JP7337300B1 - Carob polysaccharide for prevention and improvement of metabolic syndrome, method for producing the same, and use thereof - Google Patents
Carob polysaccharide for prevention and improvement of metabolic syndrome, method for producing the same, and use thereof Download PDFInfo
- Publication number
- JP7337300B1 JP7337300B1 JP2023058591A JP2023058591A JP7337300B1 JP 7337300 B1 JP7337300 B1 JP 7337300B1 JP 2023058591 A JP2023058591 A JP 2023058591A JP 2023058591 A JP2023058591 A JP 2023058591A JP 7337300 B1 JP7337300 B1 JP 7337300B1
- Authority
- JP
- Japan
- Prior art keywords
- polysaccharide
- carob
- cycarney
- fructose
- glucose
- 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.)
- Active
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 94
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 93
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 93
- 240000008886 Ceratonia siliqua Species 0.000 title claims abstract description 77
- 235000013912 Ceratonia siliqua Nutrition 0.000 title claims abstract description 77
- 208000001145 Metabolic Syndrome Diseases 0.000 title claims description 15
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 title claims description 15
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 230000006872 improvement Effects 0.000 title description 5
- 230000002265 prevention Effects 0.000 title description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 55
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 30
- 239000005715 Fructose Substances 0.000 claims abstract description 30
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229930091371 Fructose Natural products 0.000 claims abstract description 29
- 239000000470 constituent Substances 0.000 claims abstract description 28
- 239000008103 glucose Substances 0.000 claims abstract description 28
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 25
- 150000002772 monosaccharides Chemical class 0.000 claims abstract description 14
- 239000004480 active ingredient Substances 0.000 claims abstract description 10
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims abstract description 5
- 239000000203 mixture Substances 0.000 claims description 20
- 239000000284 extract Substances 0.000 claims description 19
- 235000013305 food Nutrition 0.000 claims description 15
- 239000002244 precipitate Substances 0.000 claims description 12
- 208000031226 Hyperlipidaemia Diseases 0.000 claims description 11
- 206010020772 Hypertension Diseases 0.000 claims description 11
- 206010012601 diabetes mellitus Diseases 0.000 claims description 11
- 206010010774 Constipation Diseases 0.000 claims description 10
- 208000032928 Dyslipidaemia Diseases 0.000 claims description 10
- 208000017170 Lipid metabolism disease Diseases 0.000 claims description 10
- 238000009826 distribution Methods 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 7
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 7
- 206010022489 Insulin Resistance Diseases 0.000 claims description 6
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims description 6
- 238000001641 gel filtration chromatography Methods 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 5
- 235000017367 Guainella Nutrition 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 238000010586 diagram Methods 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 description 29
- 238000011282 treatment Methods 0.000 description 24
- 238000004458 analytical method Methods 0.000 description 18
- 230000000694 effects Effects 0.000 description 18
- 210000004369 blood Anatomy 0.000 description 16
- 239000008280 blood Substances 0.000 description 16
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 15
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 15
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 15
- 235000000346 sugar Nutrition 0.000 description 14
- 241000700159 Rattus Species 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 241001465754 Metazoa Species 0.000 description 10
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 10
- 239000003814 drug Substances 0.000 description 9
- 201000001421 hyperglycemia Diseases 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 8
- 230000000968 intestinal effect Effects 0.000 description 8
- 230000009471 action Effects 0.000 description 7
- 238000005227 gel permeation chromatography Methods 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 230000008855 peristalsis Effects 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 230000011987 methylation Effects 0.000 description 6
- 238000007069 methylation reaction Methods 0.000 description 6
- 230000035515 penetration Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 208000002705 Glucose Intolerance Diseases 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 5
- 235000012000 cholesterol Nutrition 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- -1 galactomannan polysaccharide Chemical class 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000003449 preventive effect Effects 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 229940124597 therapeutic agent Drugs 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- HIMXGTXNXJYFGB-UHFFFAOYSA-N alloxan Chemical compound O=C1NC(=O)C(=O)C(=O)N1 HIMXGTXNXJYFGB-UHFFFAOYSA-N 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000000287 crude extract Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000003345 hyperglycaemic effect Effects 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 150000008163 sugars Chemical class 0.000 description 4
- 239000003826 tablet Substances 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 3
- 239000006286 aqueous extract Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 108010016268 hippuryl-histidyl-leucine Proteins 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011403 purification operation Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000012916 structural analysis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- UAOKXEHOENRFMP-ZJIFWQFVSA-N [(2r,3r,4s,5r)-2,3,4,5-tetraacetyloxy-6-oxohexyl] acetate Chemical compound CC(=O)OC[C@@H](OC(C)=O)[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](OC(C)=O)C=O UAOKXEHOENRFMP-ZJIFWQFVSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 230000003544 deproteinization Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000007446 glucose tolerance test Methods 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 235000013402 health food Nutrition 0.000 description 2
- 235000009200 high fat diet Nutrition 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 230000000291 postprandial effect Effects 0.000 description 2
- 201000009104 prediabetes syndrome Diseases 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 210000001187 pylorus Anatomy 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000013222 sprague-dawley male rat Methods 0.000 description 2
- 235000000891 standard diet Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- BEZJAQLKYOEUBD-UHFFFAOYSA-N (2,3,4,6-tetraacetyloxy-5-oxohexyl) acetate Chemical compound CC(=O)OCC(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(=O)COC(C)=O BEZJAQLKYOEUBD-UHFFFAOYSA-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
- NJVBTKVPPOFGAT-UHFFFAOYSA-N 2,3,4,5,6-pentaacetyloxyhexyl acetate Chemical compound CC(=O)OCC(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)COC(C)=O NJVBTKVPPOFGAT-UHFFFAOYSA-N 0.000 description 1
- 208000021959 Abnormal metabolism Diseases 0.000 description 1
- 244000215068 Acacia senegal Species 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000220485 Fabaceae Species 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 229920000926 Galactomannan Polymers 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- RYMZZMVNJRMUDD-UHFFFAOYSA-N SJ000286063 Natural products C12C(OC(=O)C(C)(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 RYMZZMVNJRMUDD-UHFFFAOYSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- BEZJAQLKYOEUBD-BZUAXINKSA-N [(2r,3r,4s)-2,3,4,6-tetraacetyloxy-5-oxohexyl] acetate Chemical compound CC(=O)OC[C@@H](OC(C)=O)[C@@H](OC(C)=O)[C@H](OC(C)=O)C(=O)COC(C)=O BEZJAQLKYOEUBD-BZUAXINKSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 102000011759 adducin Human genes 0.000 description 1
- 108010076723 adducin Proteins 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001142 anti-diarrhea Effects 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000015895 biscuits Nutrition 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000014510 cooky Nutrition 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 235000006694 eating habits Nutrition 0.000 description 1
- 230000002892 effect on hypertension Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960003699 evans blue Drugs 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 210000002425 internal capsule Anatomy 0.000 description 1
- 210000001596 intra-abdominal fat Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002515 isoflavone derivatives Chemical class 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 210000000713 mesentery Anatomy 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000003305 oral gavage Methods 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229930015704 phenylpropanoid Natural products 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 235000021395 porridge Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- RYMZZMVNJRMUDD-HGQWONQESA-N simvastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)C(C)(C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 RYMZZMVNJRMUDD-HGQWONQESA-N 0.000 description 1
- 229960002855 simvastatin Drugs 0.000 description 1
- 235000009561 snack bars Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
【課題】イナゴマメの莢果由来の新規な活性成分及びその用途を提供すること。【解決手段】本発明は、下記（ｉ）～（ｉｉｉ）の特徴を有する新規多糖類である。（ｉ）水に可溶。エタノールに不溶。（ｉｉ）構成単糖が、実質的にグルコース、フルクトース及びマンノースの３種からなり、その構成モル比が、グルコース：フルクトース：マンノース＝１～１０：１０～１００：１～１０である。（ｉｉｉ）構成単糖の結合様式が、下記（ａ）～（ｄ）の組み合わせからなる。（ａ）（１→４）結合グルコース （ｂ）（１→）結合フルクトース （ｃ）（１→６）結合フルクトース （ｄ）（１→３）及び（１→６）結合マンノース【選択図】なしAn object of the present invention is to provide a novel active ingredient derived from carob pods and its uses. The present invention is a novel polysaccharide having the following characteristics (i) to (iii). (i) Soluble in water. Insoluble in ethanol. (ii) The constituent monosaccharides are essentially composed of three types of glucose, fructose and mannose, and the constituent molar ratio thereof is glucose:fructose:mannose=1 to 10:10 to 100:1 to 10. (iii) The binding mode of the constituent monosaccharides is a combination of the following (a) to (d). (a) (1→4) bound glucose (b) (1→) bound fructose (c) (1→6) bound fructose (d) (1→3) and (1→6) bound mannose [Selection diagram] None
Description
本発明は、マメ科植物であるイナゴマメ（Ｃｅｒａｔｏｎｉａ ｓｉｌｉｑｕａ Ｌ．）から分離、精製して得られる新規な多糖類及びその用途に関し、さらに、この新規多糖類の製造方法に関する。 TECHNICAL FIELD The present invention relates to a novel polysaccharide obtained by separating and purifying Ceratonia siliqua L., a leguminous plant, and uses thereof, and further to a method for producing the novel polysaccharide.
イナゴマメ（Ｃｅｒａｔｏｎｉａ ｓｉｌｉｑｕａ Ｌ．）は、主に地中海地方を原産とするマメ科植物である。イナゴマメの種子からは、ガラクトマンナン多糖類のローカストビーンガムが得られ、増粘剤やゲル化剤などの食品添加物として利用されている。また、イナゴマメの莢果、すなわち、イナゴマメの莢及び果肉はキャロブ（ｃａｒｏｂ）と呼ばれ、古くから食用又は食品原料として利用されてきた。成熟した莢果は長さ１０～２５ｃｍ程度で、甘味を呈する。イナゴマメの莢果には、多糖類、セルロース及びミネラル類が多く含まれ、タンパク質や非炭水化物系の低分子化合物等が少量含まれている。 Carob (Ceratonia siliqua L.) is a leguminous plant mainly native to the Mediterranean region. Locust bean gum, which is a galactomannan polysaccharide, is obtained from carob seeds, and is used as a food additive such as a thickening agent and a gelling agent. In addition, carob pods, that is, carob pods and pulp, are called carob, and have long been used as food or food ingredients. Mature pods are about 10-25 cm long and have a sweet taste. Carob pods contain large amounts of polysaccharides, cellulose and minerals, and small amounts of proteins and non-carbohydrate low-molecular-weight compounds.
近年、イナゴマメの莢果の新たな機能に関する研究が進められており、イナゴマメの莢果（ｐｏｄ）の水抽出物が、消化管における止瀉、抗酸化、抗菌、抗潰瘍及び抗炎症作用等の複数の薬理作用を有し、潰瘍性大腸炎や胃潰瘍などの消化器疾患の予防及び治療効果を有することが報告されている。また、イナゴマメの莢果から分離されたポリフェノール化合物、フェニルプロパノイド化合物及びイソフラボン化合物がそれぞれ所定の薬理作用を奏する活性成分であることが報告されているが、イナゴマメの莢果に含まれる多糖類に関する研究はこれまでほとんどなされていなかった。 In recent years, research on new functions of carob pods has been progressing, and aqueous extracts of carob pods have multiple effects such as antidiarrheal, antioxidant, antibacterial, antiulcer and anti-inflammatory effects in the gastrointestinal tract. It has been reported that it has a pharmacological effect and has preventive and therapeutic effects on gastrointestinal diseases such as ulcerative colitis and gastric ulcer. It has also been reported that polyphenol compounds, phenylpropanoid compounds and isoflavone compounds isolated from carob pods are active ingredients exhibiting specific pharmacological actions. Very little has been done so far.
他方、メタボリックシンドローム（ＭｅｔＳ）とは、内臓脂肪型肥満をベースとして、高血圧、高血糖及び脂質代謝異常といった危険因子が集積した病態のことをいい、高血圧症、糖尿病、動脈硬化性疾患及び心血管疾患のリスク増加と強く関連することが知られている。しかしながら、今のところ、メタボリックシンドロームを統合的に治療できる医薬品はなく、メタボリックシンドロームに対する治療は、高血圧症、糖尿病又は脂質異常症といった個々の疾病や病態に応じた薬物療法のほか、運動や食生活の改善、減量が実施されているのみである。そのため、メタボリックシンドロームを統合的に治療、改善又は予防できる薬剤が期待されている。そして、イナゴマメの莢果をメタボリックシンドロームの統合的な予防、改善又は治療のために用いることについての検討はこれまでなされておらず、その有効性はまったく不明であった。 Metabolic syndrome (MetS), on the other hand, is based on visceral fat obesity and refers to a condition in which risk factors such as hypertension, hyperglycemia, and dyslipidemia are accumulated. It is known to be strongly associated with an increased risk of disease. However, at present, there are no drugs that can treat metabolic syndrome in an integrated manner, and treatment for metabolic syndrome consists of drug therapy according to individual diseases and conditions such as hypertension, diabetes, and dyslipidemia, as well as exercise and dietary habits. Only improvement and weight reduction are being implemented. Therefore, a drug that can comprehensively treat, improve, or prevent metabolic syndrome is expected. Further, no study has been made so far on using carob pods for the integrated prevention, improvement or treatment of metabolic syndrome, and the effectiveness thereof has been completely unknown.
したがって、本発明は上述した点に鑑みてなされたもので、その目的は、イナゴマメの莢果由来の新規な活性成分及びその用途を提供することにある。 Accordingly, the present invention has been made in view of the above points, and an object thereof is to provide a novel active ingredient derived from carob pods and uses thereof.
上記課題を解決するため、本発明は、下記（ｉ）～（ｉｉｉ）の特徴を有する新規多糖類である。（ｉ）水に可溶。エタノールに不溶。（ｉｉ）構成単糖が、実質的にグルコース、フルクトース及びマンノースの３種からなり、その構成モル比が、グルコース：フルクトース：マンノース＝１～１０：１０～１００：１～１０である。 In order to solve the above problems, the present invention is a novel polysaccharide having the following characteristics (i) to (iii). (i) soluble in water; Insoluble in ethanol. (ii) The constituent monosaccharides are substantially composed of glucose, fructose and mannose, and the constituent molar ratio is glucose:fructose:mannose=1-10:10-100:1-10.
（ｉｉｉ）構成単糖の結合様式が、（１→４）－Ｇｌｃ、（１→）－Ｆｒｕ、（１→６）－Ｆｒｕ、（１→３，６）－Ｍａｎの組み合わせからなる。 (iii) The binding mode of the constituent monosaccharides is a combination of (1→4)-Glc, (1→)-Fru, (1→6)-Fru, and (1→3,6)-Man.
本明細書において、Ｇｌｃはグルコースを示し、Ｆｒｕはフルクトースを示し、Ｍａｎはマンノースを示す。 As used herein, Glc indicates glucose, Fru indicates fructose, and Man indicates mannose.
また、本発明の新規多糖類は、上述した特性（ｉｉ）における構成単糖のモル比が、グルコース：フルクトース：マンノース＝６．０～７．０：８７．５～８９．０：４．５～５．５であることが好ましい。これにより、本発明の多糖類を構成するグルコース、フルクトース及びマンノースのより好ましい構成単糖のモル比が選択される。 Further, in the novel polysaccharide of the present invention, the molar ratio of the constituent monosaccharides in the property (ii) described above is glucose: fructose: mannose = 6.0-7.0: 87.5-89.0: 4.5. ~5.5 is preferred. Thereby, a more preferable molar ratio of constituent monosaccharides of glucose, fructose and mannose constituting the polysaccharide of the present invention is selected.
また、本発明の新規多糖類は、上述した特性（ｉｉ）における構成単糖のモル比が、グルコース：フルクトース：マンノース＝６．４～６．６：８８．０～８８．２：５．３～５．５であることがより好ましい。これにより、本発明の多糖類を構成するグルコース、フルクトース及びマンノースのさらに好ましい構成単糖のモル比が選択される。 In addition, in the novel polysaccharide of the present invention, the molar ratio of constituent monosaccharides in characteristic (ii) described above is glucose: fructose: mannose = 6.4 to 6.6: 88.0 to 88.2: 5.3. ~5.5 is more preferred. Accordingly, more preferable molar ratios of constituent monosaccharides of glucose, fructose and mannose which constitute the polysaccharide of the present invention are selected.
後述する実施例においては、一例として、構成モル比がグルコース：フルクトース：マンノース＝６．５：８８．１：５．４である多糖類が示されている。 In the examples described later, as an example, a polysaccharide having a constituent molar ratio of glucose:fructose:mannose=6.5:88.1:5.4 is shown.
また、本発明の新規多糖類は、さらに、下記（ｉｖ）の特徴を有することも好ましい。（ｉｖ）ゲルろ過クロマトグラフィーを用いて測定した分子量が、１×１０４～１×１０6である。これにより、本発明の多糖類として好ましい分子量が選択される。 Moreover, the novel polysaccharide of the present invention preferably further has the following characteristics (iv). (iv) has a molecular weight of 1×10 4 to 1×10 6 as measured using gel filtration chromatography; Thereby, a preferred molecular weight is selected for the polysaccharide of the present invention.
また、本発明の新規多糖類は、上述した特性（ｉｖ）における分子量が、７×１０６～８×１０６であることも好ましい。これにより、本発明の多糖類としてさらに好ましい分子量が選択される。 Also, the novel polysaccharide of the present invention preferably has a molecular weight of 7×10 6 to 8×10 6 in characteristic (iv) described above. Thereby, a more preferable molecular weight is selected for the polysaccharide of the present invention.
また、本発明の新規多糖類は、さらに、下記の特徴を有することも好ましい。分子量分布（Ｍｗ／Ｍｎ）が、１．７～１．８である。これにより、本発明の多糖類として好ましい分子量分布が選択され、分子量分布が狭く、高純度の多糖類が得られる。 Moreover, it is also preferred that the novel polysaccharide of the present invention further has the following characteristics. The molecular weight distribution (Mw/Mn) is 1.7-1.8. As a result, a preferred molecular weight distribution is selected for the polysaccharide of the present invention, and a polysaccharide with a narrow molecular weight distribution and high purity can be obtained.
後述する実施例においては、一例として、ゲルろ過クロマトグラフィーを用いて測定された分子量が７．４２８×１０６であり、分子量分布（Ｍｗ／Ｍｎ）が１．７７４の多糖類が示されている。 In Examples described later, as an example, a polysaccharide having a molecular weight of 7.428×10 6 and a molecular weight distribution (Mw/Mn) of 1.774 as measured by gel filtration chromatography is shown. .
本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」は、イナゴマメの莢果から分離、精製することにより得ることができる。 The novel polysaccharide "Cycarney" according to the present invention can be obtained by separating and purifying from carob pods.
本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」は、脂質代謝異常の改善作用、耐糖能異常の改善作用、腸蠕動運動の亢進作用及び血圧降下作用を併せて備えている。そのため、メタボリックシンドロームの危険因子である高血圧、高血糖及び脂質代謝異常を統合的に予防、改善若しくは治療できるため、統合的なメタボリックシンドロームの予防、改善若しくは治療剤として用いることができる。 The novel polysaccharide "Cycarney" according to the present invention has an action to improve dyslipidemia, an action to improve glucose intolerance, an action to enhance intestinal peristalsis, and an action to lower blood pressure. Therefore, hypertension, hyperglycemia and dyslipidemia, which are risk factors for metabolic syndrome, can be prevented, ameliorated or treated in an integrated manner.
本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」は、後述する実施例４で示すように、血中の総コレステロール及び中性脂肪の値を低下させる作用を有しており、それゆえ、脂質代謝異常、高脂血症及び動脈硬化の予防、改善若しくは治療剤として用いることができる。 The novel polysaccharide "Cycarney" according to the present invention, as shown in Example 4 below, has the effect of lowering the levels of total cholesterol and triglycerides in the blood. It can be used as a preventive, ameliorating or therapeutic agent for hyperlipidemia and arteriosclerosis.
本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」は、後述する実施例５で示すように、食後の血糖値の上昇（耐糖能異常）を抑制する作用を有しており、それゆえ、糖尿病及びインスリン抵抗性の予防、改善若しくは治療剤として用いることができる。 As shown in Example 5 below, the novel polysaccharide "Cycarney" according to the present invention has the effect of suppressing the rise in postprandial blood sugar level (impaired glucose tolerance). It can be used as a preventive, ameliorating or therapeutic agent for sex.
本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」は、後述する実施例６で示すように、アンジオテンシン変換酵素（ＡＣＥ）阻害作用を有しており、それゆえ、高血圧症の予防、改善若しくは治療剤として用いることができる。 The novel polysaccharide "Cycarney" according to the present invention has an angiotensin-converting enzyme (ACE) inhibitory action, as shown in Example 6 below, and is therefore used as a preventive, ameliorating or therapeutic agent for hypertension. be able to.
本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」は、後述する実施例７で示すように、腸蠕動運動の亢進作用を有する。それゆえ、便秘症の予防、改善若しくは治療剤として用いることができる。 The novel polysaccharide "Cycarney" according to the present invention has an effect of promoting intestinal peristalsis, as shown in Example 7 below. Therefore, it can be used as a prophylactic, ameliorating or therapeutic agent for constipation.
本発明の医薬組成物は、上述した本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」を有効成分として含有するものであり、ヒト又は動物のための医薬品として用いることができ、脂質代謝異常、高脂血症、動脈硬化、糖尿病、インスリン抵抗性、高血圧症、便秘及びメタボリックシンドロームからなる群から選択される少なくとも一つの疾病の予防、改善若しくは治療に用いられる。 The pharmaceutical composition of the present invention contains the aforementioned novel polysaccharide "Cycarney" according to the present invention as an active ingredient, and can be used as a pharmaceutical for humans or animals. at least one disease selected from the group consisting of arteriosclerosis, diabetes, insulin resistance, hypertension, constipation and metabolic syndrome.
本発明の飲食品組成物は、上述した本発明に係る新規多糖類「Ｃｙｃａｒｎｅｙ」を含有するものであり、ヒト又は動物のための食品及び医薬部外品として用いることができ、脂質代謝異常、高脂血症、動脈硬化、糖尿病、インスリン抵抗性、高血圧症、便秘及びメタボリックシンドロームからなる群から選択される少なくとも一つの疾病の予防、改善若しくは治療に用いられる。また、飲食品組成物には、サプリメント、健康食品、機能性食品、特定保健用食品及び栄養補助食品等が含まれる。 The food and drink composition of the present invention contains the above-described novel polysaccharide "Cycarney" according to the present invention, and can be used as a food and quasi-drug for humans or animals. It is used for prevention, amelioration or treatment of at least one disease selected from the group consisting of hyperlipidemia, arteriosclerosis, diabetes, insulin resistance, hypertension, constipation and metabolic syndrome. Food and drink compositions include supplements, health foods, functional foods, foods for specified health uses, dietary supplements, and the like.
本発明の新規多糖類「Ｃｙｃａｒｎｅｙ」を含む組成物を医薬品として用いる場合には、従来慣用されている方法により種々の形態に調製することができる。この場合、通常製剤用の薬理学的に許容される担体や賦形剤、滑沢剤、分散剤、崩壊剤、緩衝剤、溶剤、増量剤、保存剤、香料又は安定化剤など、医薬品の添加剤として許容されている添加剤を用いて製剤化することができる。さらに、この多糖類のバイオアベイラビリティーや安定性を向上させるために、マイクロカプセル、リポソーム製剤、微粉末化又はシクロデキストリン等を用いた包接化などの製剤技術を含むドラッグデリバリーシステムを用いることもできる。また、経口投与製剤として用いる場合には、錠剤、顆粒剤、カプセル剤又は内服用液剤等の形態で用いることができるが、消化管からの吸収に適した形態で用いることが好ましい。また、流通性や保存性などの理由から固形製剤を使用時に適当な溶剤で溶解してから用いる形態とすることもでき、液剤および半固形剤の形態で提供することも従来の製剤技術により可能である。 When the composition containing the novel polysaccharide "Cycarney" of the present invention is used as a pharmaceutical, it can be prepared into various forms by conventional methods. In this case, pharmaceutical agents such as pharmacologically acceptable carriers and excipients, lubricants, dispersants, disintegrants, buffers, solvents, fillers, preservatives, flavoring agents, or stabilizers are usually used for formulations. It can be formulated using additives that are acceptable as additives. Furthermore, in order to improve the bioavailability and stability of this polysaccharide, it is also possible to use drug delivery systems including formulation techniques such as microcapsules, liposome formulations, micronization, or inclusion using cyclodextrins and the like. can. When used as an oral preparation, it can be used in the form of tablets, granules, capsules, internal liquid preparations, etc., and is preferably used in a form suitable for absorption from the gastrointestinal tract. In addition, for reasons such as distribution and storage stability, solid formulations can be dissolved in an appropriate solvent before use, and can be provided in the form of liquid and semi-solid formulations using conventional formulation technology. is.
さらに、本発明の新規多糖類「Ｃｙｃａｒｎｅｙ」を含む飲食品組成物は、錠剤やカプセル剤、顆粒剤、シロップ剤などのサプリメント形態、清涼飲料、果汁飲料、アルコール飲料などの飲料、キャンディー、ガム、タブレット菓子、クッキー、ビスケット、チョコレート等の菓子、パン、粥、シリアル、麺類、スナックバー、ゼリー、スープ、乳製品、シロップ、甘味料や香辛料等を含む調味料等のあらゆる形態とすることができる。このように飲食品組成物として用いる際には、本発明の有効成分の効能に影響を与えない範囲において、他の有効成分や、ビタミン、ミネラル若しくはアミノ酸等の栄養素等を種々組み合わせることも可能である。また、動物用の食品組成物（飼料、ペットフード；ドライタイプ、ウェットタイプ、動物用サプリメント、動物用飲料等）として用いることも可能である。 Furthermore, food and drink compositions containing the novel polysaccharide "Cycarney" of the present invention include supplement forms such as tablets, capsules, granules and syrups, beverages such as soft drinks, fruit juices and alcoholic beverages, candies, gums, It can be in any form such as tablets, sweets such as cookies, biscuits, chocolates, breads, porridges, cereals, noodles, snack bars, jellies, soups, dairy products, syrups, seasonings including sweeteners and spices. . When used as a food or drink composition in this way, it is also possible to combine various other active ingredients, nutrients such as vitamins, minerals, amino acids, etc. within the range that does not affect the efficacy of the active ingredients of the present invention. be. It can also be used as an animal food composition (feed, pet food; dry type, wet type, animal supplement, animal drink, etc.).
上述した医薬品組成物又は飲食品組成物における、本発明の新規多糖類「Ｃｙｃａｒｎｅｙ」としての投与量又は有効摂取量は、目標とする治療効果、投与方法、投与対象及び剤形によって変化するため、特に限定されないが、一日の経口投与量としては、有効成分として約１０ｍｇ／ｋｇ体重～約２０００ｍｇ／ｋｇ体重であり、好ましくは、約５０ｍｇ／ｋｇ体重～約１０００ｍｇ／ｋｇ体重であり、これを１～３回に分割して投与することが好ましい。 The dosage or effective intake of the novel polysaccharide "Cycarney" of the present invention in the pharmaceutical composition or food and drink composition described above varies depending on the target therapeutic effect, administration method, administration subject and dosage form, Although not particularly limited, the daily oral dose of the active ingredient is about 10 mg/kg body weight to about 2000 mg/kg body weight, preferably about 50 mg/kg body weight to about 1000 mg/kg body weight. Administration in 1 to 3 divided doses is preferred.
本発明の別の態様によれば、さらに、下記のようなイナゴマメ多糖類の製造方法が提供され、当該製造方法によれば、本発明に記載のイナゴマメ多糖類を製造することができ、その純度は95％以上に達することができる。 According to another aspect of the present invention, there is further provided a method for producing a carob polysaccharide as described below, and according to the production method, the carob polysaccharide according to the present invention can be produced, and its purity can reach more than 95%.
＜イナゴマメ莢果の前処理＞
本発明の新規多糖類「Ｃｙｃａｒｎｅｙ」のイナゴマメからの分離方法について詳細に説明する。まず、イナゴマメの莢果から抽出液を得る。本発明におけるイナゴマメの莢果の抽出液とは、イナゴマメの莢果に抽出溶媒として水を加え、抽出処理を施すことによって得られた抽出液をいう。イナゴマメの莢果とは、イナゴマメの莢付果実の莢と果肉のことを意味し、莢又は果肉のいずれか一方を抽出材料として用いることも可能であるが、莢及び果肉を用いることがより好ましい。抽出処理は、採取された状態、すなわち、生の状態のイナゴマメ莢果、又は乾燥状態のイナゴマメ莢果に対して行われるが、抽出効率の向上を図るため、又は取り扱いを容易とするために種々の前処理が施されたイナゴマメ莢果に対して抽出処理を施すことも可能である。前処理としては、特に限定されないが、乾燥処理、破砕処理、粉砕処理及び／又は脱脂処理等が挙げられ、これらの前処理が施されたイナゴマメの莢果に抽出処理を施して抽出物を得てもよい。このうち、粉砕処理としては、イナゴマメの莢果の細胞構造を壊して細胞内物質の溶出を高めると共に抽出溶媒である水との接触面積を増やして抽出効率を向上させる観点から、イナゴマメの莢果を７０～３００メッシュの粉末に粉砕することが好ましい。また、脱脂処理としては、一例として、イナゴマメの莢果を２～５倍重量の９０～９５％エタノールに浸漬させた後、イナゴマメの莢果を回収することにより行われる。
<Pretreatment of carob pods>
A method for separating the novel polysaccharide "Cycarney" of the present invention from carob will be described in detail. First, an extract is obtained from the carob pod. The extract of carob pods in the present invention refers to an extract obtained by adding water as an extraction solvent to carob pods and performing an extraction treatment. Carob pods refer to the pods and pulp of a carob fruit with pods, and either the pods or the pulp can be used as the extraction material, but the pods and pulp are more preferably used. Extraction treatment is performed on the harvested state, that is, the fresh carob pods or the dried carob pods. It is also possible to apply an extraction treatment to the treated carob pods. The pretreatment is not particularly limited, but includes drying treatment, crushing treatment, pulverizing treatment and/or degreasing treatment. good too. Among these, as the pulverization treatment, from the viewpoint of breaking the cell structure of the carob pods to enhance the elution of intracellular substances and increasing the contact area with water, which is an extraction solvent, to improve the extraction efficiency, 70 carob pods were crushed. Grinding to a powder of ~300 mesh is preferred. As an example of the degreasing treatment, carob pods are immersed in 2 to 5 times the weight of 90 to 95% ethanol, and then the carob pods are recovered.
＜イナゴマメ莢果の水抽出処理＞
抽出処理としては、イナゴマメの莢果に抽出溶媒である水を加えて浸漬させ、抽出を行う。例えば、イナゴマメ莢果を含水率１０％未満の乾燥微粉末とした場合には、植物体１重量部に対し、抽出溶媒である水を２～５重量部用いて抽出することが好ましく、２～４重量部用いることがより好ましく、２～３重量部用いることがさらに好ましい。また、抽出方法としては、室温での抽出、加温抽出、加圧加熱抽出等のいずれの方法でも行うことが可能であるが、抽出温度を４０～５０℃とした加温抽出が好ましく、特に４５℃で抽出することが好ましい。抽出温度を４０～５０度、特に４５℃とすることにより、イナゴマメの莢果から本発明の多糖類の抽出が効率的に行われる。また、回収率を向上させるため、抽出処理は複数回行うことが好ましく、２～５回行うことが好ましい。抽出時間は、抽出方法、抽出材料の状態又は抽出温度等に応じて種々設定されるが、１回の抽出時間として１～５時間程度とすることが好ましく、１．５～３時間程度とすることがより好ましい。上述した抽出処理後、残渣を遠心分離、デカンテーション又はろ過等により取り除くことによりイナゴマメの莢果の抽出液が得られる。得られた抽出液は、減圧蒸留等の処理を施すことにより、濃縮液とすることも可能である。なお、抽出溶媒には水を用いるが、本発明の多糖類の抽出を妨げない範囲において、他の成分を含有させることも可能である。
<Water extraction treatment of carob pods>
As an extraction treatment, carob pods are immersed in water, which is an extraction solvent, for extraction. For example, when carob pods are made into a dry fine powder having a water content of less than 10%, it is preferable to extract using 2 to 5 parts by weight of water as an extraction solvent with respect to 1 part by weight of the plant body, and 2 to 4 parts by weight. It is more preferable to use parts by weight, and more preferably 2 to 3 parts by weight. In addition, as the extraction method, any method such as extraction at room temperature, extraction with heating, extraction with pressure and heating can be performed, but extraction with heating at an extraction temperature of 40 to 50 ° C. is preferable, especially It is preferred to extract at 45°C. By setting the extraction temperature to 40 to 50° C., particularly 45° C., the polysaccharide of the present invention can be efficiently extracted from the carob pods. In order to improve the recovery rate, the extraction treatment is preferably performed multiple times, preferably 2 to 5 times. The extraction time is variously set according to the extraction method, the state of the extraction material, the extraction temperature, etc., but it is preferable to set the extraction time for one extraction to about 1 to 5 hours, and about 1.5 to 3 hours. is more preferable. After the extraction treatment described above, the residue is removed by centrifugation, decantation, filtration, or the like to obtain an extract of carob pods. The obtained extract can be made into a concentrate by subjecting it to a treatment such as distillation under reduced pressure. Although water is used as an extraction solvent, it is also possible to contain other components as long as they do not interfere with the extraction of the polysaccharide of the present invention.
＜多糖類の沈殿生成＞
上述のようにして得られたイナゴマメの莢果の抽出液に対し、エタノール等のアルコールを添加することにより、本発明に係る新規多糖類を含有する沈殿が得られる。エタノールは、液中に不溶性の沈殿が生じる程度の量及び濃度で添加されればよいが、効率よく沈殿形成を促す観点から、具体的には、抽出液又は濃縮液と、エタノールからなる混合液中のエタノールの最終濃度が４０重量％～８０重量％となるように添加することが好ましく、エタノールの最終濃度が４０重量％～６０重量％となるように添加することが好ましく、４０重量％～５０重量％となるように添加することが特に好ましい。生成した沈殿は遠心分離処理等によって集められ、回収される。
<Precipitation of Polysaccharides>
By adding an alcohol such as ethanol to the carob pod extract obtained as described above, a precipitate containing the novel polysaccharide according to the present invention can be obtained. Ethanol may be added in such an amount and concentration as to cause an insoluble precipitate in the liquid, but from the viewpoint of efficiently promoting precipitate formation, specifically, a mixed solution consisting of an extract or concentrated solution and ethanol It is preferable to add so that the final concentration of ethanol in the medium is 40% to 80% by weight, and it is preferable to add so that the final concentration of ethanol is 40% to 60% by weight. It is particularly preferred to add 50% by weight. The generated precipitate is collected by centrifugal separation or the like and recovered.
＜タンパク質除去＞
上述のようにして得られた沈殿には、本発明に係る新規多糖類以外に、イナゴマメの莢果に元来含まれるタンパク質等の不要な成分も存在するため、これら不要な成分を除去することを目的として、さらなる分離精製操作を行ってもよい。具体的には、タンパク質除去を目的として、得られた沈殿の水分散液にクロロホルムとｎ－ブタノールの混合液からなるＳｅｖａｇｅ試薬（クロロホルム：ｎ－ブタノール＝４：１）を、水分散液：Ｓｅｖａｇｅ試薬＝４：１の割合（体積比）で加えて混合し、数十分振とうした後に静置して成層分離し、上層側の有機相を除去することが行われる。このＳｅｖａｇｅ試薬による脱タンパク処理は、複数回行うことが好ましく、２～１０回程度行うことがより好ましく、４～７回程度行うことが特に好ましい。水相を回収することにより、本発明の新規多糖類「Ｃｙｃａｒｎｅｙ」を含む画分が回収される。
<Protein removal>
In the precipitate obtained as described above, in addition to the novel polysaccharide according to the present invention, unnecessary components such as proteins originally contained in carob pods are also present, so it is necessary to remove these unnecessary components. For purposes, further separation and purification operations may be performed. Specifically, for the purpose of protein removal, a Sevage reagent consisting of a mixed solution of chloroform and n-butanol (chloroform: n-butanol = 4: 1) was added to the aqueous dispersion of the obtained precipitate, and the aqueous dispersion: Sevage Reagents are added at a ratio of 4:1 (volume ratio), mixed, shaken for several tens of minutes, left to stand for layer separation, and the organic phase on the upper layer side is removed. This deproteinization treatment with the Sevage reagent is preferably performed multiple times, more preferably about 2 to 10 times, and particularly preferably about 4 to 7 times. By collecting the aqueous phase, a fraction containing the novel polysaccharide "Cycarney" of the present invention is collected.
＜分離精製＞
また、上述のようにして得られた沈殿や上述した脱タンパク処理後の回収物には、本発明に係る新規多糖類以外に、イナゴマメの莢果由来の天然色素等の不要な成分が存在する。そのため、これら不要な成分を除去すると共に目的物質たる新規多糖類の純度を向上させることを目的として、さらなる分離精製操作を行ってもよい。特に限定されないが、具体的には、得られた沈殿又は上述した脱タンパク処理物の水分散液をマクロポーラス吸着樹脂に通し、溶出画分を回収した後、この回収画分をＤＥＡＥ－セルロースカラムに供して蒸留水で溶出させた後、この溶出画分をゲルろ過クロマトグラフィーに供して脱イオン水で溶出させ、同じ分子量の溶出ピークを有する画分を回収することにより、本発明に係る新規多糖類が単離される。各種クロマトグラフィーに用いられる担体等は、各種クロマトグラフィーに対応して適宣選択することができる。これらの分離精製操作により、分子量分布（Ｍｗ／Ｍｎ）の幅が狭く、高純度の新規多糖類「Ｃｙｃａｒｎｅｙ」を得ることができる。
<Separation and purification>
In addition to the novel polysaccharide according to the present invention, unnecessary components such as natural pigments derived from carob pods are present in the precipitate obtained as described above and the recovered material after the deproteinization treatment described above. Therefore, further separation and purification operations may be performed for the purpose of removing these unnecessary components and improving the purity of the novel polysaccharide as the target substance. Although it is not particularly limited, specifically, the obtained precipitate or the aqueous dispersion of the deproteinized product described above is passed through a macroporous adsorption resin, the eluted fraction is recovered, and this recovered fraction is subjected to a DEAE-cellulose column. and eluted with distilled water, the eluted fraction is subjected to gel filtration chromatography and eluted with deionized water, and fractions having an elution peak of the same molecular weight are collected to obtain the novel A polysaccharide is isolated. Carriers and the like used in various chromatographies can be appropriately selected according to the various chromatographies. Through these separation and purification operations, a novel polysaccharide "Cycarney" with a narrow molecular weight distribution (Mw/Mn) and high purity can be obtained.
なお、上述のようにしてイナゴマメの莢果から分離、精製された本発明の多糖類は、完全な純物質として単離されていなくてもよく、イナゴマメ原料由来の他の成分が一部含まれていてもよい。 The polysaccharide of the present invention, which has been separated and purified from carob pods as described above, may not be isolated as a completely pure substance, and may contain some other components derived from carob raw materials. may
本発明によれば、以下のような優れた効果を有する新規多糖類、並びにこれを含有する脂質代謝異常、高脂血症、動脈硬化、糖尿病、インスリン抵抗性、高血圧症、便秘及びメタボリックシンドロームからなる群から選択される少なくとも一つの疾病の予防、改善若しくは治療用の医薬組成物又は飲食品組成物を提供することができる。 According to the present invention, novel polysaccharides having the following excellent effects, and dyslipidemia, hyperlipidemia, arteriosclerosis, diabetes, insulin resistance, hypertension, constipation and metabolic syndrome containing the same It is possible to provide a pharmaceutical composition or food and drink composition for prevention, improvement or treatment of at least one disease selected from the group consisting of:
（１）血中の総コレステロール及び中性脂肪の値を低下させ、脂質代謝異常を改善することができる。
（２）食後の血糖値の上昇を抑制し、耐糖能異常を改善することができる。
（３）腸の蠕動運動を亢進させ、便秘状態を改善することができる。
（４）アンジオテンシン変換酵素（ＡＣＥ）阻害作用による血圧降下作用を有し、高血圧を改善することができる。
（５）メタボリックシンドロームを統合的に改善することができる。
（６）古来から食用とされているイナゴマメの莢果由来物を有効成分とするものであるため、人体に対する安全性が高い。
(1) It can reduce blood total cholesterol and triglyceride levels and improve dyslipidemia.
(2) It can suppress an increase in postprandial blood glucose level and improve glucose intolerance.
(3) It can enhance intestinal peristalsis and improve constipation.
(4) It has an antihypertensive effect by inhibiting angiotensin-converting enzyme (ACE), and can improve hypertension.
(5) Metabolic syndrome can be improved comprehensively.
(6) Since the active ingredient is derived from carob pods, which have been edible since ancient times, it is highly safe to the human body.
実施例により本発明をさらに説明するが、本発明の範囲はこれらに限定されるものではない。 The invention is further illustrated by examples, but the scope of the invention is not limited thereto.
本発明で用いられるイナゴマメとは、学名をＣｅｒａｔｏｎｉａ ｓｉｌｉｑｕａといい、マメ科ジャケツイバラ亜科イナゴマメ属の植物である。地中海沿岸地方を原産とする植物であるが、本発明においては、産地や栽培環境は特に限定されず、あらゆる産地及び栽培環境のイナゴマメを用いることができる。 The carob used in the present invention has a scientific name of Ceratonia siliqua, and is a plant belonging to the genus Carob, Caesalpinae, Fabaceae. Although it is a plant native to the Mediterranean region, in the present invention, the production area and cultivation environment are not particularly limited, and carob from any production area and cultivation environment can be used.
実施例１ イナゴマメ莢果の水抽出物の調製
採取後、乾燥処理されたイナゴマメ（Ｃｅｒａｔｏｎｉａ ｓｉｌｉｑｕａ）の莢付果実から種を取り除いた。このイナゴマメの莢果８ｋｇを粉砕機で粉砕して８０メッシュの微粉末状の破砕物を得た。この微粉末に対し、３倍重量の９５％エタノールを加えて脱脂処理を行った。脱脂処理後の微粉末を乾燥させた後、微粉末に脱イオン水を、微粉末：脱イオン水＝１：２．３（ｇ／ｍＬ）の割合で加え、４５℃条件下で２時間抽出を行った。この抽出操作を合計で４回行い、４回分の抽出液を合わせて５０００ｒｐｍで１０分間、遠心分離処理を行って上清を回収した。この上清を６０℃、１００Ｐａの条件下で減圧濃縮し、１２００ｇの粘稠な濃縮抽出液を得た。
Example 1 Preparation of Aqueous Extracts of Carob Pods Seeds were removed from the dry-processed podded fruits of the carob (Ceratonia siliqua) after harvesting. 8 kg of the carob pods were pulverized with a pulverizer to obtain crushed fine powder of 80 mesh. This fine powder was degreased by adding 95% ethanol of three times its weight. After drying the degreased fine powder, add deionized water to the fine powder at a ratio of fine powder: deionized water = 1:2.3 (g/mL), and extract for 2 hours at 45 ° C. did This extraction operation was performed four times in total, and the four extracts were combined and centrifuged at 5000 rpm for 10 minutes to recover the supernatant. The supernatant was concentrated under reduced pressure at 60° C. and 100 Pa to obtain 1200 g of viscous concentrated extract.
実施例２ イナゴマメ莢果の水抽出物からの多糖類の分離及び精製
実施例１で得られた濃縮抽出液１２００ｇに９５％エタノールをゆっくりと添加し、エタノールの最終濃度が４０重量％となるまでエタノールを加えた。生成した多糖類の沈殿を遠心分離処理により回収し、沈殿物を乾燥させ、イナゴマメ多糖類Cycarneyを含むイナゴマメ抽出物を得た。Cycarneyを含んだ抽出物は粗イナゴマメ多糖類、Cycarney含有量は250gである。その遠心分離後の残り部分が「Cycarney」を含まない（あるいは含有量が非常に少ない）混合物である。
Example 2 Separation and purification of polysaccharides from aqueous extract of carob pod To 1200 g of concentrated extract obtained in Example 1, slowly add 95% ethanol until the final concentration of ethanol is 40% by weight. was added. The resulting polysaccharide precipitate was collected by centrifugation and dried to obtain a carob extract containing the carob polysaccharide Cycarney. The extract containing Cycarney is crude carob polysaccharide, Cycarney content is 250g. The remainder after centrifugation is a mixture that does not contain (or contains very little) "Cycarney".
続いて、このCycarney粗抽出物２５０ｇに、２０倍重量の精製水を添加して分散させた。粗抽出物中に含まれるタンパク質を除去するため、水分散液にクロロホルムとｎ－ブタノールの混合液からなるＳｅｖａｇｅ試薬（クロロホルム：ｎ－ブタノール＝４：１）を、水分散液：Ｓｅｖａｇｅ試薬＝４：１の割合（体積比）で加えて混合し、２０分間振とうした後に静置して成層分離し、上層側の有機相を除去して水相を得た。この操作を合計で６回行い、乾燥させて、タンパク質が除去された粉末状の粗抽出物２２５ｇを得た。 Subsequently, 250 g of this Cycarney crude extract was dispersed by adding 20 times its weight of purified water. In order to remove proteins contained in the crude extract, a Sevage reagent consisting of a mixture of chloroform and n-butanol (chloroform: n-butanol = 4: 1) was added to the aqueous dispersion, and the aqueous dispersion: Sevage reagent = 4. : 1 ratio (volume ratio) and mixed, shaken for 20 minutes, allowed to stand still for layer separation, and the upper organic phase was removed to obtain an aqueous phase. This operation was performed a total of 6 times and dried to obtain 225 g of powdered crude extract from which proteins were removed.
タンパク質が除去された粗抽出物２２５ｇを１０倍量の水に分散させ、イオン交換樹脂（マクロポーラス吸着樹脂Ｄ１０１）に通してイナゴマメ莢果由来の天然色素を吸着させた。カラム容量の２倍量の蒸留水で多糖類を溶出させ、溶出画分を回収した。この溶出画分について、ＤＥＡＥ－セルロース（カラム充填材：ＤＥ５２）を用いてカラムクロマトグラフィーを行い、カラム容量の２倍量の蒸留水で順次溶出させた。さらに、この溶出画分について、ゲルろ過クロマトグラフィー（カラム充填材：Ｓｅｐｈａｄｅｘ Ｇ－１００）に供し、脱イオン水で溶出させた。回収した溶出画分のうち、分子量の同じ溶出ピークを有する画分を合わせて減圧濃縮し、凍結乾燥を行った。得られた本発明のイナゴマメ莢果由来の多糖類（以下、この多糖類を「Ｃｙｃａｒｎｅｙ」という。）の収量は１５．３ｇであり、純度は９５％であった。以降の実施例３～７では、本実施例で分離・精製して得られたＣｙｃａｒｎｅｙをサンプルとして試験を行った。 225 g of the protein-removed crude extract was dispersed in 10 times the amount of water, and passed through an ion exchange resin (macroporous adsorption resin D101) to adsorb the natural pigment derived from carob pods. The polysaccharide was eluted with distilled water twice the volume of the column, and the eluted fraction was collected. This eluted fraction was subjected to column chromatography using DEAE-cellulose (column packing material: DE52) and eluted sequentially with distilled water twice the column volume. Further, this eluted fraction was subjected to gel filtration chromatography (column packing material: Sephadex G-100) and eluted with deionized water. Among the collected elution fractions, fractions having an elution peak with the same molecular weight were combined, concentrated under reduced pressure, and lyophilized. The obtained polysaccharide derived from carob pods of the present invention (hereinafter, this polysaccharide is referred to as "Cycarney") was 15.3 g with a purity of 95%. In Examples 3 to 7 below, tests were performed using the Cycarney obtained by separation and purification in this example as a sample.
実施例３ Ｃｙｃａｒｎｅｙの構造解析
実施例２で得られた多糖類「Ｃｙｃａｒｎｅｙ」の構造解析を行った。構造解析としては、（１）ゲル浸透クロマトグラフィー（ＧＰＣ）による分子量及び分子量分布の測定、（２）赤外吸収スペクトル分析、（３）構成糖分析及び（４）メチル化分析を行った。
Example 3 Structural Analysis of Cycarney The polysaccharide "Cycarney" obtained in Example 2 was structurally analyzed. For structural analysis, (1) measurement of molecular weight and molecular weight distribution by gel permeation chromatography (GPC), (2) infrared absorption spectrum analysis, (3) constituent sugar analysis and (4) methylation analysis were performed.
（１）ゲル浸透クロマトグラフィー（ＧＰＣ）
実施例２で得られたＣｙｃａｒｎｅｙのサンプルを正確に１０．０ｍｇ秤量し、２ｍｇ／ｍＬの濃度の溶液とした。移動相には０．１ｍｏｌ／ＬのＮａＮＯ３水溶液を用い、流速は０．７ｍＬ／ｍｉｎにてゲル浸透クロマトグラフィー（ＧＰＣ）分析を行った。検出器には示差屈折計と光散乱検出器とを併用した。Ｃｙｃａｒｎｅｙの分子量測定結果を図１に示す。図１の横軸は溶出体積を、縦軸は屈折率を示している。分析の結果、平均分子量は７．４２８×１０６であった。また、重量平均分子量（Ｍｗ）と数平均分子量（Ｍｎ）との比で表される分子量分布（Ｍｗ／Ｍｎ）の値は１．７７４と小さく、実施例２でイナゴマメ莢果から分離、精製されたＣｙｃａｒｎｅｙは分子量のバラツキが少なく、純度が高いことが示された。
(1) Gel permeation chromatography (GPC)
Exactly 10.0 mg of the Cycarney sample obtained in Example 2 was weighed to obtain a solution with a concentration of 2 mg/mL. A 0.1 mol/L NaNO 3 aqueous solution was used as the mobile phase, and gel permeation chromatography (GPC) analysis was performed at a flow rate of 0.7 mL/min. A differential refractometer and a light scattering detector were used as detectors. FIG. 1 shows the results of Cycarney molecular weight measurement. The horizontal axis of FIG. 1 indicates the elution volume, and the vertical axis indicates the refractive index. As a result of analysis, the average molecular weight was 7.428×10 6 . In addition, the value of the molecular weight distribution (Mw/Mn) represented by the ratio of the weight average molecular weight (Mw) to the number average molecular weight (Mn) is as small as 1.774. Cycarney was shown to have little variation in molecular weight and high purity.
（２）赤外吸収スペクトル分析
実施例２で得られたＣｙｃａｒｎｅｙのサンプルを５ｍｇ秤量し、適量の乾燥臭化カリウム（ＫＢｒ）と一緒に粉砕して均質なタブレットを作製した。これを試料として、ＦＴＩＲ分光分析法にて４０００ｃｍ－１～４００ｃｍ－１の範囲の赤外吸収スペクトルを得た。図２に得られた赤外吸収スペクトルを示す。図２の横軸は波数を、縦軸は透過率（％）を示している。図２によれば、－ＯＨの伸縮振動を表す３４２４ｃｍ－１のピーク、－ＣＨ３の伸縮振動を表す２９３７ｃｍ－１のピークが観察され、Ｃ－Ｏ伸縮振動を表す１０１０ｃｍ－１にも強い吸収ピークが観察された。
(2) Infrared absorption spectrum analysis 5 mg of the Cycarney sample obtained in Example 2 was weighed and ground with an appropriate amount of dry potassium bromide (KBr) to prepare a homogeneous tablet. Using this as a sample, an infrared absorption spectrum in the range of 4000 cm −1 to 400 cm −1 was obtained by FTIR spectroscopic analysis. FIG. 2 shows the obtained infrared absorption spectrum. The horizontal axis in FIG. 2 indicates the wave number, and the vertical axis indicates the transmittance (%). According to FIG. 2, a peak at 3424 cm −1 representing the stretching vibration of —OH, a peak at 2937 cm −1 representing the stretching vibration of —CH 3 were observed, and a strong absorption was also observed at 1010 cm −1 representing the C—O stretching vibration. A peak was observed.
（３）構成糖分析
実施例２で得られたＣｙｃａｒｎｅｙのサンプルを５．５ｍｇ秤量してチューブに入れ、２ｍｏｌ／Ｌのトリフルオロ酢酸（ＴＦＡ）を１ｍＬ加えた後、チューブを窒素で密封し、１１０℃で１２０分間加熱してＣｙｃａｒｎｅｙを加水分解させた。引き続いて、加水分解後のサンプルを溶解し、９０ｍｇのＮａＢＨ４を加えて４時間還元反応を行った。還元後のサンプルをオーブンで乾燥させ、５ｍＬの無水酢酸を加えて、１１０℃でアセチル化処理を行った。アセチル化処理後のサンプルを酢酸エチルに溶解させてフィルターろ過した後、ガスクロマトグラフ質量分析計（ＧＣ－ＭＳ）により、構成糖の組成分析を行った。図３にＣｙｃａｒｎｅｙの構成糖分析におけるＧＣ－ＭＳトータルイオンカレント（ＴＩＣ）クロマトグラムを示す。図３の横軸は各出現ピークの時間を、縦軸は相対強度を示している。
(3) Analysis of constituent sugars 5.5 mg of the Cycarney sample obtained in Example 2 was weighed and placed in a tube, 1 mL of 2 mol/L trifluoroacetic acid (TFA) was added, and the tube was sealed with nitrogen. Cycarney was hydrolyzed by heating at 110° C. for 120 minutes. Subsequently, the sample after hydrolysis was dissolved, 90 mg of NaBH 4 was added, and reduction reaction was performed for 4 hours. The sample after reduction was dried in an oven and acetylated at 110° C. by adding 5 mL of acetic anhydride. After the acetylated sample was dissolved in ethyl acetate and filtered, composition analysis of constituent sugars was performed using a gas chromatograph-mass spectrometer (GC-MS). FIG. 3 shows a GC-MS total ion current (TIC) chromatogram in Cycarney constituent sugar analysis. The horizontal axis of FIG. 3 indicates the time of each appearing peak, and the vertical axis indicates the relative intensity.
マススペクトルライブラリとの照合により分析した結果、イナゴマメ莢果由来の多糖類「Ｃｙｃａｒｎｅｙ」の構成糖組成（モル比）は、グルコース：フルクトース：マンノース＝６．５：８８．１：５．４であった。分析結果を図４～６に示す。図４は、Ｃｙｃａｒｎｅｙの構成単糖としてグルコースを同定したスペクトルを示しており、保持時間ｔ＝１８．０７、表中の（ｒｅｐｌｉｂ）Ｄ－Ｇｌｕｃｏｓｅ，２，３，４，５，６－ｐｅｎｔａａｃｅｔａｔｅは、Ｄ－グルコース－２，３，４，５，６－ペンタアセテートを示している。図５は、Ｃｙｃａｒｎｅｙの構成単糖としてフルクトースを同定したスペクトルを示しており、保持時間ｔ＝１９．８０であり、表中の（ｍａｉｎｌｉｂ） Ｄ－Ｆｒｕｃｔｏｓｅ，１，３，４，５，６－ｐｅｎｔａａｃｅｔａｔｅは、Ｄ－フルクトース－１，３，４，５，６－ペンタアセテートを示す。また、図６は、Ｃｙｃａｒｎｅｙの構成単糖としてマンノースを同定したスペクトルを示しており、保持時間ｔ＝１９．９４、（ｒｅｐｌｉｂ）Ｄ－Ｍａｎｎｉｔｏｌ，ｈｅｘａａｃｅｔａｔｅはＤ－マンノース ヘキサアセテートを示している。 As a result of analysis by comparison with a mass spectrum library, the constituent sugar composition (molar ratio) of the carob pod-derived polysaccharide "Cycarney" was glucose: fructose: mannose = 6.5: 88.1: 5.4. . The analysis results are shown in Figures 4-6. FIG. 4 shows spectra identifying glucose as a constituent monosaccharide of Cycarney, retention time t=18.07, (replib) D-Glucose, 2,3,4,5,6-pentaacetate in the table is , D-glucose-2,3,4,5,6-pentaacetate. FIG. 5 shows the spectrum identifying fructose as the constituent monosaccharide of Cycarney, with retention time t=19.80 and (mainlib) D-Fructose, 1,3,4,5,6- pentaacetate denotes D-fructose-1,3,4,5,6-pentaacetate. FIG. 6 shows a spectrum identifying mannose as a constituent monosaccharide of Cycarney, retention time t=19.94, (replib) D-mannitol, hexaacetate indicates D-mannose hexaacetate.
（４）メチル化分析
次に、Ｃｙｃａｒｎｅｙの構成糖残基間のグリコシド結合の様式をメチル化分析によって解析した。実施例２で得られたＣｙｃａｒｎｅｙのサンプルを２０ｍｇ秤量し、６ｍＬのＤＭＳＯに添加した。ＣｙｃａｒｎｅｙがＤＭＳＯに完全に溶解するまで、マグネチックスターラーを用い、４０℃で１５時間攪拌した。メチル化処理を行ってＣｙｃａｒｎｅｙの糖鎖に存在する水酸基を完全にメチル化させた後、上述した（３）構成糖分析と同一の手順によって、加水分解処理を行い、引き続いてアセチル化処理を行った。一連の処理によって得た、部分メチル化・アセチル化物について、ガスクロマトグラフ質量分析計（ＧＣ－ＭＳ）により分析を行った。図７にＣｙｃａｒｎｅｙのメチル化分析におけるＧＣ－ＭＳトータルイオンカレント（ＴＩＣ）クロマトグラムを示す。図７の横軸は各出現ピークの時間を、縦軸は相対強度を示している。分析結果を表１に示す。表１におけるＧｌｃはグルコース、Ｆｒｕはフルクトース、Ｍａｎはマンノースを表している。
(4) Methylation Analysis Next, the mode of glycosidic bonding between constituent sugar residues of Cycarney was analyzed by methylation analysis. 20 mg of the Cycarney sample obtained in Example 2 was weighed and added to 6 mL of DMSO. Stir with a magnetic stirrer at 40° C. for 15 hours until Cycarney is completely dissolved in DMSO. After the methylation treatment is carried out to completely methylate the hydroxyl groups present in the sugar chain of Cycarney, hydrolysis treatment and then acetylation treatment are carried out according to the same procedure as in (3) Constituent sugar analysis described above. Ta. Partially methylated/acetylated products obtained by a series of treatments were analyzed by a gas chromatograph-mass spectrometer (GC-MS). FIG. 7 shows a GC-MS total ion current (TIC) chromatogram in Cycarney's methylation analysis. The horizontal axis of FIG. 7 indicates the time of each appearing peak, and the vertical axis indicates the relative intensity. The analysis results are shown in Table 1. Glc in Table 1 represents glucose, Fru fructose, and Man mannose.
イオンフラグメンテーションパターンを解析した結果、Ｃｙｃａｒｎｅｙのグリコシド結合の様式は、１→４結合グルコース、１→結合フルクトース（非還元末端フルクトース）、１→６結合フルクトース、１→３，６結合マンノースであり、多糖類の末端は主にフルクトースで構成されていることが明らかとなった。 As a result of analyzing the ion fragmentation pattern, Cycarney's glycosidic bond mode is 1→4-linked glucose, 1→linked fructose (non-reducing end fructose), 1→6-linked fructose, 1→3,6-linked mannose, and many It was clarified that the ends of sugars were mainly composed of fructose.
上述の構造解析の結果より、本発明に係るイナゴマメ莢果由来の多糖類「Ｃｙｃａｒｎｅｙ」はグルコース６．５％、フルクトース８８．１％、マンノース５．４％で構成されており、平均分子量は７．４２８×１０６であること、末端が全てフルクトースの分岐型多糖類であり、すべての分岐点はマンノースの位置にあることがわかった。下記式にＣｙｃａｒｎｅｙの多糖フラグメントを示す。グルコース（Ｇｌｃ）６５個、フルクトース（Ｆｒｕ）８８１個及びマンノース（Ｍａｎ）５４個からなるＣｙｃａｒｎｅｙの多糖フラグメントの分子量は１６２１７７となるので、この多糖類には約２４７３もの分岐があり（７４２８０００÷１６２１７７×５４）、平均すると、各分岐はマンノース１に対しグルコース１．２を有しており、残りはフルクトースから構成される。 From the results of the above structural analysis, the carob pod-derived polysaccharide "Cycarney" according to the present invention is composed of 6.5% glucose, 88.1% fructose, and 5.4% mannose, and has an average molecular weight of 7.5%. It was found to be 428×10 6 , a branched polysaccharide with all fructose ends, and all branch points at mannose positions. The following formula shows the Cycarney polysaccharide fragment. A Cycarney polysaccharide fragment consisting of 65 glucose (Glc), 881 fructose (Fru) and 54 mannose (Man) has a molecular weight of 162,177, so there are about 2,473 branches in this polysaccharide (7428,000 ÷ 162,177 × 54), on average each branch has 1.2 glucose to 1 mannose, the rest being made up of fructose.
上式中のｘ、ｙ、ｚ、ｏ、ｐ及びｑは単糖類の数を表しており、０又は正の整数である。また、式中のＲ１とＲ２は他分岐の末端位のフルクトースまたはマンノースを示し、式中のＲ３は他分岐のフルクトースを示している（Ｒ３が他分岐のマンノースである確率は非常に低いと推察される）。フラグメント全体の平均分子量は１６２１７７である。 x, y, z, o, p and q in the above formula represent the number of monosaccharides and are 0 or positive integers. In addition, R1 and R2 in the formula represent fructose or mannose at the terminal position of other branches, and R3 in the formula represents fructose of other branches (the probability that R3 is mannose of other branches is assumed to be very low. be done). The average molecular weight of the entire fragment is 162,177.
実施例４ Ｃｙｃａｒｎｅｙの血中脂質に対する作用の検討
実施例２で得たＣｙｃａｒｎｅｙを用いて、高血糖及び高脂肪モデルラットにＣｙｃａｒｎｅｙを投与したときの血中脂質に与える影響を調べた。
Example 4 Investigation of Cycarney's Action on Blood Lipids Using the Cycarney obtained in Example 2, the effects on blood lipids when Cycarney was administered to hyperglycemia and hyperlipidemia model rats were investigated.
体重１８０～２２０ｇの成熟した雄のＳＤラット（供給源：北京維通利華実験動物有限公司）を温度が２３±２℃、相対湿度が５０％～６０％、照明が１２時間周期の環境条件下にて１週間馴化飼育した。馴化飼育時には標準飼料を供し、飼料は自由摂取とし、飲水も吸水瓶からの自由飲水とした。1週間予備飼育後、実験に供した。アロキサン誘導剤を腹腔内注射する、同時に、糖尿病性脂質障害を誘発するために、糖尿病動物に高脂肪食（標準食に基づいて0.2％コレステロールと15％ラードを追加）を与え、自由に摂取させ、毎日可溶性ラード10-20ml/kgを定量に投与する。 Adult male Sprague-Dawley rats weighing 180-220 g (source: Beijing Weitong Lihua Laboratory Animal Co., Ltd.) were placed under environmental conditions with a temperature of 23±2° C., a relative humidity of 50%-60%, and a lighting cycle of 12 hours. was acclimatized for 1 week. During acclimation breeding, standard feed was provided, feed was given ad libitum, and drinking water was given ad libitum from a water absorption bottle. After preliminary breeding for 1 week, the animals were subjected to experiments. Alloxan-inducing agents were injected intraperitoneally, at the same time, diabetic animals were fed a high-fat diet (0.2% cholesterol and 15% lard based on standard diet supplemented) to induce diabetic lipid disorders and allowed ad libitum. , Administer 10-20ml/kg of soluble lard daily.
本実験はSDラット6匹ずつ、次のように７対照群に分けた。群1、蒸留水を投与した正常対照群。群2、蒸留水を投与した高血糖・高脂血症モデルコントロール群。群３-５、イナゴマメ多糖類Cycarney（イナゴマメ多糖類Cycarney含有量20％の40％エタノールで抽出したエキス）の低用量群（0.1g/kg）、中用量群（0.2g/kg）、高用量群（0.4g/kg）の投与群。群６、シンバスタチン4mg/kgを投与した陽性対照群。群７、イナゴマメ多糖類Cycarneyを含まないの40％エタノールで抽出したエキス0.4g/kg投与した溶媒対照群。各群に対して連続２１日間経口投与し、眼窩静脈から叢採血した。試薬キットを用いてTC、TGを測定し、各群のSDラットの血清TC、TG値を測定したところ、結果を表２に示した。 In this experiment, 6 SD rats each were divided into 7 control groups as follows. Group 1, normal control group administered with distilled water. Group 2, hyperglycemia/hyperlipidemia model control group to which distilled water was administered. Group 3-5, low dose group (0.1 g/kg), medium dose group (0.2 g/kg), high dose of carob polysaccharide Cycarney (extract extracted with 40% ethanol containing 20% carob polysaccharide Cycarney) Group (0.4 g/kg) administration group. Group 6, positive control group administered with 4 mg/kg of simvastatin. Group 7, vehicle control group administered 0.4 g/kg of extract extracted with 40% ethanol without carob polysaccharide Cycarney. Each group was orally administered for 21 consecutive days, and blood was collected from the orbital vein. TC and TG were measured using a reagent kit, and the serum TC and TG values of SD rats in each group were measured, and the results are shown in Table 2.
表２によれば、Ｃｙｃａｒｎｅｙは用量依存的に血清中のＴＣ（総コレステロール）及びＴＧ（中性脂肪）を低下させる作用を有している。また、イナゴマメ多糖類CycarneyのTC、TGに対する効果は、高用量群ではモデルコントロール群と比較して有意差が認められた。陽性群と比較して有意な差も示していた。イナゴマメ多糖類CycarneyのTC、TGに対する効果は、陽性（PC）群とほぼ等しかった。溶媒対照群とモデルコントロール群の間に有意差が認められなかったことと合わせ、イナゴマメ多糖類CycarneyはTCとTGを低下させる効果があると判断した。これらのことから、Ｃｙｃａｒｎｅｙが高血糖・高脂肪モデル群の脂質代謝異常を改善する効果を有することが示された。 According to Table 2, Cycarney has the effect of lowering serum TC (total cholesterol) and TG (triglyceride) in a dose-dependent manner. In addition, the effect of Cycarney, a carob polysaccharide, on TC and TG was significantly different in the high-dose group compared to the model control group. A significant difference was also shown compared to the positive group. The effect of the carob polysaccharide Cycarney on TC and TG was almost the same as that of the positive (PC) group. Combined with the fact that no significant difference was observed between the solvent control group and the model control group, we determined that the carob polysaccharide Cycarney had the effect of lowering TC and TG. From these results, it was shown that Cycarney has the effect of improving dyslipidemia in the hyperglycemia/hyperlipidemia model group.
実施例５ Ｃｙｃａｒｎｅｙの血糖値上昇抑制作用の検討
実施例２で得たＣｙｃａｒｎｅｙを用いて、高血糖及び高脂肪モデルラットにＣｙｃａｒｎｅｙを投与したときの血糖値に与える影響をグルコース負荷テストにより調べた。
Example 5 Investigation of the effect of Cycarney on blood glucose level elevation The Cycarney obtained in Example 2 was used to examine the effect on blood glucose levels when Cycarney was administered to hyperglycemia and high fat model rats by a glucose tolerance test.
体重１８０～２２０ｇの成熟した雄のＳＤラット（供給源：北京維通利華実験動物有限公司）を温度が２３±２℃、相対湿度が５０％～６０％、照明が１２時間周期の環境条件下にて１週間馴化飼育した。馴化飼育時には標準飼料を供し、飼料は自由摂取とし、飲水も吸水瓶からの自由飲水とした。1週間予備飼育後、実験に供した。アロキサン誘導剤を腹腔内注射する、同時に、糖尿病性脂質障害を誘発するために、糖尿病動物に高脂肪食（標準食に基づいて0.2％コレステロールと15％ラードを追加）を与え、自由に摂取させ、毎日可溶性ラード10-20ml/kgを定量に投与する。 Adult male Sprague-Dawley rats weighing 180-220 g (source: Beijing Weitong Lihua Laboratory Animal Co., Ltd.) were placed under environmental conditions with a temperature of 23±2° C., a relative humidity of 50%-60%, and a lighting cycle of 12 hours. was acclimatized for 1 week. During acclimation breeding, standard feed was provided, feed was given ad libitum, and drinking water was given ad libitum from a water absorption bottle. After preliminary breeding for 1 week, the animals were subjected to experiments. Alloxan-inducing agents were injected intraperitoneally, at the same time, diabetic animals were fed a high-fat diet (0.2% cholesterol and 15% lard based on standard diet supplemented) to induce diabetic lipid disorders and allowed ad libitum. , Administer 10-20ml/kg of soluble lard daily.
本実験はSDラット6匹ずつ、次のように8群に分けた。群1、蒸留水を投与した正常対照群。群2、イナゴマメ多糖類Cycarneyの中用量（0.2g/kg）を投与した正常対照群。群3、蒸留水を投与した高血糖・高脂血症モデルコントロール群。群4-6、イナゴマメ多糖類Cycarney（イナゴマメ多糖類Cycarney含有量20％の40％エタノールで抽出したエキス）の低用量群（0.1g/kg）、中用量群（0.2g/kg）、高用量群（0.4g/kg）の投与群。群7、メトホルミン200mg/kgを投与した陽性対照群。群8、イナゴマメ多糖類Cycarneyを含まないの40％エタノールで抽出したエキス、0.4g/kg投与した溶媒対照群。1日1回21日間続けます。 In this experiment, 6 SD rats were divided into 8 groups as follows. Group 1, normal control group administered with distilled water. Group 2, the normal control group receiving a moderate dose (0.2 g/kg) of the carob polysaccharide Cycarney. Group 3, hyperglycemia/hyperlipidemia model control group to which distilled water was administered. Group 4-6, low dose group (0.1 g/kg), medium dose group (0.2 g/kg), high dose of carob polysaccharide Cycarney (extract extracted with 40% ethanol containing 20% carob polysaccharide Cycarney) Group (0.4 g/kg) administration group. Group 7, positive control group administered metformin 200 mg/kg. Group 8, extract extracted with 40% ethanol without carob polysaccharide Cycarney, vehicle control group administered 0.4 g/kg. Continue once a day for 21 days.
動物は一晩断食した後、各グループに対応する薬を胃に充填する。30分後グルコース2gを与え、抗凝固採血サンプルなし時間帯0，0.5，1.0，1.5、2.0と2.5h目縁の静脉から遠心分離を収集します（3500 rpm・min-1，4℃条件下で回転速度を遠心分離10min、血清を分離します）後血糖値を検出します。結果を図８のグラフに示す。 After the animals have fasted overnight, the stomachs are filled with the drugs corresponding to each group. After 30 min, 2 g of glucose was given, and no anticoagulant blood samples were collected by centrifugation at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 h from the eyelid stasis (under 3500 rpm/min-1, 4°C conditions). Centrifuge the rotation speed at 10 min, separate the serum) and then detect the blood glucose level. The results are shown in the graph of FIG.
図８によれば、モデル群は正常対照群に比べ、耐糖能異常が認められた。モデル群と比較して、イナゴマメ多糖類Cycarney投与群は0.5hと1.0hの血糖値上昇を抑制し、陽性対照群は0.5h、1.0h、1.5h、2.0h、2.5hの血糖値上昇を抑制した。イナゴマメ多糖類Cycarneyを投与した正常群では、効果は認められなかった。また、イナゴマメ多糖類Cycarney中用量群、高用量群、陽性対照群の間にも効果の有意差は認められなかった。溶媒対照群とモデルコントロール群との間にも有意差はなかった。これらのことから、Ｃｙｃａｒｎｅｙが高血糖・高脂肪モデル群の耐糖能異常を改善する効果を有することが示された。 According to FIG. 8, impaired glucose tolerance was observed in the model group compared to the normal control group. Compared with the model group, the carob polysaccharide Cycarney-administered group suppressed the rise in blood sugar level for 0.5h and 1.0h, and the positive control group suppressed the rise in blood sugar level for 0.5h, 1.0h, 1.5h, 2.0h, and 2.5h. Suppressed. No effect was observed in the normal group receiving the carob polysaccharide Cycarney. There was also no significant difference in efficacy between the mid-dose group, the high-dose group, and the positive control group of carob polysaccharide Cycarney. There was also no significant difference between the solvent control group and the model control group. From these results, it was shown that Cycarney has the effect of improving glucose intolerance in the hyperglycemia/high fat model group.
実施例６ Ｃｙｃａｒｎｅｙのアンジオテンシン変換酵素（ＡＣＥ）阻害作用の検討
実施例２で得たＣｙｃａｒｎｅｙを用いて、Ｃｙｃａｒｎｅｙのアンジオテンシン変換酵素（ＡＣＥ）阻害作用を調べた。まず、基質溶液として、０．３ｍｏｌ／ＬのＮａＣｌを含む０．１ｍｏｌ／Ｌホウ酸緩衝液（ｐＨ８．３）を用いて、Ｈｉｐｐｕｒｙｌ－Ｈｉｓ－Ｌｅｕ（ＨＨＬ）の５ｍｍｏｌ／Ｌ溶液を調製した。サンプル濃度は、それぞれ低用量群（0.1mg / mL）、中用量群（0.2mg / mL）、高用量群（0.4mg / mL）のイナゴマメ多糖類Cycarneyであり、イナゴマメ多糖類を含まないCycarneyを採取しました。エタノール沈殿物の分離中、投与量は0.4g/kgでした。実施例２で得たＣｙｃａｒｎｅｙを適宜蒸留水に溶解し、希釈して複数の濃度の試料溶液、その中サンプル濃度は、それぞれ低用量群であった（0.1g/kg）、中用量群（0.2g/kg)と高用量群（0.4g/kg）角豆多糖類Cycarney（40％エタノール部位濃縮ディップペーストを採用、その中多糖類含量は20%）を調整した。
Example 6 Examination of Cycarney's Angiotensin-Converting Enzyme (ACE) Inhibitory Action Using the Cycarney obtained in Example 2, the angiotensin-converting enzyme (ACE) inhibitory action of Cycarney was examined. First, a 5 mmol/L solution of Hippuryl-His-Leu (HHL) was prepared using a 0.1 mol/L borate buffer (pH 8.3) containing 0.3 mol/L NaCl as a substrate solution. The sample concentrations were low dose group (0.1 mg/mL), medium dose group (0.2 mg/mL), high dose group (0.4 mg/mL) of carob polysaccharide Cycarney and Cycarney without carob polysaccharide, respectively. I took it. The dose was 0.4 g/kg during the separation of the ethanol precipitate. The Cycarney obtained in Example 2 was appropriately dissolved in distilled water and diluted to prepare sample solutions of multiple concentrations. g/kg) and high dose group (0.4g/kg) square bean polysaccharide Cycarney (adopted 40% ethanol concentrated dip paste, polysaccharide content in it is 20%).
５．０ｍｍｏｌ／ＬのＨＨＬ溶液８０μＬと試料溶液２０μＬを１．５ｍＬ容量のチューブに加えて混合し、３７℃で５分間予備加熱した。予備加熱処理後のチューブに、ＡＣＥ溶液（０．１Ｕ／ｍＬ）を２０μＬ勢いよく加え、直ちにボルテックスミキサーを用いて撹拌混合し、３７℃で３０分間反応させた。３０分後、１．０ｍｏｌ／Ｌの塩酸溶液２００μＬを添加して混合撹拌し、酵素を失活させて反応を終了させた。反応収量後の溶液について、ＨＰＬＣ分析を行い、遊離した馬尿酸を測定した。ＨＰＬＣ条件は、以下の通りであった。 80 μL of 5.0 mmol/L HHL solution and 20 μL of sample solution were added to a 1.5 mL capacity tube, mixed, and preheated at 37° C. for 5 minutes. 20 μL of ACE solution (0.1 U/mL) was vigorously added to the preheated tube, immediately stirred and mixed using a vortex mixer, and reacted at 37° C. for 30 minutes. After 30 minutes, 200 μL of 1.0 mol/L hydrochloric acid solution was added and mixed with stirring to deactivate the enzyme and terminate the reaction. The solution after reaction yield was subjected to HPLC analysis to measure liberated hippuric acid. HPLC conditions were as follows.
＜液体クロマトグラフィー条件＞
・ＨＰＬＣ使用カラム：Ｃ１８カラム（１５０ｍｍ×４．６ｍｍ）
・カラム温度：３０℃
・移動相：アセトニトリル：水：トリフルオロ酢酸溶液（５０：５０：０．０５，ｖ／ｖ）
・流速：０．４ｍＬ／ｍｉｎ
・検出波長：２２８ｎｍ
・注入量：１０μＬ
<Liquid chromatography conditions>
・HPLC used column: C18 column (150 mm × 4.6 mm)
・Column temperature: 30°C
- Mobile phase: acetonitrile: water: trifluoroacetic acid solution (50:50:0.05, v/v)
・Flow rate: 0.4 mL/min
・Detection wavelength: 228 nm
・Injection volume: 10 μL
また、ブランク対照として、試料溶液の代わりに同量のホウ酸緩衝液を用い、試験を行った。ＨＰＬＣによる馬尿酸ピーク面積に基づき、「ＡＣＥ阻害率（％）＝（Ｂ－Ａ）／Ｂ×１００」の式によりＡＣＥ阻害率（％）を求めた。ここで、「Ａ」は試料溶液を添加して反応を行ったときの馬尿酸ピーク面積、「Ｂ」はブランク対照（試料溶液の替わりにホウ酸緩衝液を用いた）の馬尿酸ピーク面積である。結果を図９のグラフに示す。 Also, as a blank control, the same amount of borate buffer was used instead of the sample solution, and the test was performed. Based on the hippuric acid peak area by HPLC, the ACE inhibition rate (%) was determined by the formula "ACE inhibition rate (%)=(BA)/B×100". Here, "A" is the hippuric acid peak area when the sample solution is added and the reaction is performed, and "B" is the hippuric acid peak area of the blank control (using borate buffer instead of the sample solution). be. The results are shown in the graph of FIG.
図９によれば、Ｃｙｃａｒｎｅｙ投与群のＡＣＥ阻害活性は、ブランク対照群と比較して有意に高いことが明らかとなった（Ｐ＜０．０１）、対照群とブランク対照群の間に有意差はなく。これらのことから、Ｃｙｃａｒｎｅｙが優れたアンジオテンシン変換酵素阻害作用を有すること、そして、このＡＣＥ阻害作用に基づく高血圧の改善効果を有することが示された。 According to FIG. 9, the ACE inhibitory activity of the Cycarney-administered group was found to be significantly higher than that of the blank control group (P<0.01), indicating a significant difference between the control group and the blank control group. Not. These results indicate that Cycarney has an excellent angiotensin-converting enzyme inhibitory action, and that it has an improvement effect on hypertension based on this ACE inhibitory action.
実施例７ Ｃｙｃａｒｎｅｙの腸蠕動運動改善作用の検討
実施例２で得たＣｙｃａｒｎｅｙを用いて、高血糖及び高脂肪モデルラットにＣｙｃａｒｎｅｙを投与したときの腸蠕動運動に与える影響を調べた。
Example 7 Examination of Cycarney's Intestinal Peristalsis Improving Action Using the Cycarney obtained in Example 2, the effect on intestinal peristalsis when Cycarney was administered to hyperglycemia and hyperlipidemia model rats was investigated.
実施例４実験動物とグループ分けの方法は、血中脂質と血糖の異常代謝の影響に関する上記の実験と同じで、各グループのラットは水を飲んで正常に食べました。各グループのラットには1 mL/100が与えられました。ＢＷインク１ｇ（アラビアゴム１００ｇ、水８００ｍＬを加え、溶液が透明になるまで煮沸し、活性化炭素を秤量した）上記の溶液に５０ｇを加え、３回煮沸した。冷却し、水を加えて容量を1000mLにした）強制経口投与。２５分後、エーテル麻酔下にてラットを安楽死させた。各個体の腹腔を開いて腸間膜を分離し、幽門部から回腸部までの腸管を切断した。回収した腸管をトレーに載せて直線状にし、腸管の長さを「小腸の全長」、幽門部からエバンスブルーのインク端までの長さを「インク進入距離」として測定した。これらの測定値に基づき、「インク進入率（％）＝｛インク進入距離（ｍｍ）／小腸の全長（ｍｍ）｝×１００」の式により、インク進入率（％）を求めた。結果を下記表５に示す。 Example 4 The experimental animals and grouping methods were the same as the above experiments on the effects of abnormal metabolism of blood lipids and blood sugar, and the rats in each group drank water and ate normally. Rats in each group were given 1 mL/100. 1 g of BW ink (100 g of gum arabic, 800 mL of water were added, boiled until the solution became clear, and the activated carbon was weighed) 50 g was added to the above solution and boiled three times. cooled and water added to bring the volume to 1000 mL) administered by oral gavage. Twenty-five minutes later, rats were euthanized under ether anesthesia. The abdominal cavity of each individual was opened, the mesentery was separated, and the intestinal tract from the pylorus to the ileum was cut. The collected intestinal tract was placed on a tray and straightened, and the length of the intestinal tract was measured as the "total length of the small intestine" and the length from the pylorus to the end of the Evans blue ink as the "ink penetration distance". Based on these measured values, the ink penetration rate (%) was determined by the formula "ink penetration rate (%)={ink penetration distance (mm)/total length of small intestine (mm)}×100". The results are shown in Table 5 below.
表５に示すように、対照群、高血糖・高脂肪モデル群と陽性対照物質投与群は、インク進入率の値が２０％代と小さく、この値は正常対照群と比較して有意差があることから、高血糖・高脂肪モデル群と陽性対照物質投与群のラットの腸の蠕動運動は鈍化していることが明らかとなった。また、高血糖・高脂肪モデル群と陽性対照物質投与群のラットは、排便状況からも便秘が観察されていた。他方、Ｃｙｃａｒｎｅｙの中用量投与群及び高用量投与群は、インク進入率の値が３５％を超えており、この値は正常対照群との有意差がなく、高血糖・高脂肪モデル群及び陽性対照物質投与群との有意差はあることがわかった。これらのことから、Ｃｙｃａｒｎｅｙが高血糖・高脂肪モデル群の腸の蠕動運動の鈍化を改善する効果を有し、Ｃｙｃａｒｎｅｙの投与によって便秘が改善され、正常対照群の腸の状態にまで回復され得ることが示された。 As shown in Table 5, the control group, the hyperglycemic/high-fat model group, and the positive control substance-administered group had a low ink penetration rate value of 20%, and this value was not significantly different from the normal control group. It was clarified that the intestinal peristalsis of rats in the hyperglycemic/high-fat model group and the positive control substance-administered group was sluggish. Constipation was also observed in the rats in the hyperglycemic/high-fat model group and the positive control substance-administered group. On the other hand, in the Cycarney medium-dose administration group and high-dose administration group, the value of the ink penetration rate exceeded 35%, and this value was not significantly different from the normal control group. It was found that there was a significant difference from the control substance administration group. From these facts, Cycarney has the effect of improving the blunting of intestinal peristalsis in the hyperglycemic/high-fat model group, and administration of Cycarney improves constipation and can restore the intestinal condition to that of the normal control group. was shown.
本発明は、上記の実施形態又は実施例に限定されるものでなく、特許請求の範囲に記載された発明の要旨を逸脱しない範囲内での種々、設計変更した形態も技術的範囲に含まれるものである。 The present invention is not limited to the above-described embodiments or examples, and includes various forms with design changes within the scope of the invention described in the claims. It is.
本発明の新規多糖類は、脂質代謝異常、高脂血症、動脈硬化、糖尿病、インスリン抵抗性、高血圧症、便秘又はメタボリックシンドロームの予防、改善若しくは治療剤として使用され、医療及び食品（機能性食品・健康食品を含む）の分野において幅広く利用されるものである。 The novel polysaccharide of the present invention is used as a preventive, ameliorating or therapeutic agent for dyslipidemia, hyperlipidemia, arteriosclerosis, diabetes, insulin resistance, hypertension, constipation or metabolic syndrome, and is used for medical and food (functional It is widely used in the field of food and health food).
Claims (7)
（ｉ）水に可溶。エタノールに不溶。
（ｉｉ）構成単糖が、グルコース、フルクトース及びマンノースの３種からなり、その構成モル比が、グルコース：フルクトース：マンノース＝６．４～６．６：８８．０～８８．２：５．３～５．５である。
（ｉｉｉ）構成単糖の結合様式が、下記（ａ）～（ｄ）の組み合わせからなる。
（ａ）（１→４）結合グルコース
（ｂ）（１→）結合フルクトース
（ｃ）（１→６）結合フルクトース
（ｄ）（１→３）及び（１→６）結合マンノース A novel polysaccharide derived from carob pods, having the following characteristics (i) to (iii).
(i) soluble in water; Insoluble in ethanol.
(ii) The constituent monosaccharides consist of three kinds of glucose , fructose and mannose, and the constituent molar ratio is glucose:fructose:mannose= 6.4-6.6:88.0-88.2:5. 3 to 5.5 .
(iii) The binding mode of the constituent monosaccharides is a combination of (a) to (d) below.
(a) (1→4) linked glucose (b) (1→) linked fructose (c) (1→6) linked fructose (d) (1→3) and (1→6) linked mannose
（ｉｖ）ゲルろ過クロマトグラフィーを用いて測定した分子量が、１×１０４～１×１０７である。 2. The novel polysaccharide according to claim 1, which further has the following characteristics (iv).
(iv) a molecular weight of 1×10 4 to 1×10 7 as determined using gel filtration chromatography;
（ｖ）分子量分布（Ｍｗ／Ｍｎ）が、１．７～１．８である。 2. The novel polysaccharide according to claim 1, which further has the following feature (v).
(v) a molecular weight distribution (Mw/Mn) of 1.7 to 1.8;
前記抽出液又はその濃縮液に対し、エタノールを最終濃度が４０～８０重量％となるように添加して沈殿を得る工程、及び、
前記沈殿から、請求項１～４のいずれか１項に記載の新規多糖類を分離する工程、を有することを特徴とする新規多糖類の製造方法。 adding water to the pods of carob (Ceratonia siliqua) to obtain an extract;
A step of adding ethanol to the extract or its concentrate to a final concentration of 40 to 80% by weight to obtain a precipitate;
A method for producing a novel polysaccharide, comprising a step of separating the novel polysaccharide according to any one of claims 1 to 4 from the precipitate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2023058591A JP7337300B1 (en) | 2023-03-31 | 2023-03-31 | Carob polysaccharide for prevention and improvement of metabolic syndrome, method for producing the same, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2023058591A JP7337300B1 (en) | 2023-03-31 | 2023-03-31 | Carob polysaccharide for prevention and improvement of metabolic syndrome, method for producing the same, and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP7337300B1 true JP7337300B1 (en) | 2023-09-01 |
Family
ID=87797929
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2023058591A Active JP7337300B1 (en) | 2023-03-31 | 2023-03-31 | Carob polysaccharide for prevention and improvement of metabolic syndrome, method for producing the same, and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7337300B1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006506464A (en) * | 2002-07-23 | 2006-02-23 | ニュートリノヴァ ニュートリション スペシャリティーズ アンド フード イングリーディエンツ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Cholesterol-lowering agents made with dietary fiber and cholesterol-lowering substances |
| JP2008127364A (en) * | 2006-11-24 | 2008-06-05 | Taiyo Kagaku Co Ltd | Marker protein expression controlling agent |
| JP2012525840A (en) * | 2009-05-08 | 2012-10-25 | ライクスユニヴェルシタイト・フローニンゲン | Gluco-oligosaccharides containing (α1 → 4) and (α1 → 6) glycosidic bonds, their use and methods of providing same |
| WO2019068466A1 (en) * | 2017-10-05 | 2019-04-11 | Pharmactive Biotech Products, S.L. | Dietary fiber composition for protecting bioactive compounds during the physiological digestion process |
| JP2022185605A (en) * | 2021-06-03 | 2022-12-15 | 株式会社ナボカルコスメティックス | Novel isoflavone compound |
-
2023
- 2023-03-31 JP JP2023058591A patent/JP7337300B1/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006506464A (en) * | 2002-07-23 | 2006-02-23 | ニュートリノヴァ ニュートリション スペシャリティーズ アンド フード イングリーディエンツ ゲゼルシャフト ミット ベシュレンクテル ハフツング | Cholesterol-lowering agents made with dietary fiber and cholesterol-lowering substances |
| JP2008127364A (en) * | 2006-11-24 | 2008-06-05 | Taiyo Kagaku Co Ltd | Marker protein expression controlling agent |
| JP2012525840A (en) * | 2009-05-08 | 2012-10-25 | ライクスユニヴェルシタイト・フローニンゲン | Gluco-oligosaccharides containing (α1 → 4) and (α1 → 6) glycosidic bonds, their use and methods of providing same |
| WO2019068466A1 (en) * | 2017-10-05 | 2019-04-11 | Pharmactive Biotech Products, S.L. | Dietary fiber composition for protecting bioactive compounds during the physiological digestion process |
| JP2022185605A (en) * | 2021-06-03 | 2022-12-15 | 株式会社ナボカルコスメティックス | Novel isoflavone compound |
Non-Patent Citations (10)
| Title |
|---|
| "ローカストビーンガム", 別冊フードケミカル, vol. 8, JPN6023025491, 1996, pages 67 - 72, ISSN: 0005090578 * |
| ACTA CIRURGICA BRASILEIRA, vol. 30(5), JPN7023002364, 2015, pages 366 - 370, ISSN: 0005090577 * |
| Acta Cirurgica Brasileira，2015年，30(5)，366-370 |
| J. PHARM. SCI. & RES., vol. 9(11), JPN7023002365, 2017, pages 2189 - 2195, ISSN: 0005090576 * |
| J. Pharm. Sci. & Res.，2017年，9(11)，2189-2195 |
| J. SEP. SCI., vol. 30, JPN6023025490, 2007, pages 557 - 562, ISSN: 0005090575 * |
| J. Sep. Sci.，2007年，30，557-562 |
| フードスタイル２１, vol. 7(6), JPN6023025493, 2003, pages 116 - 120, ISSN: 0005090579 * |
| フードスタイル２１，2003年，7(6)，116-120 |
| ローカストビーンガム，別冊フードケミカル，1996年，8，67-72 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100795327B1 (en) | Muscular tension-elevating agent | |
| JP5568806B2 (en) | Diabetes or diabetes complication preventive | |
| Kumari et al. | Onion and garlic polysaccharides: A review on extraction, characterization, bioactivity, and modifications | |
| WO2006090935A1 (en) | Acerola fruit-derived pectin and use thereof | |
| JP2006188486A (en) | Body fat accumulation-inhibiting or reducing agent | |
| KR20100134773A (en) | Compositions comprising galactomannan and a process thereof | |
| KR101363111B1 (en) | Pharmaceutical composition containing paddy husk and barley husk extract as activeingredient | |
| JP4686173B2 (en) | Processed acerola containing polyphenol and / or vitamin C | |
| US20020041904A1 (en) | Compound with alpha-glucosidase inhibiting action and method for producing the same | |
| US20100247638A1 (en) | organoleptically improved dietary fiber composition and a process thereof (teestar) | |
| JP2004091464A (en) | Obesity inhibitor | |
| JP6042800B2 (en) | Tomatoside A extraction method | |
| JP2005350453A (en) | Slightly water-soluble component of leaf of lagerstroemia speciosa l. | |
| KR20190135438A (en) | Antioxidant composition comprising mixture of the extract of Chunggukjang and Modordica charantia | |
| JP7337300B1 (en) | Carob polysaccharide for prevention and improvement of metabolic syndrome, method for producing the same, and use thereof | |
| US9737583B2 (en) | Composition for prevention or treatment of acute renal failure including herbal extract or fraction thereof as active ingredient | |
| JP2006104182A (en) | Composition for reducing body fat | |
| JP2008007473A (en) | Cassia obtusifolia l. or cassia tora l. seed extract | |
| JP2006008523A (en) | Physiologically active phenolic compound and food containing the same | |
| KR20220067467A (en) | A composition comprising the ethanol extract of Corchorus olitorius for the prevention or treatment of alcoholic liver damage | |
| KR101373493B1 (en) | Composition comprising Hizikia fusiformis for preventing and treating obesity or hyperlipidemia and atherosclerotic-vascular diseases | |
| KR101018404B1 (en) | Compositions for reducing cholesterol comprising extracts of Vaccinium oldhami Miquel leaf as an active ingredient | |
| JP2005022994A (en) | Hypoglycemic composition | |
| JP7409797B2 (en) | Promoting short-chain fatty acid production in the intestinal tract | |
| JP4612335B2 (en) | Glucose level rise inhibitor and AGE production inhibitor containing acerola seed extract and food containing them |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20230331 |
|
| A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20230331 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230626 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230721 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230807 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230822 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7337300 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |