GB2185976A - Process for the enrichment with ???6 fatty acids of a mixture of fatty acids - Google Patents
Process for the enrichment with ???6 fatty acids of a mixture of fatty acids Download PDFInfo
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- GB2185976A GB2185976A GB08602364A GB8602364A GB2185976A GB 2185976 A GB2185976 A GB 2185976A GB 08602364 A GB08602364 A GB 08602364A GB 8602364 A GB8602364 A GB 8602364A GB 2185976 A GB2185976 A GB 2185976A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/487—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
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Abstract
For the selective enrichment with fatty acids, of which the first double bond is in the 6 position, of a starting material containing fatty acids of which the first double bond is in the 6 or 9 position, the starting material is treated with a saturated solution of urea in methanol with predetermined ratios of fatty acids to urea so as to separate the fatty acids other than the ???6 polyunsaturated in the form of an insoluble inclusion complex. The enriched fractions obtained may be treated by high-performance liquid chromatography to isolate the substantially pure ???-linolenic acid. The enriched fractions, optionally recombined with the glycerol, may be used in medicaments and in nutritive compositions or may serve as starting product in the synthesis of dihomo-???-linolenic acid. <IMAGE>
Description
SPECIFICATION
Process for the enrichment with A6 fatty acids of a mixture of fatty acids
This invention relates two the selective enrichment with polyunsaturated fatty acids, of which the first double bond is in the 6 position, more especially with y-linolenic acid, of a mixture containing polyunsaturated fatty acids ofwhich the first double bond is in the 6 or 9 position.
The biological importanceof-linolenicacid (6,9,1 2-octadecatrienoic acid (A6,(1)6)) is well known. It is an essential intermediate in the metabolic process which takes place in a living organism from linoleic acid (9,1 2-octadecadienoic acid (A9,oJ6)) and which leads byway of dihomo-q-linolenic acid (5,8,11 -eicosatrienoic acid) to arachidonic acid (5,8,11,1 4-eicosatetraenoic acid). This property is utilized in its numerous dietetic, cosmetic and pharmaceutical applications. It is also known that or-linolenic acid (9,12,1 5-octadecatrienoic acid (A9,(ss3)) does not participate in this metabolic process in the same way.
The majority of vegetable oils contain the A9 polyunsaturated acids, linoleic acid (06) and a-linolenic acid (03). Avery interesting source of the A6 polyunsaturated acid, w-linolenic acid (w6), is in the seeds of fruit of the genus Ribes, particularly black currant.The seed oil of this fruit contains appreciable quantities of A9 polyunsaturated acids in the form of linoleic acid (w6) and a-linolenic acid (Z3). By way of illustration,the seed oil ofthisfruit consists oftriglycerides of the following fatty acids in percent by weight:
Fatty acids Black Red Gooseberry
currant currant
C16:0 6-7 4-5 7-8
C18:0 1-2 1-2 1
C18:1 cis 9-10 14-15 15-16 C18:1trans 0.5 0.5-1 1-2
C18:2,A9,12 47-49 41-42 39-41 C18:3,6,9,12 15-19 4-5 10-12 C18:3,A9,12,15 12-14 29-31 19-20
C18::4,A6,9,12,15, 3-4 2.5-3.5 4-5
For certain applications, particularly pharmaceutical applications, it is desirable to enrich the mixture of fatty acids emanating from these oils selectively with A6 polyunsaturated acids, particularly with y-linolenic acid. Now, the conventional method offractionating fatty acids by high-performance liquid chromatography (HPLC) does not enable -linolenic acid to be separated from a-linolenic acid.
We have surprisingly found that these two isomers may be separated by a simple method hitherto used for separating fatty acids having different degrees of saturation. In particular, the separation of unsaturated fatty acids from saturated fatty acids is described, for example, in FR-PS 1 603383, corresponding to GB-PS 1 240 513, which relates to the enrichment with -linolenic acid of a mixture of fatty acids emanating from the oil of the evening primrose (Oenothera) which moreover contains hardly any ci-linolenic acid.
The process according to the present invention is characterized in that the mixture is treated with urea dissolved in a lower alkanol in a ratio by weight of fatty acids other than the A6 polyunsaturates to urea of from 1:2.0 to 1 :4.5, in that the insoluble inclusion complex formed is separated and in that a fraction enriched with A6 polyunsaturated fatty acids, particularly with y-linolenic acid, is collected in the liquid phase.
The process is preferably carried out using a mixture offatty acids obtained by saponification of a starting material containing seed oil offruit of the genus Ribes, preferably oil of black currant (Ribes nigrum). Thus, it is possible to saponify the crude or refined oil, alternatively, directly to treatthe seeds, preferably converted into flake form.Saponification is carried out conventionally by treating the starting material with a concentrated strong base, for example sodium hydroxide, preferably in hot aqueous-alcoholic medium, advantageously containing a sequestering agent four metal ions, such as for example disodium ethylene diamine tetraacetate, separating the unsaponifiables with a solvent, for example hexane, and neutralizing the aqu eous phase, for example with hydrochloric acid in concentrated aqueous solution. After saponification,the mixture obtained is preferably protected against oxidation by addition of an antioxidant, for example 1 00to 600 ppm (parts per million) of propyl gallate or, preferably, 200 to 400 ppm ofascorbyl palmitate.
The soaps obtained during neutralization of the crude oil during refining may also be used as starting product.
The fractionation is carried out under conditions which promote the selective formation of a complex of fatty acids otherthan \6with urea in a medium inwhichthe urea is soluble, but notthe inclusion complexes formed. Asuitable medium is a lower alkanol, for example an alkanol containing from 1 to 4 carbon atoms, preferably ethanol or methanol, methanol being particularly suitable by virtue of its high dissolving power on urea. The solution is preferably saturated and should contain from 45to 50% by weight of urea.The saturated solution is advantageously prepared by dissolving the urea in the methanol, for example at around 60"C, while stirring until a transparent solution is obtained.
The quantity of urea is proportional to the total quantity of fatty acids to be eliminated from the mixture.
When the mixture offatty acids emanates from the saponification of the starting material mentioned above, a ratio by weight of starting material to urea of preferably about 1:3 is used. The quantity of methanol used is advantageously from 2 to 6 times and preferably about3 times the quantity by weight starting material used. After vigorous stirring of the mixture, it is cooled to a temperature of from Oto 1 2"C and preferably to a temperature of from 4 to 6 C over a period of from 10 to 20 h.After separation of the precipitate, for example by centrifuging, the unreacted urea is neutralized by treating the solution with an acid, for example with preferably concentrated hydrochloric acid in aqueous solution, and the fatty acids are extracted with a solvent, preferably hexane, which is then eliminated, preferably by evaporation in vacuo.
In one preferred embodiment, the fraction enriched as described above is treated once more with urea in a ratio by weight of total fatty acids to urea of from 1:1 .4to to 1:1.6. This gives a fraction containing from 92 to 96 % by weight of #6 polyunsaturated fatty acids of which from 77 to 81% consist of #-linolenicacid.
If desired, the substantially pure-γ-linolenicacid may be prepared from the fraction emanating from the second fractionation with urea by reverse high-performance liquid chromatography which enables the iinolenic acid to be separated from stearidonic acid (C18:4,#6,9,12,15).
Alternatively, a mixture containing the fatty acids emanating from saponification may be separated by chromatography and then fractionated with urea as described in the foregoing.
The free fatty acid fractions obtained in accordance with the invention may be used as such or in the form of an oil obtained by recombination with glycerol for any of the various applications of γ-linolenic acid, for oflinoIenic acid, for example in the nutritive compositions or the medicaments described in published European PatentApplications 92 085 (corresponding to GB-PA 2.118.567) and 92 076 (corresponding to US-PS 4.526.793) and administered orally, enterally, parenterally ortopically.
