Method for simultaneously preparing high-purity sphingomyelin and glycerol phosphocholine

Abstract

Disclosed in the present invention is a method for simultaneously preparing high-purity sphingomyelin and glycerol phosphocholine. The method comprises the following steps: 1) adding a degradation agent to a phospholipid mixture to degrade phospholipid, so as to obtain a sphingomyelin and glycerol phosphocholine mixture, and carrying out separation by means of a refining method to obtain a crude product; 2) carrying out column chromatography to obtain sphingomyelin having a purity higher than 98%; and 3) obtaining glycerol phosphocholine having a purity higher than 95% by means of a refining method; and 4) drying same to obtain a phospholipid satisfying the pharmaceutical standard. The solvent used in the present invention can be recycled and reused and is environmentally friendly, and the method has mild reaction conditions, good selectivity, low requirement for the raw material purity, and a relatively low production cost, and is beneficial to industrial mass production.

Description

A method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine Technical Field

[0001] The present invention relates to the field of synthetic phospholipids, and in particular to a method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine. Background Art

[0002] Egg yolk lecithin contains ingredients such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), and sphingomyelin (SM). Currently, the preparation processes for egg yolk lecithin and egg yolk PC are mature, and they are used as emulsifiers in the preparation of nutritional and drug-loaded fat emulsions. Other phospholipids, such as natural SM, have important physiological functions. SM and its metabolites participate in physiological activities such as cell growth, differentiation, and apoptosis in the body; SM is also a key component in the preparation of vincristine sulfate liposome injections. Furthermore, L-α-glycerophosphocholine (GPC), the main component of phospholipid hydrolysis, is used internationally to treat diseases such as ischemic stroke and Alzheimer's disease. Therefore, developing deep processing technology for egg yolk lecithin will help fully utilize the various components of phospholipids and develop high-value-added products.

[0003] Existing technologies for preparing high-purity SM include: Prior Art 1 (CN114989212A) obtains high-purity SM through column chromatography and recrystallization. However, the SM content in lecithin is only 3%. Preparing SM alone increases production costs and results in significant waste, making it difficult to apply to large-scale production. Prior Art 2 (CN114644650A) requires two column chromatography separations to obtain high-purity SM and egg yolk lecithin, but the operation is relatively cumbersome. As can be seen, due to the similar polarity of SM and PC, it is difficult to obtain a high-purity product (≥98%) through a single separation using existing technologies. Therefore, to address these technical challenges, the development of new SM preparation technologies to improve separation efficiency and reduce production costs has important application value.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to solve the problems of great separation difficulty and high cost in the existing natural sphingomyelin extraction and purification process.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine, characterized by comprising the following steps:

[0008] A. adding a degradation agent to the phospholipid mixture to carry out a degradation reaction to obtain an intermediate mixture 1;

[0009] B. extracting and separating the intermediate mixture 1 to obtain a refined mixture 1 from the organic phase and a refined mixture 2 from the aqueous phase;

[0010] C. further separating the refined mixture 1 to obtain a lower layer of flocculent matter;

[0011] D. purifying the lower layer of flocculent matter by column chromatography to obtain sphingomyelin;

[0012] E. subjecting the refined mixture 2 to adsorption, desalination, and recrystallization to obtain glycerophosphocholine.

[0013] The present invention discloses a method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine, comprising the following steps: 1) adding a degradation agent to a phospholipid mixture to degrade the phospholipids to obtain a sphingomyelin and glycerophosphocholine mixture, and separating the mixture through a purification method to obtain a crude product; 2) obtaining sphingomyelin with a purity exceeding 98% through column chromatography; 3) obtaining glycerophosphocholine with a purity exceeding 95% through a purification method; and 4) drying the mixture to obtain a phospholipid that meets pharmaceutical standards. The present invention utilizes solvents that can be recycled and reused, is environmentally friendly, has mild reaction conditions, good selectivity, low raw material purity requirements, and low production costs, facilitating industrialized mass production.

