Procedure for the preparation of pure phosphatides and their use in the cosmetic, pharmaceutical and alimentary fields

Abstract

Process for preparing pure phosphatides starting from mixtures of natural phosphatides, or their single components, such as soybean or egg lecithin or animal phospholipids, or from synthetic phosphatides by reacting them both with phospholipase D derived from Streptomyces hachijoense in a completely aqueous medium in the presence of defined substates containing a primary or secondary alcoholic group, and their uses thereof.

1 . A phosphatidyl-L-serine product having a fatty acid composition identical to that of soybean lecithin and a degree of peroxidation of less than 5 produced by reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase d with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction, wherein said phospholipase D is purified by eluting on an anionic cationic exchange resin at a pH of 6.2.  
     
     
         2 . A phosphatidyl-L-serine product having a fatty acid composition identical to that of egg lecithin and a degree of peroxidation of less than 5 produced by reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase D with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction, wherein said phospholipase D is purified by eluting on an anionic cationic exchange resin at a pH of 6.2.  
     
     
         3 . A phosphatidyl-L-serine product having a fatty acid composition identical to that of soybean lecithin and a degree of peroxidation of less than 5 produced by reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase D with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction.  
     
     
         4 . A phosphatidyl-L-serine product having a fatty acid composition identical to that of egg lecithin and a degree of peroxidation of less than 5 produced by reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase D with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction.  
     
     
         5 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a phosphatide of formula (I) 
       R—O—PO(OH)—O—R 1   (I) 
       wherein R is diacylglycerol and R 1  is an hydroxyl group,  
       made by the process of reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase D with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction to obtain said phosphatide according to formula (I).  
     
     
         6 . A cosmetic composition comprising a pharmaceutically acceptable carrier and a phosphatide of formula (I) 
       R—O—PO(OH)—O—R 1   (I) 
       wherein R is diacylglycerol and R 1  is an hydroxyl group,  
       made by the process of reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase D with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction to obtain said phosphatide according to formula (I).  
     
     
         7 . A food and dietary supplement comprising a carrier and a phosphatide of formula (I) 
       R—O—PO(OH)—O—R 1   (I) 
       wherein R is diacylglycerol and R 1  is an hydroxyl group,  
       made by the process of reacting a phosphatide of formula (II): 
       R—O—PO(OH)—O—R 2   (II) 
       wherein R is diacylglycerol and R 2  is CH 2 —CH 2 —NH 2  or CH 2 —CH 2 —N(CH 3 ) 3 , with a primary or secondary alcohol with a chain length of between C2 to C4, optionally substituted with one or more polar groups selected from the group consisting of amino, hydroxy and carboxy, in a single aqueous phase in the presence of an effective amount of phospholipase D with transphosphatidylation activity produced from a  Streptomyces hachijoense  strain to catalyze the reaction to obtain said phosphatide according to formula (I).  
     
     
         8 . The food and dietary supplement according to  claim 7 , wherein the phosphatide according to formula (I) is phospatidyl-L-serine and wherein the  Streptomyces hachijoense  strain used to catalyze the reaction is ATCC 19769.  
     
     
         9 . A pharmaceutical composition capable of being administered orally comprising a pharmaceutically acceptable carrier and the phosphatidyl-L-serine product according to  claim 3  or  4 .  
     
     
         10 . A cosmetic composition for topical application to the skin comprising a pharmaceutically acceptable carrier and phosphatidyl-L-serine made by the process according to  claim 3  or  4 .  
     
     
         11 . A food and dietary supplement capable of being administered orally comprising a carrier and phosphatidyl-L-serine made by the process according to  claim 3  or  4 .  
     
     
         12 . A method of treating psycho-physical stress, attention, concentration and memory deficits commonly associated with advancing age, comprising administering a therapeutically effective amount of a pharmaceutical composition according to  claim 5 .  
     
     
         13 . A method of treating dermatitis or skin with impaired physiological functions comprising applying a cosmetic composition according to  claim 6  to the skin.  
     
     
         14 . A method of treating psycho-physical stress, attention, concentration and memory deficits commonly associated with advancing age comprising administering a food and dietary supplement according to  claim 7 .  
     
     
         15 . The pharmaceutical composition according to  claim 5  in the form of a capsule, tablet or granule.  
     
     
         16 . The cosmetic composition according to  claim 6  in the form of a cream or a gel.  
     
     
         17 . A food and dietary supplement according to  claim 7  in the form of a capsule, tablet or granule.  
     
     
         18 . A food and dietary supplement according to  claim 11  in the form of a capsule, tablet or granule.  
     
     
         19 . The food and dietary supplement according to  claim 8 , wherein the phosphatide of formula (II) is selected from the group consisting of purified soybean lecithin and crude soybean lecithin.