JP2022187126A - Antiplatelet agent, platelet adhesion inhibitor, platelet aggregation inhibitor, antithrombotic agent, food composition for inhibiting platelet adhesion and/or platelet aggregation, and food composition for preventing or ameliorating thrombosis - Google Patents
Antiplatelet agent, platelet adhesion inhibitor, platelet aggregation inhibitor, antithrombotic agent, food composition for inhibiting platelet adhesion and/or platelet aggregation, and food composition for preventing or ameliorating thrombosis Download PDFInfo
- Publication number
- JP2022187126A JP2022187126A JP2021094977A JP2021094977A JP2022187126A JP 2022187126 A JP2022187126 A JP 2022187126A JP 2021094977 A JP2021094977 A JP 2021094977A JP 2021094977 A JP2021094977 A JP 2021094977A JP 2022187126 A JP2022187126 A JP 2022187126A
- Authority
- JP
- Japan
- Prior art keywords
- platelet
- platelet aggregation
- food composition
- chondroitin sulfate
- agent
- 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.)
- Pending
Links
- 208000010110 spontaneous platelet aggregation Diseases 0.000 title claims abstract description 32
- 239000000203 mixture Substances 0.000 title claims abstract description 28
- 235000013305 food Nutrition 0.000 title claims abstract description 27
- 229940127218 antiplatelet drug Drugs 0.000 title claims abstract description 18
- 208000007536 Thrombosis Diseases 0.000 title claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 12
- 239000000106 platelet aggregation inhibitor Substances 0.000 title claims abstract description 12
- 239000003146 anticoagulant agent Substances 0.000 title claims abstract description 9
- 229960004676 antithrombotic agent Drugs 0.000 title claims abstract description 9
- 101000783577 Dendroaspis angusticeps Thrombostatin Proteins 0.000 title claims abstract description 6
- 101000783578 Dendroaspis jamesoni kaimosae Dendroaspin Proteins 0.000 title claims abstract description 6
- 108010081580 platelet adhesion inhibitor Proteins 0.000 title claims abstract description 5
- 229920001287 Chondroitin sulfate Polymers 0.000 claims abstract description 104
- 229940059329 chondroitin sulfate Drugs 0.000 claims abstract description 104
- -1 chondroitin sulfate oligosaccharides Chemical class 0.000 claims abstract description 78
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 72
- 239000004480 active ingredient Substances 0.000 claims abstract description 23
- 235000000346 sugar Nutrition 0.000 claims description 46
- 239000003795 chemical substances by application Substances 0.000 claims description 21
- 150000008163 sugars Chemical class 0.000 claims description 18
- 239000000470 constituent Substances 0.000 claims description 5
- 230000037406 food intake Effects 0.000 claims description 5
- 210000001772 blood platelet Anatomy 0.000 description 65
- 210000004369 blood Anatomy 0.000 description 40
- 239000008280 blood Substances 0.000 description 39
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 210000002381 plasma Anatomy 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000000034 method Methods 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
- 239000007788 liquid Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 239000000853 adhesive Substances 0.000 description 10
- 230000001070 adhesive effect Effects 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000002994 raw material Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 102000008186 Collagen Human genes 0.000 description 9
- 108010035532 Collagen Proteins 0.000 description 9
- 229920001436 collagen Polymers 0.000 description 9
- 238000001914 filtration Methods 0.000 description 9
- 210000004623 platelet-rich plasma Anatomy 0.000 description 9
- PXBFMLJZNCDSMP-UHFFFAOYSA-N 2-Aminobenzamide Chemical compound NC(=O)C1=CC=CC=C1N PXBFMLJZNCDSMP-UHFFFAOYSA-N 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 230000002776 aggregation Effects 0.000 description 5
- 238000004220 aggregation Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000011088 calibration curve Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 4
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- QHXLIQMGIGEHJP-UHFFFAOYSA-N boron;2-methylpyridine Chemical compound [B].CC1=CC=CC=N1 QHXLIQMGIGEHJP-UHFFFAOYSA-N 0.000 description 4
- 210000000845 cartilage Anatomy 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 238000001215 fluorescent labelling Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 229910001220 stainless steel Inorganic materials 0.000 description 4
- 239000010935 stainless steel Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000002834 transmittance Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 3
- 206010047249 Venous thrombosis Diseases 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003674 animal food additive Substances 0.000 description 3
- 230000023555 blood coagulation Effects 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 235000013373 food additive Nutrition 0.000 description 3
- 239000002778 food additive Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012466 permeate Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 150000004044 tetrasaccharides Chemical class 0.000 description 3
- 206010003178 Arterial thrombosis Diseases 0.000 description 2
- 208000006820 Arthralgia Diseases 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 2
- 206010051055 Deep vein thrombosis Diseases 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000005909 Kieselgur Substances 0.000 description 2
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 2
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 2
- 208000010378 Pulmonary Embolism Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 150000002016 disaccharides Chemical group 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000005713 exacerbation Effects 0.000 description 2
- 229940097043 glucuronic acid Drugs 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 208000010125 myocardial infarction Diseases 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000036470 plasma concentration Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012465 retentate Substances 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000238366 Cephalopoda Species 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
- 102100026735 Coagulation factor VIII Human genes 0.000 description 1
- 241000777300 Congiopodidae Species 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical compound OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 description 1
- 208000008930 Low Back Pain Diseases 0.000 description 1
- 241000239205 Merostomata Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 206010033557 Palpitations Diseases 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 206010047513 Vision blurred Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 235000008452 baby food Nutrition 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
- KXKPYJOVDUMHGS-OSRGNVMNSA-N chondroitin sulfate Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003221 ear drop Substances 0.000 description 1
- 229940047652 ear drops Drugs 0.000 description 1
- 230000000001 effect on platelet aggregation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000000629 knee joint Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- FWFGVMYFCODZRD-UHFFFAOYSA-N oxidanium;hydrogen sulfate Chemical compound O.OS(O)(=O)=O FWFGVMYFCODZRD-UHFFFAOYSA-N 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
本発明は、コンドロイチン硫酸オリゴ糖を有効成分とする、抗血小板剤、血小板粘着抑制剤、血小板凝集抑制剤、抗血栓剤、血小板粘着および／または血小板凝集を抑制するための食品組成物ならびに血栓症を予防または改善するための食品組成物に関する。 The present invention provides antiplatelet agents, platelet adhesion inhibitors, platelet aggregation inhibitors, antithrombotic agents, food compositions for inhibiting platelet adhesion and/or platelet aggregation, and thrombosis, which contain chondroitin sulfate oligosaccharides as active ingredients. It relates to a food composition for preventing or ameliorating
コンドロイチン硫酸（ＣＳ）は、ウロン酸とＮ－アセチル－Ｄ－ガラクトサミンとの二糖繰り返し構造からなる長い糖鎖に硫酸基が結合した構造を持つグリコサミノグリカンの一種である。多数の硫酸基を持つことから強く負に帯電して、保水性および弾力性に富む性質を有する。生体内の分布は、軟骨のほか、皮膚などの結合組織、脳などあらゆる組織に広く見られ、細胞増殖因子や細胞外マトリックス成分と相互作用し、細胞接着、移動、増殖、分化、形態形成といった様々な細胞活動を制御しており、特に、軟骨ではクッション作用に重要な役割を果たしていることが知られている。 Chondroitin sulfate (CS) is a kind of glycosaminoglycan having a structure in which a sulfate group is bound to a long sugar chain consisting of repeating disaccharide structures of uronic acid and N-acetyl-D-galactosamine. Since it has a large number of sulfate groups, it is strongly negatively charged and has excellent water retention and elasticity properties. It is widely distributed in vivo in various tissues such as cartilage, connective tissue such as skin, and brain. It is known to regulate various cellular activities, and plays an important role in the cushioning action of cartilage in particular.
また、従来より、ＣＳは弱い血液凝固抑制作用を有することが知られ（非特許文献１）、特許文献１には、ＣＳを過硫酸化した過硫酸化ＣＳが、より高い抗血液凝固活性を有することが開示されている。 In addition, conventionally, CS has been known to have a weak blood coagulation inhibitory effect (Non-Patent Document 1). It is disclosed to have
上述のとおりＣＳあるいはその誘導物について、血液凝固抑制作用を有することは知られていたが、血小板に対する作用は知られておらず、これらを有効成分とする抗血小板剤も無かった。また、高分子のＣＳは、経口摂取してもそのほぼ全てが腸内細菌によって利用され、血中に移行しない（国際特許出願ＰＣＴ／ＪＰ２０２１／３４１４号明細書）。このことから、経口摂取の使用形態では高分子のＣＳによって血液凝固抑制作用を得ることができない。 As described above, CS or its derivatives have been known to have blood coagulation inhibitory effects, but their effects on platelets have not been known, and there have been no antiplatelet agents containing them as active ingredients. In addition, almost all high-molecular CSs are utilized by intestinal bacteria even if they are orally ingested, and do not migrate into the blood (International Patent Application PCT/JP2021/3414). For this reason, in the form of oral intake, high-molecular CS cannot have a blood coagulation inhibitory effect.
一方、本発明者らは、ＣＳを高温高圧条件下で加水分解して低分子化し、コンドロイチン硫酸オリゴ糖（ＣＳオリゴ糖）を製造する技術開発に成功している（特許第６１４６７３３号）。そして今般、本発明者らは、鋭意研究の結果、ＣＳオリゴ糖が、経口摂取された場合であっても生体内に吸収されて血中に存在すること、および血小板の粘着や血小板の凝集を抑制できることを見出した。 On the other hand, the present inventors have succeeded in developing a technique for producing chondroitin sulfate oligosaccharides (CS oligosaccharides) by hydrolyzing CS under high-temperature and high-pressure conditions to reduce its molecular weight (Patent No. 6146733). As a result of intensive research, the present inventors have recently found that CS oligosaccharides are absorbed in the body and exist in the blood even when orally ingested, and that they inhibit platelet adhesion and platelet aggregation. I have found that it can be suppressed.
すなわち、本発明は、上記課題を解決するために成されたものであって、経口摂取された場合であっても有効な、ＣＳオリゴ糖を有効成分とする抗血小板剤、血小板粘着抑制剤、血小板凝集抑制剤、抗血栓剤、血小板粘着および／または血小板凝集を抑制するための食品組成物ならびに血栓症を予防または改善するための組成物を提供することを目的とする。 That is, the present invention has been made to solve the above problems, and is effective even when orally ingested. An object of the present invention is to provide a platelet aggregation inhibitor, an antithrombotic agent, a food composition for inhibiting platelet adhesion and/or platelet aggregation, and a composition for preventing or improving thrombosis.
（１）本発明に係る抗血小板剤は、コンドロイチン硫酸オリゴ糖を有効成分とする。 (1) The antiplatelet agent according to the present invention contains chondroitin sulfate oligosaccharide as an active ingredient.
（２）本発明に係る血小板粘着抑制剤は、コンドロイチン硫酸オリゴ糖を有効成分とする。 (2) The platelet adhesion inhibitor according to the present invention contains chondroitin sulfate oligosaccharide as an active ingredient.
（３）本発明に係る血小板凝集抑制剤は、コンドロイチン硫酸オリゴ糖を有効成分とする。 (3) The platelet aggregation inhibitor according to the present invention contains chondroitin sulfate oligosaccharide as an active ingredient.
（４）本発明に係る抗血栓剤は、コンドロイチン硫酸オリゴ糖を有効成分とする。 (4) The antithrombotic agent according to the present invention contains chondroitin sulfate oligosaccharide as an active ingredient.
（５）本発明に係る剤は、経口摂取により用いられるものであってもよい。 (5) The agent according to the present invention may be used by oral ingestion.
（６）本発明において、コンドロイチン硫酸オリゴ糖の構成糖の数は、２～１２個であってもよい。 (6) In the present invention, the number of constituent sugars of the chondroitin sulfate oligosaccharide may be 2-12.
（７）本発明に係る血小板粘着および／または血小板凝集を抑制するための食品組成物は、コンドロイチン硫酸オリゴ糖を有効成分とする。 (7) The food composition for suppressing platelet adhesion and/or platelet aggregation according to the present invention contains chondroitin sulfate oligosaccharide as an active ingredient.
（８）本発明に係る血栓症を予防または改善するための食品組成物は、コンドロイチン硫酸オリゴ糖を有効成分とする。 (8) A food composition for preventing or improving thrombosis according to the present invention contains chondroitin sulfate oligosaccharide as an active ingredient.
本発明によれば、血小板のコラーゲンへの粘着または血小板の凝集を抑制することができる。すなわち、本発明によれば、血小板血栓の不必要な形成や過剰な形成を抑制することができる。このことから、血栓がその発症や悪化の要因となる疾病や不健康状態の予防や改善に寄与することができる。 According to the present invention, adhesion of platelets to collagen or aggregation of platelets can be suppressed. That is, according to the present invention, unnecessary or excessive formation of platelet thrombi can be suppressed. Therefore, it can contribute to the prevention and improvement of diseases and unhealthy conditions in which thrombus is a factor in the onset or exacerbation thereof.
有効成分であるＣＳオリゴ糖は、後述する実施例で示すように、経口摂取によって血中に移行する。よって、本発明によれば、有効成分を、簡便かつ非侵襲な使用形態である経口摂取の形態で用いて、血小板粘着または血小板凝集を抑制することができる。 CS oligosaccharide, which is an active ingredient, is transferred into the blood by oral ingestion, as shown in Examples described later. Therefore, according to the present invention, platelet adhesion or platelet aggregation can be suppressed by using an active ingredient in the form of oral intake, which is a simple and non-invasive form of use.
また、ＣＳオリゴ糖は、プラセボ二重盲検でのヒト介入試験において、経口摂取における安全性が確認されている（Mie Nishimura, Nobuyuki Miyamoto, Jun Nishihira , Daily Oral Chondroitin Sulfate Oligosaccharides for Knee Joint Pain in Healthy Subjects: A Randomized, Blinded, Placebo-Controlled Study , The Open Nutrition Journal, 2018, 12, 10-20）。よって、本発明によれば、安全性への懸念なく、血小板粘着または血小板凝集を抑制することができる。 In addition, CS oligosaccharides have been confirmed to be safe in oral intake in placebo double-blind human intervention studies (Mie Nishimura, Nobuyuki Miyamoto, Jun Nishihira, Daily Oral Chondroitin Sulfate Oligosaccharides for Knee Joint Pain in Healthy Subjects: A Randomized, Blinded, Placebo-Controlled Study, The Open Nutrition Journal, 2018, 12, 10-20). Therefore, according to the present invention, platelet adhesion or platelet aggregation can be suppressed without concern about safety.
以下、本発明について詳細に説明する。 The present invention will be described in detail below.
本発明は、抗血小板剤、血小板粘着抑制剤、血小板凝集抑制剤、抗血栓剤、血小板粘着および／または血小板凝集を抑制するための食品組成物ならびに血栓症を予防または改善するための食品組成物を提供する。以下、これらの剤および食品組成物をまとめて、あるいはこれらの剤および食品組成物のうちのいずれかを指して「本発明の剤等」あるいは「本発明に係る剤等」という場合がある。 The present invention provides antiplatelet agents, platelet adhesion inhibitors, platelet aggregation inhibitors, antithrombotic agents, food compositions for inhibiting platelet adhesion and/or platelet aggregation, and food compositions for preventing or improving thrombosis. I will provide a. Hereinafter, these agents and food compositions are collectively referred to, or either of these agents and food compositions may be referred to as "agents of the present invention, etc." or "agents, etc. of the present invention."
抗血小板剤は、血小板の働きを低下させて血栓の形成を抑制する作用を有する剤、あるいは当該作用を生体にもたらす目的で用いられる剤をいう。 An antiplatelet agent refers to an agent that has an effect of suppressing the formation of thrombi by reducing platelet activity, or an agent that is used for the purpose of bringing about such an effect to the living body.
血小板粘着抑制剤は、血小板粘着を抑制する作用を有する剤、あるいは当該作用を生体にもたらす目的で用いられる剤をいう。 A platelet adhesion inhibitor refers to an agent that has an effect of inhibiting platelet adhesion, or an agent that is used for the purpose of bringing about this effect to a living body.
血小板凝集抑制剤は、血小板凝集を抑制する作用を有する剤、あるいは当該作用を生体にもたらす目的で用いられる剤をいう。 A platelet aggregation inhibitor refers to an agent that has an effect of inhibiting platelet aggregation, or an agent that is used for the purpose of bringing about this effect to a living body.
抗血栓剤は、血栓の形成を抑制する作用を有する剤、血栓症を予防または改善する作用を有する剤、あるいは前記の作用を生体にもたらす目的で用いられる剤をいう。 An antithrombotic agent refers to an agent that inhibits the formation of thrombi, an agent that prevents or improves thrombosis, or an agent that is used for the purpose of providing the above-mentioned effects to the living body.
血小板の粘着（血小板粘着）とは、血小板が、コラーゲンや血管内皮細胞、血管内皮細胞下組織などの生体内物質に直接あるいは他因子を介して粘着することをいう。また、血小板粘着を抑制するとは、左記物質に血小板が粘着する程度（粘着した血小板数や粘着強度など）を小さくすることをいう。 Platelet adhesion (platelet adhesion) refers to adhesion of platelets to in vivo substances such as collagen, vascular endothelial cells, and vascular subendothelial tissue directly or via other factors. In addition, suppressing platelet adhesion refers to reducing the extent to which platelets adhere to the substances listed on the left (the number of adhered platelets, adhesion strength, etc.).
被験物質により血小板粘着が抑制されたか否かは、例えば、後述する実施例に示すように、コラーゲンビーズを用いた血小板粘着能検査により確認することができる。当該検査において、被験物質の存在によりビーズを通過する血小板数が増大すれば、被験物質により血小板粘着が抑制されたと判断することができる。 Whether or not the test substance inhibits platelet adhesion can be confirmed, for example, by a platelet adhesion ability test using collagen beads, as shown in Examples described later. In the test, if the number of platelets passing through the beads increases due to the presence of the test substance, it can be determined that platelet adhesion is suppressed by the test substance.
