KR20030018167A - Large-scale purification method of lipoxygenase metabolites of unsaturated fatty acid - Google Patents

Abstract

PURPOSE: Provided is a large-scale purification method of lipoxygenase metabolites of unsaturated fatty acid which inhibits abnormal growth of skin cells and skin inflammation. CONSTITUTION: The large-scale purification method of lipoxygenase metabolites of unsaturated fatty acid comprises the steps of: dissolving unsaturated fatty acid; adding lipoxygenase to the solution; adding an organic solvent thereto to extract lipoxygenase metabolites of unsaturated fatty acid; reducing the lipoxygenase metabolites of unsaturated fatty acid contained in the extract; and purifying the lipoxygenase metabolites of unsaturated fatty acid by HPLC with normal phase column and a non-polar solvent as a elution buffer.

Description

Large-scale purification method of lipoxygenase metabolites of unsaturated fatty acid}

The present invention relates to a method for mass purification of unsaturated fatty acid lipoxygenase metabolites, and more particularly, to separate and purify unsaturated fatty acid lipoxygenase metabolites known to have an effect of inhibiting skin inflammation and inhibiting abnormal skin growth. It's about how you can do it.

Fats are broken down into glycerin and fatty acids during digestion. Among these, fatty acids are known to have various types and different roles. Fatty acids are divided into saturated fatty acids, which are mostly found in animal fats, and unsaturated fatty acids, which are found in natural vegetable oils. Unsaturated fatty acid refers to fatty acids that lack 4-6 hydrogen atoms in the fatty acid molecular formula, also called vitamin F.

Such unsaturated fatty acids are known to be effective in improving reproductive function and thyroid function as well as in prostate gland trouble. In addition, emulsification of excess cholesterol in the blood and soften the joints, as well as effective in arthritis, asthma, dandruff, fatigue, migraine, kidney disease and leg pain.

Among the unsaturated fatty acids, linoleic acid, arachidonic acid and linolenic acid are part of the body's cellular structure, are essential components for the normal maintenance of brain and nerves, and synthesis of sex hormones and subsignal hormones. And intestinal beneficial bacteria propagation and the like. It is also a necessary nutrient for calm brain activity, nerve activity and healthy skin.

However, since these unsaturated fatty acids such as linoleic acid, arachidonic acid and linolenic acid are not synthesized in the body, they must be ingested through foods containing a large amount of these fatty acids, and thus they are also called essential unsaturated fatty acids.

On the other hand, some of these metabolites of unsaturated fatty acids are known to be effective in suppressing skin inflammation and inhibiting the growth of skin abnormalities, and the use as a therapeutic agent for various diseases has been proposed, but currently only a small amount from Cayman, USA It is produced by a separation method and sold at a high price, and such a small amount of separation is performed by high performance liquid chromatography (HPLC) by a reverse phase column.

In general, chromatography is classified into liquid chromatography (LC) and gas chromatography (GC) according to the type of mobile phase, and in particular, LC is a normal phase and a reverse phase depending on the type of the stationary phase support. phase). The normal phase column refers to a stationary phase having a polarity and a mobile phase having a nonpolarity, and silica gel or aluminum is used as the stationary phase. The reversed phase column means that the stationary phase is nonpolar and the mobile phase is polar, and hydrocarbon or the like is used as the stationary phase.

In the case of separating and purifying unsaturated fatty acid metabolites by HPLC using a conventional reversed phase column, the amount separated was very small, less than about 0.5 µg at a time. Therefore, in order to obtain a high yield of the material to be separated, there is a problem of repeating the column process several times after injecting a sample into the column.

Therefore, the present inventors have developed a method for separating and purifying a large amount of unsaturated fatty acid lipoxygenase metabolites, which have been proposed in various effects, such as suppressing skin inflammation and suppressing abnormal skin growth among various unsaturated fatty acid metabolites. During the study, HPLC conditions showing the best yield and purity were obtained, and the present invention was completed by developing a mass purification method including performing HPLC under these conditions.

Accordingly, an object of the present invention is to provide a method for large-scale separation and purification of unsaturated fatty acid lipoxygenase metabolites having the effect of inhibiting skin inflammation and inhibiting the growth of skin abnormalities.

In order to achieve the above object, the present invention,

1) dissolving unsaturated fatty acids;

2) reacting the unsaturated fatty acid solution by adding lipoxygenase;

3) extracting an unsaturated fatty acid lipoxygenase metabolite by adding an organic solvent to the reaction solution;

4) reducing the unsaturated fatty acid lipoxygenase metabolite contained in the extract; And

5) In a normal phase column composed of a polar fixed phase and a nonpolar mobile phase, a solution containing the reduced unsaturated fatty acid lipoxygenase metabolite is used as a mobile phase, and a nonpolar solvent is used as an elution buffer. Provided is a method for mass purification of unsaturated fatty acid lipoxygenase metabolites, comprising performing HPLC at an effluent rate of -8 ml / minutes.

Hereinafter, the present invention will be described in detail.

As used herein, the term "unsaturated fatty acid lipoxygenase metabolite" refers to a metabolite metabolized from an unsaturated fatty acid by a lipoxygenase enzyme.

Unsaturated fatty acids supplied from food are metabolized in the body by desaturation and elongation enzymes. Linoleic acid (hereinafter referred to as "LA") supplied by corn oil or safflower oil is called γ-linolenic acid (γ-linolenic acid, referred to as "GLA") by the Δ6 desaturating enzyme in the body. It is converted to dihomo-γ-linolenic acid (hereinafter referred to as DGLA) by a chain enzyme and then to arachidonic acid (hereinafter referred to as AA). GLA and AA are n-6 unsaturated fatty acids such as LA and are synthesized in the body by dietary supply of LA.

Such unsaturated fatty acids are essential for maintaining the lipid layered structure of the epidermal barrier consisting of lipid mixtures such as phospholipids, cholesterol and sphingolipids.

The present invention is characterized in providing a method for mass purification of unsaturated fatty acid lipoxygenase metabolites which is essential for maintaining the lipid layered structure of the epidermal barrier and has the effect of inhibiting skin inflammation and inhibiting abnormal proliferation of the epidermis.

In order to purify the unsaturated fatty acid lipoxygenase metabolites according to the present invention in a large amount, a step of first dissolving the unsaturated fatty acid used as a substrate is included. The unsaturated fatty acid used as the substrate is preferably selected from LA, AA and DGLA, but is not limited thereto. As a solvent for dissolving unsaturated fatty acids, ammonium hydroxide, chloroform, methanol, and the like may be used, and in particular, ammonium hydroxide is preferably used.

The dissolved unsaturated fatty acids are converted into intermediate metabolites by lipoxygenase (hereinafter referred to as 'LOX'), and in particular, it is preferable to use LOX isolated from soybean. The LOX is divided into type I-B, type I-S, type IV, and type V in the process of separating and purifying from soybean, and it is most preferable to use the type V LOX.

Intermediate metabolites produced from unsaturated fatty acids by LOX are extracted by common methods known in the art. Since the intermediate metabolite produced by LOX is a metabolite having a hydroperoxy group (-OOH), an organic solvent is used in the extraction process, and ether is most preferably used.

In order to separate the intermediate metabolite with -OOH group extracted with ether from the final metabolite with -OH group, it is necessary to reduce it using a reducing agent, and as a reducing agent, triphenylphosphine is used. Most preferred.

The mass purification method of an unsaturated fatty acid lipoxygenase metabolite according to the present invention includes a purification step by a column in addition to the enzyme reaction, extraction step and reduction step, and specifically, HPLC (normal phase column) It is desirable to purify unsaturated fatty acid lipoxygenase metabolites by performing high performance liquid chromatography. The normal phase column refers to the polarity of the stationary phase and the nonpolar phase of the mobile phase, and silica gel, aluminum, and paper are used as the stationary phase. In the present invention, μPorasil (Waters) made of silica gel was used as the stationary phase, and since the μPorasil has a feature of adsorbing a large amount of samples, the stationary phase in the column process for the mass purification method of the present invention. Used as

In addition, in the mass purification method according to the present invention, it is preferable to use a non-polar solvent, specifically, a mixed solvent of hexane, ethanol and acetic acid as an elution buffer, and more specifically, to use it in a ratio of 984: 16: 1. It is preferable to be.

On the other hand, the mass purification method according to the present invention is characterized by using a normal phase column when performing HPLC, and withdrawing the sample at a rate of 6-8 ml / minutes. In a small amount purification method using a conventional reversed phase column, the sample was discharged at a rate of 1 ml / minute, but in this case, there was a problem that a low yield of less than 0.5 μg was obtained at a time. On the other hand, when a normal column was used as in the present invention and the sample was flowed out at a rate of 6-8 ml / minute, unsaturated fatty acid lipoxygenase metabolites could be obtained in higher yield. Outflow at this rate has the advantage that large amounts of sample can be outflowed from the stationary phase in a short time.

Metabolites that can be isolated by mass purification of unsaturated fatty acid lipoxygenase metabolites according to the present invention are 13-HODE (13-Hydroxy-9,11-Octadeca-Di-Enoic acid) or 15-HETrE (15- Hydroxy-8,11,14-Eicosa-Tetra-Enoic acid), but is not limited thereto, and any metabolite converted from an unsaturated fatty acid by LOX may be included.

In the above, 13-HODE is a metabolite produced by converting LA, which is a major constituent fatty acid of the epidermis, by 15-LOX, and is known to have a physiological activity that suppresses abnormal proliferation of the epidermis.

In addition, 15-HETrE is a metabolite produced by 15-LOX from DGLA and has the function of inhibiting abnormal proliferation and inflammation of the epidermis.

In the embodiment of the present invention, 15-HETrE and 13-HODE were purified from DGLA and LA by the mass purification method. In the conventional method, 0.5 µg was purified as a result of one column purification process, whereas by the method according to the present invention, 1 mg or more was purified by one column injection.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Bulk Separation and Purification of 15-HETrE

1-1) Chemical reaction process by enzyme

A beaker containing 100 ml of Tris-HCl (0.1 M Tris-HCl, 1 mM phenol, pH 8.5) buffer was placed in a beaker with ice, and then LOX (6 mg) of soybean diluted in 10 ml of Tris-HCl buffer. / 10 mL) was added to the beaker containing the Tris-HCl buffer. The solution was continuously mixed using a stirring machine.

A solution prepared by dissolving 50 mg of unsaturated fatty acid DGLA in 6 ml of ammonium hydroxide was slowly added dropwise to the mixture, and mixed with care to avoid air bubbles using a stirrer. Thereafter, the mixture was mixed for 45 minutes to obtain a reaction solution containing an intermediate metabolite of DGLA.

1-2) Extraction Process

The pH of the reaction solution produced in Example 1-1) was titrated to 3.0 using hydrochloric acid, and mixing was continued for 10 minutes with a stirrer.

Transfer the solution adjusted to pH 3.0 to a separatory funnel, separate the ether layer from the water layer by adding several hundred ml of ether and shake it several times, and then transfer the ether layer formed on the upper layer to another beaker, and then add the same amount to the remaining water layer. The ether was added and shaken three times. The 300 ml ether solution collected by the three extractions was transferred to a separatory funnel and 100 ml distilled water was added. After shaking the funnel to which the distilled water was added to separate the ether layer and the water layer, the process of removing the lower water layer was repeated three times to obtain an extract containing the intermediate metabolite of DGLA.

1-3) Reduction Process

The extract containing the intermediate metabolite of DGLA, prepared in Example 1-2), was transferred to a beaker installed in an ice bath, and then 100 mg of triphenylphosphine was added thereto, followed by 30 minutes on ice. It was left.

The extract was transferred to a separatory funnel, washed three times with addition of 100 ml of saturated NaCl solution, and the solvent was evaporated using a rotary evaporator. The dried material was dissolved in 1 ml of 100% methanol to obtain a solution (50 mg substrate / 1 ml) containing 15-HETrE, the final metabolite.

1-4) Purification Process

100 μl (5 mg substrate portion) of the 15-HETrE-containing solution produced in Example 1-3) was taken to evaporate methanol in nitrogen gas. 100 μl of an organic solvent mixture (hexane: ethanol: acetic acid, 984: 16: 1, v / v / v) was added to the product from which the methanol was evaporated and developed on a normal phase column. As the stationary phase, μphorasil was used as the stationary phase, and a solvent in which hexane: ethanol: acetic acid was mixed at a ratio of 984: 16: 1 was used as the effluent solvent.

Meanwhile, in order to obtain 15-HETrE with maximum yield and purity, HPLC was performed by variously adjusting the outflow rate, and specific mass separation and purification conditions using HPLC were as follows. The effluent was separated and purified by changing the effluent rate at 1, 3, 6 and 8 ml / minutes under the following conditions, and the relationship between each effluent rate and the yield was shown in Table 1 below.

Column: μPorasil, Waters, particle size 10μm, 19mm × 30cm (Normalphase column)

Solvent: Hexane: Ethanol: Acetic acid (984: 16: 1, v / v / v)

Outflow rates: 1,3,6 and 8 ml / minutes, Run 100% isocratic

Guard column: Normal Phase cartridge

Sample loop: 100 μl

Running Time: 60 minutes

Outflow Rate (ml / minutes) Yield One <20 μg 3 340 µg 6 1.2mg 8 1.1mg

As shown in Table 1, 15-HETrE was obtained in the maximum yield when the sample was flowed out at a rate of 6 ml / minute, and the flow rate was 6 ml / minute even when the flow rate was 8 ml / minute. (minutes) and no significant difference in yield. However, yields decreased when the outflow rate was raised above 8 ml / minutes. Therefore, it was confirmed that the effluent rate of 6 to 8ml / minute was the best condition to obtain the unsaturated fatty acid lipoxygenase metabolite of the present invention in maximum purity and maximum yield.

<Example 2>

Bulk Separation and Purification of 13-HODE

13-HODE was purified using the same method as in Example 1 except using LA as the substrate fatty acid and adjusting the outflow rate to 6 ml / minute, and the results are shown in Table 2 below. .

Comparative Example 1

Small amount separation and purification of 15-HETrE

After obtaining the solution containing the final metabolite 15-HETrE in the same manner as in Example 1-1) to 1-3), and purified by using a reverse phase column HPLC to purify the 15-HETrE, the results It is shown in Table 2.

Specific separation and purification conditions were as follows.

Column: 0.5 μm ODS-C18, 4.6 mm × 25 cm (Reverse phase column; Beckman part No. 235329)

Solvent: Methanol: 0.08% Acetic acid (74: 26, v / v)

Outflow rate: 1 ml / minute, Run 100% isocratic

Guard column: Reverse Phase cartridge

Sample loop: 10 μl

Running Time: 60 minutes

Comparative Example 2

Small amount separation and purification of 13-HODE

Except for using LA as the substrate fatty acid 13-HODE was purified using the same method as in Comparative Example 1, the results are shown in Table 2 below.

Yield Example 1 1.2mg Example 2 1.4mg Comparative Example 1 0.5 µg Comparative Example 2 0.5 µg

As shown in Table 2, when 15-HETrE and 13-HODE were purified by the conventional small amount purification method (Comparative Examples 1 and 2), the yield was 0.5 µg at a time, whereas the mass purification method of the present invention Accordingly, when 15-HETrE and 13-HODE were purified (Examples 1 and 2), it was confirmed that the yield of 1 mg or more (1.2 and 1.4 mg, respectively) was shown at one time.

As described in detail above, the present invention provides a method for mass purification of unsaturated fatty acid lipoxygenase metabolites. By the mass purification method of this invention, unsaturated fatty acid lipoxygenase metabolites which have the effect of suppressing skin inflammation and suppressing abnormality of skin abnormality can be separated and purified in large quantities. The mass-purified metabolite may be usefully used for clinically relevant research requiring a large amount due to the nature of the research, and may be used to develop new drugs for treating various diseases.

Claims (8)

1) dissolving unsaturated fatty acids; 2) reacting the unsaturated fatty acid solution by adding lipoxygenase; 3) extracting an unsaturated fatty acid lipoxygenase metabolite by adding an organic solvent to the reaction solution; 4) reducing the unsaturated fatty acid lipoxygenase metabolite contained in the extract; And 5) In a normal phase column composed of a polar fixed phase and a nonpolar mobile phase, a solution containing the reduced unsaturated fatty acid lipoxygenase metabolite is used as a mobile phase, and a nonpolar solvent is used as an elution buffer. A method for mass purification of unsaturated fatty acid lipoxygenase metabolites, comprising performing HPLC at an effluent rate of -8 ml / minutes. The method of claim 1, wherein the unsaturated fatty acid is linoleic acid, arachidonic acid or dihomo-γ-linolenic acid. The method of claim 1, wherein the unsaturated fatty acid dissolution is by ammonium hydroxide, chloroform or methanol. The method of claim 1, wherein the lipoxygenase is isolated from soybeans. The method for mass purification of unsaturated fatty acid lipoxygenase metabolites according to claim 1, wherein the organic solvent is an ether. The method for mass purification of unsaturated fatty acid lipoxygenase metabolites according to claim 1, wherein the reduction of the extract is by triphenylphosphine. The method of claim 1, wherein the stationary phase is silica gel, aluminum or paper. 2. The method of claim 1, wherein the nonpolar solvent is a solvent in which hexane, ethanol and acetic acid are mixed in a ratio of 984: 16: 1.