CN111116770B - Centipeda minima polysaccharide and preparation method and application thereof - Google Patents
Centipeda minima polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN111116770B CN111116770B CN201911318346.5A CN201911318346A CN111116770B CN 111116770 B CN111116770 B CN 111116770B CN 201911318346 A CN201911318346 A CN 201911318346A CN 111116770 B CN111116770 B CN 111116770B
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- centipeda minima
- water
- nacl solution
- collecting
- 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 117
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 117
- 241000928504 Centipeda minima Species 0.000 title claims abstract description 84
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 150000004676 glycans Chemical class 0.000 title claims abstract 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 67
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 56
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 49
- 239000011780 sodium chloride Substances 0.000 claims abstract description 28
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 11
- 229920002678 cellulose Polymers 0.000 claims abstract description 11
- 239000001913 cellulose Substances 0.000 claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 11
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 10
- 238000010992 reflux Methods 0.000 claims abstract description 10
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 7
- 238000005238 degreasing Methods 0.000 claims abstract description 6
- 239000003472 antidiabetic agent Substances 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 21
- 238000010521 absorption reaction Methods 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
- 238000004108 freeze drying Methods 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- 235000019441 ethanol Nutrition 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 8
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 8
- 229920005654 Sephadex Polymers 0.000 claims description 7
- 239000012507 Sephadex™ Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 238000002386 leaching Methods 0.000 claims description 5
- 238000010298 pulverizing process Methods 0.000 claims description 5
- 238000005194 fractionation Methods 0.000 claims description 4
- 238000000643 oven drying Methods 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000007873 sieving Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000009826 distribution Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 235000013402 health food Nutrition 0.000 claims description 2
- 238000012869 ethanol precipitation Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 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 compound [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 abstract description 8
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 abstract description 6
- 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 abstract description 6
- 230000002401 inhibitory effect Effects 0.000 abstract description 6
- 230000003647 oxidation Effects 0.000 abstract description 5
- 238000007254 oxidation reaction Methods 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 150000001875 compounds Chemical class 0.000 abstract 1
- 230000001376 precipitating effect Effects 0.000 abstract 1
- 238000003809 water extraction Methods 0.000 abstract 1
- 150000004804 polysaccharides Chemical class 0.000 description 81
- 239000000243 solution Substances 0.000 description 20
- 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
- 239000000203 mixture Substances 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 150000002772 monosaccharides Chemical class 0.000 description 6
- 150000003254 radicals Chemical class 0.000 description 6
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 108010028144 alpha-Glucosidases Proteins 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 201000001421 hyperglycemia Diseases 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 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
- 230000004071 biological effect Effects 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000005100 correlation spectroscopy Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- 235000021257 carbohydrate digestion Nutrition 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- -1 cationic ABTS free radical Chemical class 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000002270 exclusion chromatography Methods 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- 125000003147 glycosyl group Chemical group 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
- 238000002329 infrared spectrum Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 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
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 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
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 241001633683 Centipeda <firmicute> Species 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 description 1
- 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 description 1
- 208000002249 Diabetes Complications Diseases 0.000 description 1
- 206010012655 Diabetic complications Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000414067 Inonotus obliquus Species 0.000 description 1
- 235000008708 Morus alba Nutrition 0.000 description 1
- 240000000249 Morus alba Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000195474 Sargassum Species 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 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
- 239000002253 acid Substances 0.000 description 1
- 229920001284 acidic polysaccharide Polymers 0.000 description 1
- 150000004805 acidic polysaccharides Chemical group 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
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical group OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006851 antioxidant defense Effects 0.000 description 1
- 239000008346 aqueous phase Substances 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
- 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
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000004192 high performance gel permeation chromatography Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000000291 postprandial effect Effects 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
- 238000003825 pressing Methods 0.000 description 1
- 229940079877 pyrogallol Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 239000012279 sodium borohydride Chemical class 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 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
- 238000003756 stirring Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000005406 washing Methods 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Diabetes (AREA)
- Pharmacology & Pharmacy (AREA)
- Materials Engineering (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Sustainable Development (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Mycology (AREA)
- Endocrinology (AREA)
- Emergency Medicine (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the field of plant polysaccharide, and discloses centipeda minima polysaccharide as well as a preparation method and application thereof. The structural formula of the centipeda minima polysaccharide is shown as follows. The preparation method comprises the steps of crushing the centipeda minima, carrying out ethanol reflux degreasing and decoloring, and carrying out hot water extraction to obtain a water extract; precipitating with ethanol and separating to obtain crude herba Centipedae polysaccharide; removing protein by using Sevag reagent, performing fractional separation by using cellulose DEAE-52 anion exchange resin, eluting by using water, 0.1M NaCl solution, 0.2M NaCl solution and 0.5M NaCl solution in sequence, collecting the eluted components of the 0.1M NaCl solution, and purifying by using a G-200 gel column to obtain the centipeda minima polysaccharide. The compound has the capability of efficiently removing DPPH free radicals, ABTS free radicals and superoxide free radicals, has the performances of resisting oxidation and inhibiting the activity of alpha-glycosidase, can be used as a natural antioxidant, and is applied to the preparation of hypoglycemic drugs or health-care foods.
