FR2790758A1 - SOLUBILIZATION OF POLYUNSATURATED FATTY ACIDS AND DERIVATIVES THEREOF BY FORMATION OF INCLUSION COMPLEXES WITH A CYCLODEXTRIN AND THEIR USE IN PHARMACEUTICAL, COSMETIC OR FOOD COMPOSITIONS - Google Patents

Abstract

The subject of the invention is an aqueous solution comprising at least one oleaginous substance chosen from polyunsaturated fatty acids and the salts, esters and triglycerides of polyunsaturated fatty acids, in which the said substance (s) are in the form of a complex. of inclusion in a cyclodextrin chosen from gamma-cyclodextrin and 2,6-dimethyl-cyclodextrin. The oleaginous substance (s) can be chosen from chosen from arachidonic acid (AA), cicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and their triglycerides as well as from fish.

Description

SOLUBILIZATION OF POLYUNSATURATED FATTY ACIDS AND THEIR

  DERIVATIVES BY FORMATION OF INCLUSION COMPLEXES WITH A

  CYCLODEXTRIN AND THEIR USE IN COMPOSITIONS

  PHARMACEUTICALS, COSMETICS OR FOOD.

DESCRIPTION

  Technical Field The invention relates to the solubilization and stabilization of polyunsaturated fatty acids and the corresponding salts, esters and triglycerides, by inclusion in cyclodextrins with a view to obtaining formulations which are soluble in water, can be incorporated into pharmaceutical, food compositions. or cosmetics. Polyunsaturated fatty acids such as arachidonic or eicosatetraenoic (AA) eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are long chain polyunsaturated fatty acids of the n-3 series. They are contained in seafood products, i.e. mainly in oils

  fish, but also in some micro-algae.

  They are obtained by hydrolysis-esterification of triglycerides. These acids and the corresponding triglycerides correspond to the formulas: COOH Arachidonic acid (AA) COOH X (II) Eicosapentaenoic acid (EPA) / = \ COOH (ll) Docosahexaenoic acid (DHA)

OCOR1

  OCOR2 OCOR3 Triglycerides: RI, R2, R3 represent the hydrocarbon group of a

  fatty acid such as EPA, DHA or the like.

  These n-3 series polyunsaturated fatty acids (PUFA) have very interesting properties in the prevention of certain inflammatory pathologies (psoriasis, arthritis) and cardiovascular diseases. The first epidemiological studies suggesting the existence of potentially

  beneficial of these fatty acids on cardio-

  vascular, go back to observations made in the Eskimos. Greenlandic. In this population, an almost exclusive consumer of marine mammals and fish, around 14 g of n-3 PUFA are ingested

  daily, and deaths from heart disease

  vascular are rare. The relationship between the level of consumption of fish (and a fortiori of PUFA n-3) and the incidence of cardiovascular disease has been found in Japan and in certain prospective studies. The n-3 PUFAs exert their beneficial effect by intervening at two levels: - 1) they lower the triglyceridemia (and not the cholesterolemia), this decrease resulting essentially from a synthesis of low density lipoproteins and their constituents; - 2) they inhibit the phenomena of platelet aggregation, coagulation and inflammation by playing on the synthesis of a large number of molecules (thromboxanes, prostacylins, leukotrienes, PAF, fibrinogen, PDGF, cytokines, ...). In order to limit the coronary mortality rate for the whole population, it seems prudent to recommend the

consumption of fish.

  PUFAs also have other interests, particularly in the skin and nerve structures during perinatal development. Indeed, modifications appear during a PUFA deficiency in the epidermis - increased water permeability, - cellular hyperproliferation (keratinization problem), - increase in thickness and in the number of layers,

  - eczema, irritation, erythema ...

  From a nutritional point of view, the period of perinatal development is special in that it corresponds to the most active phase of incorporation of long chain PUFAs (22: 6 n-3; i.e. 22 carbon atoms, 6 ethylenic unsaturations , the first being on carbon n-3) in the structural lipids of the central nervous system, but also the most sensitive to dietary intake of PUFAs. For the human brain, this period of rapid growth extends from the last three months of fetal life until the age of two. During the first year, the brain of the newborn will take two grams per day mainly due to the phenomenon of myelination. This implies that the food intake of PUFA must be correct

  quantitatively and qualitatively.

  Due to the low average consumption of these fatty acids in industrialized countries (200 to 400 mg / day), a number of manufacturers are starting to look into how to enrich certain foods with these fatty acids by adding them fish oils (bread, margarine, mayonnaise ...) However the incorporation of these long chain fatty acids in food or cosmetic compositions is made difficult because of the insolubility in water, the undesirable odor and

  lack of stability of these fatty acids.

  Indeed, they are sensitive to temperature and light. They are unstable and

oxidize very easily.

  State of the Prior Art To overcome these difficulties, document FR-A-2,547,829 [1] proposes a stable composition containing compounds of unsaturated fatty acids and an organic stabilizing compound corresponding to the formula:

H2COR1

I

R 2O - CH O

I he

H2C --- P-P-OCH2CH2N R3

  I O- in which R and R respectively represent fatty acid residues while R3 represents H3 or (CH3) 3. It is also possible to add a cyclodextrin to the composition, the role of which is to very strongly reduce the unpleasant odor and bitterness proper to polyunsaturated fatty acid compounds and the role of which is also to make the composition pleasant to the palate. In document FR-A-2 550 445 [2], it has been proposed to include a polyunsaturated fatty acid such as EPA in γ-cyclodextrin. For this purpose, the two compounds are mixed in the form of a paste in the presence of small volumes of water, which leads to obtaining

  a powder containing an inclusion compound y-

  cyclodextrin-EPA. Document EP-A-0 470 452 [3] describes

  also obtaining a product comprising y-

  cyclodextrin and an oleaginous substance containing

  EPA or DHA, by precipitation from a solution.

  All these techniques have the disadvantage of leading to solid products which are not suitable for incorporation into all the compositions.

  pharmaceutical, food or cosmetic.

  The present invention specifically relates to formulations in the form of aqueous solutions containing in solution at least one oleaginous substance belonging to the group of polyunsaturated fatty acids and their derivatives (salts, esters and triglycerides), which are suitable for such incorporation. DESCRIPTION OF THE INVENTION Also, the subject of the invention is an aqueous solution comprising at least one oleaginous substance chosen from polyunsaturated fatty acids and the salts, esters and triglycerides of polyunsaturated fatty acids, in which the said compound (s) ) substance (s) are in the form of an inclusion complex in a cyclodextrin chosen from y-cyclodextrin

  and 2,6-dimethyl-p-cyclodextrin.

  Remember that natural cyclodextrins are cyclic oligosaccharides

  consisting of D-glucopyranosyl units linked in α-1,4.

  Y-cyclodextrin which is a natural cyclodextrin,

  includes eight D-glycopyrnosyl units, while the -

cyclodextrin has 7.

They respond to the formula:

011 | 0

  (IV) with n = 7 for P-cyclodextrin, and

n = 8 for y-cyclodextrin.

  2,6-dimethyl 3-cyclodextrin corresponds to the formula:

4 O /, CH3

0

(V) J 7

  In the invention, 2,6-

  pure dimethyl-p-cyclodextrin (DIMEB) or the commercial product RAMEB which has on average two OCH3 groups per glucose unit but which are randomly distributed on positions 2, 3 and 6. RAMEB therefore contains statistically a small proportion of

  DIMEB pure corresponding to formula (V).

  The oleaginous substances used in the invention can be of different types and from various origins, for example from fish

or plants.

  Advantageously, the polyunsaturated fatty acids AA, EPA and DHA mentioned above are used, in particular in the form of triglycerides. It is possible to use a mixture of one or more of these acids with other saturated or unsaturated fatty acids, for example

example a fish oil.

  In this case, the solution comprises several oleaginous substances, these being constituted

with fish oil.

  The fish oil can in particular be a sardine oil or a tuna oil. Such an oil comprises unsaturated fatty acids having from 1 to 6 ethylenic unsaturations as well as fatty acids without ethylenic unsaturation. The invention also relates to a process for the preparation of an aqueous solution comprising at least one oleaginous substance chosen from polyunsaturated fatty acids and salts, esters

  and triglycerides of polyunsaturated fatty acids.

  According to a first embodiment of the process of the invention using y-cyclodextrin to dissolve these substances, the process comprises the following steps: - a) dissolving the y-cyclodextrin in water, b) adding to the solution obtained in a) the oleaginous substance (s) to be dissolved, and

  - c) separate the precipitated complex from y-

  cyclodextrin and oil-containing substance (s) of the supernatant which also contains the

complex in solution.

  Thus, an aqueous solution of the substance (s) can be obtained directly

oilseed (s).

  The precipitated complex can also be recovered, dried, then put back into an aqueous solution. Similarly, one can recover and dry the complex obtained in the supernatant and put it back

then in aqueous solution.

  Also, the method may further comprise a step of recovering and drying at least one of the complexes obtained in step c) followed by the re-solution in aqueous solution of the dried complex (s). .

  To implement the process described above

  above, we first dissolve the y-cyclodextrin in water with stirring using an amount of y-cyclodextrin which can go up to

  saturation of the solution, i.e. 230 g / l.

  In step b) which follows, the oleaginous substance (s) are added in an amount such that it corresponds to a slight excess relative to the amount of γ-cyclodextrin necessary to form the equimolar complex. It is also subjected to agitation. In step c), the supernatant is separated from the

  precipitated, for example by centrifugation.

  When operating with an excess of oleaginous substance (s), this excess is first removed before separation by centrifugation. When the complex (s) obtained are then subjected to drying, this can be carried out by lyophilization or else by a dehydration process using zeolites or

"Zéodratation".

  According to a second embodiment of the invention, intended for the inclusion of (the)

  oleaginous substance (s) in 2,6-dimethyl p-

  cyclodextrin, the process comprises the following stages: - a) dissolving 2,6-dimethyl-3-cyclodextrin in water, - b) adding to the solution obtained in a) the oil-producing substance (s) ( s) to be dissolved, and

  - c) recovering the complex solution obtained.

  As before, it is also possible to recover the complex in solution by drying the complex solution obtained in step c) and

  then put the dried complex back into aqueous solution.

  The use of 2,6-dimethyl-p-

  cyclodextrin is particularly advantageous because the complex obtained has very water solubility

high.

  The inclusion of oleaginous substances

  mentioned above in y-cyclodextrin or 2,6-dimethyl-

  P-cyclodextrin overcomes the following problems: - the polymerization of polyunsaturated fatty acids, - the migration of cis double bonds in trans, and

- peroxidation.

  The inclusion of polyunsaturated fatty acids or their triglycerides, salts and / or esters in cyclodextrins, in accordance with the invention, makes it possible to obtain aqueous formulations containing these fatty acids by eliminating and greatly reducing the problems associated with their oxidizability and their instability. Such aqueous formulations can be used to include these polyunsaturated fatty acids and / or their triglycerides, salts and esters in food compositions and in cosmetic compositions. Also, the subject of the invention is also pharmaceutical, food or cosmetic compositions comprising an aqueous solution of at least one oleaginous substance chosen from polyunsaturated fatty acids - and the salts, esters and triglycerides of polyunsaturated fatty acids, in which the (said) substance (s) are in the form of complex

  inclusion in a cyclodextrin chosen from y-

  cyclodextrin and 2,6-dimethyl-p-cyclodextrin.

  The invention will be better understood on

  reading of the description which follows, given of course

  by way of illustration and not limitation, with reference to

attached drawings.

Brief description of the drawings

  FIG. 1 is the spectrum obtained by nuclear magnetic resonance of the proton, in pyridine, of the y-cyclodextrin-fish oil complex,

precipitated in Example 1.

  Figure 2 is the nuclear magnetic resonance spectrum of the proton, in water, of the complex

  inclusion of EPA and dimethyl-f-cyclodextrin.

  FIG. 3 is the nuclear magnetic resonance spectrum of the proton, in water, of the complex

  obtained with fish oil and 2,6-dimethyl-p-

  cyclodextrin. Detailed description of the embodiments of the invention Example 1: Solubilization of fish oil using ycyclodextrin. 9.9931 g of y-cyclodextrin and 87 ml of water are introduced into a container to obtain a solution of 114.86 g / l of y-cyclodextrin, which corresponds substantially to half of the maximum solubility (230 g / l) y-cyclodextrin. The mixture is stirred on a turntable at 300 revolutions / minute until complete dissolution of the y-cyclodextrin, this

which is obtained in a few minutes.

  10 ml of crude fish oil are then added (reference SO 30 which contains at least 30% acid -3), and constant stirring is maintained at 300 revolutions / minute on a turntable for 24 hours. A white precipitate forms which settles at the bottom of the container, and an intermediate opalescent aqueous phase is observed and an oily suspension on the surface. The oily suspension is removed and the aqueous phase is separated from the precipitate by centrifugation twice successively at 1000 rpm for minutes, at room temperature. The supernatant (aqueous phase) and the

  precipitated after freezing in liquid nitrogen.

  The lyophilized products are then examined by proton nuclear magnetic resonance (1 H NMR) after dissolution in deuterated pyridine, at 298 K, using a BRUKER DRX 500 spectrometer, operating at

500.13 MHz.

  Figure 1 illustrates the nuclear magnetic resonance spectrum 'H of the precipitate. Comparison of this spectrum with that of γ-cyclodextrin alone shows the presence of signals (noted TGL in the figure) which are unambiguously attributed to the triglycerides derived from fish oil. The H NMR spectrum obtained with the supernatant

  is identical to that of figure 1.

  The amount of triglycerides included in the supernatant is quite large and the amount included

  in the precipitate is even more important.

  The solubility of the lyophilized complexes is then examined by re-solution in water. To this end, the quantity of water necessary to obtain the dissolution of 0.0846 g of lyophilized complex is added; this is 47.08 ml and it leads to an opalescent solution, the

  concentration of complex is 1.79 g / l.

It is specified that in the case of the -

  cyclodextrin, the complex formed under the same conditions has a solubility three times lower

(0.65 g / l).

  We now verify that the supernatant complex and the precipitate complex are both

  complexes of inclusion of triglycerides in the y-

  cyclodextrin. For this purpose, the lyophilized complex obtained from the supernatant and the lyophilized complex obtained from the precipitate are dissolved in a minimum volume of dimethylformamide DMF, which denatures the complex by separating the two species which constitute it. Having taken a mass of 1 g of complex, a volume of DMF of 4 ml is necessary to dissolve everything. About 60 ml of ether are added to each sample because the triglycerides are soluble therein but

  not the cyclodextrin which precipitates instantly.

  In order to properly isolate the organic phase containing the triglycerides of the precipitated γ-cyclodextrin, centrifugation is used for minutes, at 1000 revolutions / minute, at room temperature. The recovered liquid part is transferred to a separatory funnel. We wash four times

  successively with water to extract the traces of y-

  cyclodextrin possibly remained in the organic phase. This phase is dried over Na2SO4 and the bottle is capped. After about 1 hour, the samples are filtered on a frit under the vacuum of the water pump, then the organic extraction solvent is removed

(ether) on a rotary evaporator.

We thus recover the fraction of

  triglycerides that have been included in the y-

  cyclodextrin. The fluid obtained is oily in appearance.

  The mass of triglycerides recovered is 0.1178 g per 1 g of the complex originating from the supernatant and from

  0.2164 g per 1 g of the complex from the precipitate.

  The samples are stored under argon to avoid any degradation in air, before their analysis by gas chromatography, which

  will give their characteristic fatty acid profile.

  The results obtained, expressed as a percentage of the fatty acids identified are given in

the following table.

Table 1

  Oil Oil Fatty acids from

precipitated supernatant.

12: 0 0.2 0.2

14: 0 8.7 _8.7

14: 1 0.3 0.3

: 0 0.5 0.5

16: 0 18.0 _ 17.6

  16: 1 n-9 0.2 0.3 16 1 n-7 9, 8 | 9 17: 0 iso 0.4 _0.4

17: 0 0.4 __ 0.5

18 0 3, 8 3, 7

  18 1 n-9 11.4 11.4 18: 1 n-7 3, 2 3.2 18: 2 n-6 1.5 1.5

19: 0 0.4 0.4

  18: 3 n-6 0.3 0.3 18: 3 n-3 0.6 0.6 18: 4 n-3 2.2 2.2: 1 n-9 0.8 0.8: 4 n -6 (AA) 1.1 1.1

22 0 0.9 0.9

  5 n-3 EPA 21.1 20.9 22: 4 n-3 0.8 0.8

24 0 0.4 0.4

  22: 5 n-3 2.6 2.6 22: 6 n-3 (DHA) 10, 6 10.8 Total saturated 33.7 33.3 Monounsaturated 25.7 25.9 Polyunsaturated 40.8 40.8 In In this table, fatty acids are identified by their number of carbon atoms, followed by the number of ethylenic unsaturations and the location in the chain of the first unsaturation. Thus, EPA corresponds to 20: 5 n-3. The results in the table show that the oil from the supernatant has the same fatty acid composition as the oil from the precipitate. This is proof that we find the complex in the state

dissolved in the supernatant.

  EXAMPLE 2 Solubilization of Eicosapentaenoic Acid

  (EPA) in 2,6-dimethyl-p-cyclodextrin.

  10 ml of 10 mmol / l aqueous solution of 2,6-dimethyl-pcyclodextrin are prepared and 100 μl of EPA are added thereto. The heterogeneous system obtained is maintained under strong magnetic stirring for 1 night at room temperature and protected from air. A two-phase solution is thus obtained comprising a perfectly clear aqueous phase surmounted by an oily suspension. The two-phase mixture is centrifuged at 6000 revolutions / minute for 10 minutes to remove the upper phase (oily suspension

  consisting of excess fatty acid).

  The clear aqueous solution is then frozen and lyophilized. The lyophilized product is redissolved in heavy water to analyze it by

  proton nuclear magnetic resonance.

  FIG. 2 represents the spectrum obtained by 1 H NMR of the product lyophilized in heavy water. It can thus be seen that the water-soluble fraction corresponds to a 1/1 complex between cyclodextrin and

fatty acid (EPA).

  Example 3: Solubilization of fish oil in 2,6-dimethyl-pcyclodextrin. We follow the same procedure as in

  example 2 to include in 2,6-dimethyl-p-

  cyclodextrin from fish oil (SO 30).

  FIG. 3 represents the 1 H NMR spectrum obtained in solution in heavy water. As before, we see that the fraction soluble in

  water corresponds to a 1/1 complex between 2,6-

  dimethyl-p-cyclodextrin and the mixture of fatty acids

(fish oil).

  The solubility of the lyophilized complex is determined by redissolving in water by determining the amount of water necessary to dissolve 0.1875 g of lyophilized complex. It is found that only 300 μl of water suffices, which corresponds to a very high solubility of 625 g / l while the maximum solubility

  2,6-dimethyl-3-cyclodextrin is 285 g / l.

  This is very advantageous for use in food or cosmetic compositions, since the quantity of aqueous complex solution added can be limited to very low values.

REFERENCES CITED

[1]: FR-A-2 547 829.

[2]: FR-A-2 550 445.

[3]: EP-A-0 470 452.

Claims (12)

RESELL I CATIONS   1. Aqueous solution comprising at least one oleaginous substance chosen from polyunsaturated fatty acids and the salts, esters and triglycerides of polyunsaturated fatty acids, in which the said substance (s) are in the form of a complex inclusion in a selected cyclodextrin   among y-cyclodextrin and 2,6-dimethyl-p- cyclodextrin.   2. An aqueous solution according to claim 1 in which the oleaginous substance (s) are chosen from arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and their triglycerides.   3. An aqueous solution according to claim 1 comprising several oleaginous substances, in which said substances consist of a fish oil.   4. Solution according to any one of   claims 1 to 3, wherein the cyclodextrin is y-cyclodextrin.   5. Solution according to any one of   claims 1 to 3, wherein the cyclodextrin   is 2,6-dimethyl-p-cyclodextrin.   6. Method for preparing an aqueous solution according to claim 4, which comprises the following steps: - a) dissolving the y-cyclodextrin in water, - b) adding to the solution obtained in a) the (the) oleaginous substance (s) to be dissolved, and   - c) separate the precipitated complex from y-   cyclodextrin and oil-containing substance (s) of the supernatant which also contains the complex in solution.   7. The method of claim 6, which further comprises the drying of at least one of the complexes obtained in step c) followed by resetting   aqueous solution of the dried complex (s).   8. A method of preparing an aqueous solution according to - ia claim 5, which comprises the following steps: - a) dissolving 2,6-dimethyl-p-cyclodextrin in water, - b) add to the solution obtained in a) the oleaginous substance (s) to be dissolved, and   - c) recovering the complex solution obtained.   9. The method of claim 8, further comprising drying the complex solution obtained in step c) followed by resetting solution of the dried complex.   10. Food composition comprising an aqueous solution according to any one of claims 1 to 5.   11. Cosmetic composition comprising an aqueous solution according to any one of claims 1 to 5.   12. Pharmaceutical composition comprising an aqueous solution according to any one of claims 1 to 5.