한수지 48(6), 888-897, 215 Originl Artile Koren J Fish Aqut Si 48(6),888-897,215 Shewnell oneiensis PKA18 유래알긴산분해효소에의해제조된알긴산올리고당의항염증효과 김민지 배난영 박시우 김꽃봉우리 1 박지혜 박선희 안동현 * 부경대학교식품공학과 / 식품연구소, 1 부경대학교수산과학연구소 Anti-Inflmmtory Effet of Alginte Oligoshries Proue y n Alginte-Degring Enzyme from Shewnell oneiensis PKA18 on LPS-Inue RAW 264.7 Cells Min-Ji Kim, Nn-Yong e, Si-Woo rk, Koth-ong-Woo-Ri Kim 1, Ji-Hye Prk, Sun-Hee Prk n Dong-Hyun Ahn* Deprtment of Foo Siene & Tehnology/Institute of Foo Siene, Pukyong Ntionl University, usn 48513, Kore 1 Institute of Fisheries Sienes, Pukyong Ntionl University, usn 4641, Kore The nti-inflmmtory effet of lginte oligoshries on LPS-inue RAW 264.7 ells ws investigte t ifferent time points ( 6 h). The lginte oligoshries were proue y n lginte-egring enzyme from Shewnell oneiensis PKA18. The lginte oligoshries erese the proution of nitri oxie n proinflmmtory ytokines [tumor nerosis ftor-α, interleukin (IL)-1β, n IL-6] in ose-epenent mnner. The lginte oligoshries showe pek nti-inflmmtory tivity fter 36 h of inution; t tht time point, reue protein expression of NF-κ p65, inos, n COX-2 ws etete. Furthermore, the lginte oligoshrie tretment reue the formtion of er eem t 36 h ompre to smples exmine t h when the oligoshries were ministere t 5 n 25 mg/kg oy weight, s well s erml thikness n mst ell numers in histologil nlysis. These results suggest tht lginte oligoshries re promising nti-inflmmtory gent. Key wors: Alginte oligoshries, Anti-inflmmtion, Er eem, NF- kpp 서론, (Lee et l., 1992), (Kim et l., 21), (Sot et l., 1987) (Cho et l., 199) (Kim et l., 211).,,. -D-mnnuroni i (M) -L-guluroni i (G) 1,4-glyosie,, M G,,,, (Horn et l., 1999; Moen et l., 1999; Zvygintsev et l., 25).,,,, (Cho et l., 23). (Lee et l., 1996; Lee et l., 1993)., http://x.oi.org/1.5657/kfas.215.888 Koren J Fish Aqut Si 48(6) 888-897, Deemer 215 This is n Open Aess rtile istriute uner the terms of the Cretive Commons Attriution Non-Commeril Liens (http://retiveommons.org/lienses/y-n/3./) whih permits unrestrite non-ommeril use, istriution, n reproution in ny meium, provie the originl work is properly ite. Reeive 11 Novemer 215; Revise 18 Deemer 215; Aepte 22 Deemer 215 *Corresponing uthor: Tel: +82. 51. 629. 5831 Fx: +82. 51. 629. 5824 E-mil ress: hhn@pknu..kr Copyright 215 The Koren Soiety of Fisheries n Aquti Siene 888 pissn:374-8111, eissn:2287-8815
알긴산올리고당의항염증효과 889 (Kim et l., 21), (Song et l., 28), (Lee et l., 1975).,,.,. (Uo et l., 26), (rk et l., 215), (Iwmoto et l., 23).,,, (Zmor et l., 2). (mrophge) lipopolyshrie (LPS) nitri oxie (NO), interleukin-6 (IL-6), tumor nerosis ftor- (TNF- ) IL-1 ytokine (Higuhi et l., 199). rhioni i ylooxygense (COX) leukotriene, thromoxne, prostglnin NO, (Jeong et l., 214). NO ytokine, (Cho et l., 212). (Kng et l., 214). (Sunwoo et l., 213) Shewnell oneiensis PKA18. 시험균주 재료및방법 (Ulv pertus) S. oneiensis PKA18 (Sunwoo et l., 213). 조효소액제조 S. oneiensis PKA18 mrine roth (Difo, Sprks, MD, USA) 3 C 24, (Supr 3K, Hnil Siene Co., Kore) 4 1, g 3. 알긴산올리고당제조 (7%) 1:1 (w/v) 12 h -6 h., 95-1 1., 1, g, 1 min, 4, -2. thin-lyer hromtogrphy (TLC), sili gel F 254 plte smple 3 L spotting 1-utnol:eti i:wter (2:1:1 v/v/v) sili gel plte 1% 11 2. Stnr mixture ellooligoshrie, elloiose, ellotriose, ellotetrose, ellopentose, ellohexose Megzyme (Megzyme Co., Wiklow, Ireln) gluose Sigm-Alrih Chemil Co. (St. Louis, MO, USA). 세포배양 Murine RAW 264.7 (KCL 471), DMEM 1% intivte fetl ovine serum 1% peniillin-streptomyin 37, 5% CO 2. 8-9%, 2 pssges. 실험동물 8, ICR (Seongnm, Kore) 2 2 C, 5 1%, 12 h 1. ( 214-1). 세포독성측정 Prk et l. (26) MTT ssy. RAW 264.7 ell 1 1 6 ells/ml well plte 2 h, 1 g/ml LPS (1, 5, 1 g/ml)
89 김민지ㆍ배난영ㆍ박시우ㆍ김꽃봉우리ㆍ박지혜ㆍ박선희ㆍ안동현 37, 5% CO 2 inutor (MCO-15AC, Snyo, Osk, Jpn) 22 h. 5 mg/ml MTT (thizol lue tetrzolium romie, Sigm Chemil Co.) 2 h. 4, 879 g 1 (UNION 32R, Hnil Co., Inheon, Kore)., well DMSO miroplte reer(moel 55, io-r, Rihmon, CA, USA) 54 nm [optil ensity (O.D.)].. Prolifertion inex (%) = (smple O.D./ ontrol O.D.) 1 Nitri oxie 생성량측정 NO nitrite griess (Lee et l., 2). RAW 264.7 ell DMEM 2.5 1 5 ells/ml 24 well plte 5% CO 2 inutor (MCO-15AC, Snyo, Osk, Jpn) 2 h. 1 g/ml LPS 1, 5, 1 g/ml 24 h., griess (1% sulfnilmie +.1% nphthylenimine ihyrohlorie, 1:1) 1, miroplte reer (Moel 55, io-r) 54 nm. NO soium nitrite (NNO 2 ). 염증관련 ytokines 분비량측정 ytokine RAW 264.7 ell DMEM 2.5 1 5 ells/ml 24 well plte 5% CO 2 inutor (MCO-15AC, Snyo, Osk, Jpn) 18 h, 1 g/ml LPS 1, 5, 1 g/ml 12 h. TNF-, IL-6 IL-1 ytokine ELISA kit (Mouse ELISA set, D iosiene, Sn Diego, CA, USA). ELISA miroplte pture ntioy nti-mouse TNF-, IL-6 IL-1 4 oting..5% Tween 2 PST 1% FS loking. PST, miroplte 2 h. PST iotinylte nti-mouse TNF-, IL-6 etetion ntioy streptviin-horserish peroxise onjugte 1 h. IL-1, iotinylte nti-mouse IL-1 etetion ntioy 1 h, streptviin-horserish peroxise onjugte 3., PST, OPD 3. 2 N H 2 SO 4, miroplte reer (Moel 55, io-r) 49 nm. inos, COX-2 및 NF-κ 발현량측정 inos, COX-2 NF- RAW 264.7. 3 phosphte uffere sline (PS), Shee n Ash (29) inos COX-2 ytosol lysis uffer [5 mm HEPES (ph 7.4), 15 mm NCl, 5 mm EDTA, 1% eoxyholte, 5 mm phenylmethylsulfonyl fluorie, 1 g/ml protinin, 1% Triton X-1,.1% NP-4, NF- nuleus lysis uffer (1 mm HEPES, 1 mm NCl, 1.5 mm MgCl 2,.1 mm EDTA,.1 mm itiotreitol) 3 4 C lysis 15,52 g 2. CA protein ssy kit (Piere, Rokfor, IL, USA), 3 L lyste Lemmli (197) 1% SDS-PAGE. Towin et l. (1979) polyvinyliene ifluorie memrne (io-r) 1 5% skim milk tris uffere sline (TSS, ph 7.5) 2 loking. inos, COX-2 NF- nti-mouse inos, COX-2 NF- 1:5 2 TSS 3. 2 horserish peroxise nti-mouse IgG nti-rit IgG 1:2, 1 TSS 3 ECL 1-3 Gene tool (GeneGnome5, Syngene, Cmrige, UK). 귀부종측정및조직관찰 in vivo Kim et l. (22) Sriv et l. (211). 8, ICR h, (36 h) 1, 5 25 mg/kg oy weight 2 L 2.5% roton oil 2 L/er. 5 roton oil. ICR 1 mg/ml 2 L 15, 5% roton oil 2 L. 6, iethylether, 1% formlehye 72 h. hemtoxylineosin (H&E) toluiine-lue (T&)
알긴산올리고당의항염증효과 891. Eem formtion (%) = Smple / Control 1 통계처리 SAS progrm (Sttistil nlytil system V8.2, SAS Institute In., Cry, NC, USA), Dunn P<.5. 결과및고찰 반응시간에따른조효소에의한알긴산분해정도 S. oneiensis PKA18 TLC. -6 h (Fig. 1), 12, 24 h 36 h trishrie pentshrie, 48 h monoshrie. S. oneiensis PKA 18,. 세포독성측정 1, 5 1 g/ml (Fig. 2), RAW 264.7 PS. RAW 264.7 1, 5 1 g/ml. Nitri Oxie 생성량측정 NO inos -rginine -itrulline L L (Lee et l., 26).,,, NO peroxynitrite, nitrogen ioxie,, (Jeong et l., 212; Kim et l., 212). ytohrome C, poptosis inuing ftor (Hippeli n Elstner, 1999). -6 h NO RAW 264.7 LPS. 1, 5 1 g/ ml NO griess (Fig. 3). PS 5.88.13 M, LPS 55.31 1.22 M 1. NO 24 h -6 h RAW 264.7 MTT ssy. Monomer Dimer Trimer Tetrmer Pentmer Hexmer Prolifertion inex (%) 12 1 8 6 4 2 ND 1 μg/ml 5 μg/ml 1 μg/ml 1 2 3 4 5 6 7 1 PS h 12 h 24 h 36 h 48 h 6 h Time of enzyme retion Fig. 1. TLC nlysis of lginte oligoshries otine from rue lginte egring enzyme in S. oneiensis PKA 18 with vrious inution time. Retion time : lne 1, stnr; lne 2. h; lne 3, 12 h; lne 4, 24 h; lne 5, 36 h; lne 6, 48 h, lne 7, 6 h. Stnr mixture of monomer gluose, imer elluiose, trimer ellotriose, tetrmer ellotetrose, pentmer ellopentose, n hexmer ellohexose. Fig. 2. Effet of lginte ( h) n lginte oligoshrie (12, 24, 36, 48, n 6 h) on the prolifertion of RAW 264.7 ells. Cells were inute in the presene of smples (1, 5, n 1 μg/ ml) for 24 h. Cell prolifertion ws etermine y MTT ssy. Prolifertion inex (%) = (smple O.D./ ontrol O.D.) 1. ND: not signifintly ifferent. Dt represent the men ± S.D. from three experiments.