The fractions recombined with glycerol in the form of an oil are particularly suitable fortopical application in dermatology and in cosmeto-dermatology. The fractions containing the substantially pure γ-linolenic acid may also be used as starting product in the synthesis of dihomo-z-linolenic acid.
The invention is illustrated by the following Examples in which the percentage and parts are by weight, unless otherwise indicated.
Example 1
To kg of refined and deodorized black currant oil are added 63.9 kg of a 14.2% aqueous-ethanolicsol- ution of sodium hydroxide containing 95 g of disodium ethylene diaminetetraacetate. The mixture is heated to 60"C and is then stirred atthat temperature for 30 minutes. kg of water are then added and the solution is cooled to 30 C.
After addition of 79 kg of hexane and stirring for 1 hour at 30 C, the mixture is leftfor 15 minutes to separate and the upper phase containing the unsaponifiables is eliminated. kg of 32% aqueous hydrochloric acid are then added to the lower phase with stirring (to pH 1), ensuring that the temperature does not exceed 3000.
After decantation, the lower phase is eliminated and the upper phase is concentrated in a water-jetvacuum pump at 40 C.
The 28.5 kg of the fatty acid mixture obtained is added with continuous stirring to a saturated, clear solution of 90 kg of urrea in 190 kg of methanol at 60 C. The mixture is cooled to 5"C and kept at that temperature for 15 h. The solid phase formed is then separated by centrifuging whilstthe liquid phase is left standing for 4 hat 5 C.Theliquid phase is again separated from the solid phase by centrifuging. 180kg of liquid phase are thus collected.45.9 of or hexane, 39.9kg of or a 32% aqueous hydrochloric acid solution and 106 kg of water arethen added to the liquid phase and the mixture is heated to 30 C. After stirring for 1 hat 30"C, the mixture is left standing for 10 mins., afterwhich the upper phase is collected by decantation.The lower phase isthen extracted with 12 kg of hexane while stirring for 15 minutes at 3000. The mixture is left standing for 15 mins., afterwhich the upper phase is collected by decantation and combined with the preceding upper phase.
50 kg of water are then vigorously mixed with the combined phases. The mixture is then leftstandingfor3h at ambienttemperature and the upper phase containing the fatty acids is separated and dried at 4000 by evaporation in a water-jet vacuum pump.6.82 offatty acids are thus obtained (yield 22.7%, based on the oil used). The mixture has the following composition by weight, as determined by gas chromatography:
C18:1 0.6 C18:2,A9,12 22.1 C18:3,#6,9,12 55.6 C18:3,9,12,15 10.7 C18:4,#6,9,12,15, 11.0
Comparison Example
The fatty acids from black currant oil used in the previous Example are fractionated by preparative high performance liquid chromatography in theform of theirmethyl esters using reverse-phase RP-1 8 columns.
The elution of a sample having a concentration of 20% in the following solvent mixture: 67.5% methanol 22.5% ethanolll 0% water, at a rate of 150 ml/min. gives the following fractions:
1) a first fraction representing 7.6% of the mixture and containing 52% of stearidonic acid,
C18:4,A6,9,12,15;
2) a second fraction representing 25.4% ofthe mixture and containing 41% of a-linolenic acid, C18:3,A9,12,15 and 43% of y-linolenic acid, C18:3,A6,9,12; 3) a third fraction representing 45.8% of the mixture and containing 87% of linoleic acid, Cl 8:2,A9,1 2 and
4) a fourth fraction representing 21.2% of the mixture and containing 85% of saturated fatty acids.
Comparison ofthe ratios between the quantities of y-linolenic acid and a-linolenic acid calculated for the mixture obtained in accordance with Example l,forfraction 2) ofthe Comparison Example and for biack currant oil provides an indication of the selectivity of the enrichment process according to the invention::
Mixture Black Fraction
of Exam- currant 2) above
ple 1 oil % y-linolenic acid 5.2 1.3 1.1 % a-linolenic acid
It can be seen from the values indicated above that, contrary to the process according to the invention,the conventional method of chromatography does not enable the Py- and os-linolenic acids to be separated from a mixture containing them, sothatthe mixture cannot be enriched with -linolenicacid.
Example 2
A mixture of 10.2 kg of urea and 21.5 kg of methanol is heated to 60"C until a saturated, clear solution is obtained. The 6.82 kg of fatty acid mixture emanating from the fractionation according to Example 1 arethen added with stirring to that solution. The mixture is then cooled to 5"C and left standing at that temperature for 15 h. The solid phase formed is then separated by centrifuging whilst the liquid phase is left standing for 4 hat 5"C. The liquid phase is then centrifuged again and the crystals formed are eliminated.
5.9 kg of hexane, 5.1 kg of a 32% aqueous hydrochloric acid solution and 1 3.5 keg of water are then added to 23 kg of the solution obtained. The mixture heated to 30"C is then vigorously stirred for 15 minutes. It is then left standing for 10 mins. to separate and the upper phase containing the fatty acids is separated off. kg of hexane are then added to the lower phase and the mixture is vigorously stirred for 15 mins. The mixture is then left standing for 15 mins, afterwhich the upper phase is collected bydecantation and combined with the preceding upper phase.
The combined phases are then mixed with 30 kg ofwaterwhile stirring for 15 mins. The mixture is left standing for3 h, afterwhich the lower phase is eliminated bydecantation and the upper phase is collected and dried by evaporation at 4000 in a water-jet vacuum pump. 400 ppm (parts per million) of ascorbyl palmitate are then added to the4.05 of fatty acid mixture obtained (yield 13.5%, based on the mixture of fatty acids used).
The fatty acid mixture obtained has the following composition by weight, as determined by gas chromato- graphy: C18:2,A9,12 2.5 C18:3,A6,9,12 78.6 C18:3,A9,12,15 2.3 C18:4,A6,9,12,15 16.6
Example 3
Black currant seeds are converted into flakes. To 35keg of these flakes are added 150 kg of a 14.2% aqueous- ethanolic solution of sodium hydroxide containing 223 g of disodium ethylene diamine tetraacetate,the suspension heated to 600C is stirred for 1 hour and the residue formed is separated by filtration in vacuo.The filter is then rinsed and washed with a quantity of ethanol equal to the weight ofthe flakes used, afterwhich the majority of the ethanol is evaporated in the form of an azeotropic mixture. A 32% aqueous hydrochloric acid solution is then added to the residue until a pH of 1 is obtained.
After decantation, the majority of the fatty acids are separated in the form of the upper organic phase formed. 35kg of hexane are added to the lower aqueous phase which is then decanted again. The lower phase is washed a second time with kg of hexane. The combined organic phases (comprising the fatty acids and the two solutions in hexane) are then washed twice with 70 kg of water and the solvent is evapora ted at 4000 in awaterjetvacuum pump. The residue represents6.72 of of a mixture of fatty acids of which the composition as determined by gas chromatography is identical with that of the mixture obtained by saponification ofthe oil used in Example 1.The mixture is then subjected to fractionation underthe conditions of Example 1.
Example 4 A 20% solution of the mixture offatty acids emanating from the double fractionation of Example 2 is prepared in the following solvent mixture: 67.5% methanol/22.5% ethanol/10% water.
10 ml of this solution arethen injected into a preparative high-performance liquid chromatography appar- atus provided with reverse-phase RP-18 silica gel columns. The above solvent mixture is used as the mobile phase at a rate of 100 ml/min. and the quantitative identification is carried outwith a refractive index detector.
The two principal fatty acids (-IinoIenic and stearidonic) of the mixture obtained in accordance with Ex ample 2 (78.6%z-linolenic acid, 16.6% stearidonic acid) are effectively separable by the above method,the two eluted fractions obtained having the following compositions: -firstfraction: C18:3,6,9,12 49 (representing 20% ofthe mixture) C18:4,6,9,12,15 51 -second fraction: C18:3,ss6,9,12 96 (representing 80% ofthemixture) C18:3,A9,12,15 2.5 - C18:4,A6,9,12,15 1.5
The second fraction may advantageously serve as starting product in the synthesis of dihomo-y-linolenic acid.
Example 5
100 g of the fatty acid mixtures obtained in accordance with Examples 1 to 4 are reacted with 15 g of glycerolfor5 h at210 C under a pressure of 5 mm Hg. 96g oftriglycerides arethus obtained in a yield of 92%.
The oils obtained are suitablefordermatological and cosmeto-dermatological applications.
Claims (10)
1. A process for the selective enrichment with polyunsaturated fatty acids, of which the first double bond is in the 6 position, particularly with -linolenic acid, of a mixture containing polyunsaturated fatty acids of which the first double bond is in the 6 or 9 position, characterized in that the mixture is treated with urea dissolved in a lower alkanol in a ratio by weight of fatty acids otherthan the A6 polyunsaturated to urea of from 1 :2.0two to 1:4.5, in that the insoluble inclusion complex formed is separated and in that a fraction enriched with A6 polyunsaturated fatty acids, particularlywith -linolenic acid, is collected in the liquid phase.
2. A process as claimed in Claim 1, characterized in that the starting mixture of fatty acids is obtained by saponification of seeds of fruit of the genus Ribes or of an oil extracted therefrom, more particularly seeds or oil of black currant, Ribesnigrum.
3. A process as claimed in Claim 1 or 2, characterized in that the ratio of total fatty acids to urea is app- roximately 1:3.
4. A process as claimed in Claim 1, characterized in that the fraction enriched with A6 polyunsaturated fatty acids obtained is treated with urea in a ratio by weight of total fatty acids to urea of approximately 1:1.5, in thatthe insoluble inclusion complex formed is separated and in that a fraction containing from 92 to 96% by weight of A6 polyunsaturated fatty acids, of which 77 to 81% consist of Py-linolenic acid, is collected.
5. A process as claimed in any of Claims 1 to 4, characterized in that the mixture is treated with a saturated solution of urea in methanol.
6. A process as claimed in Claim 4, characterized in that the substantially pure -linolenic acid is separated from the mixture of A6 polyunsaturated fatty acids by reverse-phase high-performance liquid chromatography.
7. A process as claimed in any of Claims 1 to 6, characterized in that the mixture enriched with A6 poly unsaturated fatty acids is esterified with glycerol to obtain an oil enriched with 6 A6 polyunsaturated fatty acids, more especially with y-linolenic acid.
8. Afraction or oil containing they-linolenic acid obtained by the process claimed in any of Claims 1 to 7.
9. Acomposition containing the oil claimed in Claim 8.
10. The use of a fraction obtained in accordance with Claim 6 as starting product in the synthesis of dihomo-z-linolenic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8602364A GB2185976B (en) | 1986-01-31 | 1986-01-31 | Process for the enrichment with delta-fatty acids of a mixture of fatty acids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8602364A GB2185976B (en) | 1986-01-31 | 1986-01-31 | Process for the enrichment with delta-fatty acids of a mixture of fatty acids |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB8602364D0 GB8602364D0 (en) | 1986-03-05 |
| GB2185976A true GB2185976A (en) | 1987-08-05 |
| GB2185976B GB2185976B (en) | 1990-03-21 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8602364A Expired - Fee Related GB2185976B (en) | 1986-01-31 | 1986-01-31 | Process for the enrichment with delta-fatty acids of a mixture of fatty acids |
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| Country | Link |
|---|---|
| GB (1) | GB2185976B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009020406A1 (en) * | 2007-08-07 | 2009-02-12 | Granate Seed Limited | Methods of making lipid substances, lipid substances made thereby and uses thereof |
-
1986
- 1986-01-31 GB GB8602364A patent/GB2185976B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009020406A1 (en) * | 2007-08-07 | 2009-02-12 | Granate Seed Limited | Methods of making lipid substances, lipid substances made thereby and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2185976B (en) | 1990-03-21 |
| GB8602364D0 (en) | 1986-03-05 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 20020131 |