CN110218263B - Hericium erinaceus fungus chaff polysaccharide, preparation method and application - Google Patents
Hericium erinaceus fungus chaff polysaccharide, preparation method and application Download PDFInfo
- Publication number
- CN110218263B CN110218263B CN201910501669.1A CN201910501669A CN110218263B CN 110218263 B CN110218263 B CN 110218263B CN 201910501669 A CN201910501669 A CN 201910501669A CN 110218263 B CN110218263 B CN 110218263B
- Authority
- CN
- China
- Prior art keywords
- hericium erinaceus
- polysaccharide
- bran
- extraction
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 176
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 176
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 174
- 240000000588 Hericium erinaceus Species 0.000 title claims abstract description 149
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 235000007328 Hericium erinaceus Nutrition 0.000 claims abstract description 110
- 238000000605 extraction Methods 0.000 claims abstract description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- 238000012360 testing method Methods 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 16
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 11
- 230000004044 response Effects 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 150000003214 pyranose derivatives Chemical class 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 238000000611 regression analysis Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 238000000643 oven drying Methods 0.000 claims description 2
- 239000000049 pigment Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims description 2
- 238000007619 statistical method Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 38
- 230000003078 antioxidant effect Effects 0.000 abstract description 12
- 239000003963 antioxidant agent Substances 0.000 abstract description 11
- 238000011160 research Methods 0.000 abstract description 8
- 238000007254 oxidation reaction Methods 0.000 abstract description 6
- 230000003647 oxidation Effects 0.000 abstract description 5
- 230000001766 physiological effect Effects 0.000 abstract description 4
- 239000000835 fiber Substances 0.000 abstract description 3
- 239000013543 active substance Substances 0.000 abstract description 2
- 239000011159 matrix material Substances 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 57
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 22
- 238000010521 absorption reaction Methods 0.000 description 18
- 241000699670 Mus sp. Species 0.000 description 17
- 210000002966 serum Anatomy 0.000 description 14
- 230000006378 damage Effects 0.000 description 13
- 239000003814 drug Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 12
- 108010082126 Alanine transaminase Proteins 0.000 description 12
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 12
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 12
- 230000032683 aging Effects 0.000 description 11
- 229940079593 drug Drugs 0.000 description 11
- 235000019441 ethanol Nutrition 0.000 description 11
- -1 DPPH radicals Chemical class 0.000 description 10
- 241000233866 Fungi Species 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000008103 glucose Substances 0.000 description 10
- 230000036541 health Effects 0.000 description 10
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 9
- 239000008280 blood Substances 0.000 description 9
- 229940118019 malondialdehyde Drugs 0.000 description 9
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 9
- 229910001868 water Inorganic materials 0.000 description 9
- 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 8
- 102000019197 Superoxide Dismutase Human genes 0.000 description 7
- 108010012715 Superoxide dismutase Proteins 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 102000016938 Catalase Human genes 0.000 description 6
- 108010053835 Catalase Proteins 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000002329 infrared spectrum Methods 0.000 description 6
- 150000003254 radicals Chemical class 0.000 description 6
- 206010067125 Liver injury Diseases 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 208000029078 coronary artery disease Diseases 0.000 description 5
- 230000032050 esterification Effects 0.000 description 5
- 238000005886 esterification reaction Methods 0.000 description 5
- 230000003203 everyday effect Effects 0.000 description 5
- 231100000753 hepatic injury Toxicity 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 210000005228 liver tissue Anatomy 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 4
- 230000003712 anti-aging effect Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 238000003304 gavage Methods 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 4
- 230000019635 sulfation Effects 0.000 description 4
- 238000005670 sulfation reaction Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 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 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 230000002292 Radical scavenging effect Effects 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229910052788 barium Inorganic materials 0.000 description 3
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 3
- 150000002243 furanoses Chemical class 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000036542 oxidative stress Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000003287 bathing Methods 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 208000026758 coronary atherosclerosis Diseases 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical class O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000003859 lipid peroxidation Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- UFWWAKOWSBGGCP-UHFFFAOYSA-N pyridine;sulfurochloridic acid Chemical compound OS(Cl)(=O)=O.C1=CC=NC=C1 UFWWAKOWSBGGCP-UHFFFAOYSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 230000002000 scavenging effect Effects 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- KEQGZUUPPQEDPF-UHFFFAOYSA-N 1,3-dichloro-5,5-dimethylimidazolidine-2,4-dione Chemical compound CC1(C)N(Cl)C(=O)N(Cl)C1=O KEQGZUUPPQEDPF-UHFFFAOYSA-N 0.000 description 1
- 235000020927 12-h fasting Nutrition 0.000 description 1
- 241000222485 Agaricales Species 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000289669 Erinaceus europaeus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 241000123222 Hericium Species 0.000 description 1
- 241000959662 Hydnaceae Species 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000222383 Polyporales Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000879 anti-atherosclerotic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- XTHPWXDJESJLNJ-UHFFFAOYSA-N chlorosulfonic acid Substances OS(Cl)(=O)=O XTHPWXDJESJLNJ-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 150000002432 hydroperoxides Chemical class 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 150000004968 peroxymonosulfuric acids Chemical class 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
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000001373 regressive effect Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000005211 surface analysis Methods 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
- A61P3/06—Antihyperlipidemics
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
-
- 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)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Gastroenterology & Hepatology (AREA)
- Vascular Medicine (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Heart & Thoracic Surgery (AREA)
- Urology & Nephrology (AREA)
- Cardiology (AREA)
- Diabetes (AREA)
- Toxicology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Sustainable Development (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of antioxidant polysaccharides, and particularly relates to hericium erinaceus fungus chaff polysaccharide, a preparation method and application. The activity research aiming at the hericium erinaceus polysaccharide in the prior art shows that the polysaccharide has various physiological activities such as oxidation resistance and the like. The hericium erinaceus directly adopted as the extraction raw material has high cost, and the hericium erinaceus bran is used as the matrix residue after the hericium erinaceus are harvested, contains hericium erinaceus mycelium residues, crude fibers subjected to enzymolysis of the hericium erinaceus and other components, and has a high potential utilization value. The invention researches the activity of the hericium erinaceus bran polysaccharide, and researches show that the polysaccharide has various physiological activities and higher development value. The present disclosure further provides a sulfated hericium erinaceus polysaccharide, wherein the modified polysaccharide has improved activity, is significantly higher than the activity of hericium erinaceus polysaccharide, is an active substance with high activity and low cost, and has good development prospects.
Description
Technical Field
The invention belongs to the technical field of antioxidant polysaccharide, and particularly relates to polysaccharide extracted from hericium erinaceus bran and sulfated polysaccharide, a preparation method thereof and application thereof in preparing antioxidant and anti-aging products.
Background
The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Hericium erinaceus (Hericium erinaceum) is also known as Hericium erinaceus, Hedgehog fungus, Hericium erinaceus, and Cauliflower fungi. Since its appearance is very similar to the head of a monkey, it is named Hericium erinaceus, belonging to Basidiomycota, Agaricales, Polyporales, Hydnaceae, Hericium. The polysaccharide substances in the hericium erinaceus are extracted and researched by a research team by a microwave method, and the results show that the hericium erinaceus polysaccharide has the effects of reducing blood sugar, reducing blood fat, resisting radiation, resisting aging and the like and preventing and treating the senile diseases.
The inventor considers that the hericium erinaceus as the polysaccharide extraction raw material is expensive in manufacturing cost and has little significance for actual production. The hericium erinaceus bran is a matrix residue obtained after harvesting of the hericium erinaceus, contains hericium erinaceus mycelium residues and crude fibers subjected to enzymolysis of the hericium erinaceus, and contains a large amount of trace elements and mineral substances, so that the hericium erinaceus bran has a high potential utilization value, and needs to be developed and utilized in a targeted manner.
The edible fungus polysaccharide refers to polysaccharide substances extracted from fruiting body, mycelium, fermentation liquor or fungus chaff of edible fungus. As a biological nonspecific immunity promoter, the edible fungus polysaccharide has various biological effects of resisting tumor, virus and the like. Due to the limitations of physicochemical properties, spatial structure, and other factors, the pharmacological activity exhibited by natural polysaccharides is generally low. After proper modification, the molecular weight, spatial structure and substituent type, number and position of polysaccharide can be changed to affect the bioactivity.
Disclosure of Invention
The extraction process of the hericium erinaceus bran polysaccharide is optimized through response surface statistical analysis. The activity verification of the extracted hericium erinaceus fungus chaff polysaccharide shows that the hericium erinaceus fungus chaff polysaccharide has the effects of resisting oxidation and aging, repairing liver injury, reducing blood fat and the like. In order to improve the activity of the hericium erinaceus fungus chaff polysaccharide, the sulfated hericium erinaceus fungus chaff polysaccharide is provided, and the modified hericium erinaceus fungus chaff polysaccharide has obvious improvement on the activity in multiple aspects and has good application significance.
In order to achieve the technical effects, the present disclosure provides the following technical solutions:
in a first aspect of the disclosure, a hericium erinaceus fungus bran polysaccharide is provided, wherein the hericium erinaceus fungus bran polysaccharide is pyranose with beta-type glycosidic bond connection.
In a second aspect of the present disclosure, a preparation method of hericium erinaceus fungus chaff polysaccharide is provided, which includes the following steps: adding organic solvent into the hericium erinaceus bran solution for extraction.
Preferably, the organic solution includes, but is not limited to, ethanol.
Preferably, the mass of the organic solution is 90-98%.
Preferably, the extraction temperature is 80-95 ℃.
The third aspect of the disclosure provides sulfated hericium erinaceus bran polysaccharide which is furanose with beta-type glycosidic bond connection and has an infrared spectrum of 1262.49cm-1And 808.98cm-1There is a characteristic peak near the site.
Preferably, the preparation method of the sulfated hericium erinaceus fungus bran polysaccharide comprises the following steps: the hericium erinaceus bran polysaccharide of the first aspect is prepared by a chlorosulfonic acid-pyridine method.
In a fourth aspect of the present disclosure, there is provided a use of the sulfated hericium erinaceus bran polysaccharide of the third aspect as an antioxidant.
Preferably, the antioxidant is an antioxidant that scavenges OH or DPPH radicals.
In a fifth aspect of the present disclosure, an application of the hericium erinaceus bran polysaccharide of the first aspect or the sulfated hericium erinaceus bran polysaccharide of the third aspect in preparation of an anti-aging drug and/or a health product is provided.
Preferably, the anti-aging medicine/health care product is a medicine/health care product with SOD, GSH-Px and CAT activities and MDA content inhibition.
In a fifth aspect of the present disclosure, an application of the hericium erinaceus bran polysaccharide of the first aspect or the sulfated hericium erinaceus bran polysaccharide of the third aspect in preparation of a liver injury repair drug and/or a health product is provided.
Preferably, the liver injury repair drug/health product is an Alanine transaminase (ALT) and aspartate transaminase (AST) inhibiting type drug/health product.
In a sixth aspect of the present disclosure, there is provided an application of the hericium erinaceus bran polysaccharide of the first aspect or the sulfated hericium erinaceus bran polysaccharide of the third aspect in an anti-cardiovascular disease drug and/or a health product.
Preferably, the anti-cardiovascular disease drug/health-care product is an anti-coronary heart disease drug/health-care product or an anti-atherosclerosis drug/health-care product.
Preferably, the anti-cardiovascular disease drug/health product is Total Cholesterol (TC) and Triglyceride (TG) inhibitory drug/health product.
Compared with the prior art, the beneficial effect of this disclosure is:
1. the invention researches the activity of the hericium erinaceus bran polysaccharide, and researches show that the polysaccharide has multiple physiological activities of resisting oxidation and aging, repairing liver injury, reducing blood fat and the like, and the multiple activities mean that the polysaccharide has a wider application range and is superior to the edible fungi polysaccharide reported at present.
2. The disclosure also provides sulfated modified polysaccharide forms, which have improved activity in various aspects. Published studies show that when the hericium erinaceus polysaccharide solution reaches 15mg/ml, the clearance rate of DPPH reaches 69.2%. The research of the disclosure shows that when the sulfated hericium erinaceus bran polysaccharide is 400 mug/ml, the clearance rate can reach more than 60 percent, and the dosage is obviously lower than that of the hericium erinaceus polysaccharide. The data show that the activity of the sulfated hericium erinaceus polysaccharide is obviously higher than that of the hericium erinaceus polysaccharide, the economic cost is obviously lower than that of the hericium erinaceus polysaccharide, and the sulfated hericium erinaceus polysaccharide has good technical effects.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.
FIG. 1 is a line graph showing in vitro antioxidant capacity of sulfated and bran polysaccharides of Hericium erinaceus in example 1;
wherein, FIG. 1a is a line graph of hydroxyl radical clearance;
FIG. 1b is a line graph showing DPPH radical scavenging rate;
FIG. 1c is a reducing power line graph.
FIG. 2 is a bar graph showing the effect of Hericium erinaceus bran polysaccharide and sulfated Hericium erinaceus bran polysaccharide on the enzyme activity and lipid content of mouse brain tissue in example 1.
FIG. 3 is a bar graph showing the effect of Hericium erinaceus bran polysaccharide and sulfated Hericium erinaceus bran polysaccharide on the enzyme activity and lipid content of liver tissues of mice in example 1;
FIG. 4 is a bar graph showing the effect of Hericium erinaceus bran polysaccharide and sulfated Hericium erinaceus bran polysaccharide on biochemical indicators of the serum of the aging mice in example 1;
fig. 5 is an infrared spectrum of hericium erinaceus bran polysaccharide and sulfated hericium erinaceus bran polysaccharide in example 1.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background art, the hericium erinaceus bran, which is a substrate residue after harvesting of hericium erinaceus, contains a large amount of hericium erinaceus mycelium residues and crude fibers obtained by enzymolysis of hericium erinaceus. The edible fungus polysaccharide is known in the field to have a plurality of physiological activities, and polysaccharide substances with good activity are expected to be extracted from hericium erinaceus bran. The polysaccharide extracted from the hericium erinaceus fungus chaff has multiple effects of resisting oxidation and aging, repairing liver tissue damage, reducing blood fat and the like, and is remarkable in activity. The invention provides sulfated hericium erinaceus fungus bran polysaccharide, which is modified to improve the activity to a certain degree and has good popularization significance.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.
Example 1
1.1 treatment of Hericium erinaceus bran
Sun-drying Hericium erinaceus bran for 1-2 days, drying, oven-drying at 60 deg.C to constant weight, pulverizing, and sieving with 10-20 mesh sieve.
1.2 response surface test design
Selecting three conditions of extraction times (1, 2 and 3), ethanol concentration (75%, 85% and 95%) and extraction temperature (75 ℃, 85 ℃ and 95 ℃) to carry out RSM Design, and carrying out regression analysis on test data by adopting Design-Expert 8.0 software to determine the extraction conditions of the hericium erinaceus fungus bran polysaccharide.
TABLE 1 experimental design of response surface for extracting polysaccharide from mushroom bran
1.3 preparation of Hericium erinaceus bran polysaccharide
Weighing 1.0g of hericium erinaceus fungus chaff in each group, adding 40 times of deionized water, extracting polysaccharide and precipitating with ethanol according to corresponding extraction times, ethanol concentration and extraction temperature in the table 2, and performing 3 groups of parallel tests in each group. Adding ethanol with corresponding concentration for 24h, collecting precipitate, drying the precipitate to obtain crude Hericium erinaceus bran polysaccharide, dissolving the crude polysaccharide with deionized water, removing protein, pigment and small molecular impurities from the crude polysaccharide solution, and obtaining Hericium erinaceus bran polysaccharide.
1.4 determination of polysaccharide content
The polysaccharide content is determined by adopting a phenol-sulfuric acid method. The principle is that under the action of sulfuric acid, polysaccharide is firstly hydrolyzed into monosaccharide, and is quickly dehydrated to generate furfural derivative, and the furfural derivative reacts with phenol to generate orange yellow solution, and the orange yellow solution has characteristic absorption at 490nm and is compared and quantified with standard series.
(1) Preparation of 6% phenol solution: accurately weighing 6mL of phenol, using deionized water to fix the volume to 100mL, fully shaking up to prepare 6% phenol solution which needs to be prepared for use and needs to be placed in a brown bottle to be protected from light for storage.
(2) Preparation of standard glucose solution: accurately weighing 0.0100g glucose (dried in a 105 deg.C oven to constant weight before use) in a 100mL volumetric flask, and diluting to constant volume to obtain 100 μ g/mL glucose standard solution.
(3) Preparation of a standard curve: accurately weighing 0.1g of standard glucose (dried at 105 ℃), fixing the volume to 100mL by using deionized water, accurately sucking 10mL into a clean volumetric flask after fully dissolving, and adding deionized water to fix the volume to 100mL to obtain a standard glucose solution with the concentration of 100 mu g/mL. Diluting 100 mu g/mL standard glucose solution into glucose solutions with concentrations of 10 mu g/mL, 20 mu g/mL, 30 mu g/mL, 40 mu g/mL, 50 mu g/mL and 60 mu g/mL in sequence, respectively adding 1mL of the glucose solutions into a test tube containing 1mL of deionized water, accurately adding 2mL of deionized water into another test tube as a blank control, respectively adding 1mL of phenol solution (6%) and 5mL of concentrated sulfuric acid into 7 test tubes in sequence, fully shaking and uniformly mixing, reacting for 20min at room temperature, measuring OD value under the condition of 490nm, and drawing a regression curve by using glucose content and absorbance as horizontal and vertical coordinates respectively. The regression equation is obtained as y is 0.015x-0.0134, R2The glucose standard curve is available as 0.9972.
(4) And (3) determination of polysaccharide content: taking 2mL of polysaccharide solution to be detected in a test tube, measuring by the following method and the standard curve, measuring for three times, taking an average value, calculating the polysaccharide concentration according to a regression equation, and analyzing polysaccharide results and related equations as shown in tables 2, 3 and 4.
TABLE 2 Hericium erinaceus bran polysaccharide response surface test design and results
1.5 response surface data analysis
Through multiple regression analysis, the extraction yield of the hericium erinaceus bran polysaccharide and the coding value of the variable can be represented by a polynomial model, and a quadratic polynomial regression equation is as follows:
Y＝2.56+0.4x1+0.62x2+0.093x3+0.28x1x2+0.14x1x3-0.092x2x3-0.55x1 2-0.36x2 2-0.29x3 2
wherein Y is the predicted value of the extraction yield of the hericium erinaceus fungus bran polysaccharide, and x1、x2And x3The code values for the number of extractions, ethanol concentration and extraction temperature are indicated, respectively.
The BBD design model analysis of variance and the fit analysis of variance in the response surface analysis are shown in table 3.
TABLE 3 significance test of regressive coefficients of hericium erinaceus bran polysaccharide secondary model
Note: *: indicates significance (P < 0.05).: indicating extreme significance (P < 0.01).
As can be seen from tables 2 and 3, in the significance test of the regression coefficients of the response surface test quadratic model, the model was significant (P)<0.0001), the F value is 28.20, and the mismatch value (Rack-of-fit) is not significant (P is 0.079), which means that the model has better fitting degree and smaller test error, the test value of the extraction yield of the hericium erinaceus bran polysaccharide is consistent with the predicted value, and the coefficient R is determined20.9732, also demonstrated that the test values fit very closely to the predicted values, and analysis of variance of regression model terms indicates linear regression coefficients (x)1，x2) Two isCoefficient of the second order (x)1 2，x2 2) Are all very significant (P)<0.01), coefficient of quadratic term x3 2Is significant (P)<0.05), which shows that the extraction times, the ethanol concentration and the extraction temperature all have important influence on the yield of the hericium erinaceus fungus chaff polysaccharide, and compared with the extraction temperature, the extraction times and the ethanol concentration have more obvious influence on the extraction yield of the hericium erinaceus fungus chaff polysaccharide. In conclusion, the model has small test error and good fitting degree, and can be used for researching the optimal process for extracting the hericium erinaceus bran polysaccharide.
The optimal extraction process conditions of the hericium erinaceus bran polysaccharide calculated by the regression equation are as follows: the extraction times are 2.68 times, the ethanol concentration is 95 percent, the extraction temperature is 89.92 ℃, and the theoretical yield of the hericium erinaceus fungus chaff polysaccharide is 3.10 percent. The verification test is carried out according to the conditions, the actual factors are considered, the extraction frequency is 3 times, the ethanol concentration is 95%, the extraction temperature is 90 ℃, and under the conditions, the actual yield of the hericium erinaceus fungus chaff polysaccharide is 2.94 +/-0.11%, and the actual yield is not obviously different from the predicted value.
1.6 sulfation of Hericium erinaceus bran polysaccharide
Adopts chlorosulfonic acid-pyridine method to prepare sulfated Hericium erinaceus bran polysaccharide. Firstly preparing an esterification reagent, adding 90mL of pyridine into a triangular flask, fully stirring in an ice-water bath, and dropwise adding 15mL of chlorosulfonic acid within 1h to prepare the esterification reagent for later use.
Dissolving 3g of purified hericium erinaceus bran polysaccharide in 60mL of N, N-dimethylformamide, fully stirring, pouring into a triangular flask filled with an esterification reagent, and stirring at 50 ℃ for 2 hours at the rotating speed of 200 r/min. After the reaction is finished, cooling to room temperature, adjusting the temperature to be neutral by using a NaOH solution with the mass concentration of 10%, adding ethanol with the mass concentration of 95% with three times of volume for precipitation for 24 hours, centrifuging at 5000r/min for 5min to obtain a precipitate, dissolving the precipitate in deionized water, dialyzing for 3d, and finally carrying out vacuum freeze drying on a polysaccharide solution to obtain the sulfated hericium erinaceus bran polysaccharide.
1.7 determination of degree of substitution
(1) The reagent preparation method comprises the following steps: the method used to determine the degree of substitution of the polysaccharide was the barium chloride-gelatin method, with the method steps as given in table 4.
TABLE 4 method for preparing reagents required for the determination of degree of substitution
(2) Drawing a sulfate standard curve: accurately sucking 0.02mL, 0.04mL, 0.08mL, 0.12mL, 0.16mL and 2.00mL sulfate radical standard solutions with the concentration of 0.6mg/mL into test tubes, adding 1mol/L hydrochloric acid solution to 2mL in test tubes with the solution less than 2mL, dropwise adding 3% trichloroacetic acid 3.8mL and 1mL barium chloride-gelatin solution into each test tube, fully mixing, reacting at room temperature for 15min, and measuring the absorption value A of the reaction solution of each tube at the wavelength of 360nm1Changing 1mL barium chloride-gelatin solution to 1mL gelatin solution, and measuring the absorption value A of each test tube reaction solution by the same steps0With A1Subtract A0The obtained value is used as an ordinate, the amount of sulfate group is used as an abscissa to draw a standard curve, and a regression equation is obtained, wherein Y is 0.0016X +0.0079, and R is2The sulfate standard curve is available as 0.9948.
(3) And (3) measuring the degree of substitution of sulfated hericium erinaceus bran polysaccharide: weighing 4.5mg of sulfated hericium erinaceus fungus chaff polysaccharide into a test tube with a plug, adding 4.5mL of 1mol/L hydrochloric acid solution, sealing, and hydrolyzing in a water bath kettle at 100 ℃ for 4 h. Then dried in a drying oven at 75 ℃ to prepare a polysaccharide solution of 2mg/mL, and stored at a low temperature of 4 ℃. Taking 0.2mL of polysaccharide solution, preparing a method according to a standard curve, and measuring A of sulfated hericium erinaceus fungus bran polysaccharide1And A0And obtaining the content of sulfate groups according to a standard curve, and obtaining the substitution degree of the sulfated hericium erinaceus fungus bran polysaccharide according to the following formula.
DS＝1.62×S/(32-1.02×S)
Wherein DS is the degree of substitution; s is the percentage of sulfate radical mass in polysaccharide mass
1.8 determination of in vitro antioxidant Capacity
(1) And (3) reduction force determination: the reaction system is as follows: 2.5mL of 0.2mol/L phosphate buffer (pH 6.6), 2.5mL of 1% potassium ferricyanide solution, and 1.0mL of diluted polysaccharide solution of different concentrations. Bathing in water at 50 ℃ for 20min, adding 2.5mL of trichloroacetic acid with the mass concentration of 10% to terminate the reaction, centrifuging for 10min (3000r/min), taking 2.5mL of supernatant, adding 2.5mL of deionized water and 0.5mL of ferric trichloride with the mass concentration of 0.1%, bathing in water at 25 ℃ for 15min, and measuring the absorbance value of the reaction solution in each test tube at 700 nm.
(2) Determination of OH-scavenging ability: the reaction system is as follows: sequentially adding 1mL of 9mmol/L ferrous sulfate, 1mL of 9mmol/L salicylic acid and 1mL of diluted polysaccharide solution with different concentrations, replacing the polysaccharide solution with deionized water as a blank control, taking 1mL of hydrogen peroxide with the volume fraction of 0.03%, carrying out constant-temperature water bath on the mixture in a water bath kettle at 37 ℃ for 30min, measuring the absorbance value of each reaction solution under 510nm after centrifugation, and calculating the clearance rate of the polysaccharide to OH according to the following formula:
C＝(1-A1/A0)×100
wherein, C is OH clearance (%); a. the1Is absorbance value of polysaccharide group; a. the0Absorbance value for blank group
(3) Determination of the ability to scavenge DPPH free radicals: the reaction system is as follows: sequentially adding 2mL of DPPH ethanol solution with mass concentration of 0.1 mu mol/L and 2mL of diluted polysaccharide solutions with different concentrations, using distilled water to replace the polysaccharide solution as a blank control, carrying out thermostatic water bath on the mixture in a water bath kettle at 25 ℃ for 15min, and measuring the absorbance value of the reaction solution in each test tube at 517 nm. The clearance rate of the hericium erinaceus bran polysaccharide on DPPH free radicals is calculated according to the following formula:
C＝[1-(A1-A2)/A0]×100
wherein C is DPPH free radical scavenging rate (100%); a. the0The absorbance value of a mixed solution of DPPH ethanol solution and distilled water; a. the1The absorbance value of the mixed solution of DPPH ethanol solution and polysaccharide solution; a. the2The absorbance value of the absolute ethyl alcohol and polysaccharide solution mixed solution is shown.
1.9 in vitro antioxidant results
The more reducing substances, the more oxidation resistant. As can be seen from FIG. 1c, the reduction power of the modified sulfated Hericium erinaceus bran polysaccharide is stronger than that of the unmodified polysaccharide under the same polysaccharide concentration condition.
OH is currently a kind of active oxygen with the greatest harm to health, and too high OH can damage or kill red blood cells, degrade DNA, damage cell membranes and organelles, and further cause pathological changes of cells. As shown in FIG. 1a, the Hericium erinaceus bran polysaccharide modified by sulfation has stronger OH scavenging ability than unmodified polysaccharide.
DPPH is a stable free radical with nitrogen as the center, DPPH is widely used for detecting the in-vitro antioxidant capacity of food and drugs, and a large number of researches show that DPPH free radicals can damage myocardial cells and vascular endothelial cells and have serious damage effects on body organs. As can be seen from FIG. 1b, the DPPH radical scavenging ability of the two polysaccharides is enhanced with the increase of the concentration, and the DPPH radical scavenging ability of the modified Hericium erinaceus bran polysaccharide is significantly stronger than that of the unmodified polysaccharide.
In conclusion, the hericium erinaceus fungus chaff polysaccharide and the sulfated and modified fungus chaff polysaccharide both have strong in-vitro antioxidant capacity, the reducing power and the OH and DPPH free radical scavenging capacity of the sulfated and modified hericium erinaceus fungus chaff polysaccharide are obviously higher than those of unmodified hericium erinaceus fungus chaff polysaccharide, and the in-vitro antioxidant activity of the hericium erinaceus fungus chaff polysaccharide can be improved through sulfation modification.
1.10 in vivo anti-aging Activity test
1.10.1D-galactose model for mouse aging
As the test mice, 35 Kunming male mice (Luzhongzhi pharmaceutical Co., Ltd.) were selected, and the body weight was 20. + -.2 g. The breeding conditions are as follows: after 3 days of acclimation in the laboratory, the mice were randomly divided into 7 groups (5 per group). The ambient temperature is 22 +/-2 ℃, the ambient humidity is 60-65%, and the illumination is carried out for 12 hours every day. Grouping tests:
(1) normal group (NC): performing intragastric administration of 0.2mL of deionized water and intraperitoneal injection of 0.2mL of physiological saline every day;
(2) model group (MC): gavage 0.2mL deionized water + intraperitoneal injection 0.2mL D-galactose solution (300mg/kg) every day;
(3) positive control group (PC): intragastric administration of 0.2mL VC solution (300mg/kg) per day and intraperitoneal injection of 0.2mL D-galactose solution (300 mg/kg);
(4) HRPS high dose group: gavage 0.2mL HRPS solution (400mg/kg) every day and inject 0.2mL D-galactose solution (300mg/kg) into the abdominal cavity;
(5) HRPS low dose group: gavage 0.2mL HRPS solution (200mg/kg) every day and inject 0.2mL D-galactose solution (300mg/kg) into the abdominal cavity;
(6) SHRPS high dose group: intragastrically administering 0.2mL SHRPS solution (400mg/kg) per day and intraperitoneally injecting 0.2mL D-galactose solution (300 mg/kg);
(7) SHRPS low dose group: gavage 0.2mL SHRPS solution (200mg/kg) per day and intraperitoneal injection 0.2mL D-galactose solution (300mg/kg)
Mice in different groups are fed in feeding boxes with the same size, the frequency of changing padding materials into new materials is kept consistent, and the mice can be freely fed with feed and drinking water. After continuously feeding for 45 days, fasting for 12h, taking blood from eyeball, killing mouse by dislocation of cervical vertebra, quickly taking liver and brain samples, washing viscera with normal saline, absorbing excessive water with filter paper, respectively taking 1g of tissue samples, adding phosphate buffer solution (0.2mol/L, pH 7.4, 4 ℃) according to the proportion of 1:9, grinding, centrifuging grinding solution (8000r/min, 10min, 4 ℃) to take supernatant, and placing at-20 ℃ for standby. Meanwhile, the blood of the eyeball is centrifuged (12000r/min, 10min, 4 ℃) and the serum is taken and placed at the temperature of minus 20 ℃ for standby. The whole sampling process needs to be rapid and needs to be carried out at low temperature so as to ensure that index components in the sample are not degraded. Detection of 1.10.2 index
(1) Glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), Catalase (CAT), Malondialdehyde (MDA);
(2) serum index detection: the serum samples were analyzed by a full-automatic biochemical NYAS6905 analyzer for Alanine transaminase (ALT), aspartate transaminase (AST), Total Cholesterol (TC), and Triglycerides (TG) in mice serum.
1.11 results of animal experiments
SOD is an important active substance, can remove harmful substances generated in the metabolism process of organisms, and plays an important role in removing ROS in the organisms, thereby reducing ROS damage to body cells. The organism scavenging ability to ROS is identified by a method for detecting SOD content in each tissue and organ in the organism. GSH-Px can effectively remove H in organism2O2To convert it into H2O, converting organic hydroperoxide (ROOH) into ROH, and leading the biological membrane to be seriously damaged due to lipid peroxide generated by lipid peroxidation induced by active oxygen in the organism, thereby causing cell aging and accelerating organism aging, and GSH-Px can remove the lipid peroxide generated in the organism, thereby reducing the damage of the lipid peroxide to the cells, and therefore, the detection of the activity of the GSH-Px in the organism can directly reflect the aging degree of the organism. CAT is a terminal oxidase that scavenges hydrogen peroxide produced during cellular metabolism, which is a key enzyme in protecting cells from peroxide damage, and is widely present in the biological world, including animals, plants, and microorganisms. When a large amount of free radicals exist in a body, the free radicals and lipid generate peroxidation, MDA is a final product of oxidation reaction, MDA can damage biological macromolecules such as protein, nucleic acid and the like, excessive MDA can damage human nerves, destroy cell structures and accelerate aging of the body, and therefore the lipid peroxidation and oxidative stress degree in the body can be reflected by detecting the content level of MDA in the body.
As shown in figure 2, the sulfated hericium erinaceus bran polysaccharide can improve the effects of SOD, GSH-Px and CAT activities and MDA content in brain tissues of aged mice induced by D-galactose, and relieve oxidative stress injury.
As shown in figure 3, the sulfated hericium erinaceus bran polysaccharide can improve the effects of SOD, GSH-Px and CAT activities and MDA content in the liver tissues of aged mice induced by D-galactose, and relieve oxidative stress injury.
ALT and AST are distributed in a plurality of organ tissues of a human body, wherein the content of ALT and AST in the liver is the largest, and when liver tissue cells are damaged, ALT in the cytoplasm of the liver cells and AST in mitochondria are exuded from the cytoplasm to blood, so the damage degree of the liver tissue is generally judged by detecting the activity of ALT and AST in the blood serum in clinic. TC and TG are mainly metabolized in the liver, and when the liver is damaged, metabolic disorder is caused, hypercholesterolemia is caused, and the TC and TG content in serum is increased. The increase of TC and TG in serum can increase the incidence rate of coronary heart disease and atherosclerosis, and has great threat to the elderly.
As shown in FIG. 4, after D-galactose was intraperitoneally injected, the activities of ALT and AST in the serum of the mice in the aging model group were significantly higher than those of ALT and AST in the serum of the mice in the normal group (P <0.01), and it was found that the organ tissues of the mice were seriously damaged by D-galactose intraperitoneally injection, so that ALT in cytoplasm and AST in mitochondria were exuded into the blood. The hericium erinaceus bran polysaccharide and the sulfated hericium erinaceus bran polysaccharide with different dosages can remarkably reduce the activity of ALT and AST in serum of aging mice (P is less than 0.01), and have a certain dosage effect, which indicates that the polysaccharide has certain capacity of protecting the liver of the mice.
Continuous D-galactose injection causes the TC and TG content in the serum of the model group mice to be remarkably increased (P <0.01), which indicates that the mice have lipid metabolism disorder. After different dosages of hericium erinaceus bran polysaccharide and sulfated hericium erinaceus bran polysaccharide are perfused into the stomach, the content of TC and TG in serum can be reduced to different degrees, and certain relieving effect on liver injury is achieved. Tests show that the hericium erinaceus bran polysaccharide and the sulfated hericium erinaceus bran polysaccharide have the activity of reducing the TC and TG contents in serum, so that the incidence rate of coronary heart disease and atherosclerosis is reduced.
1.12 bond type analysis of Hericium erinaceus bran polysaccharide and sulfated Hericium erinaceus bran polysaccharide
Performing bond type analysis on the hericium erinaceus bran polysaccharide and the sulfated hericium erinaceus bran polysaccharide by using an infrared spectroscopic analysis method. Weighing 4.0mg of freeze-dried hericium erinaceus bran polysaccharide and sulfated hericium erinaceus bran polysaccharide samples, fully mixing and grinding the samples and 200mg of potassium bromide (KBr) powder in an agate mortar, tabletting, and scanning and tabletting by using a Nicolet Nexus 470 infrared spectrometer to obtain infrared chromatograms of the hericium erinaceus bran polysaccharide and the sulfated hericium erinaceus bran polysaccharide, wherein the spectrum scanning range is 500 plus one charge of 4000cm-1。
1.13 bond type analysis results of Hericium erinaceus bran polysaccharide and sulfated Hericium erinaceus bran polysaccharide
The position and shape of the spectral peak are two important features of the infrared spectrum, the position of the spectral peak is represented by wave number, the most significant feature of a certain group is represented, and the shape reflects some details of the corresponding group. Therefore, the structural characteristics of the objective polysaccharide can be estimated from the position and shape of the spectral peak in the infrared spectrum.
FIG. 5 shows the infrared spectra of Hericium erinaceus bran polysaccharide and sulfated Hericium erinaceus bran polysaccharide. At 3416.93cm-1And 3409.47cm-1The nearby absorption peak indicates the stretching vibration of the O-H bond in the polysaccharide; at 2975cm-1The nearby absorption peak indicates the methylene group (-CH) in the polysaccharide2-) C-H bond stretching vibration; at 2920cm-1The nearby absorption peak shows the stretching vibration of C-H bond in polysaccharide, and is 1630cm-1And 1417cm-1The vicinity is a characteristic absorption peak of carboxyl in the polysaccharide and a C ═ O bond in the carboxyl; at 1141.96cm-1The nearby absorption peak indicates the stretching vibration of uronic acid; the existence of the characteristic peaks can judge that the substance is a carbohydrate compound, and the hericium erinaceus fungus bran polysaccharide and the sulfated hericium erinaceus fungus bran polysaccharide are proved to have typical absorption peaks of polysaccharide and belong to carbohydrate compounds.
The polysaccharide of the fungus chaff of the hericium erinaceus is 1094.15cm-1、1141.96cm-1、1195.97cm-1There are one absorption peak and three absorption peaks, which are caused by the stretching vibration of C-O bond and O-H bond, and the sulfated Hericium erinaceus fungus chaff polysaccharide is 1046.97cm-1An absorption peak is formed, and only one absorption peak is formed nearby, so that the hericium erinaceus fungus bran polysaccharide is pyranose, and the sulfated hericium erinaceus fungus bran polysaccharide is furanose; the anomers of the sugars α -and β -are caused by angular variation of the C-H of the terminal group, the C-H bond being an equatorial bond and the α -isomer being present at 850cm-1Near the absorption peak, the C-H bond is an upright bond and is a beta-isomer at 880cm-1An absorption peak is nearby, and in conclusion, the hericium erinaceus fungus chaff polysaccharide is pyranose with beta-type glycosidic bond connection, and the sulfated hericium erinaceus fungus chaff polysaccharide is beta-typeA glycosidically linked furanose.
The infrared spectrum of the sulfated hericium erinaceus bran polysaccharide is 1262.49cm-1And 808.98cm-1The two peaks are respectively caused by asymmetric S ═ O bond and symmetric C-O-S bond stretching vibration and are both characteristic absorption peaks of thioester bonds, and the two absorption peaks do not contain hericium erinaceus bran polysaccharide, which indicates that sulfuric acid groups are connected to the hericium erinaceus bran polysaccharide modified by persulfuric acid esterification; compared with Hericium erinaceus bran polysaccharide, the sulfated Hericium erinaceus bran polysaccharide is 2920cm-1The absorption peak in the vicinity is reduced because partial sulfation agent substitution at the C-6 position results in methylene (-CH)2-) the C-H stretching vibration peak in the polymer is weakened.
The infrared spectroscopic analysis proves that the hericium erinaceus bran polysaccharide which is sulfated by the sulfate compound is successfully generated after the hericium erinaceus bran polysaccharide is modified by the sulfuric acid esterification.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.
Claims (1)
1. A method for extracting pyranose with beta-type glycosidic bond linkage from Hericium erinaceus bran comprises:
the preparation method of the beta-type glucopyranose connected with glycosidic bonds comprises the following steps: optimizing the extraction process of the hericium erinaceus bran polysaccharide through response surface statistical analysis, and adding an organic solvent into a hericium erinaceus bran solution for extraction:
the method comprises the following specific steps:
(1) treatment of hericium erinaceus bran
Sun-drying Hericium erinaceus bran for 1-2 days, drying, oven-drying at 60 deg.C to constant weight, pulverizing, and sieving with 10-20 mesh sieve;
(2) the response surface test Design selects three conditions of extraction times (1, 2 and 3), ethanol concentration (75%, 85% and 95%) and extraction temperature (75 ℃, 85 ℃ and 95 ℃) to carry out RSM Design, and Design-Expert 8.0 software is adopted to carry out regression analysis on test data so as to determine the extraction conditions of the hericium erinaceus fungus bran polysaccharide;
(3) preparation of hericium erinaceus bran polysaccharide
Weighing 1.0g of hericium erinaceus fungus chaff per group, adding 40 times of deionized water by volume, extracting polysaccharide and precipitating with ethanol, performing parallel tests on 3 groups per group, adding ethanol with corresponding concentration for 24 hours, collecting precipitate, drying the precipitate to obtain crude hericium erinaceus fungus chaff polysaccharide, dissolving the crude polysaccharide again with deionized water, and removing protein, pigment and small molecular impurities from the crude polysaccharide solution to obtain the hericium erinaceus fungus chaff polysaccharide; the optimal extraction process conditions calculated by a regression equation are as follows: the extraction times are 3 times, the ethanol concentration is 95 percent, and the extraction temperature is 90 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910501669.1A CN110218263B (en) | 2019-06-11 | 2019-06-11 | Hericium erinaceus fungus chaff polysaccharide, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910501669.1A CN110218263B (en) | 2019-06-11 | 2019-06-11 | Hericium erinaceus fungus chaff polysaccharide, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110218263A CN110218263A (en) | 2019-09-10 |
| CN110218263B true CN110218263B (en) | 2021-01-22 |
Family
ID=67816328
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910501669.1A Active CN110218263B (en) | 2019-06-11 | 2019-06-11 | Hericium erinaceus fungus chaff polysaccharide, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110218263B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115028754B (en) * | 2022-06-30 | 2023-08-11 | 上海市农业科学院 | Sulfated hericium erinaceus fruiting body beta-glucan, sulfated beta-glucan-chitosan nanoparticle and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211827A (en) * | 2014-09-16 | 2014-12-17 | 江苏农林职业技术学院 | Method for extracting polysaccharide from hericium erinaceus mushroom dregs |
| CN106478829A (en) * | 2016-10-24 | 2017-03-08 | 上海市农业科学院 | A kind of Hericium erinaceus active polysaccharide and preparation method thereof |
-
2019
- 2019-06-11 CN CN201910501669.1A patent/CN110218263B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104211827A (en) * | 2014-09-16 | 2014-12-17 | 江苏农林职业技术学院 | Method for extracting polysaccharide from hericium erinaceus mushroom dregs |
| CN106478829A (en) * | 2016-10-24 | 2017-03-08 | 上海市农业科学院 | A kind of Hericium erinaceus active polysaccharide and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| "不同提取方法猴头菇粗多糖的表征及其抗氧化活性的比较";黄越等;《食品工业科技》;20170215(第3期);第80-86页 * |
| "猴头菇胞内胞外多糖的结构、抗氧化活性和保肝护肝能力分析";崔芳源;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20170315(第3期);第36-40页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110218263A (en) | 2019-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheng et al. | Extraction, characterisation and antioxidant activity of Allium sativum polysaccharide | |
| Nie et al. | Bioactive polysaccharides from Cordyceps sinensis: Isolation, structure features and bioactivities | |
| Yu et al. | Structure, chain conformation and antitumor activity of a novel polysaccharide from Lentinus edodes | |
| Qiu et al. | Purification, structure, lipid lowering and liver protecting effects of polysaccharide from Lachnum YM281 | |
| JP5102435B2 (en) | Chitosan-containing polysaccharide, its production method and use | |
| Zhao et al. | Antioxidant and hypoglycemic effects of acidic-extractable polysaccharides from Cordyceps militaris on type 2 diabetes mice | |
| Qiao et al. | Anti-fatigue activity of the polysaccharides isolated from Ribes stenocarpum Maxim | |
| Jin et al. | Physicochemical properties of polysaccharides separated from Camellia oleifera Abel seed cake and its hypoglycemic activity on streptozotocin-induced diabetic mice | |
| Afiati et al. | The effectiveness β-glucan of shiitake mushrooms and Saccharomyces cerevisiae as antidiabetic and antioxidant in mice Sprague Dawley induced alloxan | |
| CN111671765A (en) | Application of brown algae dietary fiber in preparation of medicines, foods and/or health products for treating or preventing metabolic syndrome | |
| Xiang et al. | Structural characterization of a novel marine polysaccharide from mussel and its antioxidant activity in RAW264. 7 cells induced by H2O2 | |
| Fan et al. | Preparation, structural analysis and antioxidant activity of polysaccharides and their derivatives from Pueraria lobata | |
| CN110218263B (en) | Hericium erinaceus fungus chaff polysaccharide, preparation method and application | |
| WO2023036203A1 (en) | Cs-4 fermented mycelium heteropolysaccharide, preparation method therefor and use thereof | |
| Song et al. | Chemical structure and protective effect against alcoholic kidney and heart damages of a novel polysaccharide from Piperis Dahongpao | |
| Lin et al. | Extraction, structure and bioactivities of polysaccharides from Sanghuangporus spp.: A review | |
| CN113402624B (en) | Extracellular polysaccharide and preparation method and application thereof | |
| CN106924297A (en) | A kind of Lachnum intracellular melanin as acute liver damage medicine purposes | |
| CN108727511B (en) | Carboxymethylated mulberry polysaccharide and preparation method and application thereof | |
| Zhang et al. | Degradation of Angelica sinensis polysaccharide: Structures and protective activities against ethanol-induced acute liver injury | |
| CN115894728B (en) | Cactus milpa alta polysaccharide MAP-2 and preparation method and application thereof | |
| CN113651900B (en) | Grifola frondosa purified polysaccharide with blood lipid regulating function and preparation method thereof | |
| Song et al. | Isolation, Purification, Structural Characterization, and Hypoglycemic Activity of Polysaccharide from Trichosanthes kirilowii Maxim. Seed Shell | |
| CN116120475B (en) | Raspberry homogeneous polysaccharide RCP-90-1, and separation and purification method and application thereof as antitumor drug | |
| CN115960274B (en) | Raspberry polysaccharide and preparation method and application thereof |
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 |