CN117467030A - Pomegranate seed polysaccharide and preparation method and application thereof - Google Patents
Pomegranate seed polysaccharide and preparation method and application thereof Download PDFInfo
- Publication number
- CN117467030A CN117467030A CN202311366145.9A CN202311366145A CN117467030A CN 117467030 A CN117467030 A CN 117467030A CN 202311366145 A CN202311366145 A CN 202311366145A CN 117467030 A CN117467030 A CN 117467030A
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- seed polysaccharide
- pomegranate seed
- pomegranate
- residue
- 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 150
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 147
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 147
- 235000014360 Punica granatum Nutrition 0.000 title claims abstract description 114
- 241000219991 Lythraceae Species 0.000 title claims abstract description 98
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000004440 column chromatography Methods 0.000 claims abstract description 14
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 13
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 11
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims description 26
- 244000294611 Punica granatum Species 0.000 claims description 16
- 239000000126 substance Substances 0.000 claims description 11
- 238000005349 anion exchange Methods 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 235000013305 food Nutrition 0.000 claims description 7
- 230000036541 health Effects 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- SRBFZHDQGSBBOR-KLVWXMOXSA-N beta-L-arabinopyranose Chemical group O[C@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-KLVWXMOXSA-N 0.000 claims description 3
- 229920002678 cellulose Polymers 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 230000004957 immunoregulator effect Effects 0.000 claims description 3
- 230000002519 immonomodulatory effect Effects 0.000 claims description 2
- 230000003385 bacteriostatic effect Effects 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 17
- 210000002540 macrophage Anatomy 0.000 abstract description 17
- 230000000259 anti-tumor effect Effects 0.000 abstract description 7
- 230000007365 immunoregulation Effects 0.000 abstract description 7
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 5
- 230000000242 pagocytic effect Effects 0.000 abstract description 4
- 238000001514 detection method Methods 0.000 abstract description 3
- 230000003832 immune regulation Effects 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 24
- 239000000243 solution Substances 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 20
- 238000010828 elution Methods 0.000 description 20
- 238000012360 testing method Methods 0.000 description 13
- 230000028327 secretion Effects 0.000 description 12
- 238000010521 absorption reaction Methods 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 150000002772 monosaccharides Chemical class 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 9
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 9
- 238000000502 dialysis Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 8
- 102000003777 Interleukin-1 beta Human genes 0.000 description 7
- 108090000193 Interleukin-1 beta Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 7
- 238000002329 infrared spectrum Methods 0.000 description 7
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000003809 water extraction Methods 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 5
- 108090000695 Cytokines Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 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 description 4
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 239000000413 hydrolysate Substances 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 238000005100 correlation spectroscopy Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 3
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 3
- 150000004804 polysaccharides Polymers 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-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
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 102000003390 tumor necrosis factor Human genes 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 240000008086 Echinocereus enneacanthus Species 0.000 description 1
- 235000010837 Echinocereus enneacanthus subsp brevispinus Nutrition 0.000 description 1
- 235000006850 Echinocereus enneacanthus var dubius Nutrition 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
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108090001007 Interleukin-8 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
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- WQZGKKKJIJFFOK-UHFFFAOYSA-N alpha-D-glucopyranose Natural products OCC1OC(O)C(O)C(O)C1O WQZGKKKJIJFFOK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000012010 growth Effects 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
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 150000003214 pyranose derivatives Chemical class 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- 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
The invention discloses a novel pomegranate seed polysaccharide. Having a weight average molecular weight of 2.5X10 4 ~3.5×10 4 Da comprising 83-93% of glucose and 5-13% of arabinose, wherein the main chain residue is alpha- (1- & gt 4) -D-glucose residue, and the branched chain residue isbeta-L-arabinose residue, branching point is positioned at O-6 position of main chain residue. The preparation method comprises the steps of extracting polysaccharide extract from pomegranate seeds and purifying by column chromatography, and is simple and convenient to operate. The activity detection shows that the pomegranate seed polysaccharide can enhance the phagocytic capacity of macrophages, and promote the macrophages to secrete immune regulation, anti-tumor and anti-inflammatory factors. The pomegranate seed polysaccharide has wide application prospect in preparing products with immunoregulation, anti-tumor, anti-inflammatory or antibacterial functions.
Description
Technical Field
The invention relates to the field of natural polymers, in particular to a pomegranate seed polysaccharide, a preparation method and application thereof.
Background
Polysaccharides are a type of high molecular carbohydrates, are generally formed by polymerizing more than 10 monosaccharides through glycosidic bonds, and widely exist in various organisms, and are quite rich in variety and content. Polysaccharides, which are one of the basic molecules constituting living bodies, are widely involved in physiological activities such as proliferation, differentiation, and signal transduction of cells. Research shows that natural polysaccharide has various pharmacological activities such as anti-inflammatory, antiviral, anti-tumor, antioxidant, immunoregulation and the like, and is widely focused by researchers at home and abroad due to the advantages of wide sources, strong safety, small toxic and side effects and the like.
The pomegranate seeds are one of byproducts in the processing process of the pomegranate, and the weight of the pomegranate is about 12 percent, and the pomegranate seeds contain rich nutrients such as cellulose, fatty acid, protein, sugar and the like, and physiological active ingredients such as polyphenol, flavonoid, anthocyanin and the like beneficial to human bodies. The pharmaceutical research shows that the pomegranate seeds can be used as medicines, and the pomegranate stomach strengthening powder and the Tibetan medicine panZhu pill which are taken as the main medicines are taken as the pomegranate seeds and are carried in the traditional Chinese medicine formula of the pharmacopoeia 2020 edition of the people's republic of China. At present, the research on the pomegranate seeds at home and abroad is focused on the pomegranate seed oil, and the pomegranate seed oil has been found to contain various fatty acids, mainly including various unsaturated fatty acids such as oleic acid, palmitic acid, punica granatum acid and the like, and has the activities of resisting oxidation, resisting tumors, regulating blood fat and the like. However, intensive studies on pomegranate seed polysaccharides have not been reported at present.
Disclosure of Invention
The invention aims to develop a novel pomegranate seed polysaccharide, and a preparation method and application thereof.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
the present invention provides a polysaccharide having a weight average molecular weight of 2.5X10 4 ~3.5×10 4 Da, wherein the polysaccharide contains 83-93% of glucose and 5-13% of arabinose, the main chain residue in the structure of the polysaccharide is alpha- (1- & gt 4) -D-glucose residue, the branched chain residue is beta-L-arabinose residue, and the branch point is positioned at the O-6 position of the main chain residue.
In a preferred embodiment, the polysaccharide has a weight average molecular weight of 2.8X10 4 ~3.2×10 4 Da, wherein the Da contains 85-90% of glucose and 7-11% of arabinose.
The above-mentioned contents are the contents of the relative substances.
In the pomegranate seed polysaccharide, the connection mode of the main chain and the branched chain is shown as a formula (I),
in the formula (I), alpha-Glcp represents alpha-D-glucopyranose, and beta-Araf represents beta-L-furanose.
In the present invention, the pomegranate seed polysaccharide is also abbreviated as "PSP".
The invention also provides a preparation method of the pomegranate seed polysaccharide, and the pomegranate seed polysaccharide is obtained by extracting and purifying the pomegranate seeds.
In a preferred embodiment, the preparation method comprises the steps of:
step (1), extracting from pomegranate seeds to obtain polysaccharide extract;
step (2), removing protein and micromolecular impurities in the polysaccharide extract obtained in the step (1) to obtain crude polysaccharide;
and (3) purifying the crude polysaccharide obtained in the step (2) by anion exchange column chromatography and gel column chromatography to obtain pure polysaccharide.
Further, the step (1) adopts a water extraction and alcohol precipitation method for extraction.
Further, in the water extraction and alcohol precipitation method in the step (1), the water extraction liquid ratio is 1g to (25-35) ml, the extraction temperature is 50-70 ℃ and the extraction time is 1.5-2.5 h.
Further, in the water extraction and alcohol precipitation method in the step (1), ethanol is added into the water extraction concentrated solution until the final concentration of the ethanol is 75-85% (v/v), and the mixture is left to stand at 2-6 ℃ after the ethanol is added, so that the ethanol is fully precipitated.
Further, in the water extraction and alcohol precipitation method of the step (1), the operation of "alcohol precipitation" may be repeated several times.
Further, in the step (2), proteins and small molecular substances are removed by using trichloroacetic acid (TCA) method and dialysis. The TCA method is a common method for removing proteins by those skilled in the art. The concentration of TCA in the system of the step (2) is preferably 3-7 wt%, and the dialysis preferably has a molecular weight cut-off of about 3500.
Further, in the step (3), the anion exchange column chromatography is preferably performed using a cellulose anion exchange column, more preferably a DEAE cellulose anion exchange column, and a suitable model is selected from DEAE-52 and DEAE-32.
Further, in step (3), the anion exchange column chromatography includes the steps of: and (3) taking the solution of the crude polysaccharide to an anion exchange column, adopting a salt solution as eluent, intercepting and merging elution sections with high polysaccharide content according to an elution curve of the elution sections and absorbance, concentrating, dialyzing and freeze-drying.
Preferably, the eluent is NaCl solution, and the concentration of the NaCl solution is 0.1-0.5 mol/L.
Preferably, gradient elution is used.
Preferably, the elution flow rate is 0.5-2.0 mL/min.
Further, in the step (3), the gel column chromatography is preferably performed using a Sephadex column, more preferably a Sephadex G series column, and a suitable type is selected from the group consisting of G-50, G-75, G-100 and G-200.
Further, in the step (3), the gel column chromatography includes the steps of: preparing the polysaccharide purified by anion exchange column chromatography into solution, loading on gel column, eluting with deionized water, intercepting and combining the eluting sections with high polysaccharide content according to the eluting curve of the eluting sections and absorbance, concentrating, dialyzing, and lyophilizing to obtain pure polysaccharide. Preferably, the elution flow rate is 0.5-2.0 mL/min.
In the step (3), spin evaporation concentration is preferably adopted for concentration, and the temperature is 45-55 ℃.
Further, in step (3), the dialysis preferably has a molecular weight cut-off of about 3500.
Further, in the step (3), the preferable temperature of freeze-drying is-50 to-60 ℃.
According to the invention, a series of chemical and instrumental analysis methods are adopted to analyze the pomegranate seed polysaccharide, and the result shows that the pomegranate seed polysaccharide is a novel polysaccharide, and can be definitely composed of 83-93% of glucose and 5-13% of arabinose (based on the relative mass), and the chain structure comprises: the main chain is formed by connecting alpha- (1-4) -D-glucose, the branched beta-L-arabinose is connected with the alpha- (1-4, 6) -D-glucose of the main chain through a (1-6) glycosidic bond, and the structure does not contain a triple helix structure. The scanning electron microscope is in an irregular thick slice shape or a strip shape, the surface is smooth and compact, and a plurality of striated protrusions and folds exist.
The invention also carries out activity research on the pomegranate seed polysaccharide, and research results show that the pomegranate seed polysaccharide can enhance phagocytic capacity of macrophages, promote macrophages to release effector molecules NO and cytokines TNF-alpha and IL-1 beta, and has higher immunoregulation, anti-tumor, anti-inflammatory or antibacterial activity.
The punica granatum seed polysaccharide can be used for preparing medicines for treating tumor, resisting inflammation, inhibiting bacteria or regulating immunity.
The punica granatum seed polysaccharide can be used for preparing food, health product, daily chemical product or feed with immunoregulation, anti-inflammatory or antibacterial functions.
The invention also provides an additive for health products, foods, daily chemical products or feeds, which contains the pomegranate seed polysaccharide.
The invention also provides a medicine, a health product, a food, a daily chemical product or a feed, which contains the pomegranate seed polysaccharide or the additive.
The invention has the technical effects that:
the novel polysaccharide is extracted and purified from the pomegranate seeds, has strong immunoregulation, anti-tumor, anti-inflammatory and antibacterial activities, can enhance the phagocytic capacity of macrophages, and can obviously promote the macrophages to release effector molecules NO, and the levels of cytokines TNF-alpha and IL-1 beta are obviously improved compared with blank contrast. The pomegranate seed polysaccharide has the effects of immunoregulation, anti-tumor, anti-inflammatory and bacteriostasis, can be applied to the fields of medicines, health products, foods, daily chemical products or feeds and the like, and has a very broad prospect. In addition, the structural identification and activity research of the pomegranate seed polysaccharide are also beneficial to the full development and utilization of the pomegranate seed resources.
Drawings
Fig. 1 is a flow chart of extraction of pomegranate seed polysaccharide.
FIG. 2 is an elution profile of Granati seed polysaccharide of example (A is DEAE-52 elution; B is SephadexG-100 elution).
FIG. 3 is a GPC measurement flow out chart of pomegranate seed polysaccharide of example one.
FIG. 4 is a graph showing the GC-MS detection results of pomegranate seed polysaccharide.
FIG. 5 is an infrared spectrum of punica granatum seed polysaccharide.
FIG. 6 is a pomegranate seed polysaccharide 1 H-NMR spectrum.
FIG. 7 is a drawing of a pomegranate seed polysaccharide 13 C-NMR spectrum.
FIG. 8 is a pomegranate seed polysaccharide 1 H- 1 H COSY profile.
FIG. 9 is the HSQC spectrum of punica granatum seed polysaccharide.
Fig. 10 is an HMBC spectrum of punica granatum seed polysaccharide.
Fig. 11 is a graph of congo red test results of pomegranate seed polysaccharide.
FIG. 12 is a scanning electron micrograph of pomegranate seed polysaccharide (left, 1000; right, 5000).
FIG. 13 is a graph showing the results of the amount of TNF- α secreted by cells of punica granatum seed polysaccharide.
FIG. 14 is a graph showing the results of secretion of IL-1. Beta. By cells of punica granatum seed polysaccharide.
FIG. 15 is a graph showing the results of the cellular NO secretion amount of pomegranate seed polysaccharide.
Detailed Description
Example 1
1. Preparation of pomegranate seed polysaccharide
1.1 extraction of polysaccharide extractum
Weighing appropriate amount of semen Granati, cleaning, grinding into powder, and oven drying to constant weight. Adding distilled water according to the feed liquid ratio of 1 g:30 ml, immersing, and carrying out ultrasonic-assisted extraction for 1.5h at 60 ℃. The obtained pomegranate seed water extract is filtered by 4 layers of filter cloth and centrifuged at 4000rpm for 15min to remove suspended impurity particles. The supernatant was collected and concentrated on a rotary evaporator at 50 ℃.
Adding a certain volume of absolute ethanol into the concentrated solution to make the final concentration of the ethanol 80% (v/v), standing at 4 ℃ for 12h to fully settle, and discarding the supernatant. Repeating the above ethanol precipitation operation for 3 times, and precipitating polysaccharide thoroughly to remove partial impurities such as pigment, amino acid, lipid, etc. Removing residual solvent by suction filtration, adding ethanol into the extract for washing, removing impurities and dehydrating simultaneously to obtain the polysaccharide extract in powder form, and facilitating storage and weighing.
1.2 preparation of crude polysaccharide
Dissolving the polysaccharide extract powder with distilled water, centrifuging, and collecting supernatant. The TCA method is adopted to remove protein impurities, and the specific operation method is as follows: trichloroacetic acid is added into the polysaccharide solution to make the final concentration of the trichloroacetic acid 5wt%, the solution is uniformly mixed by shaking a shaker for 10min, and the mixture is kept stand for 12h at 4 ℃. Centrifuging at 4000rpm for 15min, and collecting supernatant. Then dialyzing with dialysis bag with molecular weight cut-off 3500 for 48h (water is replaced every 4 and 8 hours, and then water is replaced every 12 hours), removing residual TCA reagent and small molecular impurities, concentrating the dialyzed solution by rotary evaporation at 50deg.C to a proper volume, and vacuum freeze drying to obtain crude polysaccharide.
1.3 purification
Preparing a crude polysaccharide sample into a solution of 10mg/mL, filtering the solution with a 0.45 mu m filter membrane, purifying the solution by chromatography through a DEAE-52 ion exchange column, wherein the loading amount is 5mL, sequentially carrying out gradient elution by 100mL of deionized water and NaCl solutions with the concentration of 0.1, 0.3 and 0.5mol/L respectively, regulating a constant flow pump to enable the flow rate to be 1mL/min, and collecting each filtrate to be 5mL. Detecting polysaccharide content of the filtrate by phenol-sulfuric acid method, and drawing an elution curve by taking test tube number as abscissa and 490nm absorbance as ordinate. According to the elution curve, the elution sections with high polysaccharide content (as shown in the left part of FIG. 2, the elution sections corresponding to peaks a, b and c are collected), and concentrated by rotary evaporation at 50 ℃, and the concentrated solution is dialyzed by a dialysis bag with 3500 molecular weight cut-off (dialysis method is the same as 1.2), and freeze-dried at-60 ℃ for 48 hours.
The polysaccharide obtained by DEAE-52 purification was prepared as a 10mg/mL solution, and after passing through a 0.45 μm filter, the solution was subjected to secondary purification by SephadexG-100 gel column. The sample loading amount is 5mL, deionized water is used for eluting, a constant flow pump is regulated to enable the flow rate to be 1mL/min, and each pipe of filtrate is collected to be 1mL. Detecting polysaccharide content of the filtrate by phenol-sulfuric acid method, and drawing an elution curve by taking test tube number as abscissa and 490nm absorbance as ordinate. Collecting the elution sections with high content of the combined polysaccharide according to the elution curve (as shown in the right part of fig. 2, collecting the elution sections corresponding to PSP peaks of the pomegranate seed polysaccharide), concentrating by rotary evaporation at 50 ℃, dialyzing the concentrated solution by using a dialysis bag with molecular weight cut-off of 3500, and then freeze-drying (the dialysis and freeze-drying method is the same as the operation in the DEAE-52 purification step) to obtain the pure pomegranate seed polysaccharide.
The polysaccharide content in the filtrate is detected by adopting a phenol-sulfuric acid method, and the specific operation is as follows: the reaction was carried out in a ratio (volume ratio) of filtrate sample to 5% phenol to concentrated sulfuric acid=1:1:5, and after thoroughly mixing with a vortex mixer, the mixture was allowed to stand for 30 minutes, and absorbance per tube was measured at 490nm with a multifunctional microplate reader. And drawing an elution curve by taking the test tube number as an abscissa and the absorbance as an ordinate. And judging the solution containing polysaccharide according to the elution curve.
2. Structure determination of pomegranate seed polysaccharide
2.1 determination of molecular weight
Molecular weight was determined by Gel Permeation Chromatography (GPC).
The system is calibrated in advance by using an ACQUITY ARC high performance liquid chromatography system and a Waters Ultrahydrogel chromatographic column and a Waters RI differential detector, the concentration of the solution of the pomegranate seed polysaccharide is 2mg/mL, and a standard curve is drawn by taking the retention time as an abscissa and taking the logarithm of the average molecular weight of a PEG standard as an ordinate.
The GPC measurement flow out curve (FIG. 3) of the pomegranate seed polysaccharide showed a single peak, good symmetry, and a weight average molecular weight (Mw) of 2.98X10 4 Da。
2.2 analysis of monosaccharide composition
The monosaccharide composition was analyzed using gas chromatography-mass spectrometry (GC-MS).
The pomegranate seed polysaccharide is subjected to acid hydrolysis by a reference method, and the pomegranate seed polysaccharide hydrolysate and the standard monosaccharide are respectively acetylated, and the supernatant is respectively collected for GC-MS analysis.
The specific method for acid hydrolysis comprises the following steps: accurately weighing 10.0mg of punica granatum seed polysaccharide in ampoule bottle, adding 2.0ml of 2mol/L trifluoroacetic acid (TFA) solution, sealing, and hydrolyzing at 100deg.C for 6h. Removing hydrolysate by rotary evaporation at 40 ℃ to obtain the pomegranate seed polysaccharide hydrolysate.
The specific method for acetylation comprises the following steps: sequentially adding 0.5 mL pyridine and 10mg hydroxylamine hydrochloride into the pomegranate seed polysaccharide hydrolysate, sealing, oscillating at constant temperature in a fume hood at 100 ℃ for 30min, taking out, cooling to room temperature, adding 0.6mL acetic anhydride, sealing, continuing to oscillate at 100 ℃ for 30min, taking out, cooling to room temperature, and evaporating at 60 ℃ under reduced pressure and rotation. 4mL of chloroform was added for dissolution, centrifuged at 8000rpm for 5min, and the supernatant was taken for use. Standard monosaccharides (glucose, rhamnose, arabinose, fucose, xylose, mannose, galactose) were each taken 2mg in round bottom flasks, 0.6mL pyridine, 12 mg hydroxylamine hydrochloride were added separately, sealed and the subsequent steps were consistent with acetylation of pomegranate seed polysaccharide hydrolysate.
GC-MS equipment was from agilent technologies limited. The system conditions are as follows: DB-5MS chromatographic column (3 m x 0.25mm x 0.25 μm), sample inlet temperature 250 ℃, helium as carrier gas, flow rate 1mL/min, split ratio 20:1, sample volume 1 μl. Temperature program: the temperature is kept at 100 ℃ for 1min, the temperature is raised to 160 ℃ at 5 ℃/min, then the temperature is raised to 240 ℃ at 40 ℃/min, and the temperature is kept for 10min. The ion source temperature is 230 ℃, and the mass number is 30-550.
The monosaccharide composition of the punica granatum seed polysaccharide, analyzed by GC-MS (fig. 4), included glucose (88.22%) and arabinose (9.36%) on a relative mass basis.
2.3 Infrared Spectrometry analysis
Weighing 1-2 mg of pomegranate seed polysaccharide powder, placing into an agate mortar, adding 150-200 mg of dried KBr crystal, grinding uniformly, tabletting, and using an infrared spectrometer at 4000-400 cm -1 The infrared spectrum was recorded by in-range scanning.
The infrared spectrum scan results are shown in fig. 5 and table 1, which show: 3330cm -1 The strong absorption peak at the site is caused by O-H stretching vibration, which indicates that intermolecular and intramolecular hydrogen bonds exist; 2937cm -1 The weak absorption peak at the position is the absorption of C-H stretching vibration of polysaccharide; 1730cm -1 The absence of absorption peaks indicates that the structure is most likely free of uronic acid; 1633cm -1 The absorption peak at the position comes from the variable angle vibration of H-O-H, which indicates that the sample has bound water; 1418cm -1 The absorption peak at the position represents C-H angular vibration of polysaccharide; 1000-1200 cm -1 1131, 1102, 1047cm -1 The three absorption peaks indicate that the pomegranate seed polysaccharide is mainly composed of pyranose; 925 and 851cm -1 The two absorption peaks indicate that the glycosidic linkage exists in both the alpha and beta configuration.
TABLE 1 Infrared Spectroscopy analysis results of Granati seed polysaccharide
2.4 Nuclear magnetic resonance spectroscopy
Weighing 10mg of freeze-dried punica granatum seed polysaccharide sample, dissolving in appropriate amount of 99.9% D 2 O and transferred to a nuclear magnetic tube. Determination of polysaccharide Using Bruker AVANCE NEO 500 Nuclear magnetic resonance spectrometer 1 H-NMR spectrum, 13 C-NMR spectrum, and two-dimensional spectrum 1 H- 1 H COSY, HSQC and HMBC. Nuclear magnetic data were analyzed using MestReNova software.
The C, H signal assignment of the sugar residues is shown in Table 2.
TABLE 2 pomegranate seed polysaccharide 13 C and C 1 Chemical shift of H
* Is a signal masked by the solvent peak
Bonding of 1 H- 1 H COSY, HSQC spectra (FIGS. 8, 9), will 13 The anomeric signals delta 94.91, 94.82, 106.40 ppm and in the C-NMR spectrum 1 The anomeric signals delta 5.39, 5.21, 4.78ppm in the H-NMR spectra were assigned to → 4) -alpha-Glcp- (1 → 4, 6) -alpha-Glcp- (1 → and beta-Araf- (1 → respectively, which were designated as sugar residues A, B, C. Sugar residue C-H related signals by HMBC spectra (FIG. 10), respectivelyThe connection modes are A1-B4, B1-A4 and C1-B6. Combining the monosaccharide composition analysis and the infrared spectrum functional group information analysis, the repeated unit shown as the formula (II) is deduced to exist in the pomegranate seed polysaccharide.
2.5 Congo red test
2.0mL of pomegranate seed polysaccharide solution (1 mg/mL) and 2.0mL of Congo red reagent (100 mu M, which is used at present) are mixed, and a proper amount of NaOH solution (2M) is added to make the final concentration of NaOH in the mixed solution be 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5M respectively. And after standing for 10min at room temperature, scanning in a wavelength range of 400-600 nm by adopting a multifunctional enzyme-labeled instrument, and recording the maximum absorption wavelength of the mixed solution with different concentrations. Congo red reagent without polysaccharide was used as a control and the rest was the same. Distilled water was used as a blank for baseline correction.
The congo red test results are shown in fig. 11. The maximum absorption wavelength of the mixed solution of pomegranate seed polysaccharide and congo red was red shifted compared to the control group, indicating that both may form a complex. However, the phenomenon that the maximum absorption wavelength of the mixed solution is firstly increased, then rapidly decreased and then tends to be stable along with the increase of the concentration of NaOH does not occur, which indicates that the pomegranate seed polysaccharide does not contain a triple helix structure.
2.6 scanning Electron microscope analysis
And 5mg of pomegranate seed polysaccharide sample is taken and adhered on the conductive adhesive, the redundant sample is blown away by a dust ball, the nano gold film is sprayed on the surface of the sample and then is put into a scanning electron microscope, and the surface morphological characteristics of the sample are observed under different magnifications. Electron microscopy imaging at magnifications of 1000 and 5000 (fig. 12) showed that the punica granatum seed polysaccharide exhibited an irregular thick flake or strip shape, a smooth and dense surface, and many striated protrusions and wrinkles.
3. Activity study of pomegranate seed polysaccharide
3.1 the activity of the pomegranate seed polysaccharide was studied as follows:
(1) Cell culture
RAW264.7 cells are taken from a liquid nitrogen tankAfter resuscitating, the cells were cultured in DMEM complete medium containing 10% fetal bovine serum (v/v), 1% penicillin and streptomycin, and incubated at 37℃with 5% CO 2 Is cultured in a constant temperature incubator. And (3) replacing the culture solution regularly, observing the growth condition of the cells by using a microscope, and carrying out passage when the cell adhesion area reaches 70% -80%.
(2) Experimental grouping and administration
The cells in logarithmic growth phase were grown at 5X 10 4 Is seeded in 96-well plates, three replicate wells are provided per group. After 24h of cell attachment, the original culture medium is discarded, 100 mu L of DMEM culture medium (without serum) and DMEM culture medium (without serum) containing pomegranate seed polysaccharide are respectively added according to groups, and the culture is continued in a constant temperature incubator for 24h. The grouping is shown in the following table:
(3) ELISA for detecting secretion level of RAW264.7 cytokines TNF-alpha and IL-1 beta
100. Mu.L of the cultured cell culture medium per well was collected in a new well plate as a sample to be tested. The standard was subjected to double dilution (TNF-. Alpha.0, 31.25, 62.5, 125, 250, 500, 1000, 2000 pg/mL; IL-1β:0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 pg/mL) according to the method described in the instructions of the ELISA kit for TNF-. Alpha.and IL-1β (purchased from the Sysmetic bioengineering Co., ltd.), a standard curve was drawn, and the amounts of TNF-. Alpha.and IL-1β in the cell culture medium were calculated from the standard curve.
(4) Griess method for detecting NO release amount of RAW264.7 cells
Based on the step (2), the experimental group was changed to a blank group, a positive control group (1. Mu.g/mL LPS), and an experimental group (PSP with concentrations of 5, 50, 100, 200, 500. Mu.g/mL, respectively). The cultured cells were collected, lysed, centrifuged and the supernatant was retained according to the instructions of the NO detection kit (purchased from the pitaya biotechnology limited). Supernatant and Griess reagent were added to a 96-well plate at a ratio of 1:2, respectively, and mixed well. After 15min of standing, absorbance was measured at 540 and nm with a microplate reader.
(5) Data analysis
Experimental data are shown as mean ± Standard Deviation (SD), using SPSS 26.0 software for analysis of the fractional variance (ANOVA) of 4.3.2 data, using the Waller-Dunca test for analysis of the significance of the differences, P <0.01 indicates that the differences are of great significance.
3.2 Effect of Granati seed polysaccharide on macrophage secretion of cytokines
Mouse mononuclear macrophage leukemia cells (RAW 264.7) are classical cell models that mimic the immune response capacity of macrophages in vivo, and drugs stimulate macrophages to exert an immunomodulatory effect mainly through several pathways: (1) enhancing phagocytic capacity of macrophages; (2) Stimulating macrophages to secrete NO and other inflammatory factors, and eliminating microorganisms; (3) Stimulating macrophage to secrete Tumor Necrosis Factor (TNF), IL-6, IL-8 and other interleukins, and regulating the immune response of organism. Lipopolysaccharide (LPS) is an intracellular toxin that stimulates immune cells to produce an immune response, and is commonly used as a positive control in experiments. TNF-alpha and IL-1 beta are both important cytokines released during macrophage activation, can be used as an index for evaluating the immunoregulatory activity of polysaccharide, and can be used for primarily evaluating the immunoregulatory activity of the pomegranate seed polysaccharide by detecting the content of cytokines in RAW264.7 cells co-cultured with the pomegranate seed polysaccharide through a TNF-alpha and IL-1 beta enzyme-linked immunosorbent assay kit.
The secretion levels of TNF- α and IL-1β in RAW264.7 cells at various concentrations of PSP were measured by the procedure described in (one) (FIGS. 13, 14). The result shows that compared with a blank Control group (Control), PSP can obviously promote the secretion of TNF-alpha and IL-1 beta of RAW264.7 cell macrophages at a lower concentration of 50 mug/mL; wherein the secretion amounts of TNF-alpha and IL-1 beta are 1303.40pg/mL and 99.41pg/mL respectively, and 70.06% and 97.47% of the LPS positive control group are reached respectively. At a high dose concentration of 400. Mu.g/mL, there was no significant difference between TNF-. Alpha.secretion and LPS positive control, but IL-1β secretion was relatively decreased, only 78.80% of LPS, indicating that this concentration may induce RAW264.7 cells to develop resistance. The total result shows that the pomegranate seed polysaccharide can effectively promote RAW264.7 cells to release cytokines, and has higher immunoregulation, anti-tumor and anti-inflammatory activities.
3.3 Effect of Granati seed polysaccharide on macrophage secretion of NO
NO is one of main effector molecules generated by macrophages in an activated state, plays a role in destroying pathogenic microorganisms and the like, participates in various physiological functions such as blood pressure regulation, nerve signal transmission and the like, and is generally used as an important index for evaluating the immunostimulating capability of polysaccharide. The results show (FIG. 15) that compared with the blank Control group (Control), the concentration range of the pomegranate seed polysaccharide is 5-500 mug/mL, and the NO secretion of RAW264.7 cells can be obviously promotedP<0.01 Shows stronger immune response, anti-inflammatory and antibacterial activity. The cells have the strongest ability to secrete NO at a concentration of 50 mug/mL, reaching 84.64% of LPS positive control group, and then the secretion amount of NO is reduced with the increase of the concentration, which is probably that the high-concentration pomegranate seed polysaccharide causes the cells to generate drug resistance.
Example two
The second embodiment of the method for preparing pomegranate seed polysaccharide is different from the first embodiment in that:
conditions for extracting polysaccharide extract: the feed liquid ratio is 1 g:30 ml, and leaching is carried out for 2 hours at 65 ℃;
purification: when DEAE-52 ion exchange column chromatography is adopted, the flow rate of the constant flow pump is 2mL/min.
Other conditions were the same as in example one.
The polysaccharide structure was examined in the same manner as in example one, and the weight average molecular weight (Mw) of the punica granatum seed polysaccharide of example two was determined to be 3.25X10 4 Da, its monosaccharide composition includes glucose (89.60%) and arabinose (6.93%).
The infrared spectrum, nuclear magnetic resonance spectrum, congo red test and scanning electron microscope analysis test results of the pomegranate seed polysaccharide prepared in the second embodiment are basically consistent with those of the pomegranate seed polysaccharide in the first embodiment.
The activity of the pomegranate seed polysaccharide is measured by adopting the same method as that of the first embodiment, and the result shows that the pomegranate seed polysaccharide prepared in the second embodiment has the same activity as that of the first embodiment, can effectively promote RAW264.7 cells to release cytokines, and can obviously promote RAW264.7 cells to secrete NO.
Example III
The preparation method of the pomegranate seed polysaccharide in the embodiment is different from that in the embodiment:
conditions for extracting polysaccharide extract: the feed liquid ratio is 1 g:35 ml, and leaching is carried out for 2.5 hours at 55 ℃;
and in the purification step, when SephadexG-75 gel column chromatography is adopted, the flow rate of the constant flow pump is 2mL/min.
Other conditions were the same as in example one.
The polysaccharide structure was examined in the same manner as in example one, and the weight average molecular weight (Mw) of the punica granatum seed polysaccharide of example one was determined to be 2.63X10 4 Da, its monosaccharide composition includes glucose (85.62%) and arabinose (10.18%).
The infrared spectrum, nuclear magnetic resonance spectrum, congo red test and scanning electron microscope analysis test results of the pomegranate seed polysaccharide prepared in the third embodiment are basically consistent with those of the pomegranate seed polysaccharide in the first embodiment.
The activity of the pomegranate seed polysaccharide was measured by the same method as in example one, and the result shows that the pomegranate seed polysaccharide prepared in example three had the same activity as the pomegranate seed polysaccharide in example one.
Example IV
The preparation method of the fourth pomegranate seed polysaccharide is different from the first embodiment:
and in the purification step, the flow rate of a constant flow pump is 1.5mL/min when DEAE-52 ion exchange column chromatography is adopted, and the flow rate of the constant flow pump is 1.5mL/min when SephadexG-100 gel column chromatography is adopted. Other conditions were the same as in example one.
The polysaccharide structure was examined in the same manner as in example one, and the weight average molecular weight (Mw) of the punica granatum seed polysaccharide of example four was 3.05X10 4 Da, its monosaccharide composition includes glucose (90.08%) and arabinose (6.43%).
The infrared spectrum, nuclear magnetic resonance spectrum, congo red test and scanning electron microscope analysis test results of the pomegranate seed polysaccharide prepared in the fourth embodiment are basically consistent with those of the pomegranate seed polysaccharide in the first embodiment.
The activity of the pomegranate seed polysaccharide was measured by the same method as in example one, and the result shows that the pomegranate seed polysaccharide prepared in example four had the same activity as the pomegranate seed polysaccharide in example one.
