CN112876577A - Homogeneous rhizoma anemarrhenae polysaccharide and preparation method and application thereof - Google Patents
Homogeneous rhizoma anemarrhenae polysaccharide and preparation method and application thereof Download PDFInfo
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- CN112876577A CN112876577A CN202110290637.9A CN202110290637A CN112876577A CN 112876577 A CN112876577 A CN 112876577A CN 202110290637 A CN202110290637 A CN 202110290637A CN 112876577 A CN112876577 A CN 112876577A
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- Prior art keywords
- polysaccharide
- rhizoma anemarrhenae
- ethanol
- homogeneous
- aabp
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- 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.)
- Granted
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 96
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 96
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 121
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000000843 powder Substances 0.000 claims abstract description 10
- 229920002271 DEAE-Sepharose Polymers 0.000 claims abstract description 9
- 230000003078 antioxidant effect Effects 0.000 claims abstract 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 abstract description 8
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 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 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 6
- 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 6
- 238000004440 column chromatography Methods 0.000 claims abstract description 6
- 238000005238 degreasing Methods 0.000 claims abstract description 5
- 238000001556 precipitation Methods 0.000 claims abstract description 5
- 230000002218 hypoglycaemic effect Effects 0.000 claims abstract description 4
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- 239000002244 precipitate Substances 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 238000010828 elution Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 241000605445 Anemarrhena asphodeloides Species 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 241000605447 Anemarrhena Species 0.000 claims description 9
- 229930191283 anemarrhena Natural products 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 229920000869 Homopolysaccharide Polymers 0.000 claims description 7
- 241000612118 Samolus valerandi Species 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 5
- 238000000643 oven drying Methods 0.000 claims description 5
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- 239000001913 cellulose Substances 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 230000001376 precipitating effect Effects 0.000 abstract 1
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- 230000000694 effects Effects 0.000 description 12
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 229910052799 carbon Inorganic materials 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- 230000011987 methylation Effects 0.000 description 7
- 238000007069 methylation reaction Methods 0.000 description 7
- 150000002772 monosaccharides Chemical class 0.000 description 7
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 6
- 239000001257 hydrogen Substances 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical group [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 5
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000005100 correlation spectroscopy Methods 0.000 description 4
- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 4
- QELUYTUMUWHWMC-UHFFFAOYSA-N edaravone Chemical compound O=C1CC(C)=NN1C1=CC=CC=C1 QELUYTUMUWHWMC-UHFFFAOYSA-N 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000001603 reducing effect Effects 0.000 description 4
- -1 ABTS free radical Chemical class 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 238000004611 spectroscopical analysis Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 description 1
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 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 description 1
- 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 description 1
- 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 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960002632 acarbose Drugs 0.000 description 1
- XUFXOAAUWZOOIT-UHFFFAOYSA-N acarviostatin I01 Natural products OC1C(O)C(NC2C(C(O)C(O)C(CO)=C2)O)C(C)OC1OC(C(C1O)O)C(CO)OC1OC1C(CO)OC(O)C(O)C1O XUFXOAAUWZOOIT-UHFFFAOYSA-N 0.000 description 1
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 229940079877 pyrogallol Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
Images
Classifications
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- 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
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- 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
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
- C08B37/009—Konjac gum or konjac mannan, i.e. beta-D-glucose and beta-D-mannose units linked by 1,4 bonds, e.g. from Amorphophallus species; Derivatives thereof
-
- 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
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Materials Engineering (AREA)
- Diabetes (AREA)
- Emergency Medicine (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Food Science & Technology (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Sustainable Development (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a homogeneous rhizoma anemarrhenae polysaccharide, and a preparation method and application thereof. Firstly, crushing rhizome of rhizoma anemarrhenae, and leaching and degreasing the rhizome of rhizoma anemarrhenae by ethanol; extracting the degreased rhizoma anemarrhenae powder with water, precipitating with ethanol, removing protein by a Sevag method, dialyzing, and freeze-drying to obtain rhizoma anemarrhenae crude polysaccharide; redissolving the crude polysaccharide of rhizoma anemarrhenae, preparing rhizoma anemarrhenae polysaccharide with different molecular weight components by adopting gradient alcohol precipitation, and further purifying by adopting cellulose DEAE-Sepharose Fast Flow column chromatography to obtain the homogeneous polysaccharide AABP-2B of rhizoma anemarrhenae, which consists of mannose and glucose. The homogeneous rhizoma anemarrhenae polysaccharide has good antioxidant and hypoglycemic activities, and is expected to be developed into hypoglycemic drugs or health care products. In addition, the process for purifying the homogeneous rhizoma anemarrhenae polysaccharide is simple and convenient, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of separation and purification of plant active ingredients, in particular to a homogeneous rhizoma anemarrhenae polysaccharide and a preparation method and application thereof.
Background
Anemarrhena asphodeloides Bunge (Anemarrhena asphodeloides Bunge) belongs to Liliaceae perennial upright herbaceous plant, and is mainly distributed in China, Japan and other east Asia countries. Rhizoma anemarrhenae, as a traditional Chinese medicinal material, is originally recorded in Shen nong Ben Cao Jing, has the effects of nourishing yin to reduce pathogenic fire, moistening dryness and lubricating intestines and the like, and is widely used for treating diseases such as fever, cough, diabetes and the like. In addition, the pharmacological activity of the compound has been described in detail in classic traditional Chinese medicine books such as Chinese pharmacopoeia, Chinese dictionary, and Chinese materia medica. Meanwhile, the rhizoma anemarrhenae is approved as a Chinese medicinal material of 'health food' by the Wei Ji Wei of China. In recent years, with the intensive research on rhizoma anemarrhenae by numerous scholars at home and abroad, the medicinal value of rhizoma anemarrhenae is gradually developed and utilized, and the rhizoma anemarrhenae is found to have the effects of improving Alzheimer's disease, reducing blood sugar, resisting oxidation, resisting tumors and the like. The polysaccharide is one of the main active ingredients of rhizoma anemarrhenae, and studies have shown that the rhizoma anemarrhenae crude polysaccharide or the rhizoma anemarrhenae water extract has a certain effect of reducing blood sugar, but the structure of the rhizoma anemarrhenae homopolysaccharide and the studies on the research on the treatment of diabetes mellitus are rarely reported. Therefore, it is necessary to further explore the method for separating and purifying timosaccharide and the structural identification and study the hypoglycemic effect thereof, so as to provide a foundation for the development and utilization of timosaccharide.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the present invention provides a homogeneous anemarrhenae polysaccharide.
The present invention also provides a method for preparing the homogeneous polysaccharide of anemarrhena asphodeloides bunge.
The present invention further provides the use of the homogeneous anemarrhena polysaccharide.
The purpose of the invention is realized by the following scheme:
a rhizoma anemarrhenae homogeneous polysaccharide has molecular weight of 4000-.
Preferably, the molecular weight of the rhizoma anemarrhenae homopolysaccharide is 5800Da, and the molecular weight is determined by the molar ratio of 0.72: 0.28 mannose and glucose.
The rhizoma anemarrhenae homopolysaccharide has the following structural formula:
the preparation method of the rhizoma anemarrhenae homopolysaccharide comprises the following steps:
(1) pulverizing rhizoma anemarrhenae, sieving with 100 mesh sieve, reflux-defatting with ethanol, decolorizing, and oven drying; extracting rhizoma anemarrhenae polysaccharide by hot water extraction, concentrating the water extract, adding ethanol to precipitate the polysaccharide, centrifuging to collect rhizoma anemarrhenae polysaccharide precipitate, removing protein by sevag method, dialyzing, and lyophilizing to obtain rhizoma anemarrhenae crude polysaccharide;
(2) re-dissolving the rhizoma anemarrhenae crude polysaccharide in water, adding ethanol until the ethanol concentration is 60%, standing, performing centrifugal separation, collecting supernatant and precipitate, adding ethanol into the supernatant until the ethanol concentration is 80%, standing, and performing centrifugal separation to obtain precipitate, i.e. rhizoma anemarrhenae polysaccharide precipitated when the ethanol final concentration is 80%;
(3) purifying rhizoma anemarrhenae polysaccharide precipitated by ethanol with the ethanol concentration of 80% by DEAE-Sepharose Fast Flow column chromatography, performing gradient elution by sequentially adopting pure water, 0.1M NaCl, 0.2M NaCl and 0.5M NaCl solution, and collecting 0.1M NaCl elution components, thereby obtaining the rhizoma anemarrhenae homogeneous polysaccharide.
The ethanol reflux degreasing and decoloring in the step (1) refers to refluxing, degreasing and decoloring rhizoma anemarrhenae dry powder by using ethanol with the mass fraction of 70-95%, and drying in a 35-45 ℃ blast drying oven after refluxing;
the hot water leaching method in the step (1) is to leach for 2-4 times in hot water at 70-90 ℃ for 1-3 hours each time;
preferably, the water extract in the step (1) is concentrated to 1/3-1/6 of the original volume; preferably, the ethanol is added to precipitate the polysaccharide in the step (1), the ethanol is added with 3-5 times of volume of absolute ethanol, the mixture is kept stand for 12-48 hours at the temperature of 4 ℃, and the precipitate is collected after centrifugal separation;
the dialysis in the step (1) is carried out by using a dialysis bag with the molecular weight cutoff of 3500 Da;
the standing in the step (2) is preferably carried out for 12-48h at 4 ℃.
Preferably, the preparation method of the rhizoma anemarrhenae homopolysaccharide specifically comprises the following steps:
1) pulverizing rhizoma anemarrhenae, sieving with 100 mesh sieve, reflux-extracting the sieved rhizoma anemarrhenae powder with 8 times of 95% ethanol for 3 times, centrifuging, collecting precipitate, and oven drying at 40 deg.C to obtain defatted and decolorized rhizoma anemarrhenae dry powder;
2) adding 20-30 times of distilled water into the rhizoma anemarrhenae dry powder prepared in the step 1), extracting for three times at the temperature of 60-90 ℃ for 1-3 h each time, combining water extract, carrying out centrifugal separation, collecting supernatant, and concentrating under reduced pressure to one fifth of the original volume; adding 5 times of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 24 hr, centrifuging, and collecting rhizoma anemarrhenae polysaccharide precipitate;
3) removing protein from the rhizoma anemarrhenae polysaccharide prepared in the step 2) by adopting a sevag method, dialyzing, and freeze-drying to obtain crude protein-removed rhizoma anemarrhenae polysaccharide AABP;
4) redissolving the rhizoma anemarrhenae crude polysaccharide AABP prepared in the step 3) in a certain amount of water to prepare a polysaccharide solution with the concentration of 10-20 mL/mg, adding absolute ethyl alcohol until the concentration of the ethyl alcohol is 60%, standing, performing centrifugal separation, collecting precipitate and supernate, continuously adding absolute ethyl alcohol into the supernate until the concentration of the ethyl alcohol is 80%, standing, performing centrifugal separation, collecting precipitate to obtain rhizoma anemarrhenae polysaccharide AABP-2 (80% alcohol precipitation);
5) and (3) further purifying the AABP-2 prepared in the step 4) by using a DEAE-Sepharose Fast Flow ion exchange column, performing gradient elution by using pure water, 0.1M NaCl, 0.2M NaCl and 0.5M NaCl solution in sequence, collecting 0.1M NaCl elution components, concentrating, dialyzing, and freeze-drying to obtain the homogeneous rhizoma anemarrhenae polysaccharide AABP-2B.
The invention adopts a separation and purification scheme of gradient alcohol precipitation and cellulose DEAE-Sepharose Fast Flow column chromatography to prepare the rhizoma anemarrhenae homogeneous polysaccharide AABP-2B rapidly, the molecular weight of the rhizoma anemarrhenae homogeneous polysaccharide AABP-2B is 5800Da, and the rhizoma anemarrhenae homogeneous polysaccharide AABP-2B consists of mannose and glucose.
