KR100350432B1 - Derivatives of conjugated linoleic acid and method for synthesizing thereof - Google Patents

Abstract

The present invention relates to derivatives of conjugated linoleic acid (CLA), which is a highly functional substance, and a method for synthesizing the same. Phospholipids, phospholipid derivatives and CLA-containing copper and vegetable fats and fats combine the functionality of the functional groups added to the functionality of the CLA itself to provide a more functional substance, as well as reduce the toxicity from the carboxyl groups at the end of the CLA. The utility of CLA can be improved.

Description

FIELD OF CONJUGATED LINOLEIC ACID AND METHOD FOR SYNTHESIZING THEREOF

The present invention relates to conjugated linoleic acid (CLA) derivatives and methods for their synthesis.

Conjugated linoleic acid derived from a ruminant animal is a natural multifunctional fatty acid, and has effects such as anticancer, immunopotentiation, antioxidant, anticholesterologenic, antifungal, reducing body fat and promoting animal growth.

Since conjugated linoleic acid is a free fatty acid, when it is added to copper and vegetable fats and oils, the quality of these fats and oils is concerned, and this problem can be solved by synthesizing the triglyceride derivative of conjugated linoleic acid to food. Neutral fatty acid derivatives of conjugated linoleic acid have better absorption in the body than free conjugated linoleic acid, and have the additional effect of eliminating the toxic effects of carboxyl groups because the carboxyl groups are removed.

In addition, since conjugated linoleic acid is fat-soluble, there is a limit to the amount of the conjugated linoleic acid or the method to be added to the water-soluble food. In order to overcome this problem, a method of synthesizing glycolipid derivatives and phospholipid derivatives of conjugated linoleic acid can be considered. The conjugated linoleic acid derivatives thus synthesized are combined with ascorbic acid, glucose, and phospholipids, which are functional substances, and thus have higher functionality than conjugated linoleic acid.

In view of the above, it is an object of the present invention to provide a conjugated linoleic acid triglyceride derivative and a method for synthesizing the same so that conjugated linoleic acid can be added to fats and oils more effectively. By providing phospholipid derivatives and glycolipid derivatives of conjugated linoleic acid, and methods of synthesizing them, it is intended to facilitate the addition of conjugated linoleic acid to foods and to further enhance its functionality.

In order to achieve the above object, the present invention provides a copper, vegetable fat and oil containing triglyceride derivatives, ascorbic acid derivatives, glucose derivatives, cyclodextrin microcapsules, phospholipid derivatives and conjugated linoleic acid derivatives of conjugated linoleic acid The conjugated linoleic acid (hereinafter referred to as CLA) can be synthesized by alkaline isormerization from linoleic acid, as shown in Scheme 1 below, c9, t11-CLA and t10, c12-CLA. Kind is obtained.

In addition, the present invention provides a CLA derivative obtained by attaching various functional groups to the terminal carboxyl group of the CLA in order to enhance the functionality of the CLA, and a method for synthesizing such CLA derivatives.

As a method for synthesizing the triglyceride derivative of CLA, triglycerides added with CLA, and the conventional lipid lipids such as cottonseed oil, corn oil, soybean oil, sesame oil, perilla oil, fish oil, milk fat and lard There is a method of including CLA in these triglycerides by substituting some fatty acids with CLA.

The present invention first provides a method for synthesizing a triglyceride derivative of conjugated linoleic acid (hereinafter referred to as CLA-TG) in which a hydroxyl group of glycerol is ester-bonded with a carboxyl group at the CLA end. Such CLA-TG synthesis methods include chemical, enzymatic, and biological methods.

The method for synthesizing CLA-TG by chemical method is as follows.

First, 2.8 parts by weight of CLA is added to a reactor such as a round bottom flask and dried, and then 35.2 parts by weight of dried benzene is added and stirred under N ₂ to dissolve CLA. The reactor was cooled down to 0 ° C., and then 1.3 parts by weight of dried pyridine and 1.8 parts by weight of oxalyl chloride were added and stirred for 2 to 4 hours, followed by stirring for 2 to 4 hours. Rainane chloride (hereinafter referred to as CLA-Cl) is synthesized. The rf value of the synthesized CLA-Cl is about 0.8 in a hexane: ethyl acetate (5: 1) system. Such CLA-Cl may be CLA-Cl synthesized by another method using a known material. In the next step, 0.2 to 0.5 parts by weight of dry glycerol and 0.5 to 1.5 parts by weight of pyridine are added to 3 parts by weight of CLA-Cl. After reacting at room temperature for 8-15 hours, saturated sodium carbonate is added to stop the reaction. The esterified compounds were extracted with ether, concentrated to remove the solvent, and then the CLA-TG was conjugated to monoglycerides of conjugated linoleic acid using a silica gel column (hexane: ethyl acetate = 24: 1 volume ratio) or at a pressure of about 20 mmHg. (CLA-MG) and diglycerides of conjugated linoleic acid (CLA-DG).

