CN113845603B - 醇溶性均一多糖及其抗抑郁用途 - Google Patents
醇溶性均一多糖及其抗抑郁用途 Download PDFInfo
- Publication number
- CN113845603B CN113845603B CN202111215191.XA CN202111215191A CN113845603B CN 113845603 B CN113845603 B CN 113845603B CN 202111215191 A CN202111215191 A CN 202111215191A CN 113845603 B CN113845603 B CN 113845603B
- Authority
- CN
- China
- Prior art keywords
- alcohol
- asp
- soluble
- water
- polysaccharide
- 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
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 61
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 61
- 150000004676 glycans Chemical class 0.000 title claims abstract description 59
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 45
- 241001076416 Dendrobium tosaense Species 0.000 claims abstract description 34
- 239000003814 drug Substances 0.000 claims abstract description 15
- 230000001430 anti-depressive effect Effects 0.000 claims abstract description 10
- 239000000284 extract Substances 0.000 claims abstract description 9
- 239000000935 antidepressant agent Substances 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 229940005513 antidepressants Drugs 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 16
- 235000000346 sugar Nutrition 0.000 claims description 16
- 229920005654 Sephadex Polymers 0.000 claims description 15
- 238000001035 drying Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 14
- 229920000869 Homopolysaccharide Polymers 0.000 claims description 12
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 12
- 101100071254 Mus musculus Hnrnpul2 gene Proteins 0.000 claims description 12
- 239000012507 Sephadex™ Substances 0.000 claims description 12
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 12
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 11
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 11
- 238000010521 absorption reaction Methods 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 9
- 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 description 9
- 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 description 9
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 9
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 9
- 238000011068 loading method Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 238000012544 monitoring process Methods 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 8
- 229920005989 resin Polymers 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 6
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 241000233855 Orchidaceae Species 0.000 claims description 2
- 238000009835 boiling Methods 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000006187 pill Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 9
- 238000002474 experimental method Methods 0.000 abstract description 7
- 241000196324 Embryophyta Species 0.000 abstract description 6
- RTHCYVBBDHJXIQ-MRXNPFEDSA-N (R)-fluoxetine Chemical compound O([C@H](CCNC)C=1C=CC=CC=1)C1=CC=C(C(F)(F)F)C=C1 RTHCYVBBDHJXIQ-MRXNPFEDSA-N 0.000 abstract description 5
- 229960002464 fluoxetine Drugs 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 3
- 241000700159 Rattus Species 0.000 description 24
- 238000012360 testing method Methods 0.000 description 18
- 229930006000 Sucrose Natural products 0.000 description 15
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 15
- 239000005720 sucrose Substances 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 230000009182 swimming Effects 0.000 description 12
- 235000019441 ethanol Nutrition 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 238000005481 NMR spectroscopy Methods 0.000 description 9
- 230000000638 stimulation Effects 0.000 description 9
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000006399 behavior Effects 0.000 description 6
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 150000002772 monosaccharides Chemical class 0.000 description 5
- 230000000877 morphologic effect Effects 0.000 description 5
- 238000012346 open field test Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 229910021642 ultra pure water Inorganic materials 0.000 description 5
- 239000012498 ultrapure water Substances 0.000 description 5
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 229960001701 chloroform Drugs 0.000 description 4
- 230000007267 depressive like behavior Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000000105 evaporative light scattering detection Methods 0.000 description 4
- BCGWQEUPMDMJNV-UHFFFAOYSA-N imipramine Chemical compound C1CC2=CC=CC=C2N(CCCN(C)C)C2=CC=CC=C21 BCGWQEUPMDMJNV-UHFFFAOYSA-N 0.000 description 4
- 229960004801 imipramine Drugs 0.000 description 4
- 239000006286 aqueous extract Substances 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- -1 polysaccharide compound Chemical class 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 239000002390 adhesive tape Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 229940000406 drug candidate Drugs 0.000 description 2
- 239000003777 experimental drug Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012048 forced swim test Methods 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 210000003141 lower extremity Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000010445 mica Substances 0.000 description 2
- 229910052618 mica group Inorganic materials 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 235000020927 12-h fasting Nutrition 0.000 description 1
- 235000020926 24-h fasting Nutrition 0.000 description 1
- 241001075517 Abelmoschus Species 0.000 description 1
- 241000205585 Aquilegia canadensis Species 0.000 description 1