The rhizoma anemarrhenae homopolysaccharide has good antioxidant and blood sugar reducing effects, can be used as a natural antioxidant and applied to blood sugar reducing health-care food or medicine.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the process for purifying the homogeneous polysaccharide of the rhizoma anemarrhenae is simple and convenient, and is suitable for industrial production. Meanwhile, the homogeneous rhizoma anemarrhenae polysaccharide has good antioxidant and hypoglycemic activity, and is expected to be developed into hypoglycemic drugs or health care products.
Drawings
FIG. 1 is a graph showing the molecular weight distribution of Anemarrhena homogeneous polysaccharide AABP-2B prepared in example 1.
FIG. 2 is a diagram of monosaccharide composition of timosaccharide AABP-2B prepared in example 1; wherein, A is a peak appearance diagram of standard monosaccharide, and B is a monosaccharide peak appearance diagram of AABP-2B; 1 (mannose), 2 (ribose), 3 (rhamnose), 4 (galacturonic acid), 5 (glucuronic acid), 6 (glucose), 7 (galactose), 8 (arabinose), 9 (fucose).
FIG. 3 is the nuclear magnetic hydrogen spectrum of timosaccharide AABP-2B prepared in example 1.
FIG. 4 is the nuclear magnetic carbon spectrum of the timosaccharide AABP-2B prepared in example 1.
FIG. 5 is the nuclear magnetic COSY spectrum of the anemarrhena polysaccharide AABP-2B prepared in example 1.
FIG. 6 shows the nuclear magnetic HSQC spectrum of timosaccharide AABP-2B prepared in example 1.
FIG. 7 is the nuclear magnetic HMBC spectrum of the timosaccharide AABP-2B prepared in example 1.
FIG. 8 is a predicted structural diagram of the polysaccharide AABP-2B prepared in example 1.
FIG. 9 is a graph showing the antioxidant activity of polysaccharide AABP-2B prepared in example 2; a is an activity diagram for eliminating DPPH free radicals; b is an ABTS free radical scavenging activity diagram; c is the activity diagram for eliminating superoxide radical.
FIG. 10 is a graph showing that the polysaccharide AABP-2B prepared in example 3 inhibits the activity of α -glucosidase.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The reagents used in the examples are commercially available without specific reference.
Example 1: preparation of homogeneous polysaccharide AABP-2B of anemarrhena polysaccharide
(1) Extracting rhizoma anemarrhenae polysaccharide:
pulverizing 1Kg rhizoma anemarrhenae by a pulverizer, sieving with 100 mesh sieve, adding 5L 95% ethanol, refluxing at 70 deg.C for 2h, repeating for 2 times, centrifuging, and oven drying the precipitate at 45 deg.C. Adding 5L of distilled water into degreased and decolored rhizoma anemarrhenae dry powder, leaching for 3h in a water bath at 85 ℃, repeatedly extracting for 3 times, filtering, centrifuging, collecting supernatant, and concentrating the supernatant under reduced pressure to one fifth of the original volume; slowly adding 4 times of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 24 hr, centrifuging, collecting precipitate, and drying to obtain rhizoma anemarrhenae crude polysaccharide 64.5 g.
(2) Protein removal:
preparing 30g of rhizoma anemarrhenae crude polysaccharide into a polysaccharide solution of 20mg/mL by using distilled water, adding a sevag reagent to remove protein, repeating the operation for 8 times, dialyzing (3500Da), and freeze-drying to obtain the protein-removed rhizoma anemarrhenae crude polysaccharide AABP.
(3) Gradient alcohol precipitation:
dissolving anemarrhena polysaccharide AABP in distilled water again to prepare a polysaccharide solution of 10mg/mL, and slowly adding absolute ethanol into the AABP polysaccharide solution under vigorous stirring until the final concentration of the ethanol reaches 60%. The solution was then left at 4 ℃ for 24 h. Centrifuging (5000rpm, 20min), collecting precipitate and supernatant, adding anhydrous ethanol into the supernatant until the final ethanol content is 80%, standing, centrifuging, and collecting rhizoma anemarrhenae polysaccharide precipitate (AABP-2).
(4) DEAE-Sepharose Fast Flow column chromatography:
to prepare the homogeneous polysaccharides of Anemarrhena asphodeloides, AABP-2 was further purified by DEAE-Sepharose Fast Flow column chromatography. 300mg of timosaccharide AABP-2 was weighed, dissolved in 15mL of distilled water, centrifuged (10000rpm, 10min), and the supernatant was slowly added to a DEAE-Sepharose Fast Flow column (2.6X 100cm) along the wall, and elution was carried out using distilled water, 0.1, 0.2 and 0.5mol/L aqueous sodium chloride (NaCl) in this order at a Flow rate of 1.0 mL/min. The fractions of the eluate (10 mL/tube) were collected, and the absorbance at 490nm of the eluate from each tube was measured by the phenol-sulfuric acid method, and an elution curve was drawn based on the absorbance. And combining samples showing the same elution peak, concentrating, dialyzing to remove salt, and freeze-drying to obtain 0.1mol/L NaCl elution component polysaccharide, and naming the polysaccharide as AABP-2B.
(5) And (3) measuring the molecular weight:
the molecular weight of AABP-2B is 5800Da as determined by gel exclusion chromatography, and a specific molecular weight distribution diagram is shown in FIG. 1.
(6) Analysis of monosaccharide composition:
the monosaccharide composition of AABP-2B is determined by a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method. Taking 10mg of anemarrhena polysaccharide AABP-2B, hydrolyzing in an oil bath at 110 ℃ for 4h by using 4mol/L trifluoroacetic acid, decompressing and evaporating to dryness, deriving before a PMP column, extracting with dichloromethane for three times to remove redundant PMP, taking a water phase, passing the water phase through a 0.22 mu m filter membrane, and analyzing by using a high performance liquid phase. The results are shown in FIG. 2, wherein A is the peak appearance of standard monosaccharide, and B is the peak appearance of AABP-2B monosaccharide; from fig. 2, it can be seen that the timosaccharide AABP-2B is composed of mannose and glucose, and the molar ratio thereof is 0.72: 0.28.
(7) methylation analysis:
5.0mg of dry anemarrhenae polysaccharide AABP-2B was mixed with 1.5mL of anhydrous DMSO. And (3) carrying out ultrasonic treatment on the mixture until a sample is completely dissolved, then quickly adding 100mg of sodium hydroxide particles under the protection of nitrogen, slowly dropwise adding 2mL of methyl iodide into the reaction solution after reacting overnight, adding 1mL of distilled water after reacting for 2h to stop the reaction, dialyzing the mixture for 48h by using the distilled water, and freeze-drying. The methylation process was repeated until the hydroxyl (-OH) absorption peak (3200--1) And disappears in the infrared spectrum. Then, willThe full methylation rhizoma anemarrhenae polysaccharide is hydrolyzed and acetylated, and then the gas chromatography and mass spectrometry are carried out. The methylation results of the timosaccharide AABP-2B are shown in Table 1. The methylation result shows that AABP-2B contains seven glycosidic bonds which are respectively 2,4-Me2-Manp、2,3-Me2-Glcp、2,3,4,6-Me4-Glcp、2,3,4,6-Me4-Manp、2,4,6-Me3-Manp、3,4,6-Me3Manp and 2,3,6-Me3Glcp, corresponding to a molar ratio of 0.19:0.17:0.07:0.09:0.35:0.09:0.04, indicating that the 1 → 3 linked mannose is an AABP-2B structural backbone.
TABLE 1 methylation analysis of Anemarrhena asphodeloides polysaccharide AABP-2B
(8) Nuclear magnetic analysis:
collecting 60mg rhizoma anemarrhenae polysaccharide AABP-2B, and using D2Performing one-dimensional (hydrogen spectrum and carbon spectrum) and two-dimensional (COSY, HSQC and HMBC) nuclear magnetic tests after 2 times of O replacement, wherein FIG. 3 is a nuclear magnetic hydrogen spectrum diagram of anemarrhena polysaccharide AABP-2B prepared in example 1; FIG. 4 is the nuclear magnetic carbon spectrum of the timosaccharide AABP-2B prepared in example 1; FIG. 5 is a nuclear magnetic COSY spectrum of timosaccharide AABP-2B prepared in example 1; FIG. 6 is the nuclear magnetic HSQC spectrum of timosaccharide AABP-2B prepared in example 1; FIG. 7 is the nuclear magnetic HMBC spectrum of the timosaccharide AABP-2B prepared in example 1.
As shown in FIG. 3, the timosaccharide AABP-2B contains seven anomer hydrogens, and the chemical shifts delta are 4.56, 4.57, 4.91, 5.01, 5.10, 5.17 and 5.34ppm respectively. Furthermore, of AABP-2B13The C NMR spectrum (fig. 4) also contained seven anomeric carbon signal peaks in the anomeric carbon region, at δ 92.15, 99.93, 103.08, 103.18, 103.66, 103.80, and 103.97ppm, respectively; by two dimensions1H-13The C HSQC (FIG. 6) spectra show the position of the cross-peaks, classifying the cross-peaks of the anomeric hydrogen signal/anomeric carbon signal (H-1/C-1) as 4.56/103.08ppm, 4.57/103.18ppm, 4.91/99.93, 5.01/103.80ppm, 5.10/103.66ppm, 5.17/103.97ppm and 5.34/92.15ppm, and labeled as residues A, B, C, D, E, F and G, respectively.
TABLE 2 Anemarrhena asphodeloides polysaccharide AABP-2B1H and13c chemical shift Classification
In combination with nuclear magnetic hydrogen spectroscopy, carbon spectroscopy, COSY (fig. 5), HSQC, spectrogram and methylation analysis and literature reports, the carbon and hydrogen spectral chemical shift values for each glycosidic bond were assigned (table 2), with glycosidic bonds A, B, C, D, E, F and G being 3,6) - β -D-Manp- (1, 4,6) - β -D-Glcp- (1, 4) - α -D-Glcp- (1, T- α -D-Manp- (1, 3) - α -D-Manp- (1 and 2) - α -D-Manp- (1), and further the possible linkage structure of AABP-2B was deduced by HMBC spectroscopy (fig. 7) as shown in fig. 8.
Example 2: antioxidant activity of rhizoma anemarrhenae polysaccharide
The antioxidant activity of AABP-2B was evaluated by DPPH, ABTS and superoxide radical assays as follows:
and (3) detecting the activity of eliminating DPPH free radicals: mu.L of different AABP-2B polysaccharide solutions (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0mg/mL) and 100. mu.L of 0.1mM DPPH ethanol solution were added to a 96-well plate, mixed, reacted at room temperature in the dark for 30min, and the absorbance of the mixture was measured at 517nm with ascorbic acid (Vc) as a positive control, and the results are shown in graph A in FIG. 9.
Elimination of ABTS free radical activity: 2mL of ABTS (7mmol/L) aqueous solution and 2mL of potassium persulfate aqueous solution (2.45mmol/L) are mixed and reacted at room temperature in a dark place for 12 hours to obtain cation ABTS free radicals. The prepared cationic ABTS free radical solution is diluted by ethanol until the absorbance at 734nm is 0.7 +/-0.05. mu.L of an aqueous polysaccharide solution (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0mg/mL) was added to a 96-well plate, 180. mu.L of a diluted cationic ABTS free radical solution was added, the mixture was shaken, and the absorbance of the mixture was tested at 734nm with ascorbic acid (Vc) as a positive control, and the results are shown in Panel B of FIG. 9.
Superoxide radical removal: mu.L of polysaccharide aqueous solution (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0mg/mL) and 150. mu.L of Tris-HCl buffer solution (0.1mol/L) of pH 8.2 were added to a 96-well plate, mixed, incubated at room temperature in the dark for 10min, then 30. mu.L of pyrogallol aqueous solution (6mmol/L) was added, mixed, reacted in the dark for 3min, 30. mu.L of HCl (10mol/L) was added to terminate the reaction, and the absorbance of the mixture was measured at 325nm with ascorbic acid (Vc) as a positive control, and the results are shown in FIG. 9C.
The antioxidant results in fig. 9 show that the timosaccharide AABP-2B extracted by the invention has good capability of clearing DPPH, ABTS and superoxide radical.
Example 3: anemarrhena asphodeloides polysaccharide AABP-2B inhibition of alpha-glycosidase activity test
Mixing 20 μ L of rhizoma anemarrhenae polysaccharide aqueous solution (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0mg/mL) and 40 μ L of α -glucosidase aqueous solution (0.2U/mL) in 96-well plate, incubating at 37 deg.C for 10min, adding 20.0 μ L of pNPG aqueous solution (10mmol/L), mixing, reacting at 37 deg.C for 30min, and adding 100 μ L of Na2CO3The reaction was stopped with (0.2mol/L) solution, and the absorbance of the mixture was measured at 405nm, using acarbose as a positive control, and the results are shown in FIG. 10. The results show that the anemarrhena polysaccharide AABP-2B has alpha-glycosidase activity inhibition in the concentration range of 0.1-2.0mg/mL, and the inhibition activity is enhanced along with the increase of the concentration. At a concentration of 2.0mg/mL, the inhibition rate of AABP-2B was 63.54. + -. 1.88%. The rhizoma anemarrhenae polysaccharide has good activity of inhibiting the alpha-glycosidase.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be regarded as equivalent replacements, and all fall within the protection scope of the present invention.
Claims (8)
1. A homogeneous rhizoma anemarrhenae polysaccharide is characterized in that the molecular weight is 4000-8000Da, and the homogeneous rhizoma anemarrhenae polysaccharide consists of mannose and glucose.
2. The homogeneous rhizoma anemarrhenae polysaccharide of claim 1, having a molecular weight of 5800Da, and produced from a mixture of polysaccharides having a molar ratio of 0.72: 0.28 mannose and glucose.
3. The homopolysaccharide of anemarrhena according to claim 1 or 2, having the following structural formula:
4. a method for preparing the homogeneous polysaccharide of anemarrhena asphodeloides bunge according to any one of claims 1 to 3, comprising the following steps:
(1) pulverizing rhizoma anemarrhenae, sieving with 100 mesh sieve, reflux-defatting with ethanol, decolorizing, and oven drying; extracting rhizoma anemarrhenae polysaccharide by hot water extraction, concentrating the water extract, adding ethanol to precipitate the polysaccharide, centrifuging to collect rhizoma anemarrhenae polysaccharide precipitate, removing protein by sevag method, dialyzing, and lyophilizing to obtain rhizoma anemarrhenae crude polysaccharide;
(2) re-dissolving rhizoma anemarrhenae crude polysaccharide in water, gradually adding ethanol until the ethanol concentration is 60%, standing, centrifuging, collecting supernatant and precipitate, adding ethanol into the supernatant until the ethanol concentration is 80%, standing, centrifuging to obtain precipitate, which is rhizoma anemarrhenae polysaccharide precipitated when the ethanol concentration is 80%;
(3) purifying rhizoma anemarrhenae polysaccharide precipitated by ethanol with the ethanol concentration of 80% by DEAE-Sepharose Fast Flow column chromatography, performing gradient elution by sequentially adopting pure water, 0.1M NaCl, 0.2M NaCl and 0.5M NaCl solution, and collecting 0.1M NaCl elution components, thereby obtaining the rhizoma anemarrhenae homogeneous polysaccharide.
5. The method for preparing the homogeneous polysaccharide of anemarrhena asphodeloides bunge as claimed in claim 4, wherein:
the ethanol reflux degreasing and decoloring in the step (1) refers to refluxing, degreasing and decoloring rhizoma anemarrhenae dry powder by using ethanol with the mass fraction of 70-95%, and drying in a 35-45 ℃ blast drying oven after refluxing;
the hot water leaching method in the step (1) is to leach for 2-4 times in hot water at 70-90 ℃ for 1-3 hours each time;
concentrating the water extract obtained in the step (1) to 1/3-1/6 of the original volume; the step (1) of adding ethanol to precipitate the polysaccharide is to add 3-5 times of volume of absolute ethanol, stand at 4 ℃ for 12-48 hours, centrifugally separate, and collect precipitates.
6. The method for preparing the homogeneous polysaccharide of anemarrhena asphodeloides bunge as claimed in claim 4, wherein:
the standing in the step (2) is carried out for 12-48h at 4 ℃.
7. The method for preparing the homogeneous polysaccharide of anemarrhena asphodeloides bunge as claimed in claim 4, wherein:
1) pulverizing rhizoma anemarrhenae, sieving with 100 mesh sieve, reflux-extracting the sieved rhizoma anemarrhenae powder with 8 times of 95% ethanol for 3 times, centrifuging, collecting precipitate, and oven drying at 40 deg.C to obtain defatted and decolorized rhizoma anemarrhenae dry powder;
2) adding 20-30 times of distilled water into the rhizoma anemarrhenae dry powder prepared in the step 1), extracting for three times at the temperature of 60-90 ℃ for 1-3 h each time, combining water extract, carrying out centrifugal separation, collecting supernatant, and concentrating under reduced pressure to one fifth of the original volume; adding 5 times of anhydrous ethanol into the concentrated solution, standing at 4 deg.C for 24 hr, centrifuging, and collecting rhizoma anemarrhenae polysaccharide precipitate;
3) removing protein from the rhizoma anemarrhenae polysaccharide prepared in the step 2) by adopting a sevag method, dialyzing, and freeze-drying to obtain crude protein-removed rhizoma anemarrhenae polysaccharide AABP;
4) redissolving the rhizoma anemarrhenae crude polysaccharide AABP prepared in the step 3) in a certain amount of water to prepare a polysaccharide solution with the concentration of 10-20 mL/mg, adding absolute ethyl alcohol until the concentration of the ethyl alcohol is 60%, standing, performing centrifugal separation, collecting precipitate and supernate, continuously adding absolute ethyl alcohol into the supernate until the concentration of the ethyl alcohol is 80%, standing, performing centrifugal separation, collecting precipitate to obtain rhizoma anemarrhenae polysaccharide AABP-2 (80% alcohol precipitation);
5) and (3) further purifying the AABP-2 prepared in the step 4) by using a DEAE-Sepharose Fast Flow ion exchange column, performing gradient elution by using pure water, 0.1M NaCl, 0.2M NaCl and 0.5M NaCl solution in sequence, collecting 0.1M NaCl elution components, concentrating, dialyzing, and freeze-drying to obtain the homogeneous rhizoma anemarrhenae polysaccharide AABP-2B.
8. The use of the homogeneous rhizoma anemarrhenae polysaccharide according to any one of claims 1 to 3 for preparing an antioxidant and for preparing a hypoglycemic health food or medicament.
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| CN115572318A (en) * | 2022-10-23 | 2023-01-06 | 爱生泽(上海)生物科技有限公司 | Preparation method of anemarrhena oligosaccharide and application of anemarrhena oligosaccharide in cosmetics |
| CN116478309A (en) * | 2023-05-19 | 2023-07-25 | 青海大学 | Cynomorium songaricum polysaccharide, cynomorium songaricum polysaccharide compound, and preparation method and application thereof |
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| CN118126210A (en) * | 2024-03-22 | 2024-06-04 | 江汉大学 | Uniform polysaccharide of kidney bean seeds as well as preparation method and application thereof |
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