Method for simultaneously preparing high-purity sphingomyelin and choline glycerophosphate

Abstract

The invention discloses a method for simultaneously preparing high-purity sphingomyelin and glycerophosphorylcholine, which comprises the following steps: 1) adding a degradation agent into a phospholipid mixture to degrade phospholipid to obtain a sphingomyelin and glycerophosphorylcholine mixture, and separating by a refining method to obtain a crude product; 2) performing column chromatography to obtain sphingomyelin with purity higher than 98%; 3) obtaining glycerophosphorylcholine with the purity higher than 95% through a refining method; and 4) drying to obtain the phospholipid meeting the medicinal standard. The solvent used in the method can be recycled, and the method is environment-friendly, mild in reaction condition, good in selectivity, low in raw material purity requirement, relatively low in production cost and beneficial to industrial mass production.

Description

Technical Field

[0001] The 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). At present, the preparation process of egg yolk lecithin and egg yolk PC has been mature, and it is used as an emulsifier to prepare 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; at the same time, SM is also an important component for the preparation of vincristine sulfate liposome injection. In addition, L-α-glycerophosphocholine (GPC), the main component of phospholipid hydrolysis, is used abroad to treat cerebral ischemic stroke, Alzheimer's disease and other diseases. Therefore, the development of deep processing technology for egg yolk lecithin helps to make full use of the various components of phospholipids and develop high value-added products.

[0003] The existing preparation technologies for high-purity SM are as follows: Prior art 1 (CN114989212A) obtains high-purity SM by column chromatography and recrystallization, but the SM content in lecithin is only 3%. The preparation of SM alone increases production costs and causes a lot of waste, making it difficult to apply it to actual production on a large scale. Prior art 2 (CN114644650A) requires two column chromatography separations to obtain high-purity SM and egg yolk lecithin, but the operation is relatively cumbersome. It can be seen that due to the close polarity of SM and PC, it is difficult for the prior art to obtain a high-purity product (≥98%) by a single separation. Therefore, in view of the above technical difficulties, it is of great application value to develop a new SM preparation technology to improve separation effect and reduce production costs. Summary of the invention

[0004] 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.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine, characterized in that it comprises the following steps:

[0007] A. adding a degradation agent to the phospholipid mixture to perform a degradation reaction to obtain an intermediate mixture 1;

[0008] 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;

[0009] C. further separating the refined mixture 1 to obtain a lower layer of flocculent;

[0010] D. purifying the lower layer floccules by column chromatography to obtain sphingomyelin;

[0011] E. subjecting the refined mixture 2 to adsorption, desalting and recrystallization treatments to obtain glycerophosphocholine.

[0012] The 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 phospholipid to obtain a sphingomyelin and glycerophosphocholine mixture, and separating the mixture through a refining method to obtain a crude product; 2) obtaining sphingomyelin with a purity higher than 98% through column chromatography; 3) obtaining glycerophosphocholine with a purity higher than 95% through a refining method; 4) obtaining phospholipids that meet pharmaceutical standards through drying. The solvents used in the invention can be recycled and reused, are environmentally friendly, have mild reaction conditions, good selectivity, low requirements for raw material purity, and low production costs, which is conducive to industrial mass production.

[0013] Preferably, the method further comprises the following steps:

[0014] F. Drying the sphingomyelin and the glycerophosphocholine separately to obtain a sphingomyelin product and a glycerophosphocholine product at the same time.

[0015] 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 after stratification, continuing to add the 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 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.

[0016] 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.

[0017] 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 further separation is 0.5 to 24 hours; in step D, the volume ratio of halogenated alkanes, lower alcohols 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.

[0018] 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, it also includes dissolving the lower layer flocs 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.

[0019] Preferably, in step A, after the degradation reaction is completed, the operation of removing the degradation agent is also 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.

[0020] After the degradation reaction is completed, the step of removing the degradation agent should be carried out. When the degradation agent is an inorganic salt, hydrochloric acid, nitric acid, sulfuric acid, acetic acid, trifluoroacetic acid, etc. are added to adjust the pH to neutral. When the degradation agent is an enzyme, the enzyme needs to be removed by filtration or centrifugation.

[0021] 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 includes: dissolving the refined mixture 2 after desalting in a recrystallization reagent, cooling and adding seed crystals for crystallization and then washing.

[0022] Preferably, 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.

[0023] A sphingomyelin product obtained by the method for simultaneously preparing high-purity sphingomyelin and glycerophosphocholine as described above.

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

[0025] 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%.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] Compared with Chinese patents CN114644650A and CN114989212A, the high-purity SM prepared by the technical solution of this patent avoids the secondary column chromatography or other refining operations in the prior art, simplifies the preparation steps, significantly reduces the solvent consumption and preparation time, and is more suitable for industrial production. This patent combines the ester exchange method and the column chromatography method for the first time to achieve the technology of efficient enrichment of SM, and at the same time fully utilizes the raw materials to prepare high-purity L-α-glycerophosphocholine (GPC) products.

[0028] The solvents used in the present invention can be recycled and reused, are environmentally friendly, have mild reaction conditions, good selectivity, low requirements for raw material purity, and low production costs, which is conducive to industrial mass production. Through this process route, SM with a purity of not less than 98% and GPC with a purity of not less than 95% can be prepared at the same time, 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%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the technical route of Examples 1 to 3.

