분들은항산화, 항암및항아토피등에도효능을가지는것으로보고되어있다 (12,13). 오배자 (, Aphis chinensis) 는옻나무과에속하는붉나무에진딧물이기생하여생긴벌레집으로주요물질인 pyrogallol tannin은항산화 (14), 항혈전 (15), 항바이러스 (16) 및멜라닌합성억제활성 (17) 등과관련성이높고, 무색이지만산화되면짙은갈색, 황색또는갈색을나타내어천연염료로도두루사용되고있다. 황벽 (, Phellodendron amurense) 은황벽나무의노란수피로예전부터약재및염재로이용되어왔고, 황벽황색색소의주성분인 berberine은지방분해활성 (18), 피부멜라닌저해활성 (19) 및항산화활성 (20) 등에효과가있는것으로보고되어있다. 이상의연구와같이일부천연색소는색상을띰과동시에약리적작용이인정되어이미식품또는염료로활용되고있고대다수가특정색소의원료를소재로하여다양한분야에서기능성을검증하고있으나, 그원료에서추출한색소자체의특성및생리적기능성등을비교한연구는미미한실정이다. 따라서본연구에서는천연색소소재로널리이용되고있는정향, 감, 오배자, 황벽외에도치자 (, jasminoides for. grandiflora), 홍화황 (, Carthamus tinctorius L.) 및아나토 (, Bixa orellana L.) 7종의색소를이용하여항산화및항비만활성을비교 분석함으로써향후이들소재를활용한기능성식품개발에대한기초자료를제공하고자한다. 재료및방법실험재료및추출물의제조천연색소소재로정향, 감, 오배자, 황벽, 치자, 홍화황및아나토는한약건재상에서건조된것을구입하여시료로사용하였다. 천연식물소재로부터색소성분을분리하고추출물을제조하기위하여각각의건조시료 1 kg에 10 L의물을가한후 catalase(biotouchrcat200, AB Enzymes, Rajamäaki, Finland) 및 alcalase(alacalaser, Novozymes, Dittingen, Switzland) 를 1% 농도로첨가하고 45 C에서 10시간동안반응시킨다음 80 C로조절된추출기 (Cosmos660, Kyungseo, Incheon, Korea) 를이용하여 2 시간동안추출하였다. 그후추출물을여과하여농축기 (rotavapor R-220, BUCHI, Flawil, Switzerland) 로농축한다음분무건조기를이용하여분말화해실험용시료로사용하였다. 추출물제조에사용된건조시료량대비완성분말의양 (w/w) 으로부터추출수율을산정하였다. 총페놀함량측정총페놀함량은 Folin-Denis법 (21) 에따라각추출물 1 ml에 Foline-Ciocalteau 시약 1 ml를넣고 3분후 10% Na 2CO 3 용액 1 ml씩을가한다음혼합하고실온의암실에서 1시간정치하여 760 nm에서흡광도를측정하였다. 표준물질로 gallic acid(sigma-aldrich Co., St. Louis, MO, USA) 를사용하여시료와동일한방법으로분석하여얻은검량선으로부터총페놀함량을계산하였다. DPPH 및 ABTS 라디칼소거활성측정 DPPH 라디칼소거활성은 Blois(22) 의방법에따라 1,1- diphenyl-2-picrylhydrazyl(dpph) 에대한전자공여활성으로나타내었다. 즉추출물과 DPPH 용액 (5 mg/100 ml methanol) 을동량으로혼합한다음실온에서 20분간반응시킨후 525 nm에서흡광도를측정하였다. ABTS[2,2-azinobis-(3-ethylbenzo-thiazoline-6- sulphonate)] 라디칼소거활성은 Re 등 (23) 의방법에따라 7 mm의 ABTS 용액에 potassium persulfate를 2.4 mm이되도록용해시킨다음암실에서 12~16시간동안반응시킨후 415 nm에서흡광도가 1.5가되도록증류수로조정한 ABTS 용액을사용하였으며, ABTS 용액에동량의시료액을혼합하여실온에서 10분간반응시켜 415 nm에서흡광도를측정하였다. 라디칼소거활성은시료무첨가구에대한시료첨가구의흡광도비로계산하여그활성이 50% 감소되는데필요한시료의농도 (inhibition concentration, IC 50, μg/ml) 로표기하였다. FRAP법에의한환원력측정 FRAP(ferric reducing antioxidant power) 법에의한항산화활성은 Benzie와 Strain(24) 의방법에따라 300 mm acetate buffer(ph 3.6), 40 mm HCl에용해한 10 mm TPTZ(2,4,6-tripyridyl-s-triazine) 용액및 20 M ferric chloride를각각 10:1:1(v/v/v) 의비율로혼합하여 37 C의수욕상에서가온한것을 FRAP 기질액으로사용하였다. 