Isolation of stearidonic acid from fatty acid mixtures

Substantially pure stearidonic acid is isolated from a mixture of polyunsaturated fatty acids by fractionating at 25% to 35% by weight solution of fatty acids by high-performance reverse-phase liquid chromatography using a mobile phase of 75% to 95% by weight methanol and 25% to 5% by weight water. The isolated stearidonic acid is used to prepare pharmaceutical compositions which are administered to treat cardiovascular and thrombo-embolic diseases associated with platelet aggregation.

BACKGROUND OF THE INVENTION 
This invention relates to the production of stearidonic acid from a mixture 
of fatty acids in which it is present as a minor constituent. 
It is known that essential fatty acids of the n-3 series (nomenclature 
defined by the position of the first double bond from the methyl group) 
lead by a chain reaction to series 3 prostaglandins which inhibit the 
aggregation of blood platelets. 
The first element of this chain is .alpha.-linolenic acid (ALA, C 18:3 
.DELTA.9,12,15) of which the conversion into stearidonic acid (SA, C 18:4 
.DELTA.6,9,12,15) is attributable to the activity of the enzyme .DELTA.6 
desaturase which is shown to weaken with age and as a result of certain 
diseases. The synthesis of the series 3 prostaglandins is thus 
compromised. To overcome this weakness of the organism, it is proposed to 
introduce stearidonic acid directly into the body. 
Japanese patent application 86 050 752 relates to a process for the 
separation of stearidonic acid from fish oil which comprises the steps of 
converting the fatty acids present therein into their ethyl esters, 
treating the ethyl esters by molecular distillation and collecting the 
head fraction, reacting it with urea in methanol, collecting the unreacted 
fraction and, finally, separating a fraction enriched with ethyl ester 
from the stearidonic acid by two successive operations of reverse-phase 
partition chromatography. 
A purity of 85% by weight is thus obtained for a yield of 1.8%, based on 
the oil used, which represents a recovery of 48% of the stearidonic acid 
present in the starting material. 
In addition, European patent 178 442 relates to the enrichment of 
blackcurrant seed oil with .gamma.-linolenic acid (GLA, C 18:3 
.DELTA.6,9,12) by the double complexing with urea of a mixture of fatty 
acids eminating from the saponification of blackcurrant oil, followed by 
high-performance reversephase liquid chromatography. According to this 
patent, separation appeared particularly difficult, enrichment of the 
mixture only appearing possible in regard to its major constituent, 
.gamma.-linolenic acid. 
SUMMARY OF THE INVENTION 
It has been found that a fraction enriched with stearidonic acid having a 
purity of more than 90% by weight can be prepared from a mixture of fatty 
acids in which it is present without any need for molecular distillation 
of the ethyl esters and with only one chromatographic operation. 
Accordingly, the invention relates to a process for the production of 
stearidonic acid in which a mixture of fatty acids containing this acid is 
reacted with urea a fraction enriched with polyunsaturated fatty acids is 
collected, and the enriched fraction is injected into a high-performance 
reverse-phase liquid chromatography column and is eluted from this column. 
The process according to the invention is characterized in that a mixture 
of 75 to 95% by weight methanol for 25 to 5% by weight water is used as 
eluent and the fatty acids are injected into the column in the form of a 
25 to 35% by weight solution.

DETAILED DESCRIPTION OF THE INVENTION 
A starting oil containing a perceptible percentage of stearidonic acid, of 
the order of 2 to 4%, is preferably used for carrying out the process 
according to the invention. Fish oil and particularly blackcurrant seed 
oil (Ribes nigrum) are suitable in this regard. 
Fish oil contains useful proportions of stearidonic acid, eicosapentaenoic 
acid (EPA, C 20:5 .DELTA.5,8,11,14,17) and docosahexaenoic acid (DHA, 
C22:6 .DELTA.4,7,10,13,16,19) while blackcurrant seed oil is an important 
source of .gamma.-linolenic and stearidonic acids. 
The crude oils extracted, for example by solvent or by pressure, are first 
refined by degumming in known manner, for example with phosphoric acid, 
neutralization of the free fatty acids, for example with sodium hydroxide, 
decoloration, for example with a mixture of active carbon and activated 
aluminium silicate, deodorization in vacuo at a temperature of 
approximately 200.degree. C. in the case of black-currant seed oil and, 
optionally, winterizing by cooling, for example to approximately 4.C for 
about 24 hours, which enables most of the residual waxes to be eliminated 
in the case of blackcurrant seed oil. 
Fish oil is refined in the same way as blackcurrant seed oil, except that 
the deodorizing step is preferably carried out at a temperature of 
.ltoreq.180.degree. C. to avoid degradation of the polyunsaturated fatty 
acids. The starting mixture of free fatty acids is obtained by 
saponification of the refined or semi-refined oils and acidification of 
the fatty acid salts obtained or even by hydrolysis of the refined or 
semi-refined oils. Semi-refining comprises degumming and neutralization of 
the crude oils. 
The fatty acid mixture is then treated with urea in a ratio by weight of 
fatty acids to urea of 1:3 to 1:6 and preferably 1:3 to 1:4 in the 
presence of methanol in a ratio by weight of urea to methanol of 1:1.5 to 
1:3 and preferably 1:2.1 by heating the mixture to the boiling 
temperature. The reaction mixture is then cooled to a temperature of 
-5.degree. to 20.degree. C. and preferably 0.degree. to 15.degree. C., 
after which the cooled mixture is left standing at the final temperature 
for at most 15 hours and preferably for 1 to 7 hours. The precipitate 
formed is then cold-filtered, the liquid phase is recovered and the fatty 
acid mixture enriched with polyunsaturates is extracted therefrom, for 
example in a hydrochloric medium in the presence of hexane, the hexane 
being subsequently eliminated. 
The following step comprises fractionation of the fatty acid mixture 
enriched with polyunsaturates by high-performance reversed-phase .liquid 
chromatography. This type of preparative chromatography comprises 
injecting the fatty acids into the apparatus in the form of samples, 
adsorbing them through their hydrophobic part onto a column (constituting 
the stationary phase) of porous silica doped with a layer of saturated 
hydrocarbons and then selectively desorbing them by a mobile phase of 
controlled polarity in which the polar parts of the samples are dissolved 
and which constitutes the eluent. 
In the present case, the eluent consists of a water/methanol mixture 
preferably containing 85 to 90% by weight methanol for 15 to 10% by weight 
water. The mobile phase represents 1 to 3 g per liter adsorbent in the 
column and preferably 1.5 to 2 g/1. 
The fatty acids are preferably present in the injection sample in the form 
of a solution in a good solvent for the fatty acids, for example methanol. 
The concentration of the fatty acids in the injection sample is a factor 
determining the success of the separation. It is preferably of the order 
of 30% by weight for approximately 70% by weight methanol. 
It has in fact been found that better separation of the stearidonic acid is 
achieved with a more concentrated solution of fatty acids than in the case 
of state-of-the-art separation by chromatography. The effect of 
concentration in this regard is unexpected in the sense that separation 
could be expected to be facilitated by dilution. The remarkable result of 
the process according to the invention is due primarily to the suitable 
choice of the composition of the eluent and of the fatty acid 
concentration of the mobile phase. 
The invention also relates to the use of the stearidonic acid obtained by 
the process according to the invention for the production of a 
pharmaceutical composition against the cardiovascular and thrombo-embolic 
diseases associated with platelet aggregation. 
The pharmaceutical composition in question may be formulated in unit dosage 
forms according to the method of administration, for example oral, 
enteral, rectal or parenteral. For example, it may be made up in the form 
of capsules, gelatin-coated tablets, suppositories or syrups. In the case 
of enteral or parenteral administration, the compositions are formulated 
as chemically and physically stabilized, apyrogenic and sterile solutions 
or emulsions. 
The stearidonic acid may advantageously be protected against oxidation by a 
suitable antioxidant, for example ascorbyl palmitate, tocopherols, a 
mixture of such anti-oxidants or a mixture of ascorbic acid, tocopherol 
and lecithin. 
The dose administered depends on the type and seriousness of the condition 
to be treated. It may be from 0.1 to 1 g stearidonic acid per day in a 
single dose or, preferably, in 2 to 3 separate doses. 
The invention is illustrated by the following Examples in which parts and 
percentages are by weight, unless otherwise indicated. 
EXAMPLES 
In the following Examples, the fatty acids were analyzed by gas phase 
chromatography of their methyl esters. 
EXAMPLE 1 
A solution of 30.5 parts sodium hydroxide in a mixture of 101.5 parts water 
and 80.5 parts ethanol is added with thorough stirring to 100 parts 
refined blackcurrant seed oil and the mixture is heated for 30 minutes to 
the boiling temperature (approximately 80.degree. C.). The mixture is then 
cooled to 30.degree.-40.degree. C. and 100 parts 32% hydrochloric acid (pH 
1) and 166.7 parts hexane are slowly added with vigorous stirring over a 
period of 30 minutes. The mixture is then decanted. Two phases are formed. 
The upper phase is removed and the hexane is evaporated therefrom in vacuo 
at 40.degree. C. The resulting mixture constitutes the starting fatty acid 
mixture This mixture consists of the following fatty acids: 
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% 
______________________________________ 
C 16:0 7 
C 18:0 1.3 
C 18:1 cis 11.7 
C 18:2 .DELTA.9,12 47.3 
C 18:3 .DELTA.6,9,12 
16.4 
C 18:3 .DELTA.9,12,15 
13.8 
C 18:4 .DELTA.6,9,12,15 (SA) 
2.5 
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450 g urea and 945 g methanol are added to 150 g of the above fatty acid 
mixture which is then heated to the boiling temperature at which it 
becomes clear. It is then left to cool to ambient temperature and is then 
placed in a water bath at 0.degree. C. for 5 h. A precipitate is formed. 
The liquid phase is then recovered by vacuum filtration in a Buchner 
cooled beforehand to 0.degree. C., after which the fatty acids enriched 
with polyunsaturates are extracted with 0.2 pat hydrochloric acid, 0.6 
part water and 0.25 part hexane for 1 part liquid phase. Finally, the 
hexane is evaporated in vacuo at 40.degree. C. and a fatty acid mixture o 
the following composition is collected: 
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% 
______________________________________ 
C 18:2 .DELTA.6,9 2.1 
C 18:3 .DELTA.6,9,12 
80.7 
C 18:3 .DELTA.9,12,15 
2.1 
C 18:4 .DELTA.6,9,12,15 (SA) 
15.1 
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Chromatography is carried out using two columns with an internal diameter 
of 5.7 cm and a length of 30 cm arranged in series and filled with porous 
silica doped with saturated C.sub.18 hydrocarbons constituting the 
stationary phase. The columns are under 30 bar nitrogen pressure (radial 
compression) and are equipped with a preferential refractive index 
detector for quantitative identification of the fractions. The mobile 
phase consists of a methanol: water mixture containing approximately 12% 
water with a density of 0.821 to 0.824. 
10 ml samples of a solution of 30% of the fatty acid mixture in 70% 
methanol are injected so that they are adsorbed onto the column. The 
various fractions are then eluted by passing the mobile phase through the 
column at a rate of 100 ml/minute. Several fractions are collected, and 
the fraction relating to the first peak is set aside. It corresponds to 
10% of the fatty acid mixture introduced into the apparatus and contains 
92% stearidonic acid. The yield of 92% pure stearidonic acid thus rises to 
1.5% of the starting fatty acid mixture, which corresponds to 56% of the 
stearidonic acid present in the starting material. 
EXAMPLE 2 
The fraction obtained in Example 1 is reinjected as 10 ml samples of a 30% 
solution in methanol under the same conditions. The fraction relating to 
the first peak is collected; it corresponds to 60% of the fatty acid 
mixture reinjected and contains 98% stearidonic acid. 
EXAMPLE 3 
The procedure is as in Example 1, starting with a mixture of fish oil fatty 
acids fractionated with urea under the same conditions apart from the 
final standing temperature of the complex mixture with urea/liquid phase, 
which is 10.degree. C. The mixture of fatty acids has the following 
composition: 
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% 
______________________________________ 
C 18:4 .DELTA.6,9,12,15 (SA) 
16.3 
C 20:5 .DELTA.5,8,11,14,17 (EPA) 
34.9 
C 22:6 .DELTA.4,7,10,13,16,19 (DHA) 
38.6 
Others 10.2 
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The chromatography is carried out in the same way as in Example 1 in regard 
to the composition of the mobile phase and the elution rate. The only 
change is the specific charge of starting fatty acids of 1.75 g/1 
adsorbent in the column. Several fractions are collected and the fraction 
relating to the second peak, which has the following fatty acid 
composition, is set aside: 
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% 
______________________________________ 
C 18:3 .DELTA.9,12,15 (ALA) 
5 
C 18:4 .DELTA.6,9,12,15 (SA) 
86 
Others 9 
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This fraction represents 0.9% of the starting fatty acid mixture, which 
corresponds to 35% of the stearidonic acid present in the starting 
material. 
EXAMPLE 4 
It is accepted that cardiovascular and thrombo-embolic diseases are often 
associated with hyperactivity of the blood platelets. Polyunsaturated 
fatty acids of the n-3 series are considered to be prevention factors 
against thrombo-embolic disease. 
The stearidonic acid obtained in accordance with Example 2 is studied for 
its effect on platelet aggregation by comparison with .gamma.-linolenic 
acid, .alpha.-linolenic acid and eicosapentaenoic acid and by comparison 
with the fatty acid mixture obtained in accordance with Example 2 of EP 
178 442 (referred to hereinafter as concentrate). 
The blood of blood donors with an average age of 36 years is collected, an 
anticoagulant is added and the platelets are isolated. The platelets are 
then suspended in a Tyrode HEPES solution of pH 7.35 which is kept for 16 
h at 37.degree. C. in tubes closed under nitrogen and containing: 
50 .mu.M delipidized human albimin 
50 .mu.M linoleic acid (LA, C 18:2 .DELTA.9,12) 
5 .mu.M arachidonic acid (AA, C 20:4 .DELTA.5,8,11,14) 
and 5 .mu.M of the studied fatty acid. 
The platelets are incubated in this solution for 2 h at 37.degree. C., 
re-isolated and then taken up in a Tyrode HEPES solution to which 0.1% 
gelatin is added. For each test, a batch of platelet suspension serving as 
control is subjected to the same treatment except that the suspension 
contains only linoleic and arachidonic acid of which the presence does not 
affect the platelet functions. In certain tests, 10 .mu.M 
.alpha.-tocopherol is added in addition to the various fatty acids to 
compensate for the depletion of this compound caused by the 
polyunsaturated fatty acids. The platelets thus enriched are under 
conditions similar to those prevailing in blood plasma. 
Platelet aggregation is measured by Born's turbidimetric method (J. 
Physiol. 209:487, 1973). 
The concentration of the aggregating agent is calculated to obtain 
approximately 60% aggregation 4 minutes after the addition of the 
aggregating agent with the platelets in question as control. 
The results are shown in Table 1 below expressed as % aggregation and 
represent a mean the standard deviation in each case in parallel with the 
control. 
TABLE 1 
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Aggregating agent 
Arachi- Analog 
Fatty acid donic of PGH 2 
added Collagen acid (U 46619) 
Thrombin 
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SA 47 .+-. 12 
30 .+-. 6 
51 .+-. 15 
53 .+-. 12 
Control 58 .+-. 8 47 .+-. 5 
63 .+-. 3 
62 .+-. 7 
GLA 51 .+-. 12 
-- 47 .+-. 11 
57 .+-. 15 
Control 60 .+-. 9 -- 62 .+-. 11 
59 .+-. 6 
ALA 47 .+-. 12 
-- 50 .+-. 9 
54 .+-. 3 
Control 58 .+-. 9 -- 56 .+-. 7 
61 .+-. 10 
EPA 46 .+-. 12 
-- 45 .+-. 13 
59 .+-. 13 
Control 60 .+-. 9 -- 62 .+-. 11 
59 .+-. 8 
Concentrate 
50 .+-. 9 -- 57 .+-. 13 
61 .+-. 8 
Control 58 .+-. 9 -- 57 .+-. 6 
61 .+-. 10 
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Legend: -- = not tested 
The above results show that SA inhibits the platelet aggregation induced by 
collagen, arachidonic acid, the PGH2 analog or thrombin. Compared with 
GLA, ALA and EPA, SA inhibits the stimulation by thrombin more 
specifically. In addition, it was found that the presence f 10 .mu.M 
.alpha.-tocopherol did not affect platelet aggregation.