CN112250706A - Method for extracting and purifying phospholipid from clams - Google Patents
Method for extracting and purifying phospholipid from clams Download PDFInfo
- Publication number
- CN112250706A CN112250706A CN202011040174.2A CN202011040174A CN112250706A CN 112250706 A CN112250706 A CN 112250706A CN 202011040174 A CN202011040174 A CN 202011040174A CN 112250706 A CN112250706 A CN 112250706A
- Authority
- CN
- China
- Prior art keywords
- phospholipid
- clams
- extracting
- nanoparticles
- graphene oxide
- 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.)
- Granted
Links
- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 68
- 235000020639 clam Nutrition 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 40
- 241000237519 Bivalvia Species 0.000 title claims abstract description 34
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000002105 nanoparticle Substances 0.000 claims abstract description 45
- 150000002632 lipids Chemical class 0.000 claims abstract description 40
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 36
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 33
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 25
- 239000000835 fiber Substances 0.000 claims abstract description 23
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 23
- 239000000843 powder Substances 0.000 claims abstract description 14
- 239000003463 adsorbent Substances 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 9
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims abstract description 8
- 235000012239 silicon dioxide Nutrition 0.000 claims abstract description 7
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- 239000006185 dispersion Substances 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 4
- 238000007710 freezing Methods 0.000 claims abstract description 3
- 230000008014 freezing Effects 0.000 claims abstract description 3
- 238000001179 sorption measurement Methods 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims description 31
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 11
- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- 239000012535 impurity Substances 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 7
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 229910002804 graphite Inorganic materials 0.000 claims description 6
- 239000010439 graphite Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000002131 composite material Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 239000012286 potassium permanganate Substances 0.000 claims description 4
- 239000011343 solid material Substances 0.000 claims description 4
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 239000004743 Polypropylene Substances 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000001354 calcination Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 235000021588 free fatty acids Nutrition 0.000 claims description 3
- -1 polypropylene Polymers 0.000 claims description 3
- 229920001155 polypropylene Polymers 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 238000012856 packing Methods 0.000 claims description 2
- 238000010791 quenching Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- JCIBFWRKAKESTL-UHFFFAOYSA-N 2-(16-bromohexadecyl)pyridine Chemical compound BrCCCCCCCCCCCCCCCCC1=CC=CC=N1 JCIBFWRKAKESTL-UHFFFAOYSA-N 0.000 claims 1
- 230000003472 neutralizing effect Effects 0.000 claims 1
- 238000001291 vacuum drying Methods 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 11
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 18
- 150000003905 phosphatidylinositols Chemical class 0.000 description 17
- 102100029516 Basic salivary proline-rich protein 1 Human genes 0.000 description 15
- 206010036618 Premenstrual syndrome Diseases 0.000 description 15
- 229920000314 poly p-methyl styrene Polymers 0.000 description 15
- 238000011084 recovery Methods 0.000 description 14
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 13
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 13
- 150000002500 ions Chemical class 0.000 description 10
- 241000894007 species Species 0.000 description 10
- 239000002114 nanocomposite Substances 0.000 description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002441 X-ray diffraction Methods 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- QAQSNXHKHKONNS-UHFFFAOYSA-N 1-ethyl-2-hydroxy-4-methyl-6-oxopyridine-3-carboxamide Chemical compound CCN1C(O)=C(C(N)=O)C(C)=CC1=O QAQSNXHKHKONNS-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000193879 Corbicula fluminea Species 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 3
- 238000000089 atomic force micrograph Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 3
- 238000000132 electrospray ionisation Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 2
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- IZTOCYCLVRZMJR-UHFFFAOYSA-N chloroform;methanol;hydrochloride Chemical compound Cl.OC.ClC(Cl)Cl IZTOCYCLVRZMJR-UHFFFAOYSA-N 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 150000002190 fatty acyls Chemical group 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000011229 interlayer Substances 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 238000004626 scanning electron microscopy Methods 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004627 transmission electron microscopy Methods 0.000 description 2
- 241000881711 Acipenser sturio Species 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 239000004593 Epoxy Chemical group 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 238000010220 Pearson correlation analysis Methods 0.000 description 1
- 108091006622 SLC12A4 Proteins 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 102100034244 Solute carrier family 12 member 4 Human genes 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000037326 chronic stress Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 239000004567 concrete Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940090949 docosahexaenoic acid Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 1
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000020978 long-chain polyunsaturated fatty acids Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000001055 reflectance spectroscopy Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/182—Graphene
- C01B32/198—Graphene oxide
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
- C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/10—Particle morphology extending in one dimension, e.g. needle-like
- C01P2004/16—Nanowires or nanorods, i.e. solid nanofibres with two nearly equal dimensions between 1-100 nanometer
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/80—Particles consisting of a mixture of two or more inorganic phases
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/143—Preparation by elimination of some components selective absorption
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Nanotechnology (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Inorganic Chemistry (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Crystallography & Structural Chemistry (AREA)
- Materials Engineering (AREA)
- Manufacturing & Machinery (AREA)
- Molecular Biology (AREA)
- Composite Materials (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention discloses a method for extracting and purifying phospholipid from clams, which comprises the following steps: collecting clam tissues, homogenizing by using a high-speed dispersion machine, and then drying under vacuum freezing to obtain clam powder; ② chloroform methanol ultrasonic treatment is used to extract crude lipid from clam powder; thirdly, washing the crude lipid with cold acetone to obtain a crude lipid extract; and fourthly, using the compound of the silicon dioxide fiber nanoparticles and the graphene oxide nanoparticles as an adsorbent, and performing adsorption extraction on the phospholipid in the crude lipid extract by using a solid phase extraction method. The invention can effectively and reliably extract highly purified phospholipid from clams so as to carry out lipidomics analysis and phenotype analysis.
Description
Technical Field
The invention relates to a method for extracting and purifying phospholipid from clams, and belongs to the technical field of food manufacturing and detection.
Background
Clams belong to shellfish, are one of the most abundant marine mollusks, and are widely cultivated and harvested worldwide. Asian clams (Corbicula fluminea) are mainly distributed in east Asian countries, especially China, Korea and Japan. The Food and Agriculture Organization (FAO) of the united nations reports that the yield of fishing and aquaculture of clams worldwide (2018) exceeds 660 ten thousand tons, which indicates that clams are the highest yielding species of shellfish. Clams are attractive to customers in coastal areas due to their unique nutritional ingredients, and research on their nutritional ingredients, including minerals, lipids, proteins, etc., has drawn a great deal of attention from the large consumption of clams.
Recent research reports indicate that clams are rich in polyunsaturated phospholipids, which play a key role in maintaining the endogenous system and cell membranes, participating in signaling and acting as binding sites for proteins. Phospholipids are the major components of cell membranes and are composed mainly of Phosphatidic Acid (PA), Phosphatidylinositol (PI), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingomyelin (SM) and small amounts of neutral fats. The chemical structure of phospholipids consists of a glycerol backbone and a hydrophilic polar head group and two hydrophobic acyl chains. It is known that long-chain polyunsaturated fatty acyl chains (LC-PUFA), such as eicosapentaenoic acid (EPA, C20: 5) and docosahexaenoic acid (DHA, C22: 6) acyl chains, can relieve neurodegenerative diseases and improve brain function. Phospholipids of the LCPUFA structure are reported to have higher bioavailability, partitioning efficiency and functionality in reducing cardiovascular risk, etc. than ethyl esterified LC-PUFAs and glycerides of the LC-PUFA structure. However, the complexity and diversity of chemical structures present challenges to phospholipid molecular analysis.
In the fields of food industry, life sciences and medicine, scientists have prepared phospholipids by extracting purified phospholipids from substrates and have performed lipidomic and phenotypic analyses. Generally, extraction of crude total lipids using organic solvents (including methanol, dichloromethane, chloroform, etc.) presents the challenge of purifying low abundance phospholipid molecular species MS in impurities and non-polar triglycerides.
Disclosure of Invention
The invention aims to provide a method for extracting and purifying phospholipid from clams. The method can effectively and reliably extract highly purified phospholipids from clams so as to carry out lipidomics analysis and phenotype analysis.
The technical scheme of the invention is as follows: a method for extracting and purifying phospholipid from clams is characterized by comprising the following steps:
collecting clam tissues, homogenizing by using a high-speed dispersion machine, and then drying under vacuum freezing to obtain clam powder;
② chloroform methanol ultrasonic treatment is used to extract crude lipid from clam powder;
thirdly, washing the crude lipid with cold acetone to obtain a crude lipid extract;
and fourthly, using the compound of the silicon dioxide fiber nanoparticles and the graphene oxide nanoparticles as an adsorbent, and performing adsorption extraction on the phospholipid in the crude lipid extract by using a solid phase extraction method.
In the above method for extracting and purifying phospholipids from clams, in the step (iv), the preparation method of the composite of silica fiber nanoparticles and graphene oxide nanoparticles is as follows: dispersing the synthesized graphene oxide nano particles in water and carrying out ultrasonic treatment to obtain a graphene oxide solution, and uniformly dispersing for 1 h; then, adding the silica fiber nanoparticles into the dispersed graphene oxide solution according to the mass ratio of the silica fiber nanoparticles to the graphene oxide nanoparticles of 1: 1, and stirring the mixed solution for 2 hours at 120 ℃ by intense magnetic force; finally, the residue was washed three times or more by using a 50% v/v ethanol aqueous solution to remove unabsorbed silica fiber nanoparticles, to obtain a composite of silica fiber nanoparticles and graphene oxide nanoparticles.
In the method for extracting and purifying phospholipid from clams, the preparation method of the silicon dioxide fiber nanoparticles comprises the following steps: under ultrasonic treatment, after dissolving a mixture of 0.60g of urea and 1.10g of cetylpyridinium bromide (CPB) in 30mL of water for 5min, 30mL of cyclohexane and 0.94mL of isopropanol are added; then, 3mL of tetraethyl orthosilicate (TEOS) was added dropwise to the mixture using a syringe, and the solution was heated to 70 ℃ with stirring; after centrifugation at 8000g for 10min at 4 ℃, the resulting residue was washed three times with acetone, deionized water and absolute ethanol in order and dried under vacuum for 12h to obtain silica fiber nanoparticles by calcination at 500 ℃ for 4 h.
In the foregoing method for extracting and purifying phospholipids from clams, the preparation method of the graphene oxide nanoparticles comprises the following steps: mixing 360mL of sulfuric acid solution (98%) per 40mL of phosphoric acid solution (85%) to obtain a mixed acid solution, adding 18.0g of potassium permanganate and 3.0g of graphite flake into the mixed acid solution to perform reaction, after the reaction is cooled to room temperature, pouring the reaction mixture on ice, and adding 5mL of 30% H by mass concentration2O2The aqueous solution was quenched and then centrifuged, and the resulting mixture after centrifugation was neutralized with 4 wt% aqueous HCl (8,000 g at 4 ℃ for 30min), and the residual salts in the resulting solid material were removed by dialysis in ultrapure deionized water and dried under vacuum at room temperature overnight to give graphene oxide nanoparticles.
In the method for extracting and purifying phospholipid from clams, the concrete method of the step II comprises the steps of accurately weighing clam powder obtained in the step I, mixing the clam powder with chloroform-methanol mixed liquor with the volume ratio of 2:1, extracting crude lipid under ultrasonic treatment, and adding deionized water into the mixture to separate hydrophilic impurities; then, the mixture was centrifuged at 8000g for 10min, the bottom organic phase was recovered while the solid residue was extracted twice more with chloroform, and finally, the collected organic phases were combined and evaporated to dryness under a nitrogen stream using 12mL of chloroform-methanol mixture, 3.2mL of deionized water, and 10mL of chloroform per 0.3g of clam powder.
In the method for extracting and purifying phospholipid from clam, in the step (i), the rotating speed of the high-speed disperser is 1500 rpm.
In the method for extracting and purifying the phospholipid from the clams, the specific method of the step (IV) is as follows: packing the adsorbent in a sleeve, arranging two polypropylene gaskets at the bottom and the top of the sleeve to well fix the adsorbent to prepare an SPE column; and then mixing the mixture by using hexane and a volume ratio of 2:1, loading the crude lipid extract on an SPE column under vacuum, then successively activating with a chloroform/isopropanol mixture of 2:1 chloroform/isopropanol mixed solution and 0.1% formic acid ether solution by mass percentage wash SPE column, in order to remove free fatty acid and neutral lipid; finally, the reaction solution is passed through a column at a flow rate of 1.5 mL. min–1The SPE cartridge was washed with a mixture of chloroform methanol hydrochloric acid (2: 1: 0.01, v/v/v) to obtain a fraction of the objective compound, and the eluate was dried under a nitrogen stream.
Compared with the prior art, the phospholipid in the crude lipid extract is adsorbed and extracted by using a solid phase extraction method after the lipid in the clams is coarsely extracted by using a compound of silicon dioxide fiber nano particles and graphene oxide nano particles as an adsorbent, the method utilizes the characteristic that the surface of the graphene oxide contains a plurality of polar parts and the porous characteristic of the silicon dioxide fiber nano particles, uses the graphene oxide nano particles as a supporting material, compounds the silicon dioxide fiber nano particles on the graphene oxide nano particles, and selectively absorbs the phospholipid by using the adsorbent which is not subjected to solid phase extraction and is prepared from the composite material, so that the phospholipid in the clams can be more fully and reliably purified and extracted. Tests prove that the detection method can quickly and accurately detect the contents of harmful substances and flavor substances in the smoked instant sturgeon fillets, so that the quality of the products can be controlled more conveniently. The phospholipid extracted by the method can identify and quantify 35 PMS in clams at most, and obtains 53.62% of PC molecular species with EPAADHA structure.
Drawings
FIG. 1 is a schematic diagram comparing the structural characterization of GO, KCC-1 and G/KCC-1;
FIG. 2 is an XRD pattern of GO, KCC-1 and G/KCC-1;
FIG. 3 is the UVvis DRS spectra of GO, KCC-1 and G/KCC-1;
FIG. 4 is a graphical representation of the effect of pH on phospholipid recovery;
FIG. 5 is a schematic of the effect of elution solution volume on phospholipid recovery;
FIG. 6 is a graphical representation of the effect of flow rate on phospholipid recovery;
FIG. 7 is a mass spectrum of crude lipid extract (A);
FIG. 8 is a mass spectrum of acetone-washed lipid (B);
FIG. 9 is a mass spectrum of the target phospholipid (C).
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Examples are given.
1.1 materials and reagents
Graphite, 3-Aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were purchased from SigmaAldrich (St. Louis, Mo., USA). Acetonitrile and methanol were purchased from merck, darmstadt, germany, in chromatographic grade. By providing hydrogen peroxide (H)2O2) Phosphoric acid (H)3PO4) Hydrochloric acid (HCl), acetone, isopropanol, potassium permanganate (KMnO)4) And urea national chemical agents ltd (shanghai, china). Phosphatidylcholine (PC) 14: 0/14: 0, Phosphatidylethanolamine (PE) 15: 0/15: 0 and Phosphatidylinositol (PI) 16: 0/16: a representative chemical standard of 0 is available from Avanti Polar Lipids, Inc. Using MilliQ seriesUltrapure water (18.2M Ω. cm) was produced systematically (Bedford, Mass., USA).
1.2 Synthesis of composite of silica fiber nanoparticles and graphene oxide nanoparticles (hereinafter referred to as G/KCC-1)
Graphene oxide nanoparticles (hereinafter referred to as GO). And (3) adding 9: 1(360 mL: 40mL) by volume concentrated H was added2SO4/H3PO4,KMnO4The mixture of (18.0g) and graphite flakes (3.0g) may develop a slight exotherm during the reaction. After cooling to room temperature (about 25 deg.C), the mixture was poured onto ice and 5mL of 30% H2O2And (4) quenching. Then, after centrifugation, the mixture was neutralized with 4 wt% aqueous HCl (8,000 g at 4 ℃ C. for 30 min). The residual salts in the resulting solid material were removed by dialysis in ultrapure deionized water. The synthesized GO was dried under vacuum at room temperature overnight.
Synthesis of silica fiber nanoparticles (hereinafter abbreviated as KCC-1). First, a mixture of urea (0.60g) and CPB (1.10g) was dissolved in water (30mL) for 5min under sonication, followed by the addition of cyclohexane (30mL) and isopropanol (0.94 mL). TEOS (3mL) was then added dropwise to the mixture using a syringe and the solution was heated to 70 ℃ with stirring. After centrifugation at 8000g for 10min at 4 ℃, the residue was washed three times with acetone, deionized water and absolute ethanol in that order and dried under vacuum for 12 h. KCC-1 nanoparticles were obtained by calcination at 500 ℃ for 4 h.
The G/KCC-1 nanocomposite is synthesized by a direct steam-induced internal hydrolysis method. In brief, the synthesized GO nanoparticles were dispersed in water and sonicated to obtain a GO solution that was uniformly dispersed for 1 h. KCC-1 nanoparticles were then added to the GO solution at a G/KCC-1 mass ratio of 1: 1. The mixed solution was stirred vigorously magnetically at 120 ℃ for 2 h. Finally, the unabsorbed KCC-1 was removed by washing the residue three times with 50% v/v ethanol aqueous solution to obtain a G/KCC-1 nanocomposite.
1.3 characterization
Atomic Force Microscope (AFM) images were performed by tapping on a Digital Multimode V scanning probe microscope (Veeco Metrology, USA) to measure the thickness of G/KCC-1 nanocomposites. Transmission electron microscopy (TEM, JEOL Jem-2100F, Thermo Scientific, Mass.) and scanning electron microscopy (SEM, FEI survey F50, Thermo Scientific, Mass.) were used to characterize the G/KCC-1 nanohybrids. The phase composition of the G/KCC-1 nano hybrid was evaluated using X-ray diffraction (XRD) with CuK α X-ray radiation (λ 1.5406nm) (Smartlab9, Rigaku, japan). For the analysis of the elemental composition, an ultraviolet-visible diffuse reflectance spectroscopy (UV vis-DRS) spectrometer (Lambda 950, Perkin Elmer, USA) equipped with a 150mm integrating sphere attachment was used.
1.4 Experimental example of extracting purified phospholipid from clam
Clams (corbicula fluminea) were purchased from Wumei supermarket (hang state, china). Tissues were collected, homogenized using an Ultra Turrax (T25, IKA-Werka, germany) at 1500rpm, and then dried to a powder under vacuum freeze-drying (Beta 2-8LD plus, Christ, germany). 0.3g of dried clam powder was accurately weighed out again and mixed with chloroform methanol (2: 1, v/v, 12 mL). After extraction of crude lipids under sonication, deionized water (3.2mL) was added to the mixture to separate hydrophilic impurities. The mixture was then centrifuged at 8000g for 10 min. The bottom organic phase was recovered while the solid residue was extracted two more times with chloroform (10 mL). Finally, the collected organic phases were combined and evaporated to dryness under a stream of nitrogen. The crude lipids were washed with cold acetone (-20 ℃) before solid phase extraction with G/KCC-1.
The preparation method of the G/KCC-1SPE mini-column is as follows. The synthesized G/KCC1 nanocomposite (120mg, 0.8cm height) was packaged in a sleeve (1 mL). Two polypropylene gaskets were placed at the bottom and top of the cartridge to hold the adsorbent well, thus making a G/KCC-1SPE cartridge. G/KCC-1SPE cartridge by hexane and chloroform/isopropanol (2: 1, V/V) activated. The crude lipid extract (60mg) was loaded onto an SPE cartridge under vacuum. The column was then washed sequentially with chloroform/isopropanol (2: 1, v/v) and 0.1% formic acid in ether to remove free fatty acids and neutral lipids. Finally, the reaction solution is passed through a column at a flow rate of 1.5 mL. min–1The column was washed with a mixture of chloroform-methanol-hydrochloric acid (2: 1: 0.01, v/v/v) to obtain a fraction of the objective compound. The eluent is placed under nitrogenAnd (4) drying under flowing.
1.5HILIC-MS analysis
The chromatographic separation of phospholipids was carried out using a Cosmosil HILIC chromatography column (250X 4.6mm, 3 μm). Phospholipid analysis was performed using an Agilent 1100 series HPLC instrument (Agilent technologies, Palo alto, Calif.) with a mass spectrometer (4000Q-Trap, Applied biosystems Sciex, Foster, USA with an electrospray ionization (ESI) interface). In gradient mode, the target analytes retained on the column are washed with mobile phases of A (20mM ammonium acetate and 20mM acetic acid in water) and B (20mM acetic acid in ACN). The sample injection amount was set to 2. mu.L, and the flow rate was set to 0.6 mL. min–1And a column temperature of 30 ℃. Prior to each injection, the column was washed, readjusted to the initial state, and re-equilibrated for 10 minutes to achieve good reproducibility. Phospholipids were ionized by an ESI ion source in negative ion mode (500 ℃ C., -4500V). Nitrogen was controlled at a curtain pressure of 25psi, ion source gas 1(GS1)35psi and ion source gas 2(GS2)35 psi. The Declustering Potential (DP), the Entrance Potential (EP) and the ion spray voltage (IS) were set to-60V and-10V, -4500V, respectively. Analysts 1.6.3 (Applied Biosystems, california, usa) are Applied for system control, spectrum collection and data analysis.
1.6 data analysis and statistics
HILIC-MS data was analyzed by the LIPID MAPS toolkit. One-way anova was used to determine statistical differences in significance levels (p < 0.05). The correlation was checked by Pearson correlation analysis (2 tails) and visualized by R version 3.6.2. The variability present in the data sets obtained was performed by MetabioAnalyst 3.0 (McGill university of Montreal, Canada).
2 results and discussion
We designated the products obtained at each stage of the treatment process (including crude lipid extract, acetone washed lipids and target phospholipids) as a, B and C, respectively.
