CN1308334C - Extraction of phosphatidyl serine from brains of animals - Google Patents

Abstract

The present invention provides a method for extracting phosphatidylserine from animal brains, a. First, crush the dried animal brains into granules, then add acetone to the granules, stir and extract, and remove the acetone supernatant containing cholesterol and oils in the dry brain granules b. Add n-hexane or ethanol or ammonia water ethanol solution to the evaporated residue, stir and extract, remove the extract containing phospholipid complex, and evaporate the residue; c. Add ether to the evaporated residue, stir Extraction, the extract is evaporated and concentrated, then added to an alkaline aqueous solution, stirred, frozen, thawed, filtered to remove the precipitate, and further removed the remaining phospholipids, then the supernatant was adjusted to pH=3~6, diethyl ether was evaporated, and then evaporated to dryness to obtain crude Phosphatidylserine. Then dissolve the crude phosphatidylserine dry powder in n-hexane, then add sodium acetate ethanol solution, stir, let stand to separate layers, remove the lower layer containing inositol phospholipids, and evaporate the upper part of n-hexane to dryness under reduced pressure to obtain high-purity phosphatidylserine.

Description

A method for extracting phosphatidylserine from animal brain

technical field

The invention relates to a method for extracting phosphatidylserine from the brains of livestock animals, and is especially suitable for extracting phosphatidylserine from the brains of livestock animals such as cattle, sheep, rabbits, horses and donkeys.

Background technique

Bovine brain phosphatidylserine (PS) has the effects of enhancing memory and anti-senile dementia, which have been verified by animal experiments and clinical trials at home and abroad. 18(4), 207-230. However, it is difficult to extract and produce PS in batches using bovine brain as raw material. Lees M.B et al. (1981) and Abid et al. (1998) reviewed the chromatographic preparation and analysis methods of PS, and the preparation methods were extremely complicated. See Lees MB et al. preparation and Analysis of phosphatides Lipids and steroids (Lees MB et al. preparation and Analysis of phosphatides Lipids and steroids, 1981, 328-345) and Abed et al. Chromatography B Collection 1998, (717) 279-293 (Abidi, SL et al. Journal of chromatography B, 717 (1988) 279-293). Chen Su and others studied the high-pressure liquid phase preparation method. Although it can be used for reagent preparation, the output is really limited. See Chen Su et al. Chromatography B Collection 1995, (666) 178-182 (Su chen et al. Journal of chromatography B.666, 1995, 178-182). The United States uses soybean phospholipids and L-serine to synthesize PS under the action of phospholipase D, and studies the successful extraction and preparation method, see US Patent 5700668 (united states patent 5700668). However, the clinical effect of PS synthesized from soybean phospholipids is far inferior to PS extracted from bovine brain. After many repeated experiments, this method is not suitable for extracting PS from the brains of cattle and other animals.

my country's animal husbandry is developing rapidly, and a large amount of beef cattle are slaughtered. The brains of the cattle are sold at a low price along with the heads. Some are eaten, and some are thrown away. In recent years, we have been engaged in the extraction research of bovine brain PS, opened up new ways for the value-added utilization of bovine brain, and explored the successful industrial production of bovine brain PS extraction and preparation methods.

Contents of the invention

The purpose of the present invention is to provide a method for extracting phosphatidylserine, which is simple in technology and suitable for industrial production, using the brains of cattle, sheep, rabbits, horses, donkeys and other livestock animals as raw materials.

The purpose of the present invention can be achieved through the following technical measures

a. First crush the dried animal brain into granules, then add acetone to the granules, stir and extract, remove the acetone supernatant containing cholesterol and oil in the dried brain granules, and evaporate the residue to dryness;

b. Add n-hexane or ethanol or ammonia ethanol solution to the evaporated residue, stir and extract, remove the extract containing phospholipid complex, and evaporate the residue to dryness;

c. Add diethyl ether to the evaporated residue, stir and extract, add alkaline aqueous solution to the extract after evaporation, stir, freeze, thaw, filter out the precipitate, further remove the remaining phospholipids, and then adjust the supernatant to PH=3~ 6. Ether was evaporated, and then evaporated to dryness to obtain crude phosphatidylserine.

The purpose of the present invention can also be realized by the following technical measures

Dissolve the dry crude phosphatidylserine powder in step c in n-hexane, add sodium acetate ethanol solution, stir, let stand to separate layers, remove the lower layer containing inositol phospholipids, evaporate the upper part of n-hexane to dryness to obtain high-purity phosphatidylserine; or In step c, diethyl ether is distilled off, and n-hexane of approximately the same amount as the remaining solution is directly added, stirred, allowed to stand for stratification, and the lower layer containing inositol phospholipids is removed, and the upper part of n-hexane is evaporated to dryness to obtain phosphatidylserine; the aforementioned stirring extraction 3 to 5 times, 1 to 3 hours each time; the PH of the ammonia water ethanol solution and the alkaline aqueous solution is 7.5 to 11; the thawing control temperature is 4 to 7°C; the sodium acetate ethanol solution is Refers to 40-60% ethanol containing sodium acetate.

The technical measure of the present invention is to first crush the dried animal brain into particles with a size of approximately 0.2-0.4 cm, then add acetone with a volume of 3-5 times the volume to the particles, stir and extract, and remove the cholesterol and oil contained in the dry brain particles . If the solvent for extracting inositol phospholipids and PS phospholipid complexes is diethyl ether, the amount of diethyl ether added is 3 to 5 times the volume of the evaporated residue to obtain a diethyl ether extract. Most of the diethyl ether can be evaporated to make the volume of the remaining diethyl ether solution 3 to 5 times the amount of dry matter extracted with ether. Add the ether solution to Na ₂ CO ₃ aqueous solution with pH=7.5-11 to form a salt, stir for 1 hour, freeze at -10-20°C, and slowly thaw at 4-7°C. The lower precipitate was removed, and the supernatant was adjusted to pH=3-6 with 2-5% HCl. Evaporate diethyl ether and evaporate water to obtain dry powder; or distill diethyl ether and add the same amount of n-hexane as the remaining solution, stir and separate layers, take the upper n-hexane layer, and distill off n-hexane to obtain dry matter. Then add 3 to 5 times of normal hexane (or the upper normal hexane layer obtained by the last added normal hexane in the above-mentioned) to the dry matter, then add 5 to 10 times of the dry matter by volume and stir in 40 to 60% ethanol solution containing sodium acetate, After static layering, remove the lower white precipitate (mainly inositol phospholipids), take the upper n-hexane layer, evaporate to dryness to obtain phosphatidylserine, and the purity of phosphatidylserine is 90-93%.

If the solvent of the extracted inositol phospholipids and phospholipid complexes of the technical measures of the present invention is n-hexane, most of the n-hexane can be evaporated, and the volume of the remaining n-hexane solution is 3 to 5 times of the dry matter extracted by n-hexane. Impurities such as inositol phospholipids are then removed first, and then n-hexane is evaporated to obtain dry matter, which is then dissolved in ether, and finally ether is evaporated to obtain phosphatidylserine with a purity of 92-95%.

If the technical measure of the present invention removes cholesterol and grease in the brain with acetone, dry to obtain dry brain granules, then add normal hexane with a volume of 3 to 5 times the amount in the dry brain granules, stir and extract 3 to 5 times, each time 1 to 5 times. After 3 hours, the n-hexane extract was obtained, the n-hexane containing the total brain phospholipid complex was evaporated, treated with ether, and then evaporated to dryness to obtain crude phosphatidylserine with a purity of 53%. Adopting this scheme can save the use of a large amount of ethanol.

The ammoniacal liquor can be replaced by sodium hydroxide, potassium hydroxide, disodium hydrogenphosphate, dipotassium hydrogenphosphate, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium acetate or potassium acetate, but the effect is best with ammoniacal liquor. good. The n-hexane can be replaced by chloroform, methylene chloride, toluene, ether, and n-hexane is generally preferred to reduce the types of organic solvents used, reduce the construction of organic solvent recovery towers, and simplify the production process.

The present invention selects conventional organic solvent for extraction, and can realize large-scale industrialized production. Use acetone to remove cholesterol and oils, use ethanol to remove lecithin and cephalin, use n-hexane and ether to extract the phospholipid mixture with phosphatidylserine and inositol phospholipids as the main components, and then use n-hexane and alkali ethanol solvent to remove the precipitate Lower inositol phospholipids and impurities to obtain crude phosphatidylserine. Crude phosphatidylserine is dissolved in ether, and the ether solution is added to an alkaline solution to form a salt, frozen, thawed to remove precipitation, and the supernatant is adjusted to PH=3-6. Stir and extract with n-hexane, let stand to separate layers to obtain the n-hexane layer, evaporate the n-hexane to obtain high-purity phosphatidylserine. The extracted phosphatidylserine and inositol phospholipid complex can also be dissolved in ether, then dissolved in alkaline aqueous solution to freeze to remove impurities, and then use n-hexane and alkali ethanol to remove inositol phospholipids to obtain high-purity phosphatidylserine. It is also possible to use n-hexane to extract the brain particles that remove cholesterol and oil to obtain the total phospholipid complex, and then operate the same method for extracting phosphatidylserine from the phospholipid complex with phosphatidylserine and inositol phospholipids as the main components, but the purity is lower.

Detailed ways

Example 1

1. Slice fresh bovine brain (or frozen bovine brain), ventilate and dry at 60°C, or dry in the sun or dry under natural conditions. The lower the drying temperature, the better, and the best low temperature drying, broken into pieces of about 0.3cm Granules, add acetone of 4 times the volume to the dried bovine brain granules, stir and extract 4 times at room temperature, each time for 2 hours, remove the acetone supernatant, and steam the residue until there is no acetone smell;

2. Add 4 times the volume of ammonia-ethanol solution of pH=9 to the evaporated residue, stir and extract 4 times, each time for 2 hours, evaporate the residue, and the extract can be used to re-extract lecithin and cephalin;

3. Add ether to the evaporated residue to obtain an ether extract, filter and evaporate most of the ether until the volume of the remaining ether solution is 4 times that of the dry matter extracted with ether. Add it to Na ₂ CO ₃ aqueous solution with pH=8.5, stir for 1 hour, freeze at -18°C, and slowly thaw at 6°C. The light yellow supernatant and the lower part of the precipitate can be seen. After removing the precipitate, the supernatant was adjusted to pH=5 with 4% HCl, and evaporated to dryness to obtain a dry powder.

4. Dissolve the dry powder in n-hexane with a volume 4 times the volume, then add 50% ethanol containing sodium acetate with a volume 8 times the volume of the dry powder, stir, and layer at rest. It can be seen that there is a white precipitate below (the precipitate can be used to extract muscle alcohol phospholipids). Then the upper n-hexane layer was removed and evaporated to dryness under reduced pressure to obtain phosphatidylserine with a purity of 93%.

Example 2

1. If the raw materials used are fresh rabbit brains, sheep brains, horse brains, donkey brains and other livestock animal brains, the drying method of the brains is the same as that of cow brains. If the raw materials used are the dried brain particles after extracting cholesterol, it can be directly carried out according to Example 1. Start from middle 2, the same below.

Example 3

In step 2 of Example 1, adding ammonia water ethanol solution to the evaporated residue is to remove the lecithin and cephalin contained in the brain residue.

In this step, dehydrated alcohol with a volume of 4 times the volume can also be added to the brain slag, stirred and extracted for 4 hours, extracted 4 times, each time for 2 hours, and about 50% lecithin and about 25% brain can be extracted. Phospholipids and other phospholipids, but the main component is lecithin, which is conducive to the further purification of lecithin. Then use 4 times the amount of slag to extract with ammonia ethanol of PH=9, stir and extract 4 times, each 2 hours, contain cephalin in about 60% in the extract after drying, also contain 10% lecithin and other phospholipids, but The main component is cephalin, which is beneficial to the further separation of cephalin, and other steps are the same as in Example 1.

Example 4

In step 3 of Example 1, add 4 times the amount of diethyl ether to the evaporated residue for extraction. Diethyl ether can also be replaced by n-hexane, toluene, chloroform, or dichloromethane. The extraction method is the same as diethyl ether, but the extract is added to Na ₂ CO ₃ aqueous solution In this case, normal hexane etc. (toluene, chloroform, dichloromethane) must be evaporated first, then dissolved with 3 times ether, and then Na ₂ CO ₃ lye is added, other steps are the same as in Example 1.

Example 5

As in step 3 of Example 1, extract with n-hexane instead of diethyl ether, the n-hexane extract can be concentrated, and the volume of the remaining n-hexane solution is 4 times the amount of the extracted dry matter, and then add the volume to 7 times 45% ethanol containing sodium acetate in the amount of dry matter, stirred, static, layered, and the white inositol phospholipid precipitate was removed. Remove the n-hexane layer, distill off the n-hexane to obtain a dry powder, dissolve the dry powder with 4 times the volume of ether, add a Na ₂ CO ₃ aqueous solution with a pH of 8, stir for 1 hour, freeze at -15°C, and slowly thaw at 6°C to obtain a supernatant solution, adjust the pH to 5 with 3% HCl, evaporate and recover ether, and evaporate the water to obtain dry phosphatidylserine powder. Preferably, after recovery of ether by evaporation, add n-hexane to the remaining aqueous solution, stir, and stand still to obtain the n-hexane layer, then evaporate the n-hexane, and the purity of phosphatidylserine is 94%.

Example 6

1, as the method for extracting cholesterol and oils with acetone in step 1 of embodiment 1, add 4 times of normal hexane (normal hexane can be replaced by ether, methylene dichloride, chloroform, toluene) in the dried brain granules extracted by acetone , 4 times of extraction, each extraction for 2 hours, can obtain n-hexane solution, and evaporate n-hexane to obtain the total phospholipid complex in the brain.

2. As in step 3 in Example 1, the total phospholipid complex extracted above was dissolved in ether, and the following operations were as in step 3 in Example 1. After slow thawing, a large amount of lecithin and cephalin precipitates can be removed. After the supernatant is adjusted to PH=6 and diethyl ether is evaporated, the water is evaporated to obtain crude PS dry powder. The content of phosphatidylserine in the dry powder reaches 53%.

3. The above crude PS dry powder was operated according to 4 in Example 1 to obtain crude phosphatidylserine with a purity of 84%.

Claims (7)

1, a kind of method from animal brain extraction phosphatidylserine is characterized in that:

A, earlier dry animal brain is broken into particle, in particle, adds acetone again, stir extraction, remove the acetone supernatant liquor that contains cholesterol and oils in the dry cerebral particle, the residue evaporate to dryness;

B, add normal hexane or ethanol or ammoniacal liquor ethanolic soln, stir extraction, remove the extraction liquid that contains phospholipid complex, the residue evaporate to dryness to the residue of evaporate to dryness;

C, add ether to the residue of evaporate to dryness, stir extraction, extraction liquid adds alkaline aqueous solution after inspissation, stir, freeze, thaw, the filtering precipitation is further removed remaining phosphatide, then supernatant liquor is transferred PH=3～6, steamed and remove ether, evaporate to dryness gets raw phospholipid acyl Serine again.

2, a kind of method of extracting phosphatidylserine from animal brain according to claim 1, it is characterized in that raw phospholipid acyl Serine dry powder in the c operation is dissolved in the normal hexane, add the sodium-acetate ethanolic soln, stirring, standing demix, remove the lower floor of containing lipositol, top normal hexane evaporate to dryness gets high-purity phospholipid acyl Serine.

3, a kind of method of extracting phosphatidylserine from animal brain according to claim 1, it is characterized in that steaming in the c operation except that directly adding the normal hexane of roughly measuring together with surplus solution behind the ether, stirring, standing demix, remove the lower floor of containing lipositol, top normal hexane evaporate to dryness gets phosphatidylserine.

4, a kind of method from animal brain extraction phosphatidylserine according to claim 1 is characterized in that described stirring extraction is 3～5 times, each 1～3 hour.

5, according to claim 1ly a kind ofly extract the method for phosphatidylserine, it is characterized in that PH=7.5～11 of described ammoniacal liquor ethanolic soln, the PH=7.5 of described alkaline aqueous solution～11 from animal brain.

6, a kind of method from animal brain extraction phosphatidylserine according to claim 1 is characterized in that the described controlled temperature that thaws is 4～7 ℃.

7, a kind of method from animal brain extraction phosphatidylserine according to claim 2 is characterized in that described sodium-acetate ethanolic soln is meant 40～60% ethanol that contain sodium-acetate.