Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
NEW CONJUGATED LINOLENIC ACIDS AND METHODS FOR
COMMERCIAL PREPARATION AND PURIFICATION
FIELD OF THE INVENTION
The present invention relates to a method for the
preparation and purification of fatty acids which are homologues of
conjugated linoleic acids, from materials rich in alpha or gamma linolenic
acids. The method permits the transformation of approximately over two
thirds of a-linolenic acid (9Z,12Z,15Z-octadecatrienoic acid) into
9Z,11E,15Z-octadecatrienoic acid and 9Z,13E,15Z-octadecatrienoic acid.
Enrichment up to and over 40% is readily performed with urea
crystallization, Moreover, the product can be produced in over 90% purity
by simple preparative liquid chromatography. The reaction is unique in that
the reaction produces the above mentioned conjugated trienoic acids with
a high selectivity, in a short time period and in relafiively mild conditions.
The reaction also transforms gamma-linolenic acid (6Z,9Z,12Z-
octadecatrienoic acid) into 6Z,8E,12Z-octadeccatrienoic acid and
6Z,10E,12Z-octadecatrienoic acid. In all cases, geometrical isomers and
fully conjugated isomers are also produced.
BACKGROUND OF THE INVENTION
Processes for the conjugation of the double bonds of
polyunsaturated unconjugated fatty acids have found their main
application in the field of paints and varnishes. Oils comprised of
triglycerides of conjugated fatty acids are known as drying oils. Drying oils
have value because of their ability to polymerize or "dry" after they have
been applied to a surface to form tough, adherent and abrasion resistant
films. Tung oil is an example of a naturally occurring oil containing
significant levels of fully conjugafied fatty acids. Because Lung oil is
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expensive for many industrial applications, research was directed towards
finding substitutes.
In the 1930's, it was found that conjugated fatty acids were
present in oil products subjected to prolonged saponification, as originally
described by Moore (J. Biochem., 31: 142 (1937)). This finding led to the
development of several alkali isomerization processes for the production
of conjugated fatty acids from various sources of polyunsaturated fatty
acids.
The positioning of the double bonds in the hydrocarbon
chain is typically not in a conjugated, i.e., alternating double bond-single
bond-double bond, manner. For example, a-linolenic acid is an eighteen
carbon acid with three double bonds (18:3) at carbons 9, 12 and 15 in
which all three double bonds have the cis configuration, i.e., 9Z,12Z,15Z-
C18:3 acid. a-Linolenic acid is 6Z,9Z,12Z-C18:3 acid and linoleic acid is
9Z,12Z-018:2 acid (see TABLE 1 ).
TABLE 1
N°- Fatty Acid Trivial Name Structure
HOO
1 9Z,12Z,15Z-C18:3 a-Linolenic Acid
2 6Z,9Z,12Z-C18:3 y-Linolenic Acid Hoo
3' 9Z,12Z-C18:2 LinoleicAcid Hoo
Migration of double bonds (e.g., leading to conjugation)
gives rise to many positional and geometric (i.e., cis-trans) isomers.
Conjugated double bonds means two or more double bonds
which alternate with single bonds as in 1,3-butadiene. The hydrogen
atoms are on the same side of the molecule in the case of cis-structure.
The hydrogen atoms are on opposite sides of the molecule in the case of
trans-structure.
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Conjugated linoleic acid (CLA) is a general term used to
name positional and geometric isomers of linoleic acid. Linoleic acid is a
straight chain carboxylic acid having double bonds between the carbons 9
and 10, and between carbons 12 and 13. For example, one CLA positional
isomer has double bonds between carbons 9 and 10 and carbons 11 and
12 (i.e., 9Z,11E-C18:2 acid); another has double bonds between carbons
and 11 and carbons 12 and 13 (i.e., 10E,12Z-C18:2 acid), each with
Y
several possible cis-and traps-isomers (see Table 2).
10 TABLE 2
N°- Fatty Acid Trivial Name Structure
1 92,11 E-C18:2 Rumenic Acid Hoo
2 10E,12Z-C18:2 none Hoo
The 9Z,11E-C18:2 isomer has been shown to be the first
intermediate produced in the biohydrogenation process of linoleic acid by
the anaerobic rumen bacterium Butyrvibrio fibrisolvens. This reaction is
catalyzed by the enzyme X11 isomerase which converts the cis-12 double
bond of linoleic acid into a traps-11 double bond (C. R. Kepler et al., 241,
J. Biol. Chem. (1966) 1350). It has also been found that the normal
intestinal flora of rats can also convert linoleic acid to the 92,11 E-018:2
acid isomer. The reaction does not, however, take place in animals lacking
the required bacteria. Therefore, CLA is largely a product of microbial
metabolism in the digestive tract of primarily ruminants, but to a lesser
extent in other mammals and birds.
The free, naturally occurring conjugated linoleic acids (CLA)
have been previously isolated from fried meats and described as
anticarcinogens by Y. L Ha, N K. Grimm and M. W. Pariza
(Carcinogenesis, Vol.~ 8, No. 12, pp. 1881-1887 (1987)). Since then, they
have been found in some processed cheese products (Y. L. Ha, N. K.
Grimm and M. W. Pariza, J. Agric. Food Chem., Vol. 37, No. 1, pp. 75-81
(1987)). Cook et al. (U.S. Pat. 5,554,646) disclose animal feeds containing
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CLA, or its non-toxic derivatives, e.g., such as sodium and potassium salts
of CLA, as an additive in combination with conventional animal feeds or
human foods. CLA makes for leaner animal mass.
The biological activity associated with CLAs is diverse and
complex (Pariza et al., Prog. Lipid Research., Vol 40, pp. 283-298).
Anti-carcinogenic properties have been well documented, as
well as stimulation of the immune system. Administration of CLA inhibits
rat mammary tumorogenesis, as demonstrated by Ha et al., (Cancer Res.,
52:2035-s (1992)). Ha et al., (Cancer Res., 50:1097 (1990)), reported
similar results in a mouse forestomach neoplasia model. CLA has also
been identified as a strong cytotoxic agent against target human
melanoma, colorectal and breast cancer cells in vitro. A recent major
review article confirms the conclusions drawn from individual studies (Ip,
Am. J. Clin. Nutr. 66(6):1523s (1997)). In in vitro tests, CLAs were tested
for their effectiveness against the growth of malignant human melanomas,
colon and breast cancer cells. In the culture media, there was a significant
reduction in the growth of cancer cells treated with CLAs. by comparison
with control cultures. The mechanism by which CLAs exert
anticarcinogenic activity is unknown. In addition, CLAs have a strong
antioxidative effect so that, for example, peroxidation of lipids can be
inhibited (Atherosclerosis 108, 19-25 (1994)). U.S. Pat. 5,914,346
discloses the use of CLAs to enhance natural killer lymphocyte function.
U.S. Pat. 5,430,066 describes the effect of CLAs in preventing weight loss
and anorexia by immune system stimulation.
Although the mechanisms of CLA action are still obscure,
there is evidence that some components) of the immune system may be
involved, at least in vivo. U.S. Pat. 5,585,400 (Cook, et al.), discloses a
method for attenuating allergic reactions in animals mediated by type I or
IgE hypersensitivity, by administering a diet containing CLA. CLA in
concentrations of about 0.1 to about 1.0 percent was also shown to be an
effective adjuvant in preserving white blood cells. U.S. Pat. 5,674,901
(Cook, et al.), teaches that oral or parenteral administration of CLA in
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either free acid or salt form resulted in an elevation in CD-4 and CD-8
lymphocyte subpopulations associated with cell mediated immunity.
Adverse effects arising from pretreatment with exogenous tumor necrosis
factor could be alleviated indirectly by elevation or maintenance of levels
of CD-4 and CD-8 cells in animals to which CLA was administered.
CLAs have also been found to exert a profound generalized
effect on body composition, in particular, upon redirecting the partitioning
of fat and lean tissue mass. U.S. Pat. 5,554,646 and 6,020,378 teach the
use of CLAs for reducing body fat and increasing lean body mass. U.S.
Pat. 5,814,663 teaches the use of CLAs to maintain an existing level of
body fat or body weight in humans. U.S. Pat. 6,034,132 discloses the use
of CLAs to reduce body weight and treat obesity in humans. CLAs are also
disclosed in U.S. Pat. 5,804,210 to maintain or enhance bone mineral
content. EP 0 579 901 B relates to the use of CLA for avoiding loss of
weight or for reducing increases in weight or anorexia caused by
immunostimulation in humans or animals. U.S. Pat. 5,430,066 (Cook, et
al.), teaches the effect of CLA in preventing weight loss and anorexia by
immune stimulation.
CLA has been found to be an in vitro antioxidant, and in
cells, it protects membranes from oxidative attack. In relation to other
important dietary antioxidants, it quenches singlet oxygen less effectively
than (3-carotene but more effectively than a-tocopherol. It appears to act
as a chain terminating antioxidant by chain-propagating free radicals (U.S.
Pat. 6,316,645).
Skin is subject to deterioration through dermatological
disorders, environmental abuse (wind, air conditioning, central heating) or
through the normal aging process (chronoaging) which may be
accelerated by exposure of skin to sun (photoaging). In recent years the
demand for cosmetic compositions and cosmetic methods for improving
the appearance and condition of skin has grown enormously. WO
95/13806 teaches the use of a composition comprising zinc salts of 68% .
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(unconjugated) linoleic acid and 10% conjugated isomers of linoleic acid
for use in treating skin disorders.
Apart from potential therapeutic and pharmacological
applications of CLA as set forth above, there has been much excitement
regarding the use of CLA as a dietary supplement. CLA has been found to
exert a profound generalized effect on body composition, in particular
redirecting the partitioning of fat and lean tissue mass. U.S. Pat. 5,554,646
(Cook, et al.), teaches a method utilizing CLA as a dietary supplement in
which pigs, mice, and humans were fed diets containing 0.5 % CLA. In
each species, a significant drop in fat content was observed with a
concomitant increase in protein mass. It is interesting that in these
animals, increasing the fatty acid content of the diet by the addition of CLA
resulted in no increase in body weight, but was associated with a
redistribution of fat and lean tissue mass within the body. Another dietary
phenomenon of interest is the effect of CLA supplementation on feed
conversion. U.S. Pat. 5,428,072 (Cook, et al.), discloses data showing that
the incorporation of CLA into animal feed (birds and mammals) increased
the efficiency of feed conversion leading to greater weight gain in the CLA
supplemented birds and mammals. The potential beneficial effects of CLA
supplementation for food animal growers is apparent.
Another important source of interest in CLA, and one which
underscores its early commercial potential, is that it is naturally occurring
in foods and feeds consumed by humans and animals alike. In particular,
CLA is abundant in products from ruminants. For example, several studies
have been conducted in which CLA has been surveyed in various dairy
products. Aneja, et al., (J. Dairy Sci., 43: 231 [1990]) observed that
processing of milk into yogurt resulted in a concentration of CLA. Shanta,
et al. (Food Chem., 47: 257 [1993]) showed that a combined increase in
processing temperature and addition of whey increased CLA
concentration during preparation of processed cheese. In a separate
study, Shanta, et al., (J. Food Sci., 60: 695 [1995]) reported that while
processing and storage conditions did not appreciably reduce CLA
concentrations, they did not observe any increases. In fact, several studies
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have indicated that seasonal or interanimal variation can account for as
much as three fold differences in the CLA content of cows milk (Parodi, et
al., J. Dairy Sci., 60: 1550 [1977]). Also, dietary factors have been
implicated in CLA, content variation (Chin, et al., J. Food Comp. Anal., 5:
185 [1992]). Because of this variation in CLA content in natural sources,
ingestion of prescribed amounts of various foods will not guarantee that
the individual or animal will receive the optimum doses to ensure
achieving the desired nutritive effect.
Economical conjugated fatty acid production in commercial
quantities for use in domestic food animal feeds is a desirable objective in
light of the nutritional benefits realized on a laboratory scale. Preferably,
the conjugated fatty acid is produced directly from a source of raw
vegetable oil and not from expensive purified linoleic acid. Further, the
process must avoid cost generating superfluous steps, and yet result in a
safe and wholesome product palatable to animals.
Useful methodologies for the preparation of conjugated
linoleic acid (CLA) have been recently reviewed by Adlof (Ln:Yurawecz et
al. (Ed), Advances in Conjugated Linoleic Acid Research, volume 1,
AOCS Press, Champaign, II, pp 21-38 [1999]).
The usual methodology for conjugation of polyunsaturated
fatty acids ' is alkali-catalyzed isomerization. This reaction may be
performed using different bases such as hydroxides or alkoxides in
solution in appropriate alcoholic reagents. This reaction was developed in
the 1950's for the spectrophotometric estimation of polyunsaturated fatty
acids in fats and oils [AOCS official method Cd 7-58; JAOCS 30:352
(1953)].
In alkali isomerization the fatty acids are exposed to heat,
pressure and a metal hydroxide or oxide in nonaqueous or aqueous
environments, resulting in the formation of ~ conjugated isomers. Other
methods have been described which utilize metal catalysts, but which are
not as efficient for the operation of conjugated double bonds. It was found
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that isomerization could be more rapidly achieved in the presence of
higher molecular weight solvents. Kass, et al., (J. Am. Chem. Soc., 61:
4829 (1939)) and U.S. Pat. 2,487,890 teach that the replacement of
ethanol with ethylene glycol resulted in an increase in conjugation in less
time. U.S. Pat. 2,350,583 and British Patent 558,881 teach conjugation by
reacting fatty acid soaps of an oil with an excess of aqueous alkali at 200-
230°C. and increased pressure.
Dehydration of methyl ricinoleate (methyl 12-hydroxy-cis-9-
octadecenoate) (Gunstone and Said, Chem. Phys. Lipids 7, 121 [1971];
Berdeaux et al., JAOCS 74, 1011 [1997]) yields the 92,11 E-C18:2 isomer
as a major product. U.S. Pat. 5,898,074 teaches a synthetic process for
producing this fatty acid at room temperature in high yield. The tosylate or
the mesylate of the methyl ester of ricinoleic acid is formed with tosyl
chloride or mesyl chloride in a pyridine solvent or in acetonitrile and
triethyl
amine. The obtained tosylate or mesylate was reacted with diazabicyclo-
undecene in a polar, non-.hydroxylic solvent such as acetonitrile to form
the preferred isomer 9Z,11E-18:2 methyl ester in high yield. U.S. Pat.
6,160,141 discloses a synthetic process for producing conjugated
eicosanoid fatty acid from methyl lesquerolate (methyl 14-hydroxy-eis-11-
octadecenoate) at room temperature in high yield using the same
principle.
Among the processes known to effect isomerization, without
utilizing an aqueous alkali system, is a nickel-carbon catalytic method, as
described by Radlove, et al., Ind. Eng. Chem.38: 997 (1946). A variation
of this method utilizes platinum or palladium-carbon as catalysts.
Conjugated acids may also be obtained from a-hydroxy allylic unsaturated
fatty acids using acid catalyzed reduction (Yurawecz et al., JAOCS 70,
1093 [1993]) as well as by the partial hydrogenation of conjugated
acetylenic acid such as santalbic (11 E-octadec-9-ynoic) acid using
Lindlar's catalyst but the methods are limited by natural sources of such
fatty acids. Another approach using strong organic bases such as
butyllithium has been applied to both the conjugation of linoleic acid and
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the partial and full conjugation of alpha-linolenic acid (U.S. Pat.
6,316,645).
Natural fully conjugated linolenic acids have been found at
high content levels in some seed oils (Hopkins, In:Gunstone, F.D. (Ed),
Topics in Lipid Chemistry, volume 3, ELEK Science, London, pp 37-87
[1972]). For example, Takagi and Itabashi (Lipids 16, 546 [1981]) reported
calendic acid (8E,10E,12Z-C18:3 acid, 62.2%) in pot marigold seed oil,
punicic acid (9Z,11E,13Z-C18:3 acid, 83.0%) in pomegranate seed oil; a-
eleostearic acid (9Z,11E,13E-C18:3 acid) in tung (67.7%) and bitter gourd
(56.2%) seed oils; and catalpic acid (9E,11 E,13Z-C18:3 acid, 42.3%) in
catalpa seed oil, respectively.
An octadecatrienoic acid isomer whose structure has been
tentatively defined as 9Z,11E,15Z-C18:3 acid, is believed to be the first
intermediate in the biohydrogenation process of a-linolenic acid by the
anaerobic rumen bacterium Butyrvibrio fibrisolvens (C. R. Kepler and S. B.
Tove 242 J. Biol. Chem. (1967) 5686).
There thus remains a need to develop a method for the
preparation and purification of new conjugated linolenic acids.
The present invention seeks to meet these and other needs.
The present invention refers to a number of documents, the
content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention relates to a method for the
preparation and purification of fatty acids which are homologues of
conjugated linoleic acids, from natural and/or synthetic materials richin
alpha or gamma linolenic acids or both. In a preferred embodiment, the
method transforms approximately over two thirds of alpha linolenic acid
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(9Z,12Z,15Z-C18:3 acid), from a natural source such as linseed oil, into
9Z,11E,15Z and 9Z,13E,15Z C18:3 acids, producing a mixture comprising
approximately 30% of the conjugated linolenic acids. In a further
embodiment, enrichment up to and over 40% is readily performed with
urea crystallization. Moreover, the product is obtained in over 90% purity
by simple preparative liquid chromatography. The products obtained
include free fatty acids, and derivatives thereof, including, but not limited
to esters, amides, salts as well as fatty alcohols. The method of the
present invention produces the above mentioned conjugated trienoic acid
with a high selectivity, in a short time period and under relatively mild
conditions.
The present invention further relates to a method for
preparing conjugated linolenic acids comprising the steps of:
(a) blending a or a mixture of vegetable oils_ and or fats
including various concentrations of alpha or gamma and or both linolenic
acids with a base to produce a reaction mixture; and
(b) recovering said conjugated linolenic acids from the reaction
mixture.
Further scope and applicability will become apparent from
the detailed description given hereinafter. It should be understood
however, that this detailed description, while indicating preferred
embodiments of the invention, is given by way of illustration only, since
various changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art.
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BRIEF DESCRIPTION OF FIGURES
Figure 1 shows mass spectra of products resulting from the
isomerization process of alpha-linolenic acid (9Z,12Z,15Z-C18:3 acid), as
4,4-dimethyloxazoline derivatives: A, 92,11 E,15Z and 9Z,13E,15Z-C18:3;
B, 9,11,13-C18:3, C, 1 OE,12Z,14E-C18:3 and D, 11,13-CCLA (9-(6-propyl-
cyclohexa-2,4-dienyl)-nonanoic acid);
Figure 2 shows the mass sprectrum of the MTAD adducts of
cis-9, trans-11, cis-5 18:3 (A) and cis-9, traps-13, cis-15 18:3 (B) acid
methyl esters;
Figure 3 shows the thermal mechanism leading to the
formation of 11,13-CCLA [9-(6-propyl-cyclohexa-2,4-dienyl)-nonanoic acid
(Figure 1-D)] from 10E,12Z,14E-018:3 acid;
Figure 4 illustrates gas liquid chromatograms of fatty acid
methyl esters obtained after methylation of linseed oil (A), conjugated
linseed oil (B), liquid phase from urea crystallization (C), reversed-phase
liquid chromatography fraction containing about 97 % of a mixture of
9Z,11E,15Z and 9Z,13E,15Z-C18:3 acids (D), argentation liquid
chromatography fraction containing about 99+ % of a mixture of
92,11 E,15Z and 9Z,13E,15Z -C18:3 acids (E);
Figure 5 illustrates the gas liquid chromatogram of the fatty
acid methyl esters obtained after methylation of partially conjugated
evening primrose oil.
DETAILED DESCRIPTION OF THE INVENTION
The oils and fats, alone or as mixtures, containing alpha-
linolenic acid may include but are not limited to arnebia, basil, candelnut,
flax (linseed), linola, gold of pleasure, hemp, mustard, perilla, soybean, '
canola, walnut, chia, crambe, echium, hop, kiwi, pumpkin, black currant
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and purslane seed oils, or any other oil, wax, ester or amide that is rich in
linolenic acid.
The oils and fats, alone or as mixtures, containing gamma-
linolenic acid may include but are not limited to borage, evening primrose
and black currant seed oils, or any other oil, wax, ester or amide that is
rich in linolenic acid.
The disclosed method converts double bonds of a- and y-
linolenic acid isomers into partly and/or fully conjugated systems as well
as into cyclic fatty acid isomers. The process, which can be performed
both in batch and continuous modes, involves blending one or a mixture of
vegetable oils with various concentrations of alpha or gamma linolenic
acids or both or partial glycerides of such oils, or partially purified or
concentrated isomers with about 0.5 to about 10 moles of base such as
sodium hydroxide, sodium alkoxylate, sodium metal, potassium hydroxide,
potassium alkoxylate, potassium metal, and strong base resins. The
reaction proceeds at temperatures from about 20°C to about 280°C
in a
solvent, selected from commercial polyols such as propylene glycol,
glycerol and ethylene glycol, for periods ranging from about 30 seconds to
about 18 hours, depending on the base and/or the temperature and/or
solvent, and/or substrate and/or a desired expected conversion rate. After
cooling, if required, to about 20-80°C, acid is added to the reaction
mixture
to neutralize the soaps and/or remaining base in the reactor. It is preferred
to bring the pH of the contents of the reactor to a value of about 4 or less
through the addition of either a mineral or organic acid. Acids that may be
used include, but are not limited to, hydrochloric acid, sulfuric acid,
phosphoric acid and citric acid. The solvent phase (polyol + water) is
withdrawn and the remaining fatty acid rich phase can be washed with
water and/or saline solutions of variable concentrations such as sodium
chloride (5%wlw) to remove traces of acids used for acidification of the
reaction mixture. Remaining water can be removed by usual means (i.e.
centrifugation, vacuum, distillation or drying agents). As described in
Example 1, the concentration of 9Z,11E,15Z and 9Z,13E,15Z-C18:3 acid
in the product is approximately 33%. This product, as such or converted
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into derivatives, can be used in nutrition, cosmetic, nutraceutical,
biological
and/or animal feed applications.
The isomer composition of the formed fatty acid was
determined using gas-liquid chromatography coupled with a mass-
spectrometer (GC-MS) of their corresponding 4,4-dimethyloxazoline
(DMOX) derivatives. The use of derivatives is a necessary step prior to the
structural determination of fatty acids by GC-MS because the mass
spectra of fatty acid methyl esters, the usual derivatives for gas-liquid
chromatography analysis, are devoid of sufficient information for the
identification of structural isomers. This is mainly due to the high
sensitivity
of the carboxyl group to fragmentation and to double bond migration
(Christie, W.W., Gas Chromatography-Mass Spectrometry Methods for
Structural Analysis of Fatty Acids, Lipids 33:343-353 (1998)). However,
stabilization of the carboxyl group by the formation of a derivative
containing a nitrogen atom results in mass spectra that allows for the
structural determination of most fatty acids. Indeed, these fatty acid
derivatives provide diagnostic fragments that allow accurate structure
determination. The derivatives were submitted to GC-MS using a Hewlett
Packard 5890 Series II plus gas chromatograph attached to an Agilent
model 5973N MS Engine. The latter was used in the electron impact mode
at 70 eV with a source temperature of 230°C. For the DMOX derivatives,
an open tubular capillary column coated with BPX-70 (60 m×0.25
mm, 0.25 pm film; SGE, Melbourne, Australia) was used. After holding the
temperature at 60°C. for 1 minute, the oven temperature was increased
by
temperature-programming at 20°C/minute to 170°C where it was
held for
minutes, then at 5°C/minute to 210°C. where it was held for 30
minutes. Helium was the carrier gas at a constant flow-rate of 1
mL/minute, maintained by electronic pressure control.
The mass spectrum of the conjugated products of
9Z,12Z,15Z-C18:3 acid, obtained by conjugation of linseed oil, are
presented in Figure 1.
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The structural formula and mass spectrum of the DMOX
derivatives of the 9Z,11E,15Z-C18:3 acid are illustrated in Figure 1A.
DMOX has a molecular ion at m/z=331, confirming the octadecatrienoic
acid structure. The ion at m/z=262 confirms the location of the 11,15-
double bond system (by extrapolation from the known 5,9-isomer
(Berdeaux and Wolff, J. Am. Oil Chem. Soc., 73: 1323-1326 (1996)),
similarly, the molecular ion at m/z=236 confirms the location of the 9,13-
double bond system, and gaps of 12 a.m.u. between m/z=208 and 196,
and 288 and 276 verify the location of double bonds in positions 9 and 15,
respectively. Mass spectrometry does not however confirm the geometry
of the double bonds, but they have been determined according to Nichols
et al. (J. Am. Chem. Soc, 73:247-252 (1951 )) based on the Ingold theory
on the prototropic shift mechanism (Ingold, J. Chem. Soc, 1477 (1926)).
The structural formula and mass spectrum of the DMOX
derivatives of the 9,11,13-C18:3 acid are illustrated in Figure 1B. DMOX
has a molecular ion at m/z=331, confirming the octadecatrienoic acid
structure. Gaps of 12 a.m.u, between m/z=208 and 196, and 222 and 234,.
and 248 and 260 verify the location of the double bonds in positions 9 ,11
and 13, respectively. Four different minor isomers of 9,11,13-C18:3 are
present in the reaction products. The most abundant is the 9Z,11Z,13E-
C18:3 acid isomer which is known as a-eleostearic acid.
The mass spectra of the MTAD adducts of cis-9,trans-
11,cis-15 18:3 (A) and cis-9,trans-13,cis-15 18:3 (B) acid methyl esters
and presented in Figure 2.
The structural formula and mass spectrum of the DMOX
derivatives of the 10E,12Z,14E-C18:3 acid are illustrated in Figure 1C.
DMOX has a molecular ion at m/z=331, confirming the octadecatrienoic
acid structure. Gaps of 12 a.m.u. between m/z=210 and 222, and 236 and
248, and 262 and 274 verify the location of the double bonds in positions
10 ,12 and 14, respectively. The geometry of the double bonds, has been
determined according to Nichols et al. (J. Am. Chem. Soc, 73:247-252
(1951 )) based on the Ingold theory on the prototropic shift mechanism
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(Ingold, J. Chem. Soc, 1477 (1926)). The 10E,12Z,14E-C18:3 acid isomer
is prone to cyclization, thus forming thecyclohexadienyl compound (9-(6-
propyl-cyclohexa-2,4-dienyl)-nonanoic acid)) by an electrocyclization
process presented in Figure 3.
The structural formula and mass spectrum of the DMOX
derivatives of the 11,13-CCLA (9-(6-propyl-cyclohexa-2,4-dienyl)-nonanoic
acid) are illustrated in Figure 1 D. DMOX has a molecular ion at m/z=330 -
1, confirming the occurrence of a highly stabilized conjugated ion fragment
(radical in carbon 10 or 15, stabilized by resonance effect). A distinctive
ion at m/z=288 is characteristic of alpha cleavage occurring in cyclic fatty
acids (Sebedio et al. J. Am. Oil Chem. Soc., 64: 1324-1333 (1987)). The
gap of 78 atomic mass units (a.m.u.) between m/z=288 and 210 is that
expected for the cyclohexadienyl group having a conjugated double bond
system in positions 11 and 13.
The reaction progress was determined by gas-liquid
chromatography under appropriate condition as presented in Example 1.
An increase in the concentration of, for example the
9Z,11E,15Z and 9Z,13E,15Z-C18:3 acids, can be achieved using different
methods, alone or in combination. One method makes use of urea
complexation. A urea solution is prepared at a temperature ranging from
about 20 to 90°C in different solvents or mixtures thereof, selected
from
water, and/or alcohols. Complexation is performed at the same
temperature by addition of the product in a molar ratio of about 0.5 to 8,
and cooling to a temperature range of about 30°C to about -30°C,
as
required. A mixture of the above mentioned 9Z,11E,15Z and 9Z,13E,15Z-
C18:3 acids is isolated in higher concentration following treatment of the
liquid phase, obtained after separation from the solid phase using
conventional means such as filtration or centrifugation. Decomplexation is
then carried out by the addition of either a diluted organic or mineral acid.
Acids that may be used include, but are not limited to, hydrochloric acid,
sulfuric acid, phosphoric acid and citric acid. The product is obtained by
decantation or liquid-liquid extraction with an organic solvent such as but
is
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not limited to hexane, heptane, petroleum ether and ligroin. If required, the
organic solvent is eliminated (i.e. evaporation 'or distillation). A preferred
description of the present embodiment is described in Example 2.
Another method for raising the level of, for example the
92,11 E,15Z and 9Z,13E,15Z -C18:3 acids, either as free acids or
derivatives (i.e. methyl, ethyl, isopropyl, butyl, phenyl) comprises the use
of liquid chromatography using various convenient stationary phases. One
particular chromatographic method is reversed phase liquid
chromatography (i.e. ODS) for which eluents may include but are not
limited to water, acetonitrile, acetone, methanol, tetrahydrofuran, methyl-
tertbutyl ether, and combinations thereof. A detailed description of this
method is provided in Example 3.
Argentation liquid chromatography may be used to isolate
specific isomers from a complex mixture of fatty acid esters or free fatty
acids. A detailed description of this methodology applied to a mixture of
9Z,11E,15Z and 9Z,13E,15Z-018:3 acid isomers is described in Example
4.
Still another method for raising the concentration level of, for
example, a mixture of 9Z,11E,15Z and 9Z,13E,15Z-C18:3 acids, either as
free acids or derivatives (i.e, methyl, ethyl, isopropyl, butyl, phenyl) is
crystallization, either in a solvent such as, but not limited to, acetone,
methanol, pentane, or in mixtures therefor, or in the absence of a solvent
(i.e. dry fractionation). A detailed cooling program is required in order to
obtain a more concentrated product. One particular case is that of further
crystallization of urea complexes of fatty acids.
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EXPERIMENTAL
In the experimental disclosure which follows, the following
abbreviations apply: kg (kilograms); g (grams); mg (milligrams); °C
(degrees centigrade); L (liters); mL (milliliters); pL (microliters); m
(meters); cm (centimeters); mm (millimeters), pm (micrometers); NaOH
(sodium hydroxide), H2SO4 (sulfuric acid), NaCI (sodium chloride); 11,13-
CCLA (9-(6-propyl-cyclohexa-2,4-dienyl)-nonanoic acid), AgNO3 (silver
nitrate).
EXEMPLE 1
Preparation of a mixture containing high amounts of 92,11 E,15Z and
9Z,13E,15Z-C18:3 acids by conjugation of linseed oil
To commercial propylene glycol (46.48 kg) were added
NaOH (1.94 kg) at room temperature. The resulting mixture was heated at
160°C for 20 minutes into a 200 L stainless steal reactor under a
nitrogen
atmosphere and with vigorous agitation. Commercial raw linseed oil (4.19
kg) was added under a nitrogen atmosphere. The mixture was heated at
160°C for 2 hours under a nitrogen atmosphere and with vigorous
agitation. After cooling to 80°C, the reaction mixture was directly
acidified
with an aqueous solution of H2S04 (0.06 % w/w, 47.5 kg). After standing
for about 10 minutes, the top layer was washed with a NaCI aqueous
solution (5% w/w, 47.25 kg). The top layer was removed, dried and stored
at -80°C under nitrogen.
The fatty acid composition of the resulting product was
determined using high resolution gas-chromatography following
methylation of a sample (20 mg) using boron trifluoride (Metcalfe et al.,).
The analytical equipment consisted of an Agilent Technologies GLC 6890
with auto sampler. The column was a highly polar open tubular capillary
type. The following program settings were used (TABLE 3)
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TABLE 3
Injection Split mode 1:50 at 250°C
Detection Flame Ionization Detector at 250°C
Carrier Helium at 249.5 KPa at 170°C
Oven Program 60°C for 1 minute then 20°C/minute to 170°C
and 170°C throughout
for 30 minutes, then 5°Clminute 210°C throughout for 5 minutes
Column BPX-70 capillary column, 60 m X 0.25 mm i.d., 0.25 pm film
thickness
The obtained chromatogram is shown in figure 4 B. The
quantitative conversion of alpha-linolenic acid was confirmed and the
mixture comprises approximately 33 % of 92,11 E,15Z and 9Z,13E,15Z-
018:3. The fatty acid composition of the mixture is given in Table 4.
TABLE 4
Fatty Acid % Before Reaction % After Reaction
Palmitic 5.40 5.07
Stearic 4.13 3.20
Oleic 19.77 19.27
11 Z-C 18:1 0.69 0.65
Linoleic 16.47 7.16
alpha-Linolenic 53.54 0.87
92,11 E-C18:2 0.00 4.89
1 OE,12Z-018:2 0.00 4.79
11,13-CCLA 0.00 8.73
92,11 E,15Z-C 0.00 32.98
18:3
9,11,13-C18:3~ 0.00 3.73
10E,12Z,14E-C18:30.00 6.06
10,12,14- C 18:320.00 1.41
stereochemistry of the double bonds not identified
Zother stereo isomers of 10,12,14-C18:3 Acid
EXEMPLE 2
Preparation of mixtures containing high amounts of a mixture of
9Z,11E,15Z and 9Z,13E,15Z-C18:3 acid by conjugation of linseed oil.and
consecutive urea crystallization
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The top layer (3.26 kg) obtained in Example 1 was removed
and transferred into a 20 L reactor containing a solution of urea (3.26 kg)
in aqueous ethanol (95 %, v/v, 13.20 kg), prepared at 60°C under a
nitrogen atmosphere. The free fatty acids were homogenized and the
obtained mixture was cooled at 4°C for 12 h. The liquid phase (17.77
kg)
was removed from the solid phase (3.18 kg) by centrifugation and
transferred into a 100 L, stainless steal,. sight glasses reactor. An aqueous
solution of H2S04 (0.1 %, w/w, 49.12 kg) was added to the mixture and the
solution was vigorously shaken for 1 minute under a nitrogen atmosphere.
After standing for 10 minutes, the top layer was washed with an aqueous a
NaCI solution (5% w/w, 47.25 kg). The top layer was removed, dried and
stored at -80°C under nitrogen.
The solid phase (3.18 kg) was dissolved in a solution of
H2S04 (0.1 %, w/w, 49.12 kg) at 70°C and transferred into a 107 L,
stainless steal, sight glasses reactor and the solution was vigorously
shaken for 1 minute under a nitrogen atmosphere. After standing for 10
minutes, the top layer was washed in the same apparatus with an
aqueous NaCI solution (5% w/w, 47.25 kg). The top layer was removed,
dried and stored at -80°C under nitrogen.
The fatty acid composition of the resulting products was
determined using high resolution gas-chromatography following
methylation of samples (20 mg) using boron trifluoride (Metcalfe et al.,).
The analytical conditions used were the same as presented in Example 1.
The chromatogram obtained is shown in Figure 4C. The
fatty acid composition of the mixture is illustrated in Table 5.
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TABLE 5
Fatty Acid % Before % in Liquid % in Solid
Crystallization Phase Phase
Palmitic 5.07 0.58 15.41
Stearic 3.20 0.04 12.17
Oleic 19.27 17.19 27.88
11 Z-C 18:1 0.65 0.66 0.84
Linoleic . 7.16 8.50 2.60
alpha-Linolenic 0.87 0.79 0.17
92,11 E-C18:2 4.89 5.86 4.17
10E,12Z-C18:2 4.79 6.21 2.59
11,13-CCLA 8.73 10.61 1.42
92,11 E,15Z and 9Z,13E,15Z 32.98 40.74 10.88
-
C18:3
9,11,13-C18:3~ 3.73 3.54 3.17
10E,12Z,14E-C18:3 . 6.06 0.73 13.78
10,12,14- C18:3~ 1.41 1.26 1.72
~stereochemistry of the double bonds not identified
Zother stereo isomers of 10,12,14-C18:3 Acid
EXEMPLE 3
Preparation and purification of a mixture of 92,11E,15Z and 9Z,13E,15Z
C18:3 acids by reverse phase liguid chromatography.
The products obtained in Examples 1 and 2 containing a
high level of 92,11 E,15Z and 9Z,13E,15Z-C18:3 were submitted to a
preparative high performance liquid chromatograph fitted with a
preparative ODS (octadecylsilyl) column (25 cm X 6.5 cm i.d.). The mobile
phase was methanol and water (90:10, v/v, 400 mL/minute). The sample
(10 g) was injected at atmospheric pressure and the separation was
achieved in 60 minutes. The collected fractions were analyzed by gas-
liquid chromatography as presented in Example 1, and a typical gas-
chromatogram is presented in Figure 4D. The desired compounds eluted
in the first partition (partition number = 12) allowing for a purification of
about 95 %.
EXEMPLE 4
Preparation and purification of 92,11 E,15Z and 9Z,13E,15Z-C18:3 acids
by argentation liguid chromatography
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The fatty acid methyl esters prepared from the products
obtained in Examples 1 and 2, containing a high level of a mixture of
9Z,11E,15Z and 9Z,13E,15Z-C18:3, were separated using argentation thin
layer chromatography. Silica-gel plates were prepared by immersion in a
5% acetonitrile solution of AgN03 as described by Destaillats et al. (Lipids
35:1027-1032, (2000)). The developing solvent was a n-hexane/diethyl
ether (90:10, v/v) mixture. At the end of the chromatographic runs, the
plates were briefly air-dried, lightly sprayed with a solution of 2',7'-
dichlorofluorescein, and viewed under ultraviolet light (234 nm). The band
at Rf = 0.52 was scraped off and eluted several times with diethyl ether.
Complete evaporation of the combined extracts was achieved with a light
stream of dry nitrogen. The residues were dissolved in an appropriate
volume of n-hexane and analysed by gas-liquid chromatography (purity
superior to 98 %) as presented in Example 1.
EXEMPLE 5
Preparation of mixture containing 6Z, 8E,12Z, 6Z,10E,12Z- and 6Z, 9Z,12Z-
C18:3 acids by partial conjugation of borage oil
NaOH (4.30 g) was added to commercial propylene glycol
(96 g) at room temperature. The resulting mixture was heated at 160°C
for
20 minutes under a nitrogen atmosphere and with vigorous agitation.
Commercial borage oil (9.35 g) was then added under a nitrogen
atmosphere. The mixture was heated at 160°C for 1 hour under nitrogen
and with vigorous agitation. After cooling to 80°C, the reaction
mixture was
directly acidified with an aqueous solution of H2S04. After standing for 10
minutes, the top layer was washed with a 5% aqueous NaCI solution (w/w,
47.25 kg), removed, dried and stored at -80°C under nitrogen.
The fatty acid composition of the resulting products was
determined using high resolution gas-chromatography after methylation of
samples (20 mg) using boron trifluoride (Metcalfe et al.,). The analytical
conditions used were the same as presented in Example 1.
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The obtained chromatogram is shown in Figure 5. The fatty
acid composition of the mixture is given in Table 6.
TABLE 6
Fatty Acid % Before Reaction % After Reaction
Palmitic 10.34 9.55
Stearic 3.36 2.38
Oleic 15.57 13.88
11 Z-C18:1 0.57 0.52
Linoleic 39.96 30.11
?-Linolenic 22.92 5.32
7,11-CCLA 0.00 1.25
92,11 E-C18:2 0.00 6.66
1 OE,12Z-018:2 0.00 6.46
9Z-C20:1 3.69 2.60
6Z,8E,12Z and 6Z,10E,12Z- 0.00 14.50
C18:3
9Z-C22:1 2.05 1.22
7E,9Z,11 E-C18:3 0.00 1.89
Although the present invention has been described herein
above by way of preferred embodiment thereof, it can be modified without
departing from the spirit and nature of the subject invention as defined in
the appended claims.
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