CN1438215A - Method for separating and purfying ceramide using molecular ungram method - Google Patents
Method for separating and purfying ceramide using molecular ungram method Download PDFInfo
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- CN1438215A CN1438215A CN 03120819 CN03120819A CN1438215A CN 1438215 A CN1438215 A CN 1438215A CN 03120819 CN03120819 CN 03120819 CN 03120819 A CN03120819 A CN 03120819A CN 1438215 A CN1438215 A CN 1438215A
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- 238000000034 method Methods 0.000 title claims abstract description 41
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 title claims description 38
- 229940106189 ceramide Drugs 0.000 title claims description 38
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 title claims description 38
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 title claims description 38
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 title claims description 37
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 39
- 239000000178 monomer Substances 0.000 claims abstract description 38
- 238000000926 separation method Methods 0.000 claims abstract description 23
- 238000000746 purification Methods 0.000 claims abstract description 21
- 239000003960 organic solvent Substances 0.000 claims abstract description 13
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 10
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims abstract description 8
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 150000001408 amides Chemical class 0.000 claims abstract description 5
- 150000002148 esters Chemical class 0.000 claims abstract description 4
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims abstract description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 60
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 239000000284 extract Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 229920000858 Cyclodextrin Polymers 0.000 claims description 15
- 239000001116 FEMA 4028 Substances 0.000 claims description 14
- 229960004853 betadex Drugs 0.000 claims description 14
- 239000003999 initiator Substances 0.000 claims description 13
- 150000002632 lipids Chemical class 0.000 claims description 13
- 239000004005 microsphere Substances 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 11
- 238000001035 drying Methods 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 11
- 238000011010 flushing procedure Methods 0.000 claims description 9
- -1 methyl acrylic ester Chemical class 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 9
- 239000011541 reaction mixture Substances 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 7
- 229920002554 vinyl polymer Polymers 0.000 claims description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 150000001993 dienes Chemical class 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000013074 reference sample Substances 0.000 claims description 6
- 238000006116 polymerization reaction Methods 0.000 claims description 5
- JHPBZFOKBAGZBL-UHFFFAOYSA-N (3-hydroxy-2,2,4-trimethylpentyl) 2-methylprop-2-enoate Chemical compound CC(C)C(O)C(C)(C)COC(=O)C(C)=C JHPBZFOKBAGZBL-UHFFFAOYSA-N 0.000 claims description 4
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 4
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 4
- QBEDRAYIJQIKRU-UHFFFAOYSA-N N1C(=O)NC=2NC(=O)NC2C1=O.[C-]#N Chemical compound N1C(=O)NC=2NC(=O)NC2C1=O.[C-]#N QBEDRAYIJQIKRU-UHFFFAOYSA-N 0.000 claims description 4
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 4
- VVWRJUBEIPHGQF-UHFFFAOYSA-N propan-2-yl n-propan-2-yloxycarbonyliminocarbamate Chemical compound CC(C)OC(=O)N=NC(=O)OC(C)C VVWRJUBEIPHGQF-UHFFFAOYSA-N 0.000 claims description 4
- 238000005829 trimerization reaction Methods 0.000 claims description 4
- ONJMNXFNTYIEEA-UHFFFAOYSA-N benzene ethene Chemical compound C1=CC=CC=C1.C=C.C=C.C=C ONJMNXFNTYIEEA-UHFFFAOYSA-N 0.000 claims description 3
- XZKRXPZXQLARHH-UHFFFAOYSA-N buta-1,3-dienylbenzene Chemical compound C=CC=CC1=CC=CC=C1 XZKRXPZXQLARHH-UHFFFAOYSA-N 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000011065 in-situ storage Methods 0.000 claims description 3
- DBSDMAPJGHBWAL-UHFFFAOYSA-N penta-1,4-dien-3-ylbenzene Chemical compound C=CC(C=C)C1=CC=CC=C1 DBSDMAPJGHBWAL-UHFFFAOYSA-N 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 2
- 125000000864 peroxy group Chemical group O(O*)* 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 abstract 2
- VKEQBMCRQDSRET-UHFFFAOYSA-N Methylone Chemical compound CNC(C)C(=O)C1=CC=C2OCOC2=C1 VKEQBMCRQDSRET-UHFFFAOYSA-N 0.000 abstract 1
- 239000002253 acid Substances 0.000 abstract 1
- 150000001252 acrylic acid derivatives Chemical class 0.000 abstract 1
- 239000003431 cross linking reagent Substances 0.000 abstract 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 abstract 1
- 230000000379 polymerizing effect Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000012501 chromatography medium Substances 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 229960001866 silicon dioxide Drugs 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 208000001126 Keratosis Diseases 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical class CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 125000001549 ceramide group Chemical group 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- ZYASLVDNDXUOMI-UHFFFAOYSA-N n,n-dimethylmethanamine;prop-2-enoic acid Chemical compound C[NH+](C)C.[O-]C(=O)C=C ZYASLVDNDXUOMI-UHFFFAOYSA-N 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- FBCQUCJYYPMKRO-UHFFFAOYSA-N prop-2-enyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC=C FBCQUCJYYPMKRO-UHFFFAOYSA-N 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
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- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention is a method that adopts molecular print to separate and purify nervous amide, mixing the hydrophilic methyl acrolic acid monomers: methyl-acrylate, acrylic acid or one or two of acrylates with the hydrophobic monomer phenylethene and super-molecule monomer, then mixing with the cross-linking agent, the hole-making agent, the evocating agent and the sample of molecule-printed nervous amide, polymerizing, and getting rid of hole-making agent and template molecules to get the molecule-printed separating medium; adopting organic solvent to extract esters; add the esters in chromatogram column with the medium to realize the separation and purification.
Description
Technical field
The present invention relates to a kind of method that ceramide is carried out separation and purification, belong to the separating and purifying technology of ceramide.
Background technology
The ceramide English name is Ceramide, and chemical structure is N-fatty acyl group (nerve) sphingosine.Its content in keratoderma is very high, has barrier, and is bonding, preserves moisture anti-ageing and anti-allergic effects.Up-to-date studies show that, it also has activation multiple protein kinases and transcription factor participates in the function of signal conduction in the cell, thereby influences multiple physiology, pathologic processes such as cell growth, propagation, differentiation, apoptosis and damage.As a kind of novel bioactive material, ceramide has broad application prospects in medicines and health protection and cosmetic industry.
The ceramide of using in the reality is generally natural extract at present, takes from pigskin, ox brain, egg, hemocyte, plant and yeast cell etc.Tlc is mainly adopted in the ceramide separation and purification.People such as Robson have narrated the process of ceramide in this kind method separation of human keratoderma in " Journal ofLipid Research " 35 volumes in 1994, comprise that the extraction of lipid, extract hydrolysis separate with thin-layer chromatography.Concrete operations are as follows: adopt improved Bligh and Dyer method to extract lipid; Hydrolysis extract under the condition of gentleness; Extract after the hydrolysis is added on the chromatographic sheet, and chloroform/methanol/acetate is developping agent, each component is launched, and dyeing, the part that will contain ceramide scrapes from the thin-layer chromatography silica-gel plate, and chloroform/methanol elutes the ceramide component from silica gel.People such as Rupcic have narrated the sepn process of ceramide in the thin layer chromatography separated yeast in " Chemistry and Physics of Lipids " 91 volumes in 1998, comprise that fragmentation, the extraction of lipid, extract hydrolysis, the normal-phase chromatography roughing out of yeast cell separates with thin-layer chromatography.Concrete operations are as follows: utilize refiner with the yeast cell fragmentation; Adopt improved Floch method to extract lipid; Hydrolysis extract under the condition of gentleness; The normal-phase chromatography roughing out; Each component of chromatographic separation is added on the chromatographic sheet, adopts the 25 step methods of development to launch, dyeing, the part that will contain ceramide scrapes from the thin-layer chromatography silica-gel plate, and chloroform elutes the ceramide component from silica gel.As seen, adopt tlc separation and purification of nerve acid amides to need preprocessing process such as extracting solution hydrolysis and chromatogram roughing out, sepn process is loaded down with trivial details, and the cycle is long, and treatment capacity is low.
Summary of the invention
Deficiency and defective at present ceramide separation purification method, the purpose of this invention is to provide a kind of method of utilizing molecular imprinting method that ceramide is carried out separation and purification, realize that the highly selective of ceramide is separated, simplify sepn process, realize the serialization of sepn process, improve treatment capacity.
The objective of the invention is to be achieved through the following technical solutions:
A kind of molecular imprinting method that utilizes is characterized in that to the method that ceramide carries out separation and purification this method comprises the steps:
(1) with in hydrophilic monomer methacrylic acid, methyl acrylic ester, vinylformic acid or the esters of acrylic acid one or both with mix with linking agent, pore-creating agent, initiator and microsphere ceramide reference sample respectively again after beta-cyclodextrin that hydrophobic monomer vinylbenzene and supramolecule monomer contain vinyl mixes, wherein:
A. hydrophobic monomer is 0.25～1 with the ratio of hydrophilic monomer amount of substance
B. the supramolecule monomer beta-cyclodextrin that contains vinyl is 0.01～0.2 with the ratio of hydrophobic monomer amount of substance;
C. linking agent is 0.5～20 with the ratio of monomeric amount of substance;
D. microsphere is 0.01～1 with the ratio of monomeric amount of substance;
E. the pore-creating agent consumption accounts for 40～75% of reaction mixture volume content;
(2) above-mentioned reaction mixture is ultrasonic evenly mixed, join in the reactor, in-situ polymerization, polymerization temperature are controlled between 65～85 ℃, and the reaction times is 6～24 hours;
(3) after polyreaction finishes, the synthetic polymkeric substance is taken out from reactor, grind, utilize organic solvent extracting (organic solvent preferably adopts ethanol or chloroform), after the drying, screening is loaded in the gc column tube, obtains the molecular imprinting separator column;
(4) utilizing volume ratio is that 2: 1 chloroform and methanol mixed solution extracts lipid material from yeast, obtains the extract sample after the drying, dissolves in chloroform, wiring solution-forming again;
(5) above-mentioned solution is joined in the chromatographic column, remove the lipid material of non-ceramide type with the organic solvent flushing, and then use the organic solvent wash-out, collect elution fraction.
The beta-cyclodextrin that contains vinyl described in the present invention adopts single the replacement or polysubstituted acryl beta-cyclodextrin or single the replacement or polysubstituted methacryloyl beta-cyclodextrin.
Linking agent described in the present invention adopts diene and triolefin linking agent to unite use, and diene is 0.5～2 with the ratio of triolefin, and the diene linking agent adopts any in Vinylstyrene, divinyl phenylmethane and the methacrylic ester; The triolefin linking agent adopts any in trimerization isonitrile uric acid triallyl, triethylene benzene and the methacrylic ester.
Initiator described in the present invention adopts organic peroxy class or azo class material, preferably adopts dibenzoyl peroxide or Diisopropyl azodicarboxylate.
Pore-creating agent described in the present invention adopts heterogeneous ring compound, acid amides or sulfone compound, preferably adopts pyridine, dimethyl formamide or dimethyl sulfoxide (DMSO).
The used organic solvent of flushing is chloroform and normal hexane mixing solutions among the present invention, and wherein the volume percent of chloroform is 0～40%; The used organic solvent of wash-out is chloroform and normal hexane mixing solutions, and wherein the volume percent of chloroform is 50～100%, and also can adopt ethanol or methyl alcohol is elutriant.
The present invention compares with the method for present ceramide separation and purification, have the following advantages and the high-lighting effect: be microsphere with the ceramide, adopt hydrophilic monomer, hydrophobic monomer and supramolecule monomer bonded method, especially after having introduced the beta-cyclodextrin that contains vinyl, can make the synthetic separating medium have the recognition site that is complementary with ceramide molecular structure and functional group, thereby realize the highly selective separation and purification of separating medium ceramide.Compare with the tlc that adopts in the present ceramide separation and purification, adopt institute of the present invention synthetic chromatographic media to prepare by in-situ polymerization, only both can realize the high efficiency separation purifying of ceramide by last sample, flushing, wash-out and the balance of routine, can realize continuous operation, the capacity height of chromatographic column, the separation and purification process is easy, and cost is low.