Claims (9)
1. A pomegranate seed polysaccharide is characterized in that the weight average molecular weight is 2.5X10 4 ~3.5×10 4 Da, wherein contains 83-93% of glucose and 5-13% of arabinose, the main chain residue in the structure is alpha- (1- & gt 4) -D-glucose residue, the branched chain residue is beta-L-arabinose residue, and the branch point is positioned at the O-6 position of the main chain residue.
2. The pomegranate seed polysaccharide according to claim 1, characterized in that it has a weight average molecular weight of 2.8x10 4 ~3.2×10 4 Da, wherein the Da contains 85% -90% of glucose and 7% -11% of arabinose.
3. The method for preparing the pomegranate seed polysaccharide according to claim 1 or 2, wherein the pomegranate seed polysaccharide is obtained by extracting and purifying pomegranate seeds.
4. A method of preparation according to claim 3, comprising the steps of:
step (1), extracting from pomegranate seeds to obtain polysaccharide extract;
step (2), removing protein and micromolecular impurities in the polysaccharide extract obtained in the step (1) to obtain crude polysaccharide;
and (3) purifying the crude polysaccharide obtained in the step (2) by anion exchange column chromatography and gel column chromatography to obtain pure polysaccharide.
5. The method according to claim 4, wherein the anion exchange column in the step (3) is a cellulose anion exchange column, and the gel column is a sephadex column.
6. Use of the punica granatum seed polysaccharide of claim 1 or 2 for the preparation of a medicament for the treatment of tumors, anti-inflammatory, bacteriostatic or immunomodulatory.
7. Use of the pomegranate seed polysaccharide according to claim 1 or 2 for preparing food, health products, daily chemicals or feed with immunoregulatory, anti-inflammatory or bacteriostatic functions.
8. An additive for food, health products, daily chemicals or feeds, characterized by comprising the pomegranate seed polysaccharide according to claim 1 or 2.
9. A pharmaceutical, health product, food, daily chemical product or feed comprising the pomegranate seed polysaccharide of claim 1 or 2 or the additive of claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311366145.9A CN117467030A (en) | 2023-10-20 | 2023-10-20 | Pomegranate seed polysaccharide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311366145.9A CN117467030A (en) | 2023-10-20 | 2023-10-20 | Pomegranate seed polysaccharide and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117467030A true CN117467030A (en) | 2024-01-30 |
Family
ID=89634006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311366145.9A Pending CN117467030A (en) | 2023-10-20 | 2023-10-20 | Pomegranate seed polysaccharide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117467030A (en) |
-
2023
- 2023-10-20 CN CN202311366145.9A patent/CN117467030A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10835552B2 (en) | Method for preparing linseed polysaccharide having antiviral activity and immunological activity, and use of the linseed polysaccharide | |
| CN114163545B (en) | Lycium barbarum polysaccharide and application thereof in reducing blood sugar | |
| CN113278091A (en) | Porphyridium polysaccharide and preparation method and application thereof | |
| Wu et al. | Macrophage immunity promotion effect of polysaccharide LGP-1 from Guapian tea via PI3K/AKT and NF-κB signaling pathway | |
| Chen et al. | Structural characterization and biological activities of a novel polysaccharide containing N-acetylglucosamine from Ganoderma sinense | |
| CN114591448A (en) | Phellinus igniarius sporophore mannogalactan and preparation and application thereof | |
| CN113121718B (en) | Roselle polysaccharide PSGP-2 and preparation method and application thereof | |
| Zhang et al. | Preparation and structural characterization of acid-extracted polysaccharide from Grifola frondosa and antitumor activity on S180 tumor-bearing mice | |
| CN116217745B (en) | Vine tea polysaccharide, preparation method and application | |
| CN108892732B (en) | Preparation method and application of Jinchang jujube polysaccharide with immunoregulation function | |
| CN117467030A (en) | Pomegranate seed polysaccharide and preparation method and application thereof | |
| CN112794925B (en) | Amomum villosum polysaccharide and preparation method and application thereof | |
| CN114524887B (en) | Method for separating and characterizing ginseng polysaccharide with pharmaceutical activity function | |
| CN109796538B (en) | Method for improving biological activity of porphyra yezoensis polysaccharide | |
| CN112920287A (en) | Amomum villosum polysaccharide with immunoregulation effect and preparation method and application thereof | |
| CN116655820B (en) | Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics | |
| CN115746157B (en) | Delicious russula polysaccharide, and preparation method and application thereof | |
| CN116925962B (en) | Bacillus amyloliquefaciens JM033 exopolysaccharide with intestinal flora and immunoregulatory function and application thereof | |
| CN116731222B (en) | Nettle rhamnogalacturonan and preparation method and application thereof | |
| CN110894244B (en) | Structure of ground beetle polysaccharide and application thereof | |
| CN114409824B (en) | Mucor exopolysaccharide and preparation method and application thereof | |
| CN115043956B (en) | Sambucus williamsii polysaccharide, polysaccharide composition and application thereof | |
| CN116731217A (en) | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics | |
| CN116655820A (en) | Ampelopsis grossedentata acidic polysaccharide AGP-3a, extraction and separation method thereof and application thereof in preparation of anti-inflammatory cosmetics | |
| CN116284490A (en) | Wild mulberry silkworm chrysalis polysaccharide and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication |