A kind of preparation method of phosphatidic acid sodium salt
By using phospholipase D in the acetate buffer to catalyze the conversion of fatty acid chain phosphoesterylcholine to sodium phosphatate salt, and through multiple cooling and crystallization and drying steps, the problem of phosphatidic acid obtained by natural phospholipid hydrolysis is solved, and the preparation and commercial production of high-purity sodium phosphatate salt is achieved.
Patent Information
- Application Number
- CN202411933294.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-25
- Publication Date
- 2025-04-04
AI Technical Summary
The phosphatidic acid obtained by hydrolysis of natural phospholipids is not easy to purify, and the prior art is difficult to effectively solve this problem.
The sodium phosphatate salt of phosphatate choline of fatty acid chain and phospholipase D were generated by the enzyme exchange reaction of fatty acid chain phosphatate and sodium phosphatate salt in acetate buffer, and a high-purity sodium phosphatate salt was obtained through multiple cooling and crystallization and drying steps.
It realizes the preparation of high-purity sodium phosphatate salt, with mild process, easy to obtain raw materials, simple operation, low cost, and suitable for commercial production.
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Figure CN119753044A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of pharmaceutical compound synthesis, and in particular to a method for preparing phosphatidic acid sodium salt. Background Art
[0002] Phosphatidic acid is a simple phospholipid, a basic component of the cell membrane, nuclear membrane, and plastid membrane of plants and animals, and a basic substance of life. In 1927, Chibnall and Channon first isolated phosphatidic acid from cabbage leaves. In 1958, Lowell E. Hokin proved that phosphatidic acid exists in plant and animal tissues. The content of phosphatidic acid in organisms is very small, but it is an intermediate in the synthesis of glycerides and most phospholipids. At the same time, phosphatidic acid is an important second messenger in cells. Phosphatidic acid can be used in food, medicine, and cosmetics.
[0003] The preparation method of phosphatidic acid usually involves chemical synthesis or hydrolysis of natural phospholipids. The chemical synthesis method is relatively complicated and requires a series of organic synthesis steps; the phosphatidic acid obtained by hydrolysis of natural phospholipids is not easy to purify. Summary of the invention
[0004] The object of the present invention is to provide a method for preparing phosphatidic acid sodium salt, so as to solve the technical problem that phosphatidic acid obtained by hydrolysis of natural phospholipids is difficult to purify.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for preparing phosphatidic acid sodium salt, comprising the following steps:
[0007] (1) mixing fatty acid chain phosphatidylcholine, chloroform and phospholipase D acetate buffer, performing enzyme exchange reaction, and obtaining a reaction solution containing phosphatidic acid sodium salt;
[0008] (2) mixing the reaction solution containing sodium phosphatidic acid salt, the sodium chloride aqueous solution and the first alcohol compound, separating the mixture, and subjecting the obtained organic phase to a first cooling and crystallization to obtain a crude product of sodium phosphatidic acid salt;
[0009] (3) The crude sodium phosphatidic acid salt, chloroform and the second alcohol compound are mixed, and the resulting mixture is subjected to a second cooling crystallization, filtration and drying in sequence to obtain the sodium phosphatidic acid salt.
[0010] Further, the fatty acid chain phosphatidylcholine comprises one or more of dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine;
[0011] The usage ratio of the fatty acid chain phosphatidylcholine and chloroform is 100g:1-1.2L.
[0012] Furthermore, the dosage ratio of phospholipase D and fatty acid chain phosphatidylcholine in the phospholipase D acetate buffer is 1-5 mL: 1 g.
[0013] Furthermore, the usage ratio of acetate buffer and fatty acid chain phosphatidylcholine in the acetate buffer of phospholipase D is 1-2 mL: 1 g.
[0014] Furthermore, the acetate buffer of the lipase D comprises acetic acid and sodium acetate, and the pH of the acetate buffer is 5 to 6.5.
[0015] Furthermore, the temperature of the enzyme exchange reaction is 20-60° C., and the time of the enzyme exchange reaction is ≥16 h.
[0016] Furthermore, the first alcohol compound and the second alcohol compound independently contain methanol or ethanol, and the volume ratio of the reaction solution containing phosphatidic acid sodium salt, the sodium chloride aqueous solution and the first alcohol compound is 12.5:1~2:2~4; the mass ratio of the crude phosphatidic acid sodium salt, chloroform and the second alcohol compound is 1:9~10:3~4.
[0017] Furthermore, the temperature of the first cooling and crystallization is 0-5°C, and the crystallization time is ≥2h.