- 241001061264 Astragalus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 101100537302 Mus musculus Itfg1 gene Proteins 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 102000000874 Pyrin Domain-Containing 3 Protein NLR Family Human genes 0.000 description 1
- 108010001946 Pyrin Domain-Containing 3 Protein NLR Family Proteins 0.000 description 1
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000006533 astragalus Nutrition 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 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 description 1
- MAEIEVLCKWDQJH-UHFFFAOYSA-N bumetanide Chemical compound CCCCNC1=CC(C(O)=O)=CC(S(N)(=O)=O)=C1OC1=CC=CC=C1 MAEIEVLCKWDQJH-UHFFFAOYSA-N 0.000 description 1
- 229940088498 bumex Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007357 depressive behavior Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000004815 dispersion polymer Substances 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000020828 fasting Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 150000002771 monosaccharide derivatives Chemical class 0.000 description 1
- 238000000569 multi-angle light scattering Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000003901 neurotransmitter uptake inhibitor Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 230000004938 stress stimulation Effects 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 210000004233 talus Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- PNVNVHUZROJLTJ-UHFFFAOYSA-N venlafaxine Chemical compound C1=CC(OC)=CC=C1C(CN(C)C)C1(O)CCCCC1 PNVNVHUZROJLTJ-UHFFFAOYSA-N 0.000 description 1
- 229960004688 venlafaxine Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
一种醇溶性均一多糖及其抗抑郁用途,具体为铁皮石斛花醇溶性均一多糖及其制备方法和在制备抗抑郁药物中的用途。本发明是从天然植物水提物的分离组分进行多次纯化后得到一个醇溶性的均一多糖,命名为ASP(Alcohol‑solublepolysaccharide),经体内实验证实,所述的醇溶均一多糖具有显著的抗抑郁活性,且作用效果与抗抑郁上市药物氟西汀相当,可进一步开发制备抗抑郁药物。
Description
技术领域
本发明属于天然产物开发领域,涉及多糖,具体涉及一种铁皮石斛花中一种天然的醇溶 性均一多糖及其在抗抑郁方面的用途。
背景技术
抑郁症是常见的精神障碍之一,以显著而持久的心情低落为主要特征。世界卫生组织 (WHO)报告目前是世界各地的首要致残原因,也是导致全球疾病负担的一个重大因素。目前用 于治疗抑郁症的药物主要是神经递质再摄取抑制剂,如氟西汀、文拉法辛、丙咪嗪等,但这些 药物大多存在出汗、头晕、胃肠功能反应为主的副作用。因此,从天然植物中寻找副作用较小 的替代药物具有重要意义。
天然植物多糖因其毒性小,安全性高,在抑郁症治疗方面有着很大潜力。如黄芪多糖可 有效改善大鼠的抑郁样行为及海马损伤,秋葵多糖可以通过抗炎和调节肠道微生物群发挥抗抑 郁作用,金银花多糖通过抑制NLRP3炎症小体对抑郁症小鼠表现出较强的保护作用。通常天然 植物中获取多糖是通过回流提取获得水提物,再加入乙醇进行沉淀,弃去上清后收集沉淀,获 得粗多糖,随后借助柱层析方法等进行分离纯化。但这种提取方法仅关注沉淀中的水溶性多糖, 而上清液中的醇溶性多糖则被忽略。
发明内容
本发明的目的是提供天然产物中具有抗抑郁的活性成分,具体涉及一种醇溶性均一多糖 及其抗抑郁用途;尤其涉及铁皮石斛花醇溶性均一多糖及其制备方法和在制备抗抑郁药物中的 用途。
本发明是从天然植物水提物的分离组分进行多次纯化后得到一个醇溶性的均一多糖,命 名为ASP(Alcohol-soluble polysaccharide),经体内实验证实,所述的醇溶均一多糖具有显著的 抗抑郁活性,且作用效果与抗抑郁上市药物氟西汀相当,可进一步开发制备抗抑郁药物。
本发明中,所述醇溶性均一多糖来自兰科植物铁皮石斛(Dendrobium officinaleKimura et Migo)的干燥花。
本发明所述的ASP具有如下结构特征:
ASP的单糖组成为鼠李糖(Rha)、阿拉伯糖(Ara)、岩藻糖(Fuc)、甘露糖(Man)和葡萄糖 (Glc),其摩尔比为:3.88:4.69:4.84:4.92:10.75;重均分子量为3.099×104Da;糖含量为 85.33%;ASP的糖苷键主要由→3)Manp(1→、→2)Glcp(1→、→6)Manp(1→、Glcp(1→、→3)Glcp(1→、Manp(1→。
本发明所述的ASP通过下述方法制备:
步骤1)铁皮石斛(Dendrobium officinale Kimura et Migo)的干燥花,粉碎,料液比1:10, 微沸状态下回流提取三次,滤液浓缩至稠膏状,得铁皮石斛花水提物浸膏;
步骤2)将铁皮石斛花水提物浸膏以水复溶,加载至AB-8大孔树脂,以不同浓度(0%, 10%,v/v)乙醇洗脱,合并流分,减压浓缩干燥;
步骤3)将步骤2得到的干燥品以水充分溶解,边搅拌边缓慢加入4倍体积的无水乙醇 沉淀,4℃条件下静置24h,4500r/min,离心10min,收集上清液并浓缩干燥;
步骤4)将步骤3获得的干燥品加载至AB-8大孔树脂,以水洗脱,苯酚-硫酸法监测洗脱 液,合并含糖洗脱液,浓缩干燥,得铁皮石斛花醇溶性粗多糖;
步骤5)醇溶性粗多糖以水复溶后,加入Sevage试剂(氯仿∶正丁醇=5∶1,v/v),粗多糖 水溶液与Sevage试剂的体积比为4∶1,混合振荡10min,4500r/min离心10min,保留上层 清液,重复上述步骤3次,收集上层清液,浓缩,即得除蛋白的醇溶性粗多糖;
步骤6)将步骤5获得的除蛋白的醇溶性粗多糖以去离子水配制成50mg/mL的溶液,借 助Sephadex G-25葡聚糖凝胶进行初步分离纯化,以蒸馏水洗脱,苯酚-硫酸法实时监测,制作 洗脱曲线,洗脱曲线的两个吸收峰分别即ASSE-1、ASSE-2;根据洗脱曲线收集ASSE-2吸收峰 处的洗脱液,浓缩干燥后,再次加载至Sephadex G-25葡聚糖凝胶,以水洗脱,苯酚-硫酸法实 时监测,制作洗脱曲线,根据洗脱曲线收集吸收峰处的洗脱液,经透析袋流水透析48h后,冷 冻干燥,获得醇溶性均一多糖ASP。
经体内实验验证,ASP在行为绝望小鼠模型中显著降低正常小鼠在悬尾测试和强迫游泳 测试中的不动时间,表现出较好的急性抗抑郁作用;ASP在慢性不可预知刺激(CUMS)致抑郁大 鼠模型中可显著提升抑郁大鼠的蔗糖偏好指数、旷场测试中的直立次数与穿格次数,以及显著 降低强迫游泳中的不动时间,有效缓解了CUMS大鼠的抑郁样行为。
本发明所提供的ASP,可与药学上可接受的载体混合,用于制备有助于抗抑郁的保健品、 药物。
本发明的ASP可以作为活性部位与其他天然提取物与药学上可接受的赋形剂或辅料一 起用于制备药物组合物,包括但不限于片剂、颗粒剂、丸剂、胶囊、口服液等。
本发明的制备方法为条件温和、高效简便的柱层析方法,该方法普适性较强,有很高的 推广应用价值。
附图说明
图1为本发明中醇溶性粗多糖经Sephadex G-25葡聚糖凝胶洗脱曲线图;
图中:1:FASP-1,2:FASP-2;
图2为本发明中铁皮石斛花醇溶性粗多糖分离组分FASP-2经Sephadex G-25洗脱曲线 图;
图3为本发明中铁皮石斛花醇溶性均一多糖ASP的紫外可见光谱;
图4为本发明中铁皮石斛花醇溶性均一多糖ASP利用HPLC-ELSD检测图;
图5为本发明中铁皮石斛花醇溶性均一多糖ASP的GPC-RI-MALLS色图谱;
图6为本发明中铁皮石斛花醇溶性均一多糖ASP的红外色谱图;
图7为本发明中标准单糖衍生化产物的GC-MS色谱图;
图中:1:鼠李糖,2:阿拉伯糖,3:岩藻糖,4:木糖,5:甘露糖,6:葡萄糖,7: 半乳糖;
图8为本发明中铁皮石斛花醇溶性均一多糖ASP衍生化产物的GC-MS色谱图;
图中:1:鼠李糖,2:阿拉伯糖,3:岩藻糖,4:甘露糖,5:葡萄糖;
图9为本发明中铁皮石斛花醇溶性均一多糖ASP的甲基化图谱;
图中:1:→3)Manp(1→,2:→2)Glcp(1→,3:→6)Manp(1→,4:Glcp(1→,5:→3)Glcp(1→, 6:Manp(1→);
图10为本发明中铁皮石斛花醇溶性均一多糖ASP的核磁共振图;
图中:A:氢谱,B:碳谱;
图11为本发明中铁皮石斛花醇溶性均一多糖ASP的形态特征图;
图中:A~C:扫描电子显微镜(SEM)观察图(100×,500×,1000×);D:原子力显微镜(AFM) 观察图;
图12为本发明中铁皮石斛花醇溶性均一多糖ASP对行为绝望小鼠抑郁行为的影响;与 空白组比较,*P<0.05,**P<0.01,***P<0.001;
图13为本发明中铁皮石斛花醇溶性均一多糖ASP对慢性不可预知刺激致抑郁(CUMS) 大鼠的抑郁样行为的缓解作用;与空白组比较,#P<0.05,##P<0.01,###P<0.001;与模型组 比较,*P<0.05,**P<0.01,***P<0.001。
具体实施方式
实施例1
本实施例涉及一种天然植物醇溶性均一多糖(ASP)的分离制备,具体包括:取铁皮石斛 (Dendrobium officinale Kimura et Migo)的干燥花500g,粉碎,料液比1:10,温度设置为110℃, 加热回流提取三次,滤液浓缩至稠膏状得到水提物;将水提物以水复溶,加载至AB-8大孔树 脂,以不同浓度(0%,10%,v/v)乙醇洗脱,合并流分,减压浓缩干燥。随后以水充分溶解, 经4倍体积无水乙醇沉淀,离心,收集上清液并浓缩,再次加载至AB-8大孔树脂,以水洗脱, 用苯酚-硫酸法测定糖的含量,合并含糖洗脱液(与苯酚硫酸试剂反应后显色的洗脱液),浓缩干 燥,得铁皮石斛花醇溶性粗多糖。醇溶性粗多糖以水复溶后,加入Sevage试剂(氯仿∶正丁醇= 5∶1,v/v),粗多糖水溶液与Sevage试剂的体积比为4∶1,混合振荡10min,4500r/min离心 10min,保留上层清液,重复上述步骤3次,合并上层清液,浓缩,即得除蛋白的醇溶性粗多 糖;将除蛋白的粗多糖以去离子水配制成浓度为50mg/mL的溶液,借助Sephadex G-25葡聚 糖凝胶进行初步分离纯化,以蒸馏水洗脱,苯酚-硫酸法实时监测,制作洗脱曲线,根据洗脱曲 线收集吸收峰处的洗脱液,浓缩干燥,共得到两个次级组分,FASP-1与FASP-2(图1)。
用去离子水将FASP-2复溶,配制成50mg/mL的溶液,再次加载至Sephadex G-25葡聚糖凝胶,以水洗脱,苯酚-硫酸法实时监测,制作洗脱曲线,根据洗脱曲线收集吸收峰处的洗 脱液(图2),经透析袋(截留分子量:3500Da)流水透析48h后,冷冻干燥,获得醇溶性均一多 糖ASP(得率:6.06%)。
实施例2
本实施例涉及铁皮石斛花醇溶性均一多糖(ASP)的结构表征,包括:
步骤1)总糖含量测定:苯酚硫酸法测定ASP的总糖含量为85.33%。
步骤2)紫外光谱分析:通过分光光度计以扫描模式(200~500nm)测定ASP水溶液的 紫外吸收;ASP在UV-vis光谱中在260和280nm处没有吸收峰(图3),表明它不含蛋白质和核酸。
