DESCRIPTION

METHOD FOR PREPARING DOCOSAHEXAENOIC ACID ETHYL ESTER

▲ RO

BACKGROUND OF THE INVENTION:

20

Novel method for preparing docosahexaenoic acid ethyl ester or its derivatives.

Present invention relates to a novel method for the industrial preparation of docosahexaenoic acid (ethyl ester DHA) with a purity of 80% in weight or higher and free of undesirable organic residues.

25 Docosahexaenoic acid (DHA) is a polyunsaturated acid that presents a long structure of twenty two carbon atoms with six double bonds, of which the first one counting from the methyl terminal is in the third carbon-carbon bond (what is known as Omega-3 or n- 3). DHA is found naturally in triglyceride form in the fish oils and it has been object of numerous processes to obtain it.

30 DHA plays a fundamental role during growth and development of children, as it is part of neuronal structures. DHA concentrations in breast milk range from 0.07% to greater than 1.0% of total fatty acids, with a mean of about 0.34%. DHA levels in breast milk are higher if a mother's diet is high in fish. DHA has recently gained attention as a supplement for pregnant women, noting studies of improved attention and visual acuity.

35 (Beth Vincent, MHS (2005-10-31). "The Importance of DHA During Pregnancy and Breastfeeding") indicates that low levels of plasma and erythrocyte DHA were associated with poor retinal development, low visual acuity, and poor cognitive development. In that same study, alpha-hnolenic acid was shown as a source of fetal DHA, but that preformed DHA was more readily accredited. A working group from the

40 ISSFAL (International Society for the Study of Fatty Acids and Lipids) recommended 300 mg/day of DHA for pregnant and lactating women, whereas the average consumption was between 45 mg and 115 mg per day of the women in the study. DHA may also be required by individuals with inherited metabolic defects in elongation and desaturation activity, such as patients with peroxisomal disorders and some forms of

45 retinitis pigmentosa ("Role of essential fatty acids in the function of the developing nervous system" Uauy, R et al. Lipids. 1996 Mar; 31 SupplS 167-76).

DHA deficiency is associated with cognitive decline (Lukiw VJ et al."A role for docosahexaenoic acid-derived neuroprotectin Dl in neural cell survival and Alzheimer disease". J Clin Invest. 1 15(10): 2774-83). European Food Safety Authority through its Panel on Dietetic Products, Nutrition and Allergies (NDA) recently published their favorable opinion for a health claim on docosahexaenoic acid and the visual development of infants.

It is therefore clear the interest in obtaining a highly concentrate DHA without impurities.

Many of the methods for obtaining docosahexaenoic acid and its derivatives are based on purification from algae oil. Patents US5.397.591 and US5.492.938 relate to a method to obtain a concentration of DHA in triglyceride form between 20 and 35%.

Other methods for obtaining DHA are based on the extraction from the fish oil using organic solvents and subsequently using techniques of distillation, extraction or chromatography.

Using these methods it is not possible to get totally rid of traces from the organic solvents, highly undesirable. Moreover, these methods don't generally reach higher concentrations, as per example US5.130.061 patent which obtains, from crude fish oil a 30% eicosapentanoic acid (EPA) and a 40% DHA using molecular distillation.

Other methods use supercritical carbon dioxide as a chromatographic eluent and various stationary phases. Silica gel and aluminum oxide have been used in different methods as stationary phases. Patent US5.362.895 claims a method for obtaining derivatives from unsaturated fatty acids in which organic solvents as hexane is avoided. Liquid carbon dioxide is used as mobile phase but it is not possible with that method to separate eicosapentaenoic acid from docosahexaenoic acid.

That separation between EPA and DHA is the greatest difficulty when fish oils are the starting material.

SUMMARY OF THE INVENTION:

Method for obtaining docosahexaenoic acid (DHA) ethyl ester that present invention relates to presents clear advantages over the methods above mentioned. Present invention does not use any organic solvent and allows obtaining a product with at least 80% by weight purity of DHA.

There is an unmet need in the art for a product that provides a single administration of high concentrated ethyl ester DHA and the present invention by means of providing a method for obtaining high concentrate of DHA meets this need. This method is characterized by a starting point of a mixture of ethyl esters of fatty 85 acids with a content of ethyl ester DHA of 15% by weight that is subjected to a supercritical chromatography using a supercritical C0 ₂ as mobile phase and irregular porous silicon oxide impregnated with (3-aminopropyl)-triethoxysilane as stationary phase.

Applicants have discovered that taking as a starting point a raw material concentrated in 90 omega-3 fatty acids, where minimum concentration of DHA is 15% by weight, it is possible to reach concentrations of DHA 80% and more in weight. Yield of that DHA concentrate will be between 7 and 20% in weight based on the raw material used.

Stationary phase used in the supercritical chromatography object of the present invention is irregular porous silicon oxide impregnated with (3-aminopropyl)- 95 triethoxysilane in a proportion of 10% roughly and a pore size of 6 to 7 nanometers.

This is a commercial stationary phase and can be supplied by different suppliers (for instance ProntoPreg. Bischoff Analysentechnick und gerate GMBH, D-7250 Leonburg, Alemania).

Column used at industrial scale has a diameter between 50 and 60 cm and a length 100 between 260 and 280 cm, with opening and closure devices at the top and the bottom.

Stationary phase quantity ranges from 180 to 300 kg, preferably from 200 to 240 Kg and is contained between drilled plaques with filtration paper.

Supercritical C0 ₂ circulates through the column propelled with membrane pumps at a pressure ranges from 95 to 150 bars, preferably ranges from 97 to 110 bars, at a 105 temperature ranging from 40° to 60° Celsius, preferably from 45° to 50° and at a flow rate ranging from 4000 to 7000Kg/h, preferably from 5000 to 6000 Kg/h.

Mixture of ethyl esters of DHA is pumped at a discontinuous pace to the column in the current of C0 ₂ at a flow rate ranging from 0.1 to 1 Kg/s, using membrane pumps.

For every 1000 Kg of mixture of ethyl esters used, with a minimum content of 15% in 110 weight of DHA, around 150 Kg of ethyl ester of DHA of at least 80% in weight concentration is obtained.

EXAMPLE:

115 Following description is set only as an example and does not limit the scope of the invention: A cylindrical column of 540 mm diameter and 2745 mm length with opening and closure devices at the top and with drilled plaques with filtrating paper inside is used. It contains 220 Kg of stationary phase consisting in irregular porous silicon oxide 120 impregnated with (3-aminopropyl)-triethoxysilane with pore size of 6.5 nanometers (ProntoPreg, Bischoff Analysentechnick und gerate GMBH, D-7250 Leonburg, Alemania).

Supercritical C0 ₂ circulates through the column at a pressure ranging 97 to 99 bars, at a temperature ranging from 45° to 50° and at a flow rate ranging from 5000 to 6000 Kg/h. 125 With the C0 ₂ current, we pump in the column the mixture of esters at a flow rate ranging from 8 to 15 Kg in 20-30 seconds through membrane pumps.

Current pressure when exiting the column is reduced to 60 bars, which is equal to pressure condition of C0 ₂ circuit, reheating it afterwards with a steam current at 150° in a heat exchanger, evaporating the C0 _2.

130 Different fractions obtained from the column during injection time, are separated through a rotating valve with different positions.

182.75 Kg ethyl ester DHA 80.52 % in weight are obtained from 1019.6 Kg of mixture f esters with an ethyl ester content of DHA of 17.74% in weight.

Product obtained is:

Ethyl ester DHA 80 % minimum weight.

Peroxide 10 meq/Kg maximum

Anisidine 20 maximum

140