The structure and morphology of GO, KCC-1 and G/KCC-1 nanocomposites were characterized by SEM, AFM and TEM. In fig. 1 a shows a SEM image of GO, which shows a large layered structure with a smooth surface. B is an AFM image showing the thickness and surface of the GO plate. After strong oxidation of graphite, single-layer and multi-layer GO sheets with smooth surfaces and a thickness of about 0.854nm are obtained. C shows an SEM image of KCC-1 having a wrinkled structure of mesoporous KCC-1. This structure can increase the coating ability and significantly enlarge the surface area. TEM image (D) of KCC-1 shows flower-like spherical morphology of colloidal silica spheres with center-radial nanowrinkles, formed by growth of central dendritic fibers. After KCC-1 is compounded with GO, the surface of GO is covered by KCC-1 particles (E). AFM image (F) showed an increase in the thickness of G/KCC-1 (4.571 nm). The characterization results of scanning electron microscopy, atomic force microscopy and transmission electron microscopy were consistent, which could confirm the successful preparation of GO sheets coated with KCC-1 particles.
FIG. 2 depicts XRD patterns of GO, KCC-1 and G/KCC-1 nanocomposites. After oxidation, the XRD pattern of the prepared GO shows a strong diffraction peak at 10.12 ° 2 θ, which corresponds to an interlayer distance of 0.88 nm. The resulting interlayer spacing can be attributed to the presence of carboxyl, hydroxyl, and epoxy functional groups on the GO surface, indicating that graphite has been successfully oxidized to GO. The diffraction peak of the 24,25G/KCC-1 nano hybrid is 23.5 degrees, which is attributed to the diffraction peak of KCC-1 particles. The optical properties of GO, KCC-1 and G/KCC-1 nano-hybrids were studied by UVvis DRS spectroscopy (FIG. 3). The absorption peak of GO is in the range of 235 to 310 nm. The transition of the aromatic CC bond can be reflected in the absorption peak at 235 nm. Absorption peaks of the G/KCC-1 nano hybrid were observed at both 230.1nm and 271.4nm due to the formation of silane molecules and KCC-1 particles in the graphite structure and on the GO surface by the plasma.
2.2 optimization of the adsorbent Material (SPE matrix)
The G/KCC-1 nanocomposite is an excellent solid material for phospholipid SPE because it has a large surface area of carbon and silica elements. In order to completely recover PMS remaining on G/KCC-1, conditions most influential on the pH of the sample, the volume and flow rate of the elution liquid, and the like were optimized.
The effect of pH on PMS recovery was first investigated. Under very low acid and base conditions, the adsorbent will degrade and cause the KCC-1 moieties to cleave from the GO surface. Thus, the pH was adjusted to 3 to 7, and the results are shown in FIG. 4As shown. The recovery of the representative standards gradually increased as the pH increased from 3 to 6. The recovery of phospholipids was highest (75.4% -88.6%) at pH 6, and dropped dramatically as pH was further increased. Thereafter, the volume of the elution solution in the range of 1-5mL was optimized to completely elute all PMS remaining on the adsorbent. The recovery of the three phospholipid standards was gradually increased with increasing elution solution volume (figure 5). It can be observed that PI is more sensitive to the volume of the elution solution than PC and PE. When the volume was increased to 5mL, the recovery of PI increased greatly to a maximum of 69.7%. The trend of PC and PE changes relatively gradually, which means that PC and PE are relatively easy to desorb from G/KCC-1 nanocomposites compared to PI. This phenomenon also indicates that PC and PE have stronger binding ability to KCC-1 than PI. Finally, 0.5 to 2.5mL min was also tested–1To investigate its effect on PMS recovery (fig. 6). The PMS exhibits a curve in which the recovery rate increases and then decreases as the flow rate increases. At 1.5 mL/min–1The highest recovery of PC, PE and PI was in the range of 60.2% to 80.1% at the flow rate of (2). Finally, the pH of the sample was used 6, the eluent was 5mL and the flow rate was 1.5mL min–1The parameter (c) of (c).
2.3 characterization of lipids
TLC has been used for characterization of biologically active substances and to monitor the progress of the reaction by semi-quantitative or quantitative analysis. It was used in G/KCC-1SPE eluates for rapid analysis of phospholipid content. The profile of phospholipids in the clam samples and standards was analyzed. The results show that the crude lipid of clams consists of PE, PC, PI and a large number of impurities. After washing with cold acetone, some impurities were removed, indicating that cold acetone can effectively remove non-polar lipids. For the G/KCC-1SPE, the eluted fractions appeared clean as observed in TLC plates (as most of the impurities in the head and tail were eliminated.) the composition of the remaining phospholipid species was consistent with the crude lipid sample.
To fully characterize the PMS, we used HILIC-MS technique. In this study, phospholipids with different polarity heads resulted in a significant change in their retention times on HILIC columns, and thus baseline separation of the three phospholipids PC, PE and PI was achieved within a 20min run time. In the positive ion mode, phospholipids cannot be ionized simultaneously at a single time, and the signal is easily suppressed by impurities due to protonation competition, so that the ionization of phospholipids can be handled in the negative ion mode. When formic acid is added to the mobile phase, PE and PI are typically protonated as [ M-H ] -, whereas PC is readily ionized as [ M + HCOO ] -. FIG. 7 shows the-MS spectra of representative crude HILIC lipids, in which the key peaks can be grouped into three groups according to the m/z range, namely m/z 450-550, m/z600-650 and m/z 750-900. Peaks in the ranges of m/z 450-550 and m/z 750-900, respectively, can be identified as lysophospholipids and phospholipids, while peaks in the range of m/z600 to 650 are co-extracted impurities from clam samples. The crude lipids were washed with cold acetone to remove non-polar molecules and interferences and the corresponding product spectra were recorded. Compared to the spectrum of crude lipid, the phospholipid signal in FIG. 8 increased from 2.0e6 cps to 2.3e6 cps, more PMS was observed, and the impurity peaks (e.g., m/z 600.4, m/z 610.4, and m/z 627.3) increased, and decreased significantly. The spectrum of the G/KCC-1SPE eluate is recorded in FIG. 9. The phospholipid signal observed in the m/z 700-900 range was further enhanced to 2.6e6 cps. In addition, background noise is hardly seen, and most of impurities are effectively removed. The HILIC-MS results for these three samples were almost identical to those of TLC analysis. After noise filtering and centroid processing, a total of 35 ions could be effectively identified (table 1). Since the head groups of these ions have been determined by phospholipid standards, the LIPIDMAPS software can unambiguously predict the sum of carbon atoms and double bonds.
TABLE 1 identification and content of phospholipids in clam samples treated by three methods
2.4 quantification of Phospholipids
Each PMS was quantified on the basis of an external calibration model. PC 14 is based on three principles: 0/14: 0, PE 15: 0/15: 0 and PI 16: 0/16: the principle behind 0, the three phospholipid standards, used to quantify all identified PMSs, was that the ionization efficiency of the compounds in MS was largely dependent on the dipole moment, the dipole moment of the phospholipids was mainly concentrated on the head group, and the dipole moment of the fatty acyl chains was negligible. The model was established by plotting a calibration curve using the peak area and the weight coefficient of phospholipid standard concentration at five levels as 11 ×. As shown in table 2, PC 14: 0/14: linear equation of 0 is y ═ 4.76E +06x-57100, R2Is 0.9981, wherein the variables for x and y are concentrations (mg. g)–1) And peak area. Thus, the absolute content of each PMS was calculated as shown in table 1. After the G/KCC-1SPE, PC 16 was found: 0/20: 5(m/z 824.6) is most important, followed by PC 16: 0/18: 1(m/z 804.6) and PC 16: 0/22: 6(m/z 850.6). Interestingly, the PC molecular species of the epaahha structure account for a significant proportion of 53.62%. It has been reported that this species of PC molecules with EPA/DHA structure has a function in regulating the intestinal immune response, regulating the intestinal flora composition and relieving chronic stress [28 ]]. In the m/z 700-800 range, ions at m/z 716.4 and m/z 790.5 are the two most important PE molecular species, identified as [ PE 16: 0/18: 1-H]And [ PE 18: 0/22: 6-H]-. A total of 7 PI molecular species were identified, with PI 18: 0/18: 1(m/z 863.6) and PI 16: 1/18: 0(m/z 835.6) is the two PI molecular species with the highest content, respectively 1.57 and 1.06 μ g. mg–1. The results show that the G/KCC-1SPE can enrich PMS in a pure way.
TABLE 2 SPE-HILIC-MS method verification in terms of linearity, sensitivity, accuracy and recovery
2.5 analytical Properties
The performance of the G/KCC-1SPE HILIC-MS lipidomics method has been validated through correlation coefficient (R2), linear range, limit of detection (LOD), limit of quantitation (LOQ), daytime and internal validation of day precision and recovery. As shown in Table 2, the regression equations were linear in the concentration range of 0.39-200. mu.g.mL-1, and the correlation coefficient (R2) was in the range of 0.9965 to 0.9981, indicating a good linear relationship between the regression equations. Peak area (y) and target phospholipid concentration (x). LOD and LOQ are considered to be the lowest concentrations of target analyte that can be unambiguously identified and quantified, and are calculated using signal-to-noise ratios (s/n) of 3 and 10, respectively. The measured LOD and LOQ are respectively 0.19-0.51. mu.g.mL–1And 0.48-1.47. mu.g.mL–1Within the range of (1). The accuracy of the method is expressed as an intra-day accuracy and an inter-day accuracy. The day precision was evaluated by calculating the Relative Standard Deviation (RSD) of the six technical replicates, while the day precision was determined by examining the variability for three consecutive days. RSD for the intra-day precision is between 4.64% and 7.16%, and RSD for the inter-day precision is in the range of 5.78% -7.30%. By mixing at 50, 100 and 150. mu.g.mL–1And adding a standard blank matrix into the phospholipid standard substance to perform a recovery rate experiment. This value is calculated based on the proportion of standards added, which is greater than 88.19%. The result shows that the G/KCC-1SPE HILIC-MS method is reliable, accurate and effective in selective extraction from clams and lipidomics phenotype analysis.
4 conclusion
In the experimental example, G/KCC-1 nano-composite is successfully prepared and used as a self-made absorbent of a micro chromatographic column for carrying out high-selectivity SPE on the phospholipid of clams. PMS in the crude lipid was initially extracted by washing with cold acetone. The PMS was then further purified and recovered by G/KCC-1SPE in one run at optimal conditions of sample pH, elution volume and flow rate. 35 PMS in the clams are identified and quantified together, and 53.62% of PC molecular species with EPAADHA structures are obtained. The present invention provides an efficient and reliable method for selectively purifying phospholipids in clams.
Claims (7)
1. A method for extracting and purifying phospholipid from clams is characterized by comprising the following steps:
collecting clam tissues, homogenizing by using a high-speed dispersion machine, and then drying under vacuum freezing to obtain clam powder;
② chloroform methanol ultrasonic treatment is used to extract crude lipid from clam powder;
thirdly, washing the crude lipid with cold acetone to obtain a crude lipid extract;
and fourthly, using the compound of the silicon dioxide fiber nanoparticles and the graphene oxide nanoparticles as an adsorbent, and performing adsorption extraction on the phospholipid in the crude lipid extract by using a solid phase extraction method.