96 well plate에시료액 40 μl, FRAP 기질액 100 μl 및증류수 40 μl를차례로혼합하여 37 C에서 4분간반응시킨후 593 nm에서흡광도를측정하였으며, ferrous sulfate를표준물질로하여얻은표준검량선으로부터계산하였다. 소화효소저해활성측정 α-glucosidase 저해활성측정 : Watanabe와 Kawabata (25) 의방법에따라 10 mg/ml 농도의시료액 10 μl를 0.7 unit/ml α-glucosidase 효소액 50 μl와혼합하여 405 nm 에서반응전의흡광도를측정하였다. 5분간실온에방치하고기질액 5 mm pnpg(p-nitrophenyl-α-glucopyranoside) 50 μl를가하여 37 C에서 5분간반응시킨후, 다시 405 nm에서흡광도를측정하여흡광도변화로부터효소저해활성을계산하였다. α-amylase 저해활성측정 : Pancreatin 기원의 α- amylase 저해활성은 Lim 등 (26) 의방법을변형하여시료

3 천연식물로부터추출한색소성분의항산화및항비만활성 167 액 50 μl에 1 unit/ml의 α-amylase 효소액 250 μl와 50 mm potassium phosphate buffer(ph 6.9) 250 μl를혼합하여 37 C에서 10분간반응시킨후 0.5% starch를 500 μl 가하여다시 37 C에서 10분간반응시켰다. 반응액에 48 mm DNS(3,5-dinitrosalicylic acid, 30% potassium sodium tartrate in 0.5 M NaOH) 발색시약 500 μl를넣고 100 C에서 15분간끓여반응을중지시킨후냉각하고증류수 3 ml를가하여희석시켰다. 이때각 blank로는효소액첨가후기질을넣기전에 DNS 발색시약을먼저넣은것을사용하였다. 반응액은 540 nm에서흡광도를측정하여각 blank와의차이를구한후대조군과비교하여저해율을계산하였다. Lipase 저해활성측정 : Lipase 저해효과측정은 Saisuburamaniyan 등 (27) 의방법을변형하여시료액 0.25 ml에 800 unit/ml lipase 0.5 ml와 0.05 M potassium phosphate buffer(ph 6.5) 0.5 ml를혼합하여 37 C에서 15분간전처리한후 10% 로 isooctane에용해시킨 olive oil 1.25 ml를첨가하여 37 C에서 20분간진탕배양하였다. Acetone 5 ml로반응을정지시킨후 5% cupric acetate 1 ml를첨가하여혼합한뒤상온에서정치하여상층액 1 ml를취해 720 nm에서흡광도를측정하여대조군의흡광도값과비교하여저해율을계산하였다. 3T3-L1 세포에서항비만활성측정 3T3-L1 세포배양및분화 : 실험에사용된지방전구세포인 3T3-L1 세포는 American Type Culture Collection (ATCC, Manassas, VA, USA) 에서분양받아 10% bovine calf serum(bcs, Hyclone, Logan, UT, USA), 1% penicillin/streptomycin(sigma-aldrich Co.) 을첨가한 Dulbecco's modified Eagle medium(dmem, Gibco, Grand Island, NY, USA) 배지를사용하여 37 C, 5% CO 2 조건에서배양하였다. 3T3-L1 세포가 confluent 한상태가되면 10% fetal bovine serum(fbs, Hyclone), 1 μm dexamethasone (Sigma-Aldrich Co.), 0.5 mm methylisobutylxanthine (IBMX, Sigma-Aldrich Co.), 10 μg/ml insulin(gibco, Invitrogen, Carlsbad, CA, USA) 을포함하는 DMEM 배지로교환한후 3일동안지방세포분화를유도하였다. 그후 2일간격으로 10% FBS, 1% penicillin/streptomycin, 10 μg/ ml의 insulin이포함된 DMEM 배지를이용하여시료추출물과함께처리한다음분화가완성되는시점에서 insulin을포함하지않은 DMEM 배지로교환하여분화시켰다. 세포독성측정 : 시료추출물이 3T3-L1 세포의생존율에미치는영향을확인하기위해 MTT(3-4,5-dimethylthiazole-2-yl-2,5-di-phenyl-tetrazolium bromide) assay를수행하였다. 96 well plate에 3T3-L1 세포를 cells/ well 농도로 37 C, 5% CO 2 조건에서 24시간배양한후각시료추출물을농도별 (1~100 μg/ml) 로처리하여 24시간동안다시배양하였다. 그후 5 mg/ml의 MTT 시약을 10 μl씩처리하여 2시간동안배양시킨후상층액을제거하고 dimethyl sulfoxide(dmso, AMERSCO, Solon, OH, USA) 를 100 μl 넣어 10분간교반한다음 570 nm에서 ELISA reader(epoch, BioTek Instrument, Winooski, VT, USA) 로이용하여흡광도를측정하였다. 지방축적율및 triglyceride 함량측정 : 3T3-L1 세포내지방축적율은 Oil Red-O staining법으로측정하였다. 분화된세포를먼저 PBS로세척한다음 10% formalin으로 1시간동안고정하였다. 고정액을제거하고 60% isopropanol로 2회세척한후 Oil Red-O solution을첨가하여 20 분간실온에서염색하였다. 염색된세포는 100% isopropanol을첨가하여염색된지방을추출한다음 ELISA reader를이용하여 520 nm에서흡광도를측정하였다. 지방축적측정율은 control에대한백분율로나타내었다. Triglyceride 함량은 triglyceride colorimetric assay kit(cayman, Ann Arbor, MI, USA) 을이용하여확인하였다. 세포는 PBS(pH 7.4) 를이용하여 3회세척한후 lysis buffer(1% Triton X-100 in PBS) 를첨가하여스크래퍼로모은후 4 C에서 30초간 sonication 하여 10,000 rpm에서 10분간원심분리한다음상층액을분리하였다. 상층액 10 μl에 150 μl의 enzyme buffer solution을첨가하여혼합한다음실온에서 15분간반응시킨후 550 nm에서흡광도를측정하였다. 통계처리각실험은 3~5회반복실험한결과에대하여 SPSS 12.0 (SPSS, Inc., Chicago, IL, USA) 을사용하여통계처리하였으며, 각각의시료에대해평균 ± 표준편차로나타내었다. 각시료군에대한유의차검정은분산분석을한후 P<0.05 수준에서 Duncan's multiple range test에따라분석하였다. 결과및고찰천연색소소재의추출수율및총페놀화합물의함량 7종의천연식물유래색소소재의추출수율및총페놀화합물의함량을측정한결과는 Table 1과같다. 추출수율은오배자, 정향, 치자가 30% 이상으로높았으며, 감은수율이가장낮아 4% 에불과하였다. Gallic acid를기준물질로하였을때천연색소성분중총페놀화합물의함량은오배자추출물에서 2, mg/kg으로가장높게정량되었으며, 다음으로정향추출물에서 1, mg/kg으로높았다. 감, 황벽및홍화황추출물은 ~ mg/kg의범위로비슷한함량이었으며, 아나토추출물은 mg/ kg으로가장낮은함량이었다. Bae 등 (28) 은건조오배자 80% 메탄올및물추출물의총페놀함량이각각 220 mg/kg 및 42 mg/kg으로본연구의오배자추출물보다더낮은함량이라고하였는데, 이는시료의추출부위및추출용매에따른차이라생각된다. Do

4 168 황초롱 강민정 심혜진 서화진 권오운 신정혜 Table 1. Yield and total phenolic compounds contents of various color resource from natural plants ( jasminoides for. grandiflora) (Bixa orellana L.) (Diospyros kaki) (Phellodendron amurense) (Carthamus tinctorius L.) (Aphis chinensis) (Syzygium aromaticum) Yield (%) Gallic acid equiv./extract (mg/kg) ±5.76 B 95.53±1.09 A ±1.89 C ±3.93 C ±1.09 C 2,441.45±9.50 E 1,346.48±8.81 D Each value represents mean±sd, n=3. A-E Means with different letters in the same column are significantly different at P<0.05. 등 (29) 은정향종자에탄올추출물의총페놀함량이 mg/g으로보고하였고, Dong 등 (30) 은용매별정향추출물에서총페놀함량이물추출물 (23.1%) 에비해메탄올 (42.8 %) 및에테르추출물 (34.9%) 에서더높았는데, 이는정향의주요페놀화합물성분인 eugenol 및 phenylpropene이비극성인에테르나메탄올에서용이하게용출되기때문이라고보고하였다. 식물에존재하는페놀성화합물들은체내의효소단백질과같은거대분자들과결합하는성질을가지고있어항산화, 항당뇨및항비만활성과연관성이높은것으로보고되어있는데 (31,32), 본연구의오배자및정향추출물의경우다른시료추출물에비해페놀화합물이다량함유되어있으므로인체내생리활성증강에더효과적일것으로추측된다. 천연색소소재의항산화활성천연염색원료식물에대한 DPPH 및 ABTS 라디칼소거활성과환원력을측정한결과는 Table 2와같다. DPPH 라디칼소거활성은오배자추출물의 IC 50 값이 μg/ml 로가장높았으며, 다음으로정향추출물 (28.36 μg/ml), 황 Table 2. DPPH and ABTS radical scavenging activity and FRAP of various color resource from natural plants DPPH IC 50 ABTS IC 50 FRAP (FeSO4 7H 2O (μg/ml) eq µm) >900 > ±0.32 B >2,000 > ±0.95 A 71.02± ± ±7.58 E > ± ±2.94 D > ± ±2.63 C 20.50± ±0.09 1,016.42±9.60 G 28.36± ± ±2.87 F FRAP treated sample with 250 μg/ml concentration. IC 50 value in the concentration of sample required for 50% inhibition. Each value represents mean±sd, n=3. A-G Means with different letters in the same column are significantly different at P<0.05. 벽추출물 (71.02 μg/ml) 의라디칼소거활성이높았다. 반면에치자및아나토추출물의 IC 50 값은고농도로라디칼소거활성이매우낮은것으로나타났다. ABTS 라디칼소거활성은 DPPH 라디칼소거활성과유사한경향으로오배자추출물의 IC 50 값이 8.18 μg/ml로라디칼소거활성이가장높았다. 정향추출물도 IC 50 값이 μg/ml로감 (57.44 μg/ml) 및황벽 (70.16 μg/ml) 추출물에비해높은라디칼소거활성을나타내었다. 250 μg/ml 농도로조절한천연색소추출물의환원력을 FRAP법으로측정한결과, 오배자 (1, μm) 및정향 ( μm) 추출물의활성이타시료에비해월등히높았다. 특히오배자추출물은감과황벽추출물에비해서약 5배정도높은활성을나타내었다. 이에비해치자, 아나토및홍화황은낮은항산화활성을나타내었다. Han 등 (33) 은국내자생식물 23종에대한전자공여능을측정한결과오배자추출물이 70.33% 로가장높은활성을나타내는것으로보고하였고, Bae 등 (28) 은용매분획별오배자추출물의라디칼소거능을측정한결과페놀함량이높았던에틸아세테이트층에서높은소거활성이있음을보고하였는데, 이는에틸아세테이트층에오배자의주성분인 gallotannin 및 triterpenoid 화합물과같은페놀화합물이다량존재하기때문으로보고하였다. 또한 DPPH 라디칼소거활성에대한정향메탄올추출물의 IC 50 값은 μg/ ml(34), 정향에탄올추출물의 IC 50 값은 5.9 μg/ml로정향추출물도항산화활성이뛰어난식물성소재로보고되어있다 (35). 천연식물의항산화활성은페놀화합물의함량에비례하며 (36), FRAP법에의한환원력도시료의총페놀함량이높을수록활성이높다고보고되어있는데 (37), 본연구에서도총페놀및플라보노이드함량이높았던오배자와정향추출물에서라디칼소거활성과환원력이높게나타났다. 천연색소소재의소화효소저해활성식물류색소원료의 α-glucosidase, α-amylase 및 lipase 에대한저해활성을측정한결과는 Table 3과같다. α- Glucosidase에대한천연색소추출물의저해활성을 IC 50 값으로측정한결과, 감추출물이 μg/ml로저해효과가가장높았으며, 다음으로오배자추출물이 μg/ml, 정향추출물이 μg/ml 순이었다. α-amylase에대한저해활성은감추출물이 49.45% 로 positive control로사용한 acarbose(49.60%) 와유사한수준으로높은저해활성을나타내었으며, 오배자와정향추출물은각각 40.27% 및 31.91% 로 acarbose보다는낮은수준이었다. 치자및황벽추출물은 11% 이하로활성이낮았으며, 홍화황추출물은 α-amylase에대한저해활성이없는것으로나타났다. Oh 등 (38) 은산초열매에탄올및메탄올추출물의 α- glucosidase에대한억제능을분석한결과, IC 50 값은각각

5 천연식물로부터추출한색소성분의항산화및항비만활성 169 Table 3. α-glucosidase, α-amylase, and lipase inhibition activity of various color resource from natural plants α-glucosidase IC50 (μg/ml) >2,000 > ±1.60 ND >2, ± ±9.38 α-amylase (%) 9.07±1.49 A 22.25±0.58 B 49.45±0.65 E 10.97±1.65 A ND 40.27±0.94 D 31.91±0.41 C Lipase (%) 8.6±1.61 A 32.91±1.67 D 61.01±0.69 F 22.12±1.34 C ND 46.69±1.19 E 19.18±0.66 B Acarbose ±0.31 E - α-amylase and lipase inhibition activity treated sample with 1 mg/ml concentration. IC 50 value in the concentration of sample required for 50% inhibition. Each value represents mean±sd, n=5. A-F Means with different letters in the same column are significantly different at P<0.05. ND: not detected μg/ml 및 μg/ml인것으로보고하였고, Kim 등 (39) 은 11종의양치식물중꿩고비, 석위및개고사리추출물의 α-glucosidase 저해활성측정결과, IC 50 값이 12.93~19.47 μg/ml의범위로항산화활성이높았던시료에서 α-glucosidase 저해활성도우수한것으로보고하였다. Hwang과 Han(40) 은조릿대용매분획별추출물의 α- amylase 저해활성을측정한결과, 에틸아세테이트층이 56.31%, 부탄올층이 43.66% 로 acarbose보다높은저해활성을나타내었다고보고하였고, Park 등 (41) 은비타민부위별추출물을 10 mg/ml 농도로제조하여 α-amylase에대한억제효과를측정한결과, 잎의물추출물에서 54.7% 로줄기및뿌리추출물에비해높은활성을나타내었는데, 이는잎추출물이다른부위보다페놀함량이높았기때문인것으로보고하였다. Lee 등 (42) 도식물의 2차대사산물인안토시아닌, proantocyasidins 등과같은페놀성물질은 α- amylase 저해효과를가진다고보고하였다. 그러나본연구에서는페놀함량및항산화활성이높았던오배자와정향추출물보다는감추출물에서높은저해활성을나타내었는데, 감추출물은여타추출물에비해 α-glucosidase 저해활성이높고, α-amylase도 acarbose와유사한수준의높 은저해활성을보였다. 이는페놀화합물외에감에함유되어있는다른유용성분들의상호작용에의한것으로추정되며, 감추출물은탄수화물의소화속도조절에관여하여단당류의분해및흡수를지연시킴으로써혈당조절에영향을줄것으로사료된다. 천연색소추출물의 pancreatic lipase 저해활성을측정한결과감추출물이 61.01% 로가장높은활성을나타내었고, 다음으로오배자추출물이 46.69%, 아나토추출물이 32.91% 순이었다. 치자와정향추출물은 20% 미만으로활성이낮았다. Pancreatic lipase는 triacylglycerol을 2-monoacylglycerol과두분자의 fatty acid로분해하는효소로흡수된지방의체내흡수에있어매우중요한작용을하는것으로보고되어있다 (43). Kim 등 (44) 은지충이에탄올및물추출물을 5 mg/ml 농도로조절하여 lipase 저해활성을측정한결과각각 37% 및 22% 로나타났으며, Bitou 등 (45) 은 54종의해조류중 27종의해조류가 43~100% 의 lipase 저해활성을가지는것으로확인하였는데, 이는해조류에포함되어있는 tannin과같은폴리페놀화합물에의한것으로보고하였다. Daiber(46) 는폴리페놀화합물중축합형탄닌이효소저해효과를가지나카테킨은효과가없어효소활성의저해는폴리페놀의양과종류에따라달라지는것으로보고하였다. 감의주요색소물질인탄닌은 gallic acid 및그유도체에 phenol류가결합한축합형으로 (47) 감에는 300~600 mg% 정도의탄닌이함유되어있는것으로보고되어있는데 (48), 본연구에서도감추출물이여타시료에비해 lipase 저해활성이높은것으로보아감에함유되어있는탄닌물질이효소저해작용에큰영향을미쳤을것으로생각된다. 3T3-L1 세포에대한천연색소추출물의세포생존율 3T3-L1 세포에대한천연색소류의유효농도설정을위하여각시료추출물을 1~100 μg/ml 농도로조절한후세포의생존율을측정한결과는 Table 4와같다. 대조군과비교하여각시료추출물을 1 μg/ml 농도로처리시아나토추출물을제외하고는 88.95~98.17% 생존율을보여독성이적은것으로나타났다. 아나토추출물의경우 5 μg/ml Table 4. Cell viability in 3T3-L1 immature adipocytes treated with various color resource from natural plants (%) Sample concentration (μg/ml) ±4.79 * 82.90±4.23 * 91.94±6.39 * 95.08±7.33 * 98.17± ±3.12 * 89.86±2.77 * 90.89±4.19 * 78.79±2.94 * 91.54±6.09 * 90.94±8.43 * 92.18±5.19 * 82.90±3.13 * 88.37±2.69 * 89.70±4.00 * 75.64±2.26 * 88.44±5.56 * 90.24±7.19 * 91.50±6.34 * 76.13±6.32 * 86.45±2.82 * 88.49± ±7.67 * 90.50±6.54 * 86.69±6.01 * 91.43±5.03 * 71.92±5.60 * 86.17±2.70 * Means with an asterisk in the same column are significantly different of compared with control (only cultured 3T3-L1 cells) and each natural color resource extracts treated (1 100 μg/ml) cells at P<0.05.

6 170 황초롱 강민정 심혜진 서화진 권오운 신정혜 Table 5. Lipid accumulation rate of 3T3-L1 mature adipocytes treated with various color resource from natural plants (%) Concentration (μg/ml) ± ± ± ± ± ± ± ± ± ±4.00 * ± ± ± ± ± ±3.29 * 81.54±3.03 * 97.32± ± ± ±2.72 * Control was only cultured 3T3-L1 cells, and it s lipid accumulation rate 100%. Means with an asterisk in the same column are significantly different of compared with control and each natural color resource extracts treated cells at P<0.05. Table 6. Triglyceride content of 3T3-L1 mature adipocytes treated with various color resource from natural plants (%) Triglyceride contents ± ± ±4.98 * ± ± ± ±3.83 * Control was only cultured 3T3-L1 cells, and it s triglyceride contents 100%. Sample treatment concentration was 20 μg/ml. Means with an asterisk in the same column are significantly different of compared with control and each natural color resource extracts treated cells at P<0.05. 농도이상의범위에서는 80% 미만으로세포의생존율이크게감소하는경향이었고, 오배자추출물도 10 μg/ml 농도이상의범위에서는 80% 미만으로세포의생존율이감소하였는데, 특히 100 μg/ml 농도에서아나토및오배자추출물은 70.43% 및 71.92% 로시료중세포에대한독성이다른시료에비해상대적으로높았다. 3T3-L1 세포에대한천연색소추출물의지방축적율및 triglyceride 함량천연색소추출물이 3T3-L1 세포가전지방세포에서지방세포로의분화과정에작용하여 3T3-L1세포내에생성된지방의축적정도를 Oil Red O 염색법으로측정한결과를 Table 5에나타내었다. 각추출물을 5 μg/ml 농도로조절하여처리시모든시료에서지방의축적이감소되는효과를보이지않았으나, 각추출물을 10 μg/ml 농도로처리시감추출물에서 87.91% 로대조군에비해지방축적율이유의적으로낮았다. 20 μg/ml 농도로처리시지방축적율은감추출물이 81.54%, 아나토추출물이 85.70%, 정향추출물이 83.36% 로대조군에비해유의적으로지방축적이감소됨을확인하였다. 3T3-L1 세포내에생성된지방의양을정량적으로평가하기위해염색된지방을추출하여 triglyceride 함량을측정한결과 (Table 6), 지방축적율과유사한경향으로감및정향추출물에서 triglyceride 함량이유의적으로감소되었는데, 특히정향추출물이 66.11% 로가장낮았고감추출물은 88.88% 로나타났다. 그외추출물에서는대조군과비교하여유의차가없었다. 비만은지방전구세포의분화및 adipogenesis 과정에서세포내 triglyceride 축적으로발생되는데 (49), adipogenesis 과정은 PPARγ 및 C/EBPɑ와같은 adipogenic transcription factor들에의해조절되며, 지방세포내에서 triglyceride의축적을억제하거나전사인자들의활성을조절하는것이중요한비만억제치료방법으로알려져있다 (50). Ji 등 (51) 은돌미나리추출물을 1~200 μg/ml로처리하여지방세포의중성지방함량을측정한결과농도의존적으로그함량이감소되어 200 μg/ml 농도에서 80% 이상의중성지방축적억제효과가있음을보고하였다. Lee 등 (52) 은지방세포에용아초추출물을 50 μg/ml 및 100 μg/ml로처리한결과각각 58% 및 80% 의지방세포분화억제효과를나타내었는데, 이러한추출물들은 PPARγ, C/EBPɑ 및 C/EBPβ의발현을억제함으로써 lipid droplet의생성과 triglyceride의축적을감소시키는것으로추정하였다. 본실험결과감추출물과정향추출물은 3T3-L1 지방세포에대하여독성이없으며지방세포분화시관찰되는지방의축적율과지방세포내의 triglyceride 함량도억제시키는것으로확인되어비만을예방하는데도움을줄것으로사료되나, 이와관련된전사인자및단백질발현에관련된연구는좀더진행되어야할것으로생각된다. 요약정향, 감, 오배자, 황벽, 치자, 홍화황및아나토와같은천연식물로부터얻어진색소추출물의항산화및항비만활성을비교 분석하였다. 총페놀함량은오배자추출물에서 2, mg/kg 으로가장높았고, 다음으로정향추출물에서 1, mg/kg으로높았다. DPPH와 ABTS 라디칼소거활성및환원력을이용한항산화활성은오배자추출물에서가장높았다. α-glucosidase에대한저해활성은감추출물에서 (IC 50=22.83 μg/ml) 가장높았고, 다음으로오배자추출물에서높았다. α-amylase 및 lipase에대한저해활성역시감추출물처리시각각 49.45% 및 61.01% 로높은활성을보였다. 20 μg/ml 농도로처리시 3T3-L1 세포내지방축적율은감, 정향및아나토추출물처리군에서각각 81.54%, 83.36% 및 85.70% 로대조군에비해유의적으로낮았다. 3T3-L1 세포내 triglyceride 함량은정향추출물처리군이 66.11% 로가장낮았고다음으로감추출물처리군에서 88.88% 로유의적으로낮았다. 천연식물류로부터추

7 천연식물로부터추출한색소성분의항산화및항비만활성 171 출한색소원료중오배자추출물이높은항산화활성을나타내었으며, 감추출물은소화효소저해활성과 3T3-L1 세포내의지방축적억제효과를가짐으로써항비만활성을나타내는것으로판단된다. 감사의글 본논문은산업통상자원부지역특화기술융복합연구지원사업 ( 과제번호 : R ) 성과의일부이며, 지원에감사드립니다. REFERENCES 1. Boo HO, Shin JS, Hwang SJ, Bae CS, Park SH Antimicrobial effects and antioxidative activities of the cosmetic composition having natural plant pigments. Korean J Plant Res 25: Song JC, Cho WD Processed food and food colorants. Food Technology 10: Azuma K, Nakayama M, Koshika M, Ippoushi K, Yamaguchi Y, Kohata K, Ito H, Higashio H Phenolic antioxidants from the leaves of Corchorus olitorius L. J Agric Food Chem 47: Huang MT, Ho CT, Lee CY Phenolic compounds in food. In Phenolic Compounds in Food and Their Effects on Health Ⅱ. Maple Press, New York, NY, USA. p Jang JR, Kim KK, Lim SY Effects of solvent extracts from dried beet (Beta vulgaris) on antioxidant in cell systems and growth of human cancer cell lines. 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8 172 황초롱 강민정 심혜진 서화진 권오운 신정혜 35. Kim J, Kim SA, Yun WK, Kim EJ, Woo MK, Lee MS Antioxidative effect of ethanol extract for 5 kinds of spice. J Korean Soc Food Sci Nutr 33: Labuza TP Kinetics of lipid oxidation in foods. CRC Crit Rev Food Technol 2: Lee HR, Jung BR, Park JY, Hwang IW, Kim SK, Choi JU, Lee SH, Chung SK Antioxidant activity and total phenolic contents of grape juice products in the Korean market. Korean J Food Preserv 15: Oh SM, Han W, Wang MH Antioxidant and α-glucosidase inhibition activity from different extracts of Zanthoxylum schnifolium fruits. Kor J Pharmacogn 41: Kim NR, Chi LW, Lee CH Alpha-glucosidase inhibition activity of methanol extracts obtained from nine pteridophyte species native to Korea. Korean J Plant Res 26: Hwang JY, Han JS Inhibitory effects of Sasa borealis leaves extracts on carbohydrate digestive enzymes and postprandial hyperglycemia. J Korean Soc Food Sci Nutr 36: Park YH, Lim SH, Ham HJ, Jeong HN, Lee KJ, Kim KH, Kim SM Comparison of biological activities of extracts from different parts of Seabuckthorn (Hippophae rhamnoides L.). J Korean Soc Food Sci Nutr 39: Lee WY, Ahn JK, Park YK, Park SY, Kim YM, Lee HI Inhibitory effects of proanthocyanidin extracted from Distylium racemosum on α-amylase and α-glucosidase activities. Kor J Pharmacogn 35: Bitou N, Nimomiya M, Tsjita T, Okuda H Screening of lipase inhibitors from marine algae. Lipids 34: Kim DH, Kim BWE, Kim MJ, Sun WC, Jung SA, Kim HJ, Jung DH, Kim TW, Cho YJ, Ahn DH Effects of heat, ph, and gamma irradiation treatments on lipase inhibitory activity of Sargassum thunbergii ethanol extract. J Korean Soc Food Sci Nutr 41: Bitou N, Ninomiya M, Tsujita T, Okuda H Screening of lipase inhibitors from marine algae. Lipids 34: Daiber KH Enzyme inhibition by polyphenols of sorghum grain and malt. J Sci Food Agric 26: Jung JS, Park JS, Kim TK Coloration of cotton fabrics with tannins of persimmon extracts by heating process. J Korean Soc Dyers Finishers 20: No HK, Lee MH Removal of astringency in persimmons by chitosan. J Korean Soc Food Sci Nutr 27: Peter GK Obesity as a medical problem. Nature 404: Wellman NS Causes and consequences of adult obesity: health, social and economic impacts in the United States. Asia Pac J Clin Nutr 11: Ji HH, Jeong HY, Jin SJ, Kwon HJ, Kim BW Inhibition of adipocyte differentiation by methanol extracts of Oenanthe javanica seed in 3T3-L1 preadipocytes. J Life Sci 22: Lee JA, Ahn EK, Hong SS, Oh JS Anti-obesity effect of ethyl acetate extracts from Agrimonia pilosa Ledeb. in 3T3-L1 preadipocytes. J Korean Soc Food Sci Nutr 41: