[미국특허]
Process for the Preparation and Isolation of Phosphatides
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
A61K-031/661
C12P-013/02
C12P-013/00
A61P-025/28
A61P-025/00
C12N-009/16
출원번호
US-0922680
(2006-05-26)
공개번호
US-0275006
(2008-11-06)
우선권정보
IT-PD2005A000164(2005-05-30)
국제출원번호
PCT/EP06/005030
(2006-05-26)
발명자
/ 주소
Zanellato,Anna Maria
Pittarello,Mara
Gambillara,Antonio
Vaccaro,Susanna
출원인 / 주소
Zanellato,Anna Maria
Pittarello,Mara
Gambillara,Antonio
Vaccaro,Susanna
대리인 / 주소
MATHEWS, SHEPHERD, MCKAY, & BRUNEAU, P.A.
인용정보
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초록▼
Process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X═OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transph
Process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X═OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphosphatidylation reaction between phosphatidylcholine of the general formula wherein R1 and R2 and X have the above specified meanings, R3═CH2--CH2--NH2 o CH2--CH2--N+(CH3)3 and Serine in D, L or racemic form catalysed by the phospholipase D enzyme (PLD), characterised in that said reaction is carried out in a hydroalcoholic medium containing an aliphatic alcohol and in the presence of bivalent metal oxide.
대표청구항▼
1. A process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X═OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the tr
1. A process for the preparation of phosphatidylserine of formula wherein R1 and R2 independently represent a saturated, mono-unsaturated or polyunsaturated acyl C10-C30, X═OH or OM where M=alkaline or alkaline earth metal, ammonium, alkylammonium (including the inner salt) including the transphosphatidylation reaction between phosphatidylcholine of the general formula wherein R1 and R2 and X have the above specified meanings, R3═CH2--CH2--NH2 0 CH2--CH2--N+(CH3)3 and Serine in D, L or racemic form catalysed by the phospholipase D enzyme (PLD), characterised in that said reaction is carried out in a hydroalcoholic medium containing an aliphatic alcohol and in the presence of bivalent metal oxide. 2. The process according to claim 1, characterised in that said reaction occurs in a medium containing an aprotic polar solvent and in the presence of a bivalent metal oxide. 3. The process according to claim 1, characterised in that said reaction is carried out in a medium consisting of a two-phase system formed by water/organic solvent and in the presence of bivalent metal oxide. 4. The process according to claim 1 wherein the aliphatic alcohols are selected from methanol, ethanol, n-propanol, isopropanol. 5. The process according to claim 4 wherein the aliphatic alcohol is isopropanol in a concentration between 0.1 and 50% expressed as a % by volume on the volume of the starting buffer. 6. The process according to claim 5 wherein isopropanol concentration is 10%. 7. The process according to claim 2 wherein the aprotic polar solvents are selected from dimethylsulphoxide, acetonitrile, dimethylformamide and N-methyl-pyrrolidone. 8. The process according to claim 7 wherein the aprotic polar solvent is dimethylsulphoxide in a concentration between 0.1 and 50% expressed as a % by volume on the volume of the starting buffer. 9. The process according to claim 8 wherein dimethylsulphoxide concentration is 1.25%. 10. The process according to claim 3 wherein the organic solvents are selected from n-hexane, toluene, benzene and n-butanol. 11. The process according to claim 10 wherein the organic solvent is n-hexane in a concentration between 0.1 and 40% expressed as % by volume on the volume of the starting buffer. 12. The process according to claim 11 wherein n-hexane concentration is 1.25% or 2.5%. 13. The process according to claim 7 wherein the bivalent metal oxide is calcium or magnesium or zinc oxide in a concentration between 0.1 and 1M. 14. The process according to claim 13 wherein the concentration of the selected oxides is 0.33 or 0.54 M. 15. The process according to claim 1 wherein the concentration of serine ranges between 1 and 5 gg/gg of phosphatidylcholine. 16. The process according to claim 15 wherein the concentration of serine ranges between 2 and 3 gg/gg of phosphatidylcholine. 17. The process according to claim 1 wherein the phosphatidylcholine is of animal and/or vegetal origin, natural or synthetic, present in purified form or as raw material, at an starting concentration of between 10 and 500 mg/ml, preferably between 200 and 300 mg/ml. 18. The process according to claim 1 wherein the transphosphatidylation reaction occurs at a temperature of between 20�� C. and 60�� C. and preferably at 45�� C. 19. The process according to claim 1 wherein the transphosphatidylation reaction occurs at a temperature of between 20�� C. and 60�� C. and preferably at 55�� C. 20. The process according to claim 1 wherein the enzyme PLD is of fermentative origin derived from the micro-organism Streptoverticillium hachijoense, used in purified, partially purified or non-purified form. 21. The process according to claim 20 wherein the concentration of PLD that is used varies between 1 and 100 units/g of phosphatidylcholine. 22. The process according to claim 21 wherein the concentration of PLD that is used varies between 1 and 10 units/g of phosphatidylcholine. 23. The process for the purification of phosphatidylserine (PS) produced according claim 1 and involving the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with the subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 24. The process for the purification of phosphatidylserine (PS) produced according to claim 1 comprising the following steps: I) adding a saline solution of sodium chloride to the PS that has been previously filtered, with the subsequent mixing and separation of the PS; II) collection and elimination of the supernatant; III) steps I and II can be repeated modifying the starting concentration of sodium chloride solution and eliminating the supernatant; IV) adding a solution of EDTA to chelate the ions present in the solution, and subsequent mixing; V) adding a ethanol solution consisting of ethanol at a percentage of 50 to 100% or a mixture of acetone/water consisting of acetone at a percentage of 50 to 95% with subsequent mixing and sedimentation of the PS; VI) step V can be repeated modifying the percentage of ethanol; VII) adding a ethanol solution consisting of ethanol at a percentage of 90 to 100%; VIII) collection and elimination of the supernatant; IX) drying to the final product obtained. 25. The process for the purification of phosphatidylserine (PS) produced according to claim 1 comprising the following steps: I) adding a saline solution of sodium chloride to the reaction medium containing the PS that has been produced, with subsequent mixing and separation of the PS; II) collection and elimination of the subnatant; III) adding a saline solution of sodium chloride and performing ultrafiltration through a porous membrane; IV) drying the final product. 26. Pharmaceutical compositions containing phosphatidylserine produced and purified as described in claim 1. 27. Use of the phosphatidylserines produced and purified as described in claim 1 for the preparation of drugs for the prevention and treatment of pathologies linked with cerebral aging. 28. Use of the phosphatidylserines produced and purified as described in claim 1 for the preparation of food supplements. 29. Use of the phosphatidylserines produced and purified as described in claim 1 for the preparation of liposomes to use in the cosmetic field and as a system for the controlled release of drugs. 30. The process according to claim 20 wherein the enzyme PLD derived from the micro-organism Streptoverticillium hachojoense is purified involving the following steps: I) elimination of the producing agent by microfiltration with a pore size of 0.2 μm; II) ultrafiltration through filters with a molecular cut-off of 10,000 D; III) ultrafiltration through filters with membranes with a molecular cut-off of 300,000 D; IV) ultrafiltration through membranes with a molecular cut-off of 10,000 D to re-concentrate the enzyme and dialyse it against acidic buffer.
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