[0018] Furthermore, the temperature of the second cooling and crystallization is 0 to 5° C., and the crystallization time is 2 to 10 hours.
[0019] Furthermore, the drying temperature is 20-60° C., and the drying time is ≥16 h.
[0020] Beneficial effects of the present invention:
[0021] The invention converts fatty acid chain phosphatidylcholine into phosphatidic acid sodium salt under the catalysis of phospholipase D and the action of acetate buffer, and then obtains high-purity phosphatidic acid sodium salt through recrystallization.
[0022] The invention prepares phosphatidic acid sodium salt by an enzymatic method, has mild reaction conditions in the preparation process, readily available raw materials, simple operation, low cost, high product yield, and is suitable for commercial production.
[0023] The phosphatidylglycerol prepared by the invention has high purity and small process impurities. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the process of converting fatty acid phosphatidylcholine into phosphatidic acid sodium salt under the catalysis of enzyme solution, wherein R represents fatty acids with different carbon numbers (palmitic acid, oleic acid, myristic acid or stearic acid);
[0025] Figure 2 is the chromatogram of the final product prepared in Example 4;
[0026] Figure 3 This is the chromatogram of the final product prepared in Example 5. DETAILED DESCRIPTION
[0027] The present invention provides a method for preparing phosphatidic acid sodium salt, comprising the following steps:
[0028] (1) mixing fatty acid chain phosphatidylcholine, chloroform and phospholipase D acetate buffer, performing enzyme exchange reaction, and obtaining a reaction solution containing phosphatidic acid sodium salt;
[0029] (2) mixing the reaction solution containing sodium phosphatidic acid salt, the sodium chloride aqueous solution and the first alcohol compound, separating the mixture, and subjecting the obtained organic phase to a first cooling and crystallization to obtain a crude product of sodium phosphatidic acid salt;
[0030] (3) The crude sodium phosphatidic acid salt, chloroform and the second alcohol compound are mixed, and the resulting mixture is subjected to a second cooling crystallization, filtration and drying in sequence to obtain the sodium phosphatidic acid salt.
[0031] In the present invention, the fatty acid chain phosphatidylcholine comprises one or more of dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC), preferably dipalmitoyl phosphatidylcholine (DPPC) and / or dioleoyl phosphatidylcholine (DOPC).
[0032] In the present invention, the usage ratio of the fatty acid chain phosphatidylcholine and chloroform is 100g:1-1.2L, preferably 100g:1L.
[0033] In the present invention, the usage ratio of phospholipase D and fatty acid chain phosphatidylcholine in the acetate buffer of phospholipase D is 1-5 mL:1 g, preferably 2-4 mL:1 g, and more preferably 2.2-3 mL:1 g.
[0034] In the present invention, the usage ratio of acetate buffer and fatty acid chain phosphatidylcholine in the acetate buffer of phospholipase D is 1-2 mL:1 g, preferably 1.2-1.8 mL:1 g, further preferably 1.4-1.6 mL:1 g, and more preferably 1.5 mL:1 g.
[0035] The present invention preferably dissolves the fatty acid chain phosphatidylcholine in chloroform first, and then adds phospholipase D, glycerol and acetate buffer. In the present invention, phospholipase D is used as an enzyme catalyst to promote the conversion of fatty acid chain phosphatidylcholine into phosphatidic acid sodium salt; the acetate buffer solution provides a suitable pH for phospholipase D and provides a sodium source and a water source required for the generation of phosphatidic acid.
[0036] In the present invention, the acetate buffer of the lipase D comprises acetic acid and sodium acetate, and the pH of the acetate buffer is 5 to 6.5, preferably 5.5.
[0037] In the present invention, the temperature of the enzyme exchange reaction is 20-60°C, preferably 30-50°C, and more preferably 35-45°C; the time of the enzyme exchange reaction is preferably 16-30h, more preferably 20-28h, and more preferably 24h.
[0038] In the present invention, after the transesterification reaction is completed, the present invention preferably does not require post-treatment, and the obtained reaction solution containing phosphatidylcholine sodium salt is subjected to subsequent steps.
[0039] In the present invention, after obtaining a reaction solution containing phosphatidylcholine sodium salt, the reaction solution is mixed with a sodium chloride aqueous solution and a first alcohol compound, and then separated, and the obtained organic phase is subjected to a first cooling and crystallization to obtain a crude product of phosphatidic acid sodium salt.
[0040] In the present invention, the first alcohol compound and the second alcohol compound independently contain methanol or ethanol, preferably methanol; the volume ratio of the reaction solution containing phosphatidic acid sodium salt, the sodium chloride aqueous solution and the first alcohol compound is 12.5:1-2:2-4, preferably 12.5:1.2-1.8:2.5-3.5, and more preferably 12.5:1.5:3.0; the mass ratio of the crude phosphatidic acid sodium salt, chloroform and the second alcohol compound is 1:9-10:3-4, preferably 1:9.2-9.8:3.2-3.8, and more preferably 1:9.5:3.5. The present invention uses the sodium chloride aqueous solution and the first alcohol compound to remove excess glycerol. The addition of the first alcohol compound in the present invention helps to separate.
[0041] In the present invention, the mass concentration of the sodium chloride aqueous solution is 4 to 10%, preferably 4 to 8%, and more preferably 5 to 6%.
[0042] In the present invention, the reaction solution is mixed with the sodium chloride aqueous solution and the first alcohol compound, preferably by adding the first part of the sodium chloride aqueous solution and the first alcohol compound to the reaction solution, washing once, separating the liquid at rest, adding the second part of the sodium chloride aqueous solution and the first alcohol compound to the obtained lower organic phase, and stirring and washing twice. In the present invention, the amount ratio of the first part of the sodium chloride aqueous solution and the first alcohol compound to the reaction solution is added according to the above-mentioned ratio range, and the amount ratio of the second part of the sodium chloride aqueous solution and the first alcohol compound to the reaction solution is added according to the above-mentioned ratio range.
[0043] In the present invention, there is no special limitation on the amount of the reagent used in the first cooling and crystallization, as long as sufficient crystallization is ensured; the solid obtained by cooling and crystallization is the crude product of sodium phosphatidic acid salt, and the purpose of this process is to remove the remaining fatty acid chain phosphatidylcholine.
[0044] In the present invention, after obtaining the crude sodium phosphatidic acid salt, the crude sodium phosphatidic acid salt is mixed with chloroform and a second alcohol compound, and the obtained mixture is subjected to a second cooling crystallization, filtration and drying in sequence to obtain the sodium phosphatidic acid salt. The purpose of this process is to further remove the remaining fatty acid chain phosphatidylcholine.
[0045] In the present invention, the purpose of the second cooling and crystallization is to further remove excess glycerol and the remaining phosphatidylcholine in the reaction. The present invention preferably mixes the crude phosphatidylcholine glycerol salt with chloroform and the second alcohol compound, heats to 50-60°C to completely dissolve the raw materials, stirs at room temperature for 0.5h, and then performs the second cooling and crystallization; the present invention first heats up to dissolve and then cools down to crystallize to help remove impurities.
[0046] In the present invention, the temperature of the first cooling and crystallization is 0 to 5°C, preferably 1 to 4°C, and more preferably 2 to 3°C; the crystallization time is preferably 2 to 10 hours, and more preferably 4 to 6 hours.
[0047] In the present invention, the second cooling crystallization temperature is 0 to 5°C, preferably 1 to 4°C, and more preferably 2 to 3°C; the crystallization time is 2 to 10 hours, preferably 4 to 8 hours, and more preferably 5 to 6 hours.
[0048] In the present invention, the drying temperature is 20 to 60° C., preferably 30 to 50° C., and more preferably 40° C.; the drying time is preferably 16 to 30 h, more preferably 20 to 25 h, and more preferably 24 h.
[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0050] In the following examples, the chemical names corresponding to the abbreviations are:
[0051] DPPC dipalmitoylphosphatidylcholine; DPPG dipalmitoylphosphatidylglycerol;
[0052] DOPC dioleylphosphatidylcholine; DOPG dioleylphosphatidylglycerol;
[0053] DMPC dimyristoylphosphatidylcholine; DMPG dimyristoylphosphatidylglycerol;
[0054] DSPC distearate phosphatidylcholine; DSPG distearate phosphatidylcholine.
[0055] Example 1
[0056] Synthesis of DPPA sodium salt using DPPC as raw material:
[0057] 100g DPPC and 1L chloroform were added to the reactor and stirred evenly. At the same time, 220mL of phospholipase D enzyme solution and 150mL of acetate buffer (pH=5.5) were added. The stirring temperature was 45°C and the reaction was carried out for 19h. 200mL of 4% NaCl aqueous solution and 400mL of methanol were added to the obtained reaction solution (2500mL), stirred and washed once, and the liquid was separated at rest. The lower organic phase was taken, 400mL of 4% NaCl aqueous solution and 600mL of methanol were added, stirred and washed twice, and the liquid was separated at rest. 170mL of methanol was added to the organic phase and the temperature was lowered to 5°C. After crystallization for 2h, it was filtered to obtain a crude product. 720g of chloroform and 240g of methanol were added to the crude product (80g), the temperature was raised to 50°C until it was completely dissolved, stirred at room temperature for 0.5h, cooled to 5°C, stirred and crystallized for 2h, filtered, and dried in vacuo at room temperature to obtain 65g of white solid with a purity of 98.91%.
[0058] Example 2
[0059] Synthesis of DMPA sodium salt using DMPC as raw material:
[0060] 50g DMPC and 500ml chloroform were added to the reactor and stirred evenly. At the same time, 110mL of phospholipase D enzyme solution and 75mL of acetate buffer (pH=5.5) were added. The stirring temperature was 45°C and the reaction was carried out for 19h. 100mL of 4% NaCl aqueous solution and 200mL of methanol were added to the obtained reaction solution (1250mL), stirred and washed once, and the liquid was separated at rest. The lower organic phase was taken, and 200mL of 4% NaCl aqueous solution and 300mL of methanol were added and stirred and washed twice. The liquid was separated at rest. 85mL of methanol was added to the lower organic phase and the temperature was lowered to 5°C. After crystallization for 2h, it was filtered to obtain a crude product. 360g chloroform and 120g of methanol were added to the crude product (40g), and the temperature was raised to 50°C until it was completely dissolved. It was stirred at room temperature for 0.5h, cooled to 5°C, stirred and crystallized for 2h, filtered, and dried in vacuo at room temperature to obtain 31g of white solid with a purity of 98.2%.
[0061] Example 3
[0062] Synthesis of DOPA sodium salt using DOPC as raw material:
[0063] DOPC (200 g) and 2 L of chloroform were added to a reactor and stirred evenly. At the same time, 440 mL of phospholipase D enzyme solution and 300 mL of acetate buffer (pH = 5.5) were added, the temperature was raised to 45°C and stirred for reaction for 19 h. 400 mL of 4% NaCl and 800 mL of methanol were added to the obtained reaction solution, stirred and washed once, and the liquid was separated at rest; 4% NaCl (800 mL) and methanol (1.2 L) were added to the obtained organic phase, stirred and washed twice, and then the liquid was separated at rest; 3.4 L of methanol was added to the obtained organic phase, the temperature was lowered to 5°C, crystallized for 2 h, and then filtered to obtain a crude product; chloroform (1480 g) and methanol (493 g) were added to the crude product (157 g), the temperature was raised to 50°C until it was completely dissolved, stirred at room temperature for 0.5, then cooled to 5°C and stirred for crystallization for 2 h, filtered, and dried in vacuo at room temperature to obtain 140 g of a white solid with a purity of 99.69%.
[0064] Example 4
[0065] Synthesis of DSPA sodium salt using DSPC as raw material:
[0066] 300g DSPC and 3L chloroform were added to the reactor and stirred evenly. At the same time, 660mL of phospholipase D enzyme solution and 300mL of acetate buffer were added, the stirring temperature was 45°C, and the reaction was carried out for 19h; 600mL of 4% NaCl aqueous solution and 1200mL of ethanol were added to the obtained reaction solution (7500mL), stirred and washed once, the liquid was separated at rest, the lower organic phase was taken, 1200mL of 4% NaCl aqueous solution and 1800mL of ethanol were added, stirred and washed twice, the liquid was separated at rest, the lower organic phase was taken and 510mL of ethanol was added and the temperature was lowered to 5°C, and the crude product was filtered after crystallization for 2h to obtain a crude product; 2160g chloroform and 720g ethanol were added to the crude product (280g), the temperature was raised to 50°C until it was completely dissolved, stirred at room temperature for 0.5h, cooled to 5°C, stirred and crystallized for 2h, filtered, and dried in vacuo at room temperature to obtain 194g of white solid with a purity of 99.92%.
[0067] Example 5
[0068] Synthesis of DPPA sodium salt using DPPC as raw material:
[0069] DPPC (300.0 g), phospholipase D enzyme solution (660 mL), acetate buffer (300 mL) and chloroform (3.0 L) were added to a 10 L three-necked flask, stirred at 45 ° C, and reacted for 19 h; 4% NaCl (600 mL) and 1200 mL of methanol were added to the resulting reaction solution (7500 mL), washed once, and the liquid was separated at rest. The lower organic phase was taken, and then washed twice with 4% NaCl (1200 mL) and 1800 mL of methanol, and the liquid was separated at rest. 510 mL of methanol was added to the lower organic phase and cooled to 5 ° C. After crystallization for 2 h, it was filtered to obtain a crude product; 2160 g of chloroform and 720 g of methanol were added to the crude product (270 g), and the temperature was raised to 50 ° C until it was completely dissolved, stirred at room temperature for 0.5 h, cooled to 5 ° C and stirred for 2 h, filtered, and dried in vacuo at room temperature to obtain 200 g of a white solid with a purity of 99.7%.
[0070] The final products prepared in Example 4 and Example 5 were subjected to chromatographic detection, and the detection conditions were as follows: octadecylsilane bonded silica gel was used as a filler (Waters, XBridge C18, 150 mm*4.6 mm, 3.5 μm or other equivalent columns); methanol / acetic acid buffer = 8:1 (volume ratio) was used as mobile phase A; isopropanol / methanol / acetic acid buffer = 15:10:1 (volume ratio) was used as mobile phase B; the column temperature was 40° C., the flow rate was 1 mL per minute, and the injection volume was 5 μL; gradient elution was performed according to Table 1; the detector was an ELSD detector;
[0071] Table 1 Mobile phase gradient program for chromatographic detection
[0072] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.0 90 10 15.0 20 80 20.0 20 80 20.1 80 20 30.0 80 20
[0073] From the above examples, it can be seen that the present invention provides a method for preparing phosphatidic acid sodium salt. Figures 2-3 .Depend on Figures 2-3 It can be seen that the peak position is appropriate, the peak width is narrow, and the peak shape is good. The method described in the present invention can obtain high-purity sodium phosphatidic acid salt.
[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for preparing phosphatidic acid sodium salt, characterized in that: The following steps are involved: (1) mixing fatty acid chain phosphatidylcholine, chloroform and phospholipase D acetate buffer, performing enzyme exchange reaction, and obtaining a reaction solution containing phosphatidic acid sodium salt; (2) mixing the reaction solution containing sodium phosphatidic acid salt, the sodium chloride aqueous solution and the first alcohol compound, separating the mixture, and subjecting the obtained organic phase to a first cooling and crystallization to obtain a crude product of sodium phosphatidic acid salt; (3) The crude sodium phosphatidic acid salt, chloroform and the second alcohol compound are mixed, and the resulting mixture is subjected to a second cooling crystallization, filtration and drying in sequence to obtain the sodium phosphatidic acid salt.
2. The method for preparing phosphatidic acid sodium salt according to claim 1, characterized in that: The fatty acid chain phosphatidylcholine comprises one or more of dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine and distearic phosphatidylcholine; The usage ratio of the fatty acid chain phosphatidylcholine and chloroform is 100g:1-1.2L.
3. The method for preparing phosphatidic acid sodium salt according to claim 1 or 2, characterized in that: The usage ratio of phospholipase D and fatty acid chain phosphatidylcholine in the acetic acid buffer of phospholipase D is 1-5 mL:1 g.
4. The method for preparing phosphatidic acid sodium salt according to claim 3, characterized in that: The usage ratio of the acetate buffer and the fatty acid chain phosphatidylcholine in the acetate buffer of the phospholipase D is 1-2 mL: 1 g.
5. The method for preparing phosphatidic acid sodium salt according to claim 4, characterized in that: The acetate buffer of the lipase D comprises acetic acid and sodium acetate, and the pH of the acetate buffer is 5-6.
5.
6. The method for preparing the sodium phosphatidic acid salt according to claim 1, 2 or 5, characterized in that: The temperature of the enzyme exchange reaction is 20-60° C., and the time of the enzyme exchange reaction is ≥16 h.
7. The method for preparing phosphatidic acid sodium salt according to claim 6, characterized in that: The first alcohol compound and the second alcohol compound independently contain methanol or ethanol, and the volume ratio of the reaction solution containing phosphatidic acid sodium salt, the sodium chloride aqueous solution and the first alcohol compound is 12.5:1-2:2-4; the mass ratio of the crude phosphatidic acid sodium salt, chloroform and the second alcohol compound is 1:9-10:3-4.
8. The method for preparing phosphatidic acid sodium salt according to claim 1, 2 or 7, characterized in that: The temperature of the first cooling and crystallization is 0-5°C, and the crystallization time is ≥2h.
9. The method for preparing phosphatidic acid sodium salt according to claim 8, characterized in that: The temperature of the second cooling and crystallization is 0-5°C, and the crystallization time is 2-10 hours.
10. The method for preparing phosphatidic acid sodium salt according to claim 1 or 9, characterized in that: The drying temperature is 20-60° C., and the drying time is ≥16 h.