[0014] Preferably, the method further comprises the following steps:

[0015] F. Drying the sphingomyelin and the glycerophosphocholine separately to obtain a sphingomyelin product and a glycerophosphocholine product simultaneously.

[0016] Preferably, in step A, the reaction conditions of the degradation reaction include: continuous stirring at 20-60° C. and a reaction time of 0.5-8 hours; in step B, the extraction separation includes: adding an organic solvent and water to the intermediate mixture 1, stirring and mixing, standing and separating the layers, continuing to add the organic solvent to the aqueous phase and repeating the operation multiple times to obtain the refined mixture 2, and combining the organic phases extracted and separated multiple times to obtain the refined mixture 1; in step C, the further separation includes: standing until the flocs are precipitated to the bottom, and pouring the upper oil to obtain the lower flocs; in step D, the column chromatography purification includes silica gel column chromatography separation, and the composite eluent used includes a mixture of halogenated alkane-lower alcohol-water; in step E, the adsorption includes: adding an adsorbent to the refined mixture 2, stirring and mixing evenly, and then filtering and separating.

[0017] The refined mixture 1 actually contains a mixture of water-insoluble oil and solid flocs. After standing for a long period of time, the oil layer and the solid flocs will gradually separate, and the flocs will settle to the bottom.

[0018] Preferably, in step A, the mass ratio of the amount of the degradation agent to the phospholipid mixture is 1% to 20%; in step B, the volume ratio of the organic solvent added to water is 1 to 3:1 during each extraction and separation operation; in step C, the standing time for the further separation is 0.5 to 24 hours; in step D, the volume ratio of the halogenated alkane, the lower alcohol, and the water in the composite eluent is 300 to 100:100:5; in step E, the mass ratio of the amount of the adsorbent to the phospholipid mixture is 1:10 to 100.

[0019] Preferably, in step A, the phospholipid mixture includes an egg yolk phospholipid extraction mixture, and the degradation agent includes one or more of sodium methoxide, sodium ethoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, phospholipase, and lipase; in step B, the organic solvent includes one or more of butanol, amyl alcohol, chloroform, dichloromethane, ethyl acetate, hexane, octane, petroleum ether, and dimethyl carbonate; in step C, the lower layer flocculent material is dissolved with the composite eluent; in step D, the halogenated alkane in the composite eluent includes one or more of chloroform and dichloromethane, and the lower alcohol includes one or more of methanol, ethanol, and isopropanol; in step E, the adsorbent includes one or more of diatomaceous earth, alumina, silica gel, and activated carbon.

[0020] Preferably, in step A, after the degradation reaction is completed, the operation of removing the degradation agent is further included to obtain the intermediate mixture 1; in step B, the operation is repeated 2 to 3 times; in step E, the desalting includes: adding anion and cation resins to the refined mixture 2 after adsorption, stirring and mixing evenly, and then filtering and separating.

[0021] After the degradation reaction is complete, the degradation agent should be removed. When the degradation agent is an inorganic salt, the pH can be adjusted to neutral by adding hydrochloric acid, nitric acid, sulfuric acid, acetic acid, trifluoroacetic acid, etc. When the degradation agent is an enzyme, the enzyme needs to be removed by filtration or centrifugation.

[0022] Preferably, in step A, the phospholipid mixture is dissolved in a solution before the reaction; in step C, before the further separation, the refined mixture 1 is first concentrated; in step D, before the column chromatography purification is performed, the refined mixture 1 is first dissolved with the composite eluent; in step E, the recrystallization comprises: dissolving the refined mixture 2 after desalting in a recrystallization reagent, cooling and adding seed crystals for crystallization and then washing.

[0023] Preferably, in step A, the solution comprises one or more of methanol, ethanol, isopropanol, butanol, ether, n-hexane, petroleum ether, and water; in step C, the refined mixture 1 is concentrated until the solvent is evaporated to dryness; in step E, the recrystallization reagent comprises one or more of methanol, ethanol, isopropanol, and water, and the mixture is dissolved by heating under reflux, and then cooled to -20 to 20° C. for crystallization.

[0024] A sphingomyelin product obtained by the above method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine.

[0025] A glycerophosphocholine product obtained by the above method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine.

[0026] The purity of the prepared synthetic phosphatidylcholine and phosphatidylethanolamine is not less than 98%, and the residual chloroform or dichloromethane is not higher than 0.006%, the residual dimethyl carbonate is not higher than 0.5%, and the residual alcohol is not higher than 0.5%.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Compared to Chinese patents CN114644650A and CN114989212A, the high-purity SM prepared by the technical solution of this patent avoids the need for secondary column chromatography or other purification operations in the prior art, simplifies the preparation steps, significantly reduces solvent consumption and preparation time, and is more suitable for industrial production. This patent combines transesterification and column chromatography for the first time to achieve a highly efficient SM enrichment technology, while fully utilizing the raw materials to produce a high-purity L-α-glycerophosphocholine (GPC) product.

[0029] The solvents used in the present invention are all recyclable and environmentally friendly, with mild reaction conditions, good selectivity, low raw material purity requirements, and low production costs, making it suitable for industrial mass production. This process route can simultaneously produce SM with a purity of no less than 98% and GPC with a purity of no less than 95%, with residual chloroform or dichloromethane no more than 0.006%, residual dimethyl carbonate no more than 0.5%, and residual alcohol no more than 0.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a schematic diagram of the technical route of Examples 1 to 3.

[0031] Figure 2 is a thin layer chromatogram of the egg yolk lecithin after degradation in Examples 1 to 3, wherein (1) egg yolk lecithin raw material; (2) sodium methoxide was added at a mass ratio of 1% in Example 1; (3) sodium ethoxide was added at a mass ratio of 5% in Example 2; and (4) phospholipase B was added at a mass ratio of 20% in Example 3. TAG: glyceride, FFA: free fatty acid, PE: phosphatidylethanolamine, PC: phosphatidylcholine, SM: sphingomyelin, and LPC: lysophosphatidylcholine.

[0032] FIG3 is a HPLC chromatogram of SM prepared in Example 1.

[0033] FIG4 is an HPLC chromatogram of the GPC prepared in Example 1. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0035] Example 1

[0036] As shown in Figure 1, 1000 g of the phospholipid mixture was dissolved in 7 L of methanol and thoroughly stirred. 10 g of sodium methoxide was added, and the mixture was stirred at 30°C for 2 hours. Thin-layer chromatography was performed until most of the PC was degraded. As shown in Figure 2, the solution was adjusted to a pH of 7-8 with hydrochloric acid, and the solvent was evaporated under reduced pressure to obtain intermediate mixture 1. 1 L of deionized water and 1 L of n-butanol were added to intermediate mixture 1, mixed thoroughly, and allowed to stand for separation. The aqueous phase was further extracted twice with 1 L of n-butanol, and the organic phases from the three extractions were combined to obtain the combined organic phase purified mixture 1 and the aqueous phase purified mixture 2, respectively.

[0037] The purified mixture 1 was concentrated and allowed to stand at room temperature for 4 hours. The upper layer of clear oil was decanted. The lower flocculent material was dissolved with an appropriate amount of chloroform-methanol-water (300:100:5) and subjected to silica gel column chromatography. Elution with chloroform-methanol-water (100:100:5) yielded 12.4 g of SM product (98.77% purity, 41.6% overall yield), as shown in Figure 3.

[0038] 25 g of activated carbon was added to the refined mixture 2, heated to 50 ° C and stirred for 30 minutes. After filtration, anionic and cationic resins were added to the filtrate, stirred at room temperature for 2 hours and then filtered. After the filtrate was concentrated and evaporated to dryness, anhydrous methanol was added and heated under reflux to dissolve. After cooling, GPC seed crystals were added and placed at -20 ° C to cool and crystallize. After suction filtration, the crystals were washed with chilled ethanol and dried to obtain 97.1 g of GPC solid (purity 96.40%, total yield 41.6%), as shown in Figure 4.

[0039] Example 2

[0040] As shown in Figure 1, 1000g of the phospholipid mixture was dissolved in 7L of ethanol and thoroughly stirred. 50g of sodium ethoxide was added, and the temperature was maintained at 50°C with stirring for 1 hour. Thin-layer chromatography was performed until most of the PC was degraded. As shown in Figure 2, the solution was adjusted to a pH of 7-8 with glacial acetic acid, and the solvent was evaporated under reduced pressure to obtain intermediate mixture 1. 1L of deionized water and 2L of chloroform were added to intermediate mixture 1, mixed thoroughly, and allowed to stand for separation. The aqueous phase was further extracted twice with 2L of chloroform, and the organic phases from the three extractions were combined. This yielded the combined organic phase purified mixture 1 and the aqueous phase purified mixture 2, respectively.

[0041] The purified mixture 1 was concentrated and allowed to stand at room temperature for 4 hours. The upper layer of clear oil was poured out after separation. The lower layer of flocculent matter was dissolved with an appropriate amount of chloroform-ethanol-water (200:100:5) and subjected to silica gel column chromatography. Column chromatography was eluted with chloroform-ethanol-water (100:100:5) to obtain 11.2 g of SM product (purity 98.43%, total yield 37.7%).

[0042] 10 g of activated carbon was added to the refined mixture 2 and stirred at room temperature for 60 minutes. After filtration, anionic and cationic resins were added to the filtrate, stirred at room temperature for 2 hours, and then filtered. After the filtrate was concentrated and evaporated to dryness, anhydrous methanol was added and heated under reflux to dissolve. After cooling, GPC seed crystals were added and placed at -20 ° C for cooling and crystallization. After suction filtration, the crystals were washed with chilled methanol and dried to obtain 91.2 g of GPC solid (purity 95.28%, total yield 39.1%).

[0043] Example 3

[0044] As shown in Figure 1, 1000 g of a phospholipid mixture was thoroughly stirred with 1.5 L of water and 1.5 L of n-hexane. 200 g of phospholipase B was added, and the mixture was stirred at 50°C for 8 hours. Thin-layer chromatography was performed until most of the PC was degraded, as shown in Figure 2. The phospholipase was removed by filtration, and the solvent was evaporated under reduced pressure to obtain intermediate mixture 1. 1 L of deionized water and 3 L of n-hexane were added to intermediate mixture 1, mixed thoroughly, and allowed to stand for separation. The aqueous phase was extracted twice with 3 L of n-hexane, and the organic phases from the three extractions were combined to obtain the combined organic phase purified mixture 1 and the aqueous phase purified mixture 2, respectively.

[0045] The purified mixture 1 was concentrated and allowed to stand at room temperature for 4 hours. The upper layer of clear oil was poured out after separation. The lower layer of flocculent matter was dissolved with an appropriate amount of dichloromethane-methanol-water (300:100:5) and subjected to silica gel column chromatography. The column chromatography was eluted with dichloromethane-methanol-water (200:100:5) to obtain 8.49 g of SM product (purity 98.06%, total yield 28.6%).

[0046] Add 100 g of silica gel to the refined mixture 2, heat to 40 ° C and stir for 60 minutes. After filtering, add anionic and cationic resin to the filtrate, stir at room temperature for 2 hours and filter. After the filtrate is concentrated and evaporated to dryness, add 70% ethanol and heat under reflux to dissolve. After cooling, add GPC seed crystals and place at -20 ° C to cool and crystallize. After suction filtration, the crystals are washed with chilled methanol and dried to obtain 75.2 g of GPC solid (purity is 95.89%, total yield is 32.4%).

[0047] The above disclosure is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine, characterized in that: The steps include: A. adding a degradation agent to the phospholipid mixture to perform a degradation reaction to obtain an intermediate mixture 1; B. extracting and separating the intermediate mixture 1, obtaining a refined mixture 1 from the organic phase and a refined mixture 2 from the aqueous phase; C. further separating the refined mixture 1 to obtain a lower layer of flocculent; D. purifying the lower layer floccules by column chromatography to obtain sphingomyelin; E. subjecting the refined mixture 2 to adsorption, desalting and recrystallization treatments to obtain glycerophosphocholine.

2. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 1, characterized in that: The following steps are also included: F. Drying the sphingomyelin and the glycerophosphocholine separately to obtain a sphingomyelin product and a glycerophosphocholine product at the same time.

3. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 1, characterized in that: In step A, the reaction conditions of the degradation reaction include: continuous stirring at 20-60° C., and a reaction time of 0.5-8 hours; in step B, the extraction separation includes: adding an organic solvent and water to the intermediate mixture 1, stirring and mixing, standing and separating after stratification, continuing to add an organic solvent to the aqueous phase and repeating the operation for multiple times to obtain the refined mixture 2, and combining the organic phases extracted and separated for multiple times to obtain the refined mixture 1; in step C, the further separation includes: standing until the floccules are precipitated to the bottom, and pouring the upper oil to obtain the lower floccules; in step D, the column chromatography purification includes silica gel column chromatography separation, and the composite eluent used includes a mixture of halogenated alkane-lower alcohol-water; in step E, the adsorption includes: adding an adsorbent to the refined mixture 2, stirring and mixing evenly, and then filtering and separating.

4. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 3, characterized in that: In step A, the mass ratio of the amount of the degradation agent to the phospholipid mixture is 1% to 20%; in step B, the volume ratio of the organic solvent added to water is 1 to 3:1 during each extraction and separation operation; in step C, the standing time for further separation is 0.5 to 24 hours; in step D, the volume ratio of halogenated alkane, lower alcohol and water in the composite eluent is 300 to 100:100:5; in step E, the mass ratio of the amount of the adsorbent to the phospholipid mixture is 1:10 to 100.

5. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 3, characterized in that: In step A, the phospholipid mixture includes an egg yolk phospholipid extraction mixture, and the degradation agent includes one or more of sodium methoxide, sodium ethoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, phospholipase, and lipase; in step B, the organic solvent includes one or more of butanol, amyl alcohol, chloroform, dichloromethane, ethyl acetate, hexane, octane, petroleum ether, and dimethyl carbonate; in step C, it also includes dissolving the lower layer floccules with the composite eluent; in step D, in the composite eluent, the halogenated alkane includes one or more of chloroform and dichloromethane, and the lower alcohol includes one or more of methanol, ethanol, and isopropanol; in step E, the adsorbent includes one or more of diatomaceous earth, alumina, silica gel, and activated carbon.

6. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 3, characterized in that: In step A, after the degradation reaction is completed, the degradation agent is removed to obtain the intermediate mixture 1; in step B, the operation is repeated 2 to 3 times; In step E, the desalination comprises: adding anion and cation resins to the refined mixture 2 after adsorption, stirring and mixing evenly, and then filtering and separating.

7. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 3, characterized in that: In step A, the phospholipid mixture is dissolved in a solution before the reaction; in step C, before the further separation, the refined mixture 1 is first concentrated; in step D, before the column chromatography purification is performed, the refined mixture 1 is first dissolved with the composite eluent; In step E, the recrystallization comprises: dissolving the refined mixture 2 after desalination in a recrystallization agent, cooling, adding seed crystals for crystallization, and then washing.

8. The method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 7, characterized in that: In step A, the solution includes one or more of methanol, ethanol, isopropanol, butanol, ether, n-hexane, petroleum ether, and water; in step C, the refined mixture 1 is concentrated until the solvent is evaporated to dryness; in step E, the recrystallization reagent includes one or more of methanol, ethanol, isopropanol, and water, and the mixture is dissolved by heating under reflux, and then cooled to -20 to 20° C. for crystallization.

9. A sphingomyelin product obtained by the method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 1 or 2.

10. A glycerophosphocholine product obtained by the method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as claimed in claim 1 or 2.

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