CN113817076A - 一种具有免疫调节活性的点柄乳牛肝菌多糖sgp2-1及其制备方法和应用 - Google Patents
一种具有免疫调节活性的点柄乳牛肝菌多糖sgp2-1及其制备方法和应用 Download PDFInfo
- Publication number
- CN113817076A CN113817076A CN202111133125.8A CN202111133125A CN113817076A CN 113817076 A CN113817076 A CN 113817076A CN 202111133125 A CN202111133125 A CN 202111133125A CN 113817076 A CN113817076 A CN 113817076A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- sgp2
- lactobacillus
- 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.)
- Pending
Links
- 150000004676 glycans Chemical class 0.000 title claims abstract description 119
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 119
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 119
- 240000002605 Lactobacillus helveticus Species 0.000 title claims abstract description 20
- 235000013967 Lactobacillus helveticus Nutrition 0.000 title claims abstract description 20
- 229940054346 lactobacillus helveticus Drugs 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000004957 immunoregulator effect Effects 0.000 title claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 55
- 210000002540 macrophage Anatomy 0.000 claims abstract description 30
- 238000001556 precipitation Methods 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 13
- 102000019034 Chemokines Human genes 0.000 claims abstract description 11
- 108010012236 Chemokines Proteins 0.000 claims abstract description 11
- 102000004127 Cytokines Human genes 0.000 claims abstract description 10
- 108090000695 Cytokines Proteins 0.000 claims abstract description 10
- 238000005342 ion exchange Methods 0.000 claims abstract description 9
- 235000013376 functional food Nutrition 0.000 claims abstract description 6
- 229940121354 immunomodulator Drugs 0.000 claims abstract description 6
- 206010057249 Phagocytosis Diseases 0.000 claims abstract description 5
- 230000008782 phagocytosis Effects 0.000 claims abstract description 5
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 4
- 239000002955 immunomodulating agent Substances 0.000 claims abstract description 4
- 230000002584 immunomodulator Effects 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 46
- 241000186660 Lactobacillus Species 0.000 claims description 40
- 229940039696 lactobacillus Drugs 0.000 claims description 40
- 238000010828 elution Methods 0.000 claims description 28
- 239000000843 powder Substances 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000004108 freeze drying Methods 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 12
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims description 11
- 238000000502 dialysis Methods 0.000 claims description 11
