CN1583766A - Extraction of phosphatidyl serine from brains of animals - Google Patents
Extraction of phosphatidyl serine from brains of animals Download PDFInfo
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- CN1583766A CN1583766A CN 200410024225 CN200410024225A CN1583766A CN 1583766 A CN1583766 A CN 1583766A CN 200410024225 CN200410024225 CN 200410024225 CN 200410024225 A CN200410024225 A CN 200410024225A CN 1583766 A CN1583766 A CN 1583766A
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Abstract
Phosphatidylserine is extracted from animal brains by: drying animal brains, smashing them into granules, putting propanone into the granules, stirring and extracting, separating propanone supernatant liquid contained cholesterine and oils from the brain granules, steaming the residues to be dried, adding n-hexane or alcohol or ammonia water alcohol into steamed dry residues, stirring and extracting, separating extracting liquid contained phospholipid compositions, steaming the residues again, putting ether into steamed dry residues, stirring, freezing and defreezing, filtering to remove deposits, removing the remained phospholipid further, adjusting supernatant liquid pH=3-6, steaming to remove ether, and steaming to dry crude phosphatidylserine again, dissolving crude phosphatidylserine dry powders in n-hexane, adding sodium acetate alcohol liquid, stirring, settling to be laminated, removing the supernatant contained inositol phospholipid, finally, steaming to dry the n-hexane in supernatant liquid to obtain high pure phosphatidylserine under reduced pressure.
Description
Technical field
The present invention relates to the method that the livestock animals brain extracts phosphatidylserine, being particularly suitable for livestock animals brains such as ox, sheep, rabbit, horse, donkeys is the extracting method of the phosphatidylserine of raw material.
Background technology
Ox kephalin acyl Serine (PS) has hypermnesis, effects such as anti-ageing year dementia are by experimentation on animals both at home and abroad and clinical verification, referring to, Liu Ping. the pharmacology of phosphatidylserine and clinical application research progress, foreign medical science pharmacy fascicle 1981,18 (4), 207-230.But with the ox brain is raw material, and batch extracting is produced PS and had any problem.Chromatography preparation and the analytical procedure of summary such as Li Si M.B etc. (1981) and Ah's Bhide (1998) PS, preparation method and complexity thereof.Referring to Li Si MB etc., phosphatide and steroid alanysis and preparation (Lees MBet al.preparation and Analysis of phosphatides Lipids and steroids, 1981,328-345) and Ah's Bhide etc., chromatographic science B collection 1998, (717) 279-293 (Abidi, SL et al.Journal ofchromatography B, 717 (1988) 279-293). research such as Chen Su high-pressure liquid phase preparation method, though can be used for reagent preparation, output is limited really.Referring to Chen Su etc. chromatographic science B collection 1995, (666) 178-182 (Su chen et al.Journal of chromatography B.666,1995,178-182).The U.S. is synthetic PS under the Phospholipase D effect with soybean phospholipid and L-Serine, and studies and successfully extract the preparation method, referring to United States Patent (USP) 5700668 (united states patent 5700668).But this PS that can not show a candle to the extraction of ox brain with its clinical effectiveness of soybean phospholipid synthetic PS.Through many repeated experiments probatio inspectionem pecuoarems, this method also is not suitable for animal brain extraction PS such as ox.
China's livestock-raising is fast-developing, and the beef cattle amount of butchering is very big, and the ox brain is sold with the ox head cheapness, and what have is edible, and what have throws away.In the last few years, we were engaged in the extraction research of ox brain PS always, for the increment utilization of ox brain break a new path son, grope success and can extract the preparation method by industrial ox brain PS.
Summary of the invention
The purpose of this invention is to provide a kind of is that the technology of raw material is simple and be suitable for the method for the extraction phosphatidylserine of suitability for industrialized production with livestock animals brains such as ox, sheep, rabbit, horse, donkeys.
Purpose of the present invention can realize by following technical measures
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.
Purpose of the present invention also can realize by following technical measures
Raw phospholipid acyl Serine dry powder in the c operation is dissolved in the normal hexane, adds the sodium-acetate ethanolic soln, stirring, standing demix are removed the lower floor of containing lipositol, and top normal hexane evaporate to dryness gets high-purity phospholipid acyl Serine; Perhaps with steam to remove in the c operation directly add behind the ether with surplus solution roughly with the normal hexane of amount, stirring, standing demix are removed the lower floor of containing lipositol, top normal hexane evaporate to dryness gets phosphatidylserine; Foregoing stirring extraction is 3～5 times, each 1～3 hour; PH=7.5～11 of described ammoniacal liquor ethanolic soln and alkaline aqueous solution; The described controlled temperature that thaws is 4～7 ℃; Described sodium-acetate ethanolic soln is meant 40～60% ethanol that contain sodium-acetate.
Technical measures of the present invention are earlier dry animal brain to be broken into the particle that is roughly 0.2～0.4cm, and adding volume again in particle is the acetone of 3～5 times of amounts, stir extraction, remove and contain cholesterol and oils in the dry cerebral particle.If when the solvent of extracting lipositol and PS phospholipid complex was ether, the ether add-on was 3～5 times of amounts of the residue volume of evaporate to dryness, ether extracted liquid, can boil off 3～5 times of amounts of most of ether dry-matter that to make remaining diethyl ether solution volume be ether extraction.This diethyl ether solution is joined the Na of PH=7.5～11
2CO
3Salify in the aqueous solution stirred 1 hour, and-10～20 ℃ are freezed, and 4～7 ℃ slowly thaw.Remove the bottom precipitation, supernatant liquor is transferred PH=3～6 with 2～5%HCl.Steam and remove ether, evaporating water gets dry powder; Or steam except that adding behind the ether and the normal hexane of surplus solution with amount, top normal hexane layer is got in the stirring layering, boils off normal hexane and gets dry-matter.And then to 3～5 times of normal hexanes of dry-matter adding (or top normal hexane layer that the last normal hexane that adds obtains in above-mentioned), adding 40～60% ethanol liquid that 5～10 times of amount of dry matters of volume contain sodium-acetate again stirs, static layering, remove bottom white precipitate (being mainly lipositol) and get top normal hexane layer, evaporate to dryness gets phosphatidylserine, and phosphatidylserine purity is 90～93%.
Technical measures of the present invention can be steamed most of normal hexane and remove if the solvent of extractive lipositol and phospholipid complex is a normal hexane, and residue hexane solution volume is 3～5 times of normal hexane extraction dry-matter.Remove impurity such as lipositol then earlier, normal hexane steamed remove again, be dissolved in ether behind the dry-matter, steam at last and remove ether, phosphatidylserine, purity is 92～95%.
Technical measures of the present invention are if remove cholesterol and grease in the decerebrate with acetone, the dry dry cerebral particle that gets, adding volume again in dry cerebral particle is the normal hexane of 3～5 times of amounts, stir extraction 3～5 times, each 1～3 hour, get normal hexane extraction liquid, steam and remove the normal hexane that contains brain total phospholipids mixture, evaporate to dryness gets raw phospholipid acyl Serine again after ether is handled, and purity is 53%.Adopt this scheme to save and use a large amount of ethanol.
Described ammoniacal liquor available hydrogen sodium oxide, potassium hydroxide, Sodium phosphate dibasic, dipotassium hydrogen phosphate, sodium bicarbonate, saleratus, yellow soda ash, salt of wormwood, sodium-acetate or Potassium ethanoate substitute, but effect is best with ammoniacal liquor.Described normal hexane can use chloroform, methylene dichloride, toluene, ether to substitute, and generally is preferred with the normal hexane, to reduce the kind that organic solvent uses, reduces the construction of organic solvent recovery tower, simplifies production technique.
The present invention selects conventional organic solvent extraction for use, can realize large-scale industrial production.The brain grain removes de-cholesterol and oils with acetone, remove Yelkin TTS with ethanol, kephalin is the mixture of phospholipids of main component with phosphatidylserine and lipositol with normal hexane, ether extraction, remove avale lipositol and impurity with normal hexane, alkali alcohol solvent again, get raw phospholipid acyl Serine.Raw phospholipid acyl Serine is dissolved in ether, this diethyl ether solution adds salify in the basic solution, and is freezing, thaws to remove precipitation, and supernatant liquor is transferred PH=3～6.Normal hexane stirs and extracts, and standing demix gets the normal hexane layer, boils off normal hexane and gets high-purity phospholipid acyl Serine.Also phosphatidylserine and the lipositol phospholipid complex that extracts can be dissolved in ether, be dissolved in the freezing removal of impurities of alkaline aqueous solution again, and then remove lipositol, get high-purity phospholipid acyl Serine with normal hexane, alkali ethanol.The brain particle that also available normal hexane extraction removes de-cholesterol and oil gets the total phospholipids mixture, and operation is thereafter extracted the phosphatidylserine method with the phospholipid complex that with phosphatidylserine and lipositol is principal constituent, but purity is lower.
Embodiment
Embodiment 1
1, bright ox brain (or refrigerated ox brain) is cut into slices, 60 ℃ of oven dry of ventilating, but also dry under the natural condition or dry, drying temperature is low more good more, and cryodrying is best, be broken into the particle about 0.3cm, in exsiccant ox brain particle, add the acetone that volume is 4 times of amounts, stirring at normal temperature extraction 4 times, each 2 hours, remove the acetone supernatant liquor, residue steams to there not being the acetone flavor;
2, adding volume in the residue of evaporate to dryness is the ammoniacal liquor ethanolic soln of the PH=9 of 4 times of amounts, stirs extraction 4 times, and each 2 hours, residue evaporate to dryness, extracting solution can be used for extracting Yelkin TTS and kephalin again;
3, in the residue of evaporate to dryness, add ether, ether extracted liquid, filter to steam remove most of ether, till the volume of last diethyl ether solution is 4 times of dry-matter of ether extraction.It is joined the Na of PH=8.5
2CO
3In the aqueous solution, stirred 1 hour ,-18 ℃ are freezed, and 6 ℃ slowly thaw.As seen faint yellow supernatant and bottom precipitation is removed post precipitation supernatant liquor is transferred PH=5 with 4%HCl, and evaporating water gets dry powder.
4, dry powder is dissolved in the normal hexane that volume is 4 times of amounts, adding volume again is 50% ethanol that contains sodium-acetate of 8 times of dry powder amounts, stirring, static layering, visible following adularescent precipitation (precipitation can be used for extracting lipositol).Then top normal hexane layer is shifted out, evaporated under reduced pressure gets phosphatidylserine, and purity is 93%.
Embodiment 2
If 1 raw materials used be livestock animals brains such as bright rabbit brain, sheep brain, horse brain, donkey brain, the drying means of brain is with the ox brain, if raw materials used for extracting the dried brain particle after the cholesterol, can be directly by 2 beginning to do among the embodiment 1, down with.
Embodiment 3
Step 2 among the embodiment 1 adds the ammoniacal liquor ethanolic soln in the residue of evaporate to dryness, be Yelkin TTS and the kephalin that contains in the decerebrate slag in order to remove.
This step also can add volume earlier in the brain slag be the dehydrated alcohol of 4 times of amounts, stirs extraction 4 hours, extracts 4 times, each 2 hours, can extract and contain about 50% Yelkin TTS, also contain about 25% kephalin and other phospholipids, but principal constituent is a Yelkin TTS, helps being further purified of Yelkin TTS.And then, stir extraction 4 times, each 2 hours with 4 times of ammoniacal liquor extraction using alcohols of measuring PH=9 of slag, contain kephalin about 60% in the extract after doing, also contain 10% Yelkin TTS and other phosphatide, but principal constituent is a kephalin, be beneficial to the further separation of kephalin, other steps are with embodiment 1.
Embodiment 4
Step 3 among the embodiment 1 adds the ether extraction of 4 times of amounts to the residue of evaporate to dryness, and the also available normal hexane of ether, toluene or chloroform, methylene dichloride substitute, the same ether of extracting method, but extract is added Na
2CO
3During the aqueous solution, must earlier (toluene, chloroform, methylene dichloride) such as normal hexanes be evaporated, use 3 times of ether dissolutions then, add Na again
2CO
3Alkali lye, other steps are with embodiment 1.
Embodiment 5
As usefulness normal hexane extraction in embodiment 1 step 3 and without ether extraction, normal hexane extraction liquid can be concentrated, the volume of last hexane solution is 4 times of amounts of the dry-matter that it extracted, add volume then and be 7 times of amount of dry matters 45% ethanol that contains sodium-acetate, stirring, static, layering, remove the lipositol precipitation of white.Shift out the normal hexane layer, steaming removes normal hexane and gets dry powder, and the dry powder volume is 4 times of amount ether dissolutions, adds the Na of PH=8
2CO
3The aqueous solution stirred 1 hour, and-15 ℃ are freezed, and 6 ℃ slowly thaw, and got supernatant liquor, transferred PH=5 with 3%HCl, and after ether was reclaimed in evaporation, evaporating water got phosphatidylserine dry powder.After being preferably in evaporation recovery ether, add normal hexane in remaining aqueous solution, stir, the static normal hexane layer that gets steams and removes normal hexane, and phosphatidylserine purity is 94%.
Embodiment 6
1, as the method for embodiment 1 step 1 acetone extracting cholesterol and oils, the normal hexane (normal hexane can use ether, methylene dichloride, chloroform, toluene to replace) that in the dry cerebral particle of acetone extracting, adds 4 times of amounts, 4 extractings, each extracting 2 hours, can get hexane solution, steaming removes normal hexane can get total phospholipids mixture in the brain.
2, as step 3 among the embodiment 1, above-mentioned extractive total phospholipids mixture is dissolved in ether, and below operation is as step 3 among the embodiment 1.After slowly thawing, can remove a large amount of Yelkin TTS, kephalin precipitation, supernatant liquor transfers PH=6 to steam except that behind the ether, and evaporating water gets thick PS dry powder, and dry powder phosphatidylserine content reaches 53%.
3, above-mentioned thick PS dry powder can get raw phospholipid acyl Serine by 4 operations among the embodiment 1, and purity reaches 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 1 is characterized in that described sodium-acetate ethanolic soln is meant 40～60% ethanol that contain sodium-acetate.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100242257A CN1308334C (en) | 2004-06-01 | 2004-06-01 | Extraction of phosphatidyl serine from brains of animals |
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| CNB2004100242257A CN1308334C (en) | 2004-06-01 | 2004-06-01 | Extraction of phosphatidyl serine from brains of animals |
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| CN1583766A true CN1583766A (en) | 2005-02-23 |
| CN1308334C CN1308334C (en) | 2007-04-04 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559371A (en) * | 2012-02-21 | 2012-07-11 | 山东师范大学 | Method for preparing phosphatidylserine-enriched krill oil |
| CN102964379A (en) * | 2012-11-16 | 2013-03-13 | 成都圆大生物科技有限公司 | Method for preparing phosphatidylserine and phosphatidylcholine |
| CN103262893A (en) * | 2013-06-13 | 2013-08-28 | 俞祖勋 | Formula milk powder capable of improving intelligence |
| CN107242574A (en) * | 2017-06-12 | 2017-10-13 | 芜湖福民生物药业股份有限公司 | Electuary containing phosphatidylserine and preparation method thereof |
| CN107259041A (en) * | 2017-06-12 | 2017-10-20 | 芜湖福民生物药业股份有限公司 | Phosphatidylserine pressed candy and preparation method thereof |
| IT202100032252A1 (en) * | 2021-12-22 | 2023-06-22 | Fidia Farm Spa | Industrial extraction and purification process of phospholipids |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1212900B (en) * | 1983-11-17 | 1989-11-30 | Valle Francesco Della | THERAPEUTIC USE OF PHOSPHATIDYLSERINE IN DISEASES OF THE CENTRAL NERVOUS SYSTEM WITHOUT EFFECTS ON BLOOD COAGULATION |
| US5700668A (en) * | 1995-12-08 | 1997-12-23 | Italfarmaco Sud S.P.A. | Process for the industrial preparation of phosphatidylserine |
-
2004
- 2004-06-01 CN CNB2004100242257A patent/CN1308334C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559371A (en) * | 2012-02-21 | 2012-07-11 | 山东师范大学 | Method for preparing phosphatidylserine-enriched krill oil |
| CN102559371B (en) * | 2012-02-21 | 2013-09-18 | 山东师范大学 | Method for preparing phosphatidylserine-enriched krill oil |
| CN102964379A (en) * | 2012-11-16 | 2013-03-13 | 成都圆大生物科技有限公司 | Method for preparing phosphatidylserine and phosphatidylcholine |
| CN102964379B (en) * | 2012-11-16 | 2015-10-21 | 成都圆大生物科技有限公司 | A kind of method preparing phosphatidylserine and phosphatidylcholine |
| CN103262893A (en) * | 2013-06-13 | 2013-08-28 | 俞祖勋 | Formula milk powder capable of improving intelligence |
| CN107242574A (en) * | 2017-06-12 | 2017-10-13 | 芜湖福民生物药业股份有限公司 | Electuary containing phosphatidylserine and preparation method thereof |
| CN107259041A (en) * | 2017-06-12 | 2017-10-20 | 芜湖福民生物药业股份有限公司 | Phosphatidylserine pressed candy and preparation method thereof |
| IT202100032252A1 (en) * | 2021-12-22 | 2023-06-22 | Fidia Farm Spa | Industrial extraction and purification process of phospholipids |
| WO2023119129A1 (en) * | 2021-12-22 | 2023-06-29 | Fidia Farmaceutici S.P.A. | Industrial process for the extraction and purification of phospholipids |
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| CN1308334C (en) | 2007-04-04 |
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