The resulting substance is the hydroxyl group of glycerol ester-bonded with the carboxyl group of CLA to add CLA to glycerol. When all of the hydroxyl groups present in glycerol react with CLA, only CLA-TG is produced. If reacted, CLA-DG or CLA-MG is produced. CLA-TG is isolated in the product to obtain CLA-TG.

Synthesis of CLA-TG using enzymatic method is as follows.

In a reactor such as an Erlenmeyer flask, 3 parts by weight of glycerol and 1 part by weight of CLA, and 1 to 10% by weight of a mixed solvent (distilled water: isooctane in a 1: 1 volume ratio) and 5 to 30% by weight of glycerol Lipozyme is added and reacted for 1 to 10 hours at 50 ° C. and 250 rpm in a nitrogen atmosphere. Next, 7.9 parts by weight of a solvent mixed with acetone and ethanol was added to stop the reaction. Then, the solvent was removed, concentrated, and then conjugated linoleic acid using a silica gel column (hexane: ethyl acetate = 24: 1). CLA-TG can be separated from monoglycerides of conjugated linoleic acid and diglycerides of conjugated linoleic acid.

The method for producing CLA-TG by biological method is as follows.

Addition of 0.1-10% by weight of conjugated linoleic acid or conjugated linoleic acid methyl ester (hereinafter referred to as CLA-Me) to the carp feed is carried out for 1 to 10 weeks. Muscles are harvested from the cultured carp, and lipids are separated by a mixed solvent of chloroform and methanol (mixed in a volume ratio of 2: 1), and the lipid component is dissolved in 5-8 times of acetone to separate the supernatant and sediment. The supernatant contains CLA-containing neutral lipids, and the precipitate contains CLA-containing phospholipids. After the supernatant is concentrated, neutral lipids can be separated purely using a mixed solvent of silicic acid and celite (mixed in a weight ratio of 1: 1). CLA-TG can be separated from the separated neutral lipids by silica gel column or other methods. The purity of the separated CLA-TG is 96%, and the content of CLA in the neutral lipids is 5-30%.

In addition, according to the present invention by replacing some fatty acids contained in neutral lipids such as cottonseed oil, corn oil, soybean oil, sesame oil, perilla oil, fish oil, milk fat, lard with CLA to include CLA in these neutral lipids, It can increase the functionality of CLA and increase the absorption rate of CLA in the body. As described above, as a method of synthesizing copper and vegetable oil containing CLA, a conversion esterification method is mainly used, and there are a chemical method and an enzymatic method.

The method for synthesizing CLA-containing copper and vegetable oil by chemical method is as follows.

In the first step, conjugated linoleic acid methyl ester (hereinafter referred to as CLA-Me) is synthesized. That is, 300 ml of methanol in which hydrochloric acid is dissolved at a concentration of 0.05 N and 5 g of CLA are added to a reactor and mixed for 30 seconds. The reactor is connected to a reflux condenser, refluxed in a boiling water bath for 120 minutes, and cooled to room temperature. 50 ml of distilled water and 30 ml of hexane were added thereto, followed by extraction of CLA-Me with a hexane layer, followed by separation of the hexane layer. The separated hexane layer was washed several times with 20 ml of distilled water, dried over anhydrous sodium sulfate, and the like, filtered to recover the hexane layer, and concentrated to synthesize CLA-Me. The CLA-Me is a known material and can also be synthesized using CLA-Me synthesized by other methods. Then, CLA-Me can be used for vegetable oils such as cottonseed oil, soybean oil, corn oil, and animal fats such as lard and milk fat. It is added in the weight ratio of 1: 1-6: 1. Under reduced pressure (20 mmHg) and 90 ° C., sodium methoxide is added to 1 to 2 multiple moles of the number of moles of maintenance (one mole of oil is calculated as 840) and reacted for 1 to 10 hours. The reaction is then washed with water, dried with anhydrous sodium sulfate and the like, then concentrated, followed by separation of CLA-containing copper and vegetable oils using a silica gel column (hexane: ethylacetate = 24: 1). As a result of measuring the content of CLA in the contained copper and vegetable fat and oil by GC, the amount is about 10 to 70%, which varies depending on the type of lipid.

The method for synthesizing CLA-containing copper and vegetable oil by enzymatic method is as follows.

CLA-Me and triglycerides (copper and vegetable fats and oils) are mixed in a 1: 1 to 6: 1 weight ratio, and then lipozyme is added at 10% by weight of the total weight of the lipid. An organic solvent such as isooctane, hexane, and pentane is added thereto at 200 wt% of the total weight, followed by shaking at 40 to 70 ° C for 24 hours. Hexane was then added to the reaction to about 10 times the total weight to dissolve the lipids and filtered. The filtrate was concentrated and the CLA-containing neutral lipids were separated using a silica gel column (hexane: ethyl acetate = 24: 1 volume ratio) or vacuum. CLA-containing copper synthesized by this method and the content of CLA in vegetable oil or fat are about 10 to 70%. The content of CLA can be measured by GC.

According to another aspect of the present invention, a glycolipid derivative of CLA and a method for synthesizing the same are provided. Glycolipid derivatives of CLA include ascorbic acid derivatives of CLA (hereinafter referred to as CLA-Asc), D-glucose derivatives of CLA (hereinafter referred to as CLA-Glc), and cyclodextrin microcapsules of CLA (hereinafter referred to as CLA-CD). . Glycolipid derivatives of the CLA according to the present invention is a substance having a high functionality by adding the functionality of the material added with the CLA itself. Hereinafter, a method for synthesizing the glycolipid derivatives of CLA according to the present invention.

CLA-Asc is a substance in which CLA is ester-bonded to the hydroxyl group of ascorbic acid having the structure of Chemical Formula 1, and its synthesis method is as follows. The CLA can then be added at the position of all hydroxyl groups present in the ascorbic acid.

First, CLA-Cl is first synthesized in the same manner as used for chemical synthesis of CLA-TG. Since CLA-Cl is a known substance, CLA-Cl synthesized by another method may also be used. Next, 2 parts by weight of L-ascorbic acid is dissolved in 31.5 parts by weight of acetone, and 1 to 3 parts by weight of CLA-Cl. After addition of 1.6 parts by weight of pyridine and stirred for 2 to 3 hours at 25 ℃. Subsequently, 39 parts by weight of methanol is added to the reaction, the mixture is stirred for 10 to 20 minutes, the solvent is evaporated, and the remaining residue is extracted with ether. The ether layer is washed with water, dried with anhydrous sodium sulfate and ether removed in vacuo to give CLA-Asc. Synthesized CLA-Asc is a mixture of regioisomers.

CLA-Glc according to the present invention is an aldehyde-type D-glucose ester-linked having a structure as shown in the following formula (2) to the carboxyl terminal of the CLA terminal. CLA can be linked to any of the indicated 1, 2, or 3 of each hydroxyl group present in D-glucose but is mainly bound to the 3 position.

The synthesis method of CLA-Glc provided according to the present invention is as follows.

220 parts by weight of 1,2-isopropylidine-D-glucofuranos and 20 parts by weight of dried pyridine are added to the reactor and concentrated to 1/2 volume. 372 parts by weight of CLA-C1 was added thereto, followed by reaction at room temperature for 6 hours, and then the reaction was stopped with saturated sodium carbonate. The mixture was extracted with ethyl ether and chloroform, concentrated, pyridine was removed by azeotropic distillation, and the remaining residue was separated by a mixed solvent of hexane and ethyl acetate (2: 1 by volume). 10.7 parts by weight of 70% acetic acid was added to 240 parts by weight of purely isolated 1,2-isopropylidine-D-glucopyranose CLA (1,2-isopropylidine-D-glucofuranose-CLA) and 60 to 90 at 60 to 80 ° C. By stirring for a time, CLA-Glc can be synthesized.

In addition, the CLA-CD provided according to the present invention is excellent in inhibiting the oxidation of CLA as CLA is microencapsulated by α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin.

Synthesis method of CLA-CD according to the present invention is as follows.

1.4 parts by weight of CLA is dissolved in 40 parts by weight of ethanol, and 10 to 40 parts by weight of distilled water is added thereto. 1-4 weight part of (alpha)-cyclodextrins are added to this, and it stirred for 1 to 10 minutes for reaction in a 60-80 degreeC thermostat, and it cools to 4-20 degreeC over 0.5 to 5 hours with continued stirring. The reaction product is vacuum freeze-dried or spray-dried to blow off the solvent to obtain α-CD microcapsules of CLA. For β-cyclodextrin and γ-cyclodextrin, the same method as for preparing α-CD microcapsules of CLA is performed. , Β-CD microcapsules and γ-CD microcapsules of CLA can be synthesized.

CLA-Pl provided according to the present invention is a fatty acid bound to the phospholipid is substituted with CLA and has the structure as shown in the following formula (3).

CLA-Pl provided according to the present invention can be prepared by chemical, enzymatic and biological methods.

First, the chemical synthesis method of CLA-Pl is as follows.

5 parts by weight of CLA-Me was added to 5 parts by weight of lecithin, and stirred for 1 to 3 hours at 20 mmHg and room temperature. Then, the temperature was raised to 60 ° C. and sodium methoxide was added in an amount of 0.2 to 1% of the total weight. Stir for 2-5 hours. Next, wash and centrifuge with hot water several times to remove Na-CLA. After centrifugation, CLA-Pl was obtained by steam distillation at 180 ° C. and 20 mmHg for about 30 minutes.

Next, the enzymatic synthesis method of CLA-Pl is as follows.

2 parts by weight of CLA-Me is added to 2 parts by weight of lecithin and stirred for 1 to 3 hours at room temperature and 20 mmHg reduced pressure. Thereafter, the reaction temperature is increased to 60 ° C. and lipozyme is added in an amount of 5 to 30% of the total weight to react for 3 to 10 hours. Next, wash and centrifuge with hot water several times to remove Na-CLA. After centrifugation, CLA-Pl was obtained by steam distillation at 180 ° C. and 20 mmHg for about 30 minutes.

Subsequently, the method for biological synthesis of CLA-Pl is as follows.

Carp feed containing 0.1 to 10% by weight of CLA or CLA-Me is fed and the carp is cultured for 1 to 10 weeks. In the cultured carp, the total lipids in muscle are separated by a mixed solvent of chloroform and methanol (2: 1 volume ratio), and the lipids are dissolved in 5-8 volumes of acetone to prepare a supernatant containing CLA-containing neutral lipids and phospholipids containing CLA. Separate the precipitate containing. CLA-Pl can be obtained by pure separation of phospholipids using a mixture of silicic acid and celite (1: 1 weight ratio) in the separated precipitate, and the content of CLA in the obtained phospholipid derivative of CLA is 4-28%. do.

As described above, triglyceride derivatives, ascorbic acid derivatives, glucose derivatives, cyclodextrin microcapsules and phospholipid derivatives of CLA provided according to the present invention are more functional because they combine the functionality of the functional groups added to the functionality of the CLA itself. Not only can provide a high substance, but also reduces the toxicity coming from the carboxyl group at the end of the CLA, thereby increasing the utility of CLA as a food additive. Moreover, the cyclodextrin microcapsules of CLA have an excellent effect in preventing CLA oxidation by microencapsulating CLA.

Claims (17)

delete In a method for synthesizing a triglyceride derivative of conjugated linoleic acid in which a hydroxyl group of glycerol and a carboxyl group at the terminal of conjugated linoleic acid (CLA) form an ester bond, 0.2 to 0.5 parts by weight of glycerol and 0.5 to 1.5 parts by weight of pyridine were added to 3 parts by weight of conjugated linoleic acid chloride (CLA-Cl), followed by reaction at room temperature for 8 to 15 hours, followed by extracting and separating triglyceride derivatives of CLA. Chemical synthesis method of triglyceride derivatives of CLA comprising a. In a method for synthesizing a triglyceride derivative of conjugated linoleic acid in which a hydroxyl group of glycerol and a carboxyl group at the terminal of conjugated linoleic acid (CLA) form an ester bond, 3 parts by weight of glycerol, 1 part by weight of CLA, 0.03 to 0.3 parts by weight of a 1: 1 volume ratio mixed solvent of distilled water and isooctane, and 0.15 to 0.9 parts by weight of lipozyme were mixed at a temperature of 50 ° C. and 250 rpm in a nitrogen atmosphere for 1 to 10 hours. After the reaction, the method of enzymatic synthesis of triglyceride derivatives of CLA comprising the step of separating the triglyceride derivatives of CLA. In a method for synthesizing a triglyceride derivative of conjugated linoleic acid in which a hydroxyl group of glycerol and a carboxyl group at the terminal of conjugated linoleic acid (CLA) form an ester bond, Feed carp containing 0.1 to 10% by weight of conjugated linoleic acid or conjugated linoleic acid methyl ester (CLA-Me) to carp for 1 to 10 weeks; Muscles were harvested from the cultured carp to separate lipids with a 2: 1 volumetric mixed solvent of chloroform and methanol; The separated lipids are dissolved in acetone and separated into a supernatant and a precipitate; Separating the CLA-containing neutral lipids from the separated supernatant; A method for biological synthesis of triglyceride derivatives of CLA comprising the step of separating the triglyceride derivatives of CLA from the isolated CLA-containing neutral lipids. delete delete delete delete delete delete delete delete delete delete delete delete delete