23(3)-08(하배진).fm

J. Fd Hyg. Safety Vol. 23, No. 3, pp. 222~226 (2008) Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr LPS 로유도된급성간독성에대한백작약추출물의보호효과 김인덕 권륜희 허예영 이동근 이재화 이상현 하종명 하배진 * 신라대학교의생명과학대학제약공학과 The Preventive Effects of Paeoniae Radix Extract against LPS-induced Acute Hepatotoxicity In Deok Kim, Ryun Hee Kwon, Ye Young Heo, Dong Geun Lee, Jae Hwa Lee, Sang Hyeon Lee, Jong Myung Ha, and Bae Jin Ha* Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, 1-1 San, Gwaebup-dong, Sasang-gu, Busan, 617-736, Korea. (Received August 19, 2008/Revised August 29, 2008/Accepted September 1, 2008) ABSTRACT The purpose of this study was to investigate the preventive effects of Paeoniae Radix Extract (PRE) against the acute hepatotoxicity-inducing lipopolysaccharide (LPS) in the liver. PRE of 100 mg/kg concentration was intraperitoneally administered into rats at dose of 1.5 ml/kg for 20 days. On day 21, 5 mg/kg of LPS dissolved in saline was injected 4 hours before anesthetization. We examined the levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), lactate dehydrogenase (LDH) in serum of rats, superoxide dismutase (SOD) in mitochondrial fractions, and malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GPx) in liver homogenates. LPS-treatment markedly increased the levels of GOT, GPT, LDH and MDA, and significantly decreased those of SOD, CAT and GPx. But PRE-pretreatment decreased the levels of GOT, GPT, LDH and MDA, by 59.7%, 43.6%, 59.6% and 63.5%, respectively and increased those of SOD, CAT and GPx, by 85.5%, 57.8% and 62.9%, respectively. These results showed that the PRE had the preventive effects against the acute hepatotoxicityinducing LPS in the liver. Key words: Paeonia lactiflora Pallas, LPS, anti-oxidation, hepatotoxicity 서론 백작약 (Paeoniae radix) 은미나리재비과 (Ranuclaceae) 작약속 (paeonia) 에속하는약용작물로서한방에서진통, 진경, 부인약, 고혈압, 복통및염증치료제로널리사용되고있다 1). 그주된생리활성물질로서페오니프로린 (paeoniflorin) 및알비노프로린 (albinoflorin) 과같은여러모노테르펜글루코사이드 (monoterpen glucoside) 와탄닌및페놀산등이잘알려져있다 2). 백작약에관한연구로는항혈전작용 3), 항고지혈작용 4-6), 항산화작용 7,8), 혈관확장 작용 9) 그리고관상동맥질환을가진환자에서 arachidonic acid 등에대한대사작용 10) 에대해보고되었다. *Correspondence to: Bae Jin Ha, Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, 1-1 San, Gwaebup-dong, Sasang-gu, Busan, 617-736, Korea Tel: 051-999-5466, Fax: 051-999-5684 E-mail: bjha@silla.ac.kr LPS는인체내에서 O면역부위로인하여항원 (antigen, Ag) 으로작용한다. Ag는인체내에서자기방어기능으로인하여 macrophage와같은항원제시세포에의하여인식된다. LPS는주로 Toll-like receptor (TLR) 계열에의하여인식되며 CD (cluster of differentiation)-14의결합으로인체내에서방어기전을실행시킨다. LPS의주된특성은패혈성쇼크를일으키는것이며혈장속에떠돌아다니는 LPS-binding protein (LBP) 에의하여독성을일으키는첫단계에돌입한다. 인체내로들어온 LPS는 LBP에의해복합체가만들어지고이것이혈액내의 macrophage의 CD- 14에결합하게되면 TLR-4에인식되어져결합되는순간 nuclear factor kappa B (NFκB) 가활성화된다. NFκB이활성화되면 cytokine과 chemokine 등의물질이과량분비되고내재면역반응을유발시킨다. 그러나패혈성쇼크는 cytokine중에 tumor necrosis factor (TNF-α) 의과다발현으로 apoptosis가유도되어지는것으로 LPS로인한 TNFα의비정상적증가로정상세포들의사멸이일어난다 11). 이 222

The Preventive Effects of Paeoniae Radix Extract against LPS-induced Acute Hepatotoxicity 223 러한 TNF-α의증가는또한 neutrophil을활성화시키는데일조하고 neutrophil의증가로인하여 inducible nitric oxide synthase (inos) 유전자가활성화되어 NO의생성을증가시킨다 12). NO는 macrophage의활성으로인한산물로 oxygen radical과더불어강력한항미생물작용을하며 13) NO가생성될때 Ca 2+ 농도의증가로인해 superoxide anion ( ) 과 H 2 가함께생성되면산화력이강한 OH-로의연쇄반응을일으켜세포조직에영향을주어직간접적으로조직의산화를초래한다 14,15). 따라서본연구는백작약열수추출액의생리효능을체계적으로밝혀보고자 in vivo에서간독성지표와항산화효소활성지표를측정하여간독성에대한보호효과를검증하였다. 실험방법 실험동물및식이실험동물은체중 170 180 g 내외의암컷흰쥐 (Sprague- Dawley계생후 7주 ) 를샘타코 BIO KOREA( 주 ) 로부터제공받았고 7일동안적응시켰다. 실험동물은 cage에각각분리시키고고형사료와물을자유롭게섭취하도록하였다. 실험기간동안동물들은 22±1 o C의온도와 60±5% 상대습도로유지된항온항습기에서사육시켰고총 21마리의흰쥐를 7마리씩 3군으로나누었다 (Table 1.). 정상군 (NOR group) 과대조군 (CON: LPS-treated group) 은 0.9% saline 을, 시료군 (PRE: Paeoniae Radix Extract and LPS-treated group) 은백작약추출물 (100 mg/kg) 을 1.5 ml/kg씩복강내에 20일간매일투여하고 21일째되는날에대조군과시료군에서실험동물의간손상을유도하기위해 LPS를생리식염수로용해시켜 1.5 ml/kg의용량을복강내로투여하였다. LPS를투여하고절식시킨뒤 4시간후에 ether 로마취하고희생시켜서혈액을채취하고간을적출하여실험하였다. 백작약추출물의조제본실험에사용한백작약 (P. Radix) 는부산구포시장에서 구입하였다. 백작약추출물은백작약을 round flask에 200 g 을넣고증류수 2,000 ml를넣은후 8시간씩환류냉각추출한다음여과한여액을농축, 동결건조하여사용하였다. 혈액채취및간적출시료투여기간종료후실험동물을 ether 마취하에심장에서채혈하여실온에서 30분간방치한후 3000 rpm, 4 o C에서 10분간원심분리하여혈청을분리하고실험에사용할용량을각각분주하여 70 o C 냉동고에보관하여실험에사용하였다. 간은 4엽을전부적출하여 0.9% 생리식염수로세척여지로흡착한후 70 o C 냉동고에보관하여실험에사용하였다. 간무게의 5배에해당하는 PBS (Phosphate Buffer Solution 0.05 M ph 7.4) 를넣고간을균질기로균질화하여 liver homogenate로사용하였다. 간무게 10배의 solution (10 mm tris, 0.07 M saccharose, 0.1 mm EDTA, 0.2 M mannitol, in dissolved 0.1 N HCl) 을넣어균질화한후, 600 g, 4 o C에서 10분간원심분리하고상등액을다시 8,000 g, 4 o C에서 10분간원심분리하여얻은 pellet에 0.1 M phosphate buffer (ph 7.0) 5 ml 넣어 mitochondrial fraction으로사용하였다. 혈청중의 GOT, GPT와 LDH 활성측정혈청중의 GOT, GPT와 LDH의양은 Fuji dri-chem clinical chemistry analyzer (Fuji dri-chem 3500, Fujifilm, Japan) 로측정하였다. 간조직의단백질정량단백질의정량은 Lowry 등의방법 16) 에의해서 750 nm 에서흡광도를측정하고표준단백시료를 bovine serum albumin (BSA) 으로정량하였다. 간조직중 malondialdehyde (MDA) 의정량간의 MDA 수치는 Ha의방법 17) 에따라 1ml의 sodium dodecyl sulfate (SDS; 7%) 와균질화한간시료를 0.5 ml 과혼합하여 37 o C, 30분간반응시킨뒤 0.67% thiobarbituric Table 1. Experimental design of rats day 1-20 day 21 Experimental group dose of sample dose of sample NOR (7) 1.5 ml/kg of 0.9% saline, i.p 1.5 ml/kg of 0.9% saline, i.p CON (7) 1.5 ml/kg of 0.9% saline, i.p 1.5 ml/kg of LPS (5 mg/kg), i.p. PRE (7) 1.5 ml/kg of Paeoniae Radix extract (100 mg/kg ), i.p. NOR : normal group CON : LPS-treated group PRE : Paeoniae Radix extract and LPS-treated group The number of experiment animals is given in parenthesis. LPS : lipopolysaccharide i.p : intraperitoneally

224 In Deok Kim, Ryun Hee Kwon, Ye Young Heo, Dong Geun Lee, Jae Hwa Lee, Sang Hyeon Lee, Jong Myung Ha and Bae Jin Ha acid 시약을 2ml 첨가하여한시간동안끓는물에서가열하였다. 가열후즉시냉각시킨뒤부탄올 5ml을첨가하고 3000 rpm에서 10분간원심분리후상층액을취하여 535 nm의파장에서흡광도를측정하였다. 표준시약으로 1,1,4,4-tetraethoxypropane을사용하였다. 간조직내 mitochondria 분획의 superoxide dismutase (SOD) 활성측정 Beauchamp와 Fridovich의방법 18) 에따라 0.2 M K- phosphate buffer (ph 7.4) 를 672 µl, 1 mm xanthine 100 µl, 1% sodium deoxychlorate 30 µl, 1.5 mm KCN 30 µl, 0.2 mm cytochrome C 150 µl를넣은혼합액에 sample 8 µl 를넣고, xanthine oxidase 원액 10 µl를넣은후 Elisa를이용하여 550 nm에서의흡광도변화를 2분동안측정하였다. 효소의활성도는 Sigma사의 superoxide dismutase standard를표준액으로사용하여비교측정하였다. 간조직중 catalase (CAT) 의활성측정 Aebi의방법 19) 을이용하여 phosphate buffer (0.05 M ph 7.0) 1.9 ml에 sample (homogenate와 mitochondrial fraction을 800 g에서 20분간원심분리한상등액 100 µl를 buffer로 10, 20, 40, 80배희석 ) 0.1 ml와과산화수소용액 1ml를혼합하여 240 nm에서 90초동안흡광도감소를측정하였다. 간조직중 glutathione peroxidase (GPx) 의활성측정 Lawrence등의방법 20) 에준하여 0.1 M phosphate buffer (4 mm EDTA) 400 µl, 0.01 M NaN 3 70 µl, 0.01 M GSH 70 µl, 1.5 mm NADPH 70 µl, H 2 O 360 µl, GSSG-reductase (1.8 U/ml) 20 µl, sample 10 µl를혼합하여상온에서 1분간방치한후 5mM H 2 100 µl를가해잘섞은후 340 nm 에서 90초동안흡광도감소를측정하였다. 통계처리본실험에대한모든실험결과는평균치와표준편차로나타내었고, SPSS 통계 package를이용하여일원배치분산분석 (one-was analysis of variance) 을한후 p < 0.05 수 준에서 Scheffe s test에의하여각실험군의평균치간의유의성을검정하였다. 결과및고찰 혈청중 GOT, GPT와 LDH의활성변화간조직의손상은세포내부에존재하는효소가혈액으로유출되는것을측정하거나 pericentral necrosis를관찰함으로써확인할수있다. 따라서간으로부터혈액에방출된효소의활성도측정은간손상연구에있어서가장유용한방법중의하나이다. 혈청중 GOT와 GPT의상승은간손상으로인한간세포의괴사와간조직의파괴가진행됨에따라아미노기전이효소 (transaminase) 가혈중으로유리되어높게나타나는것이므로간세포의변성및괴사의지표가될수있다 22). GOT는글루탐산의아미노기를옥살로아세트산으로전이시켜주는효소로아미노기가전이되면글루탐산은 α-케토글루타르산이되고옥살로아세트산은아스파르트산이된다. GPT는글루탐산의아미노기를알라닌으로전이시켜주는효소로아미노기가전이되면글루탐산은 α-케토글루타르산이되고알라닌은피루브산이된다 23). LDH는몸안의당이분해되어피루브산이젖산로변할때작용하는효소로, 여러조직의세포중에함유되어있어서세포가파괴되면혈중으로유리되어높게나타나므로 GOT, GPT와같이간질환의지표로사용된다. 본실험에서는 LPS 투여군은대조군으로하고, LPS 투여로간손상을유도함과동시에 20일간백작약추출물을복강투여한실험군의간손상정도를살펴보기위해 GOT, GPT, LDH의혈청중의효소활성변화를측정하였다. Table 2와같이 GOT의경우대조군은 LPS의간장애유발로인하여정상군에비해약 2.21배증가하였으나, 시료군인 PRE군은대조군과비교하였을때 59.7% 감소하였다. GPT 의경우에도 LPS를투여한대조군이정상군에비해약 3.5배정도증가하였고, PRE군은대조군에비해 43.6% 로감소하는것으로나타났다. 사염화탄소는 LPS와같은간독성물질로서간세포의괴사와간조직의파괴를유도하여 GOT와 GPT를혈중으로유출되는것을증가시키는데, Park Table 2. Effects of Paeoniae Radix extract on GOT, GPT and LDH levels in serum Experimental group GOT (U/L) GPT (U/L) LDH (U/L) NOR 66.10 ± 3.20 a 24.50 ± 1.25 a 141.25 ± 8.39 a CON 146.50 ± 9.10 c 88.10 ± 2.64 b 624.00 ± 7.09 c PRE 98.50 ± 6.24 b 60.40 ± 1.91 b 336.50 ± 5.85 b NOR: normal group CON: LPS-treated group PRE: Paeoniae Radix extract and LPS-treated group Values represent means ± SD (n = 7); values sharing the same superscript letter are not significantly different from each other (p <0.05) by Scheffe s multiple range test. GOT: glutamate oxaloacetate transaminase GPT: glutamate pyruvate transaminase LDH: lactate dehydrogenase

The Preventive Effects of Paeoniae Radix Extract against LPS-induced Acute Hepatotoxicity 225 Table 3. Effects of Paeoniae Radix extract on MDA, SOD, CAT and GPx activities in liver homogenate and mitochondrial fraction Experimental group MDA (nmol/mg protein) SOD CAT GPx liver homogenate mitochondrial fraction liver homogenate liver homogenate NOR 2.86 ± 0.06 a 131.10 ± 1.98 c 302.98 ± 2.44 c 133.63 ± 4.13 b CON 6.68 ± 1.11 c 77.30 ± 2.49 a 162.83 ± 3.59 a 52.79 ± 1.65 a PRE 4.25 ± 0.82 b 25.06 ± 2.77 b 43.91 ± 4.72 b 103.70 ± 1.49 b NOR: normal group CON: LPS-treated group PRE: Paeoniae Radix extract and LPS-treated group Values represent means ± SD (n = 7); values sharing the same superscript letter are not significantly different from each other (p < 0.05) by Scheffe s multiple range test. MDA: malondialdehyde SOD: superoxide dismutase CAT: catalase GPx: glutathione peroxidase 등 24) 은 GOT와 GPT의경우사염화탄소를투여군의경우정상군과비교시 4.3, 4.5 배로증가하였으며, 천연초추출물을처리시 36%, 41% 감소시켰다고보고됨에따라본연구에서도 LPS로유도된간손상에의해증가한혈청중의 GOT와 GPT 함량이백작약추출물의처리를통해현저히저하되었음을볼수있었다. 또한, 혈청중의 LDH 활성도는 LPS를투여로정상군에비해 4.42배로현저하게증가하였으나, 백작약추출물을처리한 PRE군의경우대조군에비해 59.6% 로유의적인감소를나타내는것을알수있었다. 따라서혈중 GOT, GPT 효소활성과연관하여동일한저해효과볼수있었으며, LPS에의해유발된간손상으로부터백작약추출물의보호효과를확인할수있었다. 간조직및 mitochondrial fraction 중 MDA, SOD, CAT 및 GPx 측정항산화계효소들은대사과정중발생하는활성산소종에의해그활성이비가역적으로불활성화될수있으며, 또한지방산화에의해생성된유리기도제거하는능력이있다고보고되고있다 25,26). 생체내의항산화방어기구중에서효소적방어계의하나인 SOD는주로 mitochondria 에존재하며 superoxide radical을환원하여 H 2 를생성함으로써생체를보호한다 27,28). 생체내생명현상에서필수적인산화환원반응의일종으로생성되는 H 2 를 H 2 O와 로분해하는효소중하나가 CAT 이다. 이것은다수의 H 2 생성효소들과복합체를형성하여 peroxisome에주로분포하며 H 2 증가에따른조직손상을방어하는효과가있다고알려져있다 29,30). Glutathione은산화적인손상으로부터적혈구 (red cell) 을보호한다. 그것은 disulfide bond에의해서연결된두개의 tripeptide에의해서환원된형태 (GSH) 와산화된형태 (GSSG) 사이를순환한다. GSSG는전자근원과같은 NADPH를사용하는 flavoprotein인 glutathione reductase에의해서 GSH로환원된다. Glutathione 은호기적생명체에서해로운부산물인 hydrogen peroxide 와 organic peroxide와함께반응함으로써해독작용에서중요한역할을수행한다. 이반응에서촉매효소인 GPx는 selenium 원자가공유결합되어있는것이주목할만하다. 이효소는 H 2 와 lipid peroxide와같은 peroxides의다양한종류들을조절한다 20). Table 3는백작약추출물의투여가 LPS를투여한흰쥐의간과미토콘드리아에서의지질과산화와항산화효소의함량에미치는영향을측정한것이다. Table 3에나타난바와같이, 간조직에서의 MDA level은정상군에비해대조군이약 2.33배높게나타났고 PRE군은대조군에비해 63.5% 의유의적인지질과산화억제효과를보였다. SOD 활성정도는대조군이정상군에비해서약 1.72배정도로낮게나타났고 PRE군은대조군에비해 85.5% 의상승효과를보였다. 또한 CAT 의활성도는대조군이정상군에비해약 1.86배감소를보였으며 PRE군은대조군에비해 57.8% 의상승효과를나타냈다. GPx 활성도의경우대조군은정상군에비해약 2.21배의감소율을보였으며 PRE군은대조군에비해서 62.9% 의증가율을보였다. 또한 KIM의연구 31) 에따르면예로부터식용또는약용식물로사용되는복분자의경우 LPS로유도된산화적스트레스로인해과산화지질의증가와항산화효과가저하되었지만, LPS에의해증가된과산화지질의함량을감소와항산화효소의증가를시켰다는보고와유사하게나타났다. 따라서백작약추출물은지질과산화와항산화효과가높은것으로판단된다. 요약 본연구에서는한방에서약용으로사용하고있는미나리재비과에속하는다년생초본으로백작약의열수추출물을이용하여 LPS로유발된급성간독성에서의간보호효과를나타내는지살펴보고자하였다. 간독성이유발된쥐의혈청에서의 GOT, GPT 그리고 LDH의감소는백작약추출물이간독성으로부터간을보호하였음을보여주며, 간

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