DD275461A1 - METHOD FOR SEPARATING PHOSPHATIDE MIXTURES IN PHOSPHATIDE CLASS PREPARATES - Google Patents

Abstract

The invention relates to a process for the separation of phosphatide mixtures in Phosphatidklassenpraeparate. According to the invention, pure phosphatide class preparations (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol) are obtained by ultrafiltration on a membrane by thin-layer chromatography, the dielectric constant of the solvent system used being gradually increased under control to a value of 7.0 during the separation. The invention is applicable in the pharmaceutical as well as the food industry and in medicine.

Description

solvent system used, the dielectric constant of the solvent system used is brought to a value of 1.8 to 4.0, preferably 3.1. As a result of the subsequent ultrafiltration, the phosphatidylcholine is in the permeate, while phosphatidylethanolamine and inositol remain in the retentate. From the permeate, the phosphatidylcholine can be obtained in thin-layer chromatographic purity. Increasing the dielectric constant by further addition of the polar solvent or solvent mixture to a value of 4.0 to 7.0, preferably 5.5, is the result of the subsequent ultrafiltration, the phosphatidylethanolamine in the permeate, while the phosphatidylinositol remains in the retentate. Both phosphatides can be obtained from permeate or retentate in thin-layer chromatographic purity. If only phosphatidylinositol is to be obtained, bring the dielectric constant of the solvent system equal to a value of 4.0 to 7.0 and then performs the ultrafiltration, leaving phosphatidylinositol in the retentate.

During the separation, compliance with the dielectric constant is controlled, for example with a DK meter. Advantageously, the membrane is rinsed several times with each solvent system used after each ultrafiltration. For the selective separation of the phosphatide membranes are membranes having a molecular weight "cut off" of 1000 to 100,000, preferably from 5000 to 10,000 used. The separation by ultrafiltration is carried out at absolute pressures of 0.1 to 1 MPa, preferably at 0.2 to 0.5 MPa.

Surprisingly, it is possible to make pure phosphatide class preparations available by thin-layer chromatography using the method according to the invention. Due to the very similar chemical structure of the individual phosphatide classes, it was not to be expected that a simple change in the polarity of the solvent system used, measured by the dielectric constant, would produce such a clear separation effect. The inventive method is distinguished from the column chromatographic separation methods, which also lead to thin-layer chromatography pure Phosphatidklassenpräparaten, by a much lower material and time.

embodiments Example 1 (phosphatide enrichment)

2 to 3 g of a commercially available soy lecithin having a phosphatide content of 68.1% are dissolved in 25 ml of the corresponding mixture (hexane / acetone ratio see Table 1) from hexane and acetone and stirred at 25 ° C. for 1 hour. Subsequently, the phosphatides are precipitated by slow addition of acetone. To separate the precipitate, it is centrifuged at 2000 rpm. The phosphatide content of the samples were determined by the Phosphatidbestimmung HURST in modified form (FRANZKE, Cl., E. HOLLSTEIN and P. BRANDT, Food Ind., 19.1972, page 347) and using the factor 25,44 on the Lecithin content converted. The results are shown in Table 1.

Table 1: Phosphatidanreicherung by treatment with hexane-acetone mixtures of different composition

Experiment no. Hexane / acetone volume ratio Phosphatide content <%> 1 90/10 76 2 90/10 75 3 70/30 84 4 70/30 78 5 50/50 94 6 50/50 95 " 7 50/50 95 8th 40/60 75 " 9 Column chromatography according to 84 10 ROUSER ³¹ , modified 94

1 after two washes.

2 Lecithin already begins to stick in solvent system 2u.

3 ROUSER, G.G.KRITCHEWSKY, A.YAMAMOTO, G.SIMON, CGATTLa-S-BAUMANN, Methods in Enzymology 14,1969, page 272-318.

Run No. 9 following eluents are used sequentially: 100 ml each of chloroform, chloroform / methanol (90/10), chloroform / methanol (50/50), chloroform / methanol (10/90); the chloroform phase is discarded,

Experiment no. The following eluents are used successively: 100 ml of chloroform, 150 ml of acetone, per 100 ml

Chloroform / methanol (90/10), chloroform / methanol (50/50), chloroform / methanol (10/90); Chloroform and acetone phases are discarded.

Example 2

The phosphatide mixture used is a commercial soy phosphatide preparation enriched according to Example 1, test No. 7, from which a 1% phosphatide-n-hexane solution is prepared. The phosphatide mixture had the following composition: 40.9% phosphatidylcholine, 26.4% phosphatidylethanolamine, 27.7% phosphatidylinositol and 5% neutral fat components. A cellulose acetate membrane UF120 from VEB Zellstoff · und Zellwollewerke Wittenberge in an AMICON stirred cell type 402 is used. The results of the selective separation of a phosphatide mixture on a semipermeable membrane with an increase in the dielectric constant, which is checked by means of DK meters, are shown in Table 2 ,

Table 2: Stepwise fractionation of a phosphatide mixture

ρ = 0.15 MPa, starting solution: 100 ml of 1% phosphatide-n-hexane solution

Fraction solvent system Dielektrizi

tätskonstante

composition

(thin-layer

matographisch)

1. Permeate 2XIOOmI rinses

n-hexane

1.9

2. Permeate (100 ml), then 2 χ 100 ml rinses

n-hexane / isopropanol (80/20% by volume)

3.1

72%

28%

Phosphatidylcholine neutral fat components

3. Permeate (100ml), then 2x100ml rinses

n-hexane / isopropanol

(50/50 vol.%) 5.5

99.8% phosphatidylethanolamine ¹ '

4. Retentate

19%

81%

Phosphatidylethanolamine phosphatidylisositol

1 taking into account the detection limit of the phosphorus determination.

Example 3

It is a 1% solution of Example 1, experiment no. 7 enriched commercial Soy Phosphate Jdpräparates via a cellulose acetate membrane UF120 of VEB Zellstoff- and Zellwollewerke Wittenberge fractionated in an AMICON stirred cell, type 402 by gradually increasing the polarity of the solvent system. The same phosphatide mixture as used in Example 2 is used.

Table 3: Stepwise fractionation of a phosphatide mixture

ρ = 0.06 MPa, starting solution: 100 ml of 1% phosphatide solution in n-hexane / isopropanol (90/10% by volume)

fraction Dielectric constant Composition (thin-layer chromatographic) 1 hexane / isopropanol (90/10% by volume) 150 ml run 2.2 Sterols, sterol compounds, phosphatidylcholine, traces of phosphatidylethanolamine 2 hexane / isopropanol (80/20% by volume) 100 ml run 200 ml rinse 3.1 Traces sterol compounds phosphatidylcholine 3 hexane / isopropanol (50/50 vol.%) 100 ml run 200 ml rinse 5.5 Phosphatidylethanolamine (pure by thin-layer chromatography)

4 retentate

phosphatidylinositol

(thin layer chromatography

purely)

Example 4 The same phosphatide mixture as used in Example 2 is used. Table 4: Stepwise fractionation of a phosphatide mixture

ρ = 0.1 MPa, starting solution: 100 ml of 1% phosphatide solution in n-hexane / isobutanol (90/10% by volume)

fraction Dielectric constant Composition (thin-layer chromatographic) 1 hexane / isobutanol 300ml rinse (90 / 10vol%) 2.5 Neutral fat components, phosphatidylcholine (trace phosphatidylethanolamine) 2 hexane / isobutanol (82/18% by volume) 100 ml run 3.1 Phosphatidylcholine (thin-layer chromatographically pure)

Claims (3)

1. A process for the separation of phosphatide mixtures into phosphatide class preparations starting from enriched phosphatide mixtures prepared in a nonpolar solvent or in a mixture of several nonpolar solvents or in a mixture of one or more non-polar solvents and one or more polar solvents having a dielectric constant of not more than 2, 5, the phosphatide micelles are separated by ultrafiltration and the membrane is rinsed several times with the solvent system, characterized in that during the separation of the phosphatide mixture, the dielectric constant of the solvent system is gradually increased to 7.0 by addition of a polar solvent under control. 2. The method according to claim 1, characterized in that an alkane, for example hexane, and as the polar solvent, an alcohol, for example isopropanol or isobutanol is used as the non-polar solvent. 3. The method according to claim 1 and 2, characterized in that for separating the phosphatidylcholine from phosphatidylethanolamine and phosphatidylinositol, the dielectric constant of the solvent system to a value of 1.8 to 4.0, preferably 3.1, and for the separation of phosphatidylethanolamine from Phosphatidylinositol is brought to a value of 4.0 to 7.0, preferably 5.5. Field of application of the invention The invention relates to a process for the separation of phosphatide mixtures in Phosphatidklassenpräparate that can be used in the food industry, the pharmaceutical industry, in medicine and in biochemical research. Characterization of the known prior art Enrichment of a particular phosphatide class or recovery of pure phosphatide class preparations can be carried out by extraction methods (PARDUN, H., Fette, Soifen, Anstrichmittel 86, 1984, pages 55-62) or by column chromatography after several prepurification steps (COLACCICO, G., J. Lipid Res. 8, 1967, page 513). However, these methods allow only relatively low levels of enrichment or require a lot of time and material. EP 0049914 describes a process in which the components are separated from a mixture containing substances dissolved in a nonpolar solvent which form micelles. For this purpose, a suitable solvent system is used to control the selective micelle formation. On semipermeable membranes, the mixture is separated by pressure into permeate and retentate. Certain components in the permeate and / or retentate are enriched. Here it is possible to enrich phosphatidylcholine by using a variety of solvent mixtures and membranes in the permeate. The best results with 65% enrichment of phosphatidylcholine are obtained using hexane with 10% ethanol additive as the solvent system. The preparation is still contaminated by phosphatidylethanolamine, phosphatidic acid and sterol glycosides. The representation of thin-layer chromatographically pure phosphatide class preparations can not be shown. Object of the invention The aim of the invention is to make available, by thin-layer chromatography, pure phosphatide class preparations starting from phosphatide mixtures, saving time and money, for use in the food industry, the pharmaceutical industry, in medicine and in biochemical research. Explanation of the essence of the invention The invention is based on the object, a method for the separation of Phosphatidgemisch'en too. develop, which leads to thin-layer chromatography pure Phosphatidklassenpräparaten and is characterized by a low material and time. According to the invention, this object is achieved in that the enriched phosphatide mixture is taken up in a non-polar solvent or in a mixture of several non-polar solvents or in a mixture of one or more nonpolar solvents and one or more polar solvents having a dielectric constant of up to 2.5, the phosphatide micelles are separated by ultrafiltration and the membrane is rinsed several times with the solvent system, during which separation the dielectric constant of the solvent system is gradually increased to 7.0 by addition of a polar solvent or solvent mixture under control. As a non-polar solvent, an alkane, for example hexane, is used as the polar solvent, an alcohol, for example isopropanol or isobutanol. To separate the phosphatidylcholine from phosphatidylethanolamine and phosphatidylinositol, the procedure is such that the addition of a plarious solvent or solvent mixture to the