CN110144369A - A method of preparing high-purity lysophosphatidyl choline - Google Patents
A method of preparing high-purity lysophosphatidyl choline Download PDFInfo
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- CN110144369A CN110144369A CN201910427130.6A CN201910427130A CN110144369A CN 110144369 A CN110144369 A CN 110144369A CN 201910427130 A CN201910427130 A CN 201910427130A CN 110144369 A CN110144369 A CN 110144369A
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- low
- purity
- carbon
- lysophosphatidyl choline
- water
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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 73
- 238000000034 method Methods 0.000 title claims abstract description 54
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 81
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 72
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 28
- 230000008569 process Effects 0.000 claims abstract description 26
- 239000000741 silica gel Substances 0.000 claims abstract description 26
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 26
- 229960001866 silicon dioxide Drugs 0.000 claims abstract description 26
- 239000003480 eluent Substances 0.000 claims abstract description 25
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 23
- 239000012141 concentrate Substances 0.000 claims abstract description 22
- 239000000047 product Substances 0.000 claims abstract description 22
- 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 claims abstract description 15
- 229940083466 soybean lecithin Drugs 0.000 claims abstract description 15
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 12
- 244000068988 Glycine max Species 0.000 claims abstract description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 111
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 74
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 67
- 235000019441 ethanol Nutrition 0.000 claims description 56
- 239000007787 solid Substances 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 45
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 34
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 30
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 25
- 238000001914 filtration Methods 0.000 claims description 25
- 238000003756 stirring Methods 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 21
- 239000000706 filtrate Substances 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 15
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical class CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 claims description 12
- 102100031415 Hepatic triacylglycerol lipase Human genes 0.000 claims description 10
- 108010013563 Lipoprotein Lipase Proteins 0.000 claims description 10
- 102100037611 Lysophospholipase Human genes 0.000 claims description 9
- 108010058864 Phospholipases A2 Proteins 0.000 claims description 9
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052698 phosphorus Inorganic materials 0.000 claims description 9
- 239000011574 phosphorus Substances 0.000 claims description 9
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 6
- 239000013049 sediment Substances 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 3
- 150000002367 halogens Chemical class 0.000 claims description 3
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 23
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract description 7
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract description 7
- 239000006227 byproduct Substances 0.000 abstract description 2
- 238000011097 chromatography purification Methods 0.000 abstract description 2
- 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 28
- 239000000839 emulsion Substances 0.000 description 25
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 24
- 239000004810 polytetrafluoroethylene Substances 0.000 description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- -1 chloromethanes Alkane Chemical class 0.000 description 14
- 238000000926 separation method Methods 0.000 description 14
- 239000007788 liquid Substances 0.000 description 13
- 239000000203 mixture Substances 0.000 description 13
- 238000010907 mechanical stirring Methods 0.000 description 12
- 238000009835 boiling Methods 0.000 description 10
- 239000000499 gel Substances 0.000 description 10
- 239000003960 organic solvent Substances 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- 239000000377 silicon dioxide Substances 0.000 description 10
- 238000009736 wetting Methods 0.000 description 10
- PZNPLUBHRSSFHT-RRHRGVEJSA-N 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)COC(=O)CCCCCCCCCCCCCCC PZNPLUBHRSSFHT-RRHRGVEJSA-N 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 8
- 238000004321 preservation Methods 0.000 description 8
- 239000008347 soybean phospholipid Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 239000000787 lecithin Substances 0.000 description 5
- 235000010445 lecithin Nutrition 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 125000001095 phosphatidyl group Chemical group 0.000 description 4
- 238000007781 pre-processing Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 3
- 102000004882 Lipase Human genes 0.000 description 3
- 108090001060 Lipase Proteins 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000008588 hemolysis Effects 0.000 description 3
- 235000019421 lipase Nutrition 0.000 description 3
- 239000000376 reactant Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 2
- CWRILEGKIAOYKP-SSDOTTSWSA-M [(2r)-3-acetyloxy-2-hydroxypropyl] 2-aminoethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCCN CWRILEGKIAOYKP-SSDOTTSWSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 210000000232 gallbladder Anatomy 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000005311 nuclear magnetism Effects 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- IHNKQIMGVNPMTC-UHFFFAOYSA-N (2-hydroxy-3-octadecanoyloxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C IHNKQIMGVNPMTC-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 208000017170 Lipid metabolism disease Diseases 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000003026 anti-oxygenic effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 125000005313 fatty acid group Chemical group 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000010224 hepatic metabolism Effects 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 150000008105 phosphatidylcholines Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000011137 process chromatography Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
Abstract
The invention discloses a kind of methods for preparing high-purity lysophosphatidyl choline, and soybean lecithin is dissolved in water or calcium chloride solution, and phosphatidase is added and carries out enzyme digestion reaction；Enzyme digestion reaction product is transferred in silicagel column after pretreatment, and is repeatedly eluted using polyhalo low-carbon alkanes-low-carbon alcohols-water as eluant, eluent, collects the solution of object, high-purity lysophosphatidyl choline product is obtained after concentrate drying.The present invention is using common soybeans phosphatide as raw material, pass through enzyme digestion reaction, pretreatment and column Chromatographic purification technique prepare high-purity lysophosphatidyl choline after enzymatic hydrolysis, have the characteristics that raw material cheap and easy to get, simple process, strong operability, product yield are high, it is suitable for industrialized production, the lysophosphatidyl choline purity is high of production, by-product are few, stable product quality.
Description
Technical field
The present invention relates to the preparation methods of phospholipid metabolism product, in particular to a kind of prepare high-purity haemolysis phosphorus
The method of phosphatidylcholine.
Background technique
Lysophosphatidyl choline (lysophosphatidylcholine, abbreviation LPC) is one of metabolite of phosphatide,
Also known as lysolecithin is that phosphatidyl choline sloughs the product formed after a fatty acid chain.The emulsification of lysophosphatidyl choline
Ability, emulsion stability etc. are superior to common phospholipid prod；Furthermore lysophosphatidyl choline also has unique physiological activity, suppression
Bacterium activity and antioxygenic property.Therefore lysophosphatidyl choline is in food industry, medical industry, cosmetics industry and feed industry
In can be used as additive, medicine intermediate, pharmaceutical carrier and efficient emulsifier use.
For example, high-purity lysophosphatidyl choline is the key that one of targeting preparation auxiliary material in field of medicaments, it can also
Using the raw material as a kind of excellent antiepileptic；Because lysophosphatidyl choline is mainly in liver metabolism, in liver diseases
With obvious changes of contents can be found in hepatotoxicity wind agitation, be embodied in lysophosphatidyl choline content and be apparently higher than normal person, therefore
Lysophosphatidyl choline is also often applied to hepatic pathology and is investigated as marker, in addition lysophosphatidyl choline and diabetes,
The metabolic diseases such as atherosclerosis, dyslipidemia and cardiovascular disease are closely related, therefore the hemolytic phosphatidyl gallbladder of high-purity
Alkali has a extensive future directly as pharmaceutical intermediate, marker or as what medical auxiliary materials used.
The method for preparing lysophosphatidyl choline mainly has chemical synthesis, acid and alkali hydrolysis, enzymatic hydrolysis, physical separation and column chromatography
The methods of.These methods are each advantageous, but respectively have deficiency.Such as the material toxicity that chemical synthesis uses is big, application range
It is restricted；The hydrolysis degree of acid and alkali hydrolysis method is difficult to control；Various physical separation methods are in the purifying of target product
All encounter the difficulty for being difficult to overcome, and there are yields it is low, equipment investment is big, at high cost the problems such as；Enzymatic isolation method is easy to appear respectively
Kind side reaction, it is difficult to be directly realized by the preparation of high-purity lysophosphatidyl choline；Column chromatography depends critically upon initial feed
Purity, and in the process type, ratio and dosage of selected eluant, eluent etc. with final separation effect have direct relation, it is different
It is unique stationary phase and eluant, eluent and eluant, eluent ratio that material to be separated is usually corresponding.
Currently, the common practice of preparation high-purity lysophosphatidyl choline, is phosphatidyl choline (PC) original from high-purity
Material sets out to obtain target product.And this way is there is serious raw material dependence, used raw material are high-purity
It spends phosphatidyl choline (PC), which is obtained by the extraction means of synthesis or complexity, with high costs, it is not easy to obtain.
On the direction for preparing high-purity lysophosphatidyl choline as raw starting material using common soybeans phosphatide, Japan Patent
JP63091306A disclose it is a kind of using soybean lecithin as the preparation method of raw material, by acetone deoiling, phospholipase A2 hydrolysis, silicon
Plastic column chromatography, alcohol-water elution, finally obtaining lysophosphatidyl choline content is 87%, and lysophosphatidyl ethanolamine content is 6%
Product；Technology described in the patented technology still has that products therefrom purity is limited, it is similar to be difficult to thoroughly to remove other structures
The problem of component (such as lysophosphatidyl ethanolamine).In addition, patent CN104558021A discloses one kind from soybean or yolk
Phosphatide sets out, and directlys adopt polyhalo alkane-low-carbon alcohols-water mixtures of eluents and directly carries out column chromatography, finally obtains high-purity
The preparation method of natural lysophosphatidyl choline；But it is limited to the extremely low content of lysophosphatidyl choline in raw starting material
The total content normally about 1% or so of natural lysophosphatidyl choline (such as in soybean lecithin), method described in the technology or deposits
Not high in product yield, extraction efficiency is limited, the high problem of comprehensive preparation cost.
Disadvantages described above is worth solving.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of method for preparing high-purity lysophosphatidyl choline.
Technical solution of the present invention is as described below:
A method of preparing high-purity lysophosphatidyl choline, which is characterized in that in the presence of phosphatidase, to soybean lecithin into
Row enzyme digestion reaction, enzyme digestion reaction product are transferred in silicagel column after pretreatment, and with polyhalo low-carbon alkanes-low-carbon alcohols-
Water is that eluant, eluent is repeatedly eluted, and collects the solution of object, and the production of high-purity lysophosphatidyl choline is obtained after concentrate drying
Product.
According to the present invention of above scheme, which is characterized in that the soybean lecithin is dissolved in aqueous solution, and selects phosphatidase
A1 carries out enzyme digestion reaction.
According to the present invention of above scheme, which is characterized in that the soybean lecithin is dissolved in calcium chloride solution, and selects phosphorus
Lipase A2 carries out enzyme digestion reaction.
According to the present invention of above scheme, which is characterized in that enzyme digestion reaction product carries out pretreated process and specifically includes
Following steps: acetone is first added into enzyme digestion reaction product, is separated by filtration to obtain solid sediment after stirring and dissolving；The solid is heavy
Starch is dissolved in polyhalo low-carbon alkanes and obtains solution；The solution, which pours into low-carbon monohydric alcohol, is precipitated solid matter, crosses and filters out
Fall to be precipitated solid matter and collects filtrate；The filtrate of collection is concentrated.
According to the present invention of above scheme, which is characterized in that the polyhalo low-carbon alkanes include methylene chloride, three chloromethanes
Alkane, dichloroethanes.
According to the present invention of above scheme, which is characterized in that the low-carbon alcohols are low-carbon monohydric alcohol.
Further, the low-carbon monohydric alcohol includes methanol, ethyl alcohol, isopropanol.
According to the present invention of above scheme, which is characterized in that successively with polyhalo low-carbon alkanes, polyhalo low-carbon alkanes/
Low-carbon alcohols, polyhalo low-carbon alkanes/low-carbon alcohols/water, polyhalo low-carbon alkanes/low-carbon alcohols/water, polyhalo low-carbon alkanes/low-carbon
Alcohol/water is gradually repeatedly eluted.
Further, in multiple elution process, the polarity of eluant, eluent is gradually increased.
Further, in second of elution process, polyhalo low-carbon alkanes/low-carbon alcohols volume ratio is 5:1；?
Three times in elution process, polyhalo alkane/low-carbon alcohols/water volume ratio is 5:1:0.1；In the 4th elution process, more halogen
It is 3:1:0.1 for alkane/low-carbon alcohols/water volume ratio；In the 5th elution process, polyhalo alkane/low-carbon alcohols/water body
Product is than being 1:1:0.1.
According to the present invention of above scheme, the beneficial effect is that: the present invention is directly using common soybeans phosphatide as raw material, warp
Preprocessing process and column chromatography after enzyme digestion reaction, enzymatic hydrolysis are crossed, high-purity lysophosphatidyl choline product, preparation process are prepared
Easy to operate, raw material are cheap and easy to get, and reaction condition is mildly controllable, with short production cycle, and industrialization easy to accomplish can reduce high-purity
Spend the production cost of product phosphatidylcholines；Efficiently separating for lysophosphatidyl choline may be implemented in elution process of the invention,
The lysophosphatidyl choline purity is high of production, the yield of high production efficiency, target product is high, the overall cost of whole preparation process
It is cheap.
Detailed description of the invention
Fig. 1 is preparation flow figure of the invention.
Fig. 2 is the nuclear-magnetism phosphorus spectrogram for the high-purity lysophosphatidyl choline being prepared in the present invention.
Specific embodiment
The present invention is further described below with reference to embodiment:
As shown in Figure 1, a kind of method for preparing high-purity lysophosphatidyl choline is with soybean lecithin in the presence of phosphatidase
Raw material carries out enzyme digestion reaction, and enzyme digestion reaction product preprocessing process and carries out column chromatography after enzymatic hydrolysis, obtains high-purity haemolysis
The solution of phosphatidyl choline is repeatedly eluted using polyhalo low-carbon alkanes-low-carbon alcohols-water as eluant, eluent, sloughs solvent and true
High-purity lysophosphatidyl choline product is obtained after sky is dry.
In the present invention, soybean lecithin be commercially available liquid soy phosphatide or Powdered Soy Lecithin, the raw material be easy to get and at
This is low, greatly reduces the degree of dependence of high cost feedstocks.
The specific technical solution of the present invention is as follows:
1, enzymolysis process
During enzyme digestion reaction, soybean lecithin is dissolved, and phospholipase A1 or phospholipase A2 is selected to be digested.Specifically
: when selecting phospholipase A1, soybean lecithin is completely dissolved in aqueous solution；When selecting phospholipase A2, soybean lecithin is abundant
It is dissolved in calcium chloride solution, guarantees the stability for improving phospholipase A2.
Preferably, it is, so that the activity of phosphatidase is higher, to pass through between 40~80 °C that reaction temperature is controlled when enzyme digestion reaction
The control reaction time improves conversion ratio and reduces reactant residual, because the residual of reactant can increase the difficulty of purification.Phosphorus
The dosage of lipase are as follows: 0.1~2.0 milliliter of phosphatidase enzyme solution is added in every 100g soybean lecithin, both can guarantee that soybean lecithin carried out
Sufficient enzyme digestion reaction, and then guarantee the yield of reaction product lysophosphatidyl choline, while reducing the consumption of raw material.
2, preprocessing process after digesting
Series of preprocessing process after enzymatic hydrolysis is most important for the lysophosphatidyl choline for obtaining high-purity comprising more
The process of secondary dissolution, filtering and concentration.It is specific:
(1) acetone first is added into enzyme digestion reaction product, is separated by filtration to obtain solid sediment；
(2) solid sediment is dissolved in polyhalo low-carbon alkanes (such as methylene chloride) and obtains solution；
(3) solution, which pours into, is precipitated solid matter in low-carbon monohydric alcohol (such as ethyl alcohol), be removed by filtration solid matter be precipitated
And collect filtrate；
(4) filtrate collected is concentrated.
Preferably, the volume of acetone is 3~5 times of enzyme digestion reaction bulk product in step (1), to guarantee enzyme digestion reaction object
The speed and efficiency of dissolution；The dosage of polyhalo low-carbon alkanes in step (2) are as follows: every 10 grams of solid reactants are dissolved in 10~30
In milliliter polyhalo low-carbon alkanes, to guarantee the solute effect of solid sediment；The volume of low-carbon monohydric alcohol in step (3) is
3~5 times of polyhalo low-carbon alkanes volume in step (2), to guarantee that enzymatic hydrolysis last handling process ingredient is easier to elute.
3, column chromatography procedure
Prepare silicagel column, the solution after concentration moves in silicagel column, after using polyhalo low-carbon alkanes-low-carbon alcohols-water as eluant, eluent
It is repeatedly eluted, collects the solution of object, high-purity lysophosphatidyl choline product is obtained after concentrate drying.Specifically,
With the increase of washing steps, the polarity of eluant, eluent is gradually increased, and avoids suction of the polar component when being precipitated below by silica gel
It is attached effect and influence elution speed.
Repeatedly eluted in the present invention using polyhalo low-carbon alkanes-low-carbon alcohols-water as eluant, eluent (is five in the present embodiment
It is secondary), solid matter can sufficiently be precipitated, and not will increase the cost of elution process.Respectively with more halogen in each elution process
For low-carbon alkanes, polyhalo low-carbon alkanes/low-carbon alcohols (volume ratio 5:1), polyhalo low-carbon alkanes/low-carbon alcohols/water (volume ratio
For 5:1:0.1), polyhalo low-carbon alkanes/low-carbon alcohols/water (3:1:0.1), polyhalo low-carbon alkanes/low-carbon alcohols/water (1:1:
0.1) it is gradually handled.
The present invention sloughs solvent using the method (such as Rotary Evaporators) of vacuum distillation, or is sloughed using vacuum decompression kettle
Solvent.Since substance to be separated is mixture, the polarity of various components is different, corresponding in the eluant, eluent of various proportions
Elution speed it is different, therefore, with column chromatography progress constantly transformation three kinds of eluant, eluent components ratio, so as to reality
Existing different component efficiently separates.The present invention guarantees good component separation by the selection of above-mentioned eluant composition and ratio
Effect.
Preferably, polyhalo low-carbon alkanes include methylene chloride, chloroform, dichloroethanes；Low-carbon alcohols are low-carbon unitary
Alcohol (including methanol, ethyl alcohol, isopropanol).Further, select methanol or ethyl alcohol as low-carbon monohydric alcohol, it is low in cost, together
When compared to other alcohols be easier remove, and then guarantee final product purity.
The embodiment of several preparation high-purity lysophosphatidyl cholines is set forth below.
Embodiment 1
With 20 grams of Powdered Soy Lecithins (phosphatidylcholine content 23%) for raw material, 1.26 grams obtained, purity is the present embodiment
99.6% solid lysophosphatidyl choline.It is specific:
(1) Powdered Soy Lecithin and 38 grams is added in 250 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Water stirs 30 minutes at 50 DEG C, obtains the emulsion of soybean lecithin；0.04 gram of phospholipase A1 is dissolved in 2 grams of water, then two
It is added dropwise in the emulsion of phosphatide and water in minute, heat preservation continues to be stirred to react 3 hours at 50 DEG C；
(2) room temperature is cooled to, 200 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of chloroforms and obtains a kind of solution；The solution is poured into 300 ml methanols and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in 500 millimeters long, 30 mm dias silicagel column, the petroleum for being 60-90 DEG C with boiling point
It is spare after ether wetting；Aforementioned concentrate is transferred in silicagel column, methylene chloride (300 milliliters), methylene chloride/isopropyl are successively used
Alcohol (volume ratio 5:1,500 milliliters), methylene chloride/methanol/water (volume ratio 5:1:0.1,500 milliliters), methylene chloride/methanol/
Water (volume ratio 3:1:0.1,500 milliliters), methylene chloride/methanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and receive
Collection eluent simultaneously merges same composition, and after organic solvent is removed in concentration, solid hemolytic phosphatidyl gallbladder is dried in vacuo to obtain at 40 DEG C
Alkali.
Embodiment 2
With 20 grams of liquid soy phosphatide (phosphatidylcholine content 11%) for raw material, 0.54 gram obtained, purity is the present embodiment
99.1% solid lysophosphatidyl choline.It is specific:
(1) the liquid soy phosphatide and 38 grams are added in 250 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Water stirs 30 minutes at 50 DEG C, obtains emulsion；0.04 gram of phospholipase A1 is dissolved in 2 grams of water, then in two minutes dropwise
It is added in the emulsion of phosphatide and water, heat preservation continues to be stirred to react 2 hours at 50 DEG C；
(2) room temperature is cooled to, 200 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of dichloroethanes and obtains a kind of solution；The solution pours into 300 milliliters of isopropanols and violent
Stirring；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentration
Liquid is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, chloroform (300 milliliters), chloroform/isopropanol are successively used
(volume ratio 5:1,500 milliliters), chloroform/ethanol/water (volume ratio 5:1:0.1,500 milliliters), chloroform/ethanol/water
(volume ratio 3:1:0.1,500 milliliters), chloroform/ethanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 3
With 20 grams of Powdered Soy Lecithins (phosphatidylcholine content 23%) for raw material, 1.22 grams obtained, purity is the present embodiment
99.3% solid lysophosphatidyl choline.It is specific:
(1) add the Powdered Soy Lecithin and 58 grams of water in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick,
It is stirred 30 minutes at 50 DEG C, obtains emulsion；0.03 gram of phospholipase A1 is dissolved in 2 grams of water, is then added dropwise in two minutes
Enter into the emulsion of phosphatide and water, heat preservation continues to be stirred to react 4 hours at 50 DEG C；
(2) room temperature is cooled to, 300 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of methylene chloride and obtains a kind of solution；The solution is poured into 300 milliliters of ethyl alcohol and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, lower to be concentrated into about 30 milliliters with Rotary Evaporators, gained concentration
Liquid is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, methylene chloride (300 milliliters), methylene chloride/isopropanol are successively used
(volume ratio 5:1,500 milliliters), methylene chloride/methanol/water (volume ratio 5:1:0.1,500 milliliters), methylene chloride/methanol/water
(volume ratio 3:1:0.1,500 milliliters), methylene chloride/methanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 4
With 20 grams of liquid soy phosphatide (phosphatidylcholine content 11%) for raw material, 0.57 gram obtained, purity is the present embodiment
99.7% solid lysophosphatidyl choline.It is specific:
(1) the liquid soy phosphatide and 58 grams are added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Water stirs 30 minutes at 50 DEG C, obtains emulsion；0.03 gram of phospholipase A1 is dissolved in 2 grams of water, then in two minutes dropwise
It is added in the emulsion of phosphatide and water, heat preservation continues to be stirred to react 3 hours at 50 DEG C；
(2) room temperature is cooled to, 300 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of dichloroethanes and obtains a kind of solution；The solution is poured into 300 ml methanols and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, dichloroethanes (300 milliliters), dichloroethanes/isopropanol are successively used
(volume ratio 5:1,500 milliliters), dichloroethanes/methanol/water (volume ratio 5:1:0.1,500 milliliters), dichloroethanes/methanol/water
(volume ratio 3:1:0.1,500 milliliters), dichloroethanes/methanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 5
With 20 grams of Powdered Soy Lecithins (phosphatidylcholine content 23%) for raw material, 1.19 grams obtained, purity is the present embodiment
99.2% solid lysophosphatidyl choline.It is specific:
(1) Powdered Soy Lecithin and 78 grams is added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Water stirs 30 minutes at 50 DEG C, obtains emulsion；0.02 gram of phospholipase A1 is dissolved in 2 grams of water, then in two minutes dropwise
It is added in the emulsion of phosphatide and water, heat preservation continues to be stirred to react 5 hours at 50 DEG C；
(2) room temperature is cooled to, 400 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of chloroforms and obtains a kind of solution；The solution is poured into 300 ml methanols and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, chloroform (300 milliliters), chloroform/isopropanol are successively used
(volume ratio 5:1,500 milliliters), chloroform/methanol/water (volume ratio 5:1:0.1,500 milliliters), chloroform/methanol/water
(volume ratio 3:1:0.1,500 milliliters), chloroform/methanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 6
With 20 grams of liquid soy phosphatide (phosphatidylcholine content 11%) for raw material, 0.49 gram obtained, purity is the present embodiment
99.3% solid lysophosphatidyl choline.It is specific:
(1) the liquid soy phosphatide and 78 grams are added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Water stirs 30 minutes at 50 DEG C, obtains emulsion；0.02 gram of phospholipase A1 is dissolved in 2 grams of water, then in two minutes dropwise
It is added in the emulsion of phosphatide and water, heat preservation continues to be stirred to react 4 hours at 50 DEG C；
(2) room temperature is cooled to, 400 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of methylene chloride and obtains a kind of solution；The solution is poured into 300 milliliters of ethyl alcohol and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, methylene chloride (300 milliliters), methylene chloride/isopropanol are successively used
(volume ratio 5:1,500 milliliters), methylene chloride/ethanol/water (volume ratio 5:1:0.1,500 milliliters), methylene chloride/ethanol/water
(volume ratio 3:1:0.1,500 milliliters), methylene chloride/ethanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 7
With 20 grams of Powdered Soy Lecithins (phosphatidylcholine content 23%) for raw material, 1.24 grams obtained, purity is the present embodiment
99.0% solid lysophosphatidyl choline.It is specific:
(1) Powdered Soy Lecithin and 78 grams is added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
The CaCl of 0.4mmol/L2Solution stirs 30 minutes at 50 DEG C, obtains emulsion；0.06 gram of phospholipase A2 is dissolved in 2 grams
The CaCl of 0.4mmol/L2In solution, then it is added dropwise in two minutes in the emulsion of phosphatide and water, is kept the temperature at 50 DEG C
Continue to be stirred to react 8 hours；
(2) room temperature is cooled to, 400 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of chloroforms and obtains a kind of solution；The solution is poured into 300 ml methanols and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, methylene chloride (300 milliliters), methylene chloride/isopropanol are successively used
(volume ratio 5:1,500 milliliters), methylene chloride/ethanol/water (volume ratio 5:1:0.1,500 milliliters), methylene chloride/ethanol/water
(volume ratio 3:1:0.1,500 milliliters), methylene chloride/ethanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 8
With 20 grams of liquid soy phosphatide (phosphatidylcholine content 11%) for raw material, 0.47 gram obtained, purity is the present embodiment
99.5% solid lysophosphatidyl choline.It is specific:
(1) the liquid soy phosphatide and 78 grams are added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
The CaCl of 0.4mmol/L2Solution stirs 30 minutes at 50 DEG C, obtains emulsion；0.06 gram of phospholipase A2 is dissolved in 2 grams
The CaCl of 0.4mmol/L2In solution, then it is added dropwise in two minutes in the emulsion of phosphatide and water, is kept the temperature at 50 DEG C
Continue to be stirred to react 7 hours；
(2) room temperature is cooled to, 400 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of dichloroethanes and obtains a kind of solution；The solution is poured into 300 milliliters of ethyl alcohol and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, chloroform (300 milliliters), chloroform/isopropanol are successively used
(volume ratio 5:1,500 milliliters), chloroform/methanol/water (volume ratio 5:1:0.1,500 milliliters), chloroform/methanol/water
(volume ratio 3:1:0.1,500 milliliters), chloroform/methanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 9
With 20 grams of Powdered Soy Lecithins (phosphatidylcholine content 23%) for raw material, 1.21 grams obtained, purity is the present embodiment
99.4% solid lysophosphatidyl choline.It is specific:
(1) Powdered Soy Lecithin and 58 grams is added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
The CaCl of 0.4mmol/L2Solution stirs 30 minutes at 50 DEG C, obtains emulsion；) 0.05 gram of phospholipase A2 be dissolved in 2 grams
