Purification method of L-a-glycerophosphorylcholine
By hydrolyzing L-a-glycerolphosphorylcholine under acidic conditions and treating it with ion exchange resin, the problem of β-GPC removal was successfully solved, and efficient purification of L-a-glycerolphosphorylcholine was achieved, meeting food safety standards.
Patent Information
- Application Number
- CN202411370975.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2025-01-14
AI Technical Summary
The prior art is difficult to effectively isolate and remove β-GPC in L-a-glycerolphosphorylcholine, resulting in its content exceeding national food safety standards and it is difficult to achieve efficient removal of traditional physical methods.
By hydrolyzing L-a-glycerolphosphorylcholine and inorganic acid under acidic conditions, adjusting the temperature and time, β-glycerolphosphorylcholine is preferentially hydrolyzed, and combining ion exchange resin to remove inorganic acid and hydrolyzed impurities, β- Efficient removal of GPC.
Purification of β-GPC content in L-a-glycerolphosphorylcholine is achieved with less than 0.02%, meeting national food safety standards, and improving the purity and yield of the product.
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Figure CN119306757A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to a method for purifying La-glycerophosphorylcholine. Background Art
[0002] The structural formula of La-glycerophosphorylcholine is:
[0003]
[0004] La-Glycerylphosphocholine is a naturally occurring water-soluble phospholipid metabolic intermediate in the human body and an important neurotransmitter. Breast milk also contains a high concentration of La-Glycerylphosphocholine, which is the main source of choline required for infant growth. La-Glycerylphosphocholine can protect mitochondria, has a great protective effect on the brain, and can also promote the secretion of growth hormone.
[0005] Announcement No. 2 of 2024 issued by the Department of Food Safety Standards and Monitoring and Assessment of the National Health Commission approved La-glycerophosphorylcholine (glycerophosphorylcholine) as a new food ingredient, and stipulated that β-GPC (β-glycerophosphorylcholine), which is a positional isomer, shall not be detected, and the detection limit is 0.025g / 100g.
[0006] β-GPC is an isomer of La-glycerophosphorylcholine, with similar structure, close polarity and little difference in solubility, which makes its identification and separation very difficult.
[0007] The prior art reports a scientific method for detecting β-GPC and La-glycerophosphorylcholine using nuclear magnetic resonance phosphorus spectroscopy. Most products on the market were tested according to this method and it was found that the β-GPC content greatly exceeded 0.025%, and some were even as high as 1.43%, which seriously failed to meet national requirements. Since the structures of β-GPC and La-glycerophosphorylcholine are highly similar, the polarity is very large and highly close, and the solubility in organic solvents is limited, it is difficult to remove β-GPC from La-glycerophosphorylcholine by traditional physical methods such as column chromatography and recrystallization. The structural formula of β-GPC is:
[0008] Summary of the invention
[0009] The purpose of this application is to provide a simple and efficient method for removing β-GPC from La-glycerophosphorylcholine by chemical method, so that it meets the requirements of the food safety standards of the National Health Commission.
[0010] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:
[0011] The present application provides a method for purifying La-glycerophosphorylcholine, comprising the following steps: mixing La-glycerophosphorylcholine and an inorganic acid for hydrolysis reaction to obtain La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of less than 0.02%.
[0012]
[0013] La-glycerolphosphorylcholine is hydrolyzed under acidic conditions to generate phosphorylcholine and glycerol. The a-isomer is formed through the intermediate state of primary carbon cations, and the β-isomer is formed through the intermediate state of secondary carbon cations. By regulating the conditions of the hydrolysis, a small amount of β-isomer can be preferentially hydrolyzed under acidic conditions while the a-isomer is retained, thus achieving the purpose of removing β-glycerolphosphorylcholine from La-glycerolphosphorylcholine.
[0014] According to some embodiments of the purification method described herein, the inorganic acid includes one or more of hydrochloric acid, nitric acid and sulfuric acid.
[0015] According to some embodiments of the purification method described in the present application, the temperature of the hydrolysis reaction is 0°C to -10°C; for example, 0°C, -2°C, -5°C, -8°C, -10°C.
[0016] According to some embodiments of the purification method described herein, the temperature of the hydrolysis reaction is -2°C to -8°C.
[0017] According to some embodiments of the purification method described herein, the temperature of the hydrolysis reaction is -3°C to -6°C.
[0018] According to some embodiments of the purification method described in the present application, the hydrolysis reaction time is 10-30 min; for example, 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min, 26 min, 28 min, 30 min.