- 101100256840 Allochromatium vinosum (strain ATCC 17899 / DSM 180 / NBRC 103801 / NCIMB 10441 / D) sgpB gene Proteins 0.000 claims description 10
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 10
- 101100256841 Glossina morsitans morsitans sgp2 gene Proteins 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- 108090001005 Interleukin-6 Proteins 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 102100021943 C-C motif chemokine 2 Human genes 0.000 claims description 7
- 101710155857 C-C motif chemokine 2 Proteins 0.000 claims description 7
- 241000222455 Boletus Species 0.000 claims description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 6
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 6
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 5
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 5
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 230000002519 immonomodulatory effect Effects 0.000 claims description 5
- 230000001965 increasing effect Effects 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 239000012506 Sephacryl® Substances 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 239000002808 molecular sieve Substances 0.000 claims description 4
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 4
- 229920000742 Cotton Polymers 0.000 claims description 3
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 230000002708 enhancing effect Effects 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- OAABHEHWRQAHEJ-UHFFFAOYSA-N butan-1-ol;chloroform Chemical compound ClC(Cl)Cl.CCCCO OAABHEHWRQAHEJ-UHFFFAOYSA-N 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 12
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 abstract description 8
- 102100024333 Toll-like receptor 2 Human genes 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 6
- 230000007365 immunoregulation Effects 0.000 abstract description 4
- 241000233866 Fungi Species 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000003809 water extraction Methods 0.000 abstract description 2
- 241000589291 Acinetobacter Species 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 28
- 235000019441 ethanol Nutrition 0.000 description 20
- 238000004458 analytical method Methods 0.000 description 9
- 239000003642 reactive oxygen metabolite Substances 0.000 description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 8
- 102000004889 Interleukin-6 Human genes 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000031700 light absorption Effects 0.000 description 5
- 238000007069 methylation reaction Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 150000002772 monosaccharides Chemical class 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 4
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000011987 methylation Effects 0.000 description 4
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 229960000583 acetic acid Drugs 0.000 description 3
- 239000005482 chemotactic factor Substances 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 2
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000003832 immune regulation Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 150000003214 pyranose derivatives Chemical class 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- PUAQLLVFLMYYJJ-UHFFFAOYSA-N 2-aminopropiophenone Chemical compound CC(N)C(=O)C1=CC=CC=C1 PUAQLLVFLMYYJJ-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- 241000222382 Agaricomycotina Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 241000960391 Boletales Species 0.000 description 1
- 241001070941 Castanea Species 0.000 description 1
- 235000014036 Castanea Nutrition 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
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 206010053430 Erythrophagocytosis Diseases 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 1
- 241000363139 Hypostomus punctatus Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 241000018650 Pinus massoniana Species 0.000 description 1
- 235000011610 Pinus tabuliformis Nutrition 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000222562 Suillus Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 241000121220 Tricholoma matsutake Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000007864 aqueous solution 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
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum 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
- 210000000987 immune system Anatomy 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000017306 interleukin-6 production Effects 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- -1 saccharide compounds Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing 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
- C08B37/0003—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
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
-
- 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
Abstract
本发明属于食用菌多糖研究领域,公开了一种具有免疫调节活性的点柄乳牛肝菌多糖SGP2‑1及其制备方法和应用。SGP2‑1是由点柄乳牛肝菌子实体热水浸提和分级醇沉提取,并经离子交换柱和凝胶柱分离纯化得到的均一多糖,其主要重复单元的结构如下所示,重均分子量为150.75kDa。经体外免疫调节活性研究,SGP2‑1可被模式识别受体TLR2识别以激活巨噬细胞,显著增强小鼠单核巨噬细胞RAW264.7细胞活力及吞噬能力,促进小鼠单核巨噬细胞RAW264.7产生ROS、NO、细胞因子和趋化因子,以发挥免疫调节作用。本发明的多糖可作为潜在的免疫调节剂,用于功能性食品或药物的开发。
Description
技术领域
本发明属于食用菌多糖研究领域,特别涉及一种具有免疫调节活性的点柄乳牛肝菌多糖SGP2-1及其制备方法和应用。
背景技术