The CaCl of 0.4mmol/L2In solution, then it is added dropwise in two minutes in the emulsion of phosphatide and water, is kept the temperature at 50 DEG C
Continue to be stirred to react 10 hours；
(2) room temperature is cooled to, 300 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of dichloroethanes and obtains a kind of solution；The solution is poured into 300 milliliters of ethyl alcohol and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, methylene chloride (300 milliliters), methylene chloride/isopropanol are successively used
(volume ratio 5:1,500 milliliters), methylene chloride/ethanol/water (volume ratio 5:1:0.1,500 milliliters), methylene chloride/ethanol/water
(volume ratio 3:1:0.1,500 milliliters), methylene chloride/ethanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 10
With 20 grams of liquid soy phosphatide (phosphatidylcholine content 11%) for raw material, 0.46 gram obtained, purity is the present embodiment
99.5% solid lysophosphatidyl choline.It is specific:
(1) the liquid soy phosphatide and 58 grams are added in 500 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
The CaCl of 0.4mmol/L2Solution stirs 30 minutes at 50 DEG C, obtains emulsion；0.05 gram of phospholipase A2 is dissolved in 2 grams
The CaCl of 0.4mmol/L2In solution, then it is added dropwise in two minutes in the emulsion of phosphatide and water, is kept the temperature at 50 DEG C
Continue to be stirred to react 10 hours；
(2) room temperature is cooled to, 300 milliliters of acetone are added under stiring and continues high-speed stirred 2 hours；Filtering divides after stopping stirring
Solid is separated out, which is dissolved in 60 milliliters of methylene chloride and obtains a kind of solution；The solution is poured into 300 ml methanols and is acutely stirred
It mixes；It is removed by filtration and is formed by solid insoluble, collect filtrate, be concentrated into about 30 milliliters with Rotary Evaporators, gained concentrate
It is operated for subsequent pillar layer separation；
(3) 125 grams of dry silica gels are loaded in the silicagel column of 500 millimeters long 30 mm dia, the petroleum ether for being 60-90 DEG C with boiling point
It is spare after wetting；Aforementioned concentrate is transferred in silicagel column, methylene chloride (300 milliliters), methylene chloride/isopropanol are successively used
(volume ratio 5:1,500 milliliters), methylene chloride/methanol/water (volume ratio 5:1:0.1,500 milliliters), methylene chloride/methanol/water
(volume ratio 3:1:0.1,500 milliliters), methylene chloride/methanol/water (volume ratio 1:1:0.1,1000 milliliters) gradually elute, and collect
Eluent simultaneously merges same composition, and concentration is dried in vacuo to obtain solid lysophosphatidyl choline after removing organic solvent at 40 DEG C.
Embodiment 11
20 grams of Powdered Soy Lecithin (phosphatidyls are added in 250 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Content of choline 23%) and 58 grams of water, it is stirred 30 minutes at 50 DEG C, obtains emulsion；0.02 gram of phospholipase A1 is dissolved in 2 grams of water,
Then it is added dropwise in two minutes in the emulsion of phosphatide and water, heat preservation continues to be stirred to react at 50 DEG C.
By high performance liquid chromatography (HPLC), measures phosphatidyl choline and turn under the differential responses time in enzyme digestion reaction
The content of lysophosphatidyl choline (LPC) in rate and reaction product.
Embodiment 12
20 grams of Powdered Soy Lecithin (phosphatidyls are added in 250 milliliters of round bottom reaction flasks equipped with polytetrafluoroethylene (PTFE) mechanical stirring stick
Content of choline 23%) and 58 grams of 0.4mmol/L CaCl2Solution stirs 30 minutes at 50 DEG C, obtains emulsion；0.02 gram of phosphorus
Lipase A2 is dissolved in the CaCl of 2 grams of 0.4mmol/L2In solution, the emulsion of phosphatide and water is then added dropwise in two minutes
In, heat preservation continues to be stirred to react at 50 DEG C.
By high performance liquid chromatography (HPLC), measures phosphatidyl choline and turn under the differential responses time in enzyme digestion reaction
The content of lysophosphatidyl choline (LPC) in rate and reaction product.
In embodiment 11 and embodiment 12, HPLC method measures conversion ratio: measuring enzyme digestion reaction using high performance liquid chromatography
The content of phosphatidyl choline calculates the conversion ratio of phosphatidyl choline in the reaction system of front and back, and measures haemolysis phosphorus in reaction product
The content of phosphatidylcholine.Column condition: nova-pak@silica column length 15cm, 3.9 millimeters of column diameter, column presses 280psi, flow velocity
0.4ml/min.Detection wavelength 204mm.Mobile phase: acetonitrile: methanol: water=65:21:14 (volume ratio).
The conversion ratio of phosphatidyl choline=((PC content after PC content-reaction before reacting)/PC content before reacting) × 100%.
The conversion ratio of phosphatidyl choline is measured according to embodiment 11 and embodiment 12, the conversion ratio of phosphatidyl choline
For the core monitoring index in preparation process, react insufficient or reaction excessively can all bring it is unnecessary in later purification technique
Trouble.
The present invention is using common soybeans phosphatide as raw material, pre-processes by enzyme digestion reaction, after enzymatic hydrolysis and column Chromatographic purification technique
High-purity lysophosphatidyl choline is prepared, has the characteristics that raw material cheap and easy to get, simple process, strong operability, high income,
It is suitable for industrialized production.The lysophosphatide produced by the invention it can be seen from the nuclear-magnetism phosphorus spectrogram of Fig. 2 lysophosphatidyl choline
Phatidylcholine purity is high, by-product it is few, can be widely used in fine chemistry industry, food, medicine, cosmetics, health care product, agricultural products with
And other phosphatide deep processed product fields.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Illustrative description is carried out to the invention patent above, it is clear that the realization of the invention patent is not by aforesaid way
Limitation, as long as use the invention patent method concept and technical solution carry out various improvement, or it is not improved will this
The conception and technical scheme of patent of invention directly apply to other occasions, are within the scope of the invention.
Claims (10)
1. a kind of method for preparing high-purity lysophosphatidyl choline, which is characterized in that in the presence of phosphatidase, to soybean lecithin
Enzyme digestion reaction is carried out, enzyme digestion reaction product is transferred in silicagel column after pretreatment, and with polyhalo low-carbon alkanes-low-carbon
Alcohol-water is that eluant, eluent is repeatedly eluted, and collects the solution of object, high-purity lysophosphatidyl choline is obtained after concentrate drying
Product.
2. the method for preparation high-purity lysophosphatidyl choline according to claim 1, which is characterized in that the soybean phosphorus
Rouge is dissolved in aqueous solution, and phospholipase A1 is selected to carry out enzyme digestion reaction.
3. the method for preparation high-purity lysophosphatidyl choline according to claim 1, which is characterized in that the soybean phosphorus
Rouge is dissolved in calcium chloride solution, and phospholipase A2 is selected to carry out enzyme digestion reaction.
4. the method for preparation high-purity lysophosphatidyl choline according to claim 1, which is characterized in that enzyme digestion reaction produces
Object carries out pretreated process specifically includes the following steps: acetone is first added into enzyme digestion reaction product, filters after stirring and dissolving
Isolated solid sediment；The solid sediment is dissolved in polyhalo low-carbon alkanes and obtains solution；The solution pours into low-carbon
Solid matter is precipitated in monohydric alcohol, is removed by filtration and solid matter is precipitated and collects filtrate；The filtrate of collection is concentrated.
5. the method for preparation high-purity lysophosphatidyl choline according to claim 1 or 4, which is characterized in that described more
Halogenated low-carbon alkanes include methylene chloride, chloroform, dichloroethanes.
6. the method for preparation high-purity lysophosphatidyl choline according to claim 1, which is characterized in that the low-carbon alcohols
For low-carbon monohydric alcohol.
7. the method for preparation high-purity lysophosphatidyl choline according to claim 4 or 6, which is characterized in that described low
Carbon monohydric alcohol includes methanol, ethyl alcohol, isopropanol.
8. the method for preparation high-purity lysophosphatidyl choline according to claim 1, which is characterized in that successively with more halogen
For low-carbon alkanes, polyhalo low-carbon alkanes/low-carbon alcohols, polyhalo low-carbon alkanes/low-carbon alcohols/water, polyhalo low-carbon alkanes/low-carbon
Alcohol/water, polyhalo low-carbon alkanes/low-carbon alcohols/water are gradually repeatedly eluted.
9. the method for preparation high-purity lysophosphatidyl choline according to claim 8, which is characterized in that repeatedly eluting
In the process, the polarity of eluant, eluent is gradually increased.
10. the method for preparation high-purity lysophosphatidyl choline according to claim 9, which is characterized in that at second
In elution process, polyhalo low-carbon alkanes/low-carbon alcohols volume ratio is 5:1；In third time elution process, polyhalo alkane/
Low-carbon alcohols/water volume ratio is 5:1:0.1；In the 4th elution process, polyhalo alkane/low-carbon alcohols/water volume ratio is
3:1:0.1；In the 5th elution process, polyhalo alkane/low-carbon alcohols/water volume ratio is 1:1:0.1.
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| CN111378702A (en) * | 2020-02-11 | 2020-07-07 | 湖南中茂生物科技有限公司 | Method for extracting and separating α -GPC from soybean lecithin |
| CN112679537A (en) * | 2021-01-21 | 2021-04-20 | 江苏曼氏生物科技股份有限公司 | Method for removing free sugar and glycolipid in powdered soybean phospholipid |
| CN114540439A (en) * | 2022-01-28 | 2022-05-27 | 海南乐孕生物科技有限公司 | Extraction process of high-hydrophilicity high-activity enzymolysis soybean phospholipid |
| CN114644649A (en) * | 2020-12-21 | 2022-06-21 | 广州白云山汉方现代药业有限公司 | Method for extracting high-purity lysophosphatidylcholine from egg yolk powder |
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| CN114644649B (en) * | 2020-12-21 | 2023-10-24 | 广州白云山汉方现代药业有限公司 | Method for extracting high-purity lysophosphatidylcholine from egg yolk powder |
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