2. The method for extracting and purifying phospholipid from clams as claimed in claim 1, wherein in the step (iv), the preparation method of the compound of silica fiber nanoparticles and graphene oxide nanoparticles is as follows: dispersing the synthesized graphene oxide nano particles in water and carrying out ultrasonic treatment to obtain a graphene oxide solution, and uniformly dispersing for 1 h; then, adding the silica fiber nanoparticles into the dispersed graphene oxide solution according to the mass ratio of the silica fiber nanoparticles to the graphene oxide nanoparticles of 1: 1, and stirring the mixed solution for 2 hours at 120 ℃ by intense magnetic force; finally, the residue was washed three times or more by using a 50% v/v ethanol aqueous solution to remove unabsorbed silica fiber nanoparticles, to obtain a composite of silica fiber nanoparticles and graphene oxide nanoparticles.
3. A method for extracting and purifying phospholipid from clams as claimed in claim 2, wherein the method for preparing the silica fiber nano-particles is as follows: under ultrasonic treatment, dissolving a mixture of 0.60g of urea and 1.10g of bromohexadecylpyridine in 30mL of water for 5min, and then adding 30mL of cyclohexane and 0.94mL of isopropanol; then, 3mL of ethyl orthosilicate was added dropwise to the mixture using a syringe, and the solution was heated to 70 ℃ with stirring; after centrifugation at 8000g for 10min at 4 ℃, the resulting residue was washed three times with acetone, deionized water and absolute ethanol in order and dried under vacuum for 12h to obtain silica fiber nanoparticles by calcination at 500 ℃ for 4 h.
4. The method for extracting and purifying phospholipid from clams as claimed in claim 1, wherein the preparation method of the graphene oxide nano-particles is as follows: every 40mL of 85 mass percent phosphoric acid solution is mixed with 360mL of 98 mass percent sulfuric acid solution to obtain a mixed acid solution, 18.0g of potassium permanganate and 3.0g of graphite flake are added into the mixed acid solution for reaction, after the reaction is cooled to room temperature, the mixture obtained by the reaction is poured on ice, and 5mL of H with the mass concentration of 30 percent is used2O2Quenching the aqueous solution, then carrying out centrifugal treatment, neutralizing the mixture obtained after the centrifugal treatment by using a 4 wt% HCl aqueous solution, removing residual salt in the obtained solid material by dialysis in ultrapure deionized water, and carrying out vacuum drying at room temperature overnight to obtain the graphene oxide nano-particles.
5. A method for extracting and purifying phospholipid from clam according to claim 1, wherein the specific method of the step II comprises the steps of accurately weighing clam powder obtained in the step I, mixing the clam powder with chloroform-methanol mixed solution with a volume ratio of 2:1, extracting crude lipid under ultrasonic treatment, and adding deionized water into the mixture to separate hydrophilic impurities; then, the mixture was centrifuged at 8000g for 10min, the bottom organic phase was recovered while the solid residue was extracted twice more with chloroform, and finally, the collected organic phases were combined and evaporated to dryness under a nitrogen stream using 12mL of chloroform-methanol mixture, 3.2mL of deionized water, and 10mL of chloroform per 0.3g of clam powder.
6. The method for extracting and purifying phospholipids from clams as claimed in claim 1, which is characterized in that: in the first step, the rotating speed of the high-speed dispersion machine is 1500 rpm.
7. The method for extracting and purifying phospholipid from clams as claimed in claim 1, wherein the specific method of the step (iv) is as follows:packing the adsorbent in a sleeve, arranging two polypropylene gaskets at the bottom and the top of the sleeve to well fix the adsorbent to prepare an SPE column; and then mixing the mixture by using hexane and a volume ratio of 2:1, loading the crude lipid extract on an SPE column under vacuum, then successively activating with a chloroform/isopropanol mixture of 2:1 chloroform/isopropanol mixed solution and 0.1% formic acid ether solution by mass percentage wash SPE column, in order to remove free fatty acid and neutral lipid; finally, the reaction solution is passed through a column at a flow rate of 1.5 mL. min–1Is 2: 1: the SPE cartridge was washed with a 0.01 chloroform/methanol/hydrochloric acid mixture to obtain a fraction of the target compound, and the eluate was dried under a stream of nitrogen.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011040174.2A CN112250706B (en) | 2020-09-28 | 2020-09-28 | Method for extracting and purifying phospholipid from clams |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011040174.2A CN112250706B (en) | 2020-09-28 | 2020-09-28 | Method for extracting and purifying phospholipid from clams |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112250706A true CN112250706A (en) | 2021-01-22 |
CN112250706B CN112250706B (en) | 2023-01-24 |
Family
ID=74233819
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011040174.2A Active CN112250706B (en) | 2020-09-28 | 2020-09-28 | Method for extracting and purifying phospholipid from clams |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112250706B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114019042A (en) * | 2021-10-29 | 2022-02-08 | 浙江工商大学 | Method and device for determining content of pelteobagrus fulvidraco protease inhibitor based on graphene |
CN114019039A (en) * | 2021-10-13 | 2022-02-08 | 温州医科大学附属第二医院(温州医科大学附属育英儿童医院) | Method for selectively extracting phospholipid from plasma sample and phospholipid detection method |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1408716A (en) * | 2001-09-25 | 2003-04-09 | 上海金伴药业有限公司 | Process for preparing powdered soy bean phosphatide |