食用菌多糖具有多种功能活性,包括免疫调节、抗肿瘤、抗氧化、抗炎、降血糖血脂等,其中,免疫调节活性是多糖被研究得最广泛、最深入的生理活性,多糖可通过直接或间接方式激活巨噬细胞、T/B淋巴细胞、促进和补充干扰素、白介素、肿瘤坏死因子等多种方式来调节和改善机体的免疫系统,并与其他抗癌、抗病毒等功能相关,因此多糖在功能性食品以及药物开发上具有重要应用价值。
点柄乳牛肝菌(Suillus granulatus),别名松蘑、栗壳牛肝菌,属于担子菌门(Basidiomycota)、层菌纲(Hymenomycetes)、牛肝菌目(Boletales),是油松林地的常见外生菌根,其广泛分布于云南、四川、贵州、黑龙江等省区,点柄乳牛肝菌含有多糖、麦角固醇、人体必需氨基酸、微量元素等多种生物活性物质,具有抗肿瘤、抗氧化、抗菌、抗病毒等多种功能活性。但目前对点柄乳牛肝菌多糖的链结构解析及免疫调节活性未做深入研究。
发明内容
为了克服现有技术的缺点和不足,本发明的首要目的在于提供一种具有免疫调节活性的点柄乳牛肝菌多糖。
本发明的另一目的在于提供一种上述具有免疫调节活性的点柄乳牛肝菌多糖的制备方法;本发明以点柄乳牛肝菌子实体为研究对象,通过热水浸提和分级醇沉提取,经DEAE离子交换柱以及分子筛进行分离纯化并研究其生物活性,解析了点柄乳牛肝菌多糖SGP2-1新成分的单糖组成、糖苷键连接方式、链结构等。
本发明的再一目的在于提供一种上述具有免疫调节活性的点柄乳牛肝菌多糖的应用。
本发明的目的通过下述技术方案实现:
一种具有免疫调节活性的点柄乳牛肝菌多糖SGP2-1,该点柄乳牛肝菌多糖SGP2-1的主要重复单元的结构如下所示,重均分子量为150.75kDa:
所述点柄乳牛肝菌多糖SGP2-1的糖含量为94.84wt%。
所述点柄乳牛肝菌多糖SGP2-1主要由摩尔比为2.4:28.2:1.0的甘露糖、葡萄糖和木糖组成。
上述的点柄乳牛肝菌多糖SGP2-1的制备方法,包括以下步骤:
(1)将粉碎过筛的点柄乳牛肝菌粉末脱脂处理,干燥后得到点柄乳牛肝菌脱脂粉末;
(2)向点柄乳牛肝菌脱脂粉末中加去离子水,提取多次,过滤后合并上清液,减压浓缩,得浓缩液;
(3)浓缩液经分级醇沉、复溶沉淀、除蛋白、透析、冷冻干燥后获得粗多糖冻干粉;
(4)取粗多糖冻干粉配置成粗多糖溶液,经离子交换柱分离洗脱,苯酚硫酸法测定,收集目标组分峰洗脱产物,经浓缩、透析、冷冻干燥后获得SGP2组分;
(5)将SGP2组分经分子筛进一步分离,采用苯酚-硫酸法检测多糖含量,收集目标组分峰洗脱产物,合并后的溶液经浓缩、透析、冷冻干燥后得到点柄乳牛肝菌多糖SGP2-1。
所述步骤(1)具体按照以下步骤:将点柄乳牛肝菌子实体超微粉碎后过100目筛,得点柄乳牛肝菌粉末,按照料液比1g:20mL,向粉末中加入95vt%的乙醇,75℃提取2h,重复操作一次,过滤后收集沉淀并在60℃烘干,得到点柄乳牛肝菌脱脂粉末;
所述步骤(2)具体按照以下步骤:按照料液比1g:20-40mL,向点柄乳牛肝菌脱脂粉末中加去离子水,90℃提取2h,得水提液,重复操作2次,脱脂棉纱过滤后合并上清液,60℃减压浓缩,得浓缩液;
所述步骤(3)具体按照以下步骤:向浓缩液中加入无水乙醇,使乙醇终浓度为30vt%,在4℃静置3h沉淀不溶性多糖,以转速6000r/min离心10min收集上清液;继续往上清液中加入无水乙醇,使乙醇终浓度为80vt%,4℃下醇沉16h,在4℃以转速6000r/min离心10min收集沉淀;将沉淀物溶解于去离子水中,得到多糖溶液;向多糖溶液中加入同等体积的Sevage试剂,所述Sevage试剂为体积比为4:1的氯仿-正丁醇混合液,倾入分液漏斗中剧烈震摇30min,在4℃以转速6000r/min离心10min分层,取上清,重复除蛋白直到两相分界处无白色絮状物,并旋蒸去除有机溶剂;随后将去除蛋白的多糖溶液采用3500Da透析袋透析72h,冷冻干燥得到粗多糖。
所述步骤(4)具体按照以下步骤:将粗多糖配制成浓度为25mg/mL的溶液,经DEAE-Sepharose fast flow离子交换柱分离,依次用浓度为0、0.1、0.2、0.3、0.5、1.0M的NaCl溶液洗脱,洗脱流速为2mL/min,洗脱时间为4min/管,使用全自动部分收集器收集,采用苯酚-硫酸法检测多糖的含量,收集0.1M NaCl溶液洗脱组分,浓缩后采用3500Da透析袋透析72h,冷冻干燥得SGP2组分;
所述步骤(5)具体按照以下步骤:将SGP2组分配制成浓度为8mg/mL的溶液,经Sephacryl S-300HR凝胶渗透柱分离纯化,用0.1M NaCl溶液洗脱,洗脱流速为1mL/min,洗脱时间为8min/管,使用全自动部分收集器收集,采用苯酚-硫酸法测定多糖含量并绘制洗脱曲线,根据洗脱曲线收集第一个组分峰,再经浓缩、3500Da透析袋透析72h、冷冻干燥后得到点柄乳牛肝菌多糖SGP2-1。
上述的点柄乳牛肝菌多糖SGP2-1在制备功能性食品及免疫调节剂药物中的应用。
所述功能性食品及免疫调节剂药物具有增强巨噬细胞活力及吞噬能力,和/或增加巨噬细胞ROS及NO产生量,和/或增加巨噬细胞的细胞因子和趋化因子产生量功效的活性。
所述细胞因子包括TNF-α、IL-6;所述趋化因子包括MCP-1。
上述方法中,与传统醇沉提取方法相比,本发明的点柄乳牛肝菌多糖SGP2-1通过分级醇沉提取出不同组分的多糖,并除掉点柄乳牛肝菌中不溶性多糖(30vt%乙醇浓度醇沉),降低后续得到的可溶性多糖(80vt%乙醇浓度醇沉)分离纯化难度。
所述免疫调节剂可被模式识别受体TLR2识别,通过促进巨噬细胞的ROS和/或NO和/或细胞因子和/或趋化因子的产生量发挥其免疫调节功能。
本发明相对于现有技术具有如下的优点及有益效果:
1、本发明以点柄乳牛肝菌为研究对象,通过热水浸提和分级醇沉提取,经DEAE离子交换柱和分子筛进行分离纯化制备出点柄乳牛肝菌多糖SGP2-1,并确定其具体的活性用途;
2、与传统醇沉提取方法相比,本发明的点柄乳牛肝菌多糖SGP2-1的提取方法优势在于使用分级醇沉,不同浓度的乙醇可提取出不同组分的多糖,除去点柄乳牛肝菌中不溶性多糖,使后续得到的可溶性多糖纯度较高,达到94.84wt%。