Embodiment
The following examples will be further specified method provided by the invention.
Embodiment 1
Take by weighing hydrophobic monomer vinylbenzene, hydrophilic monomer glycidyl methacrylate, methacrylic acid and singly got the acryloyl group-beta-cyclodextrin (1: 0.5: 0.5: 0.01mol/mol/mol/mol), linking agent Vinylstyrene and trimerization isonitrile uric acid triallyl (1: 1mol/mol, linking agent/monomer 0.5: 1mol/mol); Pore-creating agent pyridine (pore-creating agent/reaction mixture 75vol%); Initiator Diisopropyl azodicarboxylate (initiator/monomer 0.01: 1mol/mol); Microsphere ceramide reference sample (microsphere/monomer 0.01: 1mol/mol), mix, in the Glass tubing of the 100 * 5.0mm that packs into, with the other end sealing, in 65 ℃ of reactions 24 hours.Polymer monolith is taken out, grind ethanol extracting, drying.With particle diameter is that 37～74 microns chromatographic media is loaded in 150 * 4.6mm chromatographic column, is connected on the chromatographic instrument.Utilize the Bligh-Dyer method, extract lipid material from yeast, (the Bligh-Dyer method is that 2: 1 chloroform and methanol mixed solution is extracting solution with volume ratio) obtains the extract sample after the drying, dissolve in chloroform again, is made into the solution that concentration is 1mg/ml.1ml solution is joined in the chromatographic column, normal hexane flushing, then, (50%: 50%vol/vol) wash-out obtains the ceramide component of wash-out to chloroform/normal hexane.
Embodiment 2
Take by weighing hydrophobic monomer vinylbenzene; hydrophilic monomer vinylformic acid and singly got methacryloyl-beta-cyclodextrin (0.5: 1: 0.1mol/mol/mol/mol); linking agent divinyl phenylmethane and trimethylammonium vinylformic acid three methanol-based propane esters (1: 1mol/mol; linking agent/monomer 1: 1mol/mol); pore-creating agent dimethyl formamide (pore-creating agent/reaction mixture 60vol%); initiator Diisopropyl azodicarboxylate (initiator/monomer 0.005: 1mol/mol); microsphere ceramide reference sample (microsphere/monomer 0.1: 1mol/mol); mix; pack in the Glass tubing of 100 * 5.0mm; with the other end sealing, in 75 ℃ of reactions 12 hours.Polymer monolith is taken out, grind ethanol extracting, drying.With particle diameter is that 37～74 microns chromatographic media is loaded in 150 * 4.6mm chromatographic column, is connected on the chromatographic instrument.Utilize the Bligh-Dyer method, the employing volume ratio is that 2: 1 chloroform and methanol mixed solution extracts lipid material from yeast, obtains the extract sample after the drying, dissolves in chloroform again, is made into the solution of 1mg/ml.1ml solution is joined in the chromatographic column, the flushing of 30% chloroform/normal hexane, then, the chloroform wash-out obtains the ceramide component of wash-out.
Embodiment 3
Take by weighing reaction monomers vinylbenzene; methacrylic acid hydroxypropyl fat; methacrylic acid and five substituent methyl acryloyl group-beta-cyclodextrins (0.5: 1: 1: 0.025mol/mol/mol/mol); linking agent allyl methacrylate(AMA) and triethylene benzene (1: 2mol/mol; linking agent/monomer 20: 1mol/mol); pore-creating agent dimethyl sulfoxide (DMSO) (pore-creating agent/reaction mixture 40vol%); initiator dibenzoyl peroxide (initiator/monomer 0.02: 1mol/mol); microsphere ceramide reference sample (microsphere/monomer 1: 1mol/mol); mix; pack in the Glass tubing of 100 * 5.0mm; with the other end sealing, in 85 ℃ of reactions 12 hours.Polymer monolith is taken out, grind extracting, drying.With particle diameter is that 37～74 microns chromatographic media is loaded in 150 * 4.6mm chromatographic column, is connected on the chromatographic instrument.Utilize the Bligh-Dyer method, the employing volume ratio is that 2: 1 chloroform and methanol mixed solution extracts lipid material from yeast, obtains the extract sample after the drying, dissolves in chloroform again, is made into the solution of 1mg/ml.1ml solution is joined in the chromatographic column, the flushing of 40% chloroform/normal hexane, then, methanol-eluted fractions obtains the ceramide component of wash-out.
Embodiment 4
Take by weighing reaction monomers vinylbenzene; the vinylformic acid formicester; vinylformic acid and five substituted acryls-beta-cyclodextrin (0.5: 1: 1: 0.05mol/mol/mol/mol); linking agent divinylbenzene and trimerization isonitrile uric acid triallyl (2: 1mol/mol; linking agent/monomer 10: 1mol/mol); pore-creating agent dimethyl formamide (pore-creating agent/reaction mixture 50vol%); initiator dibenzoyl peroxide (initiator/monomer 0.01: 1mol/mol); microsphere ceramide reference sample (microsphere/monomer 0.75: 1mol/mol); mix; pack in the Glass tubing of 100 * 5.0mm; with the other end sealing, in 85 ℃ of reactions 6 hours.Polymer monolith is taken out, grind ethanol extracting, drying.With particle diameter is that 37～74 microns chromatographic media is loaded in 150 * 4.6mm chromatographic column, is connected on the chromatographic instrument.Utilize the Bligh-Dyer method, the employing volume ratio is that 2: 1 chloroform and methanol mixed solution extracts lipid material from yeast, obtains the extract sample after the drying, dissolves in chloroform again, is made into the solution of 1mg/ml.1ml solution is joined in the chromatographic column, the flushing of 40% chloroform/normal hexane, then, ethanol elution obtains the ceramide component of wash-out.
Claims (9)
1. one kind is utilized molecular imprinting method to the method that ceramide carries out separation and purification, it is characterized in that this method comprises the steps:
(1) with in hydrophilic monomer methacrylic acid, methyl acrylic ester, vinylformic acid or the esters of acrylic acid one or both with mix with linking agent, pore-creating agent, initiator and microsphere ceramide reference sample respectively again after beta-cyclodextrin that hydrophobic monomer vinylbenzene and supramolecule monomer contain vinyl mixes, wherein:
A. hydrophobic monomer is 0.25～1 with the ratio of the amount of substance of hydrophilic monomer;
B. the supramolecule monomer beta-cyclodextrin that contains vinyl is 0.01～0.1 with the ratio of hydrophobic monomer amount of substance;
C. linking agent is 0.5～20 with the ratio of monomeric amount of substance;
D. microsphere is 0.01～1 with the ratio of monomeric amount of substance;
E. the pore-creating agent consumption is 40～75% of a reaction mixture volume content;
(2) above-mentioned reaction mixture is ultrasonic evenly mixed, join in the reactor, in-situ polymerization, polymerization temperature are controlled between 65～85 ℃, and the reaction times is 6～24 hours;
(3) after polyreaction finishes, the synthetic polymkeric substance is taken out from reactor, grind, utilize the organic solvent extracting, the screening of dry back is loaded in the gc column tube, obtains the molecular imprinting separator column;
(4) utilizing volume ratio is that 2: 1 chloroform and methanol mixed solution extracts lipid material from yeast, obtains the extract sample after the drying, dissolves in chloroform, wiring solution-forming again;
(5) above-mentioned solution is joined in the chromatographic column, remove the lipid material of non-ceramide type with the organic solvent flushing, and then use the organic solvent wash-out, collect elution fraction.
2. according to the method for the described separation and purification of claim 1, it is characterized in that: the described beta-cyclodextrin that contains vinyl adopts single the replacement or polysubstituted acryl beta-cyclodextrin or single the replacement or polysubstituted methacryloyl beta-cyclodextrin.
3. according to the method for claim 1 or 2 described separation and purification, it is characterized in that: described linking agent adopts diene and triolefin linking agent to unite use, diene is 0.5～2 with the ratio of triolefin, and the diene linking agent adopts any in Vinylstyrene, divinyl phenylmethane and the methacrylic ester; The triolefin linking agent adopts any in trimerization isonitrile uric acid triallyl, triethylene benzene and the methacrylic ester.
4. according to the method for the described separation and purification of claim 3, it is characterized in that: described initiator adopts organic peroxy class or azo class material.
5. according to the method for the described separation and purification of claim 4, it is characterized in that: described initiator adopts dibenzoyl peroxide or Diisopropyl azodicarboxylate.
6. according to the method for the described separation and purification of claim 3, it is characterized in that: described pore-creating agent adopts heterogeneous ring compound, acid amides or sulfone compound.
7. according to the method for the described separation and purification of claim 6, it is characterized in that: described pore-creating agent is pyridine, dimethyl formamide or dimethyl sulfoxide (DMSO).
8. according to the method for the described separation and purification of claim 1, it is characterized in that: the organic solvent in the step (3) adopts ethanol or chloroform.
9. according to the method for the described separation and purification of claim 1, it is characterized in that: the used organic solvent of flushing is chloroform and normal hexane mixing solutions in the step (5), wherein the volume percent 0～40% of chloroform; The used organic solvent of wash-out is chloroform and normal hexane mixing solutions, the volume percent 50～100% of chloroform wherein, and also can adopt ethanol or methyl alcohol is elutriant.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093244A (en) * | 2010-12-06 | 2011-06-15 | 天津强微特生物科技有限公司 | Method for extracting ceramide |
| CN104870525A (en) * | 2012-12-26 | 2015-08-26 | 莱雅公司 | Molecularly imprinted polymers of sol-gel type and their use as antidandruff agent |
| CN104870482A (en) * | 2012-12-26 | 2015-08-26 | 莱雅公司 | Molecularly imprinted polymers and their use as antidandruff agents |
| CN106432590A (en) * | 2016-09-21 | 2017-02-22 | 中国农业科学院农业质量标准与检测技术研究所 | Molecularly imprinted polymer with controllable sustained-release function |
| JP2017145320A (en) * | 2016-02-17 | 2017-08-24 | 公立大学法人 富山県立大学 | Template-forming polymerizable compound and curable composition thereof, and cured product thereof |
| CN113834888A (en) * | 2021-09-23 | 2021-12-24 | 大连润生康泰医学检验实验室有限公司 | Accurate detection method and kit for 4 ceramides in blood |
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2003
- 2003-03-21 CN CN 03120819 patent/CN1252037C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093244A (en) * | 2010-12-06 | 2011-06-15 | 天津强微特生物科技有限公司 | Method for extracting ceramide |
| CN104870525A (en) * | 2012-12-26 | 2015-08-26 | 莱雅公司 | Molecularly imprinted polymers of sol-gel type and their use as antidandruff agent |
| CN104870482A (en) * | 2012-12-26 | 2015-08-26 | 莱雅公司 | Molecularly imprinted polymers and their use as antidandruff agents |
| CN104870525B (en) * | 2012-12-26 | 2017-09-19 | 莱雅公司 | The molecularly imprinted polymer of sol-gel type and its purposes as anti-dandruff agent |
| JP2017145320A (en) * | 2016-02-17 | 2017-08-24 | 公立大学法人 富山県立大学 | Template-forming polymerizable compound and curable composition thereof, and cured product thereof |
| CN106432590A (en) * | 2016-09-21 | 2017-02-22 | 中国农业科学院农业质量标准与检测技术研究所 | Molecularly imprinted polymer with controllable sustained-release function |
| CN106432590B (en) * | 2016-09-21 | 2018-04-06 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of molecularly imprinted polymer with controllable sustained-release function |
| CN113834888A (en) * | 2021-09-23 | 2021-12-24 | 大连润生康泰医学检验实验室有限公司 | Accurate detection method and kit for 4 ceramides in blood |
| CN113834888B (en) * | 2021-09-23 | 2022-06-28 | 大连润生康泰医学检验实验室有限公司 | Accurate detection method and kit for 4 ceramides in blood |
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