CN114644649B - Method for extracting high-purity lysophosphatidylcholine from egg yolk powder - Google Patents
Method for extracting high-purity lysophosphatidylcholine from egg yolk powder Download PDFInfo
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- CN114644649B CN114644649B CN202011516027.8A CN202011516027A CN114644649B CN 114644649 B CN114644649 B CN 114644649B CN 202011516027 A CN202011516027 A CN 202011516027A CN 114644649 B CN114644649 B CN 114644649B
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- lysophosphatidylcholine
- yolk powder
- deoiled
- egg yolk
- alcohol
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- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 title claims abstract description 95
- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 79
- 239000000843 powder Substances 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 43
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 28
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 28
- 235000013345 egg yolk Nutrition 0.000 title claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 126
- 239000003480 eluent Substances 0.000 claims abstract description 46
- 238000001035 drying Methods 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 238000004440 column chromatography Methods 0.000 claims abstract description 28
- 238000000605 extraction Methods 0.000 claims abstract description 20
- 239000000284 extract Substances 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 19
- 239000000047 product Substances 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 17
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 239000000945 filler Substances 0.000 claims abstract description 11
- 239000000706 filtrate Substances 0.000 claims abstract description 9
- 239000000469 ethanolic extract Substances 0.000 claims abstract description 7
- 238000004108 freeze drying Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 30
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 27
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims description 26
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 22
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 17
- 239000000741 silica gel Substances 0.000 claims description 17
- 229910002027 silica gel Inorganic materials 0.000 claims description 17
- 238000010828 elution Methods 0.000 claims description 14
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000004042 decolorization Methods 0.000 claims 1
- 238000007670 refining Methods 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 6
- 238000011068 loading method Methods 0.000 abstract 1
- 230000008569 process Effects 0.000 description 21
- 239000002994 raw material Substances 0.000 description 15
- 238000011049 filling Methods 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 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 11
- 239000007858 starting material Substances 0.000 description 7
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 6
- 229940067606 lecithin Drugs 0.000 description 6
- 239000000787 lecithin Substances 0.000 description 6
- 235000010445 lecithin Nutrition 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 150000003904 phospholipids Chemical class 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- 238000000194 supercritical-fluid extraction Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 231100000086 high toxicity Toxicity 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- -1 phospholipid compound Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 1
- 101800004538 Bradykinin Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 102100035792 Kininogen-1 Human genes 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 1
- 230000004856 capillary permeability Effects 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
Abstract
The invention relates to the field of extraction and separation of natural medicines, and discloses a method for extracting high-purity lysophosphatidylcholine from egg yolk powder, which comprises the following steps: A. extracting deoiled yolk powder with alcohol, filtering to remove residue, mixing filtrates, concentrating, and drying to obtain deoiled yolk powder alcohol extract; B. completely dissolving the deoiled yolk powder ethanol extract, separating by alumina column chromatography, collecting the filtrate obtained by separation, concentrating, and drying to obtain the deoiled yolk powder extract; C. completely dissolving the deoiled yolk powder extract, loading a chromatographic column with reverse filler, eluting by using a water solution a of short-chain alcohol as an eluent, combining the eluents, concentrating and drying to obtain a crude lysophosphatidylcholine product; D. and (3) completely dissolving the crude lysophosphatidylcholine, performing secondary column chromatography, eluting by adopting an alkane-short chain alcohol system, collecting eluent, concentrating and freeze-drying to obtain a lysophosphatidylcholine product.
Description
Technical Field
The invention relates to the field of extraction and separation of natural medicines, in particular to a method for extracting high-purity lysophosphatidylcholine from egg yolk powder.
Background
Lecithin is the most commonly used phospholipid compound, so that lysolecithin is generally lysolecithin, also called Lysophosphatidylcholine (LPC), which is a degradation product produced by hydrolysis or enzymolysis of 1-or 2-ester bonds of Phosphatidylcholine (PC) under the action of phospholipase A, and part of lecithin is decomposed to produce lysophosphatidylcholine in the processes of raw material preparation, purification, storage and the like. LPC has a strong surface activity, and thus can rupture erythrocytes and other cell membranes, causing hemolysis or cell necrosis. After LPC causes hemolysis, capillary permeability is increased to cause hemorrhage, and tissue releases histamine, 5-hydroxytryptamine, epinephrine, bradykinin, etc., which indirectly affect cardiovascular and nervous system functions, and excite a series of complex pathological reactions. Therefore, the LPC content is an important quality control index of medicines, especially injection containing phospholipid auxiliary materials, and is directly related to the clinical medication safety of medicines, and the content of the medicines needs to be strictly controlled.
The lysophosphatidylcholine is mainly produced by hydrolyzing phospholipids in the processes of raw materials, auxiliary materials, preparation process, product storage and the like, and the content of the lysophosphatidylcholine is very low in nature, so that the lysophosphatidylcholine used for quality control at present is mostly obtained by directly adopting the phospholipids for hydrolysis and enzymolysis or adopting glycerol and fatty acid for synthesis, and the natural properties of the lysophosphatidylcholine are changed. Since lysophosphatidylcholine is a degradation product obtained by hydrolysis of ester bonds at the 1-or 2-position of phospholipids, it has a certain randomness, that is, lysophosphatidylcholine is a mixture of the 1-and 2-positions, and natural properties of lysophosphatidylcholine cannot be completely ensured by artificial hydrolysis, enzymolysis or synthesis means, which has a great hidden trouble in the quality control process. When the standard lysophosphatidylcholine is purchased from a hospital or abroad, most of the standard lysophosphatidylcholine is subjected to artificial hydrolysis, enzymolysis or synthesis, and the reference substance of the lysophosphatidylcholine from natural sources is very expensive due to the low content of the lysophosphatidylcholine, so that the detection cost of the enterprise products is greatly increased.
Therefore, the lysophosphatidylcholine of natural sources with low cost and stable quality is urgently needed by various large preparation enterprises in China at present, and the preparation process of the lysophosphatidylcholine of natural sources with low cost and stable quality is not reported at present. Through retrieval, 2014 of the modern pharmaceutical industry Co., ltd of Guangzhou white cloud mountain Han dynasty applies for a preparation method of lysophosphatidylcholine from natural sources, wherein egg yolk or soybean lecithin is used as a real raw material, and halogenated alkane-low carbon alcohol-water is used as an eluent for eluting twice, so that the lysophosphatidylcholine with the content of 98% -99.9% is finally obtained. The technical proposal can obtain lysophosphatidylcholine of natural source and reliable quality, but a large amount of eluent is used in the preparation process, including halogenated alkane with high toxicity. Meanwhile, the technical scheme adopts soybean or egg yolk phosphatide as a starting material, the cost of adding the soybean or egg yolk phosphatide is higher, and the content of lysophosphatidylcholine is lower, so that the LPC extraction cost is higher.
Disclosure of Invention
The invention aims to solve the technical problems of high extraction cost, low elution efficiency, high toxicity of an extraction solvent and the like of high-purity lysophosphatidylcholine in the prior art, and provides the lysophosphatidylcholine which is a natural source with lower cost and more stable quality, and the lysophosphatidylcholine with the purity of more than 98 percent is finally obtained from egg yolk powder serving as a starting material through deoiling, extraction, column chromatography separation and the like, and meanwhile, the egg yolk lecithin with the PC content of more than 70 percent can be obtained, the production of two varieties can be realized through one process, and the production cost of LPC is greatly reduced.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for extracting high-purity lysophosphatidylcholine from egg yolk powder comprises the following steps:
A. extracting deoiled yolk powder with alcohol, filtering to remove residue, mixing filtrates, concentrating, and drying to obtain deoiled yolk powder alcohol extract;
B. completely dissolving the deoiled yolk powder ethanol extract, separating by alumina column chromatography, collecting the filtrate obtained by separation, concentrating, and drying to obtain the deoiled yolk powder extract;
C. completely dissolving the deoiled yolk powder extract, eluting by using a short-chain alcohol water solution a as an eluent through a chromatographic column filled with reverse packing, combining the eluents, concentrating and drying to obtain a crude lysophosphatidylcholine product;
D. and (3) completely dissolving the crude lysophosphatidylcholine, performing secondary column chromatography, eluting by adopting an alkane-short chain alcohol system, collecting eluent, concentrating and freeze-drying to obtain a lysophosphatidylcholine product.
Preferably, in step D, the alkane-short chain alcohol system comprises: alkane and short chain alcohol in the volume ratio of 1:1, wherein the alkane comprises one or more of chloroform and methylene dichloride, and the short-chain alcohol comprises one or more of methanol, ethanol, isopropanol and the like.
Alkane and short chain alcohol 1: the system of 1 can reduce the dosage of the eluting solvent as much as possible and quicken the eluting efficiency.
Preferably, in the step D, the crude lysophosphatidylcholine is completely dissolved by chloroform, the filler for the secondary column chromatography comprises one or more of silica gel and alumina, and the mass ratio of the crude lysophosphatidylcholine to the filler for the secondary column chromatography is (1): 1-5 filled columns, the diameter-to-height ratio is 1:1 to 1:10, the pressure of the passing column is 0.3-1.0 mPa, and the temperature of the passing column is 20-40 ℃.
Preferably, in the step C, the percentage of water in the aqueous solution a of the short-chain alcohol is 5% -30%; the short chain alcohol comprises one or more of methanol, ethanol, n-butanol and isopropanol.
Preferably, in step C, the de-oiled yolk powder extract is completely dissolved with a short-chain alcohol, the short-chain alcohol including one or more of methanol, ethanol, n-butanol, isopropanol; the reverse filler comprises one or more of C18 bonded silica gel, C8 bonded silica gel and C4 bonded silica gel; the deoiled yolk powder extract and the reverse filler are mixed according to the mass ratio of 1: 1-10 filled columns, the diameter-to-height ratio is 1:1 to 1:10.
preferably, in the step C, after the crude lysophosphatidylcholine is obtained, the water solution b of the short-chain alcohol is used as an eluent, the chromatographic column filled with the reverse packing is subjected to secondary elution, the eluent is collected, concentrated, decolorized by activated carbon, filtered, concentrated, refined and freeze-dried to obtain the egg yolk lecithin product; the yolk lecithin content in the yolk lecithin product is more than 70%; in the aqueous solution b of the short-chain alcohol, the percentage of water is 0-10%; the short chain alcohol comprises one or more of methanol, ethanol, n-butanol and isopropanol.
The main purpose of this step is to elute the residual PC in the reverse column, using a lower water content short chain alcohol gives higher elution efficiency and less impurities introduced, preferably in a water content ratio of between 0 and 5%, but the LPC is completely eluted at this time, and a product with a PC content of more than 70% can be obtained with a water content of between 0 and 10%.
From the viewpoint of solvent recovery, the short-chain alcohol used for dissolving the deoiled yolk powder extract in step C should be kept uniform as well as the aqueous solution a of the short-chain alcohol and the short-chain alcohol in the aqueous solution b of the short-chain alcohol.
Preferably, in the step B, the deoiled yolk powder alcohol extract is completely dissolved by using 95% ethanol, and the deoiled yolk powder alcohol extract is prepared by mixing the deoiled yolk powder alcohol extract with alumina according to a mass ratio of 1: 1-5 filler, diameter-to-height ratio is 1:1 to 1:10, eluting with 95% ethanol, wherein the pressure of the column passing is-0.1-3.0 mPa, and the temperature of the column passing is 10-60 ℃.
The main purpose of this step is to separate out most PE (phosphatidylethanolamine) in the deoiled yolk powder alcohol extract, so this step adopts the operation of passing through an open column at normal temperature, and uses 95% ethanol as solvent and eluent, thereby not only reducing the solvent volatilization risk and the solvent recovery difficulty, but also realizing the most convenient PE separation. And detecting by using a thin-layer chromatography spot plate to determine that the deoiled yolk powder is completely eluted.
Preferably, in step a, the alcohol used for the alcohol extraction comprises one or more of methanol, ethanol and aqueous solutions thereof; the mass part ratio of the deoiled yolk powder to the alcohol is 1: 5-15 times, 2-6 times, 1-8 hours, and 15-50 ℃.
Preferably, in the step A, the deoiled yolk powder is obtained by deoiling yolk powder, and the deoiling comprises one of an acetone deoiling method and a supercritical method; when the acetone deoiling method is applied, the mass ratio of the yolk powder to the acetone is 1: 1-10 times of deoiling, wherein the deoiling temperature is 20-45 ℃, and after deoiling, merging acetone insoluble substances, and drying to obtain the deoiled yolk powder; when the supercritical method is applied, the yolk powder is placed in an extraction kettle for continuous extraction, the temperature is kept at 25-45 ℃, the CO2 flow rate is controlled at 50-120 kg/h, the pressure of the extraction kettle is 10-25 Mpa, the pressure of a separation kettle 1 is 5-20 Mpa, the pressure of a separation kettle 2 is 5-15 Mpa, and the duration is 4-10 h, so that the deoiled yolk powder is obtained.
A lysophosphatidylcholine product obtained by the method of extracting high purity lysophosphatidylcholine from egg yolk powder as described above, said lysophosphatidylcholine product having a purity of greater than 98%.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
(1) according to the technical scheme of the invention, the lysophosphatidylcholine with the purity of more than 98% can be obtained, and the nature of the lysophosphatidylcholine is ensured.
(2) Compared with the prior art, the technical scheme of the invention has obviously higher elution efficiency, and does not adopt organic solvents with larger toxicity such as halogenated alkane and the like.
(3) The technical scheme of the invention realizes the full utilization of the egg yolk powder, greatly reduces the acquisition cost of the lysophosphatidylcholine from natural sources, and can obtain the medicinal egg yolk lecithin with the PC content of more than 70 percent and meeting pharmacopoeia standards while obtaining the lysophosphatidylcholine with the purity of more than 98 percent.
Drawings
FIG. 1 is a diagram showing a process route for extracting lysophosphatidylcholine according to the technical scheme of the present invention;
FIG. 2 is a graph showing the water content during LPC elution.
Detailed Description
The present invention will be further described in detail below with reference to the accompanying drawings, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent, so that those skilled in the art can better understand the present invention and implement the present invention, but the present invention is not limited thereto. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
As shown in FIG. 1, 1kg of egg yolk powder was taken, 5L of acetone was added to carry out deoiling, deoiling was carried out 3 times at 25℃and filtration was carried out, and 726g of deoiled egg yolk powder was obtained by drying. Extracting with 3630ml 95% ethanol at 20deg.C for 2 hr, filtering, extracting the residue for 4 times, mixing filtrates, concentrating under reduced pressure, and drying to obtain deoiled yolk powder 471g. Carrying out alumina column chromatography by using 95% ethanol as an eluent, eluting until egg yolk lecithin is complete, concentrating and drying, adding methanol to dissolve to prepare a raw material liquid, filling 2.35kg C18 bonded silica gel for column chromatography (diameter-to-height ratio is 1:6), and carrying out column chromatography at a column temperature of 25 ℃ for the first time by using eluent methanol: eluting with water (85:15), wherein the column pressure in the eluting process is 2.7mPa, eluting for 8 column volumes, mixing the eluates, concentrating and drying to obtain 10.16g of crude lysophosphatidylcholine product with the purity of 96.3%, eluting with pure methanol as the eluent for the second time, wherein the column pressure in the eluting process is 1.4mPa, eluting for 10 column volumes, and mixing the eluates containing the yolk lecithin to obtain the crude yolk lecithin product. Dissolving the crude lysophosphatidylcholine in chloroform to prepare a raw material liquid, filling 12g of silica gel (diameter-to-height ratio is 1:2), and using chloroform: methanol (1:1) is used as eluent, the column pressure in the elution process is 0.3mPa, 10 column volumes are eluted, the eluents are combined, and the concentration and drying are carried out to obtain 9.65g of lysophosphatidylcholine with the purity of 99.8%.
Example 2
As shown in fig. 1, 2kg of egg yolk powder was taken, 2L of acetone was added to perform deoiling, deoiling was performed 5 times at 35 ℃, filtration was performed, and 1.48kg of deoiled egg yolk powder was obtained by drying. Extracting with 14.8L of 95% ethanol at 30deg.C for 2 hr, filtering, repeatedly extracting for 2 times, mixing filtrates, and concentrating under reduced pressure to obtain 973g of deoiled yolk powder ethanol extract. Carrying out alumina column chromatography by using 95% ethanol as an eluent, eluting until egg yolk lecithin is complete, concentrating and drying, adding ethanol to dissolve to prepare raw material liquid, filling 7kgC18 bonded silica gel for column chromatography (diameter-to-height ratio is 1:8), and carrying out column chromatography at 35 ℃ for the first time by using eluent ethanol: eluting with water (75:25), wherein the column pressure is 2.4mPa, eluting for 8 column volumes, mixing the eluates, concentrating and drying to obtain 23.68g of lysophosphatidylcholine crude product with purity of 91.6%, eluting with ethanol for the second time: eluting with water (98:2), wherein the column pressure in the eluting process is 1.2mPa, eluting for 10 column volumes, and mixing the eluents containing egg yolk lecithin to obtain crude egg yolk lecithin. Dissolving the crude lysophosphatidylcholine in chloroform to prepare a raw material liquid, filling 46g of silica gel (diameter-to-height ratio is 1:3), and using chloroform: methanol (1:1) is used as an eluent, the column pressure in the elution process is 0.4mPa, 10 column volumes are eluted, the eluents are combined, 21.08g of lysophosphatidylcholine is obtained by concentration and drying, and the purity is 99.4%.
Example 3
As shown in FIG. 1, 500g of egg yolk powder was obtained, and 5L of acetone was added thereto to deoil, and deoiled 1 time at 40℃and filtered, followed by drying to obtain 355g of deoiled egg yolk powder. Extracting with 5.32L of 95% ethanol at 35deg.C for 2 hr, filtering to remove residue, mixing filtrates, and concentrating under reduced pressure to obtain deoiled ovum gallus Domesticus flavus powder ethanol extract 231.3g. Carrying out alumina column chromatography by taking 95% ethanol as an eluent, eluting until egg yolk lecithin is complete, concentrating and drying, adding isopropanol to dissolve and prepare a raw material liquid, filling 1.2kg of C8 bonded silica gel, carrying out column chromatography (diameter-to-height ratio is 1:6), and carrying out column chromatography at a temperature of 35 ℃ for the first time by using eluent isopropanol: eluting with water (70:30), wherein the column pressure in the eluting process is 2.2mPa, eluting for 8 column volumes, mixing the eluates, concentrating and drying to obtain 6.93g of crude lysophosphatidylcholine product with purity of 85.2%, and eluting with isopropanol for the second time: eluting with water (80:20), wherein the column pressure in the eluting process is 1.1mPa, eluting for 10 column volumes, and mixing the eluents containing egg yolk lecithin to obtain crude egg yolk lecithin. Dissolving the crude lysophosphatidylcholine in chloroform to prepare a raw material liquid, filling 25g of silica gel (diameter-to-height ratio is 1:4), and using chloroform: isopropyl alcohol (1:1) is used as eluent, the column pressure in the elution process is 1.0mPa, 10 column volumes are eluted, the eluents are combined, and the concentration and drying are carried out to obtain 5.64g of lysophosphatidylcholine with the purity of 99.1%.
Example 4
As shown in FIG. 1, 5kg of yolk powder was taken and fed into a supercritical extraction vessel at 25℃and CO 2 The flow rate is 60kg/h, the pressure of the extraction kettle is 15MPa, the pressure of the separation kettle 1 is 10MPa, the pressure of the separation kettle 2 is 8MPa, and the duration is 6h, so that the deoiled yolk powder is 3.56kg. Extracting with 21.3L 95% ethanol at 25deg.C for 8 hr, filtering, repeatedly extracting for 3 times, concentrating, and drying to obtain deoiled yolk powder ethanol extract 2.02kg. Carrying out alumina column chromatography by using 95% ethanol as an eluent, eluting until egg yolk lecithin is complete, concentrating and drying, adding ethanol to dissolve to prepare raw material liquid, filling 12kg of reverse silica gel C4 for column chromatography (diameter-to-height ratio is 1:5), and carrying out column chromatography at 30 ℃ for the first time by using eluent methanol: eluting with water (90:10), wherein the column pressure is 2.9mPa, eluting for 8 column volumes, mixing the eluates, concentrating and drying to obtain 56.96g of lysophosphatidylcholine crude product, the purity is 88.9%, and eluting with ethanol for the second time: eluting with water (90:10), wherein the column pressure in the eluting process is 1.1mPa, eluting for 10 column volumes, and mixing the eluents containing egg yolk lecithin to obtain crude egg yolk lecithin. Dissolving the crude lysophosphatidylcholine in chloroform to prepare a raw material liquid, filling 170g of silica gel (diameter-to-height ratio is 1:4), and using dichloromethane at a column temperature of 20 ℃: ethanol (1:1) is used as an eluent, the column pressure in the elution process is 0.5mPa, 10 column volumes are eluted, the eluents are combined, and the concentration and drying are carried out to obtain 50.34g of lysophosphatidylcholine with the purity of 98.9%.
Example 5
As shown in FIG. 1, 10kg of yolk powder was taken and fed into a supercritical extraction vessel at a temperature of 35℃and CO 2 The flow rate is 100kg/h, the pressure of the extraction kettle is 20MPa, the pressure of the separation kettle 1 is 15MPa, the pressure of the separation kettle 2 is 12MPa, and the time lasts for 4 hours, so that 6.98kg of deoiled yolk powder is obtained. Extracting with 34.9L of 95% ethanol at 35deg.C for 4 hr, filtering, repeatedly extracting for 2 times, concentrating, and drying to obtain deoiled yolk powder 4.48kg. Performing alumina column chromatography with 95% ethanol as eluent, eluting until egg yolk lecithin is completely, concentrating, drying, adding methanol, dissolving to obtain raw material liquid, filling 31kgC18 bonded silica gel, performing column chromatography (diameter-to-height ratio of 1:6), and eluting with eluent methanol for the first time at 30deg.C: eluting with water (95:5), wherein the column pressure is 3mPa, eluting for 8 column volumes, mixing the eluates, concentrating and drying to obtain 144.39g of lysophosphatidylcholine crude product with purity of 72.6%, and eluting with isopropanol for the second time: eluting with water (90:10), wherein the column pressure in the eluting process is 1.1mPa, eluting for 10 column volumes, and mixing the eluents containing egg yolk lecithin to obtain crude egg yolk lecithin. Dissolving the crude lysophosphatidylcholine in chloroform to prepare a raw material liquid, filling 650g of neutral alumina for column chromatography (diameter-to-height ratio is 1:8), and using chloroform: methanol (1:1) is used as eluent, the column pressure in the elution process is 1mPa, 10 column volumes are eluted, the eluents are combined, and the concentration and drying are carried out to obtain 106.08g of lysophosphatidylcholine with the purity of 98.7%.
Example 6
As shown in FIG. 1, 20kg of yolk powder was taken and fed into a supercritical extraction vessel at 40℃and CO 2 The flow rate is 110kg/h, the pressure of the extraction kettle is 23MPa, the pressure of the separation kettle 1 is 17MPa, the pressure of the separation kettle 2 is 12MPa, and the duration is 3h, so that 13.4kg of deoiled yolk powder is obtained. Extracting with 160L of 95% ethanol at 45deg.C for 4 hr, filtering, concentrating, and drying to obtain 9.37kg of deoiled yolk powder ethanol extract. Performing alumina column chromatography with 95% ethanol as eluent, eluting until egg yolk lecithin is completely, concentrating, drying, adding methanol to dissolve to obtain raw material liquid, and filling 90kgC bonded silica gelColumn chromatography (diameter to height ratio 1:5) was performed at a column temperature of 35 ℃, eluting with ethanol for the first time: eluting with water (75:25), wherein the column pressure is 2.7mPa, eluting for 8 column volumes, mixing the eluates, concentrating and drying to obtain crude lysophosphatidylcholine 335g with purity of 70.4%, eluting with methanol for the second time: eluting with water (90:10), wherein the column pressure in the eluting process is 1.1mPa, eluting for 10 column volumes, and mixing the eluents containing egg yolk lecithin to obtain crude egg yolk lecithin. Dissolving the crude lysophosphatidylcholine in chloroform to prepare a raw material liquid, filling 1.6kg of neutral alumina for column chromatography (diameter-to-height ratio is 1:6), and using chloroform at a column temperature of 40 ℃: methanol (1:1) is used as an eluent, the column pressure in the elution process is 0.8mPa, 10 column volumes are eluted, the eluents are combined, and the concentration and drying are carried out to obtain 235.44g of lysophosphatidylcholine with the purity of 98.3%.
Comparative example 1
In order to fully evaluate the effect of the technology of the invention, the key steps of lysophosphatidylcholine elution were examined. In the process of extracting lysophosphatidylcholine by using egg yolk powder as a starting material, a reverse phase chromatographic column is used for extracting crude LPC, as the polarity difference between LPC and PC is small, if pure short-chain alcohol is used as an eluent, PC, LPC, SM and other components are eluted simultaneously, as the polarity of the eluent is increased, the LPC is eluted first, the LPC can be obtained in one step by utilizing the characteristic, but as the polarity is increased, the eluted LPC, PC and SM are less and less, so that LPC, PC, SM which is not eluted is left in the egg yolk lecithin, and finally the LPC content in the egg yolk lecithin exceeds the standard. Therefore, the comparative example fully examines the proportion of water in the reverse column elution process, and the examination result shows that when the water content is between 5% and 30%, high-purity LPC can be obtained and the yield is higher, and when the water content exceeds 30%, the proportion of LPC, PC and SM eluted is smaller, so that the quality of the subsequent egg yolk lecithin is influenced. According to fig. 1, it can be seen that the purity of more than 98% can be achieved, and the content of the crude lysophosphatidylcholine is at least more than 80%, so that the water content is 5-30% in consideration of the comprehensive product quality, which is key to meeting the effect of the invention in the technical scheme. The results are shown in FIG. 2.
The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent changes may be made in the claims herein without departing from the scope of the invention.
Claims (6)
1. A method for extracting lysophosphatidylcholine from egg yolk powder, comprising the steps of:
A. extracting deoiled yolk powder with alcohol, filtering to remove residue, mixing filtrates, concentrating, and drying to obtain deoiled yolk powder alcohol extract;
B. completely dissolving the deoiled yolk powder ethanol extract with 95% ethanol, separating by alumina column chromatography, collecting filtrate obtained by separation, concentrating, and drying to obtain deoiled yolk powder extract; the deoiled yolk powder alcohol extract and alumina are mixed according to the mass ratio of 1: 1-5 filler, the diameter-to-height ratio is 1: 1-1: 10, eluting with 95% ethanol;
C. completely dissolving the deoiled yolk powder extract, eluting by using a short-chain alcohol water solution a as an eluent through a chromatographic column filled with reverse packing, combining the eluents, concentrating and drying to obtain a crude lysophosphatidylcholine product; in the aqueous solution a of the short-chain alcohol, the percentage of water is 5% -30%; the short-chain alcohol is one or more of methanol, ethanol, n-butanol and isopropanol; after the crude lysophosphatidylcholine is obtained, the water solution b of short-chain alcohol is used as an eluent, the chromatographic column filled with the reverse packing is subjected to secondary elution, the eluent is collected, and the egg yolk lecithin product is obtained through concentration, activated carbon decolorization, filtration, concentration, refining and freeze-drying; in the aqueous solution b of the short-chain alcohol, the percentage of water is 0% -10%; the short-chain alcohol is one or more of methanol, ethanol, n-butanol and isopropanol;
D. completely dissolving the crude lysophosphatidylcholine, performing secondary column chromatography, eluting with alkane-short chain alcohol system, collecting eluate, concentrating, and lyophilizing to obtain lysophosphatidylcholine product; the alkane-short chain alcohol system is as follows: alkane and short chain alcohol in the volume ratio of 1:1, wherein the alkane is one or more of chloroform and dichloromethane, and the short-chain alcohol is one or more of methanol, ethanol and isopropanol.
2. The method for extracting lysophosphatidylcholine from egg yolk powder according to claim 1, wherein in the step D, chloroform is used to completely dissolve the crude lysophosphatidylcholine, the filler for the secondary column chromatography is one or more of silica gel and alumina, and the mass ratio of the crude lysophosphatidylcholine to the filler for the secondary column chromatography is 1: 1-5 filled columns, the diameter-to-height ratio is 1: 1-1: 10, the column passing pressure is 0.3-1.0 Pa, and the column passing temperature is 20-40 ℃.
3. A method of extracting lysophosphatidylcholine from egg yolk powder as claimed in claim 1, wherein in step C, the de-oiled egg yolk powder extract is completely dissolved with a short chain alcohol, the short chain alcohol being one or more of methanol, ethanol, n-butanol, isopropanol; the reverse filler is one or more of C18 bonded silica gel, C8 bonded silica gel and C4 bonded silica gel; the deoiled yolk powder extract and the reverse filler are mixed according to the mass ratio of 1: 1-10 filled columns, the diameter-to-height ratio is 1: 1-1: 10.
4. a method of extracting lysophosphatidylcholine from egg yolk powder as claimed in claim 1, wherein the egg yolk lecithin product has an egg yolk lecithin content of greater than 70%.
5. The method for extracting lysophosphatidylcholine from egg yolk powder as claimed in claim 1, wherein in the step a, the alcohol used for the alcohol extraction is one or more of methanol, ethanol and an aqueous solution thereof; the mass part ratio of the deoiled yolk powder to the alcohol is 1: 5-15, wherein the extraction times are 2-6, the extraction time is 1-8 h, and the extraction temperature is 15-50 ℃.
6. The method for extracting lysophosphatidylcholine from egg yolk powder as claimed in claim 1, wherein in the step a, the deoiled egg yolk powder is obtained by deoiling egg yolk powderThe deoiling is one of an acetone deoiling method and a supercritical method; when the acetone deoiling method is applied, the mass ratio of the yolk powder to the acetone is 1: 1-10, namely deoiling for 1-5 times, deoiling at 20-45 ℃, merging acetone insoluble substances after deoiling, and drying to obtain the deoiled yolk powder; when the supercritical method is applied, the yolk powder is placed in an extraction kettle for continuous extraction, the temperature is kept at 25-45 ℃, and CO 2 The flow rate is controlled to be 50-120 kg/h, the pressure of the extraction kettle is 10-25 mpa, the pressure of the separation kettle 1 is 5-20 mpa, the pressure of the separation kettle 2 is 5-15 mpa, and the time lasts for 4-10 h, so that the deoiled yolk powder is obtained.
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