CN115746161B - 一种改性岩藻多糖化合物及其制备方法和应用 - Google Patents
一种改性岩藻多糖化合物及其制备方法和应用 Download PDFInfo
- Publication number
- CN115746161B CN115746161B CN202211525744.6A CN202211525744A CN115746161B CN 115746161 B CN115746161 B CN 115746161B CN 202211525744 A CN202211525744 A CN 202211525744A CN 115746161 B CN115746161 B CN 115746161B
- Authority
- CN
- China
- Prior art keywords
- fucoidin
- modified
- compound
- acid
- acetylsalicylic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 98
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229960001138 acetylsalicylic acid Drugs 0.000 claims abstract description 37
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 29
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims abstract description 16
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 12
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 12
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 11
- 229940097043 glucuronic acid Drugs 0.000 claims abstract description 11
- 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 claims abstract description 10
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 claims abstract description 10
- 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 claims abstract description 9
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims abstract description 7
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims abstract description 7
- 230000003647 oxidation Effects 0.000 claims abstract description 7
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 7
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims abstract description 6
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 claims abstract description 6
- 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 claims abstract description 6
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 claims abstract description 6
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 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 5
- 229930182830 galactose Natural products 0.000 claims abstract description 5
- 229920000855 Fucoidan Polymers 0.000 claims description 39
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 24
- 150000004676 glycans Chemical class 0.000 claims description 24
- 229920001282 polysaccharide Polymers 0.000 claims description 24
- 239000005017 polysaccharide Substances 0.000 claims description 24
- 238000006731 degradation reaction Methods 0.000 claims description 19
- -1 acetylsalicylic acid imidazole ester Chemical class 0.000 claims description 18
- 230000015556 catabolic process Effects 0.000 claims description 18
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 14
- 238000005886 esterification reaction Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical group S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 238000011065 in-situ storage Methods 0.000 claims description 11
- 238000005406 washing Methods 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 8
- 230000000259 anti-tumor effect Effects 0.000 claims description 7
- 238000010525 oxidative degradation reaction Methods 0.000 claims description 7
