Process for the selective enrichment of a delta-6 and delta-9 fatty acids containing mixture with delta-6 polyunsaturated fatty acids, the enriched fractions thus obtained and their use

DESCRIPTION_COMPANY of the Swiss company SOCIETE DES PRODUITS NESTLE. SA, based in VEffEY, Switzerland

Selective enrichment method for polyunsaturated Δ6 fatty acids in a mixture containing L6 and Δ9 fatty acids, the obtained fractions obtained and their use

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65528

The invention relates to selective enrichment of polyunsaturated fatty acids whose first double bond is in position 6, mainly gamma-linoleic acid, a mixture containing polyunsaturated fatty acids whose first bond is 9 .

The biological significance of the gamma-transgenic ozone is known. (6,9,12-octadecatrienoate (Δ6, ω6)). It is an essential intermediate in the metabolic process mediated in the living organism by linoleic acid (9,12-octadecadioedioate (Δ9, ω6)) and by solubilization of diolomethacin (5,8,11-a) oxy (5,8,11,14-twenty-four tetraoio). The property is widely used in many dietary cosmetic and pharmaceutical applications. It is also known that alpsalinolenic acid (9,12,15-octadecatrienoic acid (Δ9, ω3)) does not interfere in the metabolic process.

Most vegetable oils contain polyunsaturated D9 linoleic acids (α / 6) and aliphatic, oleic acid (ω3). One source of very interesting gamma-linoleic acid D6 (ω6) is composed of 'fruit ribs of the genus RIBES, in particular GAS3IS. The coconut oil of these fruits contains a significant amount of polyunsaturated Δ9 linoleic acid (ω6) alpha-liololeate (HR). Indicatively, the coconut oil of these fruits is made up of triglycerides of the following fatty acids.

CASSIS fatty acids

C16: C 6-7

C18: C 1-2

C18: 1 CIS 9-10

C '18: 1 TRANS 0.5

C18: 2, Δ9, 12 47-49

C18: 3, Δ6, 9, 12 15-19

C18: 3, Δ9, 12, 15 12-14

C18: 4, Δ6, 9, 12, 15 3-4

Gooseberry

4-5

1-2

14-15

0,5-1

41-42

4-5

29-31

2,5-3,5

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Specifically applied, in particular pharmaceuticals, we wish to selectively enrich the fatty acid mixture derived from these oils with polyunsaturated Δ6, in particular, gamma-thiolenic acid. Where the standard method of fractionating fatty acids by high performance liquid chromatography (HFLc) does not allow the separation of gamma-linolenic acid from alpha-linolenic acid.

It is surprisingly found that we can separate these two isomers by a simple method, hitherto applied, by separating fatty acids of varying degrees of saturation. In particular, the separation of unsaturated lipoproteic acids from saturated oils is described, e.g., by U.S. Pat. France No. 1.60&gt; .pop, corresponding to Britannic No. 1,240,513, which relates to the enrichment of gamma-linolenic acid fatty acid mixture, tromomycetate from algae oil (OENOTHERA.) Which contains no other lactic acid acid. The method according to the invention is characterized in that the mixture is treated with urea in solution in lower alkanol by weight. ratio of fatty acids of other or polyunsaturated Δ6: urea 1: 2.0 to 1: 4.5, which is separated from the intrinsically insoluble content formed, and the opium is collected in the liquid phase enriched in polyunsaturated fatty acids D6, specifically gamma-linolenic acid.

In order to utilize the process we may preferably use a mixture of achieved fatty acids by saponifying a raw material containing RIBES genus load oil, preferably CASSIS oil (RIBES NIGRUM). However, we can saponize crude oil or raffinate or in a variety of ways - we treat the pellets directly, preferably under microwaves. The saponification is carried out in the classical manner by nitrogen, with the raw material having a strong dense base, e.g., sodium, preferably in a warm hydro-alcoholic medium containing advantageously metalloparasite ions, such as disodium ethylenediaminetetraacetate, separating the non-saponifiable solvent, e.g., hexavalent and decolorizing the aqueous phase, e.g., with hydrochloric acid in aqueous solution. After saponification, the solution obtained is combined

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of antioxidant, e.g., 100 to 600 mo. (parts per million) of French propyl or more preferably 200 to 400 µm. pulse ascorbus.

We can also use the achieved soap as a starting product by neutralizing crude oil during refining.

Fractionation consists in the process under conditions that allow for the selective formation of a complex of fatty acids other than Δ6 with urea in a medium where the urea is soluble but not the complex formed. A suitable medium consists of lower alkali, i.e. from 1 to 4 carbon atoms preferably ethanol, or methanol, of the last specific catalytic, owing to the high solubility of urea therein, the solution will preferably be saturated and contain 45-50% by weight of urea. The saturated solution will be advantageously produced by dissolving the urea in methanol for example at about 6 ° C with stirring until a clear solution is obtained.

The amount of urea is proportional to the amount of fatty acids in the mixture to be excluded. When the fatty acid mixture is derived from the saponification of the above-mentioned raw material, will a weighted ratio be used? raw material: urea, preferably about 1: 3. The amount of meth-

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by separation of the substrate, e.g., by centrifugation, the urea is neutralized or it is not dehydrated, the solution is treated with acid, e.g. aqueous hydrochloric acid solution, preferably concentrated, and the fatty acids extracted with solvent, preferably hexane, is then removed, preferably by evaporation in vacuo.

In a preferred embodiment, the above enriched fraction is treated once again with urea, in a weighted ratio of total fatty acids: urea, from 1: 1.4 to 1: 1.6. A fraction containing 92 to 96 wt% is thus obtained. polyunsaturated fatty acids Δ6 including 77 to 81% gamma-lyolulic acid.

If desired, gamma-lyolenic acid may be produced practically pure from the fraction derived from the second fractionation by urea by high performance liquid chromatography, which allows its separation from the acid. buccal acid (0 18: 4, AS,&gt;, 12.15).

Alternatively, the chromatographic separation of the mixture containing the fatty acids derived from saponification can then be carried out by urea fractionation of this mixture as shown above.

The obtained free fatty acid fractions according to the invention may also be used in all gamma-linoleic acid applications as such, or in the form of glycerinol recombinant oil, e.g. in the nutritional compositions or medicines described in the European Lip. Publication No. 92085 Patent Application. U.S. Patent No. 482,429). U.S. Patent No. 482,430) by orally, diarrhea, parenteral or topical (corresponding to 92076 (and

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.... Wonderful

Recombinant fractions with glycerol in the form of oil are particularly useful for topical application in dermatology and cosmetic dermatology. The fractions containing gum linolenic acid practically pure can equally serve as the starting material in the di-gamma-linolenic acid composition (DIHOMO-GAMMA-LINOLENIQUE).

The following examples illustrate the invention. In these percentages and parts are by weight except as otherwise indicated.

EXAMPLE 1.

Add to 30 x g of CASSIS raffinate oil, and a disinfected, 63.9 g / g solution of 14.2 g of sodium hydroxide containing 95 g of disodium ethylenediaminetetraacetate. The mixture is heated to 60 ° C and kept at this temperature for 30 minutes, under stirring. Then 12 g of water is added and the solution is cooled to 30 ° C.

After adding 79 x hexane / hexane and stirring for 1 h. At 30 ° C the mixture was allowed to settle for 15 minutes, and the supernatant-containing phase containing the degradable was removed. Then 30 l / g are added. of hydrochloric acid 32 ° C in the lower phase under stirring (up to PS 1), ensuring that the temperature does not exceed 3 ° C. After precipitation, the lower phase is removed and the upper phase is concentrated under vacuum at 40 ° C.

28.5 k / m are added^a of the obtained fatty acid mixture under continuous stirring in a saturated clear solution of 90 x / wt urea in 190 x / wt of methanol at 60 ° C. The mixture was cooled to 5 ° C and held at this temperature for 15 min. The entrapped solid phase was then centrifuged and the liquid phase was allowed to stand for 4 min. at 5 ° C. Separate the liquid phase of the solid phase by coatings; -^eThese are called 180 kHz liquid phase. Prepare ft1 -) stir 45&gt; 9 g of hexane 59&gt; 9 g of aqueous succinic acid in 32 # and 106 g of water and bring the mixture to 30 ° C. After stirring for 1 hour. at 30 ° C, leave to stand for 10 minutes, then the above phase is collected by decantation. The lower phase was then extracted with 12 x hexane of hexane for 15 minutes at 30 ° C. Allow the mixture to stand for 15 minutes then collect the upper phase by decanting, which is combined with the previous upper phase.

In the foregoing combined phases, there is 50 kg of water under vigorous stirring. Allow to stand, then stir for 3 hours at ambient temperature, separate the upper phase containing the fatty acids and evaporate under vacuum pump, water, at 40 ° C. Thus 6.82 g / kg of fatty acids are obtained (22.7% relative to the oil used. In a mixture the following is determined by weighted chromatography on gas:

<td>C 18</td><td> : 1</td><td> 0,6</td>

<td> 018</td><td>2, D, 9, 12</td><td> 22,1</td>

<td> 018</td><td>3, ΔS, 9, 12</td><td> 55,6</td>

<td> 018</td><td>3, Δ9, 12, 15</td><td> 10,7</td>

<td> 018</td><td>4, ΔS, 9, 12, 15</td><td> 11,0</td>

A COMPARATIVE EXAMPLE.

The fatty acids of the 0AS3IE oil were fractionated using the above by high performance preparative liquid chromatography in the form of methyl ester herein, in an RP-18 column in reverse phase.

The elution of a 20 # sample in the solvent mixture: 67.5 / methanol / 22.5 # ethanol / 10 # water, at a flow rate of 150 ml / min leads to the following fractions:

1) The first fraction represents 7.6 # of the mixture containing 52 "&gt; stearidic acid 018: 4, Δ6, 9, 12, 15"

2) a second fraction representing 25.4 # of the mixture containing

41^;alpha-lyolenic acid, C3: 3, Δ9, P2, C5 and C3 36 g, xenolenic acid, C8: 3, ΔS, 9, 12, L / C

3) a third fraction representing 45.8 # of the mixture, containing '- lyoleic acid, 018: 2, Δ9, 12 and

4) a fourth fraction represents 21.2 # of the mixture containing 85 # saturated fatty acids.

Comparison of the Relationships of Quantities of Gamma-Liolic Acid et al_nLysylenic acid calculated for the obtained mixture according to Example 1, for fraction 2) of the comparative derivative and CASSIS oil gives an indication of the selectivity of the enrichment method according to the invention:

Achieved mixture of # gamma-linoleic acid oil

Fractionated mixture according to CASSIS above 2) Del. 1

5,2 1,3 1,1 # alpha - lyulic acid

Is it certified from the above indicated values ​​that, unlike the method according to the invention, the commonly used chromatographic method does not allow the separation of gamma and alpha-lyolenic acids from a mixture containing them, and therefore no? enrich the mixture with gamma-linolenic acid.

EXAMPLE 2.

A mixture of 10.2 x / wt of urea and 21.5 x / w of methanol was heated at 6 ° C until a saturated clear solution was obtained, then 6.82 x / wt of the fatty acid mixture derived from the fractionate was added under stirring. . 1. The mixture is then cooled to 5 ° C and left at this temperature for 15 min. The solid phase formed is then dried by centrifugation, and the liquid phase is allowed to stand at 5 ° C on a gentle basis. The liquid phase is reconstituted and the formed crystals are excluded.

To 23 g / g of solution was added 5.9 g, 5.1 x aqueous hydrochloric acid / enz-J52S aqueous solution. _z IC 4 '/ 4' water 'and 13.5 x / ma water. Stir vigorously in the 0% mixture for 15 minutes. He was then allowed to settle on Yooxkai.

The upper phase containing the fatty acids is separated. Then 3 g of hexane was added to the lower phase and the mixture was vigorously stirred for 15 minutes. Allow the mixture to stand for a few minutes, then transfer the earlier phase by transfusion, which is combined with the preceding upper phase.

Combine the above combined phases with 30 liters of water under stirring for 15 minutes. Allow the mixture to stand for 3 hours, then remove the lower phase by ligation and collect the upper phase by evaporating under vacuum at 40 ° C. Then 400 pm are added. (parts

Ascorbyl palmitate (KClO) in 4.05 kg / m mixture of achieved fatty acids (yield 13.5 / 6 at reflux to the mixture of used fatty acids).

This has the following weighted composition determined by

<td>chromatography</td><td>gas</td><td>phase:</td><td> 96</td>

<td>C18: 2,</td><td>D9, 12</td><td></td><td> 2,5</td>

<td>C18: 3,</td><td>D6, 9,</td><td> 12</td><td> 78,6</td>

<td>C18: 3,</td><td>D9, 12</td><td> , 15</td><td> 2,5</td>

<td>C18: 4,</td><td>D6, 9,</td><td> 12, 15</td><td> 16,6</td>

EXAMPLE 5;

CASSIS seeds are infused in the form of flakes. Add&gt; 5 x g / ml flakes, 150 x g / g 14.2 g sodium hydroxide solution containing 223 g of ethylenediamine tetraacetate and stirred for 1 hour, heated to 60 ° C, then separated by separation. emptiness. The filter is then wetted and washed with a quantity of ethanol corresponding to the weight of the flakes used, then most of the ethanol is evaporated in the form of an azeotropic mixture. Then add to the residual aqueous solution of 32 g of hydrochloric nodule to achieve the same concentration.

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in the form of the appearance, 35 x / m "hexane was added to the resin. Wash

After transfusion, most of the higher organic phase fatty acids are separated. Lower aqueous phase and megapix-.

lower phase for the second time with hexane / aq., then wash the combined organic phases (containing the fatty acids and the two solutions in hexane) twice with 7D / hex of water in each wash, and evaporate the solvent under water pump vacuum at 40 ° C. The residue represents 6.72 kg / g of the fatty acid mixture determined by gas chromatography, similar to that obtained by saponifying the oil used in the present. 1. This is then subjected to fractionation under standard conditions. 1.

EXAMPLE 4.

Prepare a 20% solution of a fatty acid mixture derived from double fractionation in accordance with tradition. 2 in a solvent mixture: 67.5% methanol / 22.5 ethanol / 10% water.

Then 10 ml of this solution are poured into a high performance liquid chromatography apparatus equipped with a KP-18 silica gel column in an inverted phase. The above solvent mixture is used as a mobile phase at a flow rate of 100 mg / min, and quantitative certification is performed with the help of a refractive index detector.

The two main fatty acids gamma-linolenic acid and the steady state of the obtained mixture according to tradition. 2 (78.6 gm, nulic acid, 16.6% stearidonic acid) are practically separable by the above method, the obtained fractions having the following compositions:

- First fraction: 018: 3 »D6, 9, 12 (about 20% of the mixture) 018: 4, D6,9,12,15

- Second branch: C18: 3, Δ6,91 Ί 2 (representing 80% of the mixture) 018: 3, Δ9,12,15

018 : .4, Δ6.9 in the case

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Can the 2nd fraction advantageously serve as the raw material for the synthesis of dichromate / linolenic acid?

EXAMPLE 5; ',

React 100 g of the mixture of the fatty acids obtained according to Examples 1 to 4 with 15 g of glycerol at 210 ° C for 5 h, under a pressure of 5 mm H 5. . Triglycerides are thus obtained with a 92% yield. The oils obtained are useful for dermatological and cosmetic dermatological applications.