A method for preparing glycerophosphatidylcholine by alkaline method

Abstract

The present invention relates to a method for preparing glycerophosphatidylcholine by an alkaline method. The method adopts polyol and sodium ethoxide as a composite catalyst to catalyze alcohol-soluble phospholipid alcoholysis in an ethanol-n-hexane system to prepare glycerophosphatidylcholine. The method is simple, efficient, and requires less catalyst, and can significantly improve the generation rate and yield of glycerophosphatidylcholine, and has good economic benefits and industrial application prospects.

Description

Technical Field

[0001] The present invention relates to a method for preparing glycerol phosphatidylcholine by alkaline method, in particular to a method for preparing glycerol phosphatidylcholine by catalyzing alcohol-soluble phospholipid alcoholysis in an ethanol-n-hexane system using polyol and sodium ethoxide as a composite catalyst, belonging to the technical field of deep processing of oils and fats and production of health food. Background Art

[0002] Glycerylphosphatidylcholine (L-alphaglycerylphosphorylcholine, GPC for short) is a derivative of phosphatidylcholine (PC for short) molecules after the two fatty acyl groups are completely removed, and can be delivered to the brain through the blood-brain barrier. GPC can not only prevent cognitive decline caused by aging and improve memory, but also activate central functions by promoting the secretion of growth hormone and increasing the release of individual hormones, thus having a certain effect in treating Alzheimer's disease and dwarfism. GPC has become the most promising cognitive enhancer and candidate food and pharmaceutical supplement for the treatment of cognitive mental illness. However, natural sources of glycerylphosphatidylcholine are relatively rare, so it is of great significance to study and develop a method for preparing high-purity glycerylphosphatidylcholine.

[0003] The semi-synthetic methods for preparing GPC by deacylating natural PC mainly include chemical catalysis and enzyme catalysis.

[0004] In the chemical catalytic method, the catalysts of the reaction mainly include sodium methoxide, lithium methoxide, tetrabutylammonium hydroxide, choline hydroxide, isopropylamine, tert-butylamine, etc. The reaction of preparing GPC by these alkaline catalysts is generally carried out in the methanol phase. The reported chemical methods for preparing GPC have the disadvantages of product and catalyst complex, large consumption of organic reagents and high toxicity, expensive catalysts, difficulty in subsequent purification, and low efficiency of GPC production. In addition, in the reaction system with sodium ethoxide as catalyst and ethanol as medium, Zhang Xishuang et al. (Study on the Preparation of L-α-Glycerylphosphatidylcholine (L-α-GPC) by Inorganic Base Catalyzed Natural Phospholipid Transesterification [D]. Northwest University, 2014.) found that the yield of GPC was 94.9% only when the phospholipid concentration was 10.5mmol / L (about 8mg / mL), the catalyst dosage was 0.6mg / mL, that is, the mass ratio of sodium ethoxide to phospholipid was 7.5:100. However, the phospholipid concentration of the reaction was 10.5mmol / L (about 8mg / mL), the catalyst dosage was 0.6mg / mL, and the mass ratio of sodium ethoxide to phospholipid was 7.5:100. The concentration is extremely low (far below 50 mg / mL) and a large amount of sodium ethoxide is consumed, resulting in low GPC preparation efficiency, high cost, and difficulty in subsequent purification and desalting. Therefore, this method has no industrial application value; Kang Shizong et al. (Study on the process of preparing glycerol phosphatidylcholine from egg yolk powder [D]. Henan University of Technology, 2018.) found that in the ethanol-sodium ethoxide system, when the reaction conditions are that the amount of sodium ethoxide added accounts for 5% of the phospholipid mass and the material-liquid ratio is 1:8 (phospholipid concentration is 125 mg / mL), only a trace amount of GPC is produced.

[0005] Compared with the chemical catalytic method, the semi-synthetic method of preparing GPC by enzyme catalysis has the advantages of mild reaction conditions, low consumption of organic reagents, and environmental protection. However, due to the solubility of phospholipids and the high price of enzyme preparations, it has the disadvantages of low preparation efficiency and high production cost, which limits its industrial production.

[0006] At present, there is no report on using polyol and sodium ethoxide as composite catalyst to catalyze the alcoholysis of alcohol-soluble phospholipids in ethanol-n-hexane system to prepare glycerol phosphatidylcholine. Summary of the invention

[0007] In view of the scientific problem that the preparation efficiency of GPC is low, the toxicity of the reagents used is high, and ultimately affects the production cost, functionality and food safety of the product, the purpose of the present invention is to provide a method for preparing glycerophosphatidylcholine by an alkaline method to overcome the above-mentioned shortcomings, and the method can simplify the subsequent separation and purification steps of GPC, and the product can be used as a functional food raw material or a dietary supplement raw material.

[0008] The technical solution of the present invention is as follows:

[0009] A method for preparing glycerophosphatidylcholine by alkaline method, wherein the specific catalytic reaction comprises the following steps:

[0010] Step 1: Mix and dissolve the main catalyst sodium ethoxide and the co-catalyst polyol to prepare a composite catalyst;

[0011] Step 2: After the alcohol-soluble phospholipid and ethanol are stirred and dissolved, the composite catalyst and 5% ethanol volume of n-hexane are added, and the first stage is stirred and reacted for 0.5 to 1.0 h at a temperature of 40 to 55° C., and then 20% of the initial reaction volume of n-hexane is added, and the second stage is stirred and reacted for 1.0 to 2.0 h;

[0012] Step 3: After the reaction mixture obtained in step 2 is cooled to room temperature, the lower layer of ethanol solution A containing glycerol phosphatidylcholine is obtained by separation, the solution is concentrated to 25% of its volume by negative pressure desolventizing, and then an equal volume of n-hexane is added thereto for extraction twice, and finally a lower layer of ethanol solution B is obtained;

[0013] Step 4: Place the ethanol solution B in step 3 in a pressure reactor, then add deionized water with a molar amount of 1.2 times that of sodium ethoxide, introduce CO2, stir and react to obtain a reaction solution without sodium ethoxide, and then filter the solution, remove ethanol from the filtrate under negative pressure to obtain a glycerol phosphatidylcholine product.

[0014] Furthermore, in the composite catalyst, the co-catalyst polyol is one of 1,2-propylene glycol and glycerol, and the mass ratio of the co-catalyst polyol to sodium ethoxide is 2:1;

[0015] Furthermore, the mass ratio of the composite catalyst to phospholipid is 0.2:100 to 1.0:100; and the concentration of phospholipid in ethanol is 0.05 to 0.15 g / mL.

[0016] Furthermore, the alcohol-soluble phospholipid refers to a phospholipid with a phosphatidylcholine purity of more than 50%.

[0017] Beneficial Effects of the Invention

[0018] Compared with the previously reported methods, the invention is innovative in preparing glycerophosphatidylcholine in that:

[0019] (1) The catalyst raw materials used in the present invention are easy to obtain, and the 1,2-propylene glycol or glycerol therein can be used as a food additive; in step 4 of the present invention, sodium ethoxide is first hydrolyzed in a micro-water environment to generate NaOH, and further reacts with CO2 to generate NaHCO3 that is slightly soluble in ethanol, and finally most of the sodium salt product can be removed by filtration, thereby reducing the burden of GPC purification; the reaction solvent n-hexane is a common extractant in the edible oil industry, and after the liquid separation operation in step 3 of the present invention and the negative pressure desolventizing operation in step 4, its residue is extremely limited. In summary, the GPC product prepared by the present invention can ensure its food safety;

[0020] (2) The present invention uses a composite catalyst to catalyze the alcohol hydrolysis of alcohol-soluble phospholipids to obtain not only the target product glycerol phosphatidylcholine, but also a polyunsaturated fatty acid ethyl ester byproduct. The fatty acid ethyl ester byproduct is rich in mono- and poly-saturated fatty acids (taking the soybean phosphatidylcholine alcoholysis product as an example, the total content of oleic acid, linoleic acid and linolenic acid can be as high as 80% or more), has the advantages of low boiling point, easy absorption by the human body, no toxic side effects, and easy separation and purification, and has been widely used in the fields of food and medicine. Therefore, the co-production of the main and by-products of the present invention can greatly improve the benefits of the enterprise;

[0021] (3) In the dual medium of ethanol and n-hexane, the composite catalyst of the present invention can efficiently catalyze the alcoholysis of alcohol-soluble phospholipids to obtain glycerol phosphatidylcholine, which is due to the following reaction mechanism:

[0022] The reaction has serious adverse effects from trace water in the reaction medium ethanol, trace water and fatty acid in phospholipid. If there is no participation of the cocatalyst polyol, sodium ethoxide is easily hydrolyzed to generate NaOH and is consumed, and then NaOH and the fatty acid from the raw material phospholipid generate sodium fatty acid, and part of NaOH can also catalyze the hydrolysis of phospholipids to generate sodium fatty acid, and this sodium fatty acid can be wrapped in the periphery of phospholipid, fatty acid ethyl ester, lysophospholipid (phospholipid removes a fatty acyl product) as a surfactant, forming an alcohol-oil emulsification system similar to water-in-oil, so that the remaining catalyst sodium ethoxide is difficult to attack the phospholipid molecules and continue the alcoholysis reaction. Only under the condition that the reaction substrate phospholipid concentration is extremely low and the catalyst level is higher, the sodium ethoxide-ethanol system can be smoothly prepared GPC, as described in the present invention specification Zhang Xishuang etc. research report. But, under the participation of the cocatalyst polyol, the hydroxyl of polyol can form hydrogen bond with trace water and "lock" water molecule, and it can generate ester compounds with trace free fatty acids, thereby protecting the main catalyst sodium ethoxide.

[0023] In addition, the present invention adds normal hexane in two stages, and the purpose of preparing glycerophosphatidylcholine in ethanol-normal hexane dual medium is: in one stage, the normal hexane with respect to 5% of ethanol volume extracts the fatty acid ethyl ester generated by the reaction, and promotes the chemical reaction to proceed in the forward direction to generate the product mainly containing lysophosphatidylcholine, and the normal hexane of a small amount of this part has the function of reducing surface tension, thereby having a certain demulsification function, and is conducive to the alcoholysis reaction of sodium ethoxide attacking the phospholipid molecules. The second stage continues to add normal hexane with respect to 20% of ethanol volume, plays the effect of diluting the fatty acid ethyl ester concentration and extracting the newly generated fatty acid ethyl ester, and continues to promote the reaction to proceed in the forward direction, until the two fatty acyl groups of phosphatidylcholine are completely removed to generate GPC. If the one-step method adds required normal hexane, directly preparing GPC in ethanol-normal hexane dual medium, the dissolution of phosphatidylcholine in the normal hexane medium can be increased, and sodium ethoxide can only be retained in the ethanol phase to generate catalysis, and such interfacial reaction rate is far less than the rate of the homogeneous reaction of sodium ethoxide attacking the phospholipid molecules in the ethanol phase, which will greatly prolong the reaction time, and is unfavorable for production practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (A) is the HPLC spectrum of the standard glycerophosphatidylcholine; Figure 1 (B) is an HPLC spectrum of the glycerophosphatidylcholine product obtained in Example 4 of the present invention obtained according to the detection method described in the content of the present invention; Figure 1 (C) is the HPLC spectrum of the glycerophosphatidylcholine product obtained in Example 4 of the present invention after further purification according to the detection method described in the content of the present invention. Figure 1 It can be seen that the yield of the glycerophosphatidylcholine obtained in this example is 95.6%, and after further purification, a glycerophosphatidylcholine product with a purity of 99.5% can be obtained. DETAILED DESCRIPTION

[0025] For a better understanding of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope limited by the embodiments.

[0026] The reagents ethanol, sodium ethoxide, n-hexane, 1,2-propylene glycol and glycerol used in the following examples of the present invention are all analytically pure, and the purity of phosphatidylcholine in the alcohol-soluble phospholipids is 51.6%.

[0027] Example 1

[0028] 5 g of alcohol-soluble phospholipid, 0.025 g of composite catalyst (prepared with 1,2-propylene glycol according to the method of the present invention), 100 mL of anhydrous ethanol and 5 mL of n-hexane were added to a 200 mL reactor connected to a reflux cooling device, and a first-stage stirring reaction was carried out at a temperature of 40°C for 0.5 h; then 20 mL of n-hexane was added, and a second-stage stirring reaction was continued at the same temperature for 1 h; further, a glycerol phosphatidylcholine product was obtained according to the implementation method of step 3 and step 4 of the present invention; according to the following HPLC detection method, it was found that the yield of glycerol phosphatidylcholine of the product was 79.6%, and the purity of the product after further purification was 99.4%; at the same time, the n-hexane phases in step 3 were combined, demulsified and washed with 5% NaCl aqueous solution, and then the n-hexane phase was obtained by liquid separation, and finally the fatty acid ethyl ester by-product was obtained after negative pressure desolventizing, and the yield was 76.9% and the purity was 98.2% according to the following GC detection method.

[0029] HPLC detection of glycerol phosphatidylcholine product: Take a sample (about 1 mg) and dissolve it in methanol (1 mL). After filtering through a 0.22 μm organic filter membrane, detect it by liquid chromatography. The specific conditions of liquid chromatography are as follows: chromatographic column: Hypersil GOLD Silica column (4.6 mm × 250 mm, 5 μm); column temperature 35 ° C; injection volume: 50 μL. Mobile phase: methanol (phase A), methanol / water (V / V) = 8 / 1 (phase B); gradient elution; flow rate 1.0 L / min; ELSD evaporation and atomization temperatures are 40 and 65 ° C, respectively, nitrogen flow rate is 1.6 L / min, and nitrogen pressure is 35 PSI. Identification is performed by comparing the retention time of the GPC standard, and the relative content of GPC in each sample is quantitatively detected by the peak area normalization method. The content of GPC in the product is quantitatively determined by the external standard method of the GPC standard and its yield (the ratio of the number of moles of the obtained GPC to the starting raw material PC in the reaction) is calculated. The samples under each condition were processed and analyzed three times, and the average value was taken.

[0030] GC detection of fatty acid ethyl ester products: SPTM-2560 capillary column (100m×0.25mm×0.2μm); injection volume 1μL; split ratio 20:1; injection port temperature 280℃; detector temperature 250℃; FID detector. The retention time of the mixed fatty acid ethyl ester standard was compared for identification, and the internal standard method with ethyl pentadecanoate as the standard was used to quantitatively analyze the fatty acid content in the oil sample. The samples under each condition were processed and analyzed three times, and the average value was taken.

[0031] Example 2

[0032] 15 g of alcohol-soluble phospholipid, 0.3 g of composite catalyst (prepared with 1,2-propylene glycol according to the method of the present invention), 100 mL of anhydrous ethanol and 5 mL of n-hexane were added to a 200 mL reactor connected to a reflux cooling device, and a first-stage stirring reaction was carried out at a temperature of 55° C. for 1.0 h; then 20 mL of n-hexane was added, and a second-stage stirring reaction was continued for 2.0 h under the same temperature condition; further, a glycerol phosphatidylcholine product was obtained according to the implementation method of step 3 and step 4 of the present invention; according to the HPLC detection method in Example 1, it was found that the yield of glycerol phosphatidylcholine of the product was 89.8%, and the purity of the product after further purification was 99.5%; at the same time, the n-hexane phases in step 3 were combined, demulsified and washed with a 5% NaCl aqueous solution, and then the n-hexane phase was obtained by liquid separation, and finally the fatty acid ethyl ester by-product was obtained after negative pressure desolventizing, and the yield was 87.9% and the purity was 97.8% according to the GC detection method in Example 1.

[0033] Example 3

[0034] 12g of alcohol-soluble phospholipid, 0.2g of composite catalyst (prepared with glycerol according to the method of the present invention), 100mL of anhydrous ethanol and 5mL of n-hexane were added to a 200mL reactor connected to a reflux cooling device, and a first-stage stirring reaction was carried out at a temperature of 50°C for 0.8h; then 20mL of n-hexane was added, and a second-stage stirring reaction was continued for 1.5h under the same temperature condition; further, a glycerophosphatidylcholine product was obtained according to the implementation method of step 3 and step 4 of the present invention; the HPLC detection method in Example 1 found that the yield of glycerophosphatidylcholine of the product was 84.8%, and the purity of the product after further purification was 99.2%; at the same time, the n-hexane phases in step 3 were combined, demulsified and washed with 5% NaCl aqueous solution, and then the n-hexane phase was obtained by liquid separation, and finally the fatty acid ethyl ester by-product was obtained after negative pressure desolventizing, and the yield was 81.6% and the purity was 97.1% according to the GC detection method in Example 1.

[0035] Example 4

[0036] 10 g of alcohol-soluble phospholipid, 0.15 g of composite catalyst (prepared with 1,2-propylene glycol according to the method of the present invention), 100 mL of anhydrous ethanol and 5 mL of n-hexane were added to a 200 mL reactor connected to a reflux cooling device, and a first-stage stirring reaction was carried out at a temperature of 50° C. for 1.0 h; then 20 mL of n-hexane was added, and a second-stage stirring reaction was continued at the same temperature for 1.5 h; further, a glycerol phosphatidylcholine product was obtained according to the implementation method of step 3 and step 4 of the present invention; according to the HPLC detection method in Example 1, it was found that the yield of glycerol phosphatidylcholine of the product was 95.6%, and the purity of the product after further purification was 99.5%; at the same time, the n-hexane phases in step 3 were combined, demulsified and washed with 5% NaCl aqueous solution, and then the n-hexane phase was obtained by liquid separation, and finally the fatty acid ethyl ester by-product was obtained after negative pressure desolventizing, and the yield was 93.6% and the purity was 98.2% according to the GC detection method in Example 1.

[0037] In control example 1, no co-catalyst and n-hexane solvent were added, 0.5 g of sodium ethoxide was added, and the reaction was directly carried out for 2.5 h. Other conditions were the same as those in Example 4 for alcoholysis of alcohol-soluble phospholipids. Only trace amounts of glycerophosphatidylcholine were detected in the product of control example 1.

[0038] In control example 2, no promoter was added, 0.5 g of sodium ethoxide was added, and the rest was the same as in example 4. The yield of glycerophosphatidylcholine detected in the product of control example 2 was 45.6%, and the purity of glycerophosphatidylcholine after further purification was 90.5%; the yield of fatty acid ethyl ester byproduct was 41.1%, and the purity was 68.3%.

[0039] In control example 3, no n-hexane solvent was added, and the reaction was directly carried out for 2.5 hours. The other conditions were the same as those in Example 4 for alcoholysis of alcohol-soluble phospholipids. After the reaction was completed, the alcoholysis solution was extracted 3 times with 25 mL of n-hexane, and the rest was the same as in Example 4. The yield of glycerophosphatidylcholine detected in the product of control example 3 was 65.6%. After further purification, the purity of glycerophosphatidylcholine was 99.1%; the yield of fatty acid ethyl ester byproduct was 62.5%, and the purity was 70.1%.

[0040] In reference example 4, 10 g of alcohol-soluble phospholipid, 0.15 g of composite catalyst (prepared with 1,2-propylene glycol according to the method of the present invention), 100 mL of anhydrous ethanol and 25 mL of n-hexane were added to a 200 mL reactor connected to a reflux cooling device, and stirred at 50° C. for 2.5 h. The other conditions were the same as those in Example 4 for alcoholysis of alcohol-soluble phospholipid. The yield of the product glycerylphosphatidylcholine obtained in reference example 4 was 80.7%, and the purity of the product after further purification was 99.2%; the yield of the fatty acid ethyl ester byproduct was 78.2%, and the purity was 84.1%.

[0041] Comparison of the results obtained in Control Examples 1, 2, 3 and 4 with those in Example 4 shows that the present invention uses polyols and sodium ethoxide as a composite catalyst to catalyze the alcoholysis of alcohol-soluble phospholipids in an ethanol-n-hexane system to greatly improve the preparation efficiency of glycerol phosphatidylcholine, which also shows that the method is more suitable for industrial production needs.

[0042] The above specific implementation manner does not limit the technical solution of the present invention in any form. Any technical solution obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A method for preparing glycerophosphatidylcholine by an alkaline process, characterized in that: In an ethanol-n-hexane dual medium, alcohol-soluble phospholipids are catalyzed by a composite catalyst to obtain glycerol phosphatidylcholine, comprising the following steps: Step 1: Mix and dissolve the main catalyst sodium ethoxide and the co-catalyst polyol to prepare a composite catalyst; Step 2: After the alcohol-soluble phospholipid and ethanol are stirred and dissolved, the composite catalyst and a certain volume of n-hexane are added, and the first stage is stirred and reacted at a temperature of 40-55°C for 0.5-1.0 h, and then a certain volume of n-hexane is added, and the second stage is stirred and reacted for 1.0-2.0 h; Step 3: After the reaction mixture obtained in step 2 is cooled to room temperature, the lower layer of ethanol solution A containing glycerol phosphatidylcholine is obtained by separation, the solution is concentrated by negative pressure desolventizing, and then an equal volume of n-hexane is added thereto for extraction twice, and finally a lower layer of ethanol solution B is obtained; Step 4: placing the ethanol solution B in step 3 in a pressure reactor, then adding deionized water, introducing CO2, stirring and reacting to obtain a reaction solution without sodium ethanolate, and then filtering the filtrate, removing ethanol under negative pressure to obtain a glycerol phosphatidylcholine product; In the composite catalyst, the co-catalyst polyol is one of 1,2-propylene glycol and glycerol, and the mass ratio of the co-catalyst polyol to sodium ethoxide is 2:1; The mass ratio of the composite catalyst to the alcohol-soluble phospholipid is 0.5:100-2.0:100; the concentration of the alcohol-soluble phospholipid in ethanol is 0.05-0.15 g / mL; The n-hexane added before the first-stage stirring reaction in step 2 is 5% of the volume of ethanol in n-hexane; the n-hexane added before the second-stage stirring reaction is 20% of the volume of the initial reaction ethanol in n-hexane; The alcohol-soluble phospholipid refers to a phospholipid with a phosphatidylcholine purity of more than 50%.

Images