- 125000005273 2-acetoxybenzoic acid group Chemical group 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 230000032050 esterification Effects 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 5
- 238000010168 coupling process Methods 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 230000000593 degrading effect Effects 0.000 claims description 5
- 238000012869 ethanol precipitation Methods 0.000 claims description 5
- 230000002194 synthesizing effect Effects 0.000 claims description 5
- 230000009471 action Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 230000002292 Radical scavenging effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000001590 oxidative effect Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 15
- 230000004048 modification Effects 0.000 abstract description 11
- 238000012986 modification Methods 0.000 abstract description 11
- 230000006907 apoptotic process Effects 0.000 abstract description 10
- 230000005764 inhibitory process Effects 0.000 abstract description 7
- 230000002000 scavenging effect Effects 0.000 abstract description 7
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 abstract description 4
- 230000036541 health Effects 0.000 abstract description 4
- 201000005249 lung adenocarcinoma Diseases 0.000 abstract description 4
- 230000021736 acetylation Effects 0.000 abstract description 2
- 238000006640 acetylation reaction Methods 0.000 abstract description 2
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000012010 growth Effects 0.000 abstract description 2
- 239000012190 activator Substances 0.000 abstract 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 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 49
- 210000004027 cell Anatomy 0.000 description 29
- 239000000523 sample Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 238000010521 absorption reaction Methods 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 150000003254 radicals Chemical class 0.000 description 12
- 230000004071 biological effect Effects 0.000 description 9
- 150000002772 monosaccharides Chemical class 0.000 description 8
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 8
- 238000002156 mixing Methods 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 230000003078 antioxidant effect Effects 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 6
- 238000001914 filtration Methods 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 229960004889 salicylic acid Drugs 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
- 230000001028 anti-proliverative effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 229910052742 iron Inorganic materials 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- BUTHHXZGIQCRQJ-UHFFFAOYSA-N 2-acetyloxybenzoic acid;1h-imidazole Chemical compound C1=CNC=N1.CC(=O)OC1=CC=CC=C1C(O)=O BUTHHXZGIQCRQJ-UHFFFAOYSA-N 0.000 description 2
- BSYNRYMUTXBXSQ-FOQJRBATSA-N 59096-14-9 Chemical compound CC(=O)OC1=CC=CC=C1[14C](O)=O BSYNRYMUTXBXSQ-FOQJRBATSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- JUUBCHWRXWPFFH-UHFFFAOYSA-N Hydroxytyrosol Chemical compound OCCC1=CC=C(O)C(O)=C1 JUUBCHWRXWPFFH-UHFFFAOYSA-N 0.000 description 2
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000007515 enzymatic degradation Effects 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 230000006882 induction of apoptosis Effects 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- 241000512259 Ascophyllum nodosum Species 0.000 description 1
- 101500000959 Bacillus anthracis Protective antigen PA-20 Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- PNEFMUGLHZEHKK-UHFFFAOYSA-N Cl.NO.CCO Chemical compound Cl.NO.CCO PNEFMUGLHZEHKK-UHFFFAOYSA-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
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000199919 Phaeophyceae Species 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
- 108010087230 Sincalide Proteins 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 235000006092 Stevia rebaudiana Nutrition 0.000 description 1
- QFVOYBUQQBFCRH-UHFFFAOYSA-N Steviol Natural products C1CC2(C3)CC(=C)C3(O)CCC2C2(C)C1C(C)(C(O)=O)CCC2 QFVOYBUQQBFCRH-UHFFFAOYSA-N 0.000 description 1
- 239000004383 Steviol glycoside Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241001261506 Undaria pinnatifida Species 0.000 description 1
- 229940068372 acetyl salicylate Drugs 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000032683 aging 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
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- UBXYXCRCOKCZIT-UHFFFAOYSA-N biphenyl-3-ol Chemical group OC1=CC=CC(C=2C=CC=CC=2)=C1 UBXYXCRCOKCZIT-UHFFFAOYSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- UZIWRCBLXLKBID-UHFFFAOYSA-N dodecasodium sulfuric acid tetraborate Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-].[O-]B([O-])[O-].OS(O)(=O)=O UZIWRCBLXLKBID-UHFFFAOYSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182480 glucuronide Natural products 0.000 description 1
- 150000002340 glycosyl compounds Chemical class 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 229940095066 hydroxytyrosol Drugs 0.000 description 1
- 235000003248 hydroxytyrosol Nutrition 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000012844 infrared spectroscopy analysis Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 238000009160 phytotherapy Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- RYVMUASDIZQXAA-UHFFFAOYSA-N pyranoside Natural products O1C2(OCC(C)C(OC3C(C(O)C(O)C(CO)O3)O)C2)C(C)C(C2(CCC3C4(C)CC5O)C)C1CC2C3CC=C4CC5OC(C(C1O)O)OC(CO)C1OC(C1OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OCC(O)C(O)C1O RYVMUASDIZQXAA-UHFFFAOYSA-N 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 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 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 229940032084 steviol Drugs 0.000 description 1
- 229930182488 steviol glycoside Natural products 0.000 description 1
- 235000019411 steviol glycoside Nutrition 0.000 description 1
- 150000008144 steviol glycosides Chemical class 0.000 description 1
- 235000019202 steviosides Nutrition 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
- A61K36/02—Algae
- A61K36/03—Phaeophycota or phaeophyta (brown algae), e.g. Fucus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
-
- 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
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Mycology (AREA)
- Polymers & Plastics (AREA)
- Dermatology (AREA)