[0030] Figure 2The thin layer chromatograms after the degradation of egg yolk lecithin in Examples 1 to 3 are shown, wherein (1) egg yolk lecithin raw material; (2) sodium methoxide was added in a mass ratio of 1% in Example 1; (3) sodium ethoxide was added in a mass ratio of 5% in Example 2; and (4) phospholipase B was added in a mass ratio of 20% in Example 3. TAG: glyceride, FFA: free fatty acid, PE: phosphatidylethanolamine, PC: phosphatidylcholine, SM: sphingomyelin, and LPC: lysophosphatidylcholine.

[0031] Figure 3 This is the HPLC chromatogram of the SM prepared in Example 1.

[0032] Figure 4 This is the HPLC chromatogram of the GPC prepared in Example 1. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0034] Example 1

[0035] like Figure 1 As shown, 1000 g of phospholipid mixture was taken, dissolved in 7 L of methanol and stirred thoroughly, 10 g of sodium methoxide was added, the temperature was kept at 30 ° C and stirred for 2 hours, and thin layer chromatography was used to detect until most of the PC was degraded, as shown in FIG. Figure 2 As shown, the solution is adjusted to pH=7-8 with hydrochloric acid, and the solvent is evaporated under reduced pressure to obtain intermediate mixture 1. 1L of deionized water and 1L of n-butanol are added to the intermediate mixture 1 and mixed evenly, and the layers are separated. The aqueous phase is continuously extracted with 1L of n-butanol for 2 times, and the organic phases extracted 3 times are combined. The combined organic phase refined mixture 1 and the aqueous phase refined mixture 2 are obtained respectively.

[0036] The refined mixture 1 was concentrated and allowed to stand at room temperature for 4 hours. After the mixture was separated into upper and lower layers, the upper layer of clear oil was poured out. The lower layer of flocculent matter was dissolved with an appropriate amount of chloroform-methanol-water (300:100:5) and chromatographed on a silica gel column. The column chromatography was eluted with chloroform-methanol-water (100:100:5) to obtain 12.4 g of SM product (purity of 98.77%, total yield of 41.6%). Figure 3 shown.

[0037] Add 25g of activated carbon to the refined mixture 2, heat to 50°C and stir for 30 minutes. After filtering, add anion and cation resin to the filtrate, stir at room temperature for 2 hours and filter. After the filtrate is concentrated and evaporated, add anhydrous methanol 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 frozen ethanol and dried to obtain 97.1g of GPC solid (purity is 96.40%, total yield is 41.6%). Figure 4shown.

[0038] Example 2

[0039] like Figure 1 As shown, 1000 g of phospholipid mixture was taken, dissolved in 7 L of ethanol and stirred thoroughly, 50 g of sodium ethoxide was added, the temperature was kept at 50 ° C and stirred for 1 hour, and thin layer chromatography was used to detect until most of the PC was degraded, as shown in FIG. Figure 2 As shown, the solution is adjusted to pH=7-8 with glacial acetic acid, and the solvent is evaporated under reduced pressure to obtain intermediate mixture 1. 1L of deionized water and 2L of chloroform are added to the intermediate mixture 1 and mixed evenly, and the layers are separated. The aqueous phase is continuously extracted with 2L of chloroform for 2 times, and the organic phases extracted 3 times are combined. The combined organic phase refined mixture 1 and the aqueous phase refined mixture 2 are obtained respectively.

[0040] The refined mixture 1 was concentrated and allowed to stand at room temperature for 4 hours. After the mixture was separated into upper and lower layers, the upper layer of clear oil was poured out. The lower layer of flocculent matter was dissolved with an appropriate amount of chloroform-ethanol-water (200:100:5) and chromatographed on a silica gel column. The column chromatography was eluted with chloroform-ethanol-water (100:100:5) to obtain 11.2 g of SM product (purity of 98.43%, total yield of 37.7%).

[0041] Add 10g of activated carbon to the refined mixture 2 and stir at room temperature for 60 minutes. After filtering, add anionic and cationic resins to the filtrate, stir at room temperature for 2 hours and filter. After the filtrate is concentrated and evaporated, anhydrous methanol is added and heated to reflux to dissolve. After cooling, GPC seed crystals are added and placed at -20°C for cooling and crystallization. After suction filtration, the crystals are washed with frozen methanol and dried to obtain 91.2g of GPC solid (purity is 95.28%, total yield is 39.1%).

[0042] Example 3

[0043] like Figure 1 As shown, 1000 g of phospholipid mixture was taken, fully stirred with 1.5 L of water and 1.5 L of n-hexane, 200 g of phospholipase B was added, the temperature was kept at 50 ° C and stirred for 8 hours, and thin layer chromatography was used to detect until most of the PC was degraded, as shown in FIG. Figure 2 As shown. Filter to remove the phospholipase, evaporate the solvent under reduced pressure to obtain intermediate mixture 1. Add 1L of deionized water and 3L of n-hexane to the intermediate mixture 1 and mix evenly, and let it stand for stratification. The aqueous phase is continuously extracted with 3L of n-hexane for 2 times, and the organic phases extracted 3 times are combined. The combined organic phase refined mixture 1 and the aqueous phase refined mixture 2 are obtained respectively.

[0044] The refined mixture 1 was concentrated and allowed to stand at room temperature for 4 hours. After the upper and lower layers were separated, the upper layer of clear oil was poured out. 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 of 98.06%, total yield of 28.6%)

[0045] Add 100g of silica gel to the refined mixture 2, heat to 40°C and stir for 60 minutes. After filtering, add anionic and cationic resins 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 frozen methanol and dried to obtain 75.2g of GPC solid (purity is 95.89%, total yield is 32.4%).

[0046] The above disclosure is only the preferred embodiment of the present invention, which cannot be used 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 floccules; 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.

Images