JPS6160081B2 - - Google Patents
Info
- Publication number
- JPS6160081B2 JPS6160081B2 JP52132189A JP13218977A JPS6160081B2 JP S6160081 B2 JPS6160081 B2 JP S6160081B2 JP 52132189 A JP52132189 A JP 52132189A JP 13218977 A JP13218977 A JP 13218977A JP S6160081 B2 JPS6160081 B2 JP S6160081B2
- Authority
- JP
- Japan
- Prior art keywords
- protease
- chs
- comb
- chloride
- residue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 241000287828 Gallus gallus Species 0.000 claims description 20
- 108091005804 Peptidases Proteins 0.000 claims description 19
- 239000004365 Protease Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 18
- 229920002674 hyaluronan Polymers 0.000 claims description 12
- 239000012266 salt solution Substances 0.000 claims description 12
- 210000001520 comb Anatomy 0.000 claims description 9
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 5
- 102000008186 Collagen Human genes 0.000 claims description 5
- 108010035532 Collagen Proteins 0.000 claims description 5
- 229920001436 collagen Polymers 0.000 claims description 5
- 229960003160 hyaluronic acid Drugs 0.000 claims description 5
- 229920002683 Glycosaminoglycan Polymers 0.000 claims description 4
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 3
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims 3
- 229960005188 collagen Drugs 0.000 claims 1
- IRLPACMLTUPBCL-KQYNXXCUSA-N 5'-adenylyl sulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OS(O)(=O)=O)[C@@H](O)[C@H]1O IRLPACMLTUPBCL-KQYNXXCUSA-N 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000002244 precipitate Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 238000000605 extraction Methods 0.000 description 7
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 7
- 229940099552 hyaluronan Drugs 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 6
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 229920002301 cellulose acetate Polymers 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000005194 fractionation Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 239000003957 anion exchange resin Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 235000011147 magnesium chloride Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000561734 Celosia cristata Species 0.000 description 2
- 101710190411 Chalcone synthase A Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- VBDULUWSKCYWTK-HVLWPHHJSA-N (2s,3s,4s,5r,6r)-6-[(2r,3r,4r,5r)-2-acetamido-4,5,6-trihydroxy-1-oxohexan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical group CC(=O)N[C@@H](C=O)[C@H]([C@H](O)[C@H](O)CO)O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O VBDULUWSKCYWTK-HVLWPHHJSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000201841 Celosia Species 0.000 description 1
- 101710190405 Chalcone synthase B Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 241000766026 Coregonus nasus Species 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008952 bacterial invasion Effects 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229960004830 cetylpyridinium Drugs 0.000 description 1
- NEUSVAOJNUQRTM-UHFFFAOYSA-N cetylpyridinium Chemical compound CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 NEUSVAOJNUQRTM-UHFFFAOYSA-N 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Substances 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 229960004249 sodium acetate Drugs 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 125000003508 trans-4-hydroxy-L-proline group Chemical group 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Description
【発明の詳細な説明】
本発明は酸性ムコ多糖体（以下AMPSとする）
の分取法に関する。詳しくは鶏冠からAMPSを取
得するに際して、鶏冠を生のままプロテアーゼ処
理することにより天然の状態に近い性状を有する
ヒアルロン酸（以下HAとする）を選択的に、か
つ当量的に分別溶出させる方法に関しており、さ
らにはHAを溶出させた鶏冠中に残存しているコ
ンドロイチン硫酸（以下CHSとする）を、塩溶
液で処理することにより分別溶出させ次にその残
渣より酸可溶性のコラーゲン（以下CLとする）
を抽出することに関する。HAは動物の結合組織
中に広く存在する下AMPSの一種で、Ｎ−アセチ
ルヒアロビウロン酸を反復単位とする鎖状の巨大
分子であり、その水溶液は強い保水性、粘弾性を
示し、生体内では細胞間の物質の移動の制御、バ
クテリアの侵入に対する防御、細胞の増殖、分化
の制御等の重要な機能を営なんでいることが知ら
れている。またHAは医療用として眼科領域（網
膜剥離）に対して（Mod.Probl.Ophthal.、12、
370〜377、1974）、関節領域（関節炎）に対して
（Acta Vet.Scand.、17、379〜394、1976）、皮膚
領域（皮膚炎）に対して（Osped.Ital.Chir.、
19、173〜188、1968）等で使用され、その治療効
果を認められているが、いずれの場合も高分子で
あることが望ましいとされている。CHSは一般
的にCHS−Ａ、−Ｂ、−Ｃの三種の異性体に分類
され、このうちCHS−Ａ、−Ｃは軟骨中に多量に
存在するが一方、CHS−Ｂは軟骨中にはほとん
ど存在せず、皮膚、腱、大動脈等に存在する、こ
とが知られている。またその生理活性として、組
織の繊維化に一定の役割りを果していることが報
告されており、（Biochem、Ｊ、109、857〜867、
1968）、ムコ多糖の研究にはかかすことのできな
い物である。[Detailed Description of the Invention] The present invention relates to acidic mucopolysaccharide (hereinafter referred to as AMPS)
Regarding preparative methods. In detail, when obtaining AMPS from chicken combs, we will introduce a method for selectively and equivalently eluating hyaluronic acid (hereinafter referred to as HA), which has properties close to its natural state, by treating raw chicken combs with protease. Furthermore, chondroitin sulfate (hereinafter referred to as CHS) remaining in the comb from which HA has been eluted is fractionally eluted by treatment with a salt solution, and then acid-soluble collagen (hereinafter referred to as CL) is extracted from the residue. )
Concerning extracting. HA is a type of AMPS that is widely present in the connective tissue of animals, and is a chain-like macromolecule with repeating units of N-acetyl hyalobiuronic acid. Its aqueous solution exhibits strong water retention and viscoelasticity, and is bioactive. It is known that they play important functions in the body, such as controlling the movement of substances between cells, defending against bacterial invasion, and controlling cell proliferation and differentiation. HA is also used for medical purposes in the field of ophthalmology (retinal detachment) (Mod. Probl. Ophthal., 12,
370-377, 1974), for the joint area (arthritis) (Acta Vet.Scand., 17 , 379-394, 1976), for the skin area (dermatitis) (Osped.Ital.Chir.,
19 , 173-188, 1968), and their therapeutic effects have been recognized, but in all cases, polymers are considered desirable. CHS is generally classified into three isomers: CHS-A, -B, and -C. Of these, CHS-A and -C exist in large amounts in cartilage, while CHS-B is found in cartilage. It is known that it is almost absent, but exists in the skin, tendons, aorta, etc. It has also been reported that its physiological activity plays a certain role in tissue fibrosis (Biochem, J, 109 , 857-867,
(1968), which is indispensable for research on mucopolysaccharides.
さて鶏冠は高分子量HAとその大部分がCHS−
ＢであるCHSを含み、他のAMPSをほとんど含ま
ない点でその他の結合組織とは大きく異なる。こ
のような鶏冠よりの公知のHAの製造法として
は、鶏冠をミンチし、アセトン等の有機溶媒で脱
脂、変性させた後大量の水又は塩溶液で抽出する
方法（BBA156、17〜30、1968）、鶏冠をミンチ
し、アセトン等の有機溶媒で脱脂、変性後プロテ
アーゼ消化を行なつてAMPSを溶出させ（J.
Histochem.Cytochem.、６、416−424、1958）
以下公知の方法によりHAを分取する方法がある
が前者ではHAの抽出効率がいちじるしく悪くま
た収率も低いし、後者ではCHSがHAとともに溶
出してくる為にピリジンまたはアルコール存在下
における硫安分画等で代表される煩雑なHAの分
画操作が必要であるし、またこのような分画操作
に伴なつてHAの低分子化が起る。さらに両者と
もアセトン処理を必要としているが、鶏冠をアセ
トン処理するには大量のアセトンを必要とし、こ
の為工業的方法としては不向きである。 Now, cockscomb is made of high molecular weight hyaluronan and most of it is CHS-
It is very different from other connective tissues in that it contains CHS (B) and almost no other AMPS. Known methods for producing HA from chicken combs include mincing chicken combs, defatting and denaturing them with an organic solvent such as acetone, and then extracting with a large amount of water or a salt solution (BBA 156 , 17-30, (1968), minced chicken comb, defatted it with an organic solvent such as acetone, denatured it, and then digested it with protease to elute AMPS (J.
Histochem.Cytochem., 6 , 416-424, 1958)
There are methods for fractionating HA using the following known methods, but in the former, the extraction efficiency of HA is extremely poor and the yield is low, and in the latter, CHS is eluted together with HA, so ammonium sulfate is removed in the presence of pyridine or alcohol. A complicated hyaluronan fractionation operation, typified by fractionation, is required, and hyaluronan is reduced in molecular weight by such fractionation operations. Furthermore, both methods require acetone treatment, but a large amount of acetone is required to treat chicken combs with acetone, which makes them unsuitable as industrial methods.
又一般的なAMPSの分画法としてイオン交換樹
脂を用いたカラムクロマトグラフイーがあるが、
このような方法ではHAの分子量低下が著しい
し、又鶏冠中のHAが高粘性である為、工業的に
は不可能な方法である。 In addition, column chromatography using ion exchange resin is a common AMPS fractionation method.
This method is industrially impossible because the molecular weight of HA is significantly reduced and the HA in the comb is highly viscous.
さて本願発明者は鶏冠中のAMPSであるHAと
CHSを簡便に分取する方法について鋭意検討
し、先に出願した特願昭51−60574号で鶏冠を35
〜65℃で加温した後プロテアーゼ処理を行なつて
AMPSとしてHAのみを天然の状態に近い性状で
選択的に溶出させることよりなるAMPSの分取法
について述べた。しかしその後さらに検討を行な
つた結果、35〜65℃で加温することは必ずしも必
要なく鶏冠を単に生のまま脱脂変性の操作を施さ
ずにプロテアーゼ処理することによつても天然の
状態に近い性状を有するHAを選択的かつ当量的
に分別溶出させ得ること、およびこのようにして
HAを溶出させた鶏冠残渣を塩溶液で処理すると
当量的にCHSを分取できることを見出し本発明
に到達した。本発明で「生のまま」とは、脱脂、
変性処理を施さないことを意味し、保存のために
凍結する等の操作を排除する意味ではない。 Now, the inventor of the present application has discovered that HA, which is AMPS in the cock's comb,
We worked hard to find a method to easily separate CHS, and in the patent application No. 60574, filed earlier, we collected 35 chicken combs.
After heating at ~65℃, protease treatment was performed.
We have described a preparative method for AMPS that involves selectively eluating only hyaluronan as AMPS with properties close to its natural state. However, as a result of further investigation, we found that heating at 35 to 65°C is not necessarily necessary, and that simply treating the comb raw with protease without defatting and denaturing it will bring it closer to its natural state. HA having the properties can be selectively and equivalently eluted, and in this way
The present invention was achieved by discovering that CHS can be fractionated in equivalent amounts by treating chicken comb residue from which hyaluronan has been eluted with a salt solution. In the present invention, "raw" means defatted,
This means that no denaturing treatment is performed, and does not mean that operations such as freezing for preservation are excluded.
本発明の目的は鶏冠中に含まれるHA、CHS、
CL等の有用成分を高度に活用する方法を提供す
ることにあり、本目的は本発明の方法に従つて鶏
冠を処理することによつて達成することができ
る。 The purpose of the present invention is to
It is an object of the present invention to provide a method for highly utilizing useful components such as CL, and this object can be achieved by treating chicken comb according to the method of the present invention.
次に本発明を詳しく説明する。 Next, the present invention will be explained in detail.
新鮮な鶏冠を細断し、PH５〜８で35〜60℃に保
つてプロテアーゼ処理を行なつた後、液相と残渣
を分離し、液相に選択的かつ当量的にHAが溶出
するので公知の方法に従つてこれに塩化セチルピ
リジニウムを加え、生じた沈殿を塩とアルコール
を含む水溶液にとかし、さらにアルコールを加
え、生じた沈殿を分取、乾燥してHAが得られ
る。またHAを分取し、結果的にAMPSとして
CHSのみを含む鶏冠残渣を中性塩溶液で処理を
行なつた後、液相と残渣に分離し、液相を公知の
方法に従つて陰イオン交換樹脂に吸着させ、洗滌
することにより夾雑蛋白を除去し、適当な塩溶液
で溶出後、溶出液にアルコールを加え、生じた沈
殿を分取、乾燥することによりCHSが得られ
る。 It is well known that fresh chicken comb is shredded and treated with protease at pH 5-8 and kept at 35-60°C, and then the liquid phase and residue are separated, and HA is selectively and equivalently eluted into the liquid phase. Cetylpyridinium chloride is added to this according to the method of 2009, the resulting precipitate is dissolved in an aqueous solution containing salt and alcohol, alcohol is further added, and the resulting precipitate is collected and dried to obtain HA. In addition, HA is fractionated, resulting in AMPS.
After treating the comb residue containing only CHS with a neutral salt solution, it is separated into a liquid phase and a residue, and the liquid phase is adsorbed to an anion exchange resin according to a known method and washed to remove contaminant proteins. After removing and eluting with an appropriate salt solution, alcohol is added to the eluate, and the resulting precipitate is collected and dried to obtain CHS.
従来から知られているプロテアーゼを用いる
HAの抽出法では、鶏冠を脱脂、変性後のプロテ
アーゼ処理を行つたためにHAとCHSが同時に溶
出し、これを分離するのに煩雑な操作が必要であ
り、しかもHAの低分子化が余儀なくされてい
た。而るに本発明の方法に従えば、これら従来法
にみられる難点を解決し、鶏冠中の有用成分であ
るAMPSを簡便にかつ効率よく分取できる。なお
本発明の方法は従来法と異なり、鶏冠中のCLを
ほとんど変性させず、従つて、AMPSを分取した
鶏冠の残渣はさらに酸可溶性のCL製造の原料に
供することができる。 Using conventionally known proteases
In the HA extraction method, HA and CHS are eluted at the same time because the chicken comb is defatted and denatured, and then treated with protease, which requires complicated operations to separate, and furthermore, HA is inevitably reduced to a lower molecular weight. It had been. However, according to the method of the present invention, the difficulties seen in these conventional methods can be solved, and AMPS, which is a useful component in chicken comb, can be easily and efficiently separated. Note that unlike conventional methods, the method of the present invention hardly denatures CL in the chicken comb, and therefore, the residue of the chicken comb from which AMPS has been separated can be further used as a raw material for the production of acid-soluble CL.
本発明のHA抽出におけるプロテアーゼ処理は
PH５〜８の範囲において35〜60℃の間で行なう。
その際用いるプロテアーゼはコラーゲナーゼを除
いて、PH５〜８の間で活性を有する中性プロテア
ーゼであれば何れも用いることができる。PH８を
超えるPH域でのプロテアーゼ処理はHAの低分子
化およびCHSの混入を招くので好ましくなく、
PH５未満のPH域では抽出液が粘稠となつて以後の
操作が煩雑となるし、コラーゲンの溶出も起り好
ましくない。また60℃を超えるプロテアーゼ処理
ではCHSの同時溶出が起るので好ましくなく、
35℃未満でのプロテアーゼ処理では、HAの溶出
が大巾に遅れるので長時間を要することになり実
用的でない。プロテアーゼ処理時間は酵素の種
類、酸素量、処理温度によつて決まつてくる。 Protease treatment in HA extraction of the present invention is
It is carried out at a temperature of 35 to 60°C at a pH of 5 to 8.
As for the protease used in this case, any neutral protease having an activity between pH 5 and 8 can be used, except for collagenase. Protease treatment in a PH range exceeding PH8 is not preferable because it leads to lower molecular weight of hyaluronan and contamination with CHS.
In a PH range of less than 5, the extract becomes viscous, making subsequent operations complicated, and elution of collagen occurs, which is undesirable. In addition, protease treatment at temperatures exceeding 60°C is undesirable as CHS co-elutes.
Protease treatment at temperatures below 35°C is not practical, as the elution of hyaluronan is delayed considerably, requiring a long time. The protease treatment time is determined by the type of enzyme, the amount of oxygen, and the treatment temperature.
このようにしてプロテアーゼ処理によつて得た
抽出液は、HA以外の他のAMPSを含まず、また
蛋白等の夾雑物も少ないので常法に基づく単離
法、例えば塩化セチルピリジニウム沈殿およびそ
れにつづく脱セチルピリジニウム操作により容易
に高純度の高分子量HAを得ることができる。 The extract obtained by protease treatment in this way does not contain other AMPS other than HA, and has few impurities such as proteins, so it can be isolated using conventional isolation methods, such as cetylpyridinium chloride precipitation and subsequent High-purity, high-molecular-weight HA can be easily obtained by removing cetylpyridinium.
HA抽出残渣の塩溶液処理における塩溶液の塩
とは塩化ナトリウム、塩化リチウム、塩化カリウ
ム、塩化アンモニウム、塩化バリウム、塩化マグ
ネシウム、塩化カルシウム、塩化マンガン、塩化
亜鉛、塩化鉄、塩化アルミニウム、硫酸ナトリウ
ム、硫酸カリウム、硫酸マグネシウム、硫酸カル
シウム、硫酸アンモニウム、硝酸ナトリウム、硝
酸カリウム、硝酸アンモニウム、硝酸カルシウ
ム、硝酸マグネシウム、酢酸ナトリウム、酢酸ア
ンモニウム、酢酸マグネシウム等の有機塩、無機
塩のことであるがこの他中性塩であるなら何れも
用いることが出来る。工業的には安価で処理しや
すい塩化ナトリウム、塩化マグネシウム、硫酸ア
ンモニウム等が好ましい。塩溶液の濃度としては
0.02〜1.0M程度が好ましい。塩濃度が上記より
低ければ抽出時間を大巾に延長しなければなら
ず、また上記より高いと抽出液からの塩の除去が
煩雑となる。塩溶液のPHには特に制限はないが、
抽出残渣の変性を防ぐ為にはPHを５〜８の間に保
つことが好ましい。 Salts in the salt solution in salt solution treatment of HA extraction residue include sodium chloride, lithium chloride, potassium chloride, ammonium chloride, barium chloride, magnesium chloride, calcium chloride, manganese chloride, zinc chloride, iron chloride, aluminum chloride, sodium sulfate, Organic salts and inorganic salts such as potassium sulfate, magnesium sulfate, calcium sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, ammonium nitrate, calcium nitrate, magnesium nitrate, sodium acetate, ammonium acetate, magnesium acetate, and other neutral salts. You can use any of them if you have them. Industrially, sodium chloride, magnesium chloride, ammonium sulfate, etc. are preferred because they are inexpensive and easy to process. As the concentration of salt solution
Approximately 0.02 to 1.0M is preferable. If the salt concentration is lower than the above, the extraction time must be significantly extended, and if it is higher than the above, the removal of salt from the extract becomes complicated. There is no particular limit to the pH of the salt solution, but
In order to prevent denaturation of the extraction residue, it is preferable to maintain the pH between 5 and 8.
このような塩溶液処理によるHA抽出残渣より
の抽出液はCHSの他にAMPSをほとんど含まず、
また蛋白の夾雑も少ない為常法に基づく単離法、
例えば、陰イオン交換樹脂を用いて蛋白と分離
し、塩溶液で溶出後アルコールを加え、生じた沈
殿を分取、乾燥することにより容易に高純度の
CHSを得ることができる。 The extract from the HA extraction residue obtained by such salt solution treatment contains almost no AMPS in addition to CHS.
In addition, since there is little protein contamination, isolation methods based on conventional methods,
For example, by separating proteins using an anion exchange resin, eluting with a salt solution, adding alcohol, separating the resulting precipitate, and drying it, it is easy to obtain highly pure proteins.
You can get CHS.
さて本発明の方法にもとづいてHA及びCHSを
分取した鶏冠の抽出残渣はほとんど変性しておら
ず、CLの製造原料として非常に優れており、公
知の酸可溶性のCLの製造法、例えば特公昭37−
14426号、特公昭38−9295号に基づいて処理する
ことにより容易に目的物を得ることができる。 Now, the extracted residue of chicken comb from which HA and CHS were separated based on the method of the present invention is hardly denatured and is excellent as a raw material for producing CL. Kosho 37-
The desired product can be easily obtained by processing based on No. 14426 and Japanese Patent Publication No. 38-9295.
以下実施例によつて本発明の詳細を示す。 The details of the present invention will be shown below with reference to Examples.
実施例
鶏頭から切り離した後直ちに凍結させた鶏冠２
Kgを解凍し、ミンチして水10を加え、PHを7.5
に調整しプロナーゼ（科研化学〔株〕製、プロテ
アーゼの商品名）10万単位を加え、55℃に2.5時
間保つた後遠心分離し、上清液10に５％塩化セ
チルピリジニウム溶液500mlを加え生じた沈殿を
分取し、0.5Mの塩化ナトリウムを含む40％エタ
ノール水７に溶かし、さらに1/2容の95％エタ
ノールを加え、生じた沈殿を分取、乾燥して
HA11.1ｇを得た。このものはウベローデ粘度計
を用いた粘度測定で極限粘度27.5、Ｎ含量3.51
％、Ｓ含量０％で、セルロースアセテート膜によ
る電気泳動を行ないアルシアンブルー染色を行な
つた所１スポツトのみを示した。Example: Chicken crest 2 frozen immediately after being separated from the cockscomb
Thaw Kg, mince, add 10% water and adjust pH to 7.5
Add 100,000 units of pronase (product name of protease, manufactured by Kaken Kagaku Co., Ltd.), keep at 55°C for 2.5 hours, centrifuge, and add 500 ml of 5% cetylpyridinium chloride solution to supernatant 10. Collect the precipitate, dissolve it in 40% ethanol water containing 0.5M sodium chloride, add 1/2 volume of 95% ethanol, collect the resulting precipitate, and dry.
11.1 g of HA was obtained. This product has an intrinsic viscosity of 27.5 and a N content of 3.51 when measured using an Ubbelohde viscometer.
%, S content was 0%, electrophoresis was performed on a cellulose acetate membrane and Alcian blue staining showed only one spot.
HAを抽出した残渣約0.7Kgに0.05M塩化マグネ
シウム溶液10を加え、４℃において一昼夜撹拌
した後遠心分離し、上清液約10に５％塩化セチ
ルピリジニウム溶液130mlを加え、生じた沈殿を
分取し、0.5Mの塩化ナトリウムを含む40％エタ
ノール水２に溶かし、さらに1/2容の95％エタ
ノールを加え、生じた沈殿を分取、乾燥すること
によりCHS2.0ｇを得た。このものは極限粘度
1.02、Ｎ含量2.90％、Ｓ含量6.60％で、セルロー
スアセテート膜による電気泳動を行ない、アルシ
アンブルー染色を行なうとHAとは明らかに異な
る１スポツトを示した。 Add 10 ml of 0.05 M magnesium chloride solution to about 0.7 kg of the residue from which HA was extracted, stir at 4°C overnight, then centrifuge, add 130 ml of 5% cetylpyridinium chloride solution to about 10 ml of supernatant, and separate the resulting precipitate. The precipitate was collected, dissolved in 40% ethanol water containing 0.5M sodium chloride, added with 1/2 volume of 95% ethanol, and the resulting precipitate was collected and dried to obtain 2.0 g of CHS. This thing has an intrinsic viscosity
1.02, N content 2.90%, S content 6.60%, electrophoresis using a cellulose acetate membrane and Alcian blue staining showed one spot clearly different from HA.
HAおよびCHSを抽出した残渣約0.6Kgを特公昭
37−13871号に従つて塩酸酸性溶液（PH２〜３）
40を加え、ペブシン（半井化学〔株〕製１：
10000）10ｇを加えてPHを２〜３に維持しながら
室温に一昼夜保つた後、過して不溶物を除き、
液を水酸化ナトリウム溶液を用いて、PHを7.5
に調整し、生じた沈殿を分取し凍結乾燥して酸可
溶性のコラーゲン9.6ｇを得た。このものはPH２
〜３の水に可溶で、この溶解液は中和もしくは塩
析によつて白色の繊維状沈殿を生じた。またアミ
ノ酸分析によつてアミノ酸1000残基あたりグリシ
ン336.5残基、ハイドロキシプロリン106.7残基、
プロリン97.5残基を示し、典型的なコラーゲンの
アミノ酸組成を示した。 Approximately 0.6 kg of the residue from which HA and CHS were extracted was
Hydrochloric acid acidic solution (PH2-3) according to No. 37-13871
40 and pevcin (Hani Chemical Co., Ltd. 1:
10,000) was added and kept at room temperature overnight while maintaining the pH at 2 to 3, and then filtered to remove insoluble matter.
Adjust the pH of the solution to 7.5 using sodium hydroxide solution.
The resulting precipitate was collected and freeze-dried to obtain 9.6 g of acid-soluble collagen. This one has PH2
It was soluble in water of ~3, and this solution produced a white fibrous precipitate upon neutralization or salting out. In addition, amino acid analysis revealed that per 1000 amino acid residues, 336.5 glycine residues, 106.7 hydroxyproline residues,
Proline 97.5 residues were shown, indicating the amino acid composition of typical collagen.
実施例
新鮮な鶏冠２Kgを水洗後水10を加え、PHを７
に調整し、プロリシン（上田化学工業〔株〕製、
プロテアーゼの商品名）100万単位を加え、50℃
に3.5時間保つた後遠心分離し、上清液に５％塩
化セチルピリジニウム溶液500mlを加え、生じた
沈殿を分取し、0.5Mの塩化ナトリウムを含む40
％エタノール水に溶かし、更に容解液の1/2容の
エタノールを加えることによつて生じた沈殿を分
取し、乾燥することによりHA11.3ｇを得た。こ
のものは極限粘度26.5、Ｎ含量3.49％、Ｓ含量０
％でセルロースアセテート膜による電気泳動で１
スポツトを示した。HAを抽出した残渣約0.7Kgに
0.1M塩化ナトリウム水溶液10を加え、４℃で
一昼夜撹拌した後遠心分離し、液相をダウエツク
ス１×８（米国ダウケミカルズ社制、陰イオン交
換樹脂の商品名）クロル型カラム（５×50cm）に
負荷し1.0M塩化ナトリウム水溶液で洗滌後、
2.0M塩化ナトリウム水溶液で溶出し、溶出液の
２倍容の95％エタノールを加え、生じた沈殿を分
取し、乾燥することによりCHS2.1ｇを得た。こ
のものは極限粘度0.98、Ｎ含量3.0％、Ｓ含量6.7
％でセルロースアセテート膜による電気泳動で１
スポツトを示した。Example: After washing 2 kg of fresh chicken comb, add 10 g of water and adjust the pH to 7.
Prolysin (manufactured by Ueda Chemical Industry Co., Ltd.,
Add 1 million units of protease (product name) and 50°C.
After keeping for 3.5 hours, centrifuge, add 500ml of 5% cetylpyridinium chloride solution to the supernatant, collect the resulting precipitate, and add 40ml of 5% cetylpyridinium chloride solution to the supernatant.
% ethanol water and further added 1/2 volume of ethanol to the solution, the resulting precipitate was separated and dried to obtain 11.3 g of HA. This product has an intrinsic viscosity of 26.5, N content of 3.49%, and S content of 0.
% by electrophoresis on a cellulose acetate membrane.
The spot was shown. Approximately 0.7 kg of HA extracted residue
Add 10% of 0.1M sodium chloride aqueous solution, stir overnight at 4°C, centrifuge, and transfer the liquid phase to Dowex 1x8 (Dow Chemicals, USA, trade name of anion exchange resin) chloro-type column (5x50cm). After washing with 1.0M sodium chloride aqueous solution,
Elution was carried out with a 2.0M aqueous sodium chloride solution, 95% ethanol was added twice the volume of the eluate, and the resulting precipitate was collected and dried to obtain 2.1 g of CHS. This product has an intrinsic viscosity of 0.98, N content of 3.0%, and S content of 6.7.
% by electrophoresis on a cellulose acetate membrane.
The spot was shown.
Claims (1)
なく、プロテアーゼで処理してヒアルロン酸を選
択的に溶出させることよりなるヒアルロン酸の分
取法。 ２ 鶏冠を生のままプロテアーゼで処理してヒア
ルロン酸を選択的に溶出させ、次にその残渣より
塩溶液でコンドロイチン硫酸を溶出させることよ
りなる酸性ムコ多糖体の分取法。 ３ 鶏冠を生のままプロテアーゼで処理して、ヒ
アルロン酸、コンドロイチン硫酸及びコラーゲン
を選択的に溶出させることよりなる酸性ムコ多糖
体の分取法。[Scope of Claims] 1. A method for preparative separation of hyaluronic acid, which comprises treating raw chicken combs with protease to selectively elute hyaluronic acid without subjecting them to any defatting or denaturation treatment. 2. A method for preparative separation of acidic mucopolysaccharides, which comprises treating raw chicken comb with protease to selectively elute hyaluronic acid, and then eluating chondroitin sulfate from the residue with a salt solution. 3. A method for preparative separation of acidic mucopolysaccharides, which comprises treating raw chicken comb with protease to selectively elute hyaluronic acid, chondroitin sulfate, and collagen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13218977A JPS5467100A (en) | 1977-11-05 | 1977-11-05 | Separation of acid polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13218977A JPS5467100A (en) | 1977-11-05 | 1977-11-05 | Separation of acid polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5467100A JPS5467100A (en) | 1979-05-30 |
| JPS6160081B2 true JPS6160081B2 (en) | 1986-12-19 |
Family
ID=15075467
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13218977A Granted JPS5467100A (en) | 1977-11-05 | 1977-11-05 | Separation of acid polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5467100A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2007069621A1 (en) * | 2005-12-14 | 2009-05-21 | 東京化成工業株式会社 | NOVEL COMPOSITION AND METHOD FOR PRODUCING THE SAME |
| WO2011114689A1 (en) * | 2010-03-15 | 2011-09-22 | 大正製薬株式会社 | Pigment composition |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS618083A (en) * | 1984-06-23 | 1986-01-14 | 株式会社 キビ | Tatami width cutting apparatus in tatami edge sewing machine |
-
1977
- 1977-11-05 JP JP13218977A patent/JPS5467100A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS618083A (en) * | 1984-06-23 | 1986-01-14 | 株式会社 キビ | Tatami width cutting apparatus in tatami edge sewing machine |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5467100A (en) | 1979-05-30 |
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