a oft fo i = CHS OSS SABI) Studies on the evaluation of drugs containing polysaccharide (Il)-chondroitin sulfate = oa jo 2 OW ro rat ozA Ao AFINFFUARA TW 0] BIAS “HHP HES) Baw (D's ARBIAR awd. 2003, 12. 20 FHATGY ASU DASA FA aadrada 294AP ASUTA OE aaa | OSH) Bag UD ) seer | SABER ae, BAA, HPLC BA, BEBE UA aaeria ASU sa Auarada | aa 4 a77kt | 2003.3 . - 2003 . 11 PISESNTS BA SAG THE SAH SY, WAT UF SAY FA P38 BROS, MA ZA 755 GIN FAS ALS Bet. IeFoe A WHEL ENUERS Ada, A949 Gadel slo] waa] FOB AS Ha th. ES wel ala), Baal, HF So] BYso} Veh. Ae Ewo) Ws] FAM NAS 22 7BAIY4 Bole] AIA Soya Ach. lel Doll 4) WHE HONUS FAS AME US We] Sas. aa, Pez SASUESS AWIYS Aya] “IHS SF AE DQ alayy” aaa FAS QO, BUYS) Sash We I Fala Page | wo} ROM, AS] BAP) ASS 4 me RT ade alte Be art 2QUEF USTAS Qty, 2 As SHEETS QV] At JISSLNAUES BEE ZB Qdeezyosr PE VS BYARD, SUI) FSAD Me WSs s|QyEs @ EB 2 aE Se $dse Gea YA, GPC-HPLC, 271994, NMR BA, = Sze BAS AYe F GH] ZY EAS SAX-HPLCS Bao] SE = 28a. S38], SHI] APS MAGS} Bo] Vo Yapo} zy 4 Sdt8s AAARD, +S] Bd HSS |Ge FeeTH/ MSs lee] +S +R GPC-HPLCS sate] HAAAH, AP ve} AlRAa AeA S1SMAFAS ol Gs}o] Be Aa) F Pe YYoe Base BAW S MEIN. EM, BS S MAIS RY HS Hise sojage Bae FE Caco-2 ARTE Seto] BUI ATS TBA. ole] el FH] BABES Sti] PIHL|VUSSH ARTAS Wa, ae Mee SQUES KFDAREES A, HUUIde F PISS AER PISSIS Muse Saas At WIAs ala. Hiese JAUERS in vivo/ in vitro F839 BAH AM AHA 3,000~5,000 oy Y ABA SAGA Caco-2 AZT US RPS GEA. ES SAA} FAG PISER|WGES AETAS MY] AHA HAM Bt SESNUUED VRAAS Aas Idd NFL ald ASE say Et 7F40e AVG] EGY Als Alaiye.Project Summary Tile of | Studies on the evaluation of drugs containing polysaccharide] D-chondroitin sulfate Key Words | Chondrotin sulafte sodium, Evaluation, HPLC analysis, Chondroitinase Yeong Shik Kim 7 ‘Natural Products Research Institute, Institute, Project Leader. Seoul National University elise 204 = 2003, 11 Period —___} Chondroitin sulfate (CS) is a glycosaminoglycan, which is composed of an alternating sequence of sulfated and/or unsulfated residue of D-glucuronic acid (GieA) and D-N-acetylgalactosamine (GalNAc) linked by B(1->3) and p(i->4) bonds. CS is a major class of glycosaminoglycans required for the formation of proteoglycans found in the joint cartilage. It has been well established that basic damage to the arthritic cartilage involves the alteration of proteoglycans and collagen fiber. CS is being used as eye drops in medicine and supplement in osteosrthrists. Some of vitamins and cosmetics include CS as one of ingredients. The commercial CS is usuaully originated from either bovine trachea or shark cartilage and its applications are varied depending on the purity and molecular weight. This is one of the reasons why we should standardize the regulations of CS. Currently, analytical methods of CS are given in the 2nd edition of Korean Pharmaceutical Codex (KPC), but the methods are too old to discriminate the composition of various CS from different sources. Since the chemical properties of CS are polydiperse and heterogeneous, the regulation is quite different from those of small molecules, Our purpose of the research is to apply the new techniques for the analysis of CS, which are easy, consistent and sensitive. First, we prepared CS-derived disaccharides in order to be used as standards for the compositioanl analysis of CS. Twelve raw materials were provided from the FDA and six CSs as pharmaceuticals were purchased at the local drug stores. ‘They were analyzed by GPC-HPLC, electrophoresis and NMR experiments. They were also depolymerized by bacterial chondroitinase ABC and their products were analyzed using by SAX-HPLC. From these experiments, we could establish the specifi standard for quality control by KPC. Furthermore, we developed a new tion of sodium chondroitin sulfate and suggest the KFDA reference quantitative method of sodium chonéroitin sulfate in _multi~componenis pharmaceuticals including hard capsules and tablets. We could also analyze CS in the soft capsule including a lot of other components by a combination of solvent and phenol-chloroform extractions, The extract was easily analyzed by GPC-HPLC. CS in complex preparatins was first separated using anion-exchange chromatography and the eluant was analyzed by GPC-HPLC.a 4 Ala AS At Fue SNF As .. ASS ATMA Us Sl asp 3-1 BaaE, we 3-1-1 Wiles ze] as} 3-1-2 ASF EAI 7) 3-1-3 ABA) 3-1-4 G42 Ae. = 3-1-5 HPSECS 9] 8% SSE zo As] BA. 3-1-6 Agarose gel 719%. 7 3-1-7 NMR 24 — 3-1-8 PISEZAS SH HAY RW AS .. 3-1-9 Caco-2 AEH SY Maza Fp, 3-2 AYas. 3-2-1 GH AD. 3-2-2 HPSECS 8t 202. 3-2-3 Agarose gel 479%... 3-2-4 NMR 244. 3-2-5 HIBZEZ|E GH BAR AS .. 3-2-6 AVA R Nal S PASSE a BAY 3-2-7 SAPaA S PASE se] as] Ba. 3-2-8 Atal S Bh ods) 24, 3-2-9 MSEIVUER Babe EY 3-210 Caco-2 HEFE SE Whee zolds FF, 3-3 ae. A 44 APWVHEH CAS Ras} qs ASH AP MIAMHS) BEIT RAH AG PEL... Stats at. Se Ale] alAla AZ Biss sde Sls] Aas} Fete] Shel yepy Hheeszcl” A, BITSY B RESS|d CF al SERS REST Bld. ISS old AS CE N-obaY-D-Aep EAT D-SF FEA] 1308s} 148%] soppy Aad VSHs] SRM], VS VAS VA Blayrde71S Be Y PERI BE D-SHFEt AA L-oFEAS Bsa Veo, o} Se Bape HE Fopddtelejas te (1), PIEESVS BA] SA] AYR SAS] Fe, Ba AB Za Fa FY ALOE, BA 249 rsa VI] Fad AUS Ic. ay wid Sd) MSS US SFSU SIHY AIL Spaz] say ats 2, Ut Wat Fao Ad AA Ae FIA Zed oseolt SHAE A td. ae Seed Zedeaeolte24 Wea SH} dade (agarecan)l}. cheat Bd AF] WEEE Sale, AF S49] Hy Ad Sqd94 4ae Ves) ad. o] qadgts FE MESS AEH 2 HIN] 2) Vet FRAG Foley Ag gag Be wa age] AAS AMY FAM ABS se ALE Mel] gr}, LY, SEA Aaa S45 JAS A 23 Bess] daye dda Bes} Ve By 249] |S A& BARE FAA BAST (2), AFFOSY PISESUGSES Bla, H9Y Bagel oq Beal SLE ASAD A CB). HR vep Alal, Baal, sees Sa SYs\o} lc. MISE STG] FA AAS ABovine) 7) BA (tracheal Fe](Sharks) 4] AEN] PAD Roo Yala AEs Bape] Alo] wep Ales|eE E7t He +7 Ut. AAS) BISESIUYERS Bt UAH] 40,000 8] OU, BAdls ASSES PISES]AUERS Yas] F 15,0009E]4. 1 Ay, ASH lA Sek Fo] Gide] Zt + 7] deol S=7} B o] Sola - Me. lye Al BSS eP4e[gs FAS Ade Va 2 Meo] SLB. QA, PISSSVUES AVIS AFIGStAITA “As S AS BABA" AAAI HAM] VOU ), AWBe] Hash WSS ASS Fade] REA7 FA Feo] Yor, Asay BVI Es of Amey) RID YE Bl. $8), HBL sde7 Say Ve Fel zAb}SBM] BYS HPLCS SS: Aol AAS HIPS wR geo] (5). 1a Gl] AAD Qe Sh) Meh S MPa SPUE RS A BS BAYS Beste] AF TAA FAYLLA Fades AeVS F AAAS Rl alteta VFS WE APelrp. A224 HuUy-9| 7emwe aM PISENUYER] Atl Bags] Sd 8+ BR AR Bs} Yrhe Ba AAAASS Bola Yee BWA hye 7S YFLBE Bel Ass] BUH F8] FUME AISES|AGER] AFFOR EFM Paalal U4 Saas F552 th. Hhecelds qa Ace VS Ad de Sekageku SAFI Wal 2E42] CS (Oral grade ¥ Injection grade)7} Uz = wpa eh. QA, PISESNWYES] LAPHLS BRE PADIS Ba-aIy BAY} YE of8}e}3y APB] 7}Szo] Arh. ayy, las WS AYER F NSAS AIRS BH ANA SHIAS FAAS Belsj7l7 18. Say Be 4 7se] Wael Vay MssAel WA Clay] Sel Bake Hz)q PARAS olgete PYAR VPS BAsHe Wyo] aaaos wo] BEIT Meh. TY, lye WIE Ms Ale] Vole Sea + 7] ed dado Ale sole Aeote BA BGS Fe Wy Sasol Ea] Lol AA BAS SHS AGA 771 MA. Su AF Fs 3 AFP] BWA] Solsle PISSS|AYERS Sol eaztErsy ee 18a] Ve)et + GPC-HPLCS ol Setq] B48} ie} (6). GPC-HPLCE BARS a7} epy Beaqs7] Vel eA BAS FW. ae, wakes aA 7 SA 4A) HE HR Sao] MS SH BATS) OH Se Fe Qo} led QDAS AVASAT SIME PISSPNYESS) VFoH ASAI7171 a Ze VR. Baz] dager vol Wis] she Abs so}ady Base AWS wsg@ qo] We, 2 aPTIS dated Bed MISS |GyESS PAs] Asp} 34 SS 1S] ASI AS Sete AAA +e SFol Sale Art HE Bo}e ESM M4 PISSSNUS ASGE PSE VYSASHMO, MLA AWYS Gs AVE AHS alesigeh ee we PISSZMUEH A 4H FAG Adages Fag gdeszel BUESS) J Heol He APE AM lolz Veitch B ATE Ae BS BSH AVAL WF HPD Caco-2HEFS Gate] PEE Zola YESS) Batol ue Seas wm detec, A328 ATABFY us 2 Ax 3-1 ag AS 3-11. PIZESOS UE RIAs +a QA NSA Sol ASS HEB Yo PSE EYEE] Ord $39 APS AAAA, Ata, BFS Staal, 27s Hodddeal, B74 B94) BAP] Stel Aa SS Tse APS Pals Aeet Sh. EM, Hy AAW} BE ARTHMM FS Sa] ABA ASS RE JGUES URE ALVIS FUste] Ayo] ASA (Table VD. 1, UE) Ae] PIES eolY APS AF4oe o¥ysa Ve Wad Fal? (Prof. Toshihiko ToidaZ71E] 1369] BIBER UES +aapgiat (Table 2), AUS HEFLE FAAS (NZP, & 718A), Sigma Akay), Calbiochem (2 7/47] 2 Ads) CS Eze] calibration 2% Seikagaku 1H)S Fwaste. as] BFS Mee] AST BE ae USP CS reference standard # U# Seikagaku Ht AS WS CS CANE 40,000, 15,000)& A}-88h3teh. 3-1-2. AF BAI 71 Azure A, toluidine blue, Stains-All, sodium chloride, DzO& Sigma (St Louis, Mo)al4] #43492, bovine serum albumin (BSA, Santa Cruz Biotechnology), sila 24] AR (Bio-Rad, Hercules, CA), agarose (BioWhittaker Molecular Applications, Rockland, MEA] #QStMeh. 7eb ASHES A AS ALS att.HPLC 4-3 8] Amersham Pharmacia (Uppsala, Sweden] Ut AKTA purifier A}8-81913L, @3H= UNICORN software 3.18 ol Se] 2443b 4. HPSEC (High Performance Size Exclusion Chromatography) column2. += TSK-G6000 PWXL (0.78 x 30 cm, 13 u, Phenomenex, USA)3} BIOSEP C-S4000 (0.75 x 60 cm, 5 u, Phenomenex, USA)S ABST, SAX Phenosphere (0.46 x 25 cm, 5 u, Phenomenex, USA), preparative column®#% SAX-5251-N (2 x 25 em, Senshu Pak, Japan) & Ab88}Ach. #42 1.0 ml/min, UV BS4sL 210, 232, 280 nm 2798}34. NMR 300 % 500 MHz Bruker AEM AE HAMS PAFAY ASMAAATA ASKS) FVEAet. (strong anion-exchange) analytical column: 3-1-3. ARAYA 42t9) AB 0.2 e& OAs ALAS Ae] ajeye| Seizy 2a BAe ALAA, lF4 VE AVE AZAD ARS AAA. 3-1-4, GHA BE Bradford SY Meo] BSAS REFLS Fo] AS Yo Be VE a a VES BAG (DY. SU ZB Ae At CS MEd SU FS CS @ AA AS 10 mg/m] SES SH Fe) F 100 ules Aah 700 ul} HF Zt ¥F 200 ul] GAA YAY BEM Sst] 595 amd HVES SA. LEFLE AGE BSAE 1, 2, 5, 10, 20, 50, 100 ug/mi9] FEol 4A Ad VAY WYOs BAd + IES Seid BIAS Jaiate. obt 3-1-5. HPSECS SG WHER} BAY 10 mg/mio] Hu) BARS evPe Tae CS ARS Fu FE CS UY AAS 5 mem} SEZ AAYAR. SYA ARS 1000xgI1 5¥ Set Ades ¥ FSH 100 ule vle} 0.1 M NaCl @%2= 9s A121 HPLC column ( BIOSEP SEC-S4000)°] #9#14) isocratic Z@eI4] peak retention times} peak area VAR. HEFLS ‘ikagaku CS (MW 15,000%t 40,000)& APBARD ES, USP CSR PAY GE Whe.3-1-6. Agarose gel 2713 Aare A 244 1.2% agarose gel (11X14x1.5 cm)& WEA 229] CS AB 10 ul (0 mg/ml) 10 uls} 50% BS S33 SH8e] well 23 (100 ug/ 20 ude F 100 mA, 150% Set A1IGSS Bsc. ANGE F WS 0.05% azure A $F of} 208 St BEY AAS SF PASE (SH PPS ob EA=60:30:10 vivivyA Bol AS] SAAN He WES weepslct. Toluidine Blue & Stains-All 0) @ayyy 1.2% agarose (6.5*10X0.6 cm) BES 42F2] CS AB 5 ul (10 me/m)S 5 ule) 50% AY B43} [és] well] 2 (50 ug/10 ult F 50 mA, 150 2 St ANBSS AAA. AGS F AS 0.2% cetyipyridinium chloride & Rol SA ae ALA AZNB. AS F WS 0.2% toluidine blue (in oF WRSHP OME A=5049:1 v/v/v) SH 30H Sek Yor FMS BF 25 100 mI (in 50% AB) FEY Stains~All S40] YE arte Fela overnight GAS Wha. Ay F AS BSH} Yo] Ase} Salzl SMES BIRT. 3-1-7. NMR #4 SAdzt F AA 1 mis} DOF 7242] BAA 5 mg 1 mls} D204) a. SHAT 4 +54] NMR 34] (500 MHz, 7123}84)2d7-€)- 3-1-8. PISERN AY OSH BYR AE Sh HES) CS AR, SU HF CS GA ala 10 me/ml -Bhol-] 72} 100 ul ao} 800 ulo} 50 mM VHASY (PH 7.05 SHE F SESE UT ABCH 100 ul B74] 37 CHA 24a WSAAT. BAUSS BAZI71 A A LOOT SH Stk HAS F UAARs, AIL F ISA 100 ules Aero} water (pH 3.5) ¥]2) 2H Phenosphere column) #@2}gth. ALB 7 1 -F columa& water (pH 3.5) 1H CV (column volume, 4.155 ml) 8H A Aetna, aseto] 0 - 1 M NaCl (pH 3.5) linear gradient (10 CV) A944 BAS. AHA FIR AA} 0.1, 0.5, 1, 2, 5, 10, 20 me/ml $= CSE ES GcikagakuS At Ve Wee BAe F 37-12 CS SH Di-05, 6S, 4S)H4] peak areas WRT, lol web Bzho] AB ufo] Esa CS] Wee AHA. odH HEFL NZP CS SES|PRMALS olgsie] MAA + low pressure-GPC 3 HPLC-GPC%} SAX-HPLC prep column °]@-é}o] 4) RT. le BS AAA] AUA-GalNAc, AUA-GalNAc4S 3! AUA-GalNAc6S | HEELS 10 mglJa aRapgir. Was WHA Bactervides stercoris HJ-15 #4} chondroitinase AC QAE-cellulose, DEAE-cellulose, CM-Sephadex, hydroxyapatite, Hitrap heparin, Hitrap SP column chromatography# °|@éte] 4alst9s1, chondroitinase ACB UA. Baal + SE 3,968] 2 NBIES 48.9 umole/min/ngl, 27]9] HA. BAB) ¥) Dap | 5208 S7A. RELEASE MISSES] | SHS a7] Aah H ABEZTA 2 gS 13 we] 50 mM WteSe} (pH 7.044 Holy Malet chondroitinase AC 2 UnitS 7+} 4847 WeAlAt. UWsds Fa UZ 21 - SAX-HPLC prep column& AH8#}4] CS Di-OS, Di-6S, Di-4SE peak fractionation 891th. 47t9] HIELO o|PPEL FADZ F Sephadex G10 column& Fe Se Ae F SAAS lu, SAX-HPLC analytical column? 4}$3}4] ES Helapach 3-1-8. Aaa, Bh) 2 4a) S HSS ER EA FAS SAX SAY AaAGTS 50 mi tube YT 50 mio] ARH SHES AIT BHO] ABSS 45°C shaking water batholA| 72 Tesh} separate funnel‘ ‘31, n~hexane 50 ml& A7b}st Sesto} FE Set st B4. Wa FSS Bee F 1 my ae 1 mig} phenol-chioroform -isoamyl alcohol (25:24:1)& @7Hte] 208 St EeetRe. Azo] ARS 15,000 x g WA] 20% So AILE] Aaa, FSNS Aste] SH Het Was A. Syringe filter o]3}q 12H (100 ul)\S GPC-HPLCH BAYsigia, A | peak 4S APAs vlalete] Asta. AFIS AZ} Ge Ys] NZP CSS GAGAVA ARIAS of UE peck AAS Say AaLeheted, 3-1-9, Caco~2 AEFS 180 HE za F4 31-495 42] AME pH 7.4% 1% ob}, 10% FBS, 100 ug/ml 4 penicillin, streptomycin 27% DMEM “2)ol4] 5% CO2% 90% FEA] 7 14] AS F trypsin-EDTAZ Ae] F At Bas] zehp7] Ae 2.5 ~ 3.0 x10’ AEB #122 329A Transwell polycarbonate (Costar) 49+. apical side} basolateral side® 28 GALS Doha 34k AS 71Re. Apical side®) 7) HBSSE SH to}Fat 37°C] ABA. ASA ARE AMA CS, CS OVE apical So) Azlee] 3A)7 MYM F 1 Az Wyo & basolateral side] F544 CS] GS dimethylmethylene blue# °}-8-814] SRIAG. BE SAS 39 USS HAs Re PFAtspe= Baad.Table 1. ASM] TH WER NUEE IR AES cs 7a a 1 NAR 1 Porcine Trachea Hooper (Germany) ea Porcine Trachea Hooper (Germany) RAE Porcine Trachea Hooper (Germany) sy Porcine Trachea G&F Hanss Biopharma (Germany) BAP Not Labelled Seikagaku (Japan) TA Not Labelled (Korea) UA Bovine Trachea Biocorp (New Zealand) vat Bovine Trachea Biocorp (New Zealand) NAR 2 Bovine Trachea Biocorp (New Zealand) 10. HA 1 Not Labelled Biofac (Denmark) iL. HW 2 Porcine Bioiberica (Spain) 12, CAR Not Labelled Biofac (Denmark)Table 2. WHA] 79] (Toshihiko Toida AF) WE SI4ER Ae WSR BA cs aa AZ 1. MW 14298 Bovine Trachea Spain 2, MW 24909 Shark Cartilage Spain 3. MW 66451 Shark Cartilage Argentina 4. MW 30183 Shark Cartilage China 5. MW 31102 Shark Cartilage China 6. MW 31667 Shark Cartilage China 7. MW 27011 Shark Cartilage China 8. MW 24538 Shark Cartilage China 9. MW 24612 Shark Cartilage China 10. MW 28596 Shark Cartilage China 11. MW 31102 Shark Cartilage Japan 12. MW 30731 Shark Cartilage Japan 13. MW 30916 Shark Cartilage Japan3-2. AY Ast 3-2-1, da As Bradford Bol "et 1B (100 ug) Hol MS] She Gas) GES | AG AWS Table 34 YEHMNA. Akos CSE SH BAY WFAA 294 Aaa ARGA S QAWVS AA] Geo] BEER Gag 4 Be Asa + AG. ASI FSHE WISSe1AGES ded wee] BSA 2 BEPOE te GA Ge SPARS W100 ugs Ad 28 ug, A 0.01 ug Motch. HE} AMA USP FAI AWE HEATH. 3-2-1, HPSECS 2 @ 34h#=ze]d (CS) WEY Uy Sull4] fe CS MR Su alets}b] Sasa gle CS UR) Bas R Ses B7sv) Aa BIOSEP SEC-S4000 column (CV=26.507 ml) °18-8}2] HPSEC BAS +83} Ast, ASHE Figure 1} YEH. Eze] 15,0008 Seikagaku CS HES injection eo] 8]at%t 23} NZP, TAb, HAH] CS QR7} RES HAE EAS } EES UEHYYOD, NAA-1 CS WEY 8% ¥AF peak areat Abe 24 AAS lado] Sus TPH] YE Ale Belsjach. Bats] BP = ABA] BEE peake] Yetz|E Veta}et CSe] Bake SAd (oF 15-22 min)AA] Fat wh peak& YErAch. UAl, SAb, Vals] S+ol= Erb] AA RARALUY ABA] BEE peak] SES Aal, Qrt, Rab, NAk-1, H AL, CALS] 34 ABE BES peakol AEHAc QRoIx] TYR Toshihiko Toida WF A}F) CS AR Prof. Toshihiko Toidaz¥e] ASW CS ARS Ful Sse ARS ¥]528}7] Mal BIOSEP SEC-S4000 column °]-$8¢] HPSEC BAS FAIR 3, @3HE Figure Ze] UEHHSTH bovine CS #1 Spain} 2-F-o}kt NZP CS (MW 15,000) I FARRB, UES Bele YelZ) )BS= NZP CS (MW 15,000) Mla E70] c] Am CS #2, 3, 4B ASE NZ weet BIS BAe CS #3 (Argentina: JIGS] HelT ARS F BAe} -10-7Vh BLA, CS #2E We peak BES UB}. de CS #4 (China)S AM U4 Agee) 2h Suey ASS ARS va CSe) Beto] sesh, A BAGS) HER MDA ASS UTHISM, ESFLSE Seikagaku ik CS (MW 40,000) 8 USP HE] Abs Zieh. aneTable 3. HEE RYYYER Me] dda ae Sample Protein Amount (in 100 ug) 1. NAR CS @B 1/2 (Hooperiit) 2.09 0.05 2, QA CS @B (Hooperit) 1.96 + 0.07 3. RAF CS WE (Hooperit) 1.63 + 0.07 4, SAF CS @& (G&F Hanss At) 2.59 + 0.17 5. BAECS && Gioibericaitt) 0.10 0.05 6. TACS @2 uaa) 0.01 + 0.00 7. UA CS @® Biocorpit) 2.79 £ 0.10 8. VAR CS @2 (Biocorpitt) 2.46 + 0.04 9. NAF CS @8-1 (Biocorpitt) 1.04 + 0.07 10. H4} CS 1-1 (Biofacit) 1.45 + 0.07 11. HAF CS @R-1 (Bioibericaitt) 0.07 + 0.04 12. CA CS UE Biofack) 2.81 + 0.16 13, NZP CS 0.36 + 0.09 14, CA EA AA-1 2.15 + 0.05 15, AAF BU AAl 2.19 + 0.07 16. BA taal 1.19 + 0.14 17. CS A (Calbiochemiil) MW 50,000} 0.82 + 0.05 18. CS C (Calbiochemitt) MW 60,000) 0.96 + 0.02 19. CS C (Sigmait) 0.92 + 0.07 -~2-Q- CS (Hooper) R - CS (Hooper) N-CS 1/2 (Hooper) £ ~ N=GS 212 (Biocorp) é C8 (Biocorp) e V-68 (Biocorp) N © = 68 (Biofac) % HGS 172 (Blofac) a H- 8 212 (Biciberica) 8 700 E -€8 (Bioiberica) £600 8-08 (G&F Hanes ~) 2 500 T- CS (Korea) 5 Nzp cs 5 400 Seikagaku OS (MW 15000) 2 300 = Seikagaku CS (MW 40000) < 200 7 100 ° ° 5 10 15 20 25 30 35 Retention Time (min) Figure 1. HPSECS -@% SU 4-8 WESEVUIES Aes] -13-8 8 8 g & M3657 — OSH (Ching); MZOH — CSB (Ching ; MW24598 — CS#0 (China; 24612 — GS#H0 (Chine); Mave = SHH (epeny ; M311 — OSH (pend: MSDS CS #3 (apa | MSOENE NP cs Salogdaics waren, 3 8 Absorbance at 210 nm 8 Retention Tire (rin) Figure 2. HPSECE $% UY AS MSs |AEH Mes] By -14-3-2-3. Agarose gel A7]9-S Azure A 2A) ¥ 249] MEE SVUES del] LH CS ase ur az seq gy 7¥87| al agarose gel A7IGSS APAND, AHH Figure 3of Eh Heh. EAG RE ARES] ESFOS Ae Seikagaku CS (MW 15,000)9} #AHFo] AHS Hews, NZP, H4K-1, N41 CS QE AF BSEWD FA WE WES GeHARZ, CAh, HAl-2, NA}-2 CS AEH CAt, AAS] CS Bala] BW % LESH MME w CS] WS Aol} VSS YEAR, AZ : 100 ug ALCS Ae 12 ARCS 1S 22 AL- CS C2 A Figure 3. Agarose gel A7]%% - Azure A AY Toluidine Blue & Stains-All 6) G4) 22}9] HELENE Uk Eta CS USS Assy va VAs 7| He are A Ut} FE ASIEM CS Se1gs AW Ace gaa toluidine blues} Stains-All 18 SAWS ABER Figure 4). Azure A BA astWy ehse S59 ob] 2 HA}-1, NAKL CS MES] BH RE HAE We UES Gels, CAL HA2, NAH-2 CS QRH CAS] CS BAA 2 + Azure A @4%3} ha |2 RES VME A Cse] Wee] cps Abo] 7} LSS Yehieic. o] AWS] EFL SE NZP CSHB Aa. AZ : 50ug :NZP CS ALCS QR ‘At Cs 22 AL AL CS Ast 12 AE CS Bz 22 i NAH CS AR V2 :NAFCS S12 2/2. waneeoe Figure 4. Agarose gel @7]9% ~ Toluidine Blue & Stains-All @4¥i 3-2-4. NMR #4} Sud ABZ Ne CS MEY FA AVS AP Se Bee ud a Bap) 877] Ae H-NMR 24S Bsc CES), -2, Ce HAF 2, DE BAH] CS MES VENI, BE alg As} NAl-29} HAl-2, EAA AS BF RES Hale CS AWEgos Bassi. #4 Rol we NMR Ashe PSOE Byes ie3-2-5, PASESNAUERY OS EA Hol $Ssz ee he HIEESTME aE CS UES ETS 7\ Ase] SESNUAS Aes] BIW CSE AeeIsD, ase Figure 5%} Table 4°] UehASic}. 4423 NZP CS B EA, TA}, NA}-2, HAP -19 CS PAtbSe] REFLS A}99 Seikagakuit CS (MW 15,000) Baas St ARE PE GS Molsseh, Table 4. CS AS. 2 ASS VST BAY Peak Area | Percent (%) cs ae . | @i OS + 6S + 4S) | Versus Control =| 1. EAP CS Sz (Bioiberice)’ 636.00 103.5 } 2. TACS @@ (Su) | 602.87 98.1 | 3 NNcSHE> Gam) | oo | oa | 3. NAL CS 2-2 (Biocorp) i 598.58 974 4. HAL CS 12-2 (ioiberica) 615.92 100.2 | _ 5. NZP CS 705.23 | 1147 6. USP CS 581.03 | 945 7. Seikagaku CS (MW 15,000 Da) 614.67 \ 100 -17-EAt CS &1& (Btolbericay TALS &E (HERA) NAECS 81S -2 (Biocorp) 7 = = 7 = Em i» ao 8 Boe! jm io! | ° , UG A ad i A ‘| 4 as . ALI . A Hit oS 82-2 Bioiberica) nepos secs (= ¢ 1 tm 5 dae i i. i- : i. in ie “ i I. : a} * LU : LL Figure 5. SI#EE VUE ‘A: AUA~GalNAc B: AUA-GalNAc6S C: AUA-GalNAcdS QE RAB By - 18 -3-2-6. SYAStaA 2a S PIELER BY nWteoe Ft SS AA H SS] Sol VE WMISER|AVESS F SSEG/MS ENGGO=s StI, NP WIS |YYES RSES A AG4 Gol PS YYOLS FSR F GPC-HPLCH EAS 44 (Figure 6) BE ABAS AIHA (Figure 7). Fe VALS FSM »]Bel del GPC-HPLC BES Fo Sila $ ASHE IUERSY WES ABest (Figure 8). o] oh Med Bol Aste] Bap Aso] UeFE ASE Aaetach, 1000 - — CS Extracted in K-Drug (Label : 300 mg X 5) — CS Extracted in (K-Drug + CS 50 mg) / 50 ml 800 | —— CS Extracted in (K-Drug + CS 100 mg) / 50 mi —— CS Extracted in (K-Drug + CS 200 mg) / 50 mi — CS Extracted in Std NZP CS (500 mg) / 60 ml ao Std NZP CS (500 mg) / 60 mi 400 200 Absorbance at 210 nm Retention Time (min) Figure 6. NZP CSE 43a azo] Yo} eA) Slee FF GPC-HPLC -19-Peak Area 1000 800 600 400 200 © Spiked NZP CS — y= 4.92 + 52.98 Coefficients: bf] 52.98172 bit] 4.9192249143 fr? 0,983527092 0 50 100 200 Spiked NZP CS (mg/50 ml) Figure 7. S234] SF WMSE|MYES WS At BEATA aoe— CS Extracted in K-Drug (Label : 300 mg X 5) / 60 mi — CS Extracted in N-Drug (Label : 120 mg X 5) / 50 ml —— CS Extracted in H-Drug (Label : 30 mg X 5) / 50 ml —— CS Extracted in I-Drug (Label : 90 mg X 5) / 50 ml > CS Extracted in C-Drug (Label : 120 mg X ) / 50 mi “> CS Extracted in O-Drug (Label : 200 mg) / 50 mI —— CS Extracted in P-Drug (Label : 150 mg) / 50 mt — NzP CS (500 mg) / 50 ml 1400 1200 1000 800 600 Peak Area 400 200 ° 10 20 30 40 50 Retention Time (min) Figure 8. Gaga) $ WISE UYEF B4] ae3-2-7. 3494] F PASE SUYER BA XAHS} FARE} A) 19+44(CS HE: 200 mg/A)& 22H] 20 mis) Sol 2 = (et H71a CS Heel BHetc+a 10 mg/ml) syringe filter 9 3}3}o} AA YH SHI ALS AAG F, 42} 500 ule] ABS Centricon (MWCO 10,000, Millipore) 352, 2500 x g A] 20H Sek Useele FASE tube oF capS AAA 714] 1000 x gol] 3k Set UIE se Hoy Bseqoy s & 2249] 41S 100 ul GPC-HPLC# #48} (Figure 9). —— CS in X_Drug Filtered by Cenfricon Absorbance (mAU) at 210 nm Retention Time (min) Figure 9. 3444) 3 SHEE SOU ER 24] ~22-3-2-8. Ath] S$ WISHES |PYES 24 Figure 102 CS7} #4 Ate GPC! #44 aol. ARSE Fa a ZAV ¥ 10 mg/mlo] SEH 4], 45} F 100 ul4 injection 4A. Bet Jo] SNE CS] SEE CA] BH 1 me/mlela, EAL] BH 2 mg/ml +E ole. 3 8 — CS in Lyophilized C-Dru —— CS in Lyophilized E-Dru —— NZP CS (1 mg) g 8 Absorbance (mAU) at 210 nm & 8 0 5 10 15 20 25 30 35 40 45 50 Retention Time (min) Figure 10. 8a] $ HIZESWUER 24]3-2-9, IESE RS Bae By Pat Mas 2 Az dso] REFLE ASE |S YE Wietezo| ay EB) BAGS "Rage (Figure 1D). ¥aKFo] AY B AL Seikagaku CS (MW 40,000 Da)°].at Oral grade® Bago] HMA ABAGE BA Ae] a ct. USP gradeS} BF BAF} 15,000 Da AFA. NZP WE zo) y YER) 7st Saad 7s AS Ace welch — usp cs —— NzP cs — Seikagaku (MW 15000) “> Seikagaku CS (Injection Grade) —— Seikagaku CS (Oral Grade) —— Seikagaku CS (MW 40000) Absorbance at 210 nm Retention Time (min) Figure 11. GPC-HPLCo) 91% SME RUUER BEE RAG Ry =24-3-2-10. Caco-24J EFS St WSEAS He AAS WBE Zo}as] ASL GPC-HPLC| Sal BF 3,000 Darl H. Apical side AES Ae] F Caco-2 monolayerS SAR A 3A FI SA Be AS AVARS Aol slay 3% uls}s] Yo] 3rlzdo] Sshst At (Figure 12). 244, Bike. 2 PAS BER EA ol Folz|Al okt, lee ae Absexo|do] adee Fss\7|7t aa, Bu) Mae aA] REAoR Bal So] Hea + Neh AS AA seh Transport Amount (%) ° 4 2 Time (hour) Figure 12. Caco-2 27S S% HEREBY Fp) es3-3. az 3-3-1, MES UYES AS B72 AGS RES AZ GA PAG ARS Sol Ge] VHS HRS A Us BP 4s ME ZERVUUEH RES 719 SSE TELE Yet. B Ate At Z==°/94EH4] KFDA Reference Standard#4] AO + WSS 7123} = 7} USP reference standards 2322 W BEG $E9] ZEFS Az 100mg 4 24 vpo|se] ZBs-e} 100 vpolehs Azete] asec. BATS Fa ASU WIHSEWWVES BEES BH FS ASA 2 Aad East Fae 712 + sla 7d. 3-3-2. IBESPUER AR FAIS B7 FUESSIPUER ARTAS JEAFHUAID ASS 71S RAD wool pal 2 (4) 3} USP2G(NF)] Ale 2 (8) & MR (Table 5) ae BABY R 7A PEA Aol7} You, WY Ale] at YY AHS ABU] BepZol yar Z7fo] oS Hey] yep Tau BIPSs a7 VS HU AS-3] QT, VHA Fags] HA get. WA, USP Fala ABYS PISEENTUHY BATZa SUI HSS = AYO Wes SUMMERS PeY AQWS S FAM LBIIe FIR 497) | GERAAPILE BAYAN AVY 2 BH9] WP ES AO G. Gey ESS IIGES Aa7ae BY WA Berke USPWNF)Z Bhs Ao) aS SE Aloe Be Ayes WY DW) MIEeWUES Wa RAS Aalst: W Als Aza. 3-3-3. Hb) HE S MISS SGUeR BAY 7 HMESSIVUERS Wre Fiala gle] Sel, datas BH HY ya USF] HG BS ALS] Hoe Sse] V7] Me FAs qe Ad 439 BAZAS AMI NAS asl. B Arey AweSSH 2 HPLC PAGS Sho] vey wesw, ABA] AAR) Sok B8E7 Se Aloe BdM. He Wes HPLC AMS AI GSS 71E BADIA slo ald Wala Avwos Aga + Mach 3-3-4. PABELYYYER in vivo $F AWA Bt WISSSLVAYESY SHAY AAA Caco-2 METH O}-F@ in vivo AVAS BS Ge} VHoey PIBESNUES BTR AAy AU STSH Bo] HHA AALS ales. le ESe FE F Fa | bovine, porcine, avian cartilage #8 OTE BGG Bee |B) 55-70% AW) 25-38%] og Nev egegaca) a4a-ae 224 74, Soh aay B52, WES HE Al As) salwar $849) pH 65-75. (oes as, Qa ea|D mR aed aus, @aee, ousee |p use Dera 420mm absorbance 0:35 °1% sR, [2AzBS 10.0% ©] 8(105°,4hr) lye sie 0.142% layaeate 200 - 300% eana SBS 0.2496 MAES 0.50% ol st ')#420ppm) Rare 0.2436 lah '5)¥142(2ppm) lee 0.000% DEAT ses lade 35% os ABE a none GAG - turbidimetrical method AB AVS?! aeA4 4 APAWISE BYE BW qay7qm of XS} AAS AAA APL) SR BAAAdaA e+ Ad. Fa, 9 es TAMIA AAS VARS ld LAS Selo] Gas 28S Fel St MS Pas Geese PATE BY] FY PAS Sale] WBE | AAA laa WIFe] Oe FACT So] H71s aye age *] bs 10 | 20 | 30} 40 | 50} 60 | 70 | 80 | 90 }100 ad de Fa p> | ieczd edad @ Seal) eso aru) 7 > 21) eee soles] 7a el L | Aa CRRA Aa) | ]GPC-HPLC 3 a719S& o} { Jaa la 2 aa dae MRE let as Ba | 2 aa is 2 daa] BA Ae FESAX-HPLC #4] Stee eoad Staal ¥ - 28 -A5 A ATA] BEI BAF 7h. ASA 733 AEtaAaT) 34 SE (25)| & Acar) zee 39 x | 331 a4 (co-RoM, [Flaalau Taas |e aah, aa | ese] aa | a | aus leaes| eae | FE [Aag,74 Ls | § LL | UY. ASSIS Biological Pharmaceutical Bulletin 200341 12220] Wi A+ Alal aa AJB: Effects of Low Molecular Weight Chondroitin Sulfate on Type IL Collagen-Induced Arthritis in DBA/IJ Mice uy. 28A4 SEF 7190] AH BESS |YGEF KFDA HEFS ABWOEH NE AQ RQ Fade] Weed F Ad. Ee, Al Mla YR, Fe] Ra S184 NGS BIS HAIRS GB WEL Ah azn Qala Ae HE BE Heat UA ALY BSS BA F ABt ME ASS YS F VA. Stl) dol se BUSES IAS #9 US EAM 7heelth leet Bo] As FAG ad + MEA Ss E7} B2e Alt}, FA AFLE ASsa ge ol AF FSET Hel PASS SHV ERS ARAEOS BRS W REAE Aes Alc.AT BREA 1) Conrad HE (1989) Structure of heparan sulfate and dermatan sulfate. Ann N Y Acad Sci 556:18-28 2) Hascall V., Calabro, A., Midura, R., Yanagishita, M. (1994) Isolation and characterization of proteoglycan. Methods Enzymol. 230, 390-417 3) Kelly GS (1998) The role of glucossamine sulfate and chondroitin sulfates in the treatment of degenearuve joint disease. Altern, Med Rev. 3:27-39 4) 451884, Korean Pharmaceutical Codex (1998) (|S 71% 2 ABBY aA 27a) CF) AE BASF pp 1040-1042 5) Murata K and Yokohama Y (1985) Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high performance liquid chromatography. Anal. Biochem. 149:261-268 6) Choi, DW, Kim, MJ, Kim, HS, Chang, SH, Jung SG, Shin, KY, Chang, SY. (2003) A size-exclusion HPLC method for the determination of sodium chondroitin sulfate in pharmaceutical preparations. J. Pharm. Biomed Anal 31: 1229-1236 7) Bradford MM (1976) A rapid and senstive method for the quantitation of protein, Anal. Biochem. 72, 248-254 8) The United States Pharmacopeia (2003) USP 26th edtion, Pharmacopeial Convention, pp 2721-2722 -30-SE ATH] BF a \03072°F# 8561 Bars BA arse SEES] AG AID - GSES ES ape a4 aaa [passes] azaga |A% 7188 AAA Aas O OTS FFESEA PISSSNES TIA, FAY BAIA WA Bea Soe HEAT TY ty. Ee Me Aa, GAA, OE Sd EBS} Ud. B clas} Fae ae) eo ey Bol] Aw Golan or galesh Bago) ahepy A}S. He 7 keh. AAAS] BISLSIGS QE Lge] 40,000 lly, eagez A} Ssle HSE SoAS Lage] 15,0009K4. Wey, Aa Bay Ecpagsl feaaol BIA + slo] MEA) Ser} Gola + AO. las QUI Qaeesclea F Ae Ave Bue Avo) Fae + sl Val, Aa, ABA wae Besa sae azo] Bs] Nek of wea Reka she Meld. Se), Hess dHt sal eo} As) Baad aeold. lela EHGTS Teel Bh. Ha4) L|PORPE OV HHE Des, Qed de, ea Ss, Bal] (Sa ZH SS PHsle] Palas] We eal me} Asl Iz] F ea 24, GPC-HPLC, W719S4, NMR #4, ZER|eya) A428 Ade = AGHA SY 24S SAX-HPLCE Sole] CEB AGach, Healy] APE MATS 2] EA] Yoho] aya SHLAA AAD H, + sol Sel ld) IVES I|GS SVSEE/ISS Bee] F¥c}o] GPC-HPLCE olsalel ERM WHE Sole Ma FAIS ol sete] Be] Aase] Pe YgoR BAe 2 ee, BAG MPS PISS SAA] MASTS Caco-2 AETS Pate] Feast] DU SF WIS PEswA Uh. PiaO arus “A BESUUERS Us W7} 2 Ae HSE AS MNBSENAS Lo 7]B-19 Yo] Ao] FAG aA, FA, REFLS AA ot AAR BAAS ENGR FAA AL BstT4 B19] special grade 7 B= =O] aaat Ad CH ASA} 4-AaolEs 6-AMAIEA we Ble Sx zoe} 4a (chondroitinase) ABC HE ACS A}Ss}o} aa] LAA F AQIS SEH SH (A [Di-4S, ADI-6S, ADi-0S)$ HPLCS o]Béte] EA. sea, Prep TS o|ssto} 72H S) oNSHS AZHRS. MISE ad Meese CH Az1Sy (NMRS A148 ao} Beles. oBd SEREUEEY Can4 NE 87} Fa NE FAS LAMY Aviad PISSSoayess 7s AWys) ae, a 4, BUAAS Bl. | ES YYLS, SSSole tal ABC EE ACH ASA Hizcsojae dae] 17 | HPLCF Yt FA PYS EMste] BySo] Bh whe it ASHlSa SES Yrs bic. + 391 159 AVA PASSE NAYES HPLOLA We SUI BISS Ptecso|aaae| Boe Aas, ga, dea Sole. Va] a AAS Se ala} FS olFa oe, Gd AGE Yoo ye aA) VF GPU! eS Heo] BEF V7] Mo SSSolmHPts Bay WHS ol#ste] Ras at Fy] 2 Fol BAR SES APA sto] HPLC BABS ol Sse] ¥ASIAch HPLC BF} JH Abo]7) oho} GPC-HPLCS fete] Balt sict. 2 AETE 180 MES es UE) Wt eF In vitro] SFE B7G + gle YYOS ates] ALF (Caco-2)H Gs WY Je Bao] $+ YRS WUE F Vd. Caco-2 ARFE AHS] £35} BHH7} Alea isioe Be Ao] Bol SF AVE HA Se] Aes =e Ud. Mlececlas og It, HUIS BW HIESE|Y €Aass sete Pag By Hea BAe! JaatRUL B32 carbazole assayt} dimethyl methylene blue (DMMB)S °-$8}o} #3} jee Beets.O ATIMSSHF LR BEAALD 2 ate Sq aa, SHIA AISA, EE FUFT VE GZS ZS] AIUZSS FA Aged Bec Aa ARS St + RAr. F8], HPLCS 1B sey BASH WAS ATLobWIE Wel ASs|a Mou, of] ofgolrp o} obs eA AAS ANE 4] Sth. ES 7 BAW HPLCS | seq HAs Ue] Saadcde lessodyes am Ra Halls) Fave] Be] BHA +H MS Aelth HM, BkHselde PAHS Fes! Pele dez Feo] 74 ag 2 NB eIE 44) Fed + ach 0 adara ag | FUESUS ae FUSE 2a4 aaa AEE ena ae |MISSZ PUES, Fg, HPLC RY, AE BSS Keywords oA as) ~ Chondroitin sulfate sodium, evaluation, HPLC analysis, \viscosity and absorption ~33-<% S> SUSE Eo]a AEF} NMR Hole} 1. NZP CS 2. NAL UE 2/2 3. HA UB 2/2 4 EAL @& 5. TAR UR = 34 -280 NEP CS. stsNA St 3%NA REwa t : : rr