[0019] According to some embodiments of the purification method described in the present application, the hydrolysis reaction time is 12-20 minutes.
[0020] According to some embodiments of the purification method described herein, the hydrolysis reaction is carried out in a solvent.
[0021] According to some embodiments of the purification method described herein, the solvent includes water and / or ethanol.
[0022] According to some embodiments of the purification method described in the present application, the mass ratio of the La-glycerophosphorylcholine to the solvent is (2-5):10, for example, 2:10, 3:10, 4:10, 5:10.
[0023] According to some embodiments of the purification method described in the present application, the mass ratio of La-glycerophosphorylcholine to inorganic acid is 10:(0.001-0.01), for example, 10:0.001, 10:0.002, 10:0.003, 10:0.005, 10:0.008, 10:0.009, 10:0.01.
[0024] According to some embodiments of the purification method described in the present application, the purification method further comprises subjecting the hydrolysis reaction product to impurity removal treatment.
[0025] According to some embodiments of the purification method described in the present application, the impurity removal treatment includes removing inorganic acid and hydrolysis impurities from the hydrolysis reaction product through an ion exchange resin. The hydrolysis impurities described in the present application include phosphorylcholine and glycerol.
[0026] According to some embodiments of the purification method described in the present application, the ion exchange resin is a mixture of styrene cation exchange resin and styrene anion exchange resin.
[0027] The beneficial effects of the present application are as follows: the method for purifying La-glycerophosphorylcholine described in the present application comprises mixing La-glycerophosphorylcholine and an inorganic acid for hydrolysis, and first hydrolyzing β-glycerophosphorylcholine in La-glycerophosphorylcholine by regulating the temperature and time of the hydrolysis and selecting a suitable type of acid, thereby achieving the purpose of removing β-glycerophosphorylcholine and purifying La-glycerophosphorylcholine. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0029] Figure 1 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerophosphorylcholine prepared in Example 1;
[0030] Figure 2 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerophosphorylcholine prepared in Example 2;
[0031] Figure 3 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerophosphorylcholine prepared in Example 3;
[0032] Figure 4 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerophosphorylcholine prepared in Example 4;
[0033] Figure 5 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerophosphorylcholine prepared in Example 5;
[0034] Figure 6 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerylphosphorylcholine prepared in Comparative Example 1;
[0035] Figure 7 This is the nuclear magnetic resonance phosphorus spectrum of La-glycerolphosphorylcholine prepared in Comparative Example 2. DETAILED DESCRIPTION
[0036] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0037] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.
[0038] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.
[0039] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
[0040] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.
[0041] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.
[0042] The method for calculating the content of β-glycerophosphorylcholine in La-glycerophosphorylcholine in the present application is the nuclear magnetic resonance phosphorus spectrum integration.
[0043] Example 1
[0044] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 1.43% of β-GPC) and 5 ml of 1M hydrochloric acid to 1 liter of pure water, stirring and reacting at 0°C for 20 minutes, removing the hydrochloric acid and hydrolyzing impurities through styrene cation exchange resin and styrene anion exchange resin, and concentrating to obtain La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of 0.02%, and the yield of La-glycerophosphorylcholine is 94%.
[0045] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Example 1 is as follows Figure 1 shown; from Figure 1 It can be seen that the content of β-glycerophosphorylcholine is 0.02%.
[0046] Example 2
[0047] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 0.08% of β-GPC) and 5 ml of 1M hydrochloric acid to 1 liter of pure water, stirring and reacting at 0°C for 20 minutes, removing the hydrochloric acid and hydrolysis impurities therein through styrene cation exchange resin and styrene anion exchange resin, concentrating, and obtaining La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of 0.02%, wherein the yield of La-glycerophosphorylcholine is 95%.
[0048] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Example 2 is as follows Figure 2 shown; from Figure 2 It can be seen that the content of β-glycerophosphorylcholine is 0.02%.
[0049] Example 3
[0050] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 1.43% of β-GPC) and 5 ml of 1M hydrochloric acid to 1 liter of pure water, stirring and reacting at -5°C for 20 minutes, removing the hydrochloric acid and hydrolyzing impurities through styrene cation exchange resin and styrene anion exchange resin, concentrating, and obtaining La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of 0.02%, wherein the yield of La-glycerophosphorylcholine is 95%.
[0051] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Example 3 is as follows Figure 3 shown; from Figure 3 It can be seen that the content of β-glycerophosphorylcholine is 0.02%.
[0052] Example 4
[0053] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 1.43% of β-GPC) and 5 ml of 1M sulfuric acid to 1 liter of pure water, stirring and reacting at 0°C for 12 minutes, removing sulfuric acid and hydrolysis impurities through styrene cation exchange resin and styrene anion exchange resin, concentrating, and obtaining La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of 0.02%, wherein the yield of La-glycerophosphorylcholine is 90%.
[0054] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Example 4 is as follows Figure 4 shown; from Figure 4 It can be seen that the content of β-glycerophosphorylcholine is 0.02%.
[0055] Example 5
[0056] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 1.43% of β-GPC) and 5 ml of 1M phosphoric acid to 1 liter of pure water, stirring and reacting at 0°C for 20 minutes, removing phosphoric acid and hydrolyzed impurities through styrene cation exchange resin and styrene anion exchange resin, and concentrating to obtain La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of about 0.1%, and the yield of La-glycerophosphorylcholine is 82%.
[0057] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Example 5 is as follows: Figure 5 shown; from Figure 5 It can be seen that the content of β-glycerophosphorylcholine is about 0.1%, and new impurities are generated.
[0058] Comparative Example 1
[0059] The only difference between Comparative Example 1 and Example 1 is that the temperature of the hydrolysis reaction in Comparative Example 1 is 10°C.
[0060] The specific steps are:
[0061] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 1.43% of beta-GPC) and 5 ml of 1M hydrochloric acid to 1 liter of pure water, stirring and reacting at 10°C for 20 minutes, removing hydrochloric acid and hydrolysis impurities through styrene cation exchange resin and styrene anion exchange resin, and concentrating to obtain purified La-glycerophosphorylcholine.
[0062] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Comparative Example 1 is as follows: Figure 6 shown; from Figure 6 It can be seen that although the content of β-glycerophosphorylcholine is reduced or even completely disappeared, the yield of La-glycerophosphorylcholine is reduced to 68%.
[0063] Comparative Example 2
[0064] The difference between Comparative Example 2 and Example 1 is that the hydrolysis reaction time of Comparative Example 2 is 30 minutes.
[0065] The specific steps are:
[0066] A method for purifying La-glycerophosphorylcholine comprises the following steps: adding 300 g of La-glycerophosphorylcholine (containing 1.43% of β-GPC) and 5 ml of 1M hydrochloric acid to 1 liter of pure water, stirring and reacting at 0°C for 30 minutes, removing hydrochloric acid and hydrolysis impurities through styrene cation exchange resin and styrene anion exchange resin, and concentrating to obtain purified La-glycerophosphorylcholine.
[0067] The nuclear magnetic resonance phosphorus spectrum of La-glycerol phosphorylcholine prepared in Comparative Example 2 is as follows: Figure 7 shown; from Figure 7 It can be seen that although the content of β-glycerophosphorylcholine is reduced or even completely disappeared, the yield of La-glycerophosphorylcholine is reduced to 70%.
[0068] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims
1. A method for purifying La-glycerophosphorylcholine, characterized in that: The following steps are involved: La-glycerophosphorylcholine and inorganic acid are mixed for hydrolysis reaction to obtain La-glycerophosphorylcholine with a β-glycerophosphorylcholine content of less than 0.02%.
2. The purification method according to claim 1, characterized in that The inorganic acid includes one or more of hydrochloric acid, nitric acid and sulfuric acid.
3. The purification method according to claim 1, characterized in that The temperature of the hydrolysis reaction is 0°C to -10°C; Preferably, the temperature of the hydrolysis reaction is -2°C to -8°C; More preferably, the temperature of the hydrolysis reaction is -3°C to -6°C.
4. The purification method according to claim 1, characterized in that The hydrolysis reaction time is 10-30 min.
5. The purification method according to claim 1, characterized in that The hydrolysis reaction time is 12-20 minutes.
6. The purification method according to claim 1, characterized in that The hydrolysis reaction is carried out in a solvent; Preferably, the solvent comprises water and / or ethanol.
7. The purification method according to claim 6, characterized in that The mass ratio of the La-glycerophosphorylcholine to the solvent is (2-5):10; The mass ratio of the La-glycerophosphorylcholine to the inorganic acid is 10:(0.001-0.01).
8. The purification method according to claim 1, characterized in that The purification method further comprises subjecting the hydrolysis reaction product to impurity removal treatment.
9. The purification method according to claim 8, characterized in that The impurity removal treatment includes passing the hydrolysis reaction product through an ion exchange resin to remove inorganic acid and hydrolysis impurities.
10. The purification method according to claim 9, characterized in that The ion exchange resin includes a mixture of styrene cation exchange resin and styrene anion exchange resin.