CS244002B1 - Arachidic acid's methyl ester and/or ethyl ester production method - Google Patents

Abstract

Process for preparing methyl ester and / or ethyl ester erechidonic acid from livestock and fur animals rests in that the fat is obtained by extraction from the liver undergoing trensesterification followed by methyl ester and or erechidonic acid ethyl ester separated by high performance liquid Chromatography. Arachidonic acid and its esters are used in health care - dietetic additive, lower cholesterol bactericidal and antiviral effects, treatment arteroaclerosis and diabetes, etc., in veterinary medidnS - induction of egg tolerance, luteolytic effects in cattle, synchronisece ovine and in the form of esters is present vnSjSi application used in cosmetics.

Description

(54) A method for producing arachidonic acid methyl ester and / or ethyl ester

The process for producing erechidonic acid methyl ester and / or ethyl ester from livestock and fur animals is characterized by subjecting the fat extracted from the liver to trensesterification, whereupon the methyl ester or ethyl ester of erechidonic acid is separated by high performance liquid chromatography.

Arachidonic acid and its esters are used in health care - dietary additive, cholesterol lowering, bactericidal and antiviral effects, treatment of arteroaclerosis and diabetes etc., in veterinary medication - egg laying induction, luteolytic effects in cattle, ovine synchrony and ester forms used in cosmetics when applied.

The invention relates to a process for the production of erachidonic acid methyl ester and / or ethyl ester from the livers of livestock and fur animals.

Arachidonic acid plays an important role in the body, among others as a precursor of proataglandins, prostacyclin, thromboxane and leukotriene, substances that regulate various biological functions in a living organism that arise! in tissues by an oxidative metebolic process, by various enzymes.

In a healthy organism, arachidonic acid is formed mainly in the liver by a metebolic build-up of acids and 18 carbons and with fewer double bonds, namely 11nolic acid manure. A disorder of this metabolism may also be the cause of arachidonic acid deficiency in the tile.

. The effect of adding arachidonic acid or its deficiency in the diet of different animals and its application to different organs has been studied; Thus, some interesting effects of arachidonic acid have been found, for example lowering of cholesterol in the blood, inhibition of platelet aggregation, stimulation of gastric secretion, bacterial and antiviral effects, use in the treatment of erteroeclerosis and diabetes, and in other applications can also be used in cosmetics.

In veterinary medicine, the inducing effect of erachidonic acid was found in egg tolerance in hens, luteolytic effects in cattle and use in ovarian synchrony. Various preparations are known, containing essential acids for use as an additive to foods or in the form of various esters for cosmetic preparations.

Arachidonic acid has a role to play in immunoaupreel, and the metabolism of arachidonic acid has a certain relationship and relationships, which are not yet fully understood, with tumor formation.

The prior art isolation of arachidonic acid methyl ester has been carried out by column chromatography on a silver nitrate impregnated or reverse phase support, which is disadvantageous for long separation times or minor efficiencies.

Other known methods are time consuming with often unsatisfactory yields.

According to the invention, the above-mentioned drawbacks are overcome by a process for the production of erachidonic acid methyl ester and / or ethyl ester from the livers of livestock and fur animals. It is based on the fact that the fat obtained from livestock livestock livers is transesterified with methanol or ethhenol and the resulting methyl ethyl ether and / or ethyl ester of erachidonic acid is delayed by preparative high performance liquid chromatography.

According to another feature of the invention, the transesterification is carried out with an excess of methanol or ethanol under an inert gas atmosphere from the presence of an alkali metal methyl alcohol or ethyl alcohol at a temperature of 15 to 78 ° C for 3 to 15 hours.

It is further preferred to carry out trenaesterification of the fat simultaneously with its extraction with a mixture of methyl alcohol or ethanol and methyl chloride or an aromatic hydrocarbon in a volume ratio of 1: 4 to 4: 1, in the presence of an acid catalyst, preferably hydrogen chloride or p-toluenesulfonic acid at? C.

Finally, according to the last aspect of the invention, preparative high performance liquid chromatography is carried out using a polar sorbent as a stationary phase and using a mobile phase containing non-polar hydrocarbons with an addition of 0.001 to 10% by weight. polar polar solvents.

The process according to the invention is a simple and fast process for the preparation of arachidonate of methyl or ethyl in the complex of conventional extraction associated with transesterification.

The transesterification method used in this case, i.e. in an alkaline environment and in the cold associated with a simple separation by means of high-performance preparative liquid chromatography, also significantly increased the yield.

Waste material from veterinary sanitation institutes may be used as sources of raw materials, ie from dead or forced slaughter of designated large-scale animals or livers from fur animal farms which are no longer used. Said liver sources represent a very economical way of obtaining arachidonic acid methyl ester.

The process according to the invention is further described in the following examples.

He did

With the extraction cartridge, 5 g of magnesium sulfate and 5.3 g of homogenized pig liver were placed and extracted with 100 ml hexane in a Soxhlet apparatus for 6 hours. Then, the solvent was distilled off under reduced pressure on a rotary evaporator at 35 ° C and the extracted fat was further dried at 65-133 Pa at 35-40 ° C for 2 hours.

The yield of fat was 0.243 g (4.6%). To the dried fat was added 60 ml of methanol in which 0.1 g of sodium was dissolved and the mixture was stirred at 20 ° C for 6 hours. Then 1 ml of acetic acid was added and methanol was distilled off in a vacuum evaporator.

80 ml of ether and 50 ml of water were added to the residue and the ether layer separated after shaking in a separatory funnel. After extracting the aqueous layer by shaking twice with 50 ml of ether, the combined ether extracts (after washing with 10% sodium bicarbonate solution and water) were washed with magnesium sulfate.

After distilling off the ether, the residue was dried for 2 hours at 40 ° C and 65 Pa. According to gas chromatographic analysis, this mixture of methyl esters of bridge acids contained 10.33% arachidonic acid (methyl erachldonate standard) for this and other examples. All other operations and pressure equalization after distilling off the solvents under vacuum were carried out under an inert atmosphere.

Example 2

In a Soxhlet apparatus, 14.055 g of lyophilized pork liver was extracted with 120 ml of dry methylepchloride for 8 hours. After distilling off the solvent and drying the extract of Example 1, 1.695 g (12.06%) of extracted fat was obtained.

To this was added 200 ml of methanol and 100 ml of benzene, and the mixture was refluxed for 2 hours with the addition of 0.2 g of p-toluenesulfonic acid. The aseotropic mixture was then slowly distilled off through the puncture column over a period of 4 hours while occasionally delivering methanol-benzene 2: 1.

Finally, the solvent mixture was distilled off under reduced pressure, and the residue was worked up as described in Example 1. 0.86 g (51%) of the methyl ester mixture was isolated in which 8.75% arachidonic acid was determined by gas chromatography analysis.

Example 3

The Soxhlet extraction cartridge was weighed with 12.506 g of bovine liver, prepared by drying 38.076 g of fresh homogenized bovine liver under reduced pressure at 40 ° C and extracted with methanol-benzene 1: 1 for 10 hours under a nitrogen atmosphere.

After removal of the solvent by distillation and drying of the extracts of Example 1, 2.640 g of fat (6.95%) were sieved. To this was added 150 ml of methanol, in which 0.2 g of sodium was spiked, and the mixture was stirred for 12 hours at 20 ° C under a nitrogen atmosphere. Then 2 ml of acetic acid was added and by further (working as described in Example 1) 1.750 g (69%) of a mixture of methyl esters was isolated, in which 8.64% arechidonic acid was found by the above analysis.

Example 4

29.5 g of bovine liver from the animal to be slaughtered were after. homogenization dried under reduced pressure at 40 ° C. The dry residue (9.328 g) was triturated and subjected to pentane extraction in a Soxhlet apparatus for 6 hours.

The work-up described in Example 1 yielded 0.917 g of fat (9.85% in dry matter) which was first dissolved in a 50 ml methanol for 15 minutes and then, after cooling, 70 ml of methanol was added, in which 0.1 g had previously been dissolved. of sodium. After stirring at 20 ° C for 8 hours under an argon atmosphere, the mixture was treated as described in Example 1 after addition of 1 ml of acetic acid.

0.595 g (65% by weight of the starting fat) of methyl ester was obtained, in which 11.68% of arechidonic acid was detected by gas chromatographic analysis.

Example 5

6.323 g of sheep liver gave 2.257 g of dry matter on drying under similar conditions to the previous example. To this was added 200 ml of methylene chloride-methanol (2: 3) and 0.3 g of p-toluenesulfonic acid. The mixture was refluxed under nitrogen for 8 hours and filtered after cooling.

The filtrate was concentrated under reduced pressure on a rotary evaporator at 35 ° C and the residue treated as described in Example 1 and dried at 65 Pa and 35 ° C for 2 hours. 2.70% of arachldonic acid was detected by OLC analysis.

Example 6

In a Soxhlet apparatus, 255.5 g of lyophilized pork liver (in powder form) was extracted with 750 ml of hexane and 17.1 g of fat (6.7 $) was obtained after removal of the solvent and drying as described in the previous experiment.

This was first dissolved by boiling for half an hour and 200 ml of methanol, and after cooling, 100 ml of methanol were added, in which 0.5 g of sodium was dissolved.

The mixture was then stirred under argon at 20 ° C for 15 hours.

After acidification with 3 ml of acetic acid, the resulting methyl esters were isolated as described in Example 1. 0.8 g (63.56) of a methyl ester mixture was obtained in which 7.5% arachldonic acid was found by the above analysis.

Example 7

A mixture of 9.535 g of dried nutria liver, 50 ml of methylene chloride, 150 ml of methanol and 0.5 g of p-toluenesulfonic acid was heated to reflux in a 0.5 L flask for 10 hours under argon. After cooling, the mixture was filtered and worked up as described in Example 5.

Arachldonic acid was determined to be 6.60% by mass analysis of the methyl esters obtained by gas chromatography.

Example 8

Drying 4.862 g of liver from mink gave 1.848 g of dry matter. Drying was performed

Claims (1)

Drying 4.862 g of liver from mink gave 1.848 g of dry matter. Drying was carried out under reduced pressure and 40 ° C. The solid was placed in an extraction cartridge 8 with 5 g of sodium sulfate (removal of the reaction water). Extraction and transesterification were carried out for 8 hours with a mixture of 100 ml of methylene chloride and 200 ml of methanol, in which 0.25 g of p-toluenesulfonic acid was dissolved in a boiling flask. After concentration, the extract was worked up in the usual manner by extraction with ether and washing the extract with sodium bicarbonate solution and then with water (see Example 1). 0.320 g (17.3%) of the methyl ester mixture was isolated in which 6.30% arachidonic acid was determined by gas chromatography. Example 9 In a similar manner to the previous experiment, from 9.120 grams of blue tracheal liver, 3.622 grams of dry matter were first dried, followed by extraction and simultaneous transesterification (see Example 8) of 0.476 grams (14.2%) of the methyl ester mixture found in this analysis , 72% arachidonic acid. Example 10 In a Soxhlet apparatus, 25.5 g of lyophilized pork liver were extracted with 250 ml of hexane. Isolation of the extract yielded 2.25 g (9%) of fat, to which 100 ml of absolute ethanol was added, in which 0.5 g of sodium was previously dissolved. The mixture was stirred under a nitrogen atmosphere for 15 hours at 20 ° C and after addition of 3 ml of acetic acid, the resulting ethyl esters were isolated in a similar manner as described in Example 1. Analysis by gas chromatography showed 8.2% ethyl arachidonic acid ethyl ester mixture. (Standard arachidonic acid ethyl ester). Example 11 A mixture of 22.8 g of lyophilized pork liver, 150 ml of toluene, and 100 ml of absolute ethanol containing 5% hydrogen chloride was heated under reflux in a nitrogen atmosphere for 8 hours. After cooling, the suspension was filtered and the filtrate was concentrated under reduced pressure. After drying the residual oil (1.82 g, 8% liver weight), the ethyl ester mixture was analyzed by gas chromatography. 8.6 Ϊ of ethyl arachidonic acid esters were determined in the mixture. Example 12 A mixture of 5.20 g of lyophilized porcine liver, 50 ml of hexane, 50 ml of absolute ethanol and 1.24 g of aluminum chloride was heated under reflux for 5 hours under stirring under nitrogen. After cooling, the mixture was filtered, the precipitate was washed twice with 20 ml of hexane each, and the filtrate, after concentration, was treated similarly to that described in Examples 8 and 1. 0.31 g of an ethyl ester mixture was isolated in which 6.34% arachidonic acid ethyl esters were determined by gas chromatography. Example 13 A mixture of 10 g of lyophilized pork liver, 100 ml of hexane, 100 ml of absolute ethanol and 2.2 g of ketex Ostion KS was heated to boiling under stirring under nitrogen atmosphere for 8 hours. After cooling, the mixture was filtered, the precipitate was washed twice with 30 ml of hexane and the filtrate after treatment was similar to that of Example 1. 1.25 g of an ethyl ester mixture was isolated in which 6.85% arachldonic acid ethyl ester was determined. Example 14 A mobile phase of 96.9% n-pentane and 31 diethyl ether and 0.1% methanol was prepared. In an ultrasonic bath, 200 grams of 10 to 20 µm granules of gel were homogenized with a portion of the mobile phase per suspension that was poured into a preparative chromatography column. A column of 40 mk diameter and 270 mm distance was formed by axial compression, to which 2 ml of arachldonic acid methyl ester were sprayed and chromatographic separations were performed at a flow rate of 20 ml / min. According to the recorder by which the progress of the separation was monitored, they were collected. Pezy chromatographic zones containing arachldonic acid methyl ester. These solutions were concentrated on a rotary evaporator and the product was subjected to liquid and liquid analysis. gas chromatography. A higher purity was achieved by subsequent separation of these fractions with a high-performance liquid