DE1177772B - Process for obtaining a soy phosphatide emulsifier for therapeutic fat products - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Boarding school Class: A 61 k

German class: 30 h -2/30

Number: 1177 772

File number: U 4504IV a / 30 h

Filing date: April 18, 1957

Opening day: September 10, 1964

The present invention relates to a process for obtaining a soy phosphatide fraction, which is suitable as an emulsifier for fat products that can be administered intravenously in particular.

Certain fractions have already been isolated from soy phosphatides and used as emulsifiers for intravenous fat products are used. However, the fat emulsions made with them have a strong effect blood pressure lowering and are not suitable for clinical use. The antihypertensive effect this fat emulsions is caused by substances that become attached to the phosphatides when they stand Form air. In order to prevent an oxidative decomposition of the phosphatides, you have them under Stored with exclusion of oxygen at -6 ° C.

It has now been found that the constituents of the in monohydric, low molecular weight, aliphatic alcohols soluble soy phosphatide fractions by adsorption can remove aluminum oxide, magnesium oxide or activated carbon. The soy phosphatides purified in this way are stable and result in emulsions that can be administered intravenously with suitable fat products, that do not lower blood pressure.

To carry out the process according to the invention, soy phosphatides, e.g. B. commercial Soy phosphatides, such as Glidden-Lecithin RG and Glidden-Lecithin RAS, with a monovalent, low molecular weight aliphatic alcohol, such as methanol, ethanol, propanol, isopropanol or their mixtures, extracted. Ethanol is used partly because of the good yields that can be achieved with it, partly because of its Non-toxicity, if not completely removed from the product, preferred. Appropriate leads the extraction is done at about room temperature. Absolute ethanol is not necessary. Advantageous you use U. S.P. alcohol (not less than 94.9 percent by volume), If more than 30% water is used, the desired phosphatide fraction also partially falls the end. Ethanol of 85 to 100 percent by volume is therefore the most suitable.

The alcohol-soluble fraction still contains reactive ones Ingredients that, when used in an intravenous fat emulsion, include those listed above Process for obtaining a soy phosphatide emulsifier for therapeutic fat products

Applicant:

The Upjohn Company,

a corporation under the laws of the state of Delaware, Kalamazoo, Mich. (V. St. A.)

Representative:

Dr. W. Beil and A. Hoeppener,
Lawyers,
Frankfurt / M.-Höchst, Antoniterstr. 36

Named as inventor:

Curtis Erdmund Meyer, Galesburg, Mich.,

James Arthur Fancher,

Paul Edward Schurr, Kalamazoo, Mich.

(V. St. A.)

Claimed priority:

V. St. v. America April 30, 1956 (581269)

but also prevents or suppresses substantially undesirable side effects, e.g. B. chills, Back and chest pain, nausea, vomiting, dyspnea, apnea, changes in blood pressure, etc. that usually seen with the administration of therapeutic fat products.

According to a preferred embodiment of the invention, the ethanol-soluble soy phosphatide component is Stirred in about 10 parts by volume / weight of ethanol for about 30 minutes at room temperature with about 2 parts by weight of aluminum oxide. Already at 10 minutes

cause undesirable side effects. A still satisfactory adsorption is achieved,

therefore adds a fraction to the alcohol-soluble fraction. The amount of aluminum oxide is reduced to 1.5 parts by weight

Adsorbents, such as aluminum oxide, magnesium reduced, then the product obtained is not

oxide or activated charcoal (acid-washed charcoal is satisfactory. After filtering off the aluminum

should not be used). The harmful niumoxyds becomes alcohol at reduced pressure

Components are then adsorbed. The thus cleaned 50 is removed and the material so cleaned is used for production

Soy phosphatide fraction does not give just one of an emulsion in a corresponding amount

suitable emulsifier for intravenous fat products, vegetable oil added.

409 660/334

3 4

A suitable test method for the determination of the Reinhold Publishing Corp. 1951, p. 223) concerns it

clinical response of soy phosphatides is that these fatty acids are: Occurrence of an antihypertensive effect

Cats (see Geyer and coworkers in J. Lab. Palmitic acid 11.7%

Clin. Med., 34, 1949, p. 688). In the case of narcotic 5 stearic acid 4 0 ° /

sed cat observed no drop in blood pressure, so _A ___ t, -_... Λ _0; °

.... j. u ^ Ai ^ -TL-i? ι. · Ι_ arachmic acid 1.4 ⁰ / _n

iuits the examined material at the time of administration to ^{/ u}

People not to the above minor pentadecenecarboxylic acid 8.6%

appearances. , Oleic acid 5.5%

When carrying out the "cat" test, an io linoleic acid 63.3% *

Cat with the sodium salt of 5-ethyl-5- (l-methyl- q _q unsaturated 5 5 ° /

butyD-barbituric acid anesthetized. In the jugular vein * _{contains something} y ^ acid.

and a cannula is inserted into the great carotid artery. The large carotid artery is lecithin with an In R '= choline. Connected to a mercury manometer that shows the blood pressure 15 In KephalinR '= ethanolamine, serine or inositol indicates. The material to be examined, e.g. B. one (greatly simplified). Soy phosphatide fraction, is distributed via the jugular

cannula inserted and the increase in blood pressure or the total nitrogen content is therefore a benchmark

-Sink measured. If there is any blood for the phosphatides at all, if apart from these there is no nitrogen

If the pressure drops, this occurs almost immediately if the material containing it is present. Phosphatidyl inositol

after the injection. except. Also the total phosphorus content is

The exact chemical composition of the inven- tive is decisive. In addition, there is the ratio of N: P.

according to the isolated soy phosphatide fraction is It amounts to pure lecithin, phosphatidylserine and

as with all mixtures of naturally occurring phosphatidylethanolamine 1.0.

Phosphatides not certain (see also S c h o 1- 25 The percentage by weight of fatty acids that field and coworkers in J. Am. Oil. Chem. Soc, 25, saponification and iodine number of phosphatides, das 1948, p. 368). From the point of view of the reproductive neutralization equivalent and the iodine number of fatness and practicality, however, acids serve as a yardstick for the average The soy phosphatide fraction can be found as a persistent fatty acid.

Fraction of soy phosphatides can be defined, 3 ° The most important components include choline,

the first by adsorption of the unwanted serine and ethanolamine, which are in the parent compound

reactive materials that can be converted into one another. It is also clear

valuable, low molecular weight aliphatic alcohols that for a pure compound the ratio of these

soluble soy phosphatides are attached to aluminum components, e.g. B. of choline in the case of lecithin,

oxide, magnesium oxide or activated carbon produced 35 to P equals 1. Also worth the inositol is important,

secondly, since, according to the current state of knowledge, inositol can be found in a satisfactory manner

as an emulsifier for the 'in a therapeutic fat component of several structurally not yet listed

The oil used in the product is suitable and, thirdly, after Injecting Phosphatide.

tion on anesthetized cats no lowering of blood pressure The following examples explain the inventive

evokes. 40 proper procedures.

The soy phosphatide fraction isolated according to the invention can chemically are characterized in this respect, example 1 than in it compared to the total soy phosphate

tides and other fractions, the lecithin content is 300 g of a granular soy phosphatide (Glidden-

increases, the proportion of cephalin components, especially 45 lecithin RG), which in cats has a strong blood pressure

of the inositol, however, is reduced. This could lead to lowering in a Waring mixer

conclude that inositol is the unwanted active ingredient - three times for 1 to 2 minutes at room temperature

part is; At the moment, however, this is only a pure mixture of 600 ecm of absolute ethanol extracted. the

guess. The phosphatides have the following general extracts that have been filtered and reduced under

Structural formula: 5 ° pressure reduced to 250 ecm. A solid determination found 95.7 g of the material extracted

O were. 73.5 ecm of the concentrated extract, the 28 g

Solids were loaded onto a column of 900 g

pj £ Q Q j ^ aluminum oxide and with 95% aqueous

² 55 Ethanol. The following were obtained

O parliamentary groups:

H ₂ C-O-C-R
O

H ₂ C - O - P - OR '

in which R represents fatty acid residues. According to H i 1 d i t c h and Pedelty (Witteoff, The Phosphatides,

ml G ⁶⁰ No. 1 595
1016
616 1.0
6.0
1.9 No. 2 2317 2.1 No. 3 No. 4

Fraction 2 was concentrated under reduced pressure to remove the alcohol. The residue was taken up in so much water that a 1.2% solution was obtained. Two thirds of this solution

was used to make a 5% Dextrose preparation, half of which was autoclaved. Injection of the three solutions via one Jugular cannula did not reduce blood pressure in anesthetized cats.

Example 2

10 g of an alcohol-soluble fraction of soy phosphatides were dissolved in 100 cc of 95 ° / _o aqueous ethanol igeni dissolved. 20 g of aluminum oxide (Merck, for chromatographic purposes) were added and the mixture was shaken for 30 minutes. The solution was decanted and filtered. The alcohol was removed under reduced pressure and the residue was taken up in 20 ecm hexane hydrocarbons, known under the trade name "Skellysolve B". The solution was poured into 90 ecm of acetone whereupon an oil separated out. The acetone- "Skellysolve-B" mixture was decanted from the oil and the latter treated three times with acetone. The residue was freed from acetone in a vacuum desiccator. A l, 2 ° / ^e oig solution of this material in a 5 ° / "aqueous glucose solution did not result in a lowering of blood pressure when ecm cats in a dose of 0.5 to 5 per: kg was injected.

Example 3

A further 23.2 g of aluminum oxide were added to the remaining solution, the mixture was stirred and a third was removed Sample (3).

Finally, a further 21.5 g of aluminum oxide were added to the remaining solution, the mixture was stirred for 30 minutes and a fourth sample was taken (4). From each sample, there was prepared a l, 2 ° / ₀ solution in 5 ^o / _o ^e r ig dextrose forth and injected them cats.

IO relationship
of the entire
aluminum
oxyds to
Phosphatide ecm Blood pressure
modification
mm Hg ¹⁵ sample 1 0.5: 1 1.5 20; 110 Sample 2 .... 1: 1
1.5: 1
2: 1 5
5
5 -60; -30
-50; -80
0; +10; +10 Sample 3 Sample 4

Example 5

6 kg of alcohol-soluble phosphatide (lecithin Glidden RAS) were extracted by stirring with 601 15minutiges aqueous alcohol (95 ° / _0). The mixture was allowed to settle and the supernatant siphoned off. 12 kg of aluminum oxide (H ar s ha w, catalytic quality) were added to the supernatant and the mixture was stirred for 30 minutes. The mixture was allowed to settle and the supernatant filtered through alcohol

300 g of an alcohol-soluble soy phosphatide frac-30 washed Seitz filter. One received 601 solution,

tion (Glidden lecithin RAS) were on a steam bath in 1,500 cc of 95 ° / _o aqueous ethanol dissolved. The solution was then allowed to cool to room temperature. The deposited gummy precipitate was centrifuged off. 1380 ecm solution was obtained, which was divided into two parts. 700 ecm of it was placed in a refrigerator for 6 days, after which a heavy liquid was deposited. The supernatant layer was decanted off. 100 cc of it, which contained 9.3 g of solids, were mixed with 18.6 g of aluminum oxide (Merck, for chromatographic purposes). The resulting mixture was shaken for 30 minutes. After removal of the aluminum oxide and the alcohol, the remaining material was converted to the 2830 g of soy phosphatides. After removal of the alcohol in a flat still, the soy phosphatide fraction is suitable for use in a therapeutic fat product. It can also be dried, after which the emulsifier is obtained in solid form.

Example 6

In order to identify the soy phosphatide fraction isolated according to the invention and to be able to compare it with other fractions, as well as with the total soy phosphatide, it was analyzed chemically. 1,040 g of an alcohol-soluble soybean phosphatide fraction (Glidden lecithin RAS) were dissolved in 101 aqueous material is a l, 2 ° / ₀ solution in 5 ° / _o aqueous solution of 45 of ethanol (95 ° / o) and stirred for 20 minutes. Then made dextrose. The injection of 5 ecm each allowed the solution to settle. The separated kilogram did not cause a drop in blood pressure in cats
emerged. The unrefrigerated part of the original
Solution, namely 680 ml, was allowed to stand at room temperature for 1 day. An unidentifiable 50
adorned amorphous material that was filtered off.
The solution containing 82.6 g of solids was
Stirred for 30 minutes with 165.2 g of aluminum oxide.

After removal of the aluminum oxide and the alcohol is introduced from a part of the residue, a l, 2% solution in a 5 ° / _o aqueous solution of glucose forth. No decrease in blood pressure was observed after injecting 5 ml per kilogram into cats.

Example 4

1235 ecm of an alcohol solution containing 61.2 g of soy phosphatides were stirred with 30.6 g of aluminum oxide (Merck). The solution then became a Sample taken (1), which contained 1.29 g of phosphatide. 29.95 g of aluminum oxide were added to the remaining solution and stirred for 30 minutes. A second sample was then taken (2).

gummy residue would be discarded after decanting off the 10.7 liters of supernatant liquid. The alcohol was removed from part of the supernatant and the residue was analyzed. The results of this analysis are listed in the column headed "RAS / AI" in the following table. to 300 ecm of the supernatant was added to 60 g of aluminum oxide (Merck, for chromatographic purposes) and shook for 30 minutes. After removal of the aluminum oxide and the alcohol, the residue became analyzed. The analytical values determined are recorded in the following table. to 45 g of activated charcoal (Norit-SG) were added to a further 300 ecm of the supernatant and shaken for 30 minutes. Then the charcoal was filtered off; since the filtrate was still slightly colored, it was shaken again 30 minutes with another 15 g of activated charcoal. After filtering off the charcoal, a colorless filtrate remained return. The alcohol was removed in vacuo to give 2.48 g of residue. The analysis resulted in the values listed in the following table.

Analysis of various phosphatides

Glidden lecithin

RG ⁵ )

Glidden lecithin

RAS ")

RAS AF)

With

Al ₂ O,

treated

With activated charcoal
treated

Egg-

Phosphatide ')

Hanahan

Egg leci-

thin ² )

Total nitrogen, ° / ₀

Total phosphorus, ° / o

Choline,%

Inositol, ° / ₀

Ethanolamine,%

Serine, ° / ₀

Iodine number (of the whole fraction)

Ash, »/ ρ (P ₂ O ₅ )

Molar ratio N: P

Reducing substance ³ (from hydrolyzed

Material)

Fatty acids, ° / o

Iodine number (of the fatty acid)

1.15 1.23 1.38 1.44 1.45 4.15 3.45 2.44 2.48 3.01 2.84 6.31 3.00 6.23 7.16 10.60 10.04 13.85 2.41 0.207 0.08 0.01 0.04 <, 02 1.9 1.3 0.93 0.47 0.4 0.30 0.41 0.10 0.11 0.045 0.4 0.20 76.1 92.2 82.0 85.9 69.4 11.2 8.57 3.42 3.28 2.61 3.09 16.72 0.736 1,112 1.23 1.026 1.128 1,440 13.7 6.7 _ 2.16 28.3 _ 55.8 67.1 66.1 70.7 47.8 19.3 124.7 115.5 116.7 118.2 105.8 -

¹ ) Treated with Al ₂ O ₃ and activated carbon. This material also led to a decrease in blood pressure in anesthetized cats.

² ) Hanahan and coworkers, J. Biol. Chem., 192, 1951, p. 623.

³ ) As galactose. FoIin-Wu experiment carried out. J. Biol. Chem., 41, 1920, p. 367.

⁴ ) Norit-SG, American Norit Company.

⁵ ) A commercially available "solid" soy phosphatide mixture, which contains essentially all of the phosphatides found in soybeans and differs from the liquid preparations (the commercially available "lecithins") in that it has a lower oil content - around 4%.

⁶ ) A commercially available alcohol-soluble fraction of mixed soy phosphatides with a higher content of lecithin and a lower content of "cephalin" than PG.

⁷⁾ After extraction of RAS with 10 volume / weight 95 ° / "ethyl alcohol was found that only 70 ° / ₀ of the material are still about soluble. This is due to the observed reduction in the content of phosphatidylinositol and ethanolamine, which are the main components of the cephalin fraction.

The following procedures were used to determine the various data recorded in the table: Total nitrogen: Micro-Kjeldahl; Phosphorus: Fiske and Subba R 0 w, J. Biol. Chem., 66, 1925, p. 375; Choline: D. G 1 ick, J. Bio]. Chem., 156, 1944, p. 643; Ethanolamine and Serine: separated according to the method of C. Art o m, J. Biol. Chem., 157, 1947, p. 585; Determination by adapting the method according to S. M ο ο re and WH Stein, J. Biol. Chem., 211, 1954, p. 907; Inositol: Adaptation of the pyridoxine method by A kti η and coworkers (Anal. Ed.), Ind. Eng. Chem., 15, 1943, p. 141 using Sacch. carlsbergensis, ATCC 9080; Fatty acids: 2-hour hydrolysis me 2N sodium hydroxide in 50 ° / ₀ sodium ethanol and then acidifying and extracting with petroleum ether; Iodine number: USP

From the data presented in the table, it can be concluded that according to the present process obtained, purified soy phosphatide fraction a mixture of phosphatidylcholine (lecithin), Represents phosphatidylethanolamine and phosphatidylserine, in which lecithin is the main ingredient. Phosphatidyl inositol is essentially absent, which can probably be regarded as direct evidence that Phosphatidylinositol is the most reactive, or most reactive, substance found in anesthetized cats Lowering blood pressure causes, firstly, approximately a parallel between those in the various investigated The amount of materials contained and their effects and, secondly, a 50% concentrate of phosphatidylinositol was found to be extremely active.

Claims (1)

Claim: Process for obtaining a soy phosphatide emulsifier for therapeutic fat products, characterized by the fact that in soy phosphatides dissolved in a monohydric, low molecular weight, aliphatic alcohol Treated aluminum oxide, magnesium oxide or activated carbon, the precipitated solids and removed the purified soy phosphatide fraction is recovered from the solvent. important characteristic and as in 370, 1955. Documents considered: German Patent No. 617 508; Journ. American Oil Chemists Soc, 32, pp. 365 bis 409 660/394 9. 64 © Bundesdruckerei Berlin