- Alternative & Traditional Medicine (AREA)
- Botany (AREA)
- Toxicology (AREA)
- Materials Engineering (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medical Informatics (AREA)
- Birds (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
本发明公开一种改性岩藻多糖化合物及其制备方法和应用。所述改性岩藻多糖化合物由岩藻糖、鼠李糖、阿拉伯糖、半乳糖、木糖、甘露糖、葡萄糖醛酸、半乳糖醛酸、甘露糖醛酸和古洛糖醛酸组成，先采用H2O2‑VC联合氧化法制备低分子量的岩藻多糖，然后以乙酰水杨酸为接枝物，以N,N’‑羰基二咪唑为活化剂，对降解后的岩藻多糖进行乙酰化改性，得到改性岩藻多糖化合物。本发明改性岩藻多糖化合物对超氧阴离子自由基清除效果较好，对人肺腺癌细胞株A549生长具有显著抑制作用和促凋亡作用。本改性岩藻多糖化合物获取方法简单，成本较低，适于工业化生产并在临床及相关医疗保健领域应用前景广阔。
Description
技术领域
本发明涉及食品资源开发与应用以及生物、医药技术领域，具体涉及一种改性岩藻多糖化合物及其制备方法和应用。
背景技术
岩藻多糖是一种主要提取自褐藻的海洋硫酸多糖，不仅原料来源广、毒性低，还具有抗病毒、抗凝血、降血糖、抗肿瘤、抗血栓等多种生物活性。然而，天然岩藻多糖分子量由几万到几百万，黏度高、水溶性差、结构复杂、不易吸收，限制了其生物活性的发挥，同时也影响了岩藻多糖制品的深加工和应用。
结构改性是改善多糖活性、提高多糖利用度的有效方法之一，其主要途径包括多糖降解和基团修饰。将大分子多糖降解为相对小分子质量的多糖可以解决黏度高、吸收性差的问题，常见的多糖降解手段主要包括酸水解、碱水解、自由基降解、超声波降解和酶法降解。酸降解时糖苷键随机断裂，导致降解产物的分子量分布范围变宽，不易分离纯化；碱降解时的糖苷键断裂易引起多糖残基上的硫酸基团脱落，导致多糖链结构发生改变；超声波降解对实验设备的要求较高；酶法降解条件温和、效率高，但要求糖苷酶专一性高，且价格昂贵。自由基降解又称氧化降解或过氧化氢降解法，主要利用H2O2生成的羟基自由基使多糖的糖苷键发生选择性断裂，降解过程条件温和、无毒、无副产物生成，并且能保持糖环上取代基团稳定，多糖的主链结构几乎不发生变化，因此能够较大程度地保留岩藻多糖链上的硫酸基团，是岩藻多糖最理想的降解方法。
基团修饰是通过引入硫酸基、乙酰基、羧基等化学基团来改变多糖的分子结构，从而获得具有新结构的多糖衍生物。该方法在改善多糖生物活性方面发挥了关键的作用，是现阶段多糖结构改性研究中最常用的方法。乙酰化修饰对多糖固有生物活性的改善、其它功能特性的拓展具有十分重大的意义。乙酰水杨酸是一种含有乙酰基团的小分子活性化合物，在临床上具有广泛的应用。但随着应用研究的深入，乙酰水杨酸暴露出较严重的肝、肾及神经系统副反应。将乙酰水杨酸与糖基化合物进行接枝反应，不仅可以降低乙酰水杨酸的药物毒副作用，而且可以在多糖上引入乙酰基团，提高多糖生物活性。
发明内容
针对乙酰水杨酸的药物毒副作用较大和天然岩藻多糖因黏度高、水溶性差、结构复杂、不易吸收而导致其生物活性被限制发挥等现有技术存在的问题，为了降低药物的毒副作用和促进多糖生物活性的发挥，本发明提供一种改性岩藻多糖化合物及其制备方法和应用，将岩藻多糖氧化降解之后采用N,N’-羰基二咪唑原位活化耦联乙酰水杨酸酯化法得到改性岩藻多糖化合物，从而在降低药物毒副作用的同时，提升抗氧化活性和抗肿瘤作用等生物活性。
本发明解决上述技术问题的技术方案为：
一种改性岩藻多糖化合物，由按质量百分计的如下成分组成：由按质量百分计的如下成分组成：岩藻糖20-24％，鼠李糖0.9-1.4％，阿拉伯糖4.7-6.3％，半乳糖45-49％，木糖0.3-0.7％，甘露糖0.2-0.5％，葡萄糖醛酸4.5-6.1％，半乳糖醛酸0.75-1.46％，甘露糖醛酸13-17％，古洛糖醛酸0.42-0.76％；所述改性岩藻多糖化合物具有硫酸基团和乙酰水杨酸基团，硫酸基团质量占改性多糖化合物总质量的10％～20％，乙酰水杨酸基团占改性多糖化合物总质量的11％～18％，所述改性多糖化合物对超氧阴离子自由基具有显著的清除效果，对人肺腺癌细胞株A549具有明显的生长抑制和促凋亡作用。
上述改性岩藻多糖化合物的制备方法为：将岩藻多糖氧化降解之后采用N,N’-羰基二咪唑原位活化耦联乙酰水杨酸酯化法得到改性多糖化合物，具体为：以H2O2-VC联合氧化法降解岩藻多糖，制备低分子量多糖；再采用N,N’-羰基二咪唑原位活化法合成乙酰水杨酸咪唑酯；继而逐滴添加至低分子量岩藻多糖溶液中进行酯化反应，最后将所得溶液进行乙醇沉淀、洗涤、透析、冷冻干燥后获得改性岩藻多糖化合物。
进一步地，所述氧化降解的方法为H2O2-VC联合氧化法，岩藻多糖在降解剂H2O2和VC的作用下进行降解，岩藻多糖的浓度为3mg/mL～40mg/mL，降解剂H2O2和VC的浓度各为90μmol/L～270μmol/L、降解的温度为30℃～50℃、降解的时间为2～3h。
进一步地，所述的N,N’-羰基二咪唑原位活化耦联乙酰水杨酸酯化法具体为：先采用N,N’-羰基二咪唑原位活化法合成乙酰水杨酸咪唑酯；然后将所得乙酰水杨酸咪唑酯逐滴添加至氧化降解得到的低分子量岩藻多糖溶液中进行酯化反应，最后将所得溶液进行乙醇沉淀、洗涤、透析、冷冻干燥后获得改性岩藻多糖化合物。
进一步地，低分子量岩藻多糖与乙酰水杨酸的质量比为1～5:1。
本发明所得改性岩藻多糖化合物对超氧阴离子自由基具有强效的清除作用，反应浓度在125μg/mL至1000μg/mL范围内时对超氧阴离子自由基的清除率均大于90％，即能够表现出优异的抗氧化活性。
本发明的改性岩藻多糖化合物对人肺腺癌细胞株A549的生长具有明显的抑制作用，并能够诱导肿瘤细胞凋亡，即能够表现出优异的抗肿瘤作用。
上述所得改性岩藻多糖化合物能够应用于制备具有清除超氧阴离子自由基作用的日化用品、食品、保健品或药品中；或应用于制备具有抗肿瘤作用的食品、保健品或药品中。
本发明的有益效果在于：
(1)本发明首次将乙酰水杨酸作为接枝物对岩藻多糖进行基团修饰，不仅能够降低药物的毒副作用，而且提升抗氧化活性和抗肿瘤作用等生物活性，具体体现为：相同浓度的用量，本发明所得改性岩藻多糖化合物能够获得明显更高的生物活性；在同等生物活性的情况下，本发明所得改性岩藻多糖化合物所需的浓度显著下降；
(2)本发明所得改性岩藻多糖化合物是降解后的小分子多糖衍生物，具有低黏度和良好的吸收特性；
(3)本发明所得改性岩藻多糖化合物具有良好的抗氧化和抗肿瘤效果；
(4)本发明中改性岩藻多糖化合物制备简单，成本低，适于大规模生产，可辐射应用于日化用品、功能食品及保健药品等领域，社会经济效益显著。
附图说明
图1是原岩藻多糖和改性岩藻多糖化合物的异羟肟酸铁反应对比图(a.原岩藻多糖的异羟肟酸铁反应物，b.改性岩藻多糖化合物的异羟肟酸铁反应物)。
图2是标准单糖及其衍生物的液相色谱分析图。
图3是本发明改性岩藻多糖化合物水解产物的液相色谱检测图。
图4是本发明改性岩藻多糖化合物的傅里叶红外光谱。
图5是本发明改性岩藻多糖化合物对超氧阴离子自由基的清除作用。
图6是本发明改性岩藻多糖化合物对A549细胞生长的抑制作用。
图7是本发明改性岩藻多糖化合物对肿瘤细胞凋亡的诱导作用(a.未加多糖化合物的A549细胞；b.75μg/mL岩藻多糖处理的A549细胞；c.75μg/mL改性岩藻多糖化合物处理的A549细胞)。
具体实施方式
下面结合具体实施例对本发明做进一步的说明，但本发明并不限于此。
以下所述实施例中所用的材料、试剂等均能从商业渠道采购。
实施例1
H2O2-Vc联合氧化法制备低分子量岩藻多糖
(1)岩藻多糖50℃烘干，用蒸馏水溶解成10mg/mL；
(2)向100mL多糖溶液中分别加入4mL H2O2溶液(25mmol/L)和VC溶液(25mmol/L)，50℃水浴震荡2h进行降解；
(3)反应后的混合物冷却至室温，用0.01mol/L的NaOH调节溶液pH至中性；
(4)混合物经0.45μm的滤膜过滤后，转入截留分子量为3500Da的透析袋中4℃流水透析48h除去残留的H2O2和VC，然后将溶液蒸发浓缩、冷冻干燥后制得低分子量岩藻多糖。
实施例2
岩藻多糖的乙酰水杨酸改性
(1)向干燥的250mL三口烧瓶中加入1.0g乙酰水杨酸和20mL溶剂N,N-二甲基甲酰胺，接着迅速加入1.35g N,N’-羰基二咪唑，室温下恒速搅拌24h，获得合成中间体--乙酰水杨酸咪唑酯，密闭备用。
(2)向装有磁力搅拌器、恒压滴液漏斗和温度计的干燥250mL三颈烧瓶中依次加入1.2g低分子量岩藻多糖和20mL N,N-二甲基乙酰胺，并进行磁力搅拌使多糖充分溶解；
(3)通过滴液漏斗将20mL乙酰水杨酸咪唑酯缓慢滴加到低分子量岩藻多糖和N,N-二甲基乙酰胺的混合溶液中，滴加的过程中保持匀速搅拌；
(4)80℃连续搅拌8h；
(5)待反应后溶液冷却至室温，向反应液中加入4倍体积的无水乙醇，4℃沉淀过夜；
(6)8000rpm离心10min，收集沉淀，并用无水乙醇洗涤沉淀2～3次，以除去副产物和未反应掉的物质；
(7)用少量蒸馏水将沉淀物复溶，并将其在蒸馏水中透析48h(截留分子量为3500Da)，最后经过旋转蒸发、真空冷冻干燥即得改性岩藻多糖化合物。
实施例3
改性岩藻多糖化合物的成分组成测定
(1)乙酰水杨酸酯检验
依次向50mg/mL改性岩藻多糖化合物溶液中加入2.0mL盐酸羟胺-乙醇溶液(0.5mol/L)和0.4mL NaOH溶液(6mol/L)，振荡混匀后，加热至沸腾；待反应液冷却后，向其中加入4mL盐酸溶液(1mol/L)，摇匀；继续滴入3～5滴FeCl3溶液(10mg/mL)，并观察溶液颜色。以50mg/mL原岩藻多糖溶液作为对照组，重复上述操作后，发现对照组的反应溶液呈浅黄色(图1a)，为三价铁离子自身的颜色，表明原岩藻多糖中不具有能与FeCl3发生络合反应的基团。而改性岩藻多糖化合物经反应后溶液呈现明显的酒红色(图1b)，为酯类化合物经异羟肟酸铁反应生成的络合物颜色，表明化合物中存在-COOR结构，即低分子量岩藻多糖与乙酰水杨酸发生了酯化反应，标志着乙酰水杨酸成功接入到岩藻多糖的-OH基团上。
(2)总糖含量测定
准确称取干燥至恒重的岩藻糖标准品配置成0、10、20、50、100和200μg/mL的标准溶液；向离心管中分别加入1mL改性岩藻多糖化合物溶液(0.1mg/mL)或梯度浓度的岩藻糖标准溶液；继续加入1.0mL苯酚(重蒸馏试剂)溶液(50g/L)、5.0mL浓硫酸，混匀后室温下放置20min；在490nm波长下测定溶液的吸光度，并以岩藻糖的质量浓度为横坐标，吸光值为纵坐标，绘制岩藻糖标准曲线；根据岩藻糖标准曲线按照式1计算改性岩藻多糖化合物的总糖含量，式中m1——根据标准曲线计算的样品岩藻糖含量(μg/mL)，V——样品定容的体积(mL)，m——样品质量(g)。表1中结果显示改性岩藻多糖化合物的总糖含量为72.44％。
总糖含量(％)＝(m1×V×10-6)/m×100％(式1)
(3)硫酸基团含量测定
向0.5g改性岩藻多糖化合物中加入15mL盐酸(1mol/L)，密封后105℃水解4h；反应液冷却至室温后用快速定量滤纸过滤，再以少量水洗涤水解管和定量滤纸，收集所有滤液；将滤液煮沸1min，趁热逐滴加入20mL氯化钡溶液(10％，w/v)后，再煮沸3～5min；70～80℃恒温水浴陈化2h；用已烘至恒重的玻璃砂芯漏斗抽滤反应液，再用水洗涤沉淀至无氯离子；将玻璃砂芯漏斗置于105℃恒温干燥箱中烘至恒重后称重，根据式2计算样品中硫酸基团的含量，式中M——样品质量(g)，m2——玻璃砂芯漏斗烘至恒重时的质量(g)，m0——玻璃砂芯漏斗的初始质量(g)。表1中结果显示改性岩藻多糖化合物中硫酸基团的质量百分数为13.17％。
硫酸基团含量(％)＝96.06×(m2- m0)/M/233.39 (式2)
(4)糖醛酸含量测定
准确配制浓度为20、40、60、80和100μg/mL的葡萄糖醛酸标准溶液；向离心管中分别加入250μL改性岩藻多糖化合物溶液(5mg/mL)或梯度浓度的岩藻糖标准溶液，冰浴预冷20min；向离心管中加入1.5mL四硼酸钠-硫酸溶液(0.0125mol/L)，充分混匀后，沸水浴5min；将反应液迅速冷却到室温，向每个离心管中加入25μL间羟基联苯溶液(0.15％，w/v)；振荡混匀后测定OD520值，以葡萄糖醛酸的质量浓度为横坐标，以对应吸光值为纵坐标，绘制葡萄糖醛酸标准曲线；根据葡萄糖醛酸标准曲线按照式3计算改性岩藻多糖化合物的糖醛酸含量，式中c1——根据标准曲线计算的样品中葡萄糖醛酸含量(μg/mL)，V——样品定容的体积(mL)，m——样品质量(g)。表1中结果显示改性岩藻多糖化合物的糖醛酸含量为6.71％。
糖醛酸含量(％)＝(c1×V×10-6)/m×100％ (式3)
(5)乙酰水杨酸基团含量测定
精确称取质量为0.05g的改性岩藻多糖化合物样品置于水解管中，加入0.1mol/L的NaOH溶液30mL后置于100℃下密封水解2.5h。水解液冷却至室温并过滤后，稀释10倍，测定λ＝296nm处的吸光值，再以梯度浓度的水杨酸作为标准品绘制标准曲线，根据式4计算出改性岩藻多糖化合物中乙酰水杨酸的含量，式中cSA——由标准曲线计算得到的样品溶液中水杨酸的浓度(μg/mL)，V——溶液定容的体积(mL)，D——溶液稀释倍数，m——样品质量(g)，180——乙酰水杨酸相对分子质量，138——水杨酸相对分子质量。表1中结果显示改性岩藻多糖化合物的乙酰水杨酸基团含量为15.52％。
乙酰水杨酸含量＝(cSA×V×10-6)/m×D×(180/138)×100％ (式4)
表1改性岩藻多糖化合物的化学组成
(6)单糖组成测定
精确配制浓度为100μg/mL岩藻糖(Fuc)、鼠李糖(Rha)、阿拉伯糖(Ara)、半乳糖(Gal)、葡萄糖(Glc)、木糖(Xyl)、甘露糖(Man)、果糖(Fru)、核糖(Rib)、半乳糖醛酸(Gal-UA)、葡萄糖醛酸(Glc-UA)、甘露糖醛酸(Man-UA)、古罗糖醛酸(Gul-UA)标准工作液；将工作液梯度稀释后利用装配有电化学检测器的液相色谱对糖组分进行分析检测，其中的色谱参数为：Thermo ICS5000+离子色谱系统(Thermo Fisher Scientific，USA)，DionexTMCarboPacTMPA20液相色谱柱，进样量20μL，流动相为97.5％ H2O–2.5％ NaOH溶液(100mM)，流速0.5mL/min，柱温30℃。以单糖及糖醛酸标准品浓度的量为横坐标，以其峰面积为纵坐标绘制标准曲线，获得拟合方程，标准单糖及其衍生物的出峰时间及拟合方程见表2，液相色谱分析见图2。
表2标准单糖及其衍生物的出峰时间及拟合方程
同时，称取5mg改性岩藻多糖化合物于色谱瓶中，加入1mL三氟乙酸溶液(2mol/L)，密封后121℃水解2h；氮气吹干后用甲醇反复洗涤固形物，以除去三氟乙酸；用1mL去离子水溶解洗涤吹干后的固形物，0.22μm滤膜过滤后进行高效液相色谱检测，根据出峰时间确定改性岩藻多糖化合物的单糖组成类型，进而根据峰面积通过拟合方程计算其含量。改性岩藻多糖化合物的单糖及其组成结果见表3，液相色谱检测见图3，实验结果表明，改性岩藻多糖化合物的质量百分比组成为岩藻糖22.74％、鼠李糖1.23％、阿拉伯糖5.41％、半乳糖47.39％、木糖0.36％、甘露糖0.37％、半乳糖醛酸1.09％、葡萄糖醛酸5.72％、甘露糖醛酸15.13％和古洛糖醛酸0.56％。
表3改性岩藻多糖化合物的单糖组成及其浓度
实施例4
改性岩藻多糖化合物的红外光谱分析
称取10mg改性岩藻多糖化合物与100mg干燥KBr粉末经研钵充分研磨后压成薄片，在4000cm-1～500cm-1波数范围内进行红外扫描，得到傅里叶红外光谱。如图4所示，改性岩藻多糖化合物在3447.07cm-1和2990.37cm-1处是分别由O-H伸缩振动和C-H伸缩振动引起的吸收峰，是典型的多糖特征吸收峰；837.36cm-1处是硫酸基的特征吸收峰，1052.90cm-1处为吡喃糖苷键的特征吸收峰，以上各组峰均为岩藻多糖的特征吸收峰，说明改性不改变岩藻多糖的骨架结构。1452.04cm-1～1642.72cm-1范围的吸收峰代表乙酰水杨酸中苯环衍生物的振动特征吸收峰；1735.76cm-1处为乙酰水杨酸和低分子量岩藻多糖形成的乙酸酯中的C＝O特征吸收峰，1249.35cm-1处的强吸收峰为乙酸酯中C-O-C的伸缩振动峰，说明化合物中存在酯基；758.58cm-1处的特征吸收峰代表苯环的邻位取代，是改性岩藻多糖化合物区别于乙酰水杨酸和低分子量岩藻多糖的结构特征。以上结果均证明乙酰水杨酸成功接入到岩藻多糖上。
实施例5
改性岩藻多糖化合物的抗氧化活性测定
向800μL不同浓度梯度(0、125、250、500、1000μg/mL)的改性岩藻多糖化合物溶液中依次加入100μL NADH溶液(0.78mmol/L)和100μL NBT溶液(0.5mmol/L)；充分混匀后，加入100μL PMS溶液(0.1mmol/L)，再次摇匀；于560nm处测定反应液的吸光度，根据式4计算改性岩藻多糖化合物对羟基自由基的清除率，式中A0——蒸馏水空白组，Am0——无样品的对照组，Am——实验组。该反应体系以没食子酸为阳性对照。如图5所示，在作用浓度在125μg/mL至1000μg/mL范围内，改性岩藻多糖化合物对超氧阴离子自由基的清除率均大于90％，当作用浓度为1000μg/mL时，对超氧阴离子自由基的清除率为92.11±0.93％。
超氧阴离子自由基清除率(％)＝[1-(Am-Am0)/A0]×100％ (式4)
实施例6
改性岩藻多糖化合物对A549细胞的抗增殖作用
(1)取对数期的A549细胞用胰蛋白酶消化，以F-12K培养基配制成浓度为1×105个/mL的细胞悬液，取100μL接种于96孔板，置于湿润的CO2(5％)培养箱中，37℃培养24h；
(2)用培养基配制改性岩藻多糖化合物溶液，使其浓度分别为25μg/mL、50μg/mL、75μg/mL、100μg/mL、125μg/mL、150μg/mL和200μg/mL。
(3)培养A549细胞24h后，弃去培养基，替换成等体积的系列浓度的改性岩藻多糖化合物溶液，以新鲜F-12K培养液作空白对照组，每个浓度设置3个复孔，培养48h；
(4)向所有孔中加入10μL CCK-8溶液，培养箱中孵育2h；
(5)使用酶标仪测定每孔反应液在450nm处的吸光值，按式5计算样品对肿瘤细胞的增殖抑制率，式中A0——空白对照组的吸光值，An——实验组的吸光值。
增殖抑制率(％)＝(A0-An)/A0×100％ (式5)
改性岩藻多糖化合物对A549细胞的抗增殖作用如图6所示，处理48小时后A549细胞的增殖出现了明显的抑制，且改性岩藻多糖化合物对A549细胞的增殖抑制作用具有剂量依赖性，其抗增殖作用的半抑制浓度(IC50)值约为49μg/mL。
实施例7
改性岩藻多糖化合物对肿瘤细胞凋亡的诱导作用
(1)将A549细胞按2×105个/孔的浓度接种于6孔板中，37℃培养24h后弃去培养液，分别加入等体积的F-12K培养液和改性岩藻多糖化合物溶液(75μg/mL)；
(2)孵育48h后，用不含EDTA的胰酶消化液消化细胞，终止消化后收集细胞悬液，1000rpm离心3min，弃培养基；
(2)用1mL预冷的PBS洗涤细胞两次；
(3)用预冷的Binding Buffer轻轻悬浮细胞至5×105～1×106个/mL；
(4)取0.3mL细胞悬液至流式细胞管内，加入5μL Annexin V-FITC混匀，避光室温温育10min；
(5)加入5μL PI染色，混匀于黑暗下放置5min；
(6)过200目细胞筛后上流式细胞仪检测。
改性岩藻多糖化合物对A549细胞的凋亡诱导作用如图7所示：空白对照组中早期凋亡和晚期凋亡细胞很少，细胞凋亡率总和仅为4.053±0.37％；而实验组中，经过75μg/mL改性岩藻多糖化合物处理48h后，正常细胞减少，凋亡细胞增加，凋亡率为22.40±0.35％，比未经修饰的原岩藻多糖的凋亡诱导率增加11.59％。
乙酰水杨酸对超氧阴离子的半抑制浓度为706.41μg/mL(Hajihashemi S,GeunsJMC.Radical scavenging activity of steviol glycosides,steviol glucuronide,hydroxytyrosol,metformin,aspirin and leaf extract of Stevia rebaudiana.FreeRadicals and Antioxidants,2013,3(2(Suppl):S34-S41)，原岩藻多糖作用浓度为250μg/mL时，对超氧阴离子自由基的清除率为25％左右(程仕伟,陈超男,冯志彬,等.海带岩藻多糖的水提制备及其抗氧化活性研究.食品科学,2010,31(06):101-104)，而本发明改性岩藻多糖化合物于125μg/mL时对超氧阴离子自由基的清除率已达90％。
乙酰水杨酸对A549细胞增殖半抑制浓度(IC50)值为1080.96μg/mL(Vejselova D,Kutlu,HM.Inhibitory effects of salicylic acid on A549 human lungadenocarcinoma cell viability.Turkish Journal of Biology,2015,39(1):1-5)，原岩藻多糖作用浓度为200μg/mL时，对A549细胞增殖的抑制率为52.1％(Boo HJ,Hyun JH,KimSC,et al.Fucoidan from Undaria pinnatifida induces apoptosis in A549 humanlung carcinoma cells.Phytotherapy Research,2011,25(7):1082-1086)，由此能够推知原岩藻多糖的IC50值在200μg/mL左右，然而本发明改性岩藻多糖化合物的IC50值约为49μg/mL。这表明，本发明通过将乙酰水杨酸作为接枝物对岩藻多糖进行基团修饰，显著降低了对A549细胞增殖的半抑制浓度(IC50值)，与原岩藻多糖的IC50值相比，下降了75.5％，与乙酰水杨酸的IC50值相比，下降了95.5％。
通过上述对比分析能够得知，本发明通过将乙酰水杨酸作为接枝物对岩藻多糖进行基团修饰，由于协同作用，从而能够实现相同活性下显著降低了浓度(IC50值)，以及同样浓度下显著提升了生物活性。
Claims (7)
1.一种改性岩藻多糖化合物，其特征在于，由按质量百分计的如下成分组成：岩藻糖20-24%，鼠李糖0.9-1.4%，阿拉伯糖4.7-6.3%，半乳糖45-49%，木糖0.3-0.7%，甘露糖0.2-0.5%，葡萄糖醛酸4.5-6.1%，半乳糖醛酸0.75-1.46%，甘露糖醛酸13-17%，古洛糖醛酸0.42-0.76%；所述改性岩藻多糖化合物具有硫酸基团和乙酰水杨酸基团，硫酸基团质量占改性多糖化合物总质量的10%~20%，乙酰水杨酸基团占改性多糖化合物总质量的11%~18%；
所述改性岩藻多糖化合物的制备方法，将岩藻多糖氧化降解之后采用N,N’-羰基二咪唑原位活化耦联乙酰水杨酸酯化法得到改性多糖化合物，具体包括如下步骤：以H2O2-VC联合氧化法降解岩藻多糖，制备低分子量多糖；再采用N,N’-羰基二咪唑原位活化法合成乙酰水杨酸咪唑酯；继而逐滴添加至低分子量岩藻多糖溶液中进行酯化反应，最后将所得溶液进行乙醇沉淀、洗涤、透析、冷冻干燥后获得改性岩藻多糖化合物。
2.权利要求1所述的改性岩藻多糖化合物的制备方法，其特征在于，将岩藻多糖氧化降解之后采用N,N’-羰基二咪唑原位活化耦联乙酰水杨酸酯化法得到改性多糖化合物，具体包括如下步骤：以H2O2-VC联合氧化法降解岩藻多糖，制备低分子量多糖；再采用N,N’-羰基二咪唑原位活化法合成乙酰水杨酸咪唑酯；继而逐滴添加至低分子量岩藻多糖溶液中进行酯化反应，最后将所得溶液进行乙醇沉淀、洗涤、透析、冷冻干燥后获得改性岩藻多糖化合物。
3.根据权利要求2所述的改性岩藻多糖化合物的制备方法，其特征在于，所述的H2O2-VC联合氧化法，岩藻多糖在降解剂H2O2和VC的作用下进行降解，岩藻多糖的浓度为3 mg/mL~40mg/mL，降解剂H2O2和VC的浓度各为90 μmol/L~270 μmol/L、降解的温度为30℃~50 ℃、降解的时间为2~3 h。
4.根据权利要求2或3所述的改性岩藻多糖化合物的制备方法，其特征在于，所述的N,N’-羰基二咪唑原位活化耦联乙酰水杨酸酯化法具体为：先采用N,N’-羰基二咪唑原位活化法合成乙酰水杨酸咪唑酯；然后将所得乙酰水杨酸咪唑酯逐滴添加至氧化降解得到的低分子量岩藻多糖溶液中进行酯化反应，最后将所得溶液进行乙醇沉淀、洗涤、透析、冷冻干燥后获得改性岩藻多糖化合物。
5.根据权利要求4所述的改性岩藻多糖化合物的制备方法，其特征在于，低分子量岩藻多糖与乙酰水杨酸的质量比为1~5:1。
6.权利要求1所述的改性岩藻多糖化合物在制备具有清除超氧阴离子自由基作用的日化用品或药品中的应用。
7.权利要求1所述的改性岩藻多糖化合物在制备具有抗肿瘤作用的药品中的应用。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210970973.2A CN115260328A (zh) | 2022-08-14 | 2022-08-14 | 一种改性岩藻多糖化合物及其制备方法和应用 |
| CN2022109709732 | 2022-08-14 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115746161A CN115746161A (zh) | 2023-03-07 |
| CN115746161B true CN115746161B (zh) | 2024-06-07 |
Family
ID=83751916
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210970973.2A Pending CN115260328A (zh) | 2022-08-14 | 2022-08-14 | 一种改性岩藻多糖化合物及其制备方法和应用 |
| CN202211525744.6A Active CN115746161B (zh) | 2022-08-14 | 2022-11-30 | 一种改性岩藻多糖化合物及其制备方法和应用 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210970973.2A Pending CN115260328A (zh) | 2022-08-14 | 2022-08-14 | 一种改性岩藻多糖化合物及其制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN115260328A (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116120478A (zh) * | 2022-12-21 | 2023-05-16 | 大连理工大学 | 岩藻多糖铁配合物及其制备方法与应用 |
| CN117362472B (zh) * | 2023-11-13 | 2024-04-19 | 山西纳德西生物科技有限公司 | 一种改性硫酸酯多糖化合物及其制备方法 |
| CN118545704B (zh) * | 2024-07-26 | 2024-10-18 | 广东海洋大学 | 半胱氨酸与岩藻多糖荧光碳量子点的制备方法及其应用 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982339A (zh) * | 2005-12-14 | 2007-06-20 | 河南省科学院河南省发展计划委员会地理研究所 | 水杨酸与壳聚糖－2，6位接枝物及其制备方法 |
| CN102532332A (zh) * | 2011-09-09 | 2012-07-04 | 山东洁晶集团股份有限公司 | 一种从海洋褐藻中提取制备低分子岩藻多糖的方法 |
| CN102665733A (zh) * | 2009-07-27 | 2012-09-12 | Arc医疗设备股份有限公司 | 用于治疗纤维性粘连和其它疾病的包含改性岩藻多糖的药物组合物 |
| CN109970823A (zh) * | 2019-05-07 | 2019-07-05 | 青岛农业大学 | 一种岩藻寡糖及其制备方法和应用 |
-
2022
- 2022-08-14 CN CN202210970973.2A patent/CN115260328A/zh active Pending
- 2022-11-30 CN CN202211525744.6A patent/CN115746161B/zh active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982339A (zh) * | 2005-12-14 | 2007-06-20 | 河南省科学院河南省发展计划委员会地理研究所 | 水杨酸与壳聚糖－2，6位接枝物及其制备方法 |
| CN102665733A (zh) * | 2009-07-27 | 2012-09-12 | Arc医疗设备股份有限公司 | 用于治疗纤维性粘连和其它疾病的包含改性岩藻多糖的药物组合物 |
| CN102532332A (zh) * | 2011-09-09 | 2012-07-04 | 山东洁晶集团股份有限公司 | 一种从海洋褐藻中提取制备低分子岩藻多糖的方法 |
| CN109970823A (zh) * | 2019-05-07 | 2019-07-05 | 青岛农业大学 | 一种岩藻寡糖及其制备方法和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115260328A (zh) | 2022-11-01 |
| CN115746161A (zh) | 2023-03-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115746161B (zh) | 一种改性岩藻多糖化合物及其制备方法和应用 | |
| Zhang et al. | Hypoglycemic activity in vitro of polysaccharides from Camellia oleifera Abel. seed cake | |
| Zhang et al. | Structural characterization and in vitro antitumor activity of an acidic polysaccharide from Angelica sinensis (Oliv.) Diels | |
| Wang et al. | Structure characterization, modification through carboxymethylation and sulfation, and in vitro antioxidant and hypoglycemic activities of a polysaccharide from Lachnum sp. | |
| Wang et al. | Structural characterization and in vitro antitumor activity of polysaccharides from Zizyphus jujuba cv. Muzao | |
| Song et al. | Structure characterization and antitumor activity of an α β-glucan polysaccharide from Auricularia polytricha | |
| Han et al. | Structural characterization and anti-A549 lung cancer cells bioactivity of a polysaccharide from Houttuynia cordata | |
| He et al. | Structural elucidation and antioxidant activity of an arabinogalactan from the leaves of Moringa oleifera | |
| Hua et al. | Structural characterization and DPPH· radical scavenging activity of a polysaccharide from Guara fruits | |
| Yu et al. | Structural elucidation and biological activity of a novel polysaccharide by alkaline extraction from cultured Cordyceps militaris | |
| Li et al. | Extraction and properties of Ginkgo biloba leaf polysaccharide and its phosphorylated derivative | |
| Sui et al. | Extraction of polysaccharides from a species of Chlorella | |
| Zhou et al. | Extraction, structural analysis and antioxidant activity of aloe polysaccharide | |
| Jiang et al. | Structure elucidation and antitumor activity of a water soluble polysaccharide from Hemicentrotus pulcherrimus | |
| Whistler et al. | Composition and behavior of soil polysaccharides1, 2 | |
| Yao et al. | Composition and antioxidant activity of the polysaccharides from cultivated Saussurea involucrata | |
| Sun et al. | Structure characterization, antioxidant and emulsifying capacities of exopolysaccharide derived from Pantoea alhagi NX-11 | |
| Wang et al. | Structure analysis of a neutral polysaccharide isolated from green tea | |
| Xiang et al. | Antioxidant and hypoglycemic activity of tea polysaccharides with different degrees of fermentation | |
| Fan et al. | Preparation, structural analysis and antioxidant activity of polysaccharides and their derivatives from Pueraria lobata | |
| Li et al. | Relationship between heat treatment on structural properties and antitumor activity of the cold-water soluble polysaccharides from Grifola frondosa | |
| Tu et al. | A novel polysaccharide from Hericium erinaceus: Preparation, structural characteristics, thermal stabilities, and antioxidant activities in vitro | |
| CN108727509A (zh) | 一种毛竹笋壳阿拉伯半乳聚糖及其制备和用途 | |
| Wang et al. | Preparation and structural characterization of poly-mannose synthesized by phosphoric acid catalyzation under microwave irradiation | |
| CN110229243A (zh) | 一种山苦茶均一多糖及其制备方法与应用 |
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 | ||
| GR01 | Patent grant |