Description
Technical Field
The invention belongs to the field of plant polysaccharide, and particularly relates to centipeda minima polysaccharide as well as a preparation method and application thereof.
Background
Type II diabetes is a non-insulin dependent disease, a chronic disease that causes metabolic disturbance in the body due to the relative deficiency of insulin in the body caused by β -islet cell damage or resistance of target cells to insulin.
Postprandial hyperglycemia is one of the main features of type II diabetes mellitus and also one of the main inducers of diabetic complications. Delaying carbohydrate digestion is an effective method of alleviating postprandial hyperglycemia. Alpha-glucosidase is a glycoside hydrolase that hydrolyzes the terminal non-reducing 1 → 4 linked alpha-D-glucose residues, thereby releasing glucose into the blood. Thus, inhibition of α -glucosidase may reduce carbohydrate digestion and slow postprandial blood glucose levels in diabetic patients. Meanwhile, type ii diabetes is closely associated with an increase in oxidative stress. Persistent hyperglycemia will decrease the activity of antioxidant enzymes, leading to the destruction of the antioxidant defense system. Thus, agents or substances that simultaneously reduce postprandial hyperglycemia and oxidative stress may be used to treat type II diabetics.
At present, the biological activity of polysaccharide is widely researched by people, and the polysaccharide has various biological activities of improving immunity, resisting inflammation, resisting oxidation, resisting tumor, reducing blood sugar and the like. In addition, it has been shown that polysaccharides isolated from mulberry, inonotus obliquus and sargassum have both antioxidant and alpha-glucosidase inhibitory activities. Therefore, the polysaccharide can be used as a potential hypoglycemic drug or health food for diabetic patients.
Centipeda minima is a composite plant and is widely distributed in Hubei, Jiangsu Guangdong and Zhejiang in China. Studies show that the centipeda minima can be used for treating diseases such as rhinitis, tracheitis, rheumatoid arthritis and the like, and also has anti-inflammatory, antioxidant and antitumor activities. At present, the relationship between the structure and the biological activity of the centipeda minima polysaccharide is rarely reported.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide the uniform centipeda minima polysaccharide which has the functions of resisting oxidation and inhibiting the activity of alpha-glycosidase.
The invention also aims to provide a preparation method of the uniform centipeda minima polysaccharide which simultaneously has the functions of resisting oxidation and inhibiting the activity of alpha-glycosidase.
The invention further aims to provide the uniform centipeda minima polysaccharide as an antioxidant and application thereof in preparing hypoglycemic drugs or health-care food.
The purpose of the invention is realized by the following scheme:
a homogeneous polysaccharide of Centipeda minima is named as CMP-2B, and the structural formula is shown as follows:
wherein A represents 1,6-linked- β -D-Galp; b represents 1,3, 6-linked-beta-D-Manp; c represents 1, 4-linked-alpha-D-GalpA; d represents 1, 3-linked-alpha-L-Araf; e represents 1,4, 6-linked-alpha-D-Galp; f represents 1-linked-alpha-L-Araf;
the Centipeda homogeneous polysaccharide CMP-2B main chain is 1,3,6-linked-D-Manp and 1,4,6-linked-D-Galp, and the branched chains are linked with 1, 4-linked-alpha-D-GalpA, 1, 6-linked-beta-D-Galp, T-linked-alpha-L-Araf and 1, 3-linked-alpha-L-Araf.
The uniform centipeda minima polysaccharide CMP-2B has the molecular weight distribution range of 80-200KDa and the weight-average molecular weight of 126.35 KDa.
The preparation method of the uniform centipeda minima polysaccharide CMP-2B comprises the following steps:
pulverizing herba Centipedae, adding ethanol, refluxing, defatting, decolorizing, and extracting with hot water to obtain water extractive solution; carrying out alcohol precipitation separation on the water extract to obtain crude centipeda minima polysaccharide; removing protein by using Sevag reagent, carrying out fractional separation by using cellulose DEAE-52 anion exchange resin, eluting by using water, 0.1mol/L NaCl solution, 0.2mol/L NaCl solution and 0.5mol/L NaCl solution in sequence, collecting the eluted components of the 0.1mol/L NaCl solution, and purifying by using a G-200 gel column to obtain the uniform centipeda minima polysaccharide CMP-2B.
Before the centipeda minima is used, the centipeda minima is preferably cleaned and dried at the temperature of 45-60 ℃; the ground centipeda minima powder is obtained by grinding the centipeda minima powder by a grinder and then sieving the ground centipeda minima powder by a sieve of 60 to 100 meshes.
The degreasing and decoloring conditions are preferably reflux at 70-80 ℃ for 2-4 hours. The degreasing and decoloring are preferably performed three times or more by repeatedly using absolute ethyl alcohol.
The filter residue after degreasing and decoloring is preferably dried at the temperature of 45-60 ℃ for later use.
