Anticoagulant Activities of Brown Seaweed Extracts in Korea

전체 글

(1)KOREAN J. FOOD SCI. TECHNOL. Vol. 36, No. 6, pp. 1008~1013 (2004). ©The Korean Society of Food Science and Technology. ]ÚÖ "º .~ ºÂb~ .w 9 B'«*ÁBÿ>ÁRÏ+ R]®\ö. Anticoagulant Activities of Brown Seaweed Extracts in Korea Young Myoung Kim*, Dong-Soo Kim, and Yong-Suk Choi Korea Food Research Institute Effects of extraction conditions and molecular fractionation on anticoagulant activities of major brown seaweeds in Korea were investigated. Hot water extracts of C. costata, U. pinnatifida (Sporophyte), L. japonica, K. crassifolia, E. stolonifera, E. bicyclis, S. horneri, and E. kurome increaced activated partial thromboplastin time (APTT) over 190 seconds, which may be related to intrinsic pathway of blood coagulation. Hot water extract of E. Kurome (EKJ) was further fractionated by ethanol precipitation. EKJ-eim, ethanol-insoluble material of EKJ, showed higher anticoagulant activity than EKJ. EKJ-eim was further fractioned with ultrafiltration. EKJ-eim 1, (over 100 kDa) fraction showed higher APTT activity than EKJ-eim. A EKJ-eim 1 was sulfated polysaccharide consisting of fucose, xylose, mannose, galactose, glucose and, sulfate at molar ratio of 1 : 0.05 : 0.10 : 0.15 : 0.17 : 1.46. The anticoagulant activity increased as sulfate content and molecular weight increased. Key words: brown seaweed, Ecklonia kurome, anticoagulant activity, blood coagulation, APTT. *. . .wWö & & ê¯> ® (5). V~ .wBBº ^ÒöB ªÒ heparin, ²~ Ë¾ "æ~ ²ËöB ºÂ hirudin, FV W BB coumarin Ò polysine, protamine, ÒÒöB ºÂ Wî ® (6). 7öB ªç'b ô ÒÏ>º heparinf Ö && j, ËV* ÒÏ .²6~ 6²f Â., . Ã~ ¦·Ï ®b, >6V& 1-2*b Ö f 6 ®º ©b rJæ ®b(7), V .w W ²Ò~ ^B6 Öj * êB Âb $º * Û £b²Ò ¦V .w bîö & & B® ê ¯> ® (8). f &N~ öBº ÚÖ "º ~¦V . B~ BF²Ò BB ~ ¢~b .~¢ 7b .w bîj ºÂ Ï ¢ Ò~ ºÂÁ¦ï~&b, 7öB jv' W ¸~~ , î, 6, b, & , Ù, S Î¶>j ºÂNê¢ Ò~B ºÂ~ Wj Ò~&b, W Ö> 6 ºÂbj ª¶ïê ª³~ .w W 5 z' WªWj ÚÚ~ .. º J¾ *¦V " , ¢ ÿ jj æö B Ïb ¶Î Ï>Ú zb, F#öBº ® ¢¦ª~ Ï ²j ö &¦ªf ®Î&b¾ ëÏ ~~ ö ò, jò $º Òò~ öò Ï>ÚN ;î . ¾ "¾ö ö ³¦~² F>Ú ®º VËW 5 Ò W bîö & & >B ö & & B® ê¯> ® (1). 7 .~~ "Wªf ~ B cellulosef ?f " ^* 6î alginic acid, fucoidan ®b, &Ë bº " β-1,3 Öb Úê laminaran ® . 7öB fucoidan" laminaranf «·W 5 .w W Ö> ©b > ®. (2,3). "¾ö ãB >& Ëç> ~ Bzf Úÿ¦ b ¦*, "ãï, òÂ., ò.*, ÿ ãz, 7Ã ~ .B~ Ë ~¶& Ã&~º º^ (4). Ú Úö B .w >wf . wê~ Ú¶ º²ö ~ ÛB $º O·Ïj &æº ¾W wBö ~B Î"'b &F > ®ÚB Ëî~ 5 W÷~ .O 5 ~òö &. Òò 5 O» Òò þö ÒÏ òº Úö ¶ $º ·>º . ~¢ 7b *öB ò¢ «~ ¦Obj B ê >^, ê ªê r &/ &~ v þö Ò Ï~& .. *Corresponding author: Young Myoung Kim, Korea Food Research Institute, San 46-1, Baekhyun-dong, Bundang-gu, Seongnam, 463420, Korea Tel: 82-31-780-9009 Fax: 82-31-780-9099 E-mail: [email protected] 1008.

(2) ÚÖ "º .~ ºÂb~ .w W. .w 9 bî ºÂ "º .~ö Ú ®º w W bîj ºÂ~V * ~ ºÂ Ï ¢ '' Ò~B ºÂ~& . b& > º Âf ªê òö 15V~ Ã~>¢ &~ 100oCöB 2* v>~B ºÂ~& . Ò ºÂbj "(Whatman No. 41)ê 6{ ³» r ÿÖ ~ >ºÂbj áî . öêR ºÂ 5 zêR ºÂf ''~ ªê ò 15 gö 200 mL~ ''~ Ï ¢ & ê 80oCöB 4* ~~ ºÂ~&. . Ò ºÂbf " ê ÿÖ ~ ''~ öêR º Âb" zêR ºÂbj áî . "º .~öB ºÂ bî 7öB Öæ 5 « ÏW, &Ï j J~ jv' W ±~~ (UPU), î (LJW), 6(EKJ), &(EBU), Ù(HFW), S Î¶>(SHY), b(ESB) 7«~ òº >ºÂ Nêf öêR º Â ³ê¢ Ò~B ºÂ~& . ¯, Nêê ºÂf òö 15V~ Ã~>¢ &~ ºÂNê¢ 30-100oC Ò~B 2 * ºÂ r 2ß~ "æ " ê j RC ê ÿÖ ~ ºÂNêê ºÂbj áî . öêR ³êê º Âf öêR ³ê¢ 50-100% Ò~B ~~ ºÂ ê RC, ÿÖ ~ öêR ³êê ºÂbj áî . öêR ;B 5 ª¶ïê ª³ ºÂNêê >ºÂb 7öB .w W ¸~~ 90oCöB ºÂ î(LJW)f 6(EKJ)¢ 3Vï~ öêR *Ê öªÒ(3,000Üg 10 min)~ öêR &ÏW bî(ethanol soluble material, esm)" ®ÏW bî(ethanol insoluble material, eim) ¾*î . Ò W ¸f EKJ-eimf 1%(w/v, Ã~>) ³ê Ï r "V(ultrafilter cell, Amicon 8400)¢ ÒÏ~ ª¶ïê(100 kDa ç, eim 1; 10050 kDa, eim 2; 50-10 kDa, eim 3) ª³ ê ÿÖ ~ ª¶ïê ª³bj áî .. 1009. .w 9 /; .w W 7 ÚW ã(intrinsic pathway)ö V~ º WÞ 2¢Ê *(activated partial thromboplastin time; APTT)" W ã(extrinsic pathway)ö V~º * Þn *(prothrombin time; PT)f .w¶ÿªCV Sysmex CA-540j ÒÏ~ G;~& (9). ¢> 9ª ª+ C~ ïf glucose¢ standard ÒÏ~ phenol-sulphuric acid»(10)b G;~&, Wî~ ïf Lowry»(11)ö V ¢ standard bovine serum albumin(BSA, Sigma Co.)j ÒÏ ~ ;ï~& . ÖV~ ïf Dodgson" Price(12) O»ö V¢B ò¢ &>ª ê &>ª òÏ 0.2 mLö 3% TCA Ï 3.8 mLf BaCl2-J¢ Ï 1 mL¢ Î&~ 20ª* O~ ê 360 nmöB 7ê¢ G;~&b, & ¦ ïFf K2SO4¢ ÒÏ~ ·W~& . ÖÖ~ ïf Dglucuronic acid¢ &bî ÒÏ~ m-hydroxydiphenyl » (13)b ªC~& . \9 9 ª+ W W ªCf Furneaux (14)~ O»ö V¢B ò ¢ supelco vialö ê TFA &>ª ê sodium borohydride ~ö8 . Ò acetic anhydride 120oCöB 20ª * acetylation* FêÚ¢ alditol acetate *~Î r dichloromethaneö Ï*B −20oCöB &~ v gas chromatography(HP 6890) ªC~& . ¢"bº SP-2330j ÒÏ~&, 230oCöB N ªC~& . W~ {f &£~ îÆÎ" retention timej jv~ {~& , ;ïf Ú¦ &bî(myo-inositol)" &£(D-galactose, D-glucose, D-mannose, L-rhamnose, D-xylose, D-fucose, Dribose, L-arabinose)j òf ÿ¢~² ¾Ò~ ³êê ç&. Table 1. Anticoagulant activities of hot water extracts from brown seaweed1) Scientific name («) Undaria pinnatifida (1) Undaria pinnatifida (2) Costaria costata () Undaia pinnatifida (Sporophyte: æ) Undaia pinnatifida (Stalks) Hizikia fusiformis (Ù) Laminaria japonica ( î1) Laminaria. japonica ( î2) Laminaria japonica ( î 3) Kjellmaniella crassifolia (B î) Ecklonia stolonifera (b) Sargassum patens (3BÎ¶>) Eisenia bicyclis (&) Sargassum thunbergii (æÏ) Sargassum horneri (SÎ¶>) Pelvetia siliquosa (^¦V) Ecklonia kurome (¦6) Sargassum yendoi (úêJÎ¶>) 1). Habitat. Abbreviation. Wan-do Gijang Gangnung Gangnung Wando Wando Gangnung Baengnyeongdo Baengnyeongdo Gangnung Busan Yeosu Ullung-do Yeosu Yeosu Jejudo Jejudo Jejudo. UPU UPG CCG UPS UPWS HFW LJG LJB LJB 1 KCG ESB SPY EBU STY SHY PSJ EKJ SYJ. The concentration of extracts is 10 mg/mL. The activity value of >190 means over-range of coagulator.. 2). Water extract APTT (sec). PT (sec). 95.4 84.1 >190.02) >190.0 66.1 122.8 >190.0 >190.0 >190.0 >190.0 >190.0 108.5 >190.0 89.3 >190.0 79.5 >190.0 122.5. 11.8 16.2 14.4 29.9 11.8 12.5 34.7 21.9 27.4 72.9 36.6 12.7 18.9 13.0 13.8 13.7 37.3 15.5.

(3) ®"²æ B 36 ² B 6 ^ (2004). 1010. Table 2. Anticoagulant activities of ethanol and methanol extracts from brown seaweed Abbreviation1) UPU UPG CCG UPS UPWS HFW LJG LJB LJB 1 KCG ESB SPY EBU STY SHY PSJ EKJ SYJ. Methanol extract. Ethanol extract. APTT (sec). PT (sec). APTT (sec). PT (sec). 48.5 60.2 54.6 64.2 57.8 57.2 74.5 66.0 57.5 65.5 119.9 66.3 73.8 73.1 79.1 58.5 105.5 78.5. 13.9 14.2 14.1 15.3 14.9 13.1 16.1 15.6 14.5 15.4 17.8 13.5 16.2 15.5 16.7 14.0 19.7 15.9. 112.5 71.7 87.5 77.3 85.5 >190.0 72.4 79.7 50.1 62.6 107.2 136.3 175.1 120.1 72.6 77.6 98.3 118.7. 20.8 15.4 19.0 17.9 18.5 25.8 15.9 16.5 13.2 15.3 19.3 19.4 25.9 18.1 14.9 15.1 17.2 19.3. 1) Refer to Table 1. Heparin 10 U/mL: 184.0 sec (APTT) and 14.5 sec (PT).. 'j¢ ~ ~Ö~& .. Ö 5 8 ºÂ Ï ö 8 .w 9 ÚÖ "º ~¦V . B~ BF ²Ò~ BB ¢~b " .~¢ 7b '' Ï ¢ ÒÏ ~ ºÂ ºÂbö &~ .w W(APTT, PT)j G;~& (Table 1 5 2). & .Ë~ APTT 5 PT& ' ' 30.3., 11.2. &Ú >, zêR, öêR ºÂb Îv APTT 5 PT~ * Ë>î, *Ú'b APTT *f > ºÂb, öêR ºÂb, zêR ºÂb Bb .w * æ>î . Ò > ºÂb 7öB C. costata, U. pinnatifida(Sporophyte), L. japonica, K. crassifolia, E. stolonifera, E. bicyclis, S. horneri, E. kurome& 10 mg/mL ³êöB .w ~ ÚW ãö V~º APTT * 190.0. ç æ >Ú .~ 7öB Ö> .w Wj ¾æÚîb , ÚW ãö V~º PT *ê Ã&~& . Kweon (8)ö ~~ ~ Codium fragilej ºÂ Ï ¢ Ò~ ºÂ~&j r .w Wf £ÖW >Ïb ºÂ~ &j r &Ë ±~b, FVÏ ºÂ~&j r~ Wf ç&'b £~& ~&b £ÖW ºÂb~ ãÖ &" jv~ APTTº 4.3V, PTº 1.9V w* æ >Ú W ã º ÚW ãö 'Ëj "Ú w *j æÎ º Ö"f ÿ¢~&b, ~ f ª ¶ bîB ò ªê ê £ÖWb &>ª ºÂ~º © ÎN' ºÂO»b ô . Ò Yoon (15)f '« ~¢ >, ï>, zêR ºÂ~&j r wW f > ºÂb &Ë ±~b, zêR ºÂb~ Wf · ~ ~& .. Fig. 1. Yields of water and ethanol extracts of brown seaweeds. A: Water extract, B: Ethanol extract.. > ºÂ NêêÁöêR ºÂ³êê .w 9 .~7 .w W ±f 7«((UPU), î (LJW), 6(EKJ), b(ESB), &(EBU), Ù(HFW), S Î¶>(SHY))~ ¢ F;~ ºÂ Nêê 5 öêR º Â ³êê ºÂ~ .w Wj jv ¦Æ~&b , Nê 5 öêR ³êö V ºÂ >Nj Fig. 1ö ¾ æÚî . > ºÂb~ >Nf ºÂ Nê& ¸jî> > N Ã&~&b, 7öB &~ >N 14.5-19.1%. òö j~ ¸~ . >ö öêR ºÂbf öê R ºÂ ³ê& ¸jî> 6, &, bº /Ï® >N 6²~&b, î, , Ù, S Î¶>f >N 6²f ·~ . ºÂ Nê 5 öêR ³êö V ºÂb ~ .w Wf Table 3 5 4ö ¾æÚî . ¦ï";ö B ÚÚ :f ? > Nêê ºÂb~ W ¸~b , öêR ºÂ ³êê .w Wf Ô~ . > ºÂ b 7öB î~ Wf 500 µg/mL¢ r 82.2-127.0. & Ë W ±~b, rbº 6~ W 74.0-97.1. ¸~ . .w Wf Î òöB ºÂ Nê& ¸ jî> .w Wf Ã&~&b, îf 6~ ãÖ 90oCöB ºÂ ºÂb~ .w W &Ë ±~. . Yoon (15)f Pachymeniopsis ellipticaj 4, 25, 50, 70, 100 5 121oC Ò~B ºÂ~&j r .w Wf ºÂ Nê& ¸jî> Ã& ~& . ö ®ÚBê .w Wj º sulfated polysaccharide.

(4) ÚÖ "º .~ ºÂb~ .w W. 1011. Table 3. Anticoagulant activities of water extracts prepared from brown seaweed at different extraction temperatures APTT (Sec)1). PT (Sec). Extraction Temp. (oC). LJW. EKJ. ESB. EBU. HFW. SHY. UPU. LJW. EKJ. ESB. EBU. HFW. SHY. UPU. 30 40 50 60 70 80 90 100. 82.2 83.9 92.3 97.9 102.3 120.7 127.0 119.2. 74.0 78.1 84.8 88.0 90.4 96.7 97.1 90.7. 70.3 88.1 81.4 85.6 83.2 89.3 88.5 66.2. 51.0 53.8 54.3 50.6 49.7 49.6 69.2 78.4. 40.9 41.1 40.7 43.1 44.1 45.4 43.7 46.8. 61.4 67.6 74.3 72.5 81.1 88.4 90.8 89.2. 38.4 45.2 36.9 39.0 40.5 42.0 45.1 65.0. 22.5 17.8 24.4 20.7 19.4 22.1 20.6 19.1. 20.5 19.9 21.7 21.8 21.0 21.3 22.1 19.5. 18.3 19.6 19.5 19.5 19.2 17.9 17.6 13.4. 15.2 15.7 15.7 15.0 14.6 14.9 14.7 17.9. 12.8 13.0 13.5 13.9 14.0 14.4 13.7 14.4. 14.8 14.8 15.1 15.0 15.1 14.7 14.1 14.5. 10.5 10.1 10.1 10.2 10.2 10.4 10.6 11.3. The concentration of the extracts was 500 µg/mL.. 1). Table 4. Anticoagulant activities of ethanol extracts prepared from brown seaweed at different ethanol concentrations APTT (Sec)1). PT (Sec). Ethanol (%). LJW. EKJ. ESB. EBU. HFW. SHY. UPU. LJW. EKJ. ESB. EBU. HFW. SHY. UPU. 50 60 70 80 90 100. 39.7 40.2 41.5 42.3 40.6 43.0. 44.5 45.5 45.5 45.3 44.9 49.4. 40.8 41.3 43.1 42.0 43.2 44.2. 41.8 43.9 43.1 43.3 44.3 45.2. 45.0 43.5 45.5 44.1 44.4 44.9. 59.9 40.6 42.2 41.5 43.2 40.8. 46.5 55.4 46.1 39.9 44.4 42.0. 11.4 10.4 11.5 11.8 11.6 11.2. 12.1 12.3 12.3 12.4 12.2 12.6. 11.4 11.6 11.4 11.5 11.6 11.8. 12.0 12.0 12.1 12.1 12.2 12.3. 11.7 11.6 11.5 11.4 11.6 11.6. 11.0 11.2 11.3 11.0 11.3 11.3. 12..8 12.0 12.0 12.3 13.7 12.6. The concentration of the extracts was 500 µg/mL.. 1). Table 5. Anticoagulant activities of ethanol soluble and insoluble fractions of the E. kurome water extract Sample. Concentration (µg/mL). APTT (sec). 1). EKJ-esm. 20 100 500. 43.2 38.8 37.4. EKJ-eim2). 20 100 500. 48.3 185.9 190. 1). Ethanol soluble material. Ethanol insoluble material.. 2). º jv' ¸f NêöB ¾ ºÂB º V~ f ¢~ ~& .. î~ .w Wf ô (16)B : ®Ú 6 ¢ &æ rR ª³ 5 ª¶ïê ª³~ Wj ¾æÚ º ª³~ z' Wj ÚÚ~ . öêR ª³b~ .w 9 6~ > ºÂb(90oC)~ öêR &Ïbî(EKJ-esm)" ® Ï bî(EKJ-eim)ö V .w Wj Table 5ö ¾æÚ î . EKJ-eimj 20, 100 5 500 µg/mL ³ê ~ .w Wj G;~&j r APTT~ 8 48.3., 185.9. 5 190.0. çb .w * Ë>î . º APTT~ Wf ³êö jf'b W Ã&~º ©b . Ò 90oC 6 > ºÂb~ APTT& 500 µg/mL ³ê¢ r 97.0. ¾æÂ > öêR ª³~&j rº 190.0. ç. Fig. 2. Anticoagulant activities of EKJ-eim derived from the E. kurome water-extract. EKJ-eim: ethanol insoluble fractions of the E. kurome water extract; EKJ-eim 1: >100 kDa, EKJ-eim 2: 100-50 kDa, EKJ-eim 3: 50-10 kDa... b W 2V ç Ã&>îrj r > ®î . ¾ ö êR &ÏW Wª~ Wf ÿ¢ ³êöB 43.2, 38.8 5 37.4. W ~ ìî . º öêR ª³ &ÏWª &ª¶ ï² bî , Ò polyphenol êÛ~ bî "º W bîj ®ÚB .w W ©b , öêR ®ÏW bî~ ª³f ®Bb B > ~ "Wª ~ Bê& ¸j^B W Ã & ©b . Park (17)f Ó'j 0.8% HCl ºÂ ºÂbj öêR * 5 RC~ W 2V Ã&B j áî ~ þ" ?f Ö"¢ & ..

(5) ®"²æ B 36 ² B 6 ^ (2004). 1012. Table 6. Yields and chemical compositions of EKJ-eim Fractions1). Yield. Sulfate (%). Protein (%). EKJ-eim EKJ-eim 1 EKJ-eim 2 EKJ-eim 3. − 52.2 7.5 6.4. 18.6 32.1 8.1 6.5. 18.3 22.3 33.4 31.0. Uronic Total acid(%) sugar(%) 25.2 27.8 15.6 11.4. 51.1 56.4 44.6 42.9. Sugar composition2) Rhm. Fuc. Rib. Xyl. Man. Gal. Glc. N.D. N.D. 1.6 tr3). 61.7 66.5 11.5 tr. 1.1 N.D. 1.7 tr. 3.1 3.2 2.9 tr. 4.6 7.2 6.8 2.6. 8.9 10.8 1.9 tr. 20.6 12.3 73.7 97.3. 1). EKJ-eim: ethanol insoluble fractions of the E. kurome water extract, EKJ-eim 1: >100 kDa, EKJ-eim 2: 100-50 kDa, EKJ-eim 3: 50-10 kDa. N.D.: not detected. 3) Trace 2). ª¶ïê ª³b~ .w 9 6~ öêR ®ÏW bîj "V¢ Ï~ ª¶ ïê ª³ ºÂb~ .w Wf Fig. 2ö ¾æÚî. . EKJ-eimö & ª¶ïê ª³ >Nf 100 kDa ç EKJ-eim 1 52.2% " ª³îb, EKJ-eim 2, EKJ-eim 3~ >Nf '' 7.5 5 6.4% & . EKJ-eim 1~ .w Wf 500, 100 5 20 µg/mL ³êöB '' 190.0. ç, 190.0. ç 5 58.0.B EKJ-eimö j~ W Ã&~ &b¾ EKJ-eim 2 5 EKJ-eim 3~ Wf J®J 6²~& . Ò ª¶ïê Wf 100 µg/mL ³êöB &ª¶ ª³b .> W '' 190.0. ç, 65.6. 5 40.0. W 6²~ ª¶ï ª³¢> .w W ¸rj r > ®î . ª³ ~ \9 9 6~ öêR ®ÏW bî~ ª¶ïê z' WªWf Table 6ö ¾æÚî . EKJ-eim 1~ z' Wj ÚÚ ÖV& 32.1%, Wî 22.3% Ò C~ ï 56.4% &b, " Wf fucose C~ 66.5%¢ Næ~ ®î b, glucose, galactose, mannose& ²ï Ò~& . Ò fucose, xylose, mannose, galactose, glucose, sulfate~ Öjº 1 : 0.05 : 0.10 : 0.15 : 0.17 : 1.46b 2 mole~ fucoseö 3 mole~ ÖV& Ö>Ú ®º ©b . EKJ-eim 2~ fucose, mannose, glucose, sulfateö & Öjº 1 : 0.14 : 1.51 : 0.69B fucoseö & ÖV~ ï ¸j> .w W ¸ rj r > ®î . Nishino (2)f Ecklonia kurumeöB ºÂ ³ª 7 Wj ¾æÚº ª³~ ÖWjº fucose, galactose, glucuronic acid Ò sulfateº 1.00 : 0.11-0.19 : 0.02-0.07 : 1.22-1.48î ~& . Ò Nishinof Nagumo(18)º Wj ¾æÚº fucoidanj desulfation *B Wj jv~&º Cö & ÖV~ ï 6²Nö V¢B APTT 8 ·jê ~ þ" ?f Ö"¢ & .. º. £. ÚÖ "º .~ 18«~ ºÂÏ 5 Nêö V . w Wj G; Ö" , î, 6, &, b, Ù, S Î¶>~ > ºÂb~ APTT& 190.0. çb W ¸~b, r~ ºÂNêº 90oC& . 6~ öêR ®ÏWª~ .w Wf 500 µg/mLöB öêR &ÏW bîö j~ 2V ç Ã&~&b, öêR ®ÏWª~ ª ¶ïö V .w Wf 100 µg/mL ³êöB EKJ-eim 1(100 kDa ç) 190. çb EKJ-eim 2(100-50 kDa) 5. EKJ-eim 3(50-10 kDa)ö j ±~b, ª³~ z' WWªf fucose, xylose, mannose, galactose, glucose, sulfate W>Ú ®b, Öjº 1 : 0.05 : 0.10 : 0.15 : 0.17 : 1.46b 2 mole~ fucoseö 3 mole~ ÖV& Ö>Ú ®º ÖW ª j {~& .. 6Ò~ º ·>Ö¦ >Öß;BB"B~ j æö ö ~~ >¯B Ö"~ ¢¦ j æöö 6Ò ãî .. ^. ò. 1. Ito K, Hori K. Seaweed: chemical composition and potential food uses. Food Rev. Int. 5: 101-144 (1989) 2. Nishino T, Yokoyama G, Dobashi K, Fujihara M, Nagumo T. Isolation, purification, and characterization of fucose containing sulfated polysaccharides from the brown seaweed Ecklonia kurome and their blood-anticoagulant activities. Carbohydr. Res. 186: 119-129 (1989) 3. Percival E, McDowell RH. Chemistry and Enzymology of Marine Algal Polysaccharides. Academy Press Inc. London, UK (1967) 4. Richard L, Mueller MD. History of drugs for thrombotic disease. Circulation 89: 432-450 (1994) 5. Jackson CM, Nemerson Y. In Blood Coagulation. Ann. Rev. Biochem. 49: 765-771 (1980) 6. Shim YY, An JH, Cho WD, Chun H, Kim KI, Cho HY, Yang HC. Inhibitory mechanism of blood coagulation and in vivo anticoagulant activities of polysaccharides isolated from Codium fragile. J. Korean Soc. Food Sci. Nutr. 31: 917-923 (2002) 7. Jaques LB. Heparins: anionic polyelectroylte drugs. Pharmacol. Exp. Therap. 31: 99-166 (1979) 8. Kweon MH, Park MK, Ra KS, Sung HC, Yang HC. Screening of anticoagulant polysaccharides from edible plants. Agric. Chem. Biotech. 39:159-164 (1996) 9. Kim HS, Nam KH, Lee DH. Measurement of PT, APTT, and fibrinogen by automatic coagulation analyzer, sysmex CA-540. J. Clin. Pathol. Qual. Cont. 23: 247-252 (2001) 10. Dubois M, Gills KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28: 350-352 (1956) 11. Lowry OH, Rosebrough NJ, Farr L, Rindall RJ. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 256-275 (1951) 12. Dodgson KS, Price RG. A note on the determination of the ether sulphate content of sulphated polysaccharides. J. Biochem. 84: 106-107 (1962) 13. Blumenkronz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-487 (1973) 14. Furneaux RH, Miller IJ, Stevenson TT. Agaroids from New Zealand members of the Gracilariaceae (Gracilariales, Rhodophyta)-a novel dimethylated agar. Hydrobiologia 204: 645-650.

(6) ÚÖ "º .~ ºÂb~ .w W (1990) 15. Yoon JA, Yu KW, Jun WJ, Cho HY, Son YS, Yang HC. Screening of anticoagulant activity in the extracts of edible seaweeds and optimization of extraction condition. J. Korean Soc. Food Sci. Nutr. 29: 1098-1106 (2000) 16. Koo JG, Jo KS, Do JR, Woo SJ. Isolation and purification of fucoidans from Laminaria religiosa and Undaria pinnatifida in korea. J. Korean Fish. Soc. 28: 227-236 (1995). 1013. 17. Park MK, Kweon MH, Cho HY, Yang HC. Anticoagulant activity of sulfated polysaccharides isolated from Codium fragile. J. Korean Soc. Agric. Chem Biotech. 42: 140-146 (1999) 18. Nishino T, Nagumo T. The sulfate-content dependence of the anticoagulant activity of a fucan sulfate from the brown seaweed Ecklonia kurome. Carbohydr. Res. 214: 193-197 (1991) (2004j 9ú 1¢ %>; 2004j 11ú 25¢ j).

(7)