WO1991015494A1

WO1991015494A1 - Process for separating the main components of a mixture of raw deacylated phospholipids

Info

Publication number: WO1991015494A1
Application number: PCT/EP1991/000618
Authority: WO
Inventors: Giovanni Tronconi; Costantino Coccoli
Original assignee: Istituto Chemioterapico Italiano Fine Chemicals S.P.A.
Priority date: 1990-03-30
Filing date: 1991-03-28
Publication date: 1991-10-17
Also published as: IT9019895A1; IT9019895A0; IT1240614B; EP0522021A1

Abstract

A process for purifying L-alpha-glyceryl-phosphoryl-serine (GPS), L-alpha-glyceryl-phosphoryl-choline (GPC), L-alpha-glyceryl-phosphoryl-ethanolamine (GPE) from raw deacylated mixtures obtained by the deacylation of raw mixtures of the corresponding phosphatides is disclosed, which comprises the steps of: (a) separating GPS, from the raw deacylated mixtures, in the form of one of its salts with an alkli-metal or of a metal belonging to the 2nd Group, from which, pure GPS is obtained by subsequent purification, (b) purifying the raw mixture of GPC + GPE, obtained after the separation of GPS, by elution on cationic resins in their H form, firstly in a non-aqueous medium and subsequently in an aqueous medium, in order to obtain a pure mixture of GPC + GPE, from which GPC and GPE are subsequently obtained in pure form by elution on strong basic resins.

Description

"PROCESS FOR SEPARATING THE MAIN COMPONENTS OF A MIXTURE OF RAW DEACYLATED PHOSPHOLIPIDS"

The present invention relates to a process for separating the main components of a mixture of raw deacylated phosphoIipids.

Such main components which constitute the subject- matter of the instant invention are referred to by the short names "GPS", "GPC" and "GPE".

In the following disclosure the short designations "GPS", "GPC" "GPE"and "GPI"are used for the sake of reader's convenience, in order to refer to L-alpha- glyceryl-phosphoryl-serine, L-alpha-glyceryl- phosphoryl-choline, L-alpha-glyceryl-phosphoryl- ethanolamine and L-alpha-glyceryl-phosphoryl-inositole, respectively, and the short names "PS", "PC " , "PE" and "PI" are respectively used in order to refer to phosphatidyl-serine, phosphatidyl-choline, phosphatidyl-ethanolamine and phosphatidyl-inositole.

Hawthone (Biochem J. 71, 195, 1959) obtains GPS by using purified phosphatidyl-serine and performing a chromatography on strong, basic ion-exchange resins in their formate form.

The difficulty of separation is well evidenced by the Author, which sets forth that among the various eluent mixtures used, he obtains positive results only when he uses, as eluent, a mixture of sodium tetraborate and ammonium acetate with a concentration gradient. The method is only suitable for analytical purposes and is also extremely onerous, in that it requires that a very pure PS is prepared. In GB-2 058 792, pure GPC is obtained by the deacylation of pure phosphatidyl-choline. The method is very onerous because uses the phosphatide in pure form, and furthermore is unsuitable for preparing GPC, GPE and GPS from raw mixtures of the corresponding phospholipids, because it does not provide any purification (in addition to the elimination of the esters of fatty acids formed during the deacylation of pure phosphatidyl-choline).

In European patent application EP-217 765, we disclosed the preparation of GPC and GPE from raw phospholipids which do not contain PS (soy- and egglecithin). By means of such a process, the insoluble complex

(GPC+GPE)ZnCl₂ (I) is separated from the alcoholic solution of the raw deacylated mixture; then, from the aqueous solution of complex (I), zinc chloride is removed by precipitation and subsequent filtration of the

ZnCl₂.Py₂

complex (in which Py stands for pyridine), insoluble in water, with a pure solution of GPC + GPE being obtained, from which pure GPC and GPE are then separated on strong bases in OH form.

When raw phospholipids containing PS (such as, e.g., from brain, spinal medulla, from lecithins which have undergone a PC-PS enzymatic exchange, such as, e.g.: P. Comforius, Biochem. Biophys. Acta 488, 1977, page 36; Fujita: Jpn. Kokai T.K. JP-63 36, 791, 1988: CA vol. 109, 72066 t, 1988), the insoluble complexes (I) are not obtained, so that a mixture of GPC + GPE -- from which GPC and GPE would be subsequently separated -- cannot be obtained.

Therefore, the method does not make it possible GPC and GPE to be obtained with a very high purity Level, when raw phospholipids also containing PS are used.

Summing-up, to date a method for obtaining pure GPS, GPE, GPC from raw phospholipids containing the three corresponding phosphatides (PS, PE, PC), does not exist.

According to the present invention, a process is proposed, which makes it possible pure GPS, GPE, GPC to be obtained by deacylating raw phospholipids which contain meaningful amounts of PS, FE and PC.

The greatest difficulty to be faced when one tries to purify raw mixtures of GPC + GPE + GPS, consists in the separation of GPE from GPS, but, inasmuch as it has anyway been observed that GPS can be advantageously separated from raw mixtures of GPC + GPE + GPS as an insoluble salt with metals belonging to the 2nd Group or by co-precipitation with sodium or potassium sulfate or phosphate; the method according to the present invention is relatively simple and makes it possible GPC, GPE and GPS to be obtained with a high purity level, and can be easily applied to the large-scale preparation.

According to such a process, the raw, deacylated mixture containing GPC, GPE and GPS is treated with a compound selected from the following ones: chloride, bromide, acetate of one of the following metals: calcium, magnesium, barium, zinc, iron; or is treated with sodium or potassium sulfate or phosphate (obtained in situ by means of the reaction of sodium or potassium methoxide or ethoxide with sulfuric acid or phosphoric acid). In such a way, the separation of GPS from the mixture of GPC + GPE is obtained, in the form of its insoluble salt with the general approximate formula:

(GPS)_xM_y (III) in which:

GPS is the anion of L-alpha-glyceryl-phosphoryl-serine; x has the approximate values of 1, 2 and 3;

y has the approximate values of 1, 2 and 2.5;

M is: Ca, Mg, Ba, Zn, or Na**, K* *

** As briefly mentioned above, the treatment of the raw deacylated mixture with sodium or potassium sulfate or phosphate, obtained by causing the corresponding acid to react with sodium or potassium methoxide or ethoxide, leads to the co-precipitation of the sodium or potassium salt of GPS (GPS-Na₂ or GPS-K₂) with sodium or potassium sulfate or phosphate. The amount of sodium or potassium sulfate or phosphate which respectively accompanies GPS-Na₂ or GPS-K₂ depends on the conditions of precipitation. It seems anyway that in order to obtain a nearly quantitative separation of GPS, one should operate in such a way as to obtain a molar ratio of GPS-Na₂ /sodi urn sulfate or phosphate, or GPS-K₂ /potassium sulfate or phosphate, of at Least 1/12.

Inasmuch as the amount of sodium and potassium sulfate and phosphate is not fixed and well- determined, the so obtained GPS-Na₂ and GPS-K₂ will be simply referred to as GPS-Na₂** and GPS-K₂**.

The salt with the approximate general formula (III) (with M = Ca, Mg, Ba, Zn; x = 2; 3 and y = 1; 2; 2.5) is subsequently purified by precipitation from water and acetic acid, with pure GPS being then obtained after treatment with acidic ion-exchange resins, and in aqueous medium. On the contrary, the salt with approximate general formula (III) (with M = Na, K; x = 1 and y = 2) is converted into the salt with approximate general formula (III) (with M = Ca, Hg, Ba, Zn); x = 2; 3 and y = 1; 2; 2.5) from which, after purification as disclosed above, pure GPS is obtained.

From the raw solution obtained after the separation of GPS as its insoluble salt with formula (III), which contains the mixture of GPC + GPE, a pure mixture of GPC + GPE is obtained firstly by means of the purification of the raw mixture on cationic resins in their H form and in a non-aqueous medium. From the pure mixture obtained in that way, pure GPC and GPE are then obtained by elution on strong basic resins, and in an aqueous medium. If from the raw deacylated mixture containing GPC, GPE and GPS only GPC has to be obtained, the most advantageous way of operating consists in not isolating GPS, but obtaining, by purification on cationic resins in H form and in a non- aqueous medium, a pure mixture of GPC + GPE + GPS, and subsequently separating GFC from GPE + GPS on strong basic resins in an aqueous medium, as is better explained in the following.

The purification of raw deacylated mixtures of GPC + GPE + GPS or of GPC + GPE obtained by means of the deacylation of raw phosphatides containing PS, is analogous to the purification of raw, deacylated mixtures of GPC + GPE obtained from soy lecithin or egg lecithin, which we disclosed in PCT/EP 90/00717, May 4th, 1990.

Said purification exploits the amphoteric character of GFC, GPE and GPS and consists in eluting on a cationic resin in H form, in a non-aqueous solvent, the raw solution obtained from the deacylation, or the raw solution obtained after the separation of GPS, by washing the resin firstly with solvent and then water. During the elution of the raw deacylated mixture in a non-aqueous medium, GFC, GPE and GPS, as well as the other basic impurities are stably fixed to the resin, which therefore can be thoroughly washed with solvent, making it possible all the acidic and neutral impurities to be removed, without GPC, GPE and GPS being eluted.

Then, when the resin is washed with water, inasmuch as GPC, GPE and GPS are very weak bases, in an aqueous medium the equilibrium between resin-bonded bases and free bases is practically shifted in the direction of the free bases, so GPC, GPE and GPS are immediately eluted, to a practically quantitative extent. In such a way, also the impurities are removed, which have a stronger basic character than GPC, GPE and GPS, such as, e.g., traces of ethanolamine, choline and serine, because when cationic resins in H form are used, the latter are only eluted when acidic or basic eluents, or eluents consisting of salt solutions, are used, and are not eluted when water is used as the eluent.

The purification of raw mixtures of GPC + GPE + GPS or GPC + GPE (which, for the sake of convenience, are indifferently referred to in the following as "GPC/E/S") on cationic resins in H form and in a non- aqueous medium, requires well-defined operating conditions (particular couplings of resins/solvents/purification times), without which stable bonds cannot be formed between GPC/E/S and the resin, and the strong degradation (which sometimes may even be total) which GPC/E/S generally undergo in a non-aqueous medium on strong cationic resins in H form, cannot be avoided.

In general, to be able to form stable GPC/E/S- resin bonds, the carboxy resins should be equilibrated in C₂-C₄ alcohols or alcohol mixtures (ethanol, isopropanol, and so forth), and do not cause an appreciable degradation. On the contrary, when methanol is used, GPC, GPE, GPS do not form stable bonds with the carboxy resins, but are eluted together with the impurities. When sulfonic resins are used, stable bonds are formed by using C₁-C₄ alcohols or alcohol mixtures

(methanol, ethanol, and so forth). Anyway, GPC/E/S should be kept bonded to the resin for short time periods, and the minimal essential amounts of resins should be used (which amounts shall be evaluated according to the type of resins), in order to prevent

GPC/E/S from undergoing the degradation they are prone to, on the sulfonic resins and in a non-aqueous medium.

The process according to the present invention can be described as follows in its general form.

The mixture of raw phospholipids in alcoholic suspension (preferably in methanol or ethanol) is treated with an alkali-metal alkoxide (preferably sodium or potassium methoxide or ethoxide).

After the insoluble residue being filtered off, the pH of the alcoholic solution containing GPC, GPE, GPS (1) is adjusted to a value comprised within the range of from 7 to 10.5 with a mineral acid (either sulfuric or phosphoric acid). After filtration, a residue is obtained which contains GPS-Na₂ or GPS-K₂ (2), together with an alcoholic solution containing GPC, GPE (3). The residue (2) is washed with an alcohol (preferably methanol, ethanol or isopropanol), then is extracted with an alcohol (preferably methanol, ethanol o r i s o p ro pano l ) c on t a i n i ng a w a t e r pe r c e n t a g e preferably comprised within the range of from 3 to 10% (volume/volume = v/v), and the pH is adjusted to a value comprised within the range of from 5 to 7.

From the water-alcoholic extract which contains GPS-Na₂ or GPS-K₂ (4), sodium or potassium sulfate or phosphate is eliminated first by filtering off the insoluble residue, then an alcoholic solution (preferably in methanol or ethanol) of a divalent metal (preferably calcium, barium, magnesium, zinc bromide or chloride or acetate) with a concentration preferably comprised within the range of from 10 to 20% (weight/volume = w/v) is added.

The insoluble salt

(GPS)_xM_y (III) (in which M is Ca, Mg, Ba, Zn, x has the approximate values of 2 or 3, and y has the approximate values of 1, 2 and 2.5) precipitates off, and is dissolved in water, preferably at a concentration of 30-60%, and is precipitated by means of the addition of acetic acid in such an amount that a ratio of acid/water preferably comprised within the range of from 20 to 50 v/v is obtained. From the so obtained salt (III), pure GPS is obtained by elution on an acidic resin in an aqueous medium, concentration of the obtained aqueous solution down to a small volume, and precipitation with acetone, (inasmuch as GPS is a very hygroscopic solid and cannot be maintained in aqueous solution over very long time periods, because, differently from GPC, it tends to decompose, it may sometimes be more advantageous to isolate the product not in its free acid form, but in the form of its salt with a divalent metal which -- as is better evidenced in Example III -- can be easily obtained and is not hygroscopic).

The alcoholic solution (3), which contains GPC and GPE, is eluted on a cationic resin in H form equilibrated in C₁-C₄ alcohols. The resin is washed with the same solvent as used to equilibrate the same resin, until all impurities are removed. The resin is then washed with water and a pure aqueous solution of GPE + GPC is obtained, from which pure GPC and GPE a r e easily obtained by elution on basic resins in OH form.

According to an alternative route, the alcoholic solution (3) is adjusted to a neutral pH with acetic acid, hydrochloric acid, or with small amounts of acidic resin, then the solution is concentrated to a small volume, the separated oil phase is discarded and the alcoholic phase containing raw GPC + GPE is purified as disclosed above.

If only obtaining GPS is desired, the alcoholic solution (1) is adjusted to a pH value comprised within the range of from 6 to 9 with hydrochloric or acetic acid, or with a small amount of acidic resin, then an alcoholic solution (preferably in methanol or ethanol) of a salt of a divalent metal (preferably calcium, barium, magnesium, zinc bromide/chloride/acetate) at a concentration preferably comprised within the range of from 10 to 20% w/v is added, and the insoluble salt (III) (in which M is Ca, Mg, Ba, Zn; x has the approximate values of 2 or 3; and y has the values of 1, 2 and 2.5) is isolated.

The so obtained salt is purified by precipitation from water/acetic acid, and GPS is isolated as disclosed hereinabove.

If, instead of only GPS, also GPC and GPE have to be obtained, it is more advantageous to isolate GPS-Na₂ or GPS-K₂, because in that case the soLution containing GPC and GPE, which must be purified, does not contain MX2 and therefore smaller amounts of cationic resins in H form can be used for the purification.

If, on the contrary, only GPC should be obtained, in that case it is more advantageous to directly elute the alcoholic solution (1) on a cationic resin in H form and in a non-aqueous medium. In that way, a pure aqueous solution of GPC + GPE + GPS is obtained first, from which pure GPC is easily obtained (as is better explained in Example IV), because GPS is retained by the basic resins together with GPE, and the precipitation, separation and washing of the salts of formula (III) is thus avoided.

In order to better understand the characteristics and advantages of the instant invention, in the following non-limitative examples of practical embodiment thereof are given.

Example I

Preparation of GPS GPC and GPE

(a) Preparation of GPS

T h e su s pens i on o f 250 g o f r aw p ho s pholipids

(PC approximately 18%, PE approximately 18%, PS approximately 5%, PI approximately 3%) in 1.2 I of methanol containing 11.2 g of sodium methoxide is stirred at room temperature and in an anhydrous medium for 18 hours. The resulting mixture is filtered and the residue is washed with methanol (3 x 50 ml), which is combined with the filtrate. The pH of the methanolic solution (1) is adjusted to a value of 9 with 40%-H₂SO₄, the solution is kept stirred for 30 minutes, the resulting mixture is filtered and the residue is washed twice, each time with 50 ml of methanol and the methanol washes are combined with the filtrate (2). The residue (GPS- Na₂) is dissolved in 50 ml of water containing 10 ml of acetic acid, the solution is diluted with 500 ml of methanol and then is stirred at room temperature for 2 hours. The resulting mixture is filtered and the residue (Na₂SO₄) is washed twice with 50 ml of methanol each time. The methanol washes are added to the filtrate. 30 ml of a 10% (w/v) solution of zinc acetate in methanol is added, with stirring, to the water-alcoholic solution so obtained (3). The resulting mixture is stirred to room temperature for 3 hours, then is filtered. The residue is washed with methanol (2 x 50 ml). The solid residue is dissolved in water (5 ml), 200 ml of acetic acid is added, the resulting mixture is stirred at room temperature for 12 hours. The mixture is filtered, the solid residue is washed with methanol and then is dried under vacuum at 40°C over P₂O₅. 3.3 g is obtained of a mixture of salts with the approximate formula (GPS)₃Zn₂.

Computed

* Zn⁺⁺ = 13.89% = 14.46%

* GPS = 85.50% = 85.54% The solid is dissolved in water (10 ml) and is eluted on 5.0 ml of IR200 H equilibrated in water. The resin is washed with 100 ml of water. The solution is concentrated to a small volume under vacuum (approximately 8 ml), 10 ml of methanol is added and the solution is then poured in 200 ml of acetone. The mixture is stirred at room temperature for 8 hours, is filtered, the solid is washed with ethanol (2 x 20 ml), then is dried under vacuum over P₂O₅.

2.2 g of GPS (a very hygroscopic solid) is obtained.

Computed for C₆H₁₄NO₈P Found (M.W. = 259.154)

C = 27. 90 C = 27 . 81

H = 5 . 5 1 H = 5 . 44

N = 5. 35 N = 5 . 40

α D = -2.20 (c = 2.8 in H₂O)

α D = +4.52 (c = 2.1 in 1 N HCl)

1H-N.M.R. (D₂O):

δ = 3.00 - 3.90 (m2H)

δ = 3.90 - 4.15 (m3H)

δ = 4.30 - 4.70 (m4H)

(b) Preparation of GPC

The methanolic solution (2) is neutralized with acetic acid, and is concentrated down to a volume of about 350 ml. The oil phase (the bottom phase) is separated, then the methanolic phase (the upper phase) is eluted on 250 ml of IR200 H, equilibrated in methanol. The resin is washed with methanol (about 1 L) until the impurities are completely eliminated (TLC-check of eluted methanol), then is washed with water (about 800 ml). The water solution (containing pure GPC + GPE) is treated with 2 g of decolourizing charcoal, is filtered and eluted first on 120 ml of IR 93 OH resin (1) -- equilibrated in water -- by washing the resin with 200 ml of water, and then on 120 ml of resin IR 401 OH (2) -- equilibrated in water -- by washing the resin with 200 ml of water, and finally on 20 ml of IRC50 H (3) -- equilibrated in water -- by washing the resin with 40 ml of water.

Air is removed under vacuum from the aqueous solution obtained, with 10.1 g of pure GPC being obtained:

(KF = 12.1%)

A very viscous, clear Liquid:

α D = -2.8 (c = 3.1% in H₂O)

Purity (HClO₄) = 87.8%

The product crystallized from ethanol and vacuumdried over P₂O₅ is an extremely hygroscopic solid, with:

m.p. = 135-136°C.

Computed for C₈H₂₀NO₆P

Found (M.W. = 257.20)

C = 37.29 C = 37.36

H = 7.89 H = 7.84

N = 5.42 N = 5.44

(c) Preparation of GPE:

After the separation of GPC, the resin IR401 OH (2) is washed with 200 ml of an aqueous solution of acetic acid at 0.5% (which is discarded). The resin is then washed with about 500 ml of an aqueous solution of acetic acid at 5%. The washes are concentrated down to a small volume, the solution is treated with decolourizing charcoal, is filtered and is concentrated under vacuum. 9 g of pure GPE is obtained as an oil, which solidifies by resting at room temperature.

(KF = 8.2%)

Purity (HClO₄) = 91.6%

α D = -3.1 (c = 4.1% in H₂O)

By using the process of Example (I), but treating the water-alcoholic solution (3) with a methanolic solution of calcium, magnesium, barium chloride, bromide or acetate, the corresponding salts are obtained, from which GPS is obtained as disclosed above.

Example II:

Preparation of GPS

The alcoholic solution (1) of the Example (I) obtained by using 250 g of raw phospholipids is adjusted to pH 6 with glacial acetic acid. 23 ml is added of a methanolic solution of zinc chloride at 10% w/v, the solution is stirred at room temperature for 1 hour, then is filtered. The residue is suspended in methanol (100 ml), is filtered, then the residue is dissolved in water (8 ml).

200 ml of acetic acid is then added, the mixture is stirred at room temperature for 10 hours, then is filtered and the residue is washed with methanol (3 x 30 ml). The solid, with the approximate formula (GPS)₃Zn_2.5, containing about 8% of NaCl, is dissolved in water and is eluted on 60 ml of IR200 H equilibrated in water. The resin is washed with 150 ml of water. The aqueous solution is eluted on 1 ml of IR93 OH (in order to eliminate HCl), then is concentrated down to a small volume, and the process is continued as disclosed in Example (la) with 1,9 g of GPS being obtained. By using the process of Example (II), but using calcium, magnesium, barium chloride, bromide and acetate, the corresponding salts are obtained, from which GPS is subsequently obtained.

Example III

Preparation of (GPS)₂ Ca

In the water-alcoholic solution (3) of Example (I), obtained by using 500 g of raw phospholipids, 46 ml of a methanolic solution of ZnCl₂ at 10% w/v is added. The resulting mixture is stirred at room temperature for 1 hour, then is filtered and the precipitate is washed with methanol (3 x 40 ml). The solid [(GPS)₃Zn₂, containing about 3% of NaCl] is washed in water (30 ml), then is eluted on 150 ml of resin IR200 H equilibrated in water and washing the resin with 300 ml of water. The aqueous solution is treated with stirring with IR 401 (until chloride ions are eliminated, approximately 2 g), then is filtered. To the solution, 6 g of CaCO₃ is added, and the solution is stirred at room temperature for 3 hours. The mixture is filtered, is concentrated down to small volume (about 10 ml), then is diluted with 300 ml of acetic acid. The mixture is stirred for 16 hours, then is filtered. The solid is suspended again in methanol, the suspension is filtered and is dried under vacuum at 40°C over P₂O₅:

3.5 g of (GPS)₂Ca is obtained

Non-hygroscopic solid Computed for C₁₂H₂₆N₂O₁₆P₂Ca

Found (M.W. = 556.372)

C = an C = 25.90

H = 4.79 H = 4.71

N = 5.10 N = 5.03

Ca = 7.08 Ca = 7.20

α D = -12.32 (c = 2.3 in H₂O)

α D = +2.72 (c = 3.6 in 1 N HCl)

With the same process as of Example

using zinc oxide, magnesium carbonate instead of calcium carbonate, the salts with the approximate forraula:

(GPS)₃ Zn₂:

In%, computed = 14.50; found = 14.95

α D = -14.9 (c = 2.16 in H₂O)

α D = +1.88 (c = 2.28 in 1 N HCl)

(GPS)₃Mg₂:

Mg%, computed = 5.92; found = 5.60

α D = -11.87 (c = 2.03 in H₂O)

α D = +3.29 (c = 2.12 in 1 N HCl)

are obtained.

Example IV:

Preparation of GPC

The alcoholic solution (1) of Example (I), obtained by using 250 g of raw phospholipids, is eluted on 300 ml of IR200 H equilibrated in methanol. The resin is washed with 1.4 I of methanol, until the impurities are completely removed (TLC-check), then with water (1 l). The aqueous solution containing GPC + GPE + GPS is treated with 2 g of decolourizing charcoal and is then eluted on 120 ml of IR93 OH, by washing the resin with 200 ml of water, then on 120 ml of IR 401 OH by washing the resin with 200 ml of water, and finally on 20 ml of IRC50 H, by washing the resin with 40 ml of water. Water is removed from the so obtained aqueous solution, by vacuum-concentration. 11.2 g of pure GPC (KF = 15.2%) is obtained (GPS is retained together with the GPE by the resins IR93 IR401).

Claims

C l a i m s

1. Process for purifying raw deacylated mixtures containing L-aIpha-glyceryl-phosphoryl-choIine (GPC) L- alpha-glyceryl-phosphoryl-ethanolamine (GPE), L-alpha- glyceryl-phosphoryl-serine (GPS), obtained by deacylating raw mixtures of the corresponding phosphatides, by means of which GPC, GPS and GPE; or GPC or GPS are obtained; characterized in that:

(a) from the alcoholic solution of the raw deacylated mixture containing GPC + GPE + GPS, the GPS is separated in the form of an insoluble salt thereof:

(GPS)_x My

in which:

GPS is the anion of L-aIpha-glyceryl-phosphoryl- serine

M is Ca, Mg, Ba, Zn, Na, K

x has the approximate values of 1, 2 and 3 y has the approximate values of 1, 2 and 2.5;

(b) from the salt of formula (I) (with M = Ca, Mg, Ba, Zn; x = 2; 3 and y = 1; 2; 2.5), after successive purifications, pure GPS is obtained;

(c) from the alcoholic solution of the raw deacylated mixture, obtained after said separation of GPS, and which contains raw GPC + GPE, a pure mixture of GPC + GPE is obtained first by purification on cationic resins in H form and in a non-aqueous medium, then pure GPC and GPE are obtained by separation on strong basic resins in OH form.

2. Process according to claim 1, characterized in that the salts of formula (I) (with M = Na or K, x = 1 and y = 2) are obtained by coprecipitation with sodium or potassium phosphate or sulfate, formed by treating with sulfuric or phosphoric acid the raw deacylated mixture containing sodium or potassium methoxide or ethoxide.

3. Process according to claim 2, characterized in that the salts of formula (I) (GPS-Na₂ or GPS-K₂) are obtained practically free from sodium or potassium sulfate or phosphate by extraction with a water- alcoholic solution (in methanol or ethanol), with a water content of about 10% and eliminating sodium or potassium sulfate or phosphate as the insoluble residue.

4. Process according to claim 1, characterized in that the salts of formula (I) (M = Ca, Mg, Ba, Zn; x =

2; 3 and y = 1; 2; 2.5) are obtained from the raw deacylated mixtures by treatment with calcium, magnesium, barium, zinc chlorides, bromides, acetates.

5. Process according to claim 2, characterized in that the compounds of formula 1 (H = Ca, Mg, Ba, Zn, x

= 2; 3 and y = 1 ; 2; 2.5) are obtained by treatment with calcium, magnesium, barium, zinc chlorides, bromides, acetates, of the water-alcoholic solutions of the compounds of formula (I) (M = Na, K, x = 1 and y = 2).

6. Process according to claim 1, characterized in that the salts of formula (I) (with M = Ca, Mg, Ba, Zn, x = 2; 3 and y = 1; 2; 2.5) are purified by precipitation from water and acetic acid, with a ratio of water/acid preferably comprised within the range of from 20 to 50.

7. Process according to claim 6, characterized in that from the purified salt of formula (I), GPS is obtained after elution on acidic resins in aqueous medium, elimination of the solvent down to a maximum concentration of approximately 40% and subsequent precipitation in acetone.

8. Process according to claim 1, characterized in that, in order to obtain GPC only, the raw mixture of GPC + GPE + GPS is first purified on cationic resins in the H form and in a non-aqueous medium, with a pure mixture of GPC + GPE + GPS being obtained, from which pure GFC is obtained by removing GPE and GPS on strong basic resins in an aqueous medium.

9. Process according to claim 8, characterized in that the raw alcoholic solutions containing GPC + GPE or GPC + GFE + GPS are first eluted on cationic resins in H form and in a non-aqueous medium, the resin is subsequently washed with a solvent until the impurities are eliminated, and the pure mixtures of GPC + GPE or GPC + GPE + GPS are recovered by washing the resins ater.

10. Process according to claim 9, characterized in that carboxy or sulfonic resins are indifferently used and that as solvents for operations in non-aqueous medium, C₁-C₄ alcohols or their mixtures are used.