KR100750661B1 - A method for manufacturing gamma-linolenic acid complex for precaution and treatment of inflammatory, allergic and atopic diseases - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of inflammatory, allergic or atopic diseases containing the gamma linolenic acid complex as an active ingredient, and more specifically gamma linolenic acid, which is a kind of unsaturated fatty acid and an active substance such as amino acids or sugars. The present invention relates to a composition for preventing and treating atopy or atopy-related diseases containing a gamma linolenic acid complex converted by hydrogen bonding or an ester exchange reaction as an active ingredient.

Since the gamma linolenic acid complex of the present invention is excellent in water solubility, and particularly excellent in antioxidant activity and anti-inflammatory effect, it is possible to improve and restore skin barrier by supplementing deficiency of gamma linolenic acid commonly found in atopic patients, and to mediate inflammation parameters. By suppressing the production and secretion of these to exhibit an anti-inflammatory, anti-allergic effect, etc. can be usefully used as a composition for the prevention and treatment of atopic dermatitis and skin diseases associated with atopy.

Description

A method for manufacturing gamma-linolenic acid complex for precaution and treatment of inflammatory, allergic and atopic diseases}

1 is a graph showing the IR analysis of the gamma linolenic acid complex prepared according to Example 1 of the present invention,

Figure 2 is a diagram showing the test results confirming the water-soluble of the gamma linolenic acid complex prepared according to Example 1 of the present invention,

3 is a graph showing the change of the peroxide value by the heat of the gamma linolenic acid composite prepared according to Example 1 of the present invention,

Figure 4 is a graph showing the results of the antioxidant activity of the gamma linolenic acid complex prepared according to Example 1 of the present invention by ABTS radical measurement method,

Figure 5 is a graph showing the inhibitory effect on TNF-α secretion mediators of the gamma linolenic acid complex prepared according to Example 1 of the present invention,

6 is a graph showing the inhibitory effect on the synthesis of PGE-2, an inflammation mediator of the gamma linolenic acid complex prepared according to Example 1 of the present invention.

Figure 7 is a graph showing the inflammation inhibitory effect of the gamma linolenic acid complex prepared according to Example 1 of the present invention in the inflammation model of mice induced by lipopolysaccharide.

The present invention relates to a composition for the prevention and treatment of inflammatory, allergic and atopic diseases containing gamma linolenic acid complex as an active ingredient.

Atopic dermatitis is a chronic chronic dermatitis that causes severe pruritus. Although its cause has not been clearly identified to date, it is estimated that 10-20% of the world's population suffers from this dermatitis. In addition, the prevalence of atopic dermatitis continues to increase in recent decades due to an increase in allergens that cause airborne allergens due to environmental pollution, and an increase in conditions for forming dry skin. Similarly in Korea, according to a nationwide epidemiological study conducted by the Korean Pediatric Allergy and Respiratory Society in 1995, 12-24% of elementary school students and 6-8% of middle school students were diagnosed with atopic dermatitis. In 2000, the number of elementary school students increased by 24.9% and middle school students by 12.8%. Furthermore, the incidence of adult atopic dermatitis in Korea is increasing.

Atopic dermatitis was first noted in 1925 when Coca described atopic dermatitis, asthma, and fever as a result of allergic reactions to food and inhalable substances. Such atopic diseases include asthma, allergic rhinitis, allergic conjunctivitis, etc. in addition