3、经测定,点柄乳牛肝菌多糖SGP2-1的重均分子量为150.75kDa,主要由甘露糖、葡萄糖、木糖组成,摩尔比为:2.4:28.2:1.0,具有糖类化合物以及α-异构吡喃糖的特征吸收峰,并结合甲基化和核磁分析,解析SGP2-1的化学结构,确定点柄乳牛肝菌多糖SGP2-1为新物质;
4、点柄乳牛肝菌多糖SGP2-1在40-320μg/mL浓度范围内,与对照组相比,可显著增强巨噬细胞RAW264.7细胞增殖和吞噬中性红的能力;可显著促进巨噬细胞RAW264.7产生ROS和NO;可显著增加巨噬细胞RAW264.7细胞因子(TNF-α、IL-6)和趋化因子(MCP-1)等的产生量;SGP2-1在320μg/mL浓度时,可被模式识别受体TLR2识别,激活巨噬细胞并促进ROS、NO、细胞因子和趋化因子的产生,发挥其免疫调节功能。
附图说明
图1是点柄乳牛肝菌多糖SGP2-1的DEAE离子交换柱层析洗脱图;
图2是点柄乳牛肝菌多糖SGP2-1的Sephacryl S-300HR凝胶柱洗脱图;
图3是点柄乳牛肝菌多糖SGP2-1的紫外吸收光谱;
图4是点柄乳牛肝菌多糖SGP2-1的HPGPC图;
图5是点柄乳牛肝菌多糖SGP2-1的单糖组成图;
图6是点柄乳牛肝菌多糖SGP2-1的红外光谱图;
图7是点柄乳牛肝菌多糖SGP2-1的13C NMR图谱;
图8是点柄乳牛肝菌多糖SGP2-1的1H NMR图谱;
图9是点柄乳牛肝菌多糖SGP2-1的HH-COSY图谱;
图10是点柄乳牛肝菌多糖SGP2-1的HSQC图谱;
图11是点柄乳牛肝菌多糖SGP2-1的HMBC图谱;
图12是点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7的细胞毒性影响图;
图13是点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7吞噬中性红的影响图;
图14是点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7产生ROS影响(A)流式细胞仪分析图、(B)荧光显微图像;
图15是点柄乳牛肝菌多糖SGP2-1对RAW 264.7细胞培养上清(A)NO、(B)TNF-α、(C)IL-6、(D)MCP-1分泌及(E)iNOS、(F)TNF-α、(G)IL-6、(H)MCP-1mRNA表达的影响;
图16是TLR2和TLR4抗体对经点柄乳牛肝菌多糖SGP2-1诱导的巨噬细胞RAW264.7产生NO影响图;
图17是TLR2和TLR4抗体对经点柄乳牛肝菌多糖SGP2-1诱导的巨噬细胞RAW264.7产生IL-6影响图。
具体实施方式
下面结合说明书附图和具体施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。在未作特别说明的情况下,本发明所采用的试剂、设备和方法均为本技术领域常规市购的试剂、设备和常规使用的方法。
实施例1
(一)点柄乳牛肝菌多糖SGP2-1的制备
将点柄乳牛肝菌子实体超微粉碎后过100目筛,获得点柄乳牛肝菌粉末,按照料液比1g:20mL向粉末中加入95vt%的乙醇,75℃提取2h,重复操作一次,过滤后收集沉淀并在60℃烘干,得到点柄乳牛肝菌脱脂粉末;
按照料液比1g:20-40mL向点柄乳牛肝菌脱脂粉末中加去离子水,90℃提取2h,得水提液,重复操作2次,脱脂棉纱过滤后合并上清液,60℃减压浓缩,得浓缩液;
向浓缩液中加入无水乙醇,使乙醇终浓度为30vt%,在4℃静置3h沉淀不溶性多糖,离心收集上清液,所述的离心条件为以转速6000r/min离心10min;继续往上清液中加入无水乙醇,使乙醇终浓度为80vt%,4℃下醇沉16h,离心收集沉淀,所述的离心条件为在4℃以转速6000r/min离心10min,将沉淀溶解于去离子水中,得到多糖溶液;
向多糖溶液中加入同等体积的Sevage试剂(氯仿:正丁醇=4:1,v/v),倾入分液漏斗中剧烈震摇30min,离心分层,取上清,重复除蛋白直到两相分界处无白色絮状物,并旋蒸去除有机溶剂,所述的离心条件为在4℃以转速6000r/min离心10min;随后将去除蛋白的多糖溶液采用3500Da透析袋透析72h,冷冻干燥得到粗多糖。
将粗多糖配制成浓度为25mg/mL的溶液,经DEAE-Sepharose fast flow离子交换柱分离,依次用浓度为0、0.1、0.2、0.3、0.5、1.0M的NaCl溶液洗脱,洗脱流速为2mL/min,洗脱时间为4min/管,采用苯酚-硫酸法检测多糖的含量,洗脱曲线如图1所示,用全自动部分收集器收集0.1M NaCl溶液洗脱组分,浓缩后采用3500Da透析袋透析72h,冷冻干燥得到SGP2组分;
将SGP2组分配制成浓度为8mg/mL的溶液,经Sephacryl S-300HR凝胶渗透柱分离纯化,用0.1M NaCl溶液洗脱,洗脱流速为1mL/min,洗脱时间为8min/管,使用全自动部分收集器收集,采用苯酚-硫酸法测定多糖含量并绘制洗脱曲线,如图2所示,根据洗脱曲线收集第一个组分峰,再经浓缩、3500Da透析袋透析72h,冷冻干燥后得到点柄乳牛肝菌多糖SGP2-1。
二)点柄乳牛肝菌多糖SGP2-1的糖含量测定
以葡萄糖为标准品,准确称取10mg葡萄糖标品,溶于10mL蒸馏水中,使标准溶液浓度为1mg/mL,准确吸取0.05、0.04、0.03、0.02、0.01mL的标准溶液于离心管中,用蒸馏水补足至0.5mL,备用。采用苯酚硫酸法测定多糖含量,步骤如下:吸取150μL糖溶液于1.5-2mL离心管中,加入75μL6wt%苯酚,经涡旋振荡器混匀,加入375μL浓硫酸,混匀,冰上静置3min,沸水浴10min,冷却至室温,测定其在490nm处的吸光值,以葡萄糖浓度为横坐标,以吸光值为纵坐标,绘制标准曲线。将SGP2-1配制为0.1mg/mL溶液,经苯酚硫酸法步骤测定,根据标准曲线y=8.8482x+0.0996(R2=0.9999),计算得到SGP2-1糖含量为94.84wt%。
三)点柄乳牛肝菌多糖SGP2-1的紫外光谱分析
取一定量上述所得点柄乳牛肝菌多糖SGP2-1溶于蒸馏水中,制成浓度为1mg/mL的溶液,在U-2910分光光度计上扫描分析,波长范围为200-400nm,结果如图3所示,其在260nm和280nm均无特征吸收峰,表明SGP2-1中不含核酸和蛋白质。
