JPH10310555A - Separation and purification of polybasic unsaturated fatty acid ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating and purifying a polybasic unsaturated fatty acid(PUFA) ester, enabling to purify the ester of PUFA such as docosahexaenoic acid(DHA) or docosapentaenoic acid (DPA) in high efficiency at a low cost and in a high purity. SOLUTION: This method for separating and purifying a polybasic unsaturated fatty acid ester contains a process for purifying a lipid mixture containing two or more kinds of polybasic unsaturated fatty acid esters by high performance liquid chromatography. Therein, the high performance liquid chromatography uses a porous granular basic material derived from an alkyl silane as a filler.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating and purifying polyunsaturated fatty acid esters. In particular, the present invention relates to esters of polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA), which are useful as pharmaceutical and functional food ingredients.
The present invention relates to a method for separating and purifying with high purity and efficiently.

[0002]

2. Description of the Related Art In recent years, polyunsaturated fatty acids (PUFA)
Is attracting attention as a compound having a unique physiological activity. Α-linolenic acid (AL
A), γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DP)
A) and docosahexaenoic acid (DHA). PU
Research has progressed on the fact that FA is a precursor of a physiologically active substance such as prostaglandins, and various physiological actions have been reported. EPA ethyl ester derived from fish oil is about 92%
A purified product with a purity of 100% was marketed in 1990 as a drug for the indication of arteriosclerosis obliterans.

On the other hand, DHA has a cholesterol lowering action, an anticoagulant action, a carcinostatic action as its physiologically active functions.
Furthermore, the effect of improving memory and learning ability in relation to the brain metabolic system,
It has been shown to prevent senile dementia, treat Alzheimer's disease, and act as an essential growth factor for fry. Therefore, it is used as a material for health foods and baby milk, and research on pharmaceuticals is being promoted. In addition, DPA has been reported to compensate for DHA deficiency between living organisms, and is considered to have some physiologically active function. Therefore, DPA may also be used as a pharmaceutical. DPA includes (n-3) and (n-6) systems depending on the position of the unsaturated bond.
In this specification, when simply referred to as “DPA”, it refers to a (n-6) -system DPA.

[0004] These PUFAs are generally difficult compounds to be chemically synthesized. On the other hand, in nature, these exist as fatty acid residues in triglycerides and phospholipids.
Therefore, extraction and purification from natural materials have been conventionally performed. For example, EPA and DHA are contained in marine fish fish oil, and have conventionally been extracted from fish oils having relatively complex fatty acid compositions, such as sardine oil and tuna orbital oil.

[0005] In order to use PUFA as a pharmaceutical in the future, it is said that a purity of at least 95% or more, preferably 98% or more is required. In the free fatty acid state, it is difficult to purify such a reagent or pharmaceutical to a suitable level of purity. Therefore, purification is generally performed as an ester, particularly an ethyl ester.

[0006] Conventionally, a mixture containing a plurality of types of free fatty acids or ester forms thereof (hereinafter, represented by -E) is used.
As methods for separating specific fatty acids, distillation and supercritical fluid extraction methods mainly using the difference in the number of carbon atoms, low-temperature fractionation methods mainly using the difference in the number of double bonds, urea addition method, and solvent separation method In addition, chromatography using an ion exchange resin is known. By the combination of these methods, it has become possible to achieve high purification of about 90 to 92%.

In particular, as for DHA ester (DHA-E), a method combining precision distillation and preparative liquid chromatography (DHA advanced purification and extraction technology development project) has been proposed as a method aimed at high purification on an industrial scale. In addition to the research report (FY 1994-6), p.213, issued by the DHA Advanced Purification and Extraction Technology Research Association (November 1995), adsorption chromatography using silver ion-exchanged clay minerals (the same report,
p.231; Yamaguchi et al., Oil Chemistry, Vol. 40, No. 10, p. 959 (199
1)), a liquid phase extraction method using complex formation in an aqueous silver nitrate solution (the same report, p.247; Misawa et al., Proceedings of the 61st Annual Meeting of the Chemical Society of Japan, p.1245 (1991)). Is being developed. However, even with these methods, the purity of DHA-E obtained by one purification step was up to about 95%. In order to obtain DHA-E and other PUFA esters with higher purity, for example, 98% or more suitable for use as a pharmaceutical, particularly expensive equipment, reagents, raw materials and the like may be used. Combinations of complex purification operations were required.

[0008] As described above, the conventional purification method has not been satisfactory for obtaining a high-purity PUFA ester with good reproducibility at a reasonable cost on an industrial scale.

[0009]

SUMMARY OF THE INVENTION The present invention is intended to solve the above problems. The purpose is DHA, D
It is an object of the present invention to provide a method capable of purifying an ester of PUFA such as PA to high purity at high efficiency and at low cost.

[0010]

SUMMARY OF THE INVENTION The method of the present invention comprises a PUF
A method for separating and purifying A ester, comprising a step of purifying a lipid mixture containing two or more PUFA esters by high performance liquid chromatography (HPLC). Here, high performance liquid chromatography uses a porous granular substrate derivatized with an alkylsilane as a filler.

[0011] The above-mentioned porous granular base material has a size of 10 to 1000.
It may have a particle size in the range of up to μm, and a pore size in the range of 60 to 3000 Angstroms. Also, the porous particulate substrate can be octadecylsilane (ODS) derivatized silica gel having a carbon content ranging from 4% to 30%.

In the above-mentioned high performance liquid chromatography, methanol, ethanol, or an aqueous solution containing at least 80% of any of them can be used as an eluent.

The above-mentioned lipid mixture can be obtained by esterifying a cultured bacterial cell extract of a microorganism producing PUFA. The microorganism is DHA and / or DPA
Can be a microorganism that produces

By the high performance liquid chromatography, a DHA ester and / or a DPA ester having a purity of 95% or more can be obtained.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION

(Purification by HPLC) In the method of the present invention, HPL
The packing used in column C is a porous granular substrate derivatized with alkylsilane. We have found that under appropriate conditions, the filler derivatized with the alkylsilane can
It has been discovered that the difference between the number of carbon atoms and the number of unsaturated bonds of the A ester can be distinguished with a level of accuracy that is significantly higher than previously predicted, and the present invention has been completed based on this.

As the porous granular substrate, any substrate suitable as a substrate for chromatography can be used. Preferred substrates include hydrated metal oxides or hydrated metalloid oxides. Examples thereof include hydrated alumina oxide, hydrated titanium oxide, hydrated zirconium oxide, hydrated silicon oxide, hydrated tin oxide, and the like. More specifically,
Examples include silica gel, hydroxyapatite, alumina, silica-alumina, titania, diatomaceous earth, silicate glass, aluminosilicate, clay kaolin, talc, zeolite, and the like. A substrate in which glass beads are coated with silica gel fine particles or the like is also included herein. Also, polymer particles containing polystyrene, polymethyl methacrylate, and derivatives thereof can be used as a preferable substrate. Silica gel is particularly preferred. As this substrate, one having a particle diameter and a pore diameter within an appropriate range is selected. Here, the particle diameter and the pore diameter refer to values measured in a state before being derivatized with silane. This measurement, for example,
This is the average value of particles when the substrate is observed with a scanning electron microscope or the like.

The upper limit of the particle diameter is typically 1000 μm.
m, preferably 300 μm, more preferably 100 μm
It is. The lower limit of the particle size is typically 10 μm, preferably 20 μm, more preferably 40 μm. If the particle diameter is too large, the separation ability of the column tends to decrease, and it becomes difficult to highly purify the PUFA ester. If the particle size is too small, the pressure for transporting the eluent in HPLC becomes too high, and it becomes difficult to fractionate PUFA esters in large quantities, which is not practically preferable. It should be noted that an appropriate particle size varies depending on the size (inner diameter × length) of a column used for HPLC.

The upper limit of the pore size is typically 3000 angstroms, preferably 300 angstroms, more preferably 150 angstroms. The lower limit of the pore size is typically 60 Å, preferably 80 Å. The pore size is too large,
If too small, diffusion of solute PUFA ester,
It becomes difficult to optimize the holding behavior. The substrate having the above pore diameter generally has a specific surface area in the range of about 1 to 1000 m ² / g.

The porous granular substrate is derivatized with an alkyl silane. The upper limit of the chain length of the alkylsilane is typically 40 carbon atoms, preferably 30 carbon atoms, and more preferably 20 carbon atoms. The lower limit of the chain length is typically 1 carbon atom,
Preferably it has 8 carbon atoms. If the number of carbon atoms is too large, the separation ability of the column tends to decrease. In terms of availability, octadecylsilane (ODS) having an octadecyl group having 18 carbon atoms is particularly preferable.

The derivatization of the porous granular base material with the alkylsilane is performed so that the carbon content derived from the silane falls within an appropriate range. This carbon content is a value indicating the hydrophobicity of the obtained filler, and is generally measured by elemental analysis. When a polymer is used as the base material, it can be calculated stoichiometrically based on the charged amount of the derivatization reaction. The upper limit of the carbon content is typically 30%, preferably 25%, more preferably 22%.
%. The lower limit of the carbon content is typically 4%, preferably 10%, more preferably 12%. If the carbon content is too high or too low, the separation ability of the column tends to decrease.

The above derivatization reaction is carried out by a suitable silane compound having a desired alkyl group (for example, triacontyl group, eicosyl group, octadecyl group, octyl group, n-butyl group, etc.) (for example, chlorosilane compound,
Using an alkoxysilane compound) by a known method. Various silica gels derivatized with octadecyl silane (ODS) and other alkyl silanes having a particle diameter, a pore diameter, and a carbon content in the appropriate ranges described above have been obtained from, for example, YMC Corporation. It is marketed under the trade name of “YMC * GEL”.

As the eluent for HPLC, any polar solvent suitable for reverse phase chromatography can be used. Examples include methanol, ethanol, isopropanol, acetonitrile, methylene chloride, chloroform, tetrohydrofuran, combinations thereof, and combinations of these with water. Methanol, ethanol,
An aqueous solution containing 80% or more (preferably 90% or more, more preferably 95% or more) of any of them is an example of a preferred eluent. The type and mixing ratio of the solvent
Depending on the type of PUFA ester to be purified,
It is appropriately selected. Optimization of the eluent is easy for those skilled in the art.

For example, methanol or methanol of 80% or more (preferably 90% or more, more preferably 9% or more)
Aqueous solution containing DHA-E and DPA-E
Is a preferred eluent when fractionation is simultaneously performed with high purity. On the other hand, ethanol or 80% of ethanol
The above (preferably 90% or more, more preferably 95% or more) aqueous solution is a preferable eluent when only DHA-E is fractionated at high purity.

In the method of the present invention, the amount of a single charge (load) of the lipid mixture to the column packed with the filler is determined by the content of the PUFA ester to be highly purified in the lipid mixture, and It is determined depending on the content of an unnecessary compound showing a retention time close to the ester on HPLC, and the like. The amount of charge is not particularly limited, but is usually about 1 g per liter of column volume.
To about 10 g. Therefore, according to the present invention, PUFA esters can be separated and purified with sufficient practical efficiency even when applied to industrial-scale implementation.

Other operating conditions of the HPLC, such as the flow rate of the eluent, are appropriately set by those skilled in the art. As the HPLC apparatus, a commercially available appropriate apparatus can be used. Industrial scale implementations include sample dissolution tanks, sample supply tanks,
An appropriate plant including an elution tank, an eluent recovery system, and the like can be installed and performed.

For detection of the eluate from the column, ultraviolet (U
V) Any suitable means may be utilized, such as a detector, a refractive index (RI) detector, and the like.

(Preparation of Lipid Mixture Containing PUFA Ester) The range of PUFA separated and purified as an ester by the method of the present invention is not particularly limited. From the viewpoint of application as pharmaceuticals or functional foods, naturally occurring P
UFA is preferred. In particular, PUFA having 18 or more carbon atoms and having three or more unsaturated bonds is preferable. DHA,
DPA, EPA, AA, and GLA are preferred PUs
It is an example of FA.

The source of PUFA is not particularly limited as long as it contains the desired fatty acid. It is selected from animal oils such as fish oil and liver oil, vegetable oils such as fruit oil, microbial oils derived from fungi, algae and the like according to the target fatty acid. For example, as described above, DHA and EP
It is known that A is contained in fish oil in a large amount. Also, D
It is known that PA is contained in safflower oil and GLA is contained in evening primrose extract. The selection of an appropriate source is easy for those skilled in the art.

It is known that by culturing certain microorganisms, particularly marine fungi or algae, fats and oils containing one or more of DHA, EPA, AA and the like can be obtained (see, for example, Japanese Patent Application Laid-Open No. 87988 and JP-A-7-82
68, JP-A-7-75556, and JP-A-1-304
No. 892). These microbial fats and oils often have a simpler fatty acid composition than fish oils and the like, so that the separation and purification method of the present invention can be applied more efficiently. Therefore,
Cultured cells of a PUFA-producing microorganism are an example of a preferred source.

Genus Thraustochytrium and Schizochytrium
Bacteria such as the genus have been reported to accumulate lipids including DHA and DPA in their cells (J. Am. Oil Che
m. Soc., Vol. 73, No. 11, p. 1421 (1996)). In the present invention, when DHA and / or DPA are highly purified, cultured cells of the above microorganisms and microorganisms of a similar species can be advantageously used. Of course, the culture cells are not limited to those exemplified, and may be any cells as long as they are derived from a bacterial species that produces DHA or DPA.

Culture conditions such as medium composition, temperature and pH are selected according to the type of microorganism to be cultured. Selection of appropriate culture conditions is easy for those skilled in the art. For example, Thra
The genus ustochytrium and the genus Schizochytrium contain 50% artificial seawater, use a medium based on glucose and corn staples, and have a temperature near room temperature and a weakly acidic pH.
Under liquid culture under aerobic conditions, DHA and D
Accumulates lipid components including PA.

The cultured microbial cells of the microorganism are dried, if necessary, and subjected to an operation such as pulverization of the microbial cells according to a conventional method, and then lipids are extracted. When other sources such as animal and vegetable oils are used, lipids can be extracted after pretreatment according to the type of the source.

By esterifying the lipid thus extracted, a lipid mixture containing a PUFA ester is obtained. The ester group of the PUFA ester is
Typically, it is an alkyl group. Of course, it may be an alkenyl group such as vinyl, an aryl group such as phenyl, or an arylalkyl group such as benzyl. As the alkyl group, an alkyl group having 1 to 6 carbon atoms is preferable, and an alkyl group having 1 to 4 carbon atoms is more preferable.
Pharmaceutical products or functional foods to be directly administered to humans include:
Ethyl esters are particularly preferred.

Suitable reaction conditions for the esterification are known to those skilled in the art. For example, after the lipid is dissolved in an organic solvent such as hexane, an alkali (for example, ethanol containing 1N potassium hydroxide) is added, and esterification can be performed at a temperature of about 10 to 25 ° C. After the organic phase is separated, the mixture is concentrated according to a conventional method to obtain a lipid mixture containing an ester.

This lipid mixture was converted to HPL as described above.
Serve in C. According to the method of the present invention, the target PUFA ester can be obtained with high purity by one operation of HPLC. Therefore, it is not necessary to perform a preliminary purification operation after the above-mentioned esterification reaction, and HPLC can be immediately performed.

As used herein, the purity of PUFA ester refers to the purity measured by at least one of HPLC and GC. According to the present invention, typically a purity of at least 95%, preferably at least 98%, more preferably at least 99% can be achieved. Therefore, the obtained P
UFA esters can be used immediately for the production of pharmaceuticals and functional foods.

[0037]

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to these examples.

In the following examples, DHA-E
The purity of DPA-E was determined by high performance liquid chromatography (HPLC) using YMC-Pack AM-303 manufactured by YMC Corp. as an analytical column and acetonitrile / water (97.5 / 2.
5; volume ratio) at a flow rate of 1 ml / min.
0 nm). On the other hand, in the case of gas chromatography (GC), a capillary column manufactured by GL Sanence Co., Ltd., TC-70 (diameter 0.25 mm × length 30 m) is used as an analytical column, and nitrogen gas is used as a carrier gas at a flow rate of: 0.8ml
Per minute at a flow rate of 100 kPa (column head pressure), and the measurement was carried out by detecting with a FID in a temperature rising mode at a column temperature of 170 to 220 ° C. (4 ° C./min).

(Example 1) Cultured cells of Schizochytrium sp. Were dried, crushed using glass beads in a mixed solvent of chloroform and methanol, filtered, and then extracted with hexane. The lipid was obtained. The resulting lipid was esterified in hexane by the addition of ethanol containing potassium hydroxide. Post-treatment was performed according to a conventional method to obtain a lipid mixture containing a fatty acid ester. The mixture was analyzed by GC to find that DHA-
It contained about 43.7% of E and about 11.1% of DPA-E.

Octadecylsilane (O) having a particle diameter of 50 μm, a pore diameter of 120 Å, and a carbon content of 17% was used.
DS) Filler (YMC * GEL ODS-AM-120)
-S50) was used. Filled with this filler, inner diameter 40
HPLC was performed using a column having a length of 0 mm and a length of 2000 mm to separate DHA-E and DPA-E. The preparative HPLC device used was manufactured by YMC Corporation.
Using methanol as eluent, 4.4 liters /
The liquid was passed at a flow rate of 1 minute.

The column was charged with 1.42 kg of the above lipid mixture as a 10% solution in methanol. Fractions of each component were fractionated based on the retention time of each component based on the results of a preliminary study. Each fraction was evaporated and the solvent was distilled off to obtain DHA-E and DPA-E. The obtained DHA-E weighs 0.36 kg and DPA-E
Was 0.07 kg.

When the purity was analyzed by HPLC, DHA-E was 98.9%, and DPA-E was 97.
7%.

Example 2 Filler (YMC * GELO)
DS-AM-120-S50)
HPLC was performed using a column having a length of 2000 mm and a length of 2000 mm, and DHA-E and DPA-E were separated. The preparative HPLC device used was manufactured by YMC Corporation. Using a mixed solution of methanol / water (98/2; volume ratio) as an eluent, the solution was passed at a flow rate of 4.4 L / min.

1.33 kg of the same lipid mixture as in Example 1
Was charged to the column as a 10% solution in methanol. Fractions of each component were fractionated, each fraction was evaporated and the solvent was distilled off.
PA-E was obtained. 0.30 kg of the obtained DHA-E,
DPA-E was 0.07 kg. The purity of these
When analyzed by PLC, DHA-E was 99.0.
% And DPA-E were 99.7%.

Example 3 Particle size: about 20 μm, pore size: 1
ODS filler (YMC * GEL ODS-A-120-S15 / 20 Å, carbon content 17%)
HPLC was carried out using a column having an inner diameter of 6 mm and a length of 2000 mm packed with 30), and DHA-E and DPA-
E was collected. The preparative HPLC device used was LC-8A manufactured by Shimadzu Corporation. Using methanol / water (90/10; volume ratio) as an eluent, the solution was passed at a flow rate of 1 ml / min.

480 mg of the same lipid mixture as in Example 1
The column was charged as a 10% solution in methanol. Fractions of each component were fractionated, each fraction was evaporated and the solvent was distilled off, and DHA-E and DP
AE was obtained. 114.2 mg of DHA-E obtained,
DPA-E was 11.1 mg. The purity of these
When analyzed by PLC, DHA-E was 99.3.
% And DPA-E were 99.7%.

Example 4 Particle size 50 μm, pore size 12
0 Å, carbon content of about 13%, ODS filler (YMC * GEL ODS-AQ-120-S5
HPLC was carried out using a column having an inner diameter of 6 mm and a length of 2,000 mm packed with DHA-E and DPA-E.
Was collected. The preparative HPLC device used was LC-8A manufactured by Shimadzu Corporation. Using ethanol / water (95/5; volume ratio) as an eluent, the solution was passed at a flow rate of 1 ml / min.

80 mg of the same lipid mixture as in Example 1 was charged to the column as a 10% solution in methanol.
Fractions of each component were fractionated, each fraction was evaporated and the solvent was distilled off, and DHA-E and DPA-
E was obtained. 28 mg of DHA-E obtained, DPA-E
Was 5 mg. The purity was analyzed by HPLC. DHA-E was 96.5% and DPA-E was 86.5%.
It was 8.9%.

[0049]

According to the method of the present invention, DHA, DPA
Efficient, low-cost PUFA ester
It can be purified to high purity.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI // (C12P 7/64 C12R 1: 645) (71) Applicant 000001904 Suntory Limited 2-1-1 Dojimahama, Kita-ku, Osaka-shi, Osaka No. 40 (72) Inventor Ryuji Yamamura 69-1 Tai Arami, Kumiyama-cho, Kuse-gun, Kyoto Prefecture Inside YMC Separation Center (72) Inventor Yasushi Shimomura 5-28 Kokudaidai, Komatsu City, Ishikawa Pref. (72) Inventor Isao Togo 7-2-9 Migatadai, Nishi-ku, Kobe City, Hyogo Prefecture (72) Inventor Gohiro Tanaka 1-252 Nagatano-cho, Fukuchiyama City, Kyoto Inside Nagase Seikagaku Corporation (72) Inventor Toshiaki Yaguchi 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Prefecture Inside the Research Center of Suntory Limited

Claims (8)

[Claims] 1. A method for separating and purifying a polyunsaturated fatty acid ester, comprising a step of purifying a lipid mixture containing two or more polyunsaturated fatty acid esters by high performance liquid chromatography (HPLC). And wherein the high performance liquid chromatography uses a porous granular substrate derivatized with an alkylsilane as a filler. 2. The method according to claim 1, wherein the porous granular substrate is 10 to 100.
2. The composition of claim 1, having a particle size in the range of up to 0 μm and a pore size in the range of from 60 to 3000 Å.
The method described in. 3. The method according to claim 2, wherein the porous granular base material comprises 4% to 30%.
3. An octadecylsilane (ODS) derivatized silica gel having a carbon content in the range of up to 1.
The method described in. 4. The method according to claim 1, wherein said high performance liquid chromatography uses methanol, ethanol, or an aqueous solution containing at least 80% of any of them as an eluent. 5. The method according to claim 1, wherein the lipid mixture is obtained by esterifying a cultured cell extract of a microorganism producing polyunsaturated fatty acids. 6. The method according to claim 6, wherein the microorganism is docosahexaenoic acid (D
The method according to claim 5, which produces HA) and / or docosapentaenoic acid (DPA). 7. Docosahexaenoic acid (DHA) having a purity of 95% or more by the high performance liquid chromatography.
Ester and / or docosapentaenoic acid (DPA)
7. The method according to claim 1, wherein an ester is obtained. 8. The method according to claim 1, wherein the polyunsaturated fatty acid ester is an ethyl ester.