CN114106036A - Separation and extraction method of L-alpha-glycerophosphatidylcholine - Google Patents
Separation and extraction method of L-alpha-glycerophosphatidylcholine Download PDFInfo
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- 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
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- 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
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- Molecular Biology (AREA)
Abstract
The invention discloses a method for separating and extracting L-alpha-glycerophosphatidylcholine, which takes soybean phospholipid powder extract rich in Phosphatidylcholine (PC) as a starting material, obtains reaction liquid rich in L-alpha-GPC through ester exchange reaction catalyzed by strong base anion exchange resin, and obtains a target product through purification steps of concentration, liquid separation, decoloration, elution by adsorption resin and the like, wherein the purity of the target product is more than 98%. The method adopts anhydrous operation in the whole process, utilizes strong-base ion exchange resin as a catalyst, has the characteristics of short reaction time, simple and convenient operation, high product purity and the like, and is easy to realize technologization.
Description
Technical Field
The invention belongs to the technical field of compound extraction and preparation, and particularly relates to a separation and extraction method of L-alpha-glycerophosphatidylcholine.
Background
L-alpha-glycerophosphocholine (L-alpha-glycerophosphocholine, L-alpha-GPC for short) is a water-soluble phospholipid metabolite naturally existing in vivo, is a choline source for synthesizing acetylcholine and phosphatidylcholine, and has important nutrition and health care functions and medical application values; can penetrate blood brain barrier, improve cognition and memory, and improve brain health condition; recent studies show that L-alpha-GPC also has the effects of enhancing muscle sensitivity, preventing cardiovascular diseases and fatty liver, maintaining hormone levels in vivo and resisting aging;
extracting L-alpha-GPC from natural substances is a quick and effective method, but because the source way difference of raw materials is large and the distribution of impurity components is uneven, the subsequent separation and purification process is complex, the product quality is unstable, and the industrial production is difficult; in addition, the extraction method of the L-alpha-GPC comprises a total synthesis method and a catalytic hydrolysis method; the total synthesis method has the characteristics of easy separation of reaction products and high purity, is easy to realize large-scale production, but has the defects of high toxicity and instability of raw materials, long synthesis route and harsh conditions, and still needs optical and activity experiments, so that the application is still greatly limited; the catalytic hydrolysis method uses lecithin in animal and plant bodies, such as soybean powder phospholipid, as starting material, removes 2 molecules of fatty acid by catalytic hydrolysis, retains 3-position phosphatidylcholine, and separates and purifies to obtain L-alpha-GPC. The catalyst mainly comprises alkali metal alkoxide, organic amine, enzyme and the like. The metal alkoxide catalyst has the advantages of short reaction time and high conversion rate, but has the disadvantages of easiness in removing 3-bit acyl, complex later-stage separation, incapability of removing metal ions, generation of a large amount of wastewater and the like; the organic amine reaction is mild, but the production efficiency is low. The phosphatidase A1 and A2 can remove 1-position fatty acid and 2-position fatty acid respectively, and the method has the advantages of high efficiency and mild conditions, but the phospholipase is high in price, easy to inactivate, low in yield and limited in application; meanwhile, a patent reports that a gene recombination method is used for producing the phosphatidase A2, but the technology and the practical application have a larger distance;
therefore, the prior high-purity L-alpha-GPC mainly depends on import and is expensive; the domestic process is complex, the route is immature, the quality is unstable, the cost is high, and the industrial production is difficult to realize.
Disclosure of Invention
The invention provides a preparation method of L-alpha-glycerophosphatidylcholine, which takes soybean phospholipid powder extract rich in Phosphatidylcholine (PC) as a starting material, obtains reaction liquid rich in L-alpha-GPC through ester exchange reaction catalyzed by strong base anion exchange resin, and obtains a target product through purification steps of concentration, liquid separation, decoloration, elution by adsorption resin and the like, wherein the purity is more than 98%;
in order to achieve the technical purpose, the invention is realized by the following technical scheme: a separation and extraction method of L-alpha-glycerophosphatidylcholine is characterized by comprising the following steps:
s1: adding a low-carbon alcohol solvent into the soybean lecithin powder, stirring and extracting, controlling the extraction temperature and time, filtering, and collecting an extracting solution; repeating the above operation on filter residues, and combining the two obtained extracting solutions to obtain an extracting solution rich in phosphatidylcholine;
s2: adding specific strong-base anion exchange resin into the extracting solution obtained in the step S1, catalyzing to perform ester exchange reaction, controlling the reaction time, filtering after the reaction is finished, and collecting reaction liquid;
s3: adding acid into the reaction liquid in S2, adjusting the pH value of the reaction liquid to be neutral, concentrating under reduced pressure to be layered, adding petroleum ether for extraction, and collecting the L-alpha-GPC-enriched extract liquid;
s4: adding activated carbon into the extract phase obtained in the step S3 for decoloring, filtering, and further concentrating the filtrate to a small amount;
s5: loading the concentrated solution obtained in the step S4 into well-balanced macroporous adsorption resin, eluting by using low carbon alcohol, and collecting an L-alpha-GPC elution solution rich in L-alpha-GPC; further concentration until no solvent is evaporated off, a yellow viscous liquid is obtained.
S6: and (3) processing the yellow mucus obtained in the S5 by a recrystallization method or a solvent precipitation method, further carrying out centrifugal filtration, washing and vacuum drying to obtain a light yellow solid L-alpha-GPC product.
Preferably, in the step S1, the solid-to-liquid ratio of the soybean phospholipid powder to the low-carbon alcohol solvent is 1:4-7, the extraction temperature is 20-50 ℃, the extraction time is 80-120min, and the low-carbon alcohol solvent is methanol, ethanol or a mixture of the methanol and the ethanol in proportion.
Preferably, in the S2, the solid-to-liquid ratio of the strongly basic anion exchange resin to the reaction solution is 1: 2-4; the reaction time is 80-160 min.
Preferably, in S3, the acid includes but is not limited to hydrochloric acid, acetic acid, formic acid, and the solvent obtained by concentration can be recycled; the amount of petroleum ether is 3-5 times of the concentrated solution.
Preferably, in the S4, the weight ratio of the active carbon to the extract phase is 1:10-20, and the active carbon is concentrated to 1/5-1/10 of the original volume of solution.
Preferably, in the S5, the volume ratio of the concentrated solution to the macroporous adsorption resin is 1:5-10, the lower alcohol is methanol, ethanol or the mixture of the methanol and the ethanol according to the proportion, the elution speed is 1.5-3 times of the bed volume per hour, and the concentrated solvent can be recycled.
Preferably, in S6, the recrystallization solvent is one of ethanol, propanol and n-butanol, and the solvent used in the solvent precipitation method is acetone, petroleum ether, methanol or a mixture thereof.
The invention has the beneficial effects that:
according to the method for separating and extracting the L-alpha-glycerophosphatidylcholine, the strongly basic anion exchange resin is used as an ester exchange reaction catalyst to remove fatty acid, the reaction time is greatly shortened, the post-treatment is convenient, the separation and purification of the product are facilitated, the whole process adopts anhydrous operation, and further water removal operation is not needed; the optimized processes of extraction, reaction and separation can obtain high-yield and high-purity L-alpha-GPC, and the industrialization is easy to realize.
Drawings
FIG. 1 is a liquid chromatogram of GPC obtained by the solvent precipitation method of example 1;
FIG. 2 is an ESI positive ion spectrum of GPC obtained by the solvent precipitation method in example 1;
FIG. 3 is a liquid chromatogram of GPC obtained by recrystallization in example 2;
FIG. 4 is an ESI positive ion spectrum of GPC obtained by recrystallization in example 2;
FIG. 5 is a schematic process flow diagram of the present invention.
Detailed Description
In order to clearly and completely express the technical scheme and technical effect of the invention, the following embodiments are used for detailed description;
example 1
S1: extracting soybean phospholipid powder and ethanol solution at a ratio of 1:6 at 40 deg.C for 90min, and filtering to obtain first extractive solution; adding ethanol into the filter residue at a material-liquid ratio of 6:1, stirring thoroughly, extracting at 40 deg.C for 90min, filtering, and collecting the second extractive solution; the two extracts were combined to give a reddish brown extract.
S2: according to the solid-liquid ratio of the strong-base anion exchange resin to the extraction liquid of 1: 3; adding the treated strongly basic anion exchange resin into the extract obtained in S1, stirring and reacting for 100min at normal temperature, decompressing and filtering after the reaction is finished, washing the resin by a small amount of solvent, and collecting and combining light yellow reaction liquid;
s3: adding hydrochloric acid into the reaction solution after the reaction in the S2 to adjust the pH value to be neutral, distilling under reduced pressure at 40 ℃, simultaneously recovering the solvent, concentrating to be layered, adding 4 times of concentrated solution petroleum ether, and extracting for three times to obtain an extract rich in L-alpha-GPC;
s4: decolorizing the extract obtained in S3 with 1/15 weight of activated carbon at normal temperature for 20min, boiling for 5min, and filtering to obtain decolorized solution; further concentration was carried out to 1/8 of the solution.
S5: and (2) putting the concentrated solution obtained in the step (S4) into a specific macroporous adsorption resin, eluting with ethanol at the rate of 2 bed layer volumes per hour at the volume ratio of the macroporous adsorption resin to the concentrated solution of 8:1, collecting the L-alpha-GPC-enriched eluent, further concentrating until the ethanol cannot be evaporated out to obtain yellow mucus, and recovering the ethanol.
S6: the yellow mucus obtained in S5 was purified by ethanol: recrystallizing the mixed solvent with the volume ratio of the n-butanol of 2:8, placing the mixed solvent in a refrigerator for freezing at the temperature of 10 ℃ below zero overnight, separating out a large amount of precipitates, centrifuging, washing the precipitates with 20ml of acetone, and washing away the mixed solvent. Drying under vacuum at normal temperature for 24 hr to obtain yellowish solid product.
The high purity L-alpha-GPC obtained in this example was characterized by mass spectrometry and HPLC analysis to have a purity of 98.14%, a lysophosphatidylcholine LPC content of 0.19%, a water content of 0.45%, a phospholipid homologue such as phosphatidylethanolamine impurity content of less than 0.5%, and a peroxide value of 2.2, and was a slightly yellow waxy solid.
Example 2
S1: extracting soybean phospholipid powder and ethanol solution at a ratio of 1:7 at room temperature for 120min, and filtering to obtain first extractive solution; repeating the above operation on the filter residue, and collecting the second extraction liquid; the two extracts were combined to give a reddish brown extract.
S2: according to the solid-liquid ratio of the strong-base anion exchange resin to the extraction liquid of 1: 2.8; adding the treated xx strongly-basic anion exchange resin into the extract obtained in S1, stirring and reacting for 130min at normal temperature, decompressing and filtering after the reaction is finished, washing the resin by a small amount of solvent, and collecting and combining light yellow reaction liquid;
s3: adding formic acid into the reaction solution after the reaction in the S2 to adjust the pH value to be neutral, distilling under reduced pressure at 40 ℃, simultaneously recovering the solvent, concentrating to be layered, adding 4 times of concentrated solution petroleum ether, and extracting for three times to obtain an extract rich in L-alpha-GPC;
s4: decolorizing the extract obtained in S3 with 1/18 weight of activated carbon at normal temperature for 15min, boiling for 4min, and filtering to obtain decolorized solution; further concentration was carried out to 1/8 of the solution.
S5: and (2) filling the concentrated solution obtained in the step (S4) into xx macroporous adsorption resin, wherein the volume ratio of the xx macroporous adsorption resin to the concentrated solution is 10:1, eluting with ethanol at the elution speed of 1.5 times the bed volume per hour, collecting an L-alpha-GPC (layered double hydroxides) elution solution rich in L-alpha-GPC, further concentrating until the ethanol cannot be evaporated out to obtain yellow mucus, and recovering the ethanol.
S6: adding equal volume of ethanol into the yellow mucus obtained in S5, precipitating a little fine solid, adding 2 times volume of acetone into the yellow mucus, precipitating a large amount of flocculent solid, centrifuging, washing the solid with acetone to obtain light yellow solid, and drying under reduced pressure at normal temperature for 24 hours to obtain yellow waxy solid.
The high purity L- α -GPC obtained in this example was identified by mass spectrometry and analyzed by HPLC, and had a purity of 98.31%, a lysophosphatidylcholine LPC content of 0.23%, a water content of 0.39%, a content of impurities of phospholipid homologues such as phosphatidylethanolamine of less than 0.5%, and a peroxide value of 2.1, and was a pale yellow solid.
Claims (7)
1. A method for separating and extracting high-purity L-alpha-glycerophosphatidylcholine is characterized by comprising the following steps:
s1: adding a low-carbon alcohol solvent into the soybean lecithin powder, stirring and extracting, controlling the extraction temperature and time, filtering, and collecting an extracting solution; repeating the above operation on filter residues, and combining the two obtained extracting solutions to obtain an extracting solution rich in phosphatidylcholine;
s2: adding specific strong-base anion exchange resin into the extracting solution obtained in the step S1, catalyzing to perform ester exchange reaction, controlling the reaction time, filtering after the reaction is finished, and collecting reaction liquid;
s3: adding acid into the reaction liquid in S2, adjusting the pH value of the reaction liquid to be neutral, concentrating under reduced pressure to be layered, adding petroleum ether for extraction, and collecting the L-alpha-GPC-enriched extract liquid;
s4: adding activated carbon into the extract phase obtained in the step S3 for decoloring, filtering, and further concentrating the filtrate to a small amount;
s5: loading the concentrated solution obtained in the step S4 into well-balanced macroporous adsorption resin, eluting by using low carbon alcohol, and collecting an L-alpha-GPC elution solution rich in L-alpha-GPC; further concentrating until no solvent is evaporated to obtain yellow mucus;
s6: and (3) processing the yellow mucus obtained in the S5 by a recrystallization method or a solvent precipitation method, further carrying out centrifugal filtration, washing and vacuum drying to obtain a light yellow solid L-alpha-GPC product.
2. The method for separating and extracting high-purity L-alpha-glycerophosphatidylcholine according to claim 1, wherein in step S1, the solid-to-liquid ratio of soybean phospholipid powder to the low-carbon alcohol solvent is 1:4-7, the extraction temperature is 20-50 ℃, the extraction time is 80-120min, and the low-carbon alcohol solvent is methanol, ethanol or a mixture of the two in proportion.
3. The method for separating and extracting high-purity L-alpha-glycerophosphatidylcholine according to claim 1, wherein in step S2, the solid-to-liquid ratio of the strongly basic anion exchange resin to the reaction solution is 1: 2-4; the reaction time is 80-160 min.
4. The method for separating and extracting L- α -glycerophosphatidylcholine according to claim 1, wherein in step S3, the acid includes but is not limited to hydrochloric acid, acetic acid, formic acid, and the solvent obtained by concentration can be recycled; the amount of petroleum ether is 3-5 times of the concentrated solution.
5. The method for separating and extracting L- α -glycerophosphatidylcholine according to claim 1, wherein in step S4, the ratio of the amount of activated carbon to the weight of the extract phase is 1:10-20, and the extract phase is concentrated to 1/5-1/10 of the original volume of solution.
6. The method for separating and extracting high-purity L-alpha-glycerophosphatidylcholine according to claim 1, wherein in step S5, the volume ratio of the concentrated solution to the macroporous adsorbent resin is 1:5-10, the lower alcohol is methanol, ethanol or the mixture of the methanol and the ethanol according to the proportion, the elution speed is 1.5-3 times of the bed volume per hour, and the concentrated solvent can be recycled.
7. The method for separating and extracting high-purity L- α -glycerophosphatidylcholine according to claim 1, wherein in step S6, the recrystallization solvent includes but is not limited to ethanol, propanol, and n-butanol, and the solvent precipitation method uses acetone, petroleum ether, methanol, or a mixture thereof.
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| US5250719A (en) * | 1989-05-08 | 1993-10-05 | Prime European Therapeuticals S.P.A. | Process for the preparation of L-α-glycerylphosphoryl-choline and of L-α-glycerylphosphorylethanolamine |
| US5315023A (en) * | 1992-06-24 | 1994-05-24 | Chemi S.P.A. | Process for the preparation of glycerophospholipids |
| US20120244583A1 (en) * | 2011-03-24 | 2012-09-27 | Yuanfa Liu | Method for Preparing High Purity L-alpha Glycerylphosphorylcholine |
| CN102875592A (en) * | 2012-09-29 | 2013-01-16 | 常熟富士莱医药化工有限公司 | Natural L-alpha-glycero-phosphatidylcholine preparation method |
| CN105017307A (en) * | 2015-07-22 | 2015-11-04 | 沈阳天峰生物制药有限公司 | Method for preparing high-purity natural L-alpha-glycerylphosphorylcholine |
| RU2685238C1 (en) * | 2016-07-01 | 2019-04-17 | Общество с ограниченной ответственностью "ФАРМАЦЕЛЬ" | Method of producing crystalline glycerophosphatidylcholine |
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| US5250719A (en) * | 1989-05-08 | 1993-10-05 | Prime European Therapeuticals S.P.A. | Process for the preparation of L-α-glycerylphosphoryl-choline and of L-α-glycerylphosphorylethanolamine |
| US5315023A (en) * | 1992-06-24 | 1994-05-24 | Chemi S.P.A. | Process for the preparation of glycerophospholipids |
| US20120244583A1 (en) * | 2011-03-24 | 2012-09-27 | Yuanfa Liu | Method for Preparing High Purity L-alpha Glycerylphosphorylcholine |
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