四)点柄乳牛肝菌多糖SGP2-1的分子量的测定
上述所得点柄乳牛肝菌多糖SGP2-1的分子量通过Waters ACQUITY APC测得,该系统配备Waters ACQUITY APC AQ 900和ACQUITY APC AQ 450柱(2.5μm×4.6mm×150mm),柱温为35℃,流动相为NaNO3(100mM),流速为0.4mL/min,根据从不同分子量(5.2、11.6、23.8、48.6、148.0、273.0、410.0、668.0kDa)的葡聚糖标准中得到的校准曲线,估算了SGP2-1的分子量,结果如图4所示。
五)点柄乳牛肝菌多糖SGP2-1的单糖组成分析
取2mg上述所得点柄乳牛肝菌多糖SGP2-1,用1mL的2M三氟乙酸在110℃水解6h,然后用0.5M PMP衍生化,PMP衍生物在Agilent 1200系列HPLC系统(G1311A Quat泵,G1329AALS进样器,G1315D DAD检测器)上进行分析。
GC-MS条件:Eclipse XDB-C18色谱柱(250mm×4.6mm×5μm),检测温度为30℃,检测波长为250nm,流速为0.8mL/min,流动相为磷酸盐缓冲液(0.1mol/L,pH6.5)和乙腈(84:16,v:v)的混合物。
其中标准品为:甘露糖、核糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、木糖、阿拉伯糖、岩藻糖。结果如图5所示。
由GC-MS结果可知,点柄乳牛肝菌多糖SGP2-1的单糖组成主要由甘露糖、葡萄糖、木糖组成,其摩尔比为:2.4:28.2:1.0。
六)点柄乳牛肝菌多糖SGP2-1的红外光谱分析
将2mg上述所得点柄乳牛肝菌多糖SGP2-1与100mg干燥的溴化钾粉末混匀,充分研磨后压片,在4000-400cm-1的范围内进行红外光谱扫描,结果如图6所示,在3379.14cm-1、2929.28cm-1出现糖类特征吸收峰,在1155.00cm-1、1080.28cm-1、1024.36cm-1的吸收表明特征部分为吡喃糖构型,在852.43cm-1和762.07cm-1处的吸收峰表明SGP2-1存在α构型糖苷键。
七)点柄乳牛肝菌多糖SGP2-1的甲基化分析
将上述所得点柄乳牛肝菌多糖SGP2-1样品置于反应瓶中,加入DMSO,快速加入NaOH粉末,封闭,在超声作用下溶解,再加入碘甲烷反应,最后终止甲基化反应。取甲基化后的多糖,用1mL的2M三氟乙酸水解90min,旋转蒸发仪蒸干。残基加入2mL双蒸水,60mg硼氢化钠还原8h,加入冰醋酸中和,旋蒸,101℃烘箱烘干,然后加入1mL乙酸酐乙酰化100℃反应1h,冷却,然后加入3mL甲苯,减压浓缩蒸干,重复4-5次,以除去多余的醋酐。将乙酰化后的产物用3mL氯仿溶解后转移至分液漏斗,加入少量蒸馏水充分震荡后,除去上层水溶液,如此重复4次。二氯甲烷层以适量的无水硫酸钠干燥,定容10mL,分析采用GCMC-QP 2010气相色谱-质谱联用仪测定乙酰化产物样品。
GC-MS条件:RXI-5SIL MS色谱柱30m×0.25mm×0.25μm;程序升温条件为:起始温度120℃,以4℃/min升温至250℃/min;保持5min;进样口温度为250℃,检测器温度为250℃/min,载气为氦气,流速为1mL/min。结果如下表1所示。
表1-点柄乳牛肝菌多糖SGP2-1甲基化分析结果
八)点柄乳牛肝菌多糖SGP2-1的核磁共振分析
将50mg上述所得点柄乳牛肝菌多糖SGP2-1溶于0.5mL重水(D2O),充分溶解后转移至核磁管中,利用布鲁克核磁共振波谱仪进行检测。核磁共振分析结果如图7-11所示,根据图7-11的核磁图谱对各残基的各个碳和氢的化学位移值进行归属,归属结果如下表2所示。
表2-点柄乳牛肝菌多糖SGP2-1中各糖残基的氢、碳信号归属
结合单糖组成、红外光谱、甲基化和核磁共振分析得SGP2-1的基本重复单元的结构如下:
分析结果表明:SGP2-1主链主要以→4)-α-D-Glcp-(1→组成,而端基α-D-Glcp→通过O-6键连接在主链上。
实施例2
一)点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7的毒性影响
将RAW264.7细胞以5×105个/孔的密度接种于96孔板,于37℃、5%的CO2培养箱中培养24h,使其贴壁;次日弃上清,加入不同浓度的实施例1所得SGP2-1(40、80、160、320μg/mL),空白对照组和阳性对照组分别加入培养基和LPS(100ng/mL)继续培养24h;取出96孔板,弃去旧培养基,加入含有1.5%CCK-8的DMEM培养基(每200μL无血清培养基对应3μLCCK-8原液),于37℃、5%的CO2培养箱中培养1h,之后用酶标仪测定450nm吸光值,结果如图12所示,SGP2-1在40-320μg/mL浓度范围内,对巨噬细胞RAW264.7均无毒性,且促进细胞增殖。
二)点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7的中性红吞噬实验
将RAW264.7细胞以2.5×105个/孔的密度接种于96孔板,于37℃、5%的CO2培养箱中培养24h;弃去旧培养液,加入不同浓度的实施例1所得SGP2-1(40、80、160、320μg/mL),对照组和阳性对照组分别加入培养基和LPS(100ng/mL),培养24h;弃去旧培养基,加入100μL中性红溶液(0.1%),于37℃、5%的CO2培养箱中培养1h,弃去上清并用DPBS洗去残留中性红,加入100μL醋酸乙醇混合溶液(醋酸:乙醇=1:1),室温下裂解2h,之后用酶标仪测定540nm吸光值,结果如图13所示,与对照组相比,SGP2-1在40-320μg/mL浓度范围内,可显著增强巨噬细胞RAW264.7吞噬中性红的能力。
三)点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7释放活性氧(ROS)的影响
将RAW264.7细胞以1×106个/孔的密度接种于6孔板中,于37℃、5%的CO2培养箱中培养24h;弃去旧培养液,加入不同浓度的实施例1所得SGP2-1(40、80、160、320μg/mL),对照组和阳性对照组分别加入培养基和LPS(100ng/mL),培养24h后弃旧培养基,每孔加入10μMDCFA-DA避光孵育30min,通过流式细胞仪和荧光倒置显微镜检测ROS水平。结果如图14所示,SGP2-1在40-320μg/mL浓度范围内,与对照组相比,可显著促进巨噬细胞RAW264.7释放ROS。
四)点柄乳牛肝菌SGP2-1对巨噬细胞RAW264.7产生NO、细胞因子、趋化因子及相关基因表达的影响
将RAW264.7细胞以5×105个/孔的密度接种于96孔板,于37℃、5%的CO2培养箱中培养24h;弃去旧培养液,加入不同浓度的实施例1所得SGP2-1(40、80、160、320μg/mL),对照组和阳性对照组分别加入培养基和LPS(100ng/mL),培养24h后收集细胞上清液,用于测定NO、细胞因子和趋化因子的产生量。NO使用Griess试剂测定,取100μL细胞上清液与等体积Griess试剂混合,避光静置10min后用酶标仪测定542nm处吸光值。细胞因子和趋化因子的产生量及基因表达水平分别采用ELASA试剂盒和qRT-PCR技术测定,结果如图15所示,SGP2-1在40-320μg/mL浓度范围内,可显著上调iNOS、TNF-α、IL-6和MCP-1的基因表达水平,进而促进巨噬细胞RAW264.7释放NO、细胞因子(TNF-α、IL-6)和趋化因子(MCP-1)。
五)点柄乳牛肝菌多糖SGP2-1对巨噬细胞RAW264.7表面模式识别受体的识别
取RAW264.7细胞(5×105个/孔)接种于96孔板,用10μg/mL TLR2、TLR4和TLR2+TLR4抗体预处理1h,加入320μg/mL实施例1所得SGP2-1,培养24h;收集细胞培养上清液,用于测定NO和IL-6。NO使用Griess试剂测定,取100μL细胞上清液与等体积Griess试剂混合,避光静置10min后用酶标仪测定542nm处吸光值。使用ELISA试剂盒测定IL-6水平,结果如图16-17所示,SGP2-1在320μg/mL浓度下,可被模式识别受体TLR2识别,激活巨噬细胞并促进NO、细胞因子和趋化因子的产生,发挥其免疫调节功能。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
2.根据权利要求1所述的一种具有免疫调节活性的点柄乳牛肝菌多糖SGP2-1,其特征在于:所述点柄乳牛肝菌多糖SGP2-1的糖含量为94.84wt%。
3.根据权利要求1所述的一种具有免疫调节活性的点柄乳牛肝菌多糖SGP2-1,其特征在于:所述点柄乳牛肝菌多糖SGP2-1主要由摩尔比为2.4:28.2:1.0的甘露糖、葡萄糖和木糖组成。
4.根据权利要求1~3任一项所述的点柄乳牛肝菌多糖SGP2-1的制备方法,其特征在于包括以下步骤:
(1)将粉碎过筛的点柄乳牛肝菌粉末脱脂处理,干燥后得到点柄乳牛肝菌脱脂粉末;
(2)向点柄乳牛肝菌脱脂粉末中加去离子水,提取多次,过滤后合并上清液,减压浓缩,得浓缩液;
(3)浓缩液经分级醇沉、复溶沉淀、除蛋白、透析、冷冻干燥后获得粗多糖冻干粉;
(4)取粗多糖冻干粉配置成粗多糖溶液,经离子交换柱分离洗脱,苯酚硫酸法测定,收集目标组分峰洗脱产物,经浓缩、透析、冷冻干燥后获得SGP2组分;
(5)将SGP2组分经分子筛进一步分离,采用苯酚-硫酸法检测多糖含量,收集目标组分峰洗脱产物,合并后的溶液经浓缩、透析、冷冻干燥后得到点柄乳牛肝菌多糖SGP2-1。
5.根据权利要求4所述的点柄乳牛肝菌多糖SGP2-1的制备方法,其特征在于:所述步骤(1)具体按照以下步骤:将点柄乳牛肝菌子实体超微粉碎后过100目筛,得点柄乳牛肝菌粉末,按照料液比1g:20mL,向粉末中加入95vt%的乙醇,75℃提取2h,重复操作一次,过滤后收集沉淀并在60℃烘干,得到点柄乳牛肝菌脱脂粉末;
所述步骤(2)具体按照以下步骤:按照料液比1g:20-40mL,向点柄乳牛肝菌脱脂粉末中加去离子水,90℃提取2h,得水提液,重复操作2次,脱脂棉纱过滤后合并上清液,60℃减压浓缩,得浓缩液;
所述步骤(3)具体按照以下步骤:向浓缩液中加入无水乙醇,使乙醇终浓度为30vt%,在4℃静置3h沉淀不溶性多糖,以转速6000r/min离心10min收集上清液;继续往上清液中加入无水乙醇,使乙醇终浓度为80vt%,4℃下醇沉16h,在4℃以转速6000r/min离心10min收集沉淀;将沉淀物溶解于去离子水中,得到多糖溶液;向多糖溶液中加入同等体积的Sevage试剂,所述Sevage试剂为体积比为4:1的氯仿-正丁醇混合液,倾入分液漏斗中剧烈震摇30min,在4℃以转速6000r/min离心10min分层,取上清,重复除蛋白直到两相分界处无白色絮状物,并旋蒸去除有机溶剂;随后将去除蛋白的多糖溶液采用3500Da透析袋透析72h,冷冻干燥得到粗多糖。
6.根据权利要求4所述的点柄乳牛肝菌多糖SGP2-1的制备方法,其特征在于:所述步骤(4)具体按照以下步骤:将粗多糖配制成浓度为25mg/mL的溶液,经DEAE-Sepharose fastflow离子交换柱分离,依次用浓度为0、0.1、0.2、0.3、0.5、1.0M的NaCl溶液洗脱,洗脱流速为2mL/min,洗脱时间为4min/管,使用全自动部分收集器收集,采用苯酚-硫酸法检测多糖的含量,收集0.1M NaCl溶液洗脱组分,浓缩后采用3500Da透析袋透析72h,冷冻干燥得SGP2组分;
所述步骤(5)具体按照以下步骤:将SGP2组分配制成浓度为8mg/mL的溶液,经Sephacryl S-300 HR凝胶渗透柱分离纯化,用0.1M NaCl溶液洗脱,洗脱流速为1mL/min,洗脱时间为8min/管,使用全自动部分收集器收集,采用苯酚-硫酸法测定多糖含量并绘制洗脱曲线,根据洗脱曲线收集第一个组分峰,再经浓缩、3500Da透析袋透析72h、冷冻干燥后得到点柄乳牛肝菌多糖SGP2-1。
7.根据权利要求1~3任一项所述的点柄乳牛肝菌多糖SGP2-1在制备功能性食品及免疫调节剂药物中的应用。
8.根据权利要求7所述的应用,其特征在于:所述功能性食品及免疫调节剂药物具有增强巨噬细胞活力及吞噬能力,和/或增加巨噬细胞ROS及NO产生量,和/或增加巨噬细胞的细胞因子和趋化因子产生量功效的活性。
9.根据权利要求8所述的应用,其特征在于:所述细胞因子包括TNF-α、IL-6;所述趋化因子包括MCP-1。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111133125.8A CN113817076A (zh) | 2021-09-27 | 2021-09-27 | 一种具有免疫调节活性的点柄乳牛肝菌多糖sgp2-1及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111133125.8A CN113817076A (zh) | 2021-09-27 | 2021-09-27 | 一种具有免疫调节活性的点柄乳牛肝菌多糖sgp2-1及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113817076A true CN113817076A (zh) | 2021-12-21 |
Family
ID=78915570
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111133125.8A Pending CN113817076A (zh) | 2021-09-27 | 2021-09-27 | 一种具有免疫调节活性的点柄乳牛肝菌多糖sgp2-1及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113817076A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116640237A (zh) * | 2023-06-15 | 2023-08-25 | 广东医科大学 | 一种知母多糖-锌配合物的制备方法和应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111410699A (zh) * | 2020-04-08 | 2020-07-14 | 广东省微生物研究所(广东省微生物分析检测中心) | 西藏灵芝多糖glp-3及其制备方法与应用 |
-
2021
- 2021-09-27 CN CN202111133125.8A patent/CN113817076A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111410699A (zh) * | 2020-04-08 | 2020-07-14 | 广东省微生物研究所(广东省微生物分析检测中心) | 西藏灵芝多糖glp-3及其制备方法与应用 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116640237A (zh) * | 2023-06-15 | 2023-08-25 | 广东医科大学 | 一种知母多糖-锌配合物的制备方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180193373A1 (en) | Method for Preparing Linseed Polysaccharide Having Antiviral Activity and Immunological Activity, and Use of the Linseed Polysaccharide | |
CN107011453A (zh) | 一种维药恰麻古多糖及其提取方法和应用 | |
CN113817076A (zh) | 一种具有免疫调节活性的点柄乳牛肝菌多糖sgp2-1及其制备方法和应用 | |
CN112759660B (zh) | 一种香橼多糖及其提取方法与应用 | |
CN104861085B (zh) | 板栗种仁α‑1,6‑葡聚糖及其制备方法以及在抗肿瘤药物中的应用 | |
CN112358553B (zh) | 一种多糖sm-0.2m及其制备的抗肿瘤产品 | |
CN114957497B (zh) | 一种滇龙胆酸性多糖及其制备方法与应用 | |
CN113480672B (zh) | 一种类芽孢杆菌的胞外多糖及其应用 | |
CN112794925B (zh) | 一种阳春砂多糖及其制备方法和应用 | |
CN110845638B (zh) | 一种苍术寡糖的分离纯化方法 | |
CN114751995A (zh) | 一种具有抗炎药效作用的西红花花瓣多糖csp1、制备方法及其应用 | |
CN110204627B (zh) | 一种美网柄牛肝菌多糖及其制备方法和应用 | |
CN113896807A (zh) | 一种鲜地黄多糖及其制备方法和应用 | |
CN111690073B (zh) | 一种绒白乳菇多糖及其制备方法和应用 | |
CN114409824B (zh) | 毛霉胞外多糖及其制备方法和应用 | |
CN115109168B (zh) | 一种滇龙胆中性多糖及其制备方法与应用 | |
CN113717296B (zh) | 一种杜仲酸性多糖、提取方法及其在制备抗结肠癌药物中的应用 | |
CN115558035B (zh) | 一种具有免疫调节活性的天麻多糖 | |
CN115572333B (zh) | 提取红汁乳菇多糖类化合物的方法 | |
CN115141288B (zh) | 知母活性多糖、知母粗多糖及制备方法和应用 | |
CN116731222B (zh) | 荨麻鼠李半乳糖醛酸聚糖及其制备方法和应用 | |
CN112062866B (zh) | 一种猴头菌富硒多糖及其制备方法和用途 | |
CN114874346B (zh) | 一种紫丁香蘑多糖及其制备方法和应用 | |
CN110818808B (zh) | 酸性当归多糖asp3、酸性当归多糖-阿霉素共聚物纳米粒以及两者的制备方法和应用 | |
CN108530547B (zh) | 一种阿拉伯半乳聚糖kmcp及其制备方法和在制备免疫调节剂中的应用 |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211221 |