약학회지제 41 권제 5 호 565^570(1997) Yakhak Hoeji Vol. 41, No. 5 천연물로부터염증성포스포리파제 A2 저해제검색 문태철 정광원 정규찬 손건호 * 김현표 * 강삼식 ** 장현욱 * 영남대학교약학대학. '" 안동대학식품영양학과. 강원대학약학대학, * 서울대학교천연물과학연구소 (Received September 24. 1997) Screening of Inflammatory Phospholipase A2 Inhibitors from Natural Products Tae Chul Moon, Kwang Won Chung, Kyu Cham Chung, Kun Ho Son* Hyun Pyo Kim*, Sam Sik Kang** and Hyeun Wook Chang 휴 College o f P h a rm a cy, Y e u n g n a m U n iversity, K orea, * D epart. F ood & N u tr itio n, A n d o n g N a tio n a l U niversity, K orea, *College o f P h a rm a cy, K a n g w a n N a tio n a l U n iversity, K orea, * * N a tu ra l P ro d u c ts R es. In stitu te. S e o u l N a tio n a l U n iversity, K orea. Abstract High levels of extracellular phospholipase A2 (PLase A2) associated with inflammatory process in man and animal models have been extensively reported elsewhere. Thus, a logical approach to the treatment of inflammatory diseases should involve the inhibitors of PLase A2. To develope new PLase A2 inhibitors from natural products, two hundred crude drugs were screened using group 11 PLA2 inhibitory activity. Among them, methanol extract of 5 medicinal plants such as, Raphani Semen, Moutan cortex radicis, Arecae semen, Caryophylli Cortex and Betulae Cortex inhibited more than 90% of PLase Az avtivity at a concentration 2.5 Then. 10 methanol extracts sample were tmasferred into organic solvents. PLase A2 inhibitory effects were found mainly in CHCI3 and EtoAc fractions. Keywords O Inflammation. Group II Phospholipase A2 inhibitor. Natural products. Phospholipase A 2(PLase A 2) 는세균에서부터고등동물에이르기까지광범위하게, 세포막. 과립. 분비액중에존재하는효소로서, glyceropholipid외 2번위처에 ester결합을가수분해하여유러지방산과 lysophospholipid룰생성한다. 과거에는인지질의신생과대사에관련해서연구되어왔으나. 최근에는여러생러현상과병리현상에적접적으로관여함이알려졌다. 다양한생러현상과질병에관여하는 eicosanoid외전구체인 arachidonic acid는막인지질외 2번위치에결함하고있으므로 PLase A 2에의한 arachidonic acid 유러단계반응은생러활성 mediator 생성과정의율속효 " 본논문에관한문외는이저자에게로 ( 전화 ) 053-810-2811 ( 팩스 ) 053-811-3871 소임이예상되고있다. 또한최근국히미량으로혈소판을활성화시키며. 다양한생리활성및질병 ( 기관지천식, 염증. allergy, endotoxin schock 등 ) 에도직. 간접적으로관여함이알려진 PAF(Platelet Activating Factor) 의생합성과정에도 PLase A 2가관여함이밝혀져소 " 크 * arachidonic acid cascade와 PAF cycle외관련된가설이제기되었다 /' PLase A2의생화학적연구는다량의정제표품을쉽게얻을수있는췌장이나사득외효소를이용한해석이주된것이다. 최근에는실험동물을이용한염증 model 즉 gram 옴성균에의한패혈증^\ casein 유도복막염^\ 지연성과던증. allergic uveitis^^ 등과사람의질환즉, 류마치스관절염환자의관절액 "*. 결핵환자외홍막액 "\ 건선피부염환자외상피조직 ^ 과 gram 옴성균에의한 septic schock 환자의 565
문태철 정광원 정규찬 손건호 김현표 강삼식 장현욱 혈장 * 등에서높은세포외성 PLase A 2의활성과 arachidonic acid 대사산물의측적이보고되어염증반응에있어서 PLase Aj가깊이관여함이밝혀졌다. 최근 endotoxin shock rat의조직에서 group II형 PLase A 2의 m RN A level이증가되며 ****, 토끼외 chondriocyte. Rat 신장의 mesangial cell을 TNF나 IL-1 등염증성 cytokine존재하에배양했을때배양액중에세포외성 PLase A 2가방출됨이보고되어 **\ 염증반응에있어서 PLase Az외역할은더욱중요시되고있다. 따라서세계의많은연구자들이 PLase A 2의저해물질을찾아서새로운항염중제개발에힘을기울이고었다. PLase A 2 저해제의개발에는단백질, 천연물. 유기합성, 미생물. 해양생물등에서저해제외개발이많이진행되고있다.* 오 "* 무 * 그러나지금까지 PLase Az 저해제외검색에사용된 PLase A 2는예외없이췌장이나사득의 group I형 PLase A 2를효소원하여행해졌기때문에, 효소원으로서적절하지않다는문제점이제기되었다. 한편, 비만세포 (mast cell) 에서탈과립반응에도 PLase A 2의관여가시사되어즉시형 allergy 반응에있어서 PLase A 2의역활에대해최근관심이모아지고있다, 비만세포는세포막표면상에고친화성 Ig E 수용체를가져서, 항원자극에응헤서염증성 mediator 인 histamine, serotonin 등외과립내저장된내용물을방출하는동시에 prostagrandin D 2(PGD2), leucotriene C 4 (LTC4), PAF 등의지질성 mediator를생산하여방출한다. 지질성 mediator 생성은세포막인지질에서 arachidonic acid 유러에서시작되며, 자극과 coupling하여 PLase A 2가활성화되는현상으로해석되고있다. IgE를개재한비만세포의활성화에는생체막의미량성분인 lysophosphatidylserinedyso P S ) 이필수적임은이미알려져있기때문에 * * PS speific 한 PLase A 2외관여가예상되고있다. 최근즉시형 allergy반응에관여하는 PLase A 2 저해제를이용한 allergy 치료제개발이진행되고있어금후그결과가기대되고있다. 이상에서기술한것과같이 PLase A 2는각종염증성질환에직. 간접으로깊이관여하고있기때문에본연구진은지금까지천연물에서성분외분리및구조걸정에관한다년간외경험을바탕으로 PLase A 2의저해제를찾아서새로운차원의항염증제를개발하는데필요한기초적인자료를제공하려고한다. 실험방법실험재료및시약실험재료 - 본연구에사용한생약은한약도매시장에서구입하여실험재료로사용하였다. 시약 - T ris(hydroxym ethyl)am inom ethane, CaCl2 등은 Sigma 사로부터, 1,4-bis [2-(phenyloxazolyl) ]-benzene (P O P O P ), 2,5-diphenyloxazole (DPO) 는 Dojin사로부터구입하여사용하였으며, 이외의모든시약은특급품을사용하였다. 시료의조제건조한시료 100 g을 m e th and로 3시간 3회열추출하고, 이추출액을합한후농측건조하여이를 methanol 추출물로사용하였다. 각 methanol 추출물을물로현탁시킨후 «-hexane, CHCI3, ethylacetate 및 n-butanol로단계적으로분획하고마지막분획을물분확으로하여저해활성검색에사용하였다. 기질의조제 l-acyl-2-[l-*"*c]linoleoyl-sn-glycero-3-phosphatidylethanolamine 는 l-acyl-2-[l-*\d]linoleoyl-s«- glycercr3-phosphatidylcholine로부터 Plase D 에외한 base exchange방법 ** 에의해제조하였으며, linoleic acid 및 l-acyl-2- ( ] arachidonyl phosphatidylethanolamine는 Amersham사에서구업하여사용하였다. Phospholipase 활성의측정 PLase A 2 활성측정은 Ethanol/Toluene(l/1, V/V) 중에녹아있는 l-acyl-2-[l-*\]]linoleoyl-^5/i-glycero- 3-phosphoethanolamine (1,000 cpm/nmol) 을시험관에분취한후, 질소가스로용매를휘발시키고 lipid film을만든뒤, 일정량의증류수를가하여 ultrasonic cleaner(branson 2200, USA) 로초음파처리하여기질로사용하였다. PLase A 2활성은 100 m M Tris-HCl (ph 9.0), 6m M CaCU, 기질 20 nmol 및효소 (long) 및천연물추출액 25 를함유한반응액올 STC로, 지정된시간동안반응한후, 생성된 (*"C) 유리지방산을 Dole등외방법 ^"* 에따라추출하여 liquid scintillation counter Minaxi Tri-Carb, Packard, USA) 로즉정하여 PLase A 2 활성으로환산하였다. 이때, PLase Aj 활 Pharm. Soc. Korea
...Table I Inhibitory activity of methanol extract of crude drugs against group II phospholipase A2 가각갈같화국수인초활황강우명내련본담쇄초3중루곽기Korean. PLase k i Name 자Pharmaceutical Name Inhibition % 근2222068576525531454살이7자금2피45618초보그^494인8자Vol. 4L No. 5, 1997 Treminaliae Fructus Puerariae Radix Puerariae Flos Chrysamthemi Flos Euphorbiae Kansui Radix Euryales Semen Glycyrrhizae Radix Angelicae Koreanae Radix Curcumae Longae Radix Zingiberis Rhizoma Visci Herba Cassiae Semen Galli Stomachichum Corium Meliae Cortex Angelicae Tenuissimae Radix Caraganae Radix Drynariae Rhizoma Melonis Calyx Crassirhizomae Rhizoma Trichosanthis Semen Sophorae Fructus Lycii Fructus Dianthi Herba Limbricus Testudinis Plastrum Citri Unshiu Semen Rosae Laeuigatae Fructus Lonicerae Flos Platycodi Radix Raphani Semen Aloe Echinopsis Radix Saviae Radix Angelicae Gigantis Radix Arecae Pericarpium Hydnocarpi Semen Rhei Rhizoma Benincasae Semen Malvae Semen Eucommiae Cortex Aristolochiae Fructus Ephedrae Radix Viticis Fructus Liriopsis Tuber Hordei Fructus Germinatus Gossypii Semen Oystreae Testa Moutan Cortex Radicis Equiseti Herba Akebiae Caulis Myrrha Buddleiae Flos Pinelliae Tuber Bombycis Corpus Sinapis Semen Alba Chelidonii Herba Santali Album Lignum Ammomi Rotuni Fructus 38285176195632514024880293198472369070414200101309587819054515000572061047^0감강건겨결계고고골과관괄괴구글H}o 맥인판핵앵은경복회로삼귀복자화금금길내자노누단당대동두마만맥면r t 피자황!류층두자령근자동형문아설려단적통약몽하강개1 창자피목목목몰밀반백화잠자채향구댄두8^백별보복봉부비버번사산삼상색석선세소83938763075806807036253730806070044985107905731807924776451소소소쇄숙승신양애어여연영오-피457275자유천연물로부터염증성포스포리파제 A2 저해제검색 Table I Continued Korean PLase A 2 Name Pharmaceutical Name Inhibition % 36렴5모머근3부68선2자질퍼출인5편려합2갑두8령신두출자평파해78295274랑간삼242상73두수조철기육표건곡창모자복근신유맥인목업자태회생양지마초이지강엽성포0정^교자화s신,지가머배수61187465618677335황초설_K육초두응Pulsatille Radix Ampelopsis Radix Imperatae Radix Cynanchi Radix Aconiti Koreani Rhizoma Dictamni Radicis Cortex Thujae Semen Tribuli Fructus Atractylodes Rhizoma Alba Dolichoris Semen Lilii Bulbus Amydae Carapax Strychni Ignatii Semen Hoelen Hoelen Cum Radix Zedoary Aconiti Tuber 엽Spirodelae Herba Eriobotryae Folium 자Tokoro Rhizoma Arecae Semen Belamcandae Rhizoma Adenophorae Radix Torilidis Fructus Sophorae Subprostratae Radix Curni Fructus Zizyphi Spimosi Semen Notoginseng Radix Loranthi Ramulus Phytolaccae Radix 황Vagina Ovorum Mantidis Rehmanniae Radix Dendrobii Herba Acori Graminei Rhizoma Curculiginis Rhizoma Inulae Flos Asuasari Radix Tritici Semen Caesalpiniae Lignum Perillae Herba Perillae Semen Picrasmae Fructus Foeniculi Fructus Cynomorii Herba Rehmanniae Radix Preparata Cimicifugae Rhizoma Magnoiiae Flos Alpiniae Officinarum Rhizoma Artemisiae Asiaticae Herba.Hoittiyniae Herba Ligudtri Fructus.Forsythiae Fructus.Nelumbinis Semen Polygalae Japonicae Herba..Ganoderma.Acanthopanacis Cortex..Schizandrae Fructus Galla Rhois Evodiae Fructus 소소실향오오
오와위유육인일조초측편문태철 정광원 정규찬 손건호 김현표 강삼식 장현욱 Table I Continued Table 1 Continued 왕용우원원육의익옴작저적전정조중죽지차천천청초택텍토파페Korean Name 약적골송불행골규안슬방지령근황두계종이모양진천약령설자자갑분련향구엽령자지5! 지OIT 육풍선피구용인초곽호궁자지석층호피공향각구협유여엽각모피자등석부전마련산오화황목피과두백란사복사고국장측.^.Pharmaceutical Name Linderae Radix Sepiae Os Orostachys Herba Melandrii Herba Fossilia Ossis Mastodi Solanii Herba Longanae Arilus Achyranthis Radix Gleniae Radix Polygalae Radix Clematidis Radix Ulmi Pumilae Cortex Sulfur Myristicae Semen Cinnamomi Cortex Spissus Cistanchis Herba Coicis Semen Leonuri Herba Epimedii Herba Aitemisiae Capillaris Herba Cnidii Rhizoma Paeoniae Radix Polyporus Broussonetiae Fructus Halloysitum Rubrum Buthus Anthrisci Radix Sorbus Cortex Caryophilli Flos Caryophylli Cortex Gleditsiae Spina Uncariae Ramulus et Uncus Gleditsiae Fructus Stalactium Bambusae Caulis in Taeniam Bambusae Folium Auranthii Fructus Anemarrhenae Rhizoma Kochiae Fructus Plantaginis Semen Gastrodiae Rhizoma Meliae Fructus Manitis Squama Aconitii Tuber Trichosanthes Root Coptidis Rhizoma Aristolochiae Radix Aurantii Immatri Pericarpium Amomi Tsao-ko Fructus Alpiniae Katsumadaii Semen Biotae Orientalis Folium Lycopi Herba Alismatis Rhizoma Smilacis Rhizoma Cuscutae Semen Psoraleae Semen Morindae Radix Patriniae Radix Polygoni Avicularis Herba PLase k i Inhibition % Korean Name 공영발Taraxaci Herba Piperis Longi Fructus Prunellae Spica Polygoni Multiflori Radix Glauberite Rosae Rugosae Root Kalopanacis Cortex Notarchi Eachii Ovum Cyperi Rhizoma Elsholtziae Herba Scrophulariae Radix Geranii Herba Corydalis Tuber Draconis Sorguis Schizonepetae Herba Sesami Semen Picrorrhizae Rhizoma Carthami Flos Carthami Semen Betulae Cortex Scutellariae Radix Astragali Radix Polygonati Rhizoma Pharbitidis Semen 성을측정하여 90% 이상외저해를나타낼때 +4. 89-- 80% 저해일때 +3. 79-70% 저해일때 +2. 69-60% 저해를나타낼때 + 로. 그이하일때는 - 로표시하였다, 결과및고찰 한방과민간에서사용되고있는생약을무작위로 200 종을선택하여, 유기용매법으로추출한후염증반 응에직, 간접적으로관여하고었옴이알려진염중성 PLase A 2 를효소원으로사용하여저해활성을검색한 결과를 Table I 에요약하였다. Table I 외결과에 Rat platelet 로부터정제한 group II PLase A 2 를효소원으로저해활성을검색한 결과는다옴과같다. 1) 90% 이상의저헤활성을나타내는생약은다음과 같다. 노회. 목단피, 번랑자. 정향피. 화퍼 5 종류외생약에 서강한저해활성이있음을알수있었다. 2) 89*^80% 의저해활성을나타내는생약은다옴과 같다. Pharmaceutical Name PLase A 2 Inhibition C636865%720650420339060081518006감수. 감인. 금은화. 대황. 두충, 마황근, 백단향. 백 질려, 복신. 비파엽, 소목, 승마, 연자육. 작약, 천련자. 포필하한해향현혈형호135167초오석근피고수당동분부유삼자초호같개마황화피색134296인련11호홍화황흑인금기정측/. Pharm. Soc. Korea
천연물로부터염증성포스포러파제 A2 저해제검색 569 Table II Inhibitory activity of organic solvent soluble fractions of crude drugsa on group II phospholipase A2 생약 부토금작분획 % Inhibition Rate^ Methanol Hexane C H C I3 EtOAc Butanol H 2O Concentration 25 Xg/m/ "Above 90% :+4. S9-80% ;+3. 79--70% :+2. 69-60% :+. below 60% "All samples were tested duplicate 초두구. 현삼 17 중의생약이었다. Table II 결과에는 200 종외생약중활성이강한것 과약한것 10 중을선별하여유기용매법으로단계적으 로분획한후저해활성을검색한것이다. 3) PLA2 저해제캐발가능성이높은생약으로서는토 복령의 CHC 13. EtOAc 층. 금은화의 hexane. CHCI3. EtOAc 층. CHCla. EtOAc 층. 작약의 CHCI3. E- toac. Butan 이층. 노회외 methanol, hexane. CHCI3. Butan 이층. 포공영외 CH C 13. EtOAc 층등이 었다. 황금의 EtOAc. butanol. 애엽의 EtOAc, b u ta n o ls 등이었다. 평령화약회영교복은저자등은이러한방법으로금은화의 EtoAc 분획으로 부터새로운구조틀갖는 PLase A 2 저해제인 ochna- flavone 를분리하여보고하였다. 오인 금후계속적으로 PLase A 2 저해활성이강한생약을 선택하여유기용매분획법으로분리한검체를사용하여 저해제를검색하면지금까지보고된저해제와는다른 구조룰가진항염증및항알러지제의개발에필요한기 초적인자료가제공될수있으리라기대된다. 감사의말씀 본연구는선경인더스트리및보건복지부신약개발과 제에의해수행되었기에이에감사를드럽니다. 문 1) Morley. J.. Page. C. P., Mazzoni. L.. Sanjar, S. : 헌 +233.-2-+노포황+공연측애+금백엽I I +++ +3+4 4 +++ +4 +3+ +4+4 ++ Effect of ketotifen upon responses to platelet activating factor:a basic for asthma prophylaxis. Annals of Allergy. 56, 335 (1986). 2) Mallet. A. L. Cunningham. F. M. :Structural identification of platelet activating factor in psoriatic scale. Biochem. Biophys. Res. Commun., 126, 192 (1985). 3. Doebber. T. W., Wu. M.S.. Robbins. J.C.. Choy. B. M.. Chang. M. N.. Shen. T. Y. :Platelet activating factor(paf) involvement in endotoxininduced hypotension in rats. Studies with PAFreceptors antagonist Kadsurenone. Biochem. Biophys. Res. Commun., 127, 799 (1985). 4) Benveniste, J., Chignard. M.. Le Couedic, J. P.. Vargaflig. B. B. ;Biosynthesis of platelet activating factor(paf-acether). Involvement of phospholipase A 2 in the formation of PAF-acether and lyscrpaf-acether from rabbit platelet. Throm. Res., 25. 375 (1982). 5) Vadas, P.. Pruzanski. P. :Biology of diseases. Role of secretory phospholipase A 2 in the pathobiology of diseases. Lab. Invest., 55. 391 (1986). 6) Chang. H. W.. Kudo. I.. Hara. S..Inoue. K. :Extracellular phospholipase A 2 activity in peritoneal cavity of casein-treated rats. J. Biochem.. 100, 1099 (1986). 7) Hara. S.. Kudo. L. Chang. H. W.. Inoue. K. ' Purification and characterization of extracellular phospholipase A 2 from human synivial fluid in rheumatoid arthritis'' / Biochem., 105, 395 (1989).2Vol. 41 No. 5, 1997
570 문태철 정광원 정규찬 손건호 김현표 강삼식 장현욱 8) Baek, S. H., Takayama. K.. Kudo, I.. Inoue, K.. Chang. H. W. ' Detection and characterization of extracellular phospholipase A2 in pleural effusion of patients with tuberculosis. Life Sci., 49, 1095 (1991). 9) Forster, S.. Ilderton. E.. Norris. J. F. B.. Yardley. H. J. :Characterization and activity of phospholipase A2 in normal human epidermis and in lesion-free epidermis of patients with psoriasis or eczema. Br. J. Dermatol., 112. 135 (1985). 10) Nakano, T.. Ohara. O.. Teroka. H.. Arita. H. ; Group II phospholipase A2 mrna synthesis is stimulated by two distinct mechanisms in rat smooth muscle cells. FEBS Lett. 261. 171 (1990). 11) Schalkwijk. C.. Pfeilschifter, J., Marki. F.. Van den Bosch. H. :Interleukin-1 p. tumor necrosis factor and forskolin stimulate the synthesis of group II phospholipase A 2 in rat mesangial cells. Biochem. Biophys. Res. Commun., 174. 268 (1991). 12) Lanni, C.. Becker. E. L. :Inhibition of neutrophil phospholipase A 2 by p-bromophenacylbromide. nordihydroguaiaretic acid. 5. 8. 11. 14-eicosatetrayenoic acid and quercetin. Int. Archs Allergy Appl Immun., 76. 214 (1985). 13) Jacobson. P. B.. Marshall. L. A.. Sung, A.. Jacobs. R. S. Inactivation of human synovial fluid phospholipase A2 by the marine natural product. Manolide. Biochem. Pharmacol. 39. 1557 (1990). 14) Wilkerson. W. W. ;Anti-inflammatory phospholipase A2 inhibitors. Dru^ of the future 15(2). 140 (1990). 15) Nixon. J. S.. Wilkinson. S. E.. Davis. P.. Bloxham, D. P. The inhibitory profiles of hog pancreatic and human rheumatoid synovial phospholipase A2. Agents and Actions, 17. 299 (1985). 16) Fletcher. J.. Kistler, P.. Rosenberg. H.. Michaux, K. :Dantrolene and mepacrine antagonize the hemolysis of human red blood cell by halothane and bee venom phospholipase A2. Toxicol Appl. Pharmacol.. 90, 410 (1987). 17) Dennis. E. A. :Regulation of eicosanoid production :Role of phospholipase and inhibitors. Biotechnology, 5. 1294 (1987). 18) Murakami. M.. Hara. N.. Kudo. I., and Inoue. K. :Triggering of degranulation in mast cells by exogenous type II phospholipase A2. h Itntnunol, 151. 5675 (1993). 19) Horigome, K.. Tamori-Natori. Y.. Inoue. K.. Nojima, S. :Effect of serine phospholipid structure on the enhancement of concanavaline A in duced degranulation in rat mast cell /. Biochem., 100. 571 (1986). 20) Arai, H.. Inoue, K.. Nishikawa. K.. Banno, Y.. and Nojima, S. :Properities of acid phospholipase in lysosome and extracellular medium of Tetrahymena pyriformis. I Biochem.. 99. 125 (1986). 22) Chang. H. W.. Baek. S. H.. Chung. K. W., Son. K. H.. Kim. H. P. and Kang. S. S. :Inactivation of phospholipase A 2 by naturally occuring biflavonoid. ochnaflavone.. Biochem. Biophys. Res. Commun., 205. 843 (1994). Pharm. Soc. Korea