步骤3)均一性与分子量测定:ASP用超纯水配制为0.5mg/mL溶液,采用高效液相凝胶渗透色谱进行均一性检定,色谱柱为TSK-GEL G4000 SWXL凝胶柱,柱温设置为37℃,检测器为Agilent 1260型蒸发光散射(ELSD)检测器,漂移管温度设置为80℃,氮气气压为2.5Bar,Gain值为7,流动相为超纯水,流速0.5mL/min,进样量10μL。采用凝胶渗透色谱法(GPC)对ASP的分子量进行测定,色谱条件:色谱柱为Shodex OHpak SB-806HQ串联SB-804HQ, 检测器为多角度激光散射仪(MALLS)和示差检测器(RI),流动相为超纯水(含0.02%叠氮化钠), 流速为1mL/min,进样量为1mL,柱温保持在25℃。
铁皮石斛花醇溶性均一多糖ASP经HPLC-ELSD检测呈单一对称的峰型(图4),表明其为 均一多糖。ASP的分子量检测结果如图5所示,经计算ASP重均分子量(Mw)3.099×104Da,数 均分子量(Mn)2.481×104Da,Z均分子量(Mz)3.697×104Da,聚合物分散指数(PDI)为1.249。
步骤4)总糖含量测定:苯酚硫酸法测定结果显示,ASP中的总糖含量为85.3%。
步骤5)红外分析:取适量冻干后的ASP与KBr混合后研磨均匀,使用高通量的傅里叶 红外(FT-IR)光谱仪对ASP进行扫描,扫描范围4000~450cm-1,记录红外光谱图。
红外分析结果如图6所示,在3600~3200cm-1之间,3396cm-1为典型糖类的-OH伸缩振动;2361cm-1为多糖化合物中-CH2、-CH3、-CHOH中C-H单键的伸缩振动;1619cm-1为多糖化合物中C-C的伸缩振动;1409cm-1为多糖化合物中亚甲基的的弯曲振动,1107cm-1可能为C-O-C的伸缩振动;859cm-1提示可能存在α糖苷键。
步骤6)单糖组成分析:酸水解:取2mgASP溶解于1mL三氟乙酸(2mol/L),在100℃下反应6h,得到水解产物,氮气吹干后,反复加入甲醇4次置换除去残留的TFA。
衍生化:在酸水解后的ASP与单糖标准品(鼠李糖、甘露糖、木糖、半乳糖、葡萄糖、阿拉伯糖、岩藻糖)中加入5mg盐酸羟胺及1mL吡啶,封口,90℃条件下反应30min,随后 加入1mL乙酸酐,封口,90℃条件下反应30min,将反应溶剂用氮气吹干后,得到衍生化产 物。
气相质谱(GC-MS)检测:衍生化产物中加入2mL三氯甲烷,4000r/min离心10min。取上清液用于GC-MS分析。色谱条件:色谱柱为DB-5MS(30m×0.25mm×0.25μm)柱,进样 温度为250℃,分流比设置为100:1,流量为1mL/min,电离方式为EI+(70eV),检测器电压 为1333V,质量扫描范围为30~550amu。
单糖组成分析结果如图7、8所示,ASP是由鼠李糖(Rha)、阿拉伯糖(Ara)、岩藻糖(Fuc)、 甘露糖(Man)、葡萄糖(Glc)以摩尔比为0.39:0.47:0.56:4.92:10.75构成的醇溶性均一多糖。
步骤7)甲基化分析:称取40mg减压干燥的的ASP,加入4mL的无水二甲基亚砜,氮气封口,充分溶解。随后加入100mg研磨好的NaOH粉末,37℃超声2h。冰浴条件下缓慢 加入1mL碘甲烷(边加边振荡),超声反应0.5h,避光静置2h,加入1mL超纯水终止反应。 加入2mL二氯甲烷,离心取二氯甲烷层,氮气吹干。重复上述步骤2-3次。向干燥产物中加 入4mL的2mol/L三氟乙酸,100℃条件下反应3h,使用旋转蒸发仪蒸干后,加入2mL甲 醇,氮气吹干,重复三次。加入2mol/L的NaOH,100mg的硼氢化钠粉末,反应4h。滴加 醋酸值至pH呈酸性。加入甲醇2mL及1滴醋酸,减压浓缩至干,重复四次。加入醋酸酐及吡 啶各1mL,100℃反应1h,加入2mL甲醇减压浓缩至干,红外检测无羟基吸收峰。溶于2mL 三氯甲烷,GC-MS检测。
表1为ASP甲基化糖醇乙酰酯结果分析
通过甲基化分析,可以获得ASP的糖苷键类型,结果见表1及图9。GC-MS分析最终显示铁皮石斛花醇溶性均一多糖ASP由六种类型的糖残基组成,糖残基及其连接类型分别为
→3)Manp(1→、→2)Glcp(1→、→6)Manp(1→、Glcp(1→、→3)Glcp(1→、Manp(1→,其对应的摩 尔比为0.16:0.35:0.33:3.11:0.21:1.43。
步骤8)核磁共振分析:称取60mg的ASP,溶于重水,BrukerAMX-700M NMRspectrometer分析。
核磁共振结果如图10所示,通过BrukerAMX-700NMR对ASP的核磁信号进行归属。1H NMR谱显示信号范围为δ3.0-5.5ppm,13C NMR谱显示信号范围为δ50.0-110.0ppm。1H NMR谱中的化学位移小于δ5.00ppm通常为β连接残基的典型的异构体质子,而α异构体质子会出现在化学位移大于δ5.00ppm的区域。同时如13C NMR谱所示,ASP在δ90~112ppm区域 内,主峰为δ92.04,95.11和95.86ppm。进一步综合ASP的单糖组成与1H NMR谱的结果, 对光谱数据进行匹配,结果显示ASP的1H NMR在化学位移分别为δ5.32、5.14和4.55ppm, 显示存在三个异头氢信号,分别对应于13C NMR中的δ92.04、92.11和95.86ppm,表明葡萄 糖以三种不同的方式存在,即α-D-Glcp(1→、α-D-Glcp、β-D-Glcp。1H NMR中δ5.14ppm信号, 13C NMR中δ99.52、103.22ppm处信号提示可能有α-D-Manp存在。
步骤9)形态特征分析:通过扫描电子显微镜(SEM)评估ASP的形态特征。用金粉溅射干 燥的ASP粉末,并在真空条件下以100、500和1000倍的放大倍数进行观察。将ASP以超纯水溶解,配制成1μg/mL的溶液,超声2h,然后通过0.22μm过滤器过滤。取10μL ASP 溶液滴在干净的云母表面上,并在钠灯下干燥,干燥完成后氮气轻吹载有样品的云母片,然后 置于原子力显微镜(AFM)下,使用轻敲模式获得ASP的图像。
形态特征观察结果如图11所示,SEM可以获得ASP的表面特征和分子形态特征,结果 如图11A~11C所示,ASP具有光滑的表面和不规则的薄碎片形状,放大后无更清晰细节,表 明多糖分子间交联紧密,同时也提示了多糖具有较为复杂的分支结构。AFM可以提供ASP分子 在自然环境下的二维及三维图像。如图11D所示,AFM观察显示ASP为不规则的颗粒,多糖 链的厚度从0.3到2nm不等,表明分子内和分子间的范德华力和氢键使多糖分子相互连结,形 成各种支链结构和分子聚集体结构。
实施例3
本实施例按上述制备得到的铁皮石斛花醇溶性均一多糖,以行为绝望小鼠模型,观察其 急性抗抑郁作用,具体如下:
实验动物:雄性ICR小鼠,SPF级,18~25g。
实验药物:空白组(Control)给予10mL/kg.bw的生理盐水;阳性药组(Imipramine)给予15mg/kg.bw的丙咪嗪;ASP低剂量组(ASP(L))给予94mg/kg.bw的ASP;ASP高剂量组(ASP(H)) 给予234mg/kg.bw的ASP。
实验方法:将小鼠随机分为4组,每组12只,适应性喂养1周后,每天灌胃给予相应药物,持续一周,第8、9、10天分别进行旷场测试(OFT)、悬尾测试(TST)、强迫游泳(FST)测试。
旷场测试:小鼠给药的第8天进行旷场实验,将小鼠独立放于旷场箱中央,实验人员远 离旷场装置,避免干扰,录像5min,统计小鼠5min内的行进总路程。每只小鼠测试完毕后 清理旷场箱,喷洒医用酒精,擦净。
悬尾测试:在小鼠给药的第9天进行悬尾实验测试,将医用胶布粘贴在距离小鼠尾尖约 1cm处,胶布另一端则粘在悬尾装置的横杆上,控制小鼠头部与装置底部距离20cm,并保 证小鼠悬挂时无着力点,每次录像可同时测试6只小鼠,小鼠之间用黑色隔板隔离,避免相 互影响,实验人员远离实验装置,避免干扰,录像6min,统计后4min内小鼠累积不动时间。 每6只小鼠测试后清理实验装置,并喷洒医用酒精,擦净。
强迫游泳测试:在小鼠给药的第10天进行强迫游泳实验,将小鼠置于水温为23~25℃ 的圆形树脂玻璃桶中,通过控制水量保证小鼠后肢无法着力于桶底部,实验人员远离实验装置, 避免干扰,录像6min,统计后4min内小鼠的累积不动时间。每次同时录像小鼠6只,每 6只结束时换水,再进行下一组测试。
统计学处理:试验数据以mean±sem表示,采用单因素方差分析进行组间差异比较,当 P值小于0.05时视为具有显著性差异。
试验结果如图12所示,各组小鼠旷场实验行进总路程均无显著性差异,表明所有试验 药物均无神经兴奋或抑制作用。与空白组相比,给予丙咪嗪(阳性药)后,小鼠的悬尾及游泳不 动时间分别降低了38.89%(P<0.01)和53.96%(P<0.001)。与空白组相比,经103mg/kg及 290mg/kg的ASP处理后小鼠强迫游泳不动时间分别降低了43.43%(P<0.001)、35.05(P< 0.01),悬尾不动时间则分别降低了11.63%(P=0.070)、4.17%(P=0.335),这表明ASP对在行 为绝望小鼠中表现出一定抗抑郁效果。
实施例4
本实施例按上述制备得到的铁皮石斛花醇溶性均一多糖,以慢性不可预知刺激(CUMS) 致抑郁大鼠模型,观察其抗抑郁作用,具体如下:
实验动物:雄性SD大鼠,SPF级,160~180g。
实验药物:空白组(Control)给予10mL/kg.bw的生理盐水;模型组(CUMS)给予10mL/kg.bw的生理盐水;CUMS+阳性药组(CUMS+FLX)给予10mg/kg.bw的盐酸氟西汀;
CUMS+ASP低剂量组(CUMS+ASP(L))给予65mg/kg.bw的ASP;CUMS+ASP高剂量组(CUMS+ASP(H))给予162mg/kg.bw的ASP。
实验方法:大鼠适应性喂养1周后,经72h蔗糖水消耗训练后,进行糖水偏好水平测试。根据糖水偏好指数均匀分组,每组9只。其中,空白组与其余各组大鼠隔离饲养,不接受任何造模刺激,而其余各组大鼠每天接受1~2种不可预知刺激,共刺激28天,每种刺激不连续出现,每天刺激前1h灌胃给药。刺激包括:通宵照明,白噪音1h、震荡30min、潮湿环 境24h、频闪照明过夜、禁食24h、禁水24h、夹尾1min、45°倾斜饲养24h、低温游泳5min、 束缚2h。在应激刺激期间,除了禁食禁水和蔗糖水平测试外,自由饮食饮水,28天刺激结束 后,依次进行蔗糖偏好测试、旷场测试、强迫游泳测试。
蔗糖偏好测试:首先对大鼠进行蔗糖溶液适应训练,在笼架上放置两瓶相同体积的1% 蔗糖水溶液,24h后更换为一瓶蔗糖水溶液和一瓶纯水,再经过24h后,全部更换为两瓶纯 水。禁食禁水12h后,在笼架上放置相同体积的一瓶蔗糖水溶液和一瓶纯水,测试蔗糖偏好基 线12h。并按照以下公式进行计算:糖水偏好指数(%)=蔗糖水溶液消耗体积/(蔗糖水溶液消耗 体积+纯水消耗体积)×100%。
旷场测试:将大鼠独立放于旷场箱中央,远离旷场装置,避免干扰,录像5min,统计大鼠5min内的穿格次数(Crossingnumber)与直立次数(Rear number)。每只大鼠测试完毕后 清理旷场箱,喷洒医用酒精,擦净。
强迫游泳测试:将大鼠放置于盛有23~25℃水的圆形树脂玻璃桶(直径20cm,高度50cm)中,使大鼠后肢无法着地被迫游泳,录像5min,记录5min内大鼠累积不动时间,其 不动标准为除为避免没入水中的主动运动外,无其他行为。
统计学处理:试验数据以mean±sem表示,采用单因素方差分析进行组间差异比较,当 P值小于0.05时视为具有显著性差异。
试验结果如图13所示,与空白组相比,CUMS组大鼠蔗糖偏好指数、旷场穿格次数及直立次数均分别降低31.62%(P<0.001)、33.76%(P<0.01)、44.47%(P<0.001),且游泳不动时间增加34.16%(P<0.05),说明CUMS大鼠造模成功。与CUMS组相比,给予阳性药氟西 汀后,CUMS大鼠蔗糖偏好指数、旷场穿格次数及直立次数均分别提升27.18%(P<0.001)、45.62%(P<0.05)、57.63%(P<0.05),且游泳不动时间减少34.74%(P<0.001),表明氟西汀可有效缓解CUMS大鼠的抑郁样行为。同样地,与CUMS组相比,给予65mg/kg及162mg/kg 的ASP后,CUMS大鼠蔗糖偏好指数分别增加了49.72%(P<0.001)和40.55%(P<0.001); 旷场穿格次数则分别升高了63.96%(P<0.001)和41.24%(P<0.05),直立次数也分别提升了61.02%(P<0.05)和52.54%(P<0.05);游泳不动时间分别降低了32.07%(P<0.01)和23.56%(P<0.05),表明ASP可有效缓解CUMS大鼠的抑郁样行为。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式 对其进行局部调整,本发明的保护范围以权利要求书为准且不由上述具体实施所限,在其范围 内的各个实现方案均受本发明之约束。
Claims (6)
1.一种醇溶性的均一多糖ASP,其特征在于,其组成为鼠李糖(Rha)、阿拉伯糖(Ara)、岩藻糖(Fuc)、甘露糖(Man)和葡萄糖(Glc),其摩尔比为:3.88:4.69:4.84:4.92:10.75;重均分子量为3.099×104 Da;糖含量为85.33%;ASP的糖苷键由→3)Manp(1→、→2)Glcp(1→、→6)Manp(1→、Glcp(1→、→3)Glcp(1→、Manp(1→组成。
2.根据权利要求1所述的醇溶性的均一多糖ASP,其特征是,来自兰科植物铁皮石斛的干燥花。
3.一种制备如权利要求1或2所述醇溶性的均一多糖ASP的方法,其特征在于,包括:
步骤1)铁皮石斛的干燥花,粉碎,料液比1:10,微沸状态下回流提取三次,滤液浓缩至稠膏状,得铁皮石斛花水提物浸膏;
步骤2)将铁皮石斛花水提物浸膏以水复溶,加载至AB-8大孔树脂,以浓度为0%v/v、10%v/v乙醇洗脱,合并流分,减压浓缩干燥;
步骤3)将步骤2得到的干燥品以水充分溶解,边搅拌边缓慢加入4倍体积的无水乙醇沉淀,4 ℃条件下静置24 h,4500 r/min,离心10 min,收集上清液并浓缩干燥;
步骤4)将步骤3获得的干燥品加载至AB-8大孔树脂,以水洗脱,苯酚-硫酸法监测洗脱液,合并含糖洗脱液,浓缩干燥,得铁皮石斛花醇溶性粗多糖;
步骤5)醇溶性粗多糖以水复溶后,加入Sevage试剂,粗多糖水溶液与Sevage试剂的体积比为4∶1,混合振荡10 min,4500 r/min离心10 min,保留上层清液,重复上述步骤3次,收集上层清液,浓缩,即得除蛋白的醇溶性粗多糖;
所述的Sevage试剂为:氯仿∶正丁醇=5∶1,v/v;
步骤6)将步骤5获得的除蛋白的醇溶性粗多糖以去离子水配制成50 mg/mL的溶液,借助Sephadex G-25葡聚糖凝胶进行初步分离纯化,以蒸馏水洗脱,苯酚-硫酸法实时监测,制作洗脱曲线,洗脱曲线的两个吸收峰分别即ASSE-1、ASSE-2;根据洗脱曲线收集ASSE-2吸收峰处的洗脱液,浓缩干燥后,再次加载至Sephadex G-25葡聚糖凝胶,以水洗脱,苯酚-硫酸法实时监测,制作洗脱曲线,根据洗脱曲线收集吸收峰处的洗脱液,经透析袋流水透析48 h后,冷冻干燥,获得醇溶性均一多糖ASP。
4.一种根据权利要求1或2所述醇溶性的均一多糖ASP或权利要求3所述方法制备得到的醇溶性的均一多糖ASP的应用,其特征在于,将其用于制备抗抑郁药物。
5.根据权利要求4所述的应用,其特征是,具体为:与药学上可接受的载体混合,用于制备有助于抗抑郁的保健品、药物组合物。
6.根据权利要求5所述的应用,其特征是,所述的药物组合物,包括片剂、颗粒剂、丸剂、胶囊、口服液。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111215191.XA CN113845603B (zh) | 2021-10-19 | 2021-10-19 | 醇溶性均一多糖及其抗抑郁用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111215191.XA CN113845603B (zh) | 2021-10-19 | 2021-10-19 | 醇溶性均一多糖及其抗抑郁用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113845603A CN113845603A (zh) | 2021-12-28 |
CN113845603B true CN113845603B (zh) | 2022-08-09 |
Family
ID=78978852
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111215191.XA Active CN113845603B (zh) | 2021-10-19 | 2021-10-19 | 醇溶性均一多糖及其抗抑郁用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113845603B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115073620B (zh) * | 2022-05-10 | 2023-03-28 | 延边大学 | 两种具有抗补体活性的均一射干多糖及其制备方法和应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111978424A (zh) * | 2020-08-27 | 2020-11-24 | 上海应用技术大学 | 一种一步纯化铁皮石斛水溶性多糖的方法 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3081320A (en) * | 1960-11-08 | 1963-03-12 | Baxter Laboratories Inc | Method of purifying phosphatides |
CN104740337B (zh) * | 2014-12-31 | 2017-10-17 | 浙江济公缘药业有限公司 | 一种铁皮石斛提取物及其制备方法和应用 |
KR101739518B1 (ko) * | 2015-08-20 | 2017-05-26 | 비알 컨설팅 인코포레이티드 | 황칠나무의 추출물을 포함하는 조성물 |
CN110559394A (zh) * | 2018-06-06 | 2019-12-13 | 捷通国际有限公司 | 包含金钗石斛的组合物及其使用方法 |
CN109363075A (zh) * | 2018-10-11 | 2019-02-22 | 佛山科学技术学院 | 一种石斛花仙草多糖面条及其制备方法 |
CN111777692B (zh) * | 2020-08-17 | 2022-02-08 | 山东聚胜生物科技有限公司 | 一种茅莓分级多糖、制备方法和在抗抑郁中的应用 |
AU2020103186A4 (en) * | 2020-11-02 | 2021-01-14 | Sichuan Agricultural University | Preparation Method and Application of Dendrobium Nobile Polysaccharide Modified by Low-temperature Plasma |
CN112778435A (zh) * | 2021-03-18 | 2021-05-11 | 广东工业大学 | 一种双水相萃取分离纯化铁皮石斛多糖的方法 |
-
2021
- 2021-10-19 CN CN202111215191.XA patent/CN113845603B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111978424A (zh) * | 2020-08-27 | 2020-11-24 | 上海应用技术大学 | 一种一步纯化铁皮石斛水溶性多糖的方法 |
Also Published As
Publication number | Publication date |
---|---|
CN113845603A (zh) | 2021-12-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Structural characterization of a novel polysaccharide from Lepidium meyenii (Maca) and analysis of its regulatory function in macrophage polarization in vitro | |
Zhang et al. | A fructan from Anemarrhena asphodeloides Bunge showing neuroprotective and immunoregulatory effects | |
Yin et al. | Separation, structure characterization, conformation and immunomodulating effect of a hyperbranched heteroglycan from Radix Astragali | |
EP2989156B1 (en) | Phytoglycogen nanoparticles and methods of manufacture thereof | |
Ma et al. | Structural characterization and antiviral effect of a novel polysaccharide PSP-2B from Prunellae Spica | |
Khawas et al. | In vivo cough suppressive activity of pectic polysaccharide with arabinogalactan type II side chains of Piper nigrum fruits and its synergistic effect with piperine | |
Deng et al. | Structural characterization and hypolipidemic activities of purified stigma maydis polysaccharides | |
US11547719B2 (en) | Acetylation of aloe polysaccharides | |
WO2021143595A1 (zh) | 一种低分子量金耳葡糖醛酸-木甘聚糖及其制备方法和应用 | |
CN113845603B (zh) | 醇溶性均一多糖及其抗抑郁用途 | |
CN114163545B (zh) | 一种枸杞多糖及其在降血糖中的应用 | |
Chen et al. | Structural characterization and biological activities of a novel polysaccharide containing N-acetylglucosamine from Ganoderma sinense | |
Wang et al. | Structural elucidation of a novel polysaccharide from Ophiopogonis Radix and its self-assembly mechanism in aqueous solution | |
Ma et al. | Isolation of an acidic polysaccharide from the flowers of Leucosceptrum canum Smith and its immunomodulatory activity evaluation | |
CN114591448A (zh) | 一种桑树桑黄子实体甘露半乳聚糖及其制备和用途 | |
JPH0539305A (ja) | アストラガルス・メンブラナセウスから抽出した免疫 変調性多糖類及びこれらを含有する薬剤組成物 | |
WO2023036203A1 (zh) | Cs-4发酵菌丝体杂聚多糖及其制备方法与用途 | |
Zhang et al. | A zinc-modified Anemarrhena asphodeloides polysaccharide complex enhances immune activity via the NF-κB and MAPK signaling pathways | |
CN111040042B (zh) | 一种枸杞多糖、制备方法及其应用 | |
Wang et al. | Structural elucidation and immunomodulatory activities in vitro of type I and II arabinogalactans from different origins of Astragalus membranaceus | |
CN115028752B (zh) | 一种均一的水溶性多糖及其制备方法和应用 | |
CN112755104A (zh) | 一种抗癫痫药物组合物及其制备方法和应用 | |
CN112076175A (zh) | 一种蛹虫草多糖泡腾片、制备方法及应用 | |
JPWO2003048212A1 (ja) | グリコーゲンを物理化学的に製造する方法およびこの方法で得られるグリコーゲン | |
CN101838337B (zh) | 一种由25个果糖和1个葡萄糖构成的果聚糖 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |