Method and system for purifying saturated fatty acid acyl phosphatidylcholine

Abstract

The invention belongs to the technical field of biochemical engineering and separation and purification, and particularly relates to a method and a system for purifying saturated fatty acid acyl phosphatidylcholine, the saturated fatty acid is palmitic acid and / or stearic acid, firstly, poly N-isopropylacrylamide (PNIPAM) with carboxyl at the tail end and phosphatidylserine (PS) are grafted through an amidation reaction, and then, the saturated fatty acid acyl phosphatidylcholine is obtained. The PS-PNIPAM grafted copolymer, saturated fatty acid acyl phosphatidylcholine and TPGS are co-assembled in a water phase, and high-purity saturated fatty acid acyl phosphatidylcholine solid is obtained through temperature gradient treatment, centrifugal washing, dissolving and filtering; the purification system comprises a grafting reaction unit, a ternary blending unit, a temperature-sensitive induction separation purification unit and other modular units, efficient purification and continuous production of the saturated fatty acid acyl phosphatidylcholine are achieved, and the purification efficiency and the industrial applicability are remarkably improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of bio-chemical engineering and separation and purification technology, and particularly relates to a saturated fatty acid acyl phosphatidylcholine purification method and system. BACKGROUND

[0002] Saturated fatty acid acyl phosphatidylcholine (dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine) is an important class of phospholipid compounds, which is widely used in the fields of liposome drug carriers (such as anticancer drug doxorubicin liposome), cell membrane model construction, cosmetic emulsifiers and food additives, etc. Distearoyl phosphatidylcholine (DSPC, 1,2-Distearoyl-sn-glycero-3-PC) is an important member of the phosphatidylcholine (PC) family, two saturated stearic acid (C18:0) acyl chains are connected to the sn1 and sn2 positions of the glycerol skeleton through an ester bond, and the phosphocholine group serves as the hydrophilic head, which endows it with the typical amphiphilic characteristic. The phase transition temperature (Tc) of DSPC is as high as 55℃, which is significantly higher than the physiological temperature (37℃), so that it forms a highly ordered gel state lipid bilayer at room temperature, and is widely used in the fields of liposome drug delivery system, vaccine adjuvant, biomimetic material of biological membrane and cosmetic sustained-release carrier, etc. The purity of DSPC directly affects the stability of the liposome, the drug encapsulation efficiency and the biocompatibility. Dipalmitoyl phosphatidylcholine (DPPC, 1,2-Dipalmitoyl-sn-glycero-3-PC) is a pharmaceutical excipient and a lung surfactant, and the purity thereof directly affects the drug delivery efficiency and clinical safety. The industrial purification requires the purity of the target product to be ≥99.8%, and the crude product synthesized by chemical synthesis often contains similar impurities of phospholipids, so the traditional purification method faces many challenges. The traditional purification method relies on the adsorption difference of silica gel, ion exchange resin and other fillers to realize separation, although the purity is high, but there are significant defects. For example, the cost of the filler is high, and it is easily affected by the viscosity and polarity of the sample, which leads to the decrease of column efficiency; the process cycle is long (several hours are needed for single separation), the solvent consumption is large, and it is difficult to meet the needs of continuous production; it is difficult to scale up, and the problems of uniformity of filler loading and pressure control lead to poor batch repeatability. The solvent extraction method separates different phospholipids in organic solvents according to their solubility differences, which has the following defects: solvent residue risk, which needs to be removed by multi-stage distillation, increasing energy consumption and cost; limited selectivity, which is not good for separating saturated / unsaturated fatty acid acyl phosphatidylcholine with similar structures; environmental pollution problem, a large amount of organic solvents such as chloroform and methanol are used, which does not meet the trend of green chemistry.

[0003] Therefore, it is urgent to develop an efficient, environmentally friendly and suitable for industrial production saturated fatty acid acyl phosphatidylcholine purification method. SUMMARY

[0004] In response to the deficiencies in the prior art, the present application provides a method and system for purifying saturated fatty acid acylphosphatidylcholine, wherein the saturated fatty acid is palmitic acid and / or stearic acid, and the terminal carboxyl-containing PNIPAM is grafted with phosphatidylserine (PS) through an amidation reaction, and the PS-PNIPAM graft copolymer is mixed with saturated fatty acid acylphosphatidylcholine and TPGS in an aqueous phase. High-purity saturated fatty acid acylphosphatidylcholine solid is obtained through temperature-sensitive induction treatment, centrifugal washing, and dissolution and filtration; the system includes a grafting reaction module, a mixing module, a temperature-sensitive induction separation and purification module, etc., to realize the full-process production from grafting reaction to finished product drying.

[0005] In a first aspect, the present application provides a method for purifying saturated fatty acid acylphosphatidylcholine, comprising the following steps:

[0006] Grafting poly (N-isopropylacrylamide) containing a carboxyl group at the end onto phosphatidylserine through an amidation reaction to obtain a graft copolymer of poly (N-isopropylacrylamide) and phosphatidylserine;

[0007] The obtained poly (N-isopropylacrylamide) and phosphatidylserine graft copolymer is uniformly mixed with saturated fatty acid acylphosphatidylcholine and D-α-tocopheryl polyethylene glycol succinate in an aqueous phase to obtain a complex;

[0008] The complex is treated by thermosensitive induction and centrifuged to obtain a precipitate, and the precipitate is dissolved, filtered, rotary evaporated, and vacuum dried to obtain a high-purity saturated fatty acid acylphosphatidylcholine solid.

[0009] In one feasible embodiment, the poly (N-isopropylacrylamide) containing a terminal carboxyl group is prepared by the following method: using N-isopropylacrylamide as a monomer, thioglycolic acid as a chain transfer agent, and ammonium persulfate as an initiator, conducting an aqueous solution polymerization reaction at 65-70° C. under nitrogen protection for 4-6 hours, and then dialyzing and freeze-drying to obtain the obtained product.

[0010] In one feasible embodiment, the mass ratio of N-isopropylacrylamide, ammonium persulfate, thioglycolic acid and water is 100:(0.1-2):(0.5-5):1000.

[0011] For a feasible embodiment, the amidation reaction is carried out at room temperature in a weakly acidic environment for 2-3 hours with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as condensing agents, the reaction solution is purified by ultrafiltration to obtain a poly N-isopropyl acrylamide and phosphatidylserine graft copolymer aqueous solution, and then freeze-dried to obtain a poly N-isopropyl acrylamide and phosphatidylserine graft copolymer, the mass ratio of the poly N-isopropyl acrylamide and phosphatidylserine is (5-20):1, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.

[0012] For a feasible embodiment, the preparation method of the complex is as follows: methanol solution of saturated fatty acid acyl phosphatidylcholine, ethanol solution of D-α-tocopherol polyethylene glycol succinate are simultaneously dropped into the poly N-isopropyl acrylamide and phosphatidylserine graft copolymer aqueous solution, vortex mixed for 30-60 seconds, and the mass ratio of the poly N-isopropyl acrylamide and phosphatidylserine graft copolymer, saturated fatty acid acyl phosphatidylcholine and D-α-tocopherol polyethylene glycol succinate is 1:(1-5):(0.05-0.2).

[0013] For a feasible embodiment, the warm-sensitive induction treatment includes: (a) treatment in a 40-45℃ water bath for 10-15 minutes, (b) immediate cooling to less than 10℃, and standing for 10-15 minutes.

[0014] For a feasible embodiment, the centrifugation condition is centrifugation at 4℃, 12000-15000 rpm for 10-15 minutes.

[0015] For a feasible embodiment, the dissolution filtration includes adding ethanol to the precipitate and ultrasonic treatment, vacuum suction filtration through an organic phase-preferred PVDF filter membrane, and recycling of the filter cake.

[0016] For a feasible embodiment, the rotary evaporation temperature is 30-40℃, and the vacuum drying time is 30-40 minutes.

[0017] In a second aspect, the application provides a system for the purification method of the saturated fatty acid acyl phosphatidylcholine.

[0018] The grafting reaction module: the terminal carboxyl-containing poly N-isopropyl acrylamide is grafted with phosphatidylserine through amidation reaction to obtain a poly N-isopropyl acrylamide and phosphatidylserine graft copolymer;

[0019] Mixing module: the obtained poly-N-isopropyl acrylamide and phosphatidylserine graft copolymer is mixed with saturated fatty acid acyl phosphatidylcholine, D-alpha-tocopherol polyethylene glycol succinate in an aqueous phase to obtain a complex;

[0020] Temperature-sensitive induction separation and purification module: the complex is treated by temperature-sensitive induction, centrifuged to obtain a precipitate, the precipitate is dissolved and filtered, rotary evaporated, and vacuum dried to obtain high-purity saturated fatty acid acyl phosphatidylcholine solid.

[0021] The present application first grafts poly-N-isopropyl acrylamide with carboxyl-terminated ends and phosphatidylserine through an amidation reaction; the poly-N-isopropyl acrylamide and phosphatidylserine graft copolymer is mixed with saturated fatty acid acyl phosphatidylcholine and D-alpha-tocopherol polyethylene glycol succinate in an aqueous phase to obtain a mixed solution; the mixed solution is treated by a temperature gradient, centrifuged and washed, and dissolved and filtered to obtain high-purity saturated fatty acid acyl phosphatidylcholine solid, solving the problems of large use of organic solvents, long separation period, and poor purification effect in the prior art.

[0022] N-isopropylacrylamide (PNIPAM) has a lower critical solution temperature (LCST) around 32°C. Below the LCST, the amide groups on the PNIPAM molecular chain form hydrogen bonds with water molecules, and the molecule is in a hydrophilic stretched state and dissolves in water; when the temperature is higher than the LCST, the hydrogen bonds are broken, the hydrophobic interaction is enhanced, and the molecular chain is curled and shrunk to precipitate from water. Phosphatidylserine and saturated fatty acid acylphosphatidylcholine both contain phosphatidyl groups. The saturated fatty acid chains (palmitic acid / stearic acid) of phosphatidylserine and saturated fatty acid acylphosphatidylcholine have similar straight-chain alkane structures in the hydrophobic tail, which are easy to form ordered complexes through hydrophobic interaction (van der Waals force, alkyl chain packing). In the poly N-isopropylacrylamide and phosphatidylserine graft copolymer, phosphatidylserine can enhance the binding force with saturated fatty acid acylphosphatidylcholine through the specific interaction of phosphatidyl groups, while D-α-tocopheryl polyethylene glycol succinate wraps saturated fatty acid acylphosphatidylcholine in a hydrophobic microenvironment, reduces its direct contact with dissolved oxygen in the water phase, reduces the risk of oxidation, inhibits non-specific adsorption, makes it difficult for impurities to enter the complex, and significantly improves the purification selectivity. When the temperature is higher than the LCST of PNIPAM, the PNIPAM segment changes from a hydrophilic random coil to a hydrophobic tight micellar state, and its hydrophobic core directly contacts with the long chain of saturated fatty acid, forming a hydrophobic-hydrophobic synergistic effect. At this time, the phosphatidyl group of phosphatidylserine acts as an "anchor point" to fix the phosphatidyl group of saturated fatty acid acylphosphatidylcholine in the poly N-isopropylacrylamide and phosphatidylserine graft copolymer through backbone homology, forming a "phosphatidyl-phosphatidyl" directional docking. When the temperature is lower than the LCST of PNIPAM, PNIPAM dissolves due to strong hydrophilicity, and saturated fatty acid acylphosphatidylcholine self-aggregates and precipitates due to hydrophobic interaction. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a HPLC chart of distearoylphosphatidylcholine.

[0024] Figure 2 is a HPLC chart of dipalmitoylphosphatidylcholine.

[0025] Figure 3 is a NMR chart of distearoylphosphatidylcholine.

[0026] Figure 4 is a NMR chart of dipalmitoylphosphatidylcholine.

[0027] Figure 5 is a schematic diagram of the chemical reaction for preparing the graft-modified PNIPAM. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below in conjunction with embodiments. However, it should not be understood as the scope of the present application being limited to the following examples. Any other embodiments obtained by those of ordinary skill in the art without creative efforts under the premise of not departing from the method idea of the present application shall fall within the scope of the present application.

[0029] In the present application, the terms used in the present application are merely for the purpose of describing specific embodiments, and are not intended to limit the present application.

[0030] As used in the present application, the singular forms "is", "an", "any" and "the" are intended to include the plural forms, unless the context clearly indicates otherwise.

[0031] In addition, in the present application, the terms "first", "second" appear only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0032] The following will specifically describe a saturated fatty acid acyl phosphatidylcholine purification method provided by the present application in conjunction with different embodiments.

[0033] Embodiment 1:

[0034] A dipalmitoyl phosphatidylcholine purification method comprises the following steps:

[0035] 1. Dissolve NIPAM in deionized water, add mercaptoacetic acid, stir and dissolve. Add ammonium persulfate, and remove oxygen by nitrogen for 15 minutes. Under the protection of nitrogen, react in a 65°C constant temperature water bath for 5 hours. After the reaction solution is cooled to room temperature, it is transferred to a dialysis bag and dialyzed with deionized water. The dialysate is freeze-dried (-50°C, 24 hours) to obtain white powder-like terminal carboxyl PNIPAM, which is sealed and stored. The mass ratio of poly N-isopropyl acrylamide, ammonium persulfate, mercaptoacetic acid and water is 100:1:1:1000.

[0036] 2. Weigh the carboxyl-terminated poly-N-isopropyl acrylamide (PNIPAM) into a beaker, add ultrapure water, and stir magnetically at room temperature until completely dissolved (about 30 min) to form a clear and transparent solution. Weigh the phosphatidylserine and add N,N-dimethylformamide (DMF) to a concentration of 10 mg / L, and vortex for 5 min. Transfer the prepared PNIPAM aqueous solution to a conical flask, slowly add the phosphatidylserine solution while stirring magnetically (speed 200-300 rpm), and mix uniformly to form a light yellow transparent dispersion. Add EDC (1-ethyl-3-(3-dimethylaminopropyl) and NHS (N-hydroxysuccinimide) to the reaction system. Seal the conical flask, place it in a magnetic stirrer at room temperature (25±2°C) and avoid light for 2.5 h (stirring speed maintained at 200 rpm). After the reaction is completed, transfer the reaction solution to an ultrafiltration centrifuge tube (molecular weight cutoff 10000), centrifuge at 4°C and 8000 rpm for 30 min to remove unreacted small molecules and condensing agents. Collect the filtrate in the ultrafiltration centrifuge tube and freeze-dry it to obtain the poly-N-isopropyl acrylamide and phosphatidylserine graft copolymer. The mass ratio of poly-N-isopropyl acrylamide and phosphatidylserine is 10:1. As shown in Figure 5 wherein n = 150-300, R1 and R2 are fatty acid acyl chains on the glycerol backbone of phosphatidylserine.

[0037] 3. Dissolve dipalmitoyl phosphatidylcholine in methanol. Dissolve TPGS in ethanol. Simultaneously drop the above two solutions into the aqueous solution of poly-N-isopropyl acrylamide and phosphatidylserine graft copolymer (10 mg / mL), vortex mix for 40 seconds while dropping, and stand for 10 minutes to stabilize the system to form a ternary complex. The mass ratio of poly-N-isopropyl acrylamide and phosphatidylserine graft copolymer, dipalmitoyl phosphatidylcholine and D-alpha-tocopheryl polyethylene glycol succinate is 1:3:0.1.

[0038] 4. Preheat the constant temperature water bath to 40°C in advance, transfer the mixed solution to a centrifuge tube, and place it in the water bath for 10-15 minutes with the opening. Immediately transfer the centrifuge tube to an ice bath (0-4°C) and stand for 15 minutes. The system shows obvious solid-liquid stratification. Place the centrifuge tube in a centrifuge and centrifuge at 4°C and 12000 rpm for 15 minutes. Carefully pour the supernatant and retain the sediment at the bottom of the tube.

[0039] 5. Add ethanol to the precipitate and sonicate for 10 minutes to dissolve the dipalmitoyl phosphatidylcholine. The solution is vacuum filtered through a 0.22 μm PVDF filter and the filtrate is collected. The filter cake (PS-PNIPAM) is recovered after washing with pH 7.4 PBS. The filtrate is transferred to a rotary evaporator and the ethanol is evaporated under reduced pressure at 30°C. The residual solid is dried in a vacuum oven (30°C, < 100 mbar) for 30 minutes to obtain high purity dipalmitoyl phosphatidylcholine as a white solid. The purity of the dipalmitoyl phosphatidylcholine is shown in Figure 1 as a HPLC profile, Figure 2 Figure 2 as a NMR profile. Figure 4

[0040] Example 2:

[0041] A method for purifying a dipalmitoyl phosphatidylcholine, comprising the steps of:

[0042] 1. Dissolve NIPAM in deionized water and add mercaptoacetic acid, stirring to dissolve. Add ammonium persulfate and deoxygenate under nitrogen for 15 minutes. React in a constant temperature water bath at 70°C for 4 hours under nitrogen protection. After the reaction solution is cooled to room temperature, it is transferred to a dialysis bag and dialyzed with deionized water. The dialysate is freeze-dried (-50°C, 24 hours) to obtain carboxyl-terminated PNIPAM in the form of white powder, which is stored in a sealed container. The mass ratio of poly N-isopropyl acrylamide, ammonium persulfate, mercaptoacetic acid and water is 100:0.1:0.5:1000.

[0043] 2. Weigh the carboxyl-terminated poly N-isopropyl acrylamide (PNIPAM) and place it in a beaker. Add ultrapure water and stir magnetically at room temperature until it is completely dissolved (about 30 min) to form a clear and transparent solution. Weigh the phosphatidylserine and add N,N-dimethylformamide (DMF) to a concentration of 10 mg / L, and vortex for 5 min. Transfer the prepared aqueous PNIPAM solution to a conical flask and slowly add the phosphatidylserine solution while stirring magnetically (speed 200-300 rpm). After mixing well, a light yellow transparent dispersion is formed. Add EDC (1-ethyl-3-(3-dimethylaminopropyl)) and NHS (N-hydroxysuccinimide) to the reaction system. Seal the conical flask and place it in a magnetic stirrer at room temperature (25±2°C) in the dark for 2 h (stirring speed maintained at 200 rpm). After the reaction is completed, transfer the reaction solution to an ultrafiltration centrifuge tube (molecular weight cutoff 10000) and centrifuge at 4°C and 8000 rpm for 30 min to remove unreacted small molecules and condensing agents. Collect the filtrate in the ultrafiltration centrifuge tube and freeze-dry it to obtain the poly N-isopropyl acrylamide and phosphatidylserine graft copolymer. The mass ratio of poly N-isopropyl acrylamide and phosphatidylserine is 5:1.

[0044] ​3. Dissolve distearoylphosphatidylcholine in methanol. Dissolve TPGS in ethanol. Add the two solutions into the aqueous solution of poly(N-isopropylacrylamide) grafting with phosphatidylserine (10 mg / mL) simultaneously drop by drop, vortex mix for 30 seconds, and let the system stand for 10 minutes to form ternary complex. The mass ratio of poly(N-isopropylacrylamide) grafting with phosphatidylserine, distearoylphosphatidylcholine and D-a-tocopheryl polyethylene glycol succinate is 1:1:0.05.

[0045] 4. Preheat the constant temperature water bath to 40℃ in advance. Transfer the mixed solution into a centrifuge tube and put it into the water bath with the opening for 10-15 minutes. Immediately transfer the centrifuge tube into an ice bath (0-4℃) and let it stand for 15 minutes. The system shows obvious solid-liquid stratification. Put the centrifuge tube into a centrifuge and centrifuge at 4℃, 15000 rpm for 10 minutes. Carefully pour the supernatant and reserve the sediment at the bottom of the tube.

[0046] 5. Add ethanol to the sediment and ultrasonically treat for 10 minutes to dissolve distearoylphosphatidylcholine. Vacuum filter the solution through a 0.22 μm PVDF filter membrane and collect the filtrate. Wash the filter cake (PS-PNIPAM) with pH 7.4 PBS and then recover it. Transfer the filtrate into a rotary evaporator and evaporate ethanol under reduced pressure at 35℃. Dry the residual solid in a vacuum drying oven (30℃, ≤100 mbar) for 35 minutes to obtain high-purity distearoylphosphatidylcholine white solid. As shown in FIG. 1, it is the HPLC chart of distearoylphosphatidylcholine, Figure 1 As shown in FIG. 2, it is the NMR chart of distearoylphosphatidylcholine. Figure 3

[0047] Example 3:

[0048] A method for purifying dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, comprising the following steps:

[0049] 1. Dissolve NIPAM in deionized water and add mercaptoacetic acid, stirring to dissolve. Add ammonium persulfate and remove oxygen by nitrogen for 15 minutes. Under nitrogen protection, react in a 70℃ constant temperature water bath for 6 hours. After the reaction solution is cooled to room temperature, transfer it into a dialysis bag and dialyze with deionized water. Freeze-dry (-50℃, 24 hours) the dialysate to obtain white powdery end-carboxyl PNIPAM, which is sealed and stored. The mass ratio of poly(N-isopropylacrylamide), ammonium persulfate, mercaptoacetic acid and water is 100:2:5:1000.

[0050] ​2. Weigh the carboxyl-terminated poly N-isopropyl acrylamide (PNIPAM) into a beaker, add ultrapure water, and stir magnetically at room temperature until completely dissolved (about 30 min) to form a clear and transparent solution. Weigh the phosphatidylserine and add N,N-dimethylformamide (DMF) to a concentration of 10 mg / L, and vortex for 5 min. Transfer the prepared PNIPAM aqueous solution to a conical flask, slowly add the phosphatidylserine solution while stirring magnetically (speed 200-300 rpm), and mix uniformly to form a light yellow transparent dispersion. Add EDC (1-ethyl-3-(3-dimethylaminopropyl) and NHS (N-hydroxysuccinimide) to the reaction system. Seal the conical flask, place it in a magnetic stirrer at room temperature (25±2°C) in the dark for 3 h (stirring speed maintained at 200 rpm). After the reaction is completed, transfer the reaction solution to an ultrafiltration centrifuge tube (molecular weight cut-off 10000), centrifuge at 4°C and 8000 rpm for 30 min to remove unreacted small molecules and condensing agents. Collect the filtrate in the ultrafiltration centrifuge tube, and freeze-dry it to obtain the poly N-isopropyl acrylamide and phosphatidylserine graft copolymer. The mass ratio of poly N-isopropyl acrylamide and phosphatidylserine is 20:1.

[0051] 3. Dissolve dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine in methanol. Dissolve TPGS in ethanol. Simultaneously, drop the above two solutions into the poly N-isopropyl acrylamide and phosphatidylserine graft copolymer aqueous solution (10 mg / mL) while vortex mixing for 60 seconds. Let the system stand for 10 minutes to stabilize, and form a ternary complex. The mass ratio of poly N-isopropyl acrylamide and phosphatidylserine graft copolymer, saturated fatty acid acyl phosphatidylcholine, and D-α-tocopherol polyethylene glycol succinate is 1:5:0.2.

[0052] 4. Preheat the constant-temperature water bath to 45°C in advance, transfer the mixed solution to a centrifuge tube, and place it in the water bath for 10-15 minutes with the opening. Immediately transfer the centrifuge tube to an ice bath (0-4°C) and let it stand for 15 minutes. The system shows obvious solid-liquid stratification. Place the centrifuge tube in a centrifuge and centrifuge at 4°C and 12000 rpm for 15 minutes. Carefully pour off the supernatant and retain the sediment at the bottom of the tube.

[0053] 5. Add ethanol to the sediment and ultrasonicate for 10 minutes to dissolve the dipalmitoyl phosphatidylcholine and distearoyl phosphatidylcholine. Vacuum filter the solution through a 0.22 μm PVDF filter membrane, and collect the filtrate. Wash the filter cake (PS-PNIPAM) with pH 7.4 PBS and recover it. Transfer the filtrate to a rotary evaporator, evaporate the ethanol under reduced pressure at 40°C. Dry the residual solid in a vacuum drying oven (30°C, ≤100 mbar) for 40 minutes to obtain high-purity distearoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine white solids.

[0054] Comparative Example 1:

[0055] A method for purifying distearoylphosphatidylcholine comprises the following steps:

[0056] 1. Dissolve the crude DSPC in a mixed solvent of chloroform and isopropanol (4:1, v / v), heat to 50°C and stir until completely dissolved; add activated carbon and diatomaceous earth (10%), stir for 30 minutes, and filter to remove pigments, metal ions and partial oxidation products.

[0057] 2. Multi-stage gradient elution chromatography: using reverse phase C18 bonded silica gel column (particle size 10 μm, pore size ), column temperature was constant at 30°C;

[0058] Gradient 1 (0-15 min): acetonitrile:methanol:water = 70:20:10 (v / v), eluting polar impurities;

[0059] Gradient 2 (15-30 minutes): acetonitrile:methanol:water = 60:35:5, eluting the main component of DSPC (retention time 20-25 minutes);

[0060] Gradient 3 (30-40 minutes): acetonitrile:methanol:water = 40:55:5, eluting non-polar impurities;

[0061] 3. The eluate was concentrated and transferred to a ternary mixed solvent system of ethanol: tert-butanol: water (6:3:1, v / v);

[0062] Gradient temperature controlled crystallization:

[0063] Primary crystallization: cool down to 5°C at a rate of 2°C / min and keep at this temperature for 2 hours to precipitate primary crystals of DSPC;

[0064] Secondary crystallization: continue cooling to 20°C at a rate of 0.5°C / min and let stand for 12 hours to obtain high-purity DSPC crystals; the crystals are washed three times with pre-cooled ethanol and vacuum freeze-dried (50°C, 10Pa) to obtain distearoylphosphatidylcholine.

[0065] Comparative Example 2:

[0066] A method for purifying dipalmitoylphosphatidylcholine comprises the following steps:

[0067] 1. Gradient elution procedure:

[0068] Initial eluent: chloroform (100%), methanol was gradually added in the following ratios: chloroform:methanol = 95:5 → 90:10 → 85:15 (v / v).

[0069] 2. Dissolve the crude DPPC product in 5 mL of chloroform and evenly load it onto the top of a silica gel column using a dropper. Elute using a gradient, collecting 50 mL of the eluate. Monitor by TLC (developing solvent: chloroform-methanol-water = 65:25:4, iodine vapor color development). Combine the eluates containing DPPC and rotary evaporate to dryness to obtain a pale yellow solid.

[0070] 3. Dissolve the solid purified by column chromatography in hot anhydrous ethanol (60-70°C) and stir until completely dissolved. Slowly cool the hot solution to room temperature and then place it in a 4°C refrigerator for 12-24 hours to allow DPPC crystals to fully precipitate. Filter using a Buchner funnel and wash the crystals 2-3 times with cold ethanol (4°C) to remove residual mother liquor. Place the filter cake in a vacuum drying oven at 40°C for 6-8 hours to obtain dipalmitoylphosphatidylcholine.

[0071] According to the general rules of Part IV of the Chinese Pharmacopoeia, the high performance liquid chromatography method was used to detect dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Figure 1 and Figure 2 The purity of phosphatidylcholine obtained from the test results is shown in Table 1.

[0072] Table 1 Test results of purified saturated fatty acid acylphosphatidylcholine in Examples and Comparative Examples

[0073]

[0074]

[0075] The test results show that the purity of the saturated fatty acid acylphosphatidylcholine in Examples 1-3 is superior to that in Comparative Examples 1-2. This is because the poly (N-isopropylacrylamide) and phosphatidylserine graft copolymer in the examples specifically bind to the saturated fatty acid acylphosphatidylcholine, and D-α-tocopheryl polyethylene glycol succinate, as an amphiphilic molecule, not only stabilizes the complex but also repels nonspecifically adsorbed impurities through its polyethylene glycol chain, further improving the purity.

[0076] 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 technical principles 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 purifying saturated fatty acid acylphosphatidylcholine, wherein the saturated fatty acid is palmitic acid and / or stearic acid, characterized in that: The following steps are involved: Grafting poly (N-isopropylacrylamide) containing a carboxyl group at the end onto phosphatidylserine through an amidation reaction to obtain a graft copolymer of poly (N-isopropylacrylamide) and phosphatidylserine; The obtained poly (N-isopropylacrylamide) and phosphatidylserine graft copolymer is uniformly mixed with saturated fatty acid acylphosphatidylcholine and D-α-tocopheryl polyethylene glycol succinate in an aqueous phase to obtain a complex; The complex is treated by thermosensitive induction and centrifuged to obtain a precipitate, and the precipitate is dissolved, filtered, rotary evaporated, and vacuum dried to obtain a high-purity saturated fatty acid acylphosphatidylcholine solid.

2. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The poly N-isopropylacrylamide containing a carboxyl group at the end is prepared by the following method: using N-isopropylacrylamide as a monomer, thioglycolic acid as a chain transfer agent, and ammonium persulfate as an initiator, performing aqueous solution polymerization at 65-70° C. under nitrogen protection for 4-6 hours, and then dialyzing and freeze-drying to obtain the obtained product.

3. A method for purifying saturated fatty acid acylphosphatidylcholine according to claim 2, characterized in that, The mass ratio of the N-isopropylacrylamide, ammonium persulfate, thioglycolic acid and water is 100:(0.1-2):(0.5-5):1000.

4. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The amidation reaction uses 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as condensing agents, reacts at room temperature in a weakly acidic environment for 2-3 hours, and the reaction solution is purified by ultrafiltration to obtain an aqueous solution of a poly(N-isopropylacrylamide)-phosphatidylserine) graft copolymer. The aqueous solution is then freeze-dried to obtain a poly(N-isopropylacrylamide)-phosphatidylserine) graft copolymer. The mass ratio of the poly(N-isopropylacrylamide)-phosphatidylserine is (5-20):1, and the molar ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the N-hydroxysuccinimide is 1:

1.

5. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The preparation method of the complex comprises: simultaneously dropping a methanol solution of saturated fatty acid acylphosphatidylcholine and an ethanol solution of D-α-tocopheryl polyethylene glycol succinate into an aqueous solution of poly N-isopropylacrylamide and phosphatidylserine graft copolymer, vortex mixing for 30-60 seconds, and the mass ratio of poly N-isopropylacrylamide to phosphatidylserine graft copolymer, saturated fatty acid acylphosphatidylcholine and D-α-tocopheryl polyethylene glycol succinate is 1:(1-5):(0.05-0.2).

6. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The temperature-sensitive induction treatment comprises: (a) treating in a 40-45° C. water bath for 10-15 minutes, and (b) immediately cooling to less than 10° C. and standing for 10-15 minutes.

7. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The centrifugation conditions are: centrifugation at 4°C and 12000-15000 rpm for 10-15 minutes.

8. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The dissolution and filtration comprises adding ethanol to the precipitate for ultrasonic treatment, vacuum filtration through an organic phase-friendly PVDF filter membrane, and recycling the filter cake.

9. The method for purifying saturated fatty acid acylphosphatidylcholine according to claim 1, wherein The rotary evaporation temperature is 30-40° C., and the vacuum drying time is 30-40 minutes.

10. A system for the purification method of a saturated fatty acid acylphosphatidylcholine according to any one of claims 1 to 9, characterized in that: include: Grafting reaction module: Grafting poly (N-isopropylacrylamide) containing a carboxyl group at the end with phosphatidylserine through an amidation reaction to obtain a graft copolymer of poly (N-isopropylacrylamide) and phosphatidylserine; Mixing module: uniformly mixing the obtained poly (N-isopropylacrylamide) and phosphatidylserine graft copolymer with saturated fatty acid acylphosphatidylcholine and D-α-tocopheryl polyethylene glycol succinate in an aqueous phase to obtain a complex; Thermosensitive induction separation and purification module: The complex is treated with thermosensitive induction and centrifuged to obtain a precipitate, and the precipitate is dissolved, filtered, rotary evaporated, and vacuum dried to obtain a high-purity saturated fatty acid acylphosphatidylcholine solid.

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