CN102391111A

CN102391111A - Method for producing docosahexaenoic acid ethyl ester (DHA-EE)

Info

Publication number: CN102391111A
Application number: CN2011103246902A
Authority: CN
Inventors: 赵永俊
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-10-24
Filing date: 2011-10-24
Publication date: 2012-03-28

Abstract

The invention provides a method for producing docosahexaenoic acid ethyl ester (DHA-EE). The method includes the following steps: (a) improving the purity of the DHA-EE in material fish oil to 80%-83% from original 70(plus or minus 5)% by molecular distillation, wherein the actual yield achieves 50%-70%; (b) conducting carbamide clathration on the fish oil intermediate with DHA-EE purity of 80%-83% which is obtained from the step (a), thus improving the DHA-EE purity to 89%-92%, wherein the actual yield achieves 40%-50%; (c) by industrial preparative chromatography, purifying the fish oil with DHA-EE purity of 89%-92% which is obtained from the step (b), thus improving the DHA-EE purity to above 98%, wherein the actual yield achieves above 80%. The method combines the advantages of the existing DHA purification method, maximally avoids the defects such as the change of chemical or pharmacodynamic property in each independent operation and finally can realize the industrial production of high-purity DHA-EE medical material with purity being up to above 98%.

Description

The method of a kind of production docosahexenoic acid ethyl ester (DHA-EE)

Technical field

the present invention relates to a kind of separation method of compound, relate more specifically to the method for a kind of production high purity docosahexenoic acid ethyl ester (DHA-EE).

Background technology

docosahexenoic acid (DHA); Be that a kind of extremely important but human body self can not the synthetic long chain polyunsaturated fatty acids; DHA is mainly derived from marine fishes and some other marine animal and plants; Through these marine fishes are separated with marine animal and plant, operation such as extraction, can make the raw material fish oil that contains a certain amount of DHA, the main source of Here it is relevant dietary supplements of the common DHA in market.But the content of DHA relatively low (about 30%) in these raw material fish oil; Purity is not high enough, and contains some sfass and other pufass (like timnodonic acid, EPA) etc.; Can not reach the requirement of DHA far away as bulk drug or chemical intermediate; Therefore, exploitation prepares the method and the technology of high purity unsaturated fatty acids (like DHA) from raw material fish oil, caused domestic and international scientist's extensive attention.

are present; The method of having reported of from fish oil, separating and prepare DHA-EE is a lot; Mainly comprise following several kinds: urea adduct method, low-temperature freezing, metal salt precipitate method, vacuum distillation method, supercritical extraction, HPLC and chromatography etc.; These methods respectively have relative merits; But most of method still rests on the hierarchy of skill of pigment, SUV and a certain amount of sfas in the simple removal raw material fish oil, can't prepare highly purified DHA-EE, in addition; Though can obtain the DHA-EE of higher degree like preparative chromatography or CO 2 supercritical chromatography etc., high because of cost, need shortcomings such as specific installation (like utmost point cyrogenic equipment), industriallization difficulty can't be able to use.Therefore, the development simple controllable, running cost is low and be applicable to that the method for preparing high purity DHA-EE of suitability for industrialized production is of crucial importance and crucial.

Summary of the invention

for overcoming the problems referred to above of the prior art, the invention provides a kind of can be from raw material fish oil the method for production high purity (greater than 98%) docosahexenoic acid ethyl ester.

The technical scheme that the present invention adopts is: the method for a kind of production docosahexenoic acid ethyl ester (DHA-EE) may further comprise the steps:

(a) adopting molecular distillation method, is that the DHA-EE purity in the raw material fish oil of 70 (± 5) % is brought up to 80%-83% with DHA-EE purity, and actual recovery reaches 50%-70%;

(b) adopt the chemical process of the plain inclusion of urea, with the DHA-EE purity of gained in the step (a) be the fish oil of 80%-83% through the plain inclusion of urea, make its DHA-EE purity bring up to 89%-92%, actual recovery reaches 40%-50%;

(c) adopting the preparation of industrialization chromatography, is 89%-92% with the DHA-EE purity of gained in the step (b)

The fish oil of brings up to more than 98% its DHA-EE purity through the preparative hplc purifying, and actual recovery is not less than 80%.

further; In step (b); In the aqueous solution of absolute ethyl alcohol or absolute ethyl alcohol, the DHA-EE of gained in the step (a) is carried out the urea inclusion and handle, removing most of sfas and other impurity, but do not influence chemistry or the pharmacodynamic properties of DHA-EE.

further, in the urea inclusion of step (b) was handled, the volume ratio of water was less than 15% in the aqueous solution of absolute ethyl alcohol.

wherein, in the urea inclusion of step (b) was handled, the amount of water directly had influence on the effect of inclusion, and then further has influence on the purity of gained DHA-EE.When not adding water, promptly carry out inclusion with absolute ethyl alcohol, when the amount of water account for absolute ethyl alcohol and water TV 15% the time, the DHA-EE bullion can't dissolve and layering occur.In step (a), short-path distillation does not influence chemistry or the pharmacodynamic properties of DHA; In step (c), the chromatogram purification method in this patent method be meant can be suitable for large-scale industrial production from kilogram to tonne high purity DHA product.

compared with prior art; The present invention has advantage: the method for production high purity docosahexenoic acid ethyl ester provided by the present invention (DHA-EE); Present Research to from raw material fish oil, separating the DHA-EE method has both at home and abroad effectively utilized more existing purification technique advantages, has combined the advantage of molecular distillation method, the plain inclusion method of urea and large-scale preparative chromatography fully; And existing shortcoming when having avoided each method to use separately; It is low to have reached a kind of running cost on the whole, is fit to the combined method of suitability for industrialized production high purity DHA-EE, and can obtains higher yield.

Description of drawings

Fig. 1 is the GC collection of illustrative plates of raw material fish oil;

Fig. 2 be purity shown in Fig. 1 raw material fish oil through behind the molecular distillation method purifying the GC collection of illustrative plates;

Fig. 3 is the GC collection of illustrative plates after the raw material fish oil of purity shown in Fig. 2 passes through the plain inclusion method of urea purifying;

Fig. 4 is the GC collection of illustrative plates after the raw material fish oil of purity shown in Fig. 3 passes through the preparative chromatography purifying.

Embodiment

are made further specific descriptions below in conjunction with specific embodiment to the present invention.

Raw material:

Gc (GC) collection of illustrative plates of the DHA-EE raw material fish oil that present embodiment adopts is as shown in Figure 1; As can be seen from the figure; The content of DHA-EE in the DHA-EE raw material (RT:33.681min) is lower; Be 72.33%, also contain multiple other impurity simultaneously, have a plurality of single foreign matter contents to surpass 5%.The integration content of each composition is as shown in table 1 below in the raw material.

Table 1

The first step purifying: molecular distillation method

Adopt the VKL70-4 type short-path distillation appearance of German VTA company, evaporation area is 0.043m ² , built-in condensing surface area is 0.065m ² , adopt the roller type knifing WRS of system (standard type) and three fens discharger collection techniques, built-in 250mL needle type valve, treatment capacity is 0.1-2.0kg/h.

The concrete operations of molecular distillation method purifying DHA-EE of the present invention are following: at first check air tight; Open heating thermal oil switch and water of condensation then; Temperature regulation is 76 ℃, in cold-trap, adds liquid nitrogen, adds 100mL absolute ethanol washing equipment then; After equipment for washing finishes, it is 106 ℃ with attemperation; Open the one-level pump, pressure is adjusted to 0.05mbr; After treating that temperature, pressure is stable, adding 250mL purity is 72.33% raw material fish oil, manual regulation sample introduction valve, slowly blowing; After treating that weight two-phase proportion (cut ratio) is stable, rotate three fens bottles, collect two cuts.According to said method, handle 1.6kg DHA-EE continuously.After sample preparation is intact, close the one-level pump, from three fens discharger vacuum breakers.At this moment, accomplish single flash, collect two components, weigh, sealing is preserved and is the short-path distillation cut one time.DHA-EE in the raw material fish oil can reach 82.72% (RT:33.904min) through a short-path distillation after cut purity, and main single foreign matter content all has been reduced to below 5%, shown in the GC collection of illustrative plates among Fig. 2 and each the component integration in the following table 2.Yield can reach 78%.

under the same conditions; If through not reaching desired purity behind the short-path distillation; Then can use the heavy constituent behind the above-mentioned short-path distillation to distill through quadratic component; DHA-EE purity will further improve like this, and major impurity content list is assorted can be reduced to below 2%, and yield can reach 52%.

Table 2

The second step purifying: the plain inclusion method of urea

concrete operations step is following: the purity after the first step short-path distillation purification process that in reaction kettle, adds 0.75kg is 82.72% DHA-EE, adds 5.25kg urea element, and adds 12.75L absolute ethyl alcohol, 2.25L water; To reaction kettle vacuum nitrogen filling gas, triplicate; Heat temperature raising to 55 ℃ left and right sides stirring and dissolving stirred in two hours and is cooled to-25 ℃, freeze overnight.After spending the night, from reaction kettle, take out reactant, the low temperature suction filtration is drained the collection mother liquor and is reclaimed subsequent use.Filter residue soaks 1-2min with-40 ℃ of normal hexanes (6*1.25L) washing, repeats 5-6 back and collects washing lotion and filter residue, and is subsequent use; In the washing lotion of above-mentioned collection, add 5% salt solution of 2.5L, mixing leaves standstill separatory, stays organic layer.Add 5% salt solution of 1.25L in the organic layer, leave standstill separatory, stay organic layer; Use anhydrous sodium sulfate drying, suction filtration washs filter residue twice with normal hexane; Solvent evaporated on the Rotary Evaporators obtains the DHA-EE 330g behind the plain inclusion of urea, and purity is 89.35% (RT:33.869min); The GC collection of illustrative plates can be referring to Fig. 3 and table 3 with each component integration, and yield is 44%.

Table 3

It is that about 80 DHA-EE is that raw material carries out the plain inclusion of urea that purity behind the short-path distillation is adopted in ; Can DHA-EE purity be improved 8-10 percentage point, and except the impurity of content 6.2% (RT:31.572min), other foreign matter contents are all very low; It is thus clear that carry out the plain inclusion of urea behind the short-path distillation again; Improved yield greatly, reduced production cost, more easy realization of industrial production.Simultaneously, in the DHA-EE sample after above method is handled, major impurity is single, more helps the application of next step preparative hplc.

The 3rd step purifying: preparation of industrialization chromatography

single-column lock out operation step: adopt the DAC-HB800 of Hanbon Sci. & Tech. Co., Ltd. (800 * 1300mm) dynamic axial compression preparative hplc post; The NU3000C ultraviolet-visible(light)detector, YMC-Pack ODS-AQ reverse phase filler experimentizes.Using earlier NP7300C to prepare pump will be that 89.35% sample injects preparation system through the purity that above two-step purifying operation obtains, and applied sample amount is 650g.Adopt anti-phase methanol-water (88%-12% adds 0.1%TFA) eluent system to carry out the constant gradient wash-out.Under the ultraviolet detection wavelength of 210nm, the eluent of collecting concentrates through Rotary Evaporators, obtains 575g DHA-EE sample altogether, and yield is 88.5%.Shown in the GC collection of illustrative plates and component integration such as Fig. 4 and table 4 of gained high purity DHA-EE.DHA-EE purity behind the preparative hplc purifying can reach 98.55% (RT:33.768min).

Table 4

are above to be illustrated specific embodiment of the present invention; But protection content of the present invention is not only limited to above embodiment; In the technical field, the common knowledge of a GPRS just can be carried out diversified change in its technological main idea scope under of the present invention.

Claims

1. method of producing docosahexenoic acid ethyl ester (DHA-EE) is characterized in that may further comprise the steps:

(a) adopting molecular distillation method, is that the DHA-EE purity in the raw material fish oil of 70 (± 5) % is brought up to 80%-83% with DHA-EE purity, and actual recovery reaches 50%-70%;

(c) adopt the preparation of industrialization chromatography, with the DHA-EE purity of gained in the step (b) be the fish oil of 89%-92% through the preparative hplc purifying, its DHA-EE purity is brought up to more than 98%, actual recovery reaches more than 80%.

2. the method for production docosahexenoic acid ethyl ester according to claim 1 (DHA-EE); It is characterized in that: in step (b); DHA-EE to gained in the step (a) in the aqueous solution of absolute ethyl alcohol or absolute ethyl alcohol carries out the processing of urea inclusion, to remove most of sfas and other impurity.

3. the method for production docosahexenoic acid ethyl ester according to claim 2 (DHA-EE) is characterized in that: in the urea inclusion of step (b) was handled, the volume ratio of water was less than 15% in the aqueous solution of absolute ethyl alcohol.