The hot water leaching conditions are preferably aqueous leaching at 60-90 ℃ for 2-4 hours.
Preferably, the alcohol precipitation separation is to add absolute ethyl alcohol into the water extract, then to stand for 8-24 hours at-4 ℃, to perform centrifugation, and to collect the crude centipeda minima polysaccharide.
The operation of removing the protein specifically comprises the following steps: dissolving the crude polysaccharide in water, adding Sevag reagent, shaking, centrifuging, collecting supernatant, repeatedly removing protein for 8-10 times, dialyzing, and freeze drying to obtain the crude polysaccharide of the protein-removed centipeda minima.
Before the fractionation, the crude centipeda minima polysaccharide without protein is preferably dissolved in water, and is centrifuged for 6-10min at the speed of 8000-10000r/min, and the supernatant is taken and then fractionated by cellulose DEAE-52 anion exchange resin.
And (3) purifying by using a G-200 gel column, preferably dissolving the product after the fractionation in water, centrifuging, purifying the supernatant by using a Sephadex G-200 gel column, eluting by using water, detecting by using a phenol-sulfuric acid method, collecting the first component, concentrating, dialyzing, and freeze-drying to obtain the purified uniform centipeda minima polysaccharide CMP-2B.
Preferably, the preparation method of the uniform centipeda minima polysaccharide CMP-2B specifically comprises the following steps:
(1) pretreatment of centipeda minima: cleaning herba Centipedae with water, oven drying at 45-60 deg.C, pulverizing with pulverizer, sieving to obtain herba Centipedae powder, adding anhydrous ethanol, refluxing at 70-80 deg.C for 2-4 hr, filtering, defatting and decolorizing the residue with ethanol twice, filtering, and oven drying at 45-60 deg.C;
(2) extracting crude polysaccharide: adding water with the weight of 20-30 times of that of the filter residue obtained in the step (1), leaching for 2-4 hours at the temperature of 60-90 ℃, centrifuging, filtering and concentrating to obtain a centipeda minima water extract; adding anhydrous ethanol into the water extract, standing at-4 deg.C for 8-16 hr, centrifuging, and collecting crude polysaccharide of herba Centipedae;
(3) protein removal: dissolving the crude polysaccharide obtained in the step (2) in water, adding Sevag reagent, shaking, centrifuging, collecting supernatant, repeatedly removing protein for 8-10 times, dialyzing, and freeze-drying to obtain the protein-removed crude polysaccharide of centipeda minima;
(4) polysaccharide purification: dissolving the protein-removed centipeda minima polysaccharide obtained in the step (3) by water, centrifuging for 6-10min at the speed of 8000-10000r/min, carrying out fractional separation on the supernatant by cellulose DEAE-52 anion exchange resin, eluting by water, 0.1mol/L, 0.2mol/L and 0.5mol/L NaCl solution in sequence, collecting the eluted components of the 0.1mol/L NaCl solution, concentrating, dialyzing, and freeze-drying; dissolving the uniform polysaccharide in water, centrifuging, purifying the supernatant by a Sephadex G-200 gel column, eluting with water, collecting the eluate, concentrating, dialyzing, and freeze-drying to obtain the uniform polysaccharide CMP-2B of centipeda minima.
Preferably, the centrifugation conditions in step (2) are both: centrifuging for 5-8min at the rotating speed of 6000-; in addition, anhydrous ethanol is added into the water extract to ensure that the ethanol concentration in the water extract of the centipeda minima reaches 75-85% (v/v) preferably.
Preferably, the cut-off molecular weight of the dialysis bag in the step (3) is 3500-8000 Da.
Preferably, in the step (4), the polysaccharide is primarily purified by cellulose DEAE-52 anion exchange resin, eluted by water, 0.1mol/L, 0.2mol/L and 0.5mol/L NaCl solutions in sequence, detected by a phenol-sulfuric acid method, an elution curve is drawn, 0.1mol/L NaCl solution elution components are collected according to an absorption peak of the elution curve, and the product is obtained by concentration, dialysis and freeze drying; dissolving the product in water, further purifying with Sephadex G-200 gel column, eluting with water, detecting with phenol-sulfuric acid method, drawing elution curve, obtaining two absorption peaks according to the elution curve, combining and collecting the first absorption peak, concentrating, dialyzing, and freeze drying to obtain the final product. The gel chromatogram of the polysaccharide CMP-2B only has a uniform and symmetrical absorption peak detected by high performance gel exclusion chromatography, and the molecular weight distribution is 80-200KDa according to the retention time calculation.
The uniform centipeda minima polysaccharide CMP-2B is an acidic polysaccharide and contains six glycosidic bonds of 1,3,6-linked-D-Manp,1,4,6-linked-D-Galp,1, 4-linked-alpha-D-GalpA, 1, 6-linked-beta-D-Galp, T-linked-alpha-L-Araf and 1, 3-linked-alpha-L-Araf. The CMP-2B has the capability of efficiently eliminating DPPH free radicals, ABTS free radicals and superoxide free radicals, has the performances of resisting oxidation and inhibiting the activity of alpha-glycosidase, can be used as a natural antioxidant, and is applied to the preparation of hypoglycemic drugs or health-care foods.
Drawings
FIG. 1 shows the analysis results of monosaccharide composition of the polysaccharide CMP-2B prepared in example 1, wherein A is a standard monosaccharide profile; b is the monosaccharide composition analysis spectrum of the uniform polysaccharide CMP-2B of the centipeda minima. The labels in the figure are: man-mannose, Rha-rhamnose, GalA-galacturonic acid, Glc-glucose, Gal-galactose, Ara-arabinose, Fuc-fucose.
FIG. 2 is an infrared spectrum of the polysaccharide CMP-2B prepared in example 1.
FIG. 3 shows the preparation of polysaccharide CMP-2B of example 11H NMR spectrum.
FIG. 4 shows the preparation of polysaccharide CMP-2B of example 113C NMR spectrum.
FIG. 5 is a COSY spectrum of the polysaccharide CMP-2B prepared in example 1.
FIG. 6 is an HSQC spectrum of the polysaccharide CMP-2B prepared in example 1.
FIG. 7 is a HMBC profile of the polysaccharide CMP-2B prepared in example 1.
FIG. 8 is a graph of the antioxidant activity of polysaccharide CMP-2B prepared in example 1; 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. 9 is a graph showing the inhibition of α -glucosidase activity by polysaccharide CMP-2B prepared in example 1.
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 reagents used in the examples are commercially available without specific reference.
Example 1: preparation of centipeda minima polysaccharide CMP-2B
(1) Polysaccharide extraction:
taking 1000g of cleaned and dried centipeda minima, crushing the cleaned and dried centipeda minima by a crusher, sieving the crushed centipeda minima by a 60-mesh sieve, adding 6L of absolute ethyl alcohol, refluxing for two hours under the condition of 70 ℃ water bath, repeatedly refluxing, degreasing and decoloring for two times, filtering, drying filter residues in a 45 ℃ blast oven, adding 20L of water, leaching for 4 hours under the condition of 80 ℃ water bath, centrifuging, filtering, concentrating to 2.5L, slowly adding 10L of absolute ethyl alcohol with the volume being four times of that of the filter residues at the rotating speed of 500r/min, standing for 12 hours under the condition of-4 ℃, centrifuging, collecting precipitates, washing for 3 times by the absolute ethyl alcohol and acetone in sequence, and freeze-drying the precipitates to obtain 23.6g of crude polysaccharide of the centipeda minima.
(2) Protein removal:
and (2) dissolving 20g of the prepared crude centipeda minima polysaccharide in 300mL of water, stirring, carrying out centrifugal separation, adding a Sevag reagent into the supernatant, oscillating, standing, carrying out centrifugal separation, taking the supernatant, adding the Sevag reagent, repeating for 10 times, dialyzing the supernatant, concentrating, and carrying out freeze drying to obtain the crude centipeda minima polysaccharide without proteins.
(3) Polysaccharide purification:
dissolving 2g of crude polysaccharide without protein in 100mL of water, centrifuging for 8min at the rotating speed of 8000r/min, taking supernatant, carrying out primary separation by using cellulose DEAE-52 anion exchange resin, eluting by using water, 0.1mol/L, 0.2mol/L and 0.5mol/L NaCl aqueous solution in sequence, detecting by using a phenol-sulfuric acid method, drawing an elution curve, collecting 0.1mol/L NaCl solution elution components according to an absorption peak of the elution curve, concentrating, dialyzing, and freeze-drying to obtain the centipeda minima polysaccharide CMP-2; dissolving CMP-2 in water, centrifuging, taking supernatant, further purifying by a Sephadex G-200 gel column, eluting by water, detecting by a phenol-sulfuric acid method, drawing an elution curve, obtaining two absorption peaks according to the elution curve, merging and collecting the first absorption peak, concentrating, dialyzing, freeze-drying to obtain 107mg of centipeda minima polysaccharide CMP-2B, and detecting by high performance gel exclusion chromatography, wherein CMP-2B only has one uniform and symmetrical absorption peak on a gel chromatogram, which indicates that the uniform component polysaccharide is uniform.
(4) Primary structure analysis of polysaccharide CMP-2B:
a. the average molecular weight of CMP-2B was determined to be 126.35KDa by high performance gel permeation chromatography.
b. Completely hydrolyzing 5mg of polysaccharide with trifluoroacetic acid, performing PMP pre-column derivatization, extracting with chloroform, passing the aqueous phase through a 0.22 μm filter membrane, and analyzing with high performance liquid chromatography. The monosaccharide composition results are shown in fig. 1, and the centipeda minima polysaccharide CMP-2B comprises four monosaccharides, namely mannose, galacturonic acid, galactose and arabinose, in a molar ratio of 0.27:0.12:0.42: 0.17.
