DE1949399B - Glycol phosphatide mixtures and their production - Google Patents

Description

The known cephalin fractions contain, in addition to lecithins, essentially phosphatidylcolamine when obtained from vegetable material, and phosphatidylcolamine and phosphatidylserine when obtained from animal sources. Depending on the specific starting material, these cephalins have different fatty acid residues with generally up to 24 carbon atoms. The cephalin fractions preferred for therapeutic purposes contain phosphatidylcolamines or phosphatidylserines, which have a large percentage of unsaturated, in particular polyunsaturated, fatty acid residues. Because of the strong reactivity, especially of the polyunsaturated fatty acids, their isolation is not easy. They are generally obtained from the extraction of rock phosphatides with lower alcohols.

The cephalins or the phosphatide fractions containing them can, inter alia, be natural Emulsifiers are used as they are in addition to the lipophilic Fatty acid residues still have the hydrophilic olcerolphosphoric acid group in addition to the hydrophilic colamine or serine residues. All· however, the emulsifier effect is relatively small, which is »$ can be attributed to the fact that the free amino group is bound intramolecularly by the phosphoric acid group.

Because of the presence of the unsaturated fatty acids, which are valuable for physico-chemical and biological reasons, cephalin-containing phosphatide mixtures are also used as medicaments. As emulsifiers for intravenous fat feeding , however, they should only be used with caution, since the kephalins are activators of blood clotting as such. It has also been found that the cephalin-containing phosphatides previously used for intravenous fat feeding can cause unpleasant side effects such as fever and chills. As has now been recognized, these adverse effects must be attributed primarily to the amino group of the kephalins.

From the United States patent 3 301 881 a method is already known in which durui treatment aqueous phosphatide emulsions with organic acid anhydrides the ktphalin in whole or in part was acylated, creating the primary amino group

ao was converted into an acid amide. This is supposed to the phosphatides become more compatible and can be more easily dispersed in aqueous media. However, their ability to emulsify is no better than that of the non-acylated cephalins. Eb would be therefore desirable if one could use such emulsifiers that are not only poor in cephalins but in which these substances are completely absent, but it has been shown that pure lecithins do not have adequate emulsifying properties. The complete separation of the cephalins therefore does not solve the problem.

The invention is based on the knowledge that

by deamination of phosphatidylcolamines or phosphatidylserines for the above Purpose of use suitable and well tolerated emulsifiers can be obtained.

The invention relates to glycol phosphatide mixtures of the general formula

H ₂ CO- R ¹

HC-O —R ²
H ₂ C - O - P - O - CH ₂ CHOH

O OH X

where R ¹ and R ^{2 represent} fatty acid residues with 10 to 24 carbon atoms occurring in vegetable and animal phosphatides and X is a hydrogen atom or the - COOH group.

Glycol phosphatide mixtures in which the fatty acid residues are unsaturated or polyunsaturated are preferred.

The invention also relates to a process for the preparation of the glycol phosphatide mixtures of the general formula given above, which is characterized in that a cooled aqueous nitrous acid is added to a solution of phosphatidylcolamine and / or phosphatidylisite in an organic solvent at room temperature or under low heating the phosphate compounds diazotized by diazotization and hydrolysis are isolated in a manner known per se

The fatty acids of plant and animal cells It is well known that they always have an even number of carbon atoms and are exceptionally rare straight-chain nature (Wolfgang Langenbeck

Organic Chemistry Textbook, 13th / 14th edition, 1053, Steinkopf-Verlag Dresden and Leipzig; Advances in Medicine, Vol. 83, S, 517-519, 1965). The phosphatides with unsaturated fatty acids, to which essentially oleic acid and linoleic acid, Linolenic acid and arachidonic acid are found in both vegetable and animal (both in warm-blooded animals as well as fish) fats and oils are widely used. They are because of their physiological metabolic effects of importance, which is why the glycol phosphatides according to the invention with unsaturated or polyunsaturated fatty acid residues are particularly valuable.

The new glycol phosphatide mixtures, which can also be called desaminokephalins, are, in contrast to the cephalins, completely alcohol-soluble. At the same time, they also have a significantly greater solubility in water than the Phosphatidylcolamine and Phosphu'.idylserine. Since they no longer have the disruptive coagulation activity of the cephalins, the new glycol phosphatides are ideally suited for intravenous fat nutrition. At the same time, they can also serve as more effective oil-in-water P-emulsifiers for the production of fat preparations. Due to the reactive free _a5 hydroxyl group, the glycol phosphatides are also ■ jv.r the production of other compounds substituted on the OH group, e.g. B. ester compounds, suitable and therefore also valuable intermediates for novel drugs. The composition of the fatty acids varies depending on the number of cephalin raw materials used. Mad In general, the proportion of fatty acids with ώ .. carbon numbers 16 and 18 predominates, which also applies to unsaturated fatty acids. In Kephalinen from egg yolk which Glycolphosphatide produced therefrom contain, among other things, C ₂₀ - and CJJ fatty acids in the brain as Ausgangssuhstanz also C ^ fatty acids.

Compared to the cephalins, the deamination to the glycol phosphatides is so-called Improved emulsifying power, as the following simple experiments can easily see.

A simple shake test to test the suitability as an emulsifier for an oil-in-water emulsion is used carried out as follows:

1 g of a phosphatide is dissolved in 10 g of sunflower oil in a 100 ml shaking cylinder by heating in a water bath to 70 ° C. while shaking. After cooling to 25 ^° C., 90 ml of distilled water are added. Water at 25 C was added and the phases were emulsified by sharp up and down movements of the cylinder every 30 seconds. The number of shakes necessary to form an emulsion that is stable for at least 30 seconds is determined.

Soy rock phosphatide

de-oiled raw soy phos phatid

alcohol-soluble fraction from soy rock phosphatide ..

Commercial product

According to the present invention

Number of shakes substance

10 16 40

1 1 1 With careful exposure to nitrous acid according to the method of the invention, e.g. in the cold, the polyunsaturated fatty acid residues of the phosphatidyl compounds are not chemically changed either. The spectral analytical and gas chromatographic investigations showed that the characteristic IR band of the kephalin at 9.8 μ has disappeared without the double bonds having changed as a result of the treatment.

The action of nitrous acid is preferably carried out in such a way that an aqueous solution is obtained a salt of nitrous acid and an acid salt of an organic or inorganic acid used and this aqueous solution is added dropwise with stirring to the cephalin-containing organic solution. This mild form of action of nitrous acid enables the reaction at room temperature or under low heating even without danger to the steric form of the unsaturated fatty acid residues perform. Alcohols are suitable as solvents for the cephalin-containing starting substances or chlorinated hydrocarbons with 1 to 4 carbon atoms. Hydrocarbon solvents are also suitable. When carrying out the deamination reaction of the phosphatidylcolamines according to the invention and / or Phosphatidylserine has particularly proven to be chloroform as a very useful solvent

proven.

The deamination process according to the invention enables the glycol phosphatides to be easily isolated, as their great alcohol solubility facilitates the extraction and separation of other accompanying phosphatides facilitated.

In practice, the process according to the invention for the preparation of the glycol phosphatides proceeds in such a way that either pure cephalin, as can be obtained, for example, by column chromatographic separation of cephalin-rich phosphatide fractions on silica gel columns by elution with chloroform-methanol mixtures, or a cephalin-containing phosphatide mixture - oil-containing or deoiled - in an alcohol or chlorinated hydrocarbon with 1 to 4 carbon atoms or in a hydrocarbon solvent such as petroleum ether, hexane, cyclohexane. Benzene or mixtures of these solvents is dissolved, this solution is then dissolved with cooling, at room temperature or higher temperatures with the aqueous solution of nitrous acid or successively or simultaneously with an aqueous solution of an alkali or alkaline earth metal salt of nitrous acid and an acidic salt of an inorganic or organic acid, preferably sodium dihydrogen phosphate. Sodium hydrogen citrate or potassium hydrogen phthalate is added and the mixture is stirred in the absence of air until the free minogroups belonging to the cephalin phosphatides can no longer be detected with ninhydrin reagent. Thereafter, when using alcohol, the solvent is completely or partially evaporated in vacuo and preferably replaced by a chlorinated hydrocarbon. To remove the water-soluble components, the solution can be washed out two to three times with water. After evaporation of the solvent under nitrogen results i a yellow to amber plastic product.

The particular advantage of the procedure using saline solutions is based on the fact that that it is possible in the diazotization of the free

i 949 399W

Amino group to do without the acid completely ζμ and to control the release of nitrous acid from the nitrite by acid salts in situ so that a Rearrangement of those present in the Pbosplwtiden Fatty acids to trans fatty acids are practically absent. Also a change in the fatty acid composition of the treated products does not occur, in particular, the content of polyunsaturated remains Fatty acids, especially poly-eu fatty acids with their essential properties, through the process unchanged.

As a rule, the diazotization will be carried out up to the quantitative removal of the primary NH ₂ group of the kephalin; but it is also easily possible to work with less than the amounts of sodium nitrite and acid salt specified in the examples, partial removal of the nitrogen from the cephalin molecule being achieved, which already leads to a good distribution of these phosphatide products in aqueous media.

Another advantage of the present process of the application is the fact that the cephalin-free Products have favorable properties, e.g. B. significantly better acid numbers and phosphorus values than the corresponding starting products, as can be seen from the table below.

Table 1

substance Iodine number Acid number 0 /
/ n
phosphorus Commercially available raw
phosphatide 86.5 20.3 2.1 According to the procedure of
Registration disaminated 87.2 9.4 2.34 Deoiled rock phosphatide 70.3 30.1 3.1 According to the procedure of
Registration disaminated 77.4 5.5 3.28 Alcohol-insoluble soy
phosphatide fraction ... 66.7 32.3 3.0 According to the procedure of
Registration disaminated 70.2 14.4 3.23 Alcohol soluble soy
phosphatide fraction ... 85.0 18.0 3.1 According to the procedure of
Registration disaminated 86.7 4.0 3.12

For the reaction with the nitrous acid according to the invention, the pure cephalin compounds or the phosphatide mixtures containing them can be used. The lecithins still contained in these cannot interfere with the reaction, because they are not attacked by the nitrous acid under the mild reaction conditions, since they do not contain any primary amino groups.

In the investigation of the new glycid phosphatides obtained by deamination of phosphatidylcolamine according to the process according to the invention, iron fatty acids were determined in the proportion of soy phosphatide as the starting material in the gas chromatographic investigation:

Table II

Fatty acid composition of phosphatidyl glycol from soy phosphatide

Lauric acid 0.1%

Myristic acid 0.2%

Palmitic acid 26.0%

Stearic acid 2.0%

Oleic acid 7.0%

Linoleic acid 60.0%

Linolenic acid 4.5%

Arachidic acid 0.2%

100.0%

In the case of a mixture of phosphatidylcolamine and phosphatidylserine obtained from egg, after the following fatty acid composition is determined after deamination:

'i'. Belle III

Fatty acid composition of glycol phosphatide from egg cephalin (mixture of colamin and serine

Cephalin)

Capric acid 1.2%

Lauric acid 0.4%

Myristic acid 2.0%

Myristoleic acid 0.2%

Palmitic acid 22.6%

Palmit oleic acid 0.7%

Stearic acid 23.0%

oleic acid 17.2%

Linoleic acid 16.0%

Linolenic acid 2.0%

Arachidic acid 0.4%

Arachidonic acid 12.8%

Behenic acid 1.5%

100.0% Example 1

200 g of pure cephalin ( phosphatidylcolamine), produced from alcohol-insoluble soy phosphatide fraction by column chromatographic separation on silica gel with chloroform-methanol 80:20 as the eluent, are dissolved in a mixture of 1000 ml of chloroform and 1000 ml of methanol and, with stirring and introduction of N _2, with a Solution of 125 g of sodium nitrite in 200 ml of distilled water. Water added.

The mixture is heated to 37 ° C. in a water bath. A solution of 230 g of sodium dihydrogen phospha ¹ : times 2 water in 200 ml of distilled water is then slowly left. Add water dropwise. After a total of 2 hours of stirring at 37 ° C., all of the kephalin is diazotized and no amino group, ie no more kephalin, can be detected by thin-layer chromatography with Ninhydriii reagent.

For work-up, the insolubles are decanted and the residue is washed out with a little chloroform

So and discarded. The chloroform solutions are transferred to a separatory funnel and, if necessary, water is added until two Form layers. The lower chloroform-containing layer is separated and washed with about 400 ml of water

it was washed and again separated from the water phase in a separating funnel. After evaporation of the solvent in vacuo under N ₈ , 190 g of olymolphosphatide remain.

Example 1 Residue decanted, the residue with a little

^v Washed out chloroform and this chloroform

500 g of alcohol-insoluble soybean phosphatide fragments are added to the decanted solution. The combined ion, as obtained by de-oiling commercial solutions, is then _{stirred with 500 ml of water under N 1} soy phosphatide with acetone and then S, the solution is allowed to settle briefly in the separating funnel and extract the de-oiled product several times and then the lower chloroform layer of with ethanol in the heat as a residue remains separated from the laundry water. The gas is washed out (cephalin content about 30%) in a mixture of chloroform solution and repeated twice with 400 ml of 700 ml of chloroform and 500 ml of methanol under water. Finally, the chloroform stirring and the introduction of N, are dissolved and a solution of 69 g of NaNO in 80 ml of distilled water is removed from the solution in vacuo under nitrogen. Water released. There remains 1.8 kg (- 90%) of one set. A yellow-brown, plastic phosphatide mixture is then added to this mixture while stirring, a solution of 138 g of NaH ₁ PO ₄ · 1 hydrate in 100 ml having the following characteristics: JZ - = 78th SZ dist. Add water dropwise. whereupon the reaction mixture = 5.5. P - 3.3 ° /. In the emulsification test. as is still wetter at room temperature under N ₁ , it is described in Example 2. was already after will. After 3.5 hours, a sample of the substance shows a rotation, compared to 16 for the starting material, obtained after thin-layer chromatographic separation to a stable emulsion. Silica gel plates in the mobile phase chloroform-methanol-water (65: 25: 4) and after spraying example 4 with ninhydrin reagent that no more cephalin * e

is present in the mixture. The solution is made up of 1 kg of an alcohol-soluble phosphatide fraction.

decant from the separated semi-solid phosphate - as can be done by de-oiling commercially available raw

and for so long with dist. Water until the phosphatides are mixed with acetone and then again

have formed two layers. To do this, approximately 150 ml of the de-oiled phosphatide mixture must be extracted

Water required. The water phase (upper phase) »5 can be obtained with alcohol, is in 31

is separated in separating funnels and discarded. Dissolved ethanol and stirring and passing in

After adding about 200 ml of water it becomes good N with a solution of 120 g of NaNO in 120 ml

mixed and again from the water phase of water and a solution of 200 g of NaH PO _1, · 1 H _t O

severed. Should the second water phase still noticeably add 200 ml of water. Both salts are taking

borrowed to be acidic, is heated again with water 30 in dist. Water brought into solution. The

washed. After evaporation of the solvent reaction mixture wild at 50 C with stirring and

in vacuo under N, result from the chloroform with the introduction of N, heated. After about 1 hour

phase 400 g of a phosphatide mixture with the following is the entire cephalin dia / otiert. The reaction

Kemvahlen: JZ 70. SZ 14. P 3.2 "/ _0. The solution is decanted from the residue and the

instead of the cephalin, the new glycol phosphatide residue was washed with a little ethanol. Through

contains. Distillation in vacuo under N, the alcohol becomes

The improved emulsification of this product distilled off to about 700 ml and left to residue

shows the following experiment: about 1.51 chloroform added. The solution will be

Mix 1 g of the glycolphosphatide-containing phosphatide two to three times with about 300 ml of water, then wash it by stirring and heating to 60 40 and discard the washing water. the Dissolved in 10 g of sunflower oil up to 70 C. After chloroform solution is in vacuo under N, cooling to 20 C, the mixture is evaporated in a. There remain 950 g (-95 °,) of a kephalin 100 ml shaking cylinder transferred and 90 ml of distilled water. free. Glycol phosphatide-containing mixture was added to water at 20 ° C. Through a sharp back with the following germ counts: JZ - 82. Rotation of the cylinder by 90 every 30 seconds 45 SZ - 4. P ^ 3.2 * “.

the two phases are emulsified. Is measured

the number of turns required, example 5

to obtain an emulsion stable for 30 seconds.

For the cephalin-free product, only two 1 kg of commercially available raw soybean phosphatide are required

relationships required, while the use of 5 ° in 1.21 of dichloroethane and 600 ml of ethanol untei starting material, ie kephalinhaltigen aiko- N ₂ protection and dissolved with stirring at a Temholunlöslichen phosphatide, even after 50 DRE p ^{erature of} 50 ° C successively with a solution no emulsion was obtained. of 100 g of NaNO ₂ in 120 ml of water and 170 {

NaH ₂ PO ₄ -IH ₂ O in 175 ml of water was added. In Example 55 it is convenient to use the sodium hydrogen phosphate

2 kg should only be slowly added dropwise by repeated treatment with the solution. After stirring soybean rock phosphatide from the oil for 2.5 hours of acetone, the reaction mixture is cephalin-free. The solution is then decanted in a mixture of 41 chloroform and cooling to room temperature, stirring and protection of an inert solution, the semi-solid back pressure gas (Nj) is dissolved and a solution of 250 g 60 with dichloroethane is added the washing liquid NaNO ₂ in 220 ml warm dist. Water added. to the main solution and washes the dichloroethane. The reaction mixture is ^{warmed to 50 0} C and ethanol solution two to three degrees with about 250 m each dropwise with a solution of 460 g of NaH ₂ PO ₄ · water. After the 1 H ₂ O solution has been distilled off in 450 ml of warm distilled water. Water added. means in vacuo to N ₁ 950 g of oil-containing remain After a total of 2.5 hours back stirring at 50 ⁰ C 65 kephalinfreies Rohphosphatid. For stable: under N ₂ the reaction product was free of cephalin. Emulsion according to that listed under Example 2 After cooling to room temperature, the test is only required one turn against the chloroform-methanol solution of the inorganic 10 in the case of the commercially available rock phosphatide.

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9 ^W 10

Example 6 Example 8

1 kg of an alcohol-insoluble soy phosphatide fak '180 g egg phosphatide, which is made from fresh egg yolk

tiön, as it was also used in Example 2, initially by treatment with Aeetoä for Ent *

are removed with stirring and protection of an inert gas of the neutral lipids and cholesterol «and

Ii dissolved in hexane and sew heating to 5O ⁰ C intestine by extracting the oil-free residue with

with a solution of 200 g of sodium nitrate in 325 ml of chloroform-methanol 2: 1 and washing out the

Water added. Water-soluble components according to J. FoUh and Mit 'are added to the reaction mixture.

A solution of 400 g of sodium dihydro workers (J. Biol. Chem. 191, p. 833, 1951) was prepared dropwise

gene phosphate in 550 ml of water. Naeh all in all it was both serine phosphatide and

2.5 hours of vigorous stirring is the phosphatide containing colamine phosphatide are in a mixture

cephalin-free fraction. The reaction product of 1 1 of chloroform and 11 of methanol is dissolved and

iweekohnerwerae cool in the refrigerator and, stirring and warming to 37 ° C, one after the other

then decant the supernatant solution. The with a solution of 120 g sodium dihydrogen

The residue is washed out repeatedly with hexane, 15 phosphate and 60 g sodium nitrite in 200 ml each

the hexane solutions are combined and then about water is added. After 3.5 hours of stirring that is

Shaken out three times with 300 ml of water each time. The reaction product is free of ninhydrin-positive phos-

Aqueous phases are separated off, if necessary phatiden. The chloroformhahige solution is of

borrowed by brief centrifugation, and discarded; separated from the water phase formed and still one

Hexane solutions shaken out in a vacuum from the solvent ao up to twice with 200 ml of water each time,

freed. 750 g of a phosphatide mixture result. After the solvent has been distilled off in the

that instead of the cephalin, the GlyGolphosphatid vacuum under nitrogen, 169 g of a cephalin-

contains. free, containing the corresponding glycol phosphatides

B e s ο 'e 1 7 tending phosphatide mixture back.

500 g of an alcohol-soluble phosphatide fraction, Example 9 which laughs the procedure of the German patent specification

1047597 was obtained, in a mixture 500 g of an alcohol-soluble phosphatide fraction of 700 ml of chloroform and 500 ml of methanol (obtained as described in Example 4), and post-stirring and heating at 5O ⁰ C 30 1.51 Ethanol dissolved and mixed at room temperature with a solution of 70 g of sodium nitrite with an aqueous solution cooled to 0 ° C in 200 ml of water and a slurry of nitrous acid which nitrite from 200 g of sodium 185 g of potassium hydrogen phthalate in 100 ml of water . L-dissolves in a mixture of 800 ml of water sets. After 7 hours, the reaction mixture and 250 ml of ethanol are cephalin-free by the dropwise addition of 470 ml. After cooling, the excess hydrochloric acid produced under ice-cooling is decanted from the insoluble material that has become. After stirring for 3 hours under nitrogen residue, it was rewashed with chloroform, the washing protection showed the phosphatide mixture no longer reacting liquid with the chloroform-methanol solution to ninhydrin reagent. By adding some chloroform and repeatedly with about 120 ml of water, two layers are formed, one of which is washed. Then the chloroform solution is separated in the lower 40 and evaporated with water after washing vacuum under nitrogen. Yield: will. After evaporation of the solvent in 450 g of a glycol phosphatide-containing alcohol vacuum under nitrogen protection, 470 g of a soluble phosphatide mixture result. Glycol phosphatide-containing mixture.

2387

Claims (5)

Patent claims: 1. Glycolphosphftide according to the general formula H, C - O - R ' HC-O —R ² i
H _a C-O-P-O-CH ₂ CHOH / N I O OH X where R ¹ and R ^{2 represent} fatty acid residues with 10 to 24 carbon atoms occurring in vegetable and animal phosphatides and X is a hydrogen atom or the - COOH group. 2. glycol phosphatide mixtures according to claim 1, characterized in that the fatty acid residues are unsaturated or polyunsaturated. 3. Process for the preparation of the glycol phosphatide mixtures according to claim 1 or 2, characterized characterized in that a solution is obtained at room temperature or with gentle heating of phosphatidylcolamine and / or phosphatidylserine in an organic solvent chilled aqueous nitrous acid is added and the diazotization and hydrolysis are deaminated Phosphatide compounds isolated in a manner known per se. 4. The method according to claim 3, characterized in that instead of nitrous acid an aqueous solution of a salt of nitrous acid and an acidic salt of an inorganic one or organic acid is used. 5. The method according to claim 4, characterized in that the solvent used is chloroform and instead of nitrous acid, an aqueous solution of sodium nitrite and sodium dihydrogen phosphate used. fl