Journal of Life Science 2006 Vol. 16. No. 2. 328~332 cjls 백자인추출물에의한 pancreatic lipase 의저해효과 김민수 김보연 박찬선 윤병대 안순철 1 오원근 안종석 * 한국생명공학연구원, 1 부산대학교의과대학미생물및면역학교실 Received February 24, 2006 /Accepted March 28, 2006 Inhibitory Effect of Thujae orientalis Semen Extract on Pancreatic Lipase Activity. Min Soo Kim, Bo Yeon kim, Chan Sun Park, Byung Dae Yoon, Soon Cheol Ahn 1, Won Keun Oh and Jong Seog Ahn*. Korea Research Institute of Bioscience and Biotechnology, 1 Department of Microbiology and Immunology, College of Medicine, Pusan National University The possible presence of inhibitors of pancreatic lipase (tricaylglycerol acylhydrolase EC 3.1.1.3) was screened from Korean traditional edible or medicinal herbs. Among tested herbs, Arecae pericarpium, Mucunae Caulis, Rhus javanica, Thujae orientalis were shown to have strong inhibitory effect against pancreatic lipase. Thujae orientalis was finally selected as a candidate for pancreatic lipase inhibitor. The extract of Thujae orientalis was showed selective inhibition on porcine pancreatic lipase activity. Active inhibitors, TF1, TF2, TF3, were purified from an extract of Thujae orientalis, using chloroform extraction, followed by successive chromatography in silica gel and LH20 and high performance liquid chromatography (HPLC). The IC 50 values of TF1, TF2, TF3 and orlistat were 44.7, 98.7, 46.1 and 27.6 μg /ml, respectively. And also the TF2 and orlistat were shown to be inhibitory effect on the differentiation of preadipocyte NIH3T3 L1 cells at a concentration of 10 μg /ml. Key words Pancreatic lipase inhibitor, Thujae orientalis 서 국내산업의발달및소득수준의향상과더불어식생활, 식습관등라이프스타일 (lifestyle) 의서구화됨에따라만성병이나성인병환자가급증하고있으며, 그기반이되는것중의하나가비만이다. 비만은단지성인에게만국한되는것이아니라근간에는소아비만과청소년비만으로까지문제가대두되면서그심각성을더해가고있는실정이며, 국내보건협회도이미비만을만성질병으로서규정하고있는실정이다. 비만은체질량지수 (Body Mass Index, BMI) 가 30 BMI 이상인경우를의미하며, 25~30 BMI인경우는과체중으로정의하고있으며, BMI는체중을키의제곱으로나눈값 (kg/m 2) 으로계산한다 [19]. 비만은체내에지방조직이과다하게축적되어있는상태를의미하며, 비만의가장큰원인은고에너지나고지방을함유한음식의섭취및운동부족으로인한체중의증가나체내지방이축적이지만 [5,6], 최근에는신경내분비계통의이상, 약물, 유전적요인및생화학적이상반응에의해서도유발되는것으로보고되고있다 [2,11]. 세계보건기구 (WHO) 에따르면, 현재전세계적으로과체중및비만에해당되는사람들의숫자는 12억명에이르며, 미국의경우성인인구의약 65% 가과체중에해당된다고발표하였으며 [9], 건강을위협하는만성질병으로규정하고 론 *Corresponding author *Tel:82428604312, Fax:82428604595 *Email : jsahn@kribb.re.kr 있다 [21]. 또한우리나라의경우 2001년국민건강심층분석보고서에의하면체질량지수 25 BMI 이상인비만인구가 20세미만의경우 11.2%, 20세이상인경우 31% 로비만유병률이점차증가하는추세로파악되고있다 [1]. 이러한비만현상은단순히외형상의문제뿐만아니라체중증가와더불어당뇨, 동맥경화, 심혈관질환, 고혈압, 고지혈증등심각한성인병을유발하는것으로보고되어있다 [7,10,14,16,20]. 최근에는비만치료제개발을위한많은연구가진행되고있으며, 그중에하나가췌장지방분해효소저해제 (pancreatic lipase inhibitor) 이다. Pancreatic lipase는 triglyceride를 2monoacylglycerol과 fatty acid로분해하는 key enzyme으로작용한다 [3]. 대표적인 pancreatic lipase inhibitor는 Streptomyces toxitricini로부터유래된 lipstatin의유도체인 tetrahydrolipstatin (Orlistat, Ro 180647) 으로서섭취된지방의약 30% 를저해할정도로효능이가장우수한것으로알려져있으며 [8,12,13,17], 현재의약품으로시판중이다. 그러나이와같은효능에도불구하고 tetrahydrolipstatin 은위장장애, 과민증, 담즙분비장애, 지용성비타민흡수억제등의부작용이있는것으로알려져있다 [15]. 따라서최근에는부작용이없는식품및, 천연물로부터 pancreatic lipase inhibitor의개발을위한연구가진행되고있다 [18,22]. 본연구에서는 pancreatic lipase의활성을저해하는물질을탐색하고자국내의식용또는약용식물의추출물을대상으로 lipase 활성에대한저해능력을측정하고그중에서저해능이우수한백자인 (Thujae orientalis) 추출물에대한결과
Journal of Life Science 2006, Vol. 16. No. 2 329 를보고하고자한다. 재료및방법실험재료및시약본실험에사용한생약재료인백자인은측백나무의종자로서대전의일산약품 ( 주 ) 에서구입하였다. 활성물질의분리및확인에사용된 silica gel (Kiesegel 60, paticle size : 0.04 0~0.063 nm) 은 Merck사 (Darmstadt, Germany), ODS RP18 (ODSA, 120A, S150 μm ) 은 YMCGEL사 (Tokyo, Japan) 로부터구입하였고, TLC에이용된 procoated silica gel plates 60F 254 (0.25mm and 0.5 mm in thickness) 와 ODS RP18 F 254S (25DCPlatten 5 10 cm) 는 Merck사 (Darmstadt, Germany) 로부터구입하였다. 각분리단계에이용된유기용매는덕산약품공업 ( 주 ) 의고순도제품을사용하였고, HPLC 용매는 Burdick & Jackson (Muskegon, USA) 을사용하였다. pancreatic lipase assay에사용한 tributylin은 Fluka (St. Gallen, Switzerland) 에서, pancreatic lipase (Type VIS, porcine pancreas), gum arabic은 Sigma (St. Louis, USA) 제품을사용하였다. 그외에사용된시약은모두특급및 1급시약을사용하였다. Pancreatic lipase 활성저해물질의탐색 10 mm CaCl 2, 200 mm NaCl을포함한 5% gum arabic 용액 100 ml을제조하여 9 ml을취한후, 여기에 tributylin 1 ml을섞어초음파장치내에서유화시킨후, 2% 한천배지에혼합하여평판배지를제조하였다. 5 mm 두께의한천배지에지름 6 mm의구멍을만들고, 메탄올에녹인시료와 pancreatic lipase 용액을섞어 3분간반응시켜넣은후 4시간후투명환 (clear zone) 의형성크기를측정하여 pancreatic lipase 에대한저해활성을측정하였다. Pancreatic lipase 활성및저해능측정 Pancreatic lipase의활성및저해능은 Bitou 등 [3] 의방법에따라 triolein으로부터분리되는 oleic acid를측정함으로써결정하였다. 기질은 0.1 M NaCl이첨가된 0.1 M Ntris(hydroxymethyl)methyl2aminoethanesulfonic acid (ph 7.0) 9 ml에 90 μmol triolein, 45 mg gum arabic과 9.45 μmol taurocholic acid을넣고초음파로 5분동안처리하여제조하였다. Lipase 활성측정을위한반응용액은효소용액 (pancreatic lipase, 1500 U/ml) 15 μl, 식물추출물 5 μl, 기질용액 180 μl을섞어최종반응용액의부피는 200 μl되도록제조한후, ph 7.0, 반응온도 37 에서 30분간반응시켰다. 효소반응후생성된 oleic acid의정량은 Zapf 등 [22] 의방법에따라결정하였다. 효소반응후반응용액 0.2 ml에 2%(v/v) methanol이포함된 chloroform/heptane(1:1) 용액 3 ml을첨가하여 10분동안교반한후, 10분동안원심분리 (2,000 g) 하여수층을제거하였다. 여기에 copper 반응액 1 ml을첨가하여다시 10분동안교반한후, 10분동안원심분리 (2,000 g) 하여추출된 oleic acid와 copper salt가포함된유기용매층 1 ml을취하여 0.05%(w/v) 3tertbutyl4hydroxyanisole이포함된 0.1%(w/v) bathocuprione 용액 1 ml을첨가한후, 480 nm에서흡광도를측정하여 oleic acid를정량함으로써효소활성을측정하였다. NIH3T3 L1 세포의분화및분화능측정실험에사용된 NIH3T3 L1 세포 (preadipocyte) 는 ATCC 에서분양받아 10% FBS, 50 U/ml penicillin, 50 μg /ml streptomycin이첨가된 Dulbecco's modified Eagle's medium (DMEM) 에접종하여 37 에서 5% CO 2 세포배양기에서배양하였다. NIH3T3 L1 세포를 phosphate buffered saline (PBS) 용액으로세척한후, 5 10 4 cells/ml을 12 wellflat plate에배양하였다. 48시간동안배양후 10% FBS, 0.5 mm dexamethasone (DEXA), 10 mg/ml insulin, 1 μm 3isobutyl1methyxanthine (IBMX) 이첨가된배지에서 48시간동안배양하고, 이후 2일간격으로 6일동안 10% FBS가첨가된배지에 10 μg /ml insulin과시료를첨가하여분화를유도하였다. NIH3T3 L1 세포의분화정도는역상현미경으로관찰하여세포내밝은색의지방과립이형성되는것을확인하였고, 세포의분화정도는 Oil red O로염색하여측정하였다. 60% triethylphosphate 100 ml에 500 mg Oil red O를녹인후여과하여 Oil red O stock solution을제조하고, Oil red O stock solution 12 ml에증류수 8 ml을혼합하여실험에사용하였다. 세포배양 8일후분화된세포를관찰하기위하여배지를제거하고 10% formaldehyde를처리하여 1시간동안고정하였다. PBS 용액으로두번세척하고건조시킨후, 1 ml Oil red O 용액으로처리하여 3시간동안염색하였다. 다시 PBS 용액으로세척하고건조시킨후, isopropanol 을처리하여염색된지방과립을추출하여 microplate reader (Beckman, USA) 로 510 nm에서흡광도를측정함으로써세포의분화정도를결정하였다. 결과및고찰 Pancreatic lipase 저해활성물질의탐색및선정본실험실에서보유하고있는식용및약용식물의추출물을대상으로 pancreatic lipase의활성저해물질을탐색하였다. 각각의식물은 methanol로추출하여 1 mg/ml이되도록제조한후, pancreatic lipase 용액과섞어 3분간반응시킨후, tributylin을함유한평판배지의구멍에넣고 4시간후투명환 (clear zone) 의크기를측정하여 pancreatic lipase의 activ
330 생명과학회지 2006, Vol. 16. No. 2 ity를측정하여투명환의크기가작은시료를 1차탐색하여 13종의식물을선정하였다. 선정한각각의식물추출물을 methanol, chloroform, buthanol, H 2O 층으로용매분획하여 1 mg/ml 농도가되도록시료를제조하여 pancreatic lipase 에대한저해능을조사하였다. 그결과 methanol 추출물의경우, pancreatic lipase에대한저해효과는 Arecae pericarpium( 대복피 ), Mucunae Caulis( 계혈등 ), Rhus javanica( 오배자 ), Thujae orientalis( 백자인 ) 이우수한저해활성을나타냈다. 대부분의추출물은 chloroform 분획에활성이있는것으로나타났으며, 오배자등은 buthanol 분획에서도활성이있었다 (Table 1). Table 1의결과에따라 lipase에대한저해활성이우수한대복피, 계혈등, 오배자, 백자인의 methanol 추출물과 orlistat를 1 mg/ml로제조하여각시료가 Chemobacterium sp., Candida sp., Trichoderma sp., Rhizopus sp., 기원의 lipase와 porcine pancreatic lipase에대한선택적저해활성여부를조사하였다 (Table 2). Orlistat의경우, Candida sp. 유래 lipase 나 porcine pancreatic lipase에대해상대적으로강한활성을나타냈으며, 미생물에서유래하는 lipase의종류에따라제어 Table 1. Inhibition of pancreatic lipase activity by herbal plants extracts Samples Solvent Amomum kravahn ( 백두구 ) Areca catechu ( 빈랑 ) Arecae pericarpium ( 대복피 ) Caesalpinia sappan ( 소목 ) Cinnamomi ramulus ( 계지 ) Coptis japonica ( 황련 ) Magnolia liliflora ( 신이화 ) Mucunae Caulis ( 계혈등 ) Nelumbo nucifera ( 연자육 ) Rubus coreanus ( 복분자 ) Rhus javanica ( 오배자 ) Thujae orientalis ( 백자인 ) Zedoariae rhizoma ( 봉출 ) MtOH Chloroform Buthanol H 2O Inhibitory effect :, very strong;, strong;, weak;, none 활성의차이를나타내지만, 선택적제어활성은크지않은것으로판단된다. 그러나본실험에서선정한대복피의경우에서는미생물유래 lipase보다는 porcine pancreatic lipase에대하여강한저해활성을나타냄으로써선택적제어효과가있는것으로사료된다. 또한복분자추출물의경우에서는 Chemobacterium sp. 와 porcine pancreatic lipase에서, 백자인의경우에서는 Chemobacterium sp., Candida sp. 유래 lipase와 porcine pancreatic lipase에서상대적으로우수한저해효과를나타내었다. 이와같은결과를근거로본실험에서는 lipase에대한활성이가장우수한것으로판단되는백자인을대상으로 pancreatic lipase에대한저해활성연구를진행하였다. Pancreatic lipase 저해활성물질의탐색백자인으로부터 pancreatic lipase 활성물질을분리하기위하여 Fig. 1에서나타낸바와같이백자인 200g을 6일동안메탄올 1 L에침지하여 3회추출하고진공회전농축기로감압, 농축하여 39.6g를회수하고이를 chloroform, buthanol, H 2O 로각각분획하였다. Panreatic lipase에대해강한저해활성을나타내는 chloroform 층을감압농축하여 21.7g를획득한후, 이를 nhexane/chloroform(1/1, v/v) 조건에서 silica gel column chromatography(kiesegel 60, paticle size : 0.040~0.063 nm) 를실시하여활성물질 2.4g을용출하였다. 얻어진활성분획을 chloroform /nhexane/methanol(2/3/1) 의용매조건으로 Sephadex LH20 column chromatography 를수행하여활성분획 0.74 g을획득하였다. 활성물질의순도를높이기위하여 HPLC(YMC, ODSH80, 250 20mm, 30% CH 3CN(0.01% TFA), flow rate 2 ml/min) 를수행하여최종적으로활성물질 TF1, Tf2, TF3 각각 3.5mg, 24. mg, 1.3mg을분리하였다. 백자인추출물의 pancreatic lipase에대한저해효과최종분리된활성물질 TF1, TF2, TF3 및 orlistat를최종반응농도별 (1~250 μg /ml) 로제조하여 porcine pancreatic lipase에대한저해효과를측정하였다 (Fig. 2). Fig. 2에나타난바와같이활성물질및 orlistat의처리농도가증가함에따 Table 2. Selective inhibition of lipase activity by herbal plants extracts Samples Inhibition(%) a Lipase source Chemobacterium sp. Candida sp. Trichoderma sp. Rhizopus sp. Porcine Orlistat Arecae pericarpium Mucunae caulis Rubus coreanus Thujae orientalis 72.8 98.8 57.9 58.6 84.3 64.3 50.6 48.7 36.1 96.7 a Inhibition(%) means relative enzyme activity to a control of no treatment. 55.9 48.6 36.7 28.6 51.1 81.4 60.8 66.4 54.0 85.2 86.7 88.9 42.3 36.3 99.4
Journal of Life Science 2006, Vol. 16. No. 2 331 Thujae orientalis (200 g) Methanol extraction Chloroform extraction Silica gel column chromatography eluted with nhexane/chloroform(1/1) LH20 column chromatography HPLC TF1, TF2, TF3 eluted with chloroform/nhexane/methanol(2/3/1) YMC column(odsh80, 250 20mm), flow rate 2 ml/min, 254nm eluted with CN3CN/water(3/7, 0.01% TFA) Fig. 1. Isolation procedure of pancreatic lipase inhibitor from Thujae orientalis. Relative Lipase Activity (%) 100 80 60 40 20 0 TF 1 TF 2 TF 3 Orlistat 0 1 5.0 12.5 25 50 125 250 Final concentration of Inhibitors (µg/ml) Fig. 2. Inhibitory effect of Thujae orientalis extracts on lipase activity : Lipase activity was measured using porcine pancreatic lipase and Inhibitory effect was shown as the lowering of relative activity(%) against the lipase activity of control. 라 lipase에저해효과도증가함을알수있었다. Porcine pancreatic lipase에대한저해효과는 TF2보다 TF1, TF3가다소우수한것으로나타났으나, 대조구로사용한 orlistat 보다는다소저해효과가낮은것으로조사되었다. 이때각활성물질 TF1, TF2, TF3 및 orlistat의 porcine pancreatic lipase에대한 IC 50 값은각각 44.7, 98.7, 46.1 및 27.6 μg /ml이었다. 이와같은결과로보아본실험에서탐색한백자인추출물 TF1, TF2, TF3은비록시판되고있는 orlistat보다는 lipase에대한활성저해능을다소떨어지더라도좀더순수하게정제된후에는그활성이더욱높아질것으로예상된다. 백자인추출물의지방세포분화에대한저해효과 백자인추출물 TF1, TF2, TF3 및 orlistat의농도를 10 μg /ml 제조한후, 미분화상태전구지방세포인 NIH3T3 L1의 Table 3. Effect of Thujae orientalis extract and orlistat on adipocyte differentiation analyzed by Oil red O staining and extraction method Treatment Absorbance(510 nm) a Percent (%) b Control c TF1 TF2 TF3 Orlistat 3.14 2.41 0.78 1.89 0.75 100 76.75 24.84 60.19 23.88 a NIH3T3 L1 was stained with Oil red O for 3 hr. The dye containing lipid droplet was extracted with isopropanol and its absorbance was determined at 510 nm. b Percent(%) : absorbance ratio to control. c Control : no treatment 지방세포로의분화에미치는영향을조사하였다 (Table 3). NIH3T3 L1이지방세포로분화되는정도의측정은지방세포내 lipid droplet 형성정도로측정하였다. 즉지방세포내 lipid droplet을 Oil red O 시약으로염색하여 isopropanol로용출시켜 510 nm에서흡광도를측정한결과, Table 3에서보는바와같이 TF1, TF3를처리한실험구에서는대조구에비해지방세포분화능이각각 76.75, 60.19% 로나타났다. 반면, TF2를처리한실험구에서는 24.84% 의지방세포분화도를나타내었으며, orlistat를처리한실험구에서는 23.88% 의분화도를나타내는것으로조사되었으므로 TF2 및 orlistat 의경우지방세포분화저해정도가우수함을확인할수있었다. 따라서활성물질 TF2가지방세포분화의지표로사용되는 lipid droplet의형성을억제함으로써지방세포분화를저해하는효과도가지고있음을확인할수있었다. 요 Pancreatic lipase의활성을저해하는물질을탐색하고자국내의식용또는약용식물의추출물을대상으로 lipase 활성에대한저해능력이있는식물추출물은탐색한결과대복피, 계혈등, 오배자및백자인의식물추출물을선정하였다. Pancreatic lipase에대한저해활성이우수한백자인으로부터클로로포름용매추출, 실리카겔컬럼크로마토그라피, 세파덱스 LH20 컬럼크로마토그라피와고속액체크로마토그라피를실시하여 pancreatic lipase에대한저해활성물질로 TF1, TF2, TF3를분리하였다. 이들을대상으로 porcine pancreatic lipase에대한저해효과를측정한결과, 활성물질 TF1, TF2, TF3 및 orlistat의 porcine pancreatic lipase에대한 IC 50 값은각각 44.7, 98.7, 46.1 및 27.6 μg /ml인것으로나타났다. 활성물질 TF2와 orlistat의경우에는 10 μg /ml의농도에서 NIH3T3 L1의지방세포로의분화에도억제효과가있음을입증하였다. 약
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