c. Mixing 2mg dried Centipeda minima polysaccharide CMP-2B with KBr powder, pressing into granules at 4000-400cm-1The FT-IR measurement is performed in the frequency range of (a). The results are shown in FIG. 2 at 3388cm-1The absorption peak is the free stretching vibration absorption peak of polysaccharide hydroxyl, and is 2935cm-1The absorption peak at (A) belongs to the stretching vibration of the C-H bond. 1608cm-1Contains absorption peaks, which indicate that the centipeda minima polysaccharide CMP-2B contains uronic acid, and the result is consistent with the analysis of monosaccharide composition.
d. Dissolving 5mg of dried Centipeda minima polysaccharide CMP-2B in 2mL of anhydrous DMSO completely, adding 100mg of NaOH and 2mL of iodomethane, reacting in the dark for 12 hours under the protection of nitrogen, adding 1mL of water to stop the reaction, dialyzing for 24 hours with running water, extracting for 2 times with chloroform, evaporating chloroform phase to dryness to obtain methylated polysaccharide, and obtaining the methylated polysaccharide in an infrared spectrum of 3200-3600cm-1No hydroxyl characteristic absorption peak is formed, and the complete methylation of the polysaccharide is proved. Trifluoroacetic acid hydrolyzes methylated polysaccharide, sodium borohydride reduces, acetylates, and finally GC-MS analysis is carried out, and the result is shown in Table 1.
TABLE 1 methylation analysis of Centipeda minima polysaccharide CMP-2B
Methylation results show that the CMP-2B polysaccharide contains six different types of glycosidic bonds, which are respectively: 1,3,6-linked-D-Manp,1,4,6-linked-D-Galp,1,4-linked- α -D-GalpA,1,6-linked- β -D-Galp, T-linked- α -L-Araf and 1,3-linked- α -L-Araf.
e. Drying 40mg of Centipeda minima polysaccharide CMP-2B in a vacuum drying oven for 24 hours, D2NMR analysis was carried out after O substitution 2 times. The one-dimensional and two-dimensional NMR spectra of CMP-2B polysaccharide are shown in FIGS. 3-7, wherein FIG. 3 is1H NMR spectrum, FIG. 4 is13C NMR spectrum, COSY spectrum, HSQC spectrum, HMBC spectrum and HMBC spectrum are shown in figure 5 and 7 respectively. According to said polysaccharide1H NMR、13C NMR, COSY and HSQC spectra and results of previous research summarize six different types of glycosidic bonds contained in CMP-2B and their corresponding chemical shift signals in Table 2, chemical shifts delta 4.50/102.67ppm, 4.58/103.44ppm, 5.01/99.01ppm, 5.07/106.93, 5.16/97.53 and 5.18/109.28ppm of H1/C1 of Centipeda-N-aminothion CMP-2B represent the chemical shifts of anomeric and anomeric carbons of 1, 6-linked-beta-D-Galp, 1,3, 6-linked-beta 1-D-Manp, 1, 4-linked-beta 0-D-GalpA, 1, 3-linked-beta 4-L-Araf, 1,4, 6-linked-beta 6-D-Galp and T-linked-beta 7-L-Araf, respectively. Furthermore, the signal peak at δ 170-. In the HMBC map of CMP-2B (FIG. 7), the order of linkage between glycosidic linkages can be determined. C-1 of the sugar group 1,3, 6-linked-. beta.2-D-Manp and H-4 of the sugar group 1,4, 6-linked-. beta.8-D-Galp have a cross peak, C-1 of the sugar group 1,4, 6-linked-. beta.9-D-Galp and H-3 of the sugar group 1,3, 6-linked-. beta.3-D-Manp have a cross peak, C-1 of the sugar group 1, 6-linked-. beta.5-D-Galp and H-6 of the sugar group 1,3, 6-linked-. beta.D-Manp are related, C-1 of the sugar group 1, 3-linked-. alpha. -L-Araf and H-6 of the sugar group 1, 6-linked-. beta.D-Galp overlap, C-1 of the sugar group 1, 4-linked-. alpha. -D-GalpA and 1, the H-3 of 3-linked-. alpha. -L-Araf had a cross peak, and the C-1 of T-linked-. alpha. -L-Araf and the H-6 of 1,4, 6-linked-. alpha. -D-Galp had a cross peak.
In conclusion, the polysaccharide takes 1,3, 6-linked-beta-D-Manp and 1,4, 6-linked-alpha-D-Galp residues as main chains, and other glycosyl groups are branched chains and are connected with the main chain glycosyl residues in a direct or indirect connection mode.
TABLE 2 preparation of Centipeda minima polysaccharide CMP-2B1H and13chemical shift of CClass table
f. In combination with the above analysis, it was determined that the structural formula of Centipeda minima polysaccharide CMP-2B is shown below:
example 2: antioxidant activity experiment of centipeda minima polysaccharide CMP-2B
The antioxidant activity of the polysaccharide is evaluated by adopting DPPH and ABTS methods and superoxide radical experiments respectively, and the specific steps are as follows:
DPPH free radical scavenging: mu.L of CMP-2B aqueous solution (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL) and 150. mu.L of DPPH ethanol solution (60. mu. mol/L) were mixed in a 96-well plate, and 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 FIG. 8A.
Elimination of ABTS free radicals: 2mL of an aqueous ABTS (7mmol/L) solution (mixed with 2mL of an aqueous potassium persulfate solution (2.45mmol/L) and reacted at room temperature in the dark for 12 hours to produce cationic ABTS free radicals, the produced cationic ABTS free radical solution was diluted with ethanol to an absorbance of 0.7. + -. 0.05 at 734nm, 20. mu.L of an aqueous polysaccharide solution (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL) was added to a 96-well plate, 180. mu.L of the diluted cationic ABTS free radical solution was added, the mixture was shaken and the absorbance at 734nm was measured, with ascorbic acid (Vc) as a positive control, and the results are shown in FIG. 8B.
Superoxide radical removal: mu.L of polysaccharide aqueous solution (0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL) and 150. mu.L of Tris-HCl buffer solution (0.1mol/L) with 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, as shown in FIG. 8C.
The result shows that the centipeda minima polysaccharide CMP-2B has stronger capability of clearing DPPH, ABTS and superoxide radical.
Example 3: experiment for inhibiting activity of alpha-glycosidase by centipeda minima polysaccharide CMP-2B
Adding 20 μ L polysaccharide water solution (0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/mL) and 40 μ L α -glucosidase water solution (0.2U/mL) into 96-well plate, mixing, culturing at 37 deg.C for 10min, adding 20.0 μ L pNPG water solution (10mmol/L), mixing, reacting at 37 deg.C for 30min, and adding 100 μ L 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. 11.
The results show that the centipeda minima polysaccharide CMP-2B has alpha-glycosidase activity inhibition in the concentration range of 0.1-3.0mg/mL, and the inhibition activity is enhanced along with the increase of the concentration. The inhibition rate of CMP-2B was 90.03. + -. 1.49% at a concentration of 3.0 mg/mL. The centipeda minima polysaccharide has good alpha-glycosidase activity inhibition.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The uniform centipeda minima polysaccharide is named as CMP-2B and is characterized by being prepared by the following method:
pulverizing herba Centipedae, adding ethanol, refluxing, defatting, decolorizing, and extracting with hot water at 60-90 deg.C for 2-4 hr to obtain water extractive solution; adding absolute ethyl alcohol into the water extract to ensure that the concentration of the ethyl alcohol in the water extract of the centipeda minima reaches 75-85 percent, and carrying out alcohol precipitation separation to obtain crude centipeda minima polysaccharide; removing protein by using Sevag reagent, carrying out fractional separation by using cellulose DEAE-52 anion exchange resin, eluting by using water, 0.1mol/L NaCl solution, 0.2mol/L NaCl solution and 0.5mol/L NaCl solution in sequence, collecting 0.1mol/L NaCl solution elution components, purifying by using G-200 gel column, combining and collecting a first absorption peak to obtain the uniform centipeda minima polysaccharide CMP-2B.
2. The homogeneous centipeda minima polysaccharide according to claim 1, characterized in that:
the uniform centipeda minima polysaccharide CMP-2B has the molecular weight distribution range of 80-200KDa and the weight-average molecular weight of 126.35 KDa.
3. A method for preparing homogeneous centipeda minima polysaccharide according to claim 1 or 2, characterized in that it comprises the following steps:
pulverizing herba Centipedae, adding ethanol, refluxing, defatting, decolorizing, and extracting with hot water at 60-90 deg.C for 2-4 hr to obtain water extractive solution; adding absolute ethyl alcohol into the water extract to ensure that the concentration of the ethyl alcohol in the water extract of the centipeda minima reaches 75-85 percent, and carrying out alcohol precipitation separation to obtain crude centipeda minima polysaccharide; removing protein by using Sevag reagent, carrying out fractional separation by using cellulose DEAE-52 anion exchange resin, eluting by using water, 0.1mol/L NaCl solution, 0.2mol/L NaCl solution and 0.5mol/L NaCl solution in sequence, collecting 0.1mol/L NaCl solution elution components, purifying by using G-200 gel column, combining and collecting a first absorption peak to obtain the uniform centipeda minima polysaccharide CMP-2B.
4. The method for preparing uniform centipeda minima polysaccharide according to claim 3, wherein the method comprises the following steps:
the degreasing and decoloring conditions are reflux at 70-80 ℃ for 2-4 hours.
5. The method for preparing uniform centipeda minima polysaccharide according to claim 3, wherein the method comprises the following steps:
adding absolute ethyl alcohol into the ethanol precipitation separation point water extract, standing for 8-24 hours at-4 ℃, centrifuging, and collecting crude centipeda minima polysaccharide;
the operation of removing the protein specifically comprises the steps of dissolving the crude polysaccharide in water, adding a Sevag reagent, shaking, centrifuging, collecting supernatant, repeatedly removing the protein for 8-10 times, dialyzing, and freeze-drying to obtain the crude polysaccharide of the protein-removed centipeda minima.
6. The method for preparing uniform centipeda minima polysaccharide according to claim 3, wherein the method comprises the following steps:
before the fractionation, the crude centipeda minima polysaccharide without protein is dissolved in water, the solution is centrifuged for 6 to 10min at the speed of 8000-10000r/min, and the supernatant is taken and fractionated by cellulose DEAE-52 anion exchange resin;
and (3) purifying by using a G-200 gel column, namely dissolving the product after the fractionation in water, centrifuging, purifying the supernatant by using a Sephadex G-200 gel column, eluting by using water, detecting by using a phenol-sulfuric acid method, collecting the first component, concentrating, dialyzing, and freeze-drying to obtain the purified uniform centipeda minima polysaccharide CMP-2B.
7. The method for preparing uniform centipeda minima polysaccharide according to claim 3, which is characterized by comprising the following steps:
(1) pretreatment of centipeda minima: cleaning herba Centipedae with water, oven drying at 45-60 deg.C, pulverizing with pulverizer, sieving to obtain herba Centipedae powder, adding anhydrous ethanol, refluxing at 70-80 deg.C for 2-4 hr, filtering, defatting and decolorizing the residue with ethanol twice, filtering, and oven drying at 45-60 deg.C;
(2) extracting crude polysaccharide: adding water with the weight of 20-30 times of that of the filter residue obtained in the step (1), leaching for 2-4 hours at the temperature of 60-90 ℃, centrifuging, filtering and concentrating to obtain a centipeda minima water extract; adding anhydrous ethanol into the water extractive solution to make ethanol concentration in the water extractive solution of herba Centipedae reach 75-85%, standing at-4 deg.C for 8-16 hr, centrifuging, and collecting crude polysaccharide of herba Centipedae;
(3) protein removal: dissolving the crude polysaccharide obtained in the step (2) in water, adding Sevag reagent, shaking, centrifuging, collecting supernatant, repeatedly removing protein for 8-10 times, dialyzing, and freeze-drying to obtain the protein-removed crude polysaccharide of centipeda minima;
(4) polysaccharide purification: dissolving the protein-removed centipeda minima polysaccharide obtained in the step (3) by water, centrifuging for 6-10min at the speed of 8000-10000r/min, carrying out fractional separation on the supernatant by cellulose DEAE-52 anion exchange resin, eluting by water, 0.1mol/L, 0.2mol/L and 0.5mol/L NaCl solution in sequence, collecting the eluted components of the 0.1mol/L NaCl solution, concentrating, dialyzing, and freeze-drying; dissolving the uniform polysaccharide in water, centrifuging, purifying the supernatant through a Sephadex G-200 gel column, eluting with water, collecting the first elution component, concentrating, dialyzing, and freeze-drying to obtain the uniform polysaccharide CMP-2B of the centipeda minima.
8. The method for preparing uniform centipeda minima polysaccharide according to claim 7, wherein the method comprises the following steps:
the centrifugation conditions in the step (2) are as follows: centrifuging for 5-8min at the rotating speed of 6000-;
the cut-off molecular weight of the dialysis bag in the step (3) is 3500-8000 Da;
in the step (4), preliminarily purifying the polysaccharide by using cellulose DEAE-52 anion exchange resin, eluting by using water, 0.1mol/L NaCl solution, 0.2mol/L NaCl solution and 0.5mol/L NaCl solution in sequence, detecting by using a phenol-sulfuric acid method, drawing an elution curve, collecting 0.1mol/L NaCl solution elution components according to an absorption peak of the elution curve, concentrating, dialyzing, and freeze-drying to obtain a product; and dissolving the product in water, centrifuging, further purifying the supernatant by a Sephadex G-200 gel column, eluting with water, detecting by a phenol-sulfuric acid method, drawing an elution curve, obtaining two absorption peaks according to the elution curve, merging and collecting the first absorption peak, concentrating, dialyzing, and freeze-drying to obtain the final product.
9. Use of the homogeneous centipeda minima polysaccharide according to claim 1 or 2 for the preparation of an antioxidant.
10. Use of the homogeneous centipeda minima polysaccharide according to claim 1 or 2 in the preparation of hypoglycemic drugs and health foods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911318346.5A CN111116770B (en) | 2019-12-19 | 2019-12-19 | Centipeda minima polysaccharide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911318346.5A CN111116770B (en) | 2019-12-19 | 2019-12-19 | Centipeda minima polysaccharide and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111116770A CN111116770A (en) | 2020-05-08 |
| CN111116770B true CN111116770B (en) | 2021-08-10 |
Family
ID=70500156
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911318346.5A Active CN111116770B (en) | 2019-12-19 | 2019-12-19 | Centipeda minima polysaccharide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111116770B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000247829A (en) * | 1999-02-24 | 2000-09-12 | Mikimoto Pharmaceut Co Ltd | Cosmetic, unregulated drug, medicine and food |
| JP2000344653A (en) * | 1999-06-04 | 2000-12-12 | Kanebo Ltd | Eraser for active oxygen and skin cosmetic |
| CN108653355A (en) * | 2018-03-17 | 2018-10-16 | 启东创绿绿化工程有限公司 | A kind of Herba Centipedae extract preparation method with anti-inflammatory effect |
| CN110194808A (en) * | 2019-06-12 | 2019-09-03 | 陈敏 | A kind of plant essence polysaccharide and preparing the application in skin-lightening cosmetic |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170055172A (en) * | 2015-11-11 | 2017-05-19 | 주식회사 코리아나화장품 | Cosmetic Composition Comprising Extracts of Centipeda minima |
| KR102004366B1 (en) * | 2017-09-06 | 2019-07-29 | 주식회사 아미코스메틱 | A cosmetic composition comprising centipeda minima extract |
-
2019
- 2019-12-19 CN CN201911318346.5A patent/CN111116770B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000247829A (en) * | 1999-02-24 | 2000-09-12 | Mikimoto Pharmaceut Co Ltd | Cosmetic, unregulated drug, medicine and food |
| JP2000344653A (en) * | 1999-06-04 | 2000-12-12 | Kanebo Ltd | Eraser for active oxygen and skin cosmetic |
| CN108653355A (en) * | 2018-03-17 | 2018-10-16 | 启东创绿绿化工程有限公司 | A kind of Herba Centipedae extract preparation method with anti-inflammatory effect |
| CN110194808A (en) * | 2019-06-12 | 2019-09-03 | 陈敏 | A kind of plant essence polysaccharide and preparing the application in skin-lightening cosmetic |
Non-Patent Citations (2)
| Title |
|---|
| 鹅不食草煎液对小鼠肝损伤的保护作用;钱妍;《中国药业》;20040625;第13卷(第6期);第25-26页 * |
| 鹅不食草的研究进展;冉茂莲;《中南药学》;20190801;第17卷(第11期);第1874-1879页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111116770A (en) | 2020-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100544743C (en) | The extracting method of Radix Codonopsis polysaccharide | |
| Yang et al. | Isolation, purification, structural characterization, and hypoglycemic activity assessment of polysaccharides from Hovenia dulcis (Guai Zao) | |
| CN108727509B (en) | Moso bamboo shoot shell arabinogalactan and preparation and application thereof | |
| CN104945531B (en) | Gentian acidity uniform polysaccharide and purification method and application thereof | |
| Li et al. | Isolation and structural characterization of a neutral polysaccharide from the stems of Dendrobium densiflorum | |
| CN112876577B (en) | Homogeneous rhizoma anemarrhenae polysaccharide and preparation method and application thereof | |
| CN110128562B (en) | An antitumor fructus Psoraleae polysaccharide, its extraction and separation method, and its application in preparing antitumor drugs | |
| CN111116771B (en) | Polysaccharide extracted from Undaria pinnatifida and application thereof in preparation of alpha-glucosidase activity inhibition drugs | |
| CN111793141B (en) | Pleurotus citrinopileatus mycelium polysaccharide and preparation method and application thereof | |
| CN110229243A (en) | A kind of mountain herb mixtures tea homogeneous polysaccharide and the preparation method and application thereof | |
| CN113024685A (en) | Low-molecular-weight Dictyophora indusiata (Vent. Ex pers) Fisch trum-Dictyophora (Vent. Ex pers) Fisch trum et Schott polysaccharide, and preparation method and application thereof | |
| Tu et al. | A novel polysaccharide from Hericium erinaceus: Preparation, structural characteristics, thermal stabilities, and antioxidant activities in vitro | |
| CN114591448A (en) | Phellinus igniarius sporophore mannogalactan and preparation and application thereof | |
| CN110606900B (en) | Method for separating and purifying fructus Jujubae polysaccharide with antioxidant effect | |
| CN107012184B (en) | Angelica dahurica polysaccharide extracted by enzyme method, preparation method and application thereof | |
| Zhou et al. | Extraction, purification and antioxidant activity of Juglans regia shell polysaccharide | |
| CN111808209B (en) | Selenium-rich pleurotus citrinopileatus mycelium polysaccharide and preparation and application thereof | |
| Fan et al. | Structure, physicochemical properties, antioxidant, and hypoglycemic activities of water‐soluble polysaccharides from millet bran | |
| CN110204627B (en) | Phlebopus portentosus polysaccharide and preparation method and application thereof | |
| CN111116770B (en) | Centipeda minima polysaccharide and preparation method and application thereof | |
| CN112062866A (en) | Hericium erinaceus selenium-rich polysaccharide and preparation method and application thereof | |
| CN114316077A (en) | Preparation method and application of sea cucumber polysaccharide | |
| CN114805624B (en) | Cortex moutan polysaccharide and preparation method and application thereof | |
| Shuxiu et al. | Immunoactivities of the polysaccharides from Morus alba, Chlamydomonas mexicana and Poria cocos | |
| CN113621085A (en) | Preparation method of dandelion root polysaccharide sulfation modification product and application of dandelion root polysaccharide sulfation modification product |
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 |