血小板の凝集（血小板凝集）とは、血小板同士が直接あるいは他因子を介して凝集して凝集塊を形成することをいう。また、血小板凝集を抑制するとは、凝集塊を形成する程度（凝集した血小板数など）を小さくすることをいう。 Aggregation of platelets (platelet aggregation) refers to aggregation of platelets directly or via other factors to form aggregates. In addition, to inhibit platelet aggregation means to reduce the degree of formation of aggregates (the number of aggregated platelets, etc.).
被験物質により血小板凝集が抑制されたか否かは、例えば、後述する実施例に示すように、光透過法による血小板凝集能検査により確認することができる。当該検査において、血小板刺激物質（アデノシン二リン酸など）添加時の光透過率（凝集率）が被験物質の存在により小さくなれば、被験物質により血小板凝集が抑制されたと判断することができる。 Whether or not platelet aggregation is inhibited by the test substance can be confirmed, for example, by a platelet aggregation ability test using a light transmission method, as shown in Examples described later. In the test, if the light transmittance (aggregation rate) when a platelet-stimulating substance (adenosine diphosphate, etc.) is added becomes smaller due to the presence of the test substance, it can be determined that platelet aggregation is inhibited by the test substance.
血小板粘着および血小板凝集が起こると血栓が形成される。形成された血栓が血管に詰まると、そこから先の血流が著しく悪くなり、組織に重大な障害を引き起こす。例えば、心臓や脳、肺の血管に血栓が詰まると、心筋梗塞、脳梗塞、肺塞栓症を引き起こし、生命に危険をもたらす。このような、血管が血栓で閉塞することにより発症する疾病を血栓症という。血栓症は、動脈血栓症（脳梗塞、心筋梗塞、末梢動脈血栓症など）と静脈血栓症（深部静脈血栓症、術後深部静脈血栓症、肺塞栓、エコノミークラス症候群など）とに分けられる場合がある。 A thrombus is formed when platelet adhesion and aggregation occur. When the formed thrombus clogs the blood vessel, the blood flow from there is significantly impaired, causing serious damage to the tissue. For example, clogging of blood vessels in the heart, brain, and lungs can lead to myocardial infarction, cerebral infarction, and pulmonary embolism, which are life-threatening. A disease caused by such occlusion of a blood vessel by a thrombus is called thrombosis. When thrombosis is divided into arterial thrombosis (cerebral infarction, myocardial infarction, peripheral arterial thrombosis, etc.) and venous thrombosis (deep vein thrombosis, postoperative deep vein thrombosis, pulmonary embolism, economy class syndrome, etc.) There is
また、疾病まではいかずとも、正常状態よりも血栓の量が増加すると、動脈硬化、痴呆、血圧異常、めまい、肩こり、頭痛、腰痛、関節痛、目のかすみ、不眠、動悸、息切れ、不整脈、皮膚のくすみ、くまなどの不健康状態を引き起こす。 In addition, even if the amount of blood clots increases beyond normal conditions, arteriosclerosis, dementia, abnormal blood pressure, dizziness, stiff shoulders, headaches, lower back pain, arthralgia, blurred vision, insomnia, palpitations, shortness of breath, arrhythmia, etc. Causes unhealthy conditions such as dull skin and dark circles.
ＣＳオリゴ糖は、血小板粘着を抑制、あるいは血小板凝集を抑制できることから、血小板血栓の生成を抑制することができる。よって、ＣＳオリゴ糖は、抗血小板剤や抗血栓剤の有効成分とすることができる。 Since CS oligosaccharide can suppress platelet adhesion or platelet aggregation, it can suppress the formation of platelet thrombi. Therefore, CS oligosaccharides can be used as active ingredients of antiplatelet agents and antithrombotic agents.
すなわち、本発明の剤等は、血小板血栓がその発症または悪化の要因となる疾患や不健康状態を予防または改善する用途の剤や食品組成物として用いることができる。本発明は、血小板粘着を抑制することまたは血小板凝集を抑制することに意義のあるすべての用途に用いることができる。 That is, the agent, etc. of the present invention can be used as an agent or food composition for preventing or improving diseases or unhealthy conditions in which platelet thrombosis is a factor in the onset or exacerbation of these conditions. The present invention can be used in all applications where inhibition of platelet adhesion or inhibition of platelet aggregation is significant.
本発明において、ＣＳオリゴ糖とは、ウロン酸とＮ－アセチル－Ｄ－ガラクトサミンとの二糖繰り返し構造からなる糖鎖に硫酸基が結合した構造（コンドロイチン硫酸構造）をもち、かつ、構成糖の個数が、コンドロイチン硫酸よりも少ないものをいう。ＣＳオリゴ糖の構成糖の個数は、単糖よりも多く、コンドロイチン硫酸よりも少ない数であればよく、例えば、２糖、３糖、４糖、５糖、６糖、７糖、８糖、９糖、１０糖、１１糖、１２糖、１３糖、１４糖、１５糖、１６糖、１７糖、１８糖、１９糖、２０糖・・・４８糖などを挙げることができる。これらのうち、構成糖の個数が２～１２個のＣＳオリゴ糖は、腸管膜の透過性が高いことから（特許第６１４６７３３号明細書：段落［００８１］－［００８６］、［図１８］）、経口摂取により生体に吸収されやすいと考えられる。よって、２～１２糖のＣＳオリゴ糖を有効成分とすれば、本発明の剤等を、経口摂取により簡便に用いられる形態とすることができる。 In the present invention, the CS oligosaccharide has a structure (chondroitin sulfate structure) in which a sulfate group is bound to a sugar chain consisting of a repeating disaccharide structure of uronic acid and N-acetyl-D-galactosamine, and The number is less than that of chondroitin sulfate. The number of constituent sugars of the CS oligosaccharide should be larger than that of monosaccharides and smaller than that of chondroitin sulfate. 9 sugars, 10 sugars, 11 sugars, 12 sugars, 13 sugars, 14 sugars, 15 sugars, 16 sugars, 17 sugars, 18 sugars, 19 sugars, 20 sugars, . Among these, CS oligosaccharides having 2 to 12 constituent sugars are highly permeable to the intestinal membrane (Patent No. 6146733: paragraphs [0081]-[0086], [Fig. 18]). , is considered to be readily absorbed by the body through oral ingestion. Therefore, if a CS oligosaccharide having 2 to 12 sugars is used as an active ingredient, the agent or the like of the present invention can be made into a form that can be easily used by oral ingestion.
ＣＳオリゴ糖は、化学合成して得てもよく、高分子のＣＳを公知の方法により分解して低分子化することにより得てもよい。ＣＳを低分子化する方法としては、例えば、温度Ｔが１７５℃≦Ｔ≦２２０℃でありかつ圧力Ｐが５ＭＰａ≦Ｐ≦２５ＭＰａ高温高圧条件で加水分解する方法（特許第６１４６７３３号公報）や、塩酸などの酸により加水分解する方法（Ｃｉｆｏｎｅｌｌｉ、Ｃａｒｂｏｈｙｄｒａｔｅ Ｒｅｓ．、第２巻、第１５０－１６１頁、１９６６年）、コンドロイチナーゼＡＢＣやコンドロイチナーゼＡＣＩＩ、精巣ヒアルロニダーゼ、ＣＳ分解酵素（特開平９－１６８３８４号公報）などの酵素により脱離分解あるいは加水分解する方法、ｐＨ２．５～１２．０のＣＳ水溶液を１００～１６０℃未満の水熱条件下に５～２０分未満保つ方法（特開２０１０－７７２５６号公報）を例示することができる。 The CS oligosaccharide may be obtained by chemical synthesis, or by decomposing high-molecular CS by a known method to reduce the molecular weight. As a method for reducing the molecular weight of CS, for example, a method of hydrolyzing under high-temperature and high-pressure conditions where the temperature T is 175° C.≦T≦220° C. and the pressure P is 5 MPa≦P≦25 MPa (Japanese Patent No. 6146733), A method of hydrolysis with an acid such as hydrochloric acid (Cifonelli, Carbohydrate Res., vol. -168384)), a method of keeping an aqueous CS solution of pH 2.5 to 12.0 under hydrothermal conditions of 100 to 160 ° C. for 5 to 20 minutes or less (Japanese Patent Application Laid-Open 2010-77256) can be exemplified.
原料のＣＳは、動物の軟骨等から抽出して得てもよく、市販品を用いてもよい。ＣＳの由来としては、例えば、エイやサメ、ウシ、クジラ、ウサギ、ヒツジ、カブトガニ、ブタ、イカ、ニワトリなどの動物を挙げることができるが、いずれの生物であってもよい。また、ＣＳが有する構造としては、例えば、グルクロン酸－Ｎ－アセチル－Ｄ－ガラクトサミン４－硫酸構造（いわゆるコンドロイチン硫酸Ａ構造）や、グルクロン酸－Ｎ－アセチル－Ｄ－ガラクトサミン６－硫酸構造（いわゆるコンドロイチン硫酸Ｃ構造）、グルクロン酸２－硫酸－Ｎ－アセチル－Ｄ－ガラクトサミン６－硫酸構造（いわゆるコンドロイチン硫酸Ｄ構造）、グルクロン酸－Ｎ－アセチル－Ｄ－ガラクトサミン４，６－硫酸構造（いわゆるコンドロイチン硫酸Ｅ構造）、上記の構造において、グルクロン酸がエピマー化してイズロン酸である構造（いわゆるコンドロイチン硫酸Ｂ構造）を挙げることができるが、これらのいずれであってもよい。 CS as a raw material may be obtained by extracting from animal cartilage or the like, or a commercially available product may be used. Examples of the origin of CS include animals such as rays, sharks, cows, whales, rabbits, sheep, horseshoe crabs, pigs, squids, and chickens, but may be any organism. Further, the structure of CS includes, for example, glucuronic acid-N-acetyl-D-galactosamine 4-sulfate structure (so-called chondroitin sulfate A structure) and glucuronic acid-N-acetyl-D-galactosamine 6-sulfate structure (so-called chondroitin sulfate C structure), glucuronic acid 2-sulfate-N-acetyl-D-galactosamine 6-sulfate structure (so-called chondroitin sulfate D structure), glucuronic acid-N-acetyl-D-galactosamine 4,6-sulfate structure (so-called chondroitin Sulfuric acid E structure), and in the above structure, a structure in which glucuronic acid is epimerized to form iduronic acid (so-called chondroitin sulfate B structure), but any of these may be used.
本発明の剤は、ヒトもしくは動物の生体、または生体から採取した血小板を含む細胞や組織・器官に投与することにより使用することができる。投与方法としては、例えば、経口、皮下注射、静脈内注射、筋肉内注射、髄腔内注射、舌下投与、口腔粘膜投与、経直腸投与、経膣投与、点眼、点耳、経鼻、経口吸入、噴霧吸入、経皮などを例示することができる。本発明の剤の形態としては、有効成分であるＣＳオリゴ糖のみからなるもののほか、適当な賦形剤や担体と合わせてなる、医薬品や医薬部外品、サプリメント、食品添加剤、飼料添加物、化粧品などの形態を挙げることができる。係る医薬品や医薬部外品、サプリメント、食品添加剤、飼料添加物、化粧品は、当業者に公知の方法で製造することができる。 The agent of the present invention can be used by administering it to a human or animal living body, or to a cell, tissue or organ containing platelets collected from the living body. Examples of administration methods include oral administration, subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, oral mucosal administration, transrectal administration, transvaginal administration, eye drops, ear drops, intranasal administration, and oral administration. Examples include inhalation, spray inhalation, transdermal and the like. Forms of the agent of the present invention include those consisting only of CS oligosaccharide, which is an active ingredient, as well as pharmaceuticals, quasi-drugs, supplements, food additives, and feed additives, which are combined with appropriate excipients and carriers. , cosmetics, and the like. Such pharmaceuticals, quasi-drugs, supplements, food additives, feed additives, and cosmetics can be produced by methods known to those skilled in the art.
医薬品や医薬部外品、サプリメント、食品添加剤、飼料添加物におけるＣＳオリゴ糖の含有量は、その剤型により異なるが、例えば、乾燥質量を基準として０．００１～９０質量％、０．０１～８５質量％、０．１～８０質量％などとすることができる。 The content of CS oligosaccharides in pharmaceuticals, quasi-drugs, supplements, food additives, and feed additives varies depending on the dosage form, but is, for example, 0.001 to 90% by mass, based on the dry mass, 0.01 ~85% by mass, 0.1~80% by mass, and the like.
また、化粧品におけるＣＳオリゴ糖の含有量は、その剤型により異なるが、例えば、乾燥質量を基準として０.０００１～８０質量％、０.００１～６０質量％、０．０１～５０質量％などとすることができる。 In addition, the content of CS oligosaccharides in cosmetics varies depending on the formulation, but for example, 0.0001 to 80% by mass, 0.001 to 60% by mass, 0.01 to 50% by mass, etc. based on the dry mass. can be
本発明の食品組成物の形態としては、有効成分であるＣＳオリゴ糖のみからなるもののほか、菓子や飲料、加工食品、健康食品、乳幼児食品などの通常の飲食物の形態を挙げることができる。飲食物の形態とする場合は、通常の製造過程で、有効成分を添加して製造することができる。この場合の食品組成物には、人用の飲食物のみならず、家畜、競走馬、愛玩動物などの飼料、ペットフード、飲料など（動物用飼料等）も包含する。動物用飼料等は、対象が動物である以外は飲食物とほぼ等しいことから、本明細書における食品組成物に関する記載は、動物用飼料等についても同様に当てはめることができる。 Examples of the form of the food composition of the present invention include those consisting only of CS oligosaccharide, which is an active ingredient, as well as ordinary food and drink forms such as confectionery, beverages, processed foods, health foods, and infant foods. In the case of making it into the form of food or drink, it can be produced by adding an active ingredient in a normal production process. Food compositions in this case include not only food and drink for humans, but also feeds for livestock, racehorses, pets, etc., pet foods, beverages, etc. (animal feeds, etc.). Animal feeds and the like are almost the same as food and drink except that the object is an animal, so the description regarding food compositions in this specification can be similarly applied to animal feeds and the like.
食品組成物におけるＣＳオリゴ糖の含有量は、飲食物の形態により異なるが、例えば、乾燥質量を基準として０．００１～９９質量％、０．０１～８０質量％、１～８０質量％などとすることができる。１日当たりの摂取量は、１回で摂取してもよいが、数回に分けて摂取してもよい。 The content of the CS oligosaccharide in the food composition varies depending on the form of food and drink, but is, for example, 0.001 to 99% by mass, 0.01 to 80% by mass, 1 to 80% by mass, etc. based on the dry mass. can do. The daily intake may be taken in one time, or may be taken in several divided doses.
以下、本発明について各実施例に基づいて説明する。本発明の技術的範囲は、これらの実施例によって示される特徴に限定されない。 Hereinafter, the present invention will be described based on each embodiment. The scope of the invention is not limited to the features illustrated by these examples.
本実施例において、「％」は、特段の記載のない限り質量％（（ｗ／ｗ）％）を表す。また、単位の「Ｍ」はmol/Lを意味する。本実施例において、「コンドロイチン硫酸オリゴ糖（ＣＳオリゴ糖）の常用者」は、ＣＳオリゴ糖を約３００ｍｇ／日の摂取量で約１２ヶ月間、毎日経口摂取している者を被験者とした。「ＣＳオリゴ糖の非常用者」は、ＣＳオリゴ糖の摂取経験が無い者、または摂取経験があるとしても前回の摂取から３０日以上経過している者を被験者とした。血小板粘着能および血小板凝集能の測定は株式会社保健科学研究所にて行った。健常者における基準値は当該測定機関にて設定された値である。 In the present examples, "%" represents % by mass ((w/w) %) unless otherwise specified. Also, the unit "M" means mol/L. In this example, "regular users of chondroitin sulfate oligosaccharides (CS oligosaccharides)" were subjects who orally ingested CS oligosaccharides at an intake of about 300 mg/day every day for about 12 months. "Never-use CS oligosaccharides" subjects were those who had never taken CS oligosaccharides, or those who had taken CS oligosaccharides for 30 days or more after their last intake. The platelet adhesive ability and platelet aggregation ability were measured by Health Science Laboratory Co., Ltd. The standard value for healthy subjects is the value set by the measuring institution.
＜実施例１＞被験物質の調製
（１）高分子のコンドロイチン硫酸
エイの軟骨２８０ｋｇを斜軸ニーダーに入れ、攪拌しながらパパイン３００ｇを投入して、５５℃で３時間反応させた。９２℃に１０分間保つことにより失活処理したのち、金網を通過させて粗ろ過し、ろ液を回収した。ろ液を５０℃に冷却し、ろ過助剤として珪藻土１０ｋｇを加えてよく攪拌した後、加圧型ろ過装置フィルタープレスを用いて清澄ろ過し、淡黄色清明なろ液を得た。ろ液を分画分子量１３０００の限外ろ過膜を備えた濾過装置に供して、適宜加水しながら１２時間透析した。内液（透析保持液）を回収して９２℃で加熱殺菌した後、スプレードライヤーにより噴霧乾燥し、白色粉末１０．２ｋｇを得て、これを粗精製コンドロイチン硫酸とした。
<Example 1> Preparation of Test Substance (1) Polymer Chondroitin Sulfate 280 kg of ray cartilage was placed in an oblique kneader, 300 g of papain was added while stirring, and reacted at 55°C for 3 hours. After inactivation treatment was carried out by keeping at 92° C. for 10 minutes, coarse filtration was performed by passing through a wire mesh, and the filtrate was recovered. The filtrate was cooled to 50° C., 10 kg of diatomaceous earth was added as a filter aid, and the mixture was thoroughly stirred, followed by clarification filtration using a pressurized filter press to obtain a pale yellow clear filtrate. The filtrate was subjected to a filtration apparatus equipped with an ultrafiltration membrane with a molecular weight cutoff of 13,000, and dialyzed for 12 hours while adding water appropriately. The internal liquid (dialysis retentate) was recovered, heat sterilized at 92° C., and then spray-dried with a spray dryer to obtain 10.2 kg of white powder, which was used as crude chondroitin sulfate.
粗精製コンドロイチン硫酸１０ｇをイオン交換水５００ｍＬに溶解し、活性炭５ｇを加え、４℃で一晩攪拌した。続いて、ろ過助剤として珪藻土を加えて清澄ろ過し、ろ液を回収した。エタノールを終濃度７０（ｖ／ｖ）％となるよう加え、よく攪拌したのち４℃で一晩静置した。白色沈殿をガラスフィルターでろ過して集め、冷却した７０（ｖ／ｖ）％エタノールで洗った。これを４００ｍＬのイオン交換水に溶解し、エタノールを終濃度８０（ｖ／ｖ）％となるよう加え、よく攪拌したのち４℃で一晩静置した。白色沈殿をガラスフィルターでろ過して集め、冷却した８０（ｖ／ｖ）％エタノールで洗った。これを４００ｍＬのイオン交換水に溶解し、エタノールを終濃度９０（ｖ／ｖ）％となるよう加え、よく攪拌したのち４℃で一晩静置した。白色沈殿をガラスフィルターでろ過して集め、別に冷却した９０（ｖ／ｖ）％エタノールで洗った。これをエタノール中で良く攪拌したのち、白色沈殿をガラスフィルターでろ過して集め、デシケーターで乾燥して、精製したコンドロイチン硫酸の白色粉末３．８ｇを得た。これを高分子のコンドロイチン硫酸とした。 10 g of crude chondroitin sulfate was dissolved in 500 mL of ion-exchanged water, 5 g of activated carbon was added, and the mixture was stirred overnight at 4°C. Subsequently, diatomaceous earth was added as a filter aid, followed by clarification filtration, and the filtrate was recovered. Ethanol was added to a final concentration of 70 (v/v) %, stirred well, and then allowed to stand overnight at 4°C. A white precipitate was collected by filtration through a glass filter and washed with cold 70 (v/v)% ethanol. This was dissolved in 400 mL of ion-exchanged water, ethanol was added to a final concentration of 80 (v/v)%, the mixture was stirred well, and then allowed to stand overnight at 4°C. A white precipitate was collected by filtration through a glass filter and washed with cold 80 (v/v)% ethanol. This was dissolved in 400 mL of ion-exchanged water, ethanol was added to a final concentration of 90 (v/v)%, the mixture was stirred well, and allowed to stand overnight at 4°C. A white precipitate was collected by filtration through a glass filter and washed separately with chilled 90 (v/v)% ethanol. After thoroughly stirring this in ethanol, the white precipitate was collected by filtration through a glass filter and dried in a desiccator to obtain 3.8 g of purified white powder of chondroitin sulfate. This was designated as high molecular weight chondroitin sulfate.
（２）ＣＳオリゴ糖およびＣＳ４糖
高分子のコンドロイチン硫酸を、１７５～２２０℃かつ５～２５ＭＰａの高温高圧条件で加水分解して、オリゴ糖のコンドロイチン硫酸（ＣＳオリゴ糖）を得た。具体的には、まず、特許第６１４６７３３号公報に記載のオリゴ糖製造装置（図１）を用意した。すなわち、当該装置は、図１に示すように、蒸留水を収容する水容器、高圧ポンプＡ（ミルフロー制御容量ポンプ Ｍ１５０ パルスレスＣ２４－Ｚ３、日機装）、ヒーター（電気ヒーター）、センサーＡ、原料を収容する原料容器、高圧ポンプＢ（ミルフロー制御容量ポンプ Ｍ１５０ パルスレスＣ２３－Ｘ１、日機装）、混合Ｔ字管、反応部（内腔径０．５ｍｍのステンレス３１６製配管（内腔の空間体積４６８００ｍｍ３～１９１０００ｍｍ３）からなる）、センサーＢ、センサーＣ、ウォーターバス、センサーＤ、背圧弁（Ｈｉｇｈ Ｐｒｅｓｓｕｒｅ／Ｂａｃｋ Ｐｒｅｓｓｕｒｅ ２６－１７６２－６６－３１４、ＴＥＳＣＯＭ）および反応生成物を収容する生成物容器を備えるものであり、水容器、原料容器および生成物容器はすべてステンレス製配管でつながれた構造である。反応部の入り口温度をセンサーＢ、出口温度をセンサーＣで確認する。圧力はセンサーＤにより確認する。
(2) CS oligosaccharide and CS tetrasaccharide A high-molecular weight chondroitin sulfate was hydrolyzed under high temperature and pressure conditions of 175 to 220°C and 5 to 25 MPa to obtain an oligosaccharide chondroitin sulfate (CS oligosaccharide). Specifically, first, an oligosaccharide manufacturing apparatus (FIG. 1) described in Japanese Patent No. 6146733 was prepared. That is, as shown in FIG. 1, the device contains a water container containing distilled water, a high-pressure pump A (Millflow control capacity pump M150 pulseless C24-Z3, Nikkiso), a heater (electric heater), a sensor A, and raw materials. Raw material container, high-pressure pump B (Millflow control capacity pump M150 pulseless C23-X1, Nikkiso), mixing T-shaped tube, reaction part (piping made of stainless steel 316 with a bore diameter of 0.5 mm (inner cavity volume 46800 mm 3 to 191000 mm 3 )), sensor B, sensor C, water bath, sensor D, back pressure valve (High Pressure/Back Pressure 26-1762-66-314, TESCOM) and a product container containing the reaction product. The water container, raw material container and product container are all connected by stainless steel pipes. Check the inlet temperature of the reaction section with sensor B and the outlet temperature with sensor C. Pressure is checked by sensor D.
本実施例１（１）に記載の方法により調製した粗精製コンドロイチン硫酸１０ｋｇを５００Ｌの水に溶解し、Ｂｒｉｘ２．０に調整して原料液とした。脱気した蒸留水を水容器に入れ、高圧ポンプＡにより連続的に送液してヒーターで加熱した。原料液を原料容器に入れ、高圧ポンプＢにより連続的に送液した。加熱された蒸留水と常温の原料液とは、反応部入口の混合Ｔ字管で混合され、反応部にて、水と原料液に含まれるコンドロイチン硫酸とを反応させて加水分解を行った。反応条件は、反応部の温度が１７５～２２０℃、反応部の圧力が５～２５ＭＰａ、反応時間が８．８秒間とした。続いて、ステンレス製配管をウォーターバスで直接冷却することにより反応を速やかに終了させた。その後、背圧弁によりステンレス製配管内の圧力を下げて、反応生成物を生成物容器に収容した。なお、蒸留水の流量は原料液の流量の３倍以上とした。 10 kg of crude chondroitin sulfate prepared by the method described in Example 1(1) was dissolved in 500 L of water and adjusted to Brix 2.0 to obtain a raw material solution. Degassed distilled water was placed in a water container, continuously fed by a high-pressure pump A, and heated by a heater. A raw material liquid was placed in a raw material container and continuously fed by a high-pressure pump B. The heated distilled water and the raw material liquid at room temperature were mixed in a mixing T-tube at the inlet of the reaction section, and the water and chondroitin sulfuric acid contained in the raw material liquid were reacted in the reaction section to perform hydrolysis. The reaction conditions were a temperature of 175 to 220° C. in the reaction section, a pressure in the reaction section of 5 to 25 MPa, and a reaction time of 8.8 seconds. Subsequently, the stainless steel pipe was directly cooled in a water bath to quickly terminate the reaction. After that, the pressure in the stainless steel pipe was lowered by a back pressure valve, and the reaction product was stored in the product container. The flow rate of distilled water was three times or more the flow rate of the raw material liquid.
反応生成物を分画分子量３０００の限外ろ過膜を備えた濾過装置に供して限外濾過し、外液（透過液）を回収して濃縮した。これを陰イオン交換担体NuviaQ（Bio Rad）を充填したイオン交換クロマトグラフィーに供して精製し、続いて、Biogel P-6（Bio Rad）に通して脱塩処理した後、凍結乾燥して、コンドロイチン硫酸オリゴ糖（ＣＳオリゴ糖）の粉末を得た。また、濃縮後の外液を、Biogel P-6（Bio Rad）を充填したカラムを用いたゲル濾過クロマトグラフィーに供して分画し、四糖画分を分取した。これらを、上記と同様に精製、脱塩処理および凍結乾燥し、コンドロイチン硫酸構造を有する４糖（ＣＳ４糖）の粉末を得た。 The reaction product was subjected to ultrafiltration using a filtration device equipped with an ultrafiltration membrane with a molecular weight cut off of 3000, and the external liquid (permeate liquid) was recovered and concentrated. This was subjected to ion exchange chromatography packed with an anion exchange carrier NuviaQ (Bio Rad) for purification, then passed through Biogel P-6 (Bio Rad) for desalting, freeze-dried, and chondroitin A powder of oligosaccharide sulfate (CS oligosaccharide) was obtained. In addition, the concentrated external solution was fractionated by gel filtration chromatography using a column filled with Biogel P-6 (Bio Rad) to collect a tetrasaccharide fraction. These were purified, desalted and freeze-dried in the same manner as above to obtain a tetrasaccharide (CS4sugar) powder having a chondroitin sulfate structure.
＜実施例２＞ＣＳオリゴ糖の血中移行の確認
（１）血漿の取得
ＣＳ４糖を文献（河原ら、応用糖質科学、２０２１年、in press）に従い精製した後、超純水に溶解して水溶液とした。ＣＳ４糖１０００ｍｇを含有する水溶液を、健康な成人男性ボランティア２名（被験者１、２）に経口投与した。なお、被験者１は、ＣＳオリゴ糖の服用を２４時間中止した常用者であり、被験者２は非常用者である。投与前と投与から２時間または３時間後とに、エチレンジアミン四酢酸（ＥＤＴＡ）を終濃度１ｍｇ／ｍＬになるように添加した１０ｍＬ容量のシリンジ(テルモ)に血液を採取した。採血後ただちに１０回ほどシリンジを転倒混和した後、１５ｍＬ容量の遠心チューブに血液を移し、１５分間静置した。これを遠心分離（２３７０×ｇ、１５分）し、上清（血漿）を分離回収して、－２０℃で冷凍保存した。被験者１から取得した血漿を検体１、被験者２から取得した血漿を検体２とした。
<Example 2> Confirmation of blood migration of CS oligosaccharides (1) Obtaining plasma After purifying CS tetrasaccharide according to the literature (Kawahara et al., Applied Glycoscience, 2021, in press), it was dissolved in ultrapure water. was used as an aqueous solution. An aqueous solution containing 1000 mg of CS4 sugar was orally administered to two healthy adult male volunteers (subjects 1 and 2). Subject 1 is a regular user who stopped taking CS oligosaccharides for 24 hours, and Subject 2 is a non-user. Before administration and 2 hours or 3 hours after administration, blood was collected into a 10 mL syringe (Terumo) to which ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 1 mg/mL. Immediately after blood collection, the syringe was inverted about 10 times to mix, and then the blood was transferred to a 15 mL centrifuge tube and allowed to stand for 15 minutes. This was centrifuged (2370×g, 15 minutes), and the supernatant (plasma) was separated and collected and stored frozen at -20°C. The plasma obtained from Subject 1 was designated Sample 1, and the plasma obtained from Subject 2 was designated Sample 2.
（２）サンプル液の調製
解凍した血漿５００μＬを、分画分子量１００００の限外ろ過膜を備えた遠心濃縮チューブに分取し、２ＭのＮａＣｌ水溶液５００μＬを加え、ピペッティングで攪拌した。その後、遠心分離（８０００×ｇ、２０分、１０℃）を行い、透過液を回収した。保持液約２００μＬに等量の１ＭのＮａＣｌ水溶液を加え、ピペッティングで攪拌し、遠心分離（８０００×ｇ、１５分、１０℃）を行い、透過液を回収した。同様の操作を合計４回繰り返し行って透過液を回収し、合計約１７００μＬの透過液を得た。以上の操作により、血漿中に存在するＣＳ４糖の９９％以上を透過液中に回収したと考えられる。透過液を減圧遠心濃縮（５１０×ｇ、１０時間、室温、２０Ｐａ）した後、凍結乾燥した。乾燥物に３００μＬの超純水を加え、ボルテックスにて溶解してサンプル液とした。
(2) Preparation of
（３）ＣＳ４糖の蛍光標識
参考文献（Bigge et al, Analytical Biochemistry, 1995）に従い、サンプル液中の糖の還元末端を２－アミノベンズアミド（２ＡＢ）により蛍光標識した。ただし、水溶液中で還元アミノ化反応を行うため、還元剤として２－ピコリンボラン（２ＰＢ）を用いた。すなわち、３０％酢酸ジメチルスルホキシド溶液に、５０ｍｇ／ｍＬとなるよう２ＡＢを、１１０ｍｇ／ｍＬとなるよう２ＰＢを、それぞれ加えタッピングで攪拌、溶解し、蛍光標識液とした。サンプル液を１００μＬ分取し、蛍光標識液を１００μＬ加え、タッピングで攪拌後、軽度の遠心分離に供して壁面に付着した液を落とした。これを６５℃で１時間置くことにより反応させた。続いて、室温にて放冷後、超純水を１００μＬ加えボルテックスミキサーで攪拌した。過剰に加えた２ＡＢの除去のため、クロロホルム５００μＬを加えボルテックスミキサーで攪拌後、遠心分離に供して水層と有機溶媒層を分離し、ピペットで吸引して有機溶媒層を廃棄した。同様の操作を合計５回行って、水層を回収した。水層を孔径０．２２μｍのメンブレンフィルターGLCTD-MCE1322（島津ジーエルシー）で濾過して有形物を除去し、濾液を回収して血漿サンプルとした。
(3) Fluorescent Labeling of CS4 Sugar According to the reference (Bigge et al, Analytical Biochemistry, 1995), the reducing end of the sugar in the sample solution was fluorescently labeled with 2-aminobenzamide (2AB). However, since the reductive amination reaction is performed in an aqueous solution, 2-picoline borane (2PB) was used as a reducing agent. That is, 2AB and 2PB were added to a 30% dimethyl sulfoxide solution of acetate at 50 mg/mL and 2PB at 110 mg/mL, respectively, and stirred and dissolved by tapping to obtain a fluorescent labeling solution. 100 μL of the sample solution was taken, 100 μL of the fluorescent labeling solution was added, and after stirring by tapping, the solution adhering to the wall surface was removed by mild centrifugation. This was allowed to react by placing it at 65° C. for 1 hour. Subsequently, after standing to cool at room temperature, 100 μL of ultrapure water was added and stirred with a vortex mixer. To remove excess 2AB, 500 μL of chloroform was added, stirred with a vortex mixer, centrifuged to separate the aqueous layer and the organic solvent layer, and the organic solvent layer was discarded by aspirating with a pipette. The same operation was performed a total of 5 times, and the aqueous layer was recovered. The aqueous layer was filtered through a membrane filter GLCTD-MCE1322 (Shimadzu GLC) having a pore size of 0.22 μm to remove solid matter, and the filtrate was recovered to be used as a plasma sample.
また、ＣＳ４糖を超純水に溶解して希釈列を作製し、同様に蛍光標識して、これを検量線用サンプルとした。 In addition, CS4 sugar was dissolved in ultrapure water to prepare a dilution series, fluorescently labeled in the same manner, and used as a sample for the calibration curve.
（４）糖濃度の測定
検量線用サンプルを下記条件の高速液体クロマトグラフィー（ＨＰＬＣ）に供した。得られたクロマトグラムにおいてＣＳ４糖のピーク面積を測定し、検量線を作製した。その結果を図２に示す。図２に示すように、近似曲線は原点を通り、傾き６９７４の直線であり、その決定係数（Ｒ２）は０．９９９９であった。このことから、本実施例における蛍光標識手法は、サンプルのＣＳ４糖１００ｎｇから１ｍｇにおいて非常に良い線形性を有することが確認された。
《ＨＰＬＣ分析条件》
システム：株式会社島津製作所 プロミネンス
検出器：蛍光検出器（励起波長330 nm、検出波長 420 nm）
カラム：ゲルろ過カラムSuperdex 30 Increase 10/300 GL (Cytiva)
移動相：５０ｍＭ重炭酸アンモニウム水溶液
流速：０．５ｍＬ／分
カラム温度：室温
サンプル注入量：１００μＬ
(4) Measurement of Sugar Concentration A calibration curve sample was subjected to high performance liquid chromatography (HPLC) under the following conditions. The CS4 sugar peak area was measured in the obtained chromatogram to prepare a calibration curve. The results are shown in FIG. As shown in FIG. 2, the approximated curve was a straight line passing through the origin and having a slope of 6974, and its coefficient of determination (R 2 ) was 0.9999. From this, it was confirmed that the fluorescence labeling method in this example has very good linearity in the sample of 100 ng to 1 mg of CS4 sugar.
<<HPLC analysis conditions>>
System: Shimadzu Corporation Prominence detector: Fluorescence detector (excitation wavelength 330 nm, detection wavelength 420 nm)
Column: Gel
Mobile phase: 50 mM ammonium bicarbonate aqueous solution Flow rate: 0.5 mL/min Column temperature: Room temperature Sample injection volume: 100 μL
続いて、血漿サンプルを同条件のＨＰＬＣに供してクロマトグラムを得た。その結果を図３に示す。図３に示すように、検体１、２のどちらにおいても、投与後のみＣＳ４糖に相当する溶出位置（保持時間２５．５分）に強いピークが観測された。ＣＳ４糖に相当する溶出位置のピーク面積を、図３において塗り潰しで示す部分に区切って測定した。続いて、投与後の当該ピーク面積から投与前の当該ピーク面積を減じて実質ピーク面積を算出した。この実質ピーク面積を図２の検量線に当てはめてＣＳ４糖の血漿中濃度を算出した。その結果、検体１の投与２時間後でＣＳ４糖の血漿中濃度は１４０ｎｇ／ｍＬであり、検体２の投与３時間後で１７４ｎｇ／ｍＬであった。すなわち、ＣＳ４糖を経口投与した後の血漿中には、投与前と比較して比較的大量のＣＳ４糖が存在していた。これらの結果から、経口投与したＣＳオリゴ糖は、腸管より吸収されて血中に移行し、血中に存在していることが明らかになった。 Subsequently, plasma samples were subjected to HPLC under the same conditions to obtain chromatograms. The results are shown in FIG. As shown in FIG. 3, in both specimens 1 and 2, a strong peak was observed at the elution position (retention time 25.5 minutes) corresponding to CS tetrasaccharide only after administration. The peak area at the elution position corresponding to CS4 sugar was measured by dividing into the filled portions in FIG. Subsequently, the peak area before administration was subtracted from the peak area after administration to calculate the real peak area. The plasma concentration of CS4 sugar was calculated by applying this real peak area to the calibration curve in FIG. As a result, the plasma concentration of CS4 sugar was 140 ng/mL 2 hours after administration of sample 1 and 174 ng/mL 3 hours after administration of sample 2. That is, a relatively large amount of CS4 sugar was present in the plasma after oral administration of CS4 sugar compared to before administration. These results revealed that orally administered CS oligosaccharides were absorbed from the intestinal tract, transferred to the blood, and existed in the blood.
＜実施例３＞血小板粘着能に対する効果：ＣＳ４糖添加時の評価
（１）全血の取得
ＣＳ４糖を生理食塩水に溶解し、各濃度のＣＳ４糖溶液を調製した。ＣＳオリゴ糖の常用者１名（成人男性）を被験者とした。サイズ２１Ｇの翼状針を用いて被験者腕静脈に穿刺し、真空採血管に数ｍＬの血液を採取したのちこれを捨て、４本の新しい５ｍＬクエン酸含有真空採血管に等圧となるまで採血した。採血後すみやかに採血管を転倒混和したのち、シリンジを用いてＣＳ４糖溶液または生理食塩水５０μＬを注入し、再度転倒混和した。ＣＳ４糖溶液は、添加したＣＳ４糖の終濃度が、５０、２５０および５００μｇ／ｍＬとなるよう全血に添加した。これを４℃で冷蔵保存し、血小板粘着能の測定に供した。
<Example 3> Effect on platelet adhesive ability: evaluation when adding CS4 sugar (1) Obtaining whole blood CS4 sugar was dissolved in physiological saline to prepare CS4 sugar solutions of various concentrations. One CS oligosaccharide user (adult male) was used as a subject. A butterfly needle of size 21G was used to puncture the subject's brachial vein, several mL of blood was collected in vacuum blood collection tubes, then discarded, and blood was collected in four new 5 mL citric acid-containing vacuum blood collection tubes until equal pressure was achieved. . Immediately after blood collection, the blood collection tube was mixed by inversion, then 50 μL of CS4 sugar solution or physiological saline was injected using a syringe, and mixed by inversion again. The CS4 saccharide solution was added to whole blood so that the final concentrations of added CS4 saccharide were 50, 250 and 500 μg/mL. This was refrigerated at 4° C. and used for platelet adhesion measurement.
（２）血小板粘着能の測定
コラーゲンビーズ法により血小板粘着能を測定した。すなわち、本実施例３（１）のクエン酸含有全血をシリンジに充填し、コラーゲンビーズカラム（プラスチックビーズ表面にコラーゲンを結合させたもの、型番Lot 0012-4D、アイエスケー）に、シリンジポンプ（シリンジポンプMODEL 777、アイエスケー）を用いて流速０．７５ｍＬ／分で１２０秒間（１．５ｍＬ）通液させた。自動血球計数器（型番XE-2100、シスメックス）を用いて、カラム通過前後の血液の血小板数を計測し、下記式１により血小板粘着能を算出した。その結果を図４に示す。なお、このように測定・算出した血小板粘着能の健常者における基準値は２７．０～５９．０％である。
式１：血小板粘着能（％）＝｛（カラム通過前の血小板数－通過後の血小板数）／カラム通過前の血小板数｝×１００
(2) Measurement of platelet adhesive ability Platelet adhesive ability was measured by the collagen beads method. That is, the citric acid-containing whole blood of Example 3 (1) was filled in a syringe, and a collagen bead column (collagen-bound plastic bead surface, model number Lot 0012-4D, ISK) was added to a syringe pump ( Using a syringe pump MODEL 777, ISK, the solution was passed through at a flow rate of 0.75 mL/min for 120 seconds (1.5 mL). Using an automatic hemocytometer (model number XE-2100, Sysmex), the number of blood platelets before and after passing through the column was measured, and the platelet adhesion capacity was calculated by the following formula 1. The results are shown in FIG. The reference values for the platelet adhesion capacity measured and calculated in this way for healthy subjects are 27.0 to 59.0%.
Formula 1: Platelet adhesive capacity (%) = {(number of platelets before passing through column - number of platelets after passing) / number of platelets before passing through column} x 100
図４に示すように、血小板粘着能はＣＳ４糖の濃度が高いほど小さくなった。すなわち、血中におけるＣＳ４糖の存在量が大きいほど、血小板のコラーゲンへの粘着反応が抑制された。また、ＣＳ４糖を添加しない場合（０μｇ／ｍＬ）において特に明らかなように、ＣＳオリゴ糖の常用者の血液では、健常者における基準値と比較して、もともとの血小板粘着能が顕著に小さかった。これらの結果から、ＣＳオリゴ糖は、血中において血小板のコラーゲンへの粘着を抑制できる、極めて高い効果を有することが明らかになった。 As shown in FIG. 4, the platelet adhesive ability decreased with increasing CS4 sugar concentration. That is, the larger the amount of CS4 sugar present in the blood, the more the platelet adhesion reaction to collagen was suppressed. In addition, as was particularly evident when CS tetrasaccharide was not added (0 μg/mL), the blood of regular users of CS oligosaccharide had significantly lower original platelet adhesion capacity than the reference value for healthy subjects. . These results demonstrate that CS oligosaccharides are extremely effective in suppressing the adhesion of platelets to collagen in blood.
＜実施例４＞血小板粘着能に対する効果：服用中止・服用開始時の評価
実施例３で、ＣＳオリゴ糖の常用者血液の血小板粘着能が、健常者における基準値と比較して顕著に低かったことから、ＣＳオリゴ糖の服用中止および服用開始による血小板粘着能の変化を検討した。
<Example 4> Effect on platelet adhesive ability: evaluation at the time of discontinuation and initiation of administration In Example 3, the platelet adhesive ability of regular CS oligosaccharide users' blood was significantly lower than the reference value in healthy subjects. Therefore, changes in platelet adhesive ability due to discontinuation and initiation of administration of CS oligosaccharides were examined.
すなわち、ＣＳオリゴ糖の常用者１名と非常用者１名と（いずれも成人男性）を被験者とし、試験開始時に両者から採血した。続いて、常用者についてはＣＳオリゴ糖の服用（経口摂取）を中止し、８日間経過後に再度採血した。また、非常用者についてはＣＳオリゴ糖の服用（ＣＳオリゴ糖２００ｍｇ（１錠当たりＣＳオリゴ糖５０ｍｇ含有の錠剤４錠）／日を水とともに毎日経口摂取）を開始し、６日間経過後に再度採血した。採血は、サイズ２１Ｇの翼状針を用いて被験者腕静脈に穿刺し、真空採血管に数ｍＬの血液を採取したのちこれを捨て、１本の新しい５ｍＬクエン酸含有真空採血管に等圧となるまで採血した。採血後すみやかに採血管を転倒混和したのち、４℃で冷蔵保存した。これらのクエン酸含有全血について、実施例３（２）に記載の方法により血小板粘着能を測定した。その結果を図５に示す。 That is, one regular CS oligosaccharide user and one non-user (both adult males) were used as subjects, and blood was collected from both subjects at the start of the test. Subsequently, for the regular users, the administration (oral intake) of the CS oligosaccharide was discontinued, and blood was collected again after 8 days had passed. In addition, emergency patients started taking CS oligosaccharide (200 mg of CS oligosaccharide (4 tablets containing 50 mg of CS oligosaccharide per tablet)/day taken orally with water every day), and blood was collected again after 6 days. did. Blood collection is performed by puncturing the subject's brachial vein using a size 21G butterfly needle, collecting several mL of blood in a vacuum blood collection tube, discarding it, and placing it in a new 5 mL citric acid-containing vacuum blood collection tube to equal pressure. Blood was collected until Immediately after blood collection, the blood collection tube was mixed by inversion, and then stored in a refrigerator at 4°C. The platelet adhesion ability of these citrate-containing whole bloods was measured by the method described in Example 3(2). The results are shown in FIG.
図５に示すように、常用者血液の血小板粘着能は、試験開始時に８．１％であったのが、ＣＳオリゴ等の服用中止８日目には２５．５％であった。すなわち、血小板粘着能は、ＣＳオリゴ糖の服用中止後に顕著に増大した。 As shown in FIG. 5, the platelet adhesion capacity of regular user's blood was 8.1% at the beginning of the test, but it was 25.5% on the 8th day after stopping CS oligo. That is, the platelet adhesion capacity increased significantly after withdrawal of CS oligosaccharides.
一方、非常用者血液の血小板粘着能は、試験開始時に２８．３％であったのが、ＣＳオリゴ等の服用開始６日目には２１．６％であった。すなわち、血小板粘着能は、ＣＳオリゴ糖の服用開始後に顕著に低下した。 On the other hand, the platelet adhesiveness of the emergency blood was 28.3% at the start of the test, but it was 21.6% on the 6th day after the start of administration of CS oligo and the like. That is, the platelet adhesion ability decreased significantly after starting administration of CS oligosaccharide.
これらの結果から、血小板粘着能は、ＣＳオリゴ糖を経口摂取していると低くなり、経口摂取していないと高くなることが明らかになった。すなわち、ＣＳオリゴ糖は、経口摂取により血小板のコラーゲンへの粘着を抑制できる、極めて高い効果を有することが明らかになった。 These results revealed that the platelet adhesive ability decreased when CS oligosaccharide was orally ingested and increased when not orally ingested. In other words, it was revealed that CS oligosaccharide has an extremely high effect of suppressing adhesion of platelets to collagen when orally ingested.
＜実施例５＞血小板凝集能に対する効果
ＣＳオリゴ糖の常用者１名と非常用者１名と（いずれも成人男性）を被験者とし、実施例３（１）に記載の方法によりクエン酸含有全血を取得して、添加したＣＳ４糖の終濃度が０、５０、２５０および５００μｇ／ｍＬとなるよう加えた後、４℃で冷蔵保存した。
<Example 5> Effect on platelet aggregation ability One regular CS oligosaccharide user and one emergency CS oligosaccharide user (both adult men) were used as subjects, and the citric acid-containing total Blood was obtained, added with CS4sugar at final concentrations of 0, 50, 250 and 500 μg/mL, and stored in a refrigerator at 4°C.
光透過法により血小板凝集能を測定した。すなわち、クエン酸含有全血を室温で１時間静置後、遠心分離（１９０×ｇ、１０分）して上清を多血小板血漿（ＰＲＰ）として回収した。また、その沈渣部を遠心分離（１４００×ｇ、１０分）して上清を乏血小板血漿（ＰＰＰ）として回収した。ＰＲＰを２００μＬずつ２本のキュベットに分注し、３７℃で１分間インキュベートした。血小板刺激物質としてアデノシン二リン酸（ＡＤＰ）を終濃度が１μＭおよび３μＭとなるよう各キュベットに添加した。ＰＰＰ、ＰＲＰ、ＡＤＰを上記濃度に添加したＰＲＰについて、血小板凝集能測定装置（MCM HEMA TRACER 313M、エム・シー・メディカル）に供して光透過率を測定した。ＰＲＰ、ＰＰＰそれぞれの光透過率を０％、１００％とし、ＡＤＰを添加したＰＲＰの光透過率を百分率に換算して、これを血小板凝集率（％）とした。続いて、下記式２により血小板凝集能を算出した。その結果を図６に示す。なお、このように測定・算出した血小板凝集能の健常者における基準値は１８．７～６８．７％である。
式２：血小板凝集能（％）＝３μＭのＡＤＰ添加ＰＲＰにおける最大血小板凝集率－１μＭのＡＤＰ添加ＰＲＰにおける最大血小板凝集率
Platelet aggregation ability was measured by light transmission method. That is, citrate-containing whole blood was allowed to stand at room temperature for 1 hour, centrifuged (190×g, 10 minutes), and the supernatant was collected as platelet-rich plasma (PRP). In addition, the sediment portion was centrifuged (1400×g, 10 minutes) and the supernatant was collected as platelet poor plasma (PPP). 200 μL of PRP was dispensed into two cuvettes and incubated at 37° C. for 1 minute. Adenosine diphosphate (ADP) was added to each cuvette as a platelet stimulant to final concentrations of 1 μM and 3 μM. PPP, PRP, and PRP to which ADP was added at the above concentrations were subjected to a platelet aggregation measuring device (MCM HEMA TRACER 313M, MC Medical) to measure light transmittance. The light transmittance of PRP and PPP was set to 0% and 100%, respectively, and the light transmittance of ADP-added PRP was converted into a percentage, which was defined as the platelet aggregation rate (%). Subsequently, the platelet aggregation ability was calculated by the following formula 2. The results are shown in FIG. The reference value for platelet aggregation in healthy subjects measured and calculated in this manner is 18.7 to 68.7%.
Equation 2: Platelet aggregation capacity (%) = maximum platelet aggregation rate in 3 μM ADP-added PRP - maximum platelet aggregation rate in 1 μM ADP-added PRP
図６に示すように、ＣＳオリゴ糖の常用者および非常用者のいずれの血液においても、ＣＳ４糖の濃度が高いほど、血小板凝集能の値が小さくなった。すなわち、血中におけるＣＳ４糖の存在量が大きいほど、血小板の凝集反応が抑制された。この結果から、ＣＳオリゴ糖は、血中において血小板の凝集を抑制できることが明らかになった。 As shown in FIG. 6, in the blood of both regular users and non-users of CS oligosaccharides, the higher the concentration of CS4 sugar, the lower the value of platelet aggregation ability. That is, the platelet aggregation reaction was inhibited as the amount of CS4 sugar present in the blood increased. These results demonstrate that CS oligosaccharide can suppress platelet aggregation in blood.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2021094977A JP2022187126A (en) | 2021-06-07 | 2021-06-07 | Antiplatelet agent, platelet adhesion inhibitor, platelet aggregation inhibitor, antithrombotic agent, food composition for inhibiting platelet adhesion and/or platelet aggregation, and food composition for preventing or ameliorating thrombosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2021094977A JP2022187126A (en) | 2021-06-07 | 2021-06-07 | Antiplatelet agent, platelet adhesion inhibitor, platelet aggregation inhibitor, antithrombotic agent, food composition for inhibiting platelet adhesion and/or platelet aggregation, and food composition for preventing or ameliorating thrombosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2022187126A true JP2022187126A (en) | 2022-12-19 |
Family
ID=84525375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2021094977A Pending JP2022187126A (en) | 2021-06-07 | 2021-06-07 | Antiplatelet agent, platelet adhesion inhibitor, platelet aggregation inhibitor, antithrombotic agent, food composition for inhibiting platelet adhesion and/or platelet aggregation, and food composition for preventing or ameliorating thrombosis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2022187126A (en) |
-
2021
- 2021-06-07 JP JP2021094977A patent/JP2022187126A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5048673B2 (en) | A kind of extract that prevents or treats thrombotic diseases | |
| JP5728234B2 (en) | Pharmaceutical composition for use in the treatment or prevention of bone and joint diseases | |
| JP6755980B2 (en) | High-purity heparin and its manufacturing method | |
| JPH02502006A (en) | Sulfated polysaccharide with anti-metastatic activity | |
| JP2004107295A (en) | Histamine release inhibitor | |
| KR20170063836A (en) | Method and composition for producing enhanced anti-inflammatory/anti-catabolic and regenerative agents from autologous physiological fluid | |
| JPH03243601A (en) | Mucopolysaccharide composition having ability to control blood coagulation and preparation thereof | |
| Pawlaczyk et al. | Animal models of peritoneal dialysis: thirty years of our own experience | |
| JPS5837001A (en) | Production of hyaluronic acid | |
| US20050196410A1 (en) | Seaweed extract composition for treatment of inflammation | |
| CN101647776A (en) | Doxofylline venous injection with small volume as well as preparation method and quality control method thereof | |
| JP2022187126A (en) | Antiplatelet agent, platelet adhesion inhibitor, platelet aggregation inhibitor, antithrombotic agent, food composition for inhibiting platelet adhesion and/or platelet aggregation, and food composition for preventing or ameliorating thrombosis | |
| JPWO2016052509A1 (en) | Renal failure progression inhibitor, renal failure prevention agent, and indoxyl sulfate production inhibitor | |
| Dong et al. | Pharmacological effect of panax notoginseng saponins on cerebral ischemia in animal models | |
| CN104725532B (en) | A kind of method of chondroitin sulfate and dermatan sulfate content in accurate quantification control heparin/heparan | |
| Morgan et al. | Severe peripheral neuropathy complicating legionnaires' disease. | |
| CN115297871A (en) | Method for producing enhanced anti-inflammatory/anti-catabolic agents from autologous physiological fluids | |
| WO2023171648A1 (en) | Antihypertensive drug and antihypertensive food composition | |
| JP2583183B2 (en) | Process for producing bioactive glycogen and bioactivity | |
| JP4599480B2 (en) | Infectious immune function enhancer, metabolic function promoter, biological function deterioration prevention / amelioration agent, and functional foods containing these | |
| Valadao et al. | Evaluation of peritoneal fluid in horses with experimental endotoxemia | |
| JP2011528058A (en) | Method for producing glycosaminoglycan | |
| CN113509464B (en) | Uric acid adsorbent in intestinal cavity and application and preparation method thereof | |
| CN103372024A (en) | An inhibitor of the production of advanced glycation end products | |
| CN110898080B (en) | Application of horseshoe crab plasma in promoting growth and development |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20240311 |