CN106616022A (en) * | 2016-12-05 | 2017-05-10 | 陈庆功 | Carassius auratus feed |
CN106928908A (en) * | 2017-02-19 | 2017-07-07 | 广州市芯检康生物科技有限公司 | A kind of new aeroge multifunctional material and preparation method thereof |
US20180360854A1 (en) * | 2017-06-16 | 2018-12-20 | Institute of Rheological Functions of Food | Uses of ether phospholipids in treating diseases |
CN110726769A (en) * | 2019-11-21 | 2020-01-24 | 浙江工商大学 | Rapid detection method for phospholipid |
-
2020
- 2020-09-28 CN CN202011040174.2A patent/CN112250706B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1408716A (en) * | 2001-09-25 | 2003-04-09 | 上海金伴药业有限公司 | Process for preparing powdered soy bean phosphatide |
CN106616022A (en) * | 2016-12-05 | 2017-05-10 | 陈庆功 | Carassius auratus feed |
CN106928908A (en) * | 2017-02-19 | 2017-07-07 | 广州市芯检康生物科技有限公司 | A kind of new aeroge multifunctional material and preparation method thereof |
US20180360854A1 (en) * | 2017-06-16 | 2018-12-20 | Institute of Rheological Functions of Food | Uses of ether phospholipids in treating diseases |
CN110726769A (en) * | 2019-11-21 | 2020-01-24 | 浙江工商大学 | Rapid detection method for phospholipid |
Non-Patent Citations (3)
Title |
---|
GONGSHUAI SONG等,: ""Titania-coated fibrous silica (TiO2/KCC-1) core-shell microspheres based solid-phase extraction in clam (Corbicula fluminea) using hydrophilic interaction liquid chromatography and mass spectrometry"", 《FOOD RESEARCH INTERNATIONAL》 * |
姜家良: ""SiO2-GO 复合纳米颗粒改性 PVDF 超滤膜的制备与性能研究"", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 * |
王鹏鹏等,: ""氧化石墨烯/二氧化硅复合材料的制备及其在吸附领域应用研究进展"", 《人工晶体学报》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114019039A (en) * | 2021-10-13 | 2022-02-08 | 温州医科大学附属第二医院(温州医科大学附属育英儿童医院) | Method for selectively extracting phospholipid from plasma sample and phospholipid detection method |
CN114019042A (en) * | 2021-10-29 | 2022-02-08 | 浙江工商大学 | Method and device for determining content of pelteobagrus fulvidraco protease inhibitor based on graphene |
Also Published As
Publication number | Publication date |
---|---|
CN112250706B (en) | 2023-01-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Lu et al. | Integrated metabonomics analysis of the size-response relationship of silica nanoparticles-induced toxicity in mice | |
CN112250706B (en) | Method for extracting and purifying phospholipid from clams | |
Song et al. | Lipidomics phenotyping of clam (Corbicula fluminea) through graphene/fibrous silica nanohybrids based solid-phase extraction and HILIC-MS analysis | |
DE69919981T2 (en) | PROCESS FOR THE ISOLATION, RECOVERY AND CLEANING OF NON-POLAR EXTRACTS | |
Shen et al. | Lipidomic profiling of dried seahorses by hydrophilic interaction chromatography coupled to mass spectrometry | |
Shi et al. | Magnetic covalent organic framework material: synthesis and application as a sorbent for polycyclic aromatic hydrocarbons | |
Song et al. | Titania-coated fibrous silica (TiO2/KCC-1) core-shell microspheres based solid-phase extraction in clam (Corbicula fluminea) using hydrophilic interaction liquid chromatography and mass spectrometry | |
Jiang et al. | Liquid–solid extraction coupled with magnetic solid-phase extraction for determination of pyrethroid residues in vegetable samples by ultra fast liquid chromatography | |
Babincová et al. | Antioxidant properties of carboxymethyl glucan: comparative analysis | |
Zhang et al. | Magnetic retrieval of chitosan: Extraction of bioactive constituents from green tea beverage samples | |
Pereira et al. | Arsenic containing medium and long chain fatty acids in marine fish oil identified as degradation products using reversed-phase HPLC-ICP-MS/ESI-MS | |
Huang et al. | Performance of metal-organic framework MIL-101 after surfactant modification in the extraction of endocrine disrupting chemicals from environmental water samples | |
US20160272661A1 (en) | Purified phosphatidylcholine product made by a process employing magnetic nanoparticles | |
Tay et al. | Magnetic nanoparticle assisted dispersive liquid–liquid microextraction for the determination of 4-n-nonylphenol in water | |
CN110658280A (en) | Method for detecting bisphenol compounds based on magnetic metal organic framework composite material | |
Liu et al. | The determination of pesticides in tea samples followed by magnetic multiwalled carbon nanotube-based magnetic solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry | |
Li et al. | Thiol-rich polyhedral oligomeric silsesquioxane-modified magnetic nanoparticles for the highly efficient separation and preconcentration of Cd (II) and Pb (II) in food and water prior to ICP-OES determination | |
Schlappack et al. | Sulfonated halloysite nanotubes as a novel cation exchange material for solid phase extraction of toxic pyrrolizidine alkaloids | |
Yuan et al. | Polypyrrole-modified magnetic multi-walled carbon nanotube-based magnetic solid-phase extraction combined with dispersive liquid–liquid microextraction followed by UHPLC-MS/MS for the analysis of sulfonamides in environmental water samples | |
Du et al. | Root exudates as natural ligands that alter the properties of graphene oxide and environmental implications thereof | |
Kraffe et al. | Evidence of a tetradocosahexaenoic cardiolipin in some marine bivalves | |
CN111474248A (en) | Method for determining four preservatives in cosmetics | |
Salem Jr et al. | Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines | |
CN114019039A (en) | Method for selectively extracting phospholipid from plasma sample and phospholipid detection method | |
Moghadam et al. | Dendrimer-modified magnetic nanoparticles as a sorbent in dispersive micro-solid phase extraction for preconcentration of metribuzin in a water sample |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |