At present, the preparation methods of GPC at home and abroad include direct extraction of animal organs, chemical synthesis, chemical hydrolysis, and phospholipase hydrolysis.
1) Direct extraction of animal organs
In order to obtain GPC, the method of extracting from bovine pancreas was first used. Crush the bovine pancreas, take 0.5 g of bovine pancreas tissue fluid, add 2 mL of purified water to obtain a tissue suspension, then add 40 mL of sodium hydroxide solution (0.1 mol/L), transfer it to a conical flask, add anhydrous Ethanol and ether (volume ratio 3:1) were extracted, and the ether layer was separated to obtain an anhydrous ethanol extract, which was boiled and filtered. The filtrate is added with petroleum ether for secondary extraction and separation, and the extract in absolute ethanol is suction filtered and dried to obtain the crude GPC product. This method has complicated operation process and low product yield, and has been replaced by other methods.
2) Chemical synthesis method
Applying the principle of epoxy ring-opening reaction, it is proposed to use ( R)-( + ) condensed glycerol ( ( R)-( + ) Glycidol) and phosphorylcholine for nucleophilic addition in the presence of isopropylamine, and then pass through the ion exchange resin layer The preparation of GPC by analysis is simple, but the total yield is less than 50%. In view of the instability of glycidol (2,3-epoxy-1-propanol), it needs to be frozen to avoid self-condensation, and its purity must be determined before use. , usually purified by distillation under reduced pressure. 2-Chloro-2-oxo-1,3,2-dioxaphosphalane was condensed with isopropylidene glycerol in an inert solvent at 0-10°C; the obtained condensate was condensed with triethylamine at room temperature Under the ring-opening condensation, the crude GPC is obtained; then it is concentrated by cation exchange resin, and then crystallized to obtain GPC.
3) Chemical hydrolysis method
GPC was prepared by alcoholysis of egg yolk phospholipid, the solvent was removed by rotary evaporation, and then extracted with ether to obtain a mixture of GPC and glycerophosphoryl ethanolamine, and then purified by silica gel column to obtain refined GPC. Using tetrabutylammonium hydroxide as a catalyst to hydrolyze lecithin to obtain relatively pure GPC, but it is necessary to remove the catalyst and recrystallize and purify the product. The whole process is complicated and the cost is high. The GPC was prepared by hydrolysis of lecithin, and the by-products were precipitated by mercuric chloride to achieve the purpose of purifying GPC. Excess mercury ions were removed by hydrogen sulfide and barium carbonate. This method was less efficient, and the obtained GPC not only contained unreacted eggs. Phospholipids, and a large amount of toxic mercury ions remain.
The egg yolk lecithin was dissolved in ether, stirred at room temperature for 2 h, the resulting precipitate was washed with ether, dissolved in methanol, and diatomite was added to make GPC diatomite mixed powder, which was mixed with 4- Dimethylaminopyridine was mixed, chloroform was added, cooled in an ice-water bath, sonicated at 50°C for 48 h, filtered to remove diatomaceous earth, the supernatant was concentrated and passed through a silica gel column, and the obtained active ingredients were sprayed with Hodamine 6G and DittmerLester-reagent After color development, concentrated and purified by Sephadex column to obtain high-purity GPC.
4) Phospholipase hydrolysis method
The GPC is prepared by an enzymatic hydrolysis method, and belongs to the technical field of lipid development and application. GPC was prepared by hydrolyzing soybean powder phospholipid with phospholipase A1 in an aqueous system, and the GPC yield reached 95%. Lipozyme IM and phospholipase A2 were used to co-catalyze the hydrolysis of phosphatidylcholine in a microemulsion system for 48 h to prepare GPC, and the highest yield was 94%. Phospholipase A1 catalyzed the hydrolysis of PC80, and the product GPC yield was 86% after 24 h. The adsorption method was used to prepare immobilized phospholipase A1 to catalyze the preparation of GPC from phosphatidylcholine, and the GPC yield of this method could reach 85%. reused many times.