892 김민지ㆍ배난영ㆍ박시우ㆍ김꽃봉우리ㆍ박지혜ㆍ박선희ㆍ안동현 Nitri Oxie (μm) 7 6 5 4 3 2 1 F g A C D e Time of enzyme retion, 5-1 g/ml ( h)., 36 h 1 g/ml 3.93.88 M LPS 44% NO. 24 h trishrie pentshrie, 48 monoshrie NO. Yng et l. (21) NO, NO. 염증관련 ytokines 분비량측정 1 μg/ml 5 μg/ml 1 μg/ml PS LPS h 12 h 24 h 36 h 48 h 6 h LPS TNF-, IL-6 IL-1. IL-1, TNF- (Dinrello et l., 199; Stnkiewiz et l., 22)., IL-6 T (Liu et l., 1998)., ytokine. (Fig. 4), 1, 5 1 g/ml ytokine. TNF- -6 E f D E e Fig. 3. Inhiitory effet of lginte ( h) n lginte oligoshries (12, 24, 36, 48, n 6 h) on the proution of nitri oxie in LPS-inue RAW 264.7 ells. Cells were inute in the presene of LPS (1 μg/ml) lone or in omintion with smples (1, 5, n 1 μg/ml) for 24 h. The ulture mei of the trete ells were use to mesure NO levels. Mens with ifferent letters (- : 1 μg/ml, A-F : 5 μg/ml, -g : 1 μg/ml) ove the rs re signifintly ifferent (P<.5). h 1-5 g/ml, 12-36 h 1 g/ml LPS 3%. IL-1 12 h, 24 h. 1 g/ml 24-6 h, 36 h 1 5 g/ml, 3% 4%. IL-6 LPS 557.52 18.48 pg/ml,, 24-6 h,, 48-6 h 1 g/ml 6%. Yoon et l. (27) TNF- IL-6. Yng et l. (21), IL-1, (1-3 kd).,, 36 h. inos, COX-2 발현에미치는영향 inos inos NO,,, (Guh n Mkmn, 21). COX-2 ytokine,,, prostglnin E 2 (PGE 2 ) (Chun n Surh, 24). inos COX- 2 western lot (Fig. 5). NO pro-inflmmtory ytokine 36 h inos COX-2., RAW 264.7 LPS (1 g/ml) inos COX-2 1 g/ml, 5 g/ml 1 g/ml. 36 h NO inos, COX-2 COX-2. NF-κ p65 활성화에미치는영향 LPS, TNF- NF- p65 inos
알긴산올리고당의항염증효과 893 TNF-α (pg/ml) 3 25 2 15 1 A 1 µg/ml 5 µg/ml 1µg/mL C C D IL-1 β (pg/ml) 4 35 3 25 2 15 A A A C D C 1 µg/ml 5 µg/ml 1µg/mL C 1 5 E e PS LPS h 12 h 24 h 36 h 48 h 6 h Time of enzyme retion 5 E PS LPS h 12 h 24 h 36 h 48 h 6 h Time of enzyme retion 6 5 A 1 µg/ml 5 µg/ml 1µg/mL IL-6 (pg/ml) 4 3 2 CD D C 1 e E e PS LPS h 12 h 24 h 36 h 48 h 6 h Time of enzyme retion Fig. 4. Inhiitory effet of lginte ( h) n lginte oligoshries (12, 24, 36, 48, n 6 h) on the TNF-α, IL-1β, n IL-6 in RAW 264.7 ells. Cells were trete with the inite onentrtions of smples (1, 5, n 1 μg/ml) in the presene or sene of LPS (1 μg/ml) for 24 h. Culture superntnts were then isolte n nlyze using ELISA kit for ytokines. Mens with ifferent letters (-e : 1 μg/ml, A-E : 5 μg/ml, -e : 1 μg/ml) ove the rs re signifintly ifferent (P<.5). COX-2. NF- p65 (Tk n Firestein, 21). 36 h NF- p65 western lot. (Fig. 5), NF- p65, 5 g/ml 4%. NF- p65., LPS NF- signling. 귀부종억제효과및조직관찰 h 36 h 1, 5 25 mg/kg 2 L roton oil. Croton oil (Towin
894 김민지ㆍ배난영ㆍ박시우ㆍ김꽃봉우리ㆍ박지혜ㆍ박선희ㆍ안동현 LPS (1 μg/ml) Smple (μg/ml) + - + + + - - 1 5 1 inos COX-2 NF-κ p65 β-tin A 1.2 Density Rtio 1..8.6.4 inos Density Rtio 1.2 1..8.6.4 e COX-2 C Density Rtio 1.2 1..8.6.4 NF-κ p65.2. LPS Smple (μg/ml) + - + + + - - 1 5 1.2. LPS Smple (μg/ml) + - + + + - - 1 5 1.2. LPS Smple (μg/ml) + - + + + - - 1 5 1 Fig. 5. Effet of lginte oligoshrie (36 h) on LPS-inue inos, COX-2, n NF-κ p65 expression in RAW 246.7 ells. The levels of inos, COX-2 in the ytosoli protein n the p65 suunit of NF-κ in nuler protein were etermine y western lot nlysis. RAW 264.7 ells were trete with the inite onentrtions of lginte oligoshrie (1, 5, n 1 μg/ml) n LPS (1 μg/ml) for 18 h or 3 min n the proteins were etete using speifi ntioies. et l., 1995). prenisolone, % Eem formtion 12 1 8 6 4 2 f Fig. 6. Inhiition of lginte ( h) n lginte oligoshrie (36 h) ginst roton oil-inue mouse er eem. Mens with ifferent letters ove the rs re signifintly ifferent (P<.5). Control 1 5 1 5 25 1 5 25 mg/kg Prenisolone h smple 36 h smple e ortiosteroi roton oil (Tk n Firestein, 21). Croton oil (Fig. 6), h 36 h, 1 mg/kg w 5 1 mg/kg w 36 h h., 36 h 25 mg/kg oy weight positive ontrol 5 mg/kg. h 36 h. (Fig. 7), roton oil h 36 h prenisolone, mst ell., trishrie pentshrie, S. oneiensis PKA18
895 알긴산 올리고당의 항염증 효과 Control Prenisolone h 36 h Non -inflme 1 H&E 2 T& Fig. 7. Photomirogrph of trnsverse setions of mie ers sensitize with topil pplition of roton oil 5% (v/v) in etone or vehile etone stine with hemtoxylin-eosin n toluiine-lue exmine uner light mirosopy (mgnifition: 2). Tretments: vehile 2% Tween 8, prenisolone.8 mg/er, h smple 2 μl/er, n 36 h smple 2 μl/er. The numers 1 n 2 inite ermis n epiermis, respetively. The rrow inites mst ell. 산 분해효소는 알긴산의 고점도 문제 해결과 함께 알긴산 올리 고당의 항염증 활성도 증진시켜 산업적으로 활용 가능성이 높 을 것으로 사료된다. 사 사 이 논문은 부경대학교 자율창의학술연구비(215년)에 의하 여 연구되었습니다. Referenes rk SW, Kim KWR, Kim MJ, Kng K, Pk WM, Ahn NK, Choi YU, Prk JH, e NY, Lim SM n Ahn DH. 215. Antioxint effet of enzymti hyrolyste from Srgs sum thunergii using Virio rssostree PKA 12 rue enzyme. Miroiol iotehnol Lett 43, 15-111. http:// x.oi.org/1.414/ml.151.13. Cho HJ, Shim JH, So HS n Yoon JH. 212. Mehnism unerlying the nti-inflmmtory tion of pietnnol inue y lipopolyshrie. J Koren So Foo Si Nutr 41, 12261234. http://x.oi.org/1.3746/jkfn.212.41.9.1226. Cho KJ, Lee YS n Ryu H. 199. Antitumor effet n immunology tivity of sewees towr srom-18. ull Kor Fish So 23, 345-352. Cho M, Kim Y n Rhim JH. 23. Degrtion of lginte solution y using γ-irrition n orgin i. Koren J Foo Si Tehnol 35, 67-71. Chun KS n Surh YJ. 24. Signl trnsution pthwys regulting ylooxygense-2 expression: potentil moleulr trgets for hemoprevention. iohem Phrmol 15, 18911. http://x.oi.org/1.116/j.p.24.5.31. Dinrello CA, Enres S, Meyni SN, Meyni M n Hellerstein MK. 199. Interleukin-1, norexi, n ietry ftty is. Ann N Y A Si 587, 332-338. Guh M n Mkmn N. 21. LPS inution of gene expression in humn monoytes. Cell Signl 13, 85-94. http:// x.oi.org/1.116/s898-6568()149-2. Higuhi M, Hisghi N, Tki H n Osw T. 199. Cytolyti mehnism of tivte mrophges. Tumor nerosis ftor n L-rginine epenent mehnisms t synergistilly s the mjor ytolyti mehnism of tivte mrophges. J Immunol 144, 1425-1432. Hippeli S n Elstner EF. 1999. Inhiition of iohemil moel retions for inflmmtory proesses y plnt extrts; review on reent evelopments. Free Ri Res 31, 81-87. http://x.oi.org/1.18/1715769931361. Horn SJ, Moen E n Ostgr K. 1999. Diret etermintion lginte ontent in rown lge y ner infr-re (NIR) spetrosopy. J Appl Phyol 11, 9-13. http://x.oi. org/1.123/a:18249954. Iwmoto Y, XU X, Tmur T, ODA T n Murmtsu T. 23. Enzymtilly epolymerize lginte oligomers tht use ytotoxi ytokine proution in humn mononuler ells. iosi iotehnol iohem 67, 258-263. http://x.oi. org/258-263. 1.1271/.67.258.
896 김민지ㆍ배난영ㆍ박시우ㆍ김꽃봉우리ㆍ박지혜ㆍ박선희ㆍ안동현 Jeong DH, Kng K, Kim KWR, Kim MJ n Ahn DH. 214. Anti-inflmmtory tivity of Srgssum mirnthum wter extrt. J Appl iol Chem 57, 227-234. http://x.oi. org/1.3839/j.214.36. Jeong HR, Sung MS, Kim YH, Hm HM, Choi YM n Lee JS. 212. Anti inflmmtory tivity of Slvi pleei R. r. lef through Heme Oxygense-μinution in LPS-stimulte RAW 264.7 mrophges. J Koren So Foo Si Nutr 41, 888-894. http://x.oi. org/1.3746/jkfn.212.41.7.888. Kng K, Kim KWR, Kim MJ, rk SW, Pk WM, Kim R, Ahn NK, Choi YU n Ahn DH. 214. Anti-inflmmtory tivity of n ethnol extrt of Lminri jponi root on lipopolyshrie-inue inflmmtory responses in RAW 264.7 ells. Koren J Foo Si Tehnol 46, 729-733. Kim DW, Chi YS, Son KH, Chng HW, Kim JS, Kng SS n Kim HP. 22. Effets of sophorflvnone G, prenylte flvonoi from Sophor flvesens, on ylooxygense-2 n in vivo inflmmtory response. Arh Phrm Res 25, 329-335. http://x.oi. org/1.17/f2976635. Kim JH, Kim YH, Kim SK, Kim W n Nm SW. 211. Properties n inustril pplitions of sewee polyshries-egring enzymes from the mrine miroorgnisms. Koren J Miroiol iotehnol 39, 189-199. Kim OJ, Lee DG, Lee SM, Lee SJ, Do HJ, Prk HJ, Kim A, Lee JH n H MJ. 21. Isoltion n hrteristis of lginte-egring Methyloterium sp. HJM27. Kor J Miroiol iotehnol 38, 144-15. Kim YS, Lee SJ, Hwng JW, Kim EH, Prk PJ n Jeong JH. 212. Anti-inflmmtory effets of extrts from Ligustrum ovlifolium H. leves on RAW 264.7 mrophges. J Koren So Foo Si Nutr 41, 125-121. http://x.oi. org/1.3746/jkfn.212.41.9.125. Lemmli UK. 197. Clevge of struturl proteins uring the ssemly of the he of teriophge T4. Nture 227, 68-685. http://x.oi.org/1.138/22768. Lee HJ, Lee CW, Choi MS, Son DJ n Hong JT. 26. Effets of estheti essentil oils on LPS-inue nitri oxie genertion in murin mrophge RAW 264.7 ells. J So Cosmet Si Kore 32, 111-116. Lee HS, Choi MS, Lee YK, Prk SH n Kim YJ. 1996. A stuy on the evelopment of high-fier supplements for the ieti ptients- Effet of sewee supplementtion on the lipi n gluose metolism in streptozotoin inue ieti rts. J Nutr Helth 29, 296-36. Lee JK, Choi HS, Yoon SK n Kim WJ. 1993. Effet of extrtion temperture on some qulity of se tngle extrt. J Koren So Foo Nutr 22, 771-776. Lee SR, Cho HO n Prk SK. 1975. Extrtion yiel n qulity ttriutes of gr from omesti sewees oring to vrious pretretment. Koren J Foo Si Tehnol 7, 19-114. Lee ST, Jeong YR, H MH, Kim SH, yun MW n Jo SK. 2. Inution of nitri oxie n TNF-α y herl plnt extrts in mouse mrophges. J Koren So Foo Si Nutr 29, 342-348. Lee YS, Kim DS, Ryu H n Lee SH. 1992. Antitumor n immunomoulting effets of sewees towr srom-18 ell. J Kor So Foo Nutr 21, 544-55. Liu M, Li J, Kong F, Lin J n Go Y. 1998. Inution of immunomoulting ytokines y new polyshrie-peptie omplex from ulture myeli of Lentinus eoes. Immunophrmology 4, 187-198. Moen E, Lrsen, Ostgr K n Jensen A. 1999. Alginte stility uring high slt preservtion of Asohyllum noosum. J Appl Phyol 11, 21-25. http://x.oi. org/1.123/a:1877412543. Prk YM, Won JH, Yun KJ, Ryu JH, Hn YN, Choi SK n Lee KT. 26. Preventive effet of Ginkgo ilo extrt (G) on the lipopolyshrie-inue expressions of inuile nitri oxie synthse n ylooxygense-2 vi suppression of nuler ftor-κ in RAW 264.7 ells. iol Phrm ull 29, 985-99. http://x.oi.org/1.1248/p.29.985. Sriv RA, Arrun MK, Oliveir RC, Menezes KD, Leite GO, Kerntopf MR, Cost JG, Roh J, Tomé AR, Cmpos AR n Menezes IR. 211. Topil nti-inflmmtory effet of Cryor orieum Wittm. (Cryoree) fruit pulp fixe oil on mie er eem inue y ifferent irritnt gents. J Ethnophrmol 136, 54-51. http://x.oi. org/1.116/j.jep.21.7.2. Sot M, Colin GM, Dvi J, Mills L n rin J. 1987. Estimtion of meioenthi nemtoe iversity y non speilists. Mr Pollut ull 18, 646-649. http://x.oi. org/1.116/25-326x(87)9398-5. Shee MS n Ash VV. 29. Criospermum hlium ethnol extrt inhiits LPS inue COX-2, TNF-α n inos expression, whih is meite y NF-κ regultion, in RAW264.7 ells. J Ethnophrmol 124, 39-44. http:// x.oi.org/ 1.116/j.jep.29.4.2. Song EJ, Kim AR, Kim MJ, Lee SY, Kim KWR, Prk JG, Kim JH, Lee JW, yun MW n Ahn DH. 28. Effet of gmm irrition on egring n ntioxition tivity of lginte n rrgeenn. Foo Eng Prog 12, 58-64. http://x.oi.org/ 1.3746/jkfn.28.37.3.357. Stnkiewiz W, Drowski MP, Chilowski A n Plus T. 22. Cellulr n ytokine immunoregultion in ptients with hroni ostrutive pulmonry isese n ronhil sthm. Meitors Inflmm 11, 37-312. Sunwoo C, Kim KWR, Kim DH, Jung SA, Kim HJ, Jeong DH, Jung HY, Kng K, rk S W, Lim SM, Hong YK n Ahn DH. 213. Optimiztion of Conitions for the Proution n Properties of Alginte-egring Crue Enzyme from Shewnell oneiensis PKA 18. Koren J Miroiol iotehnol 41, 372 378. http://x.oi.org/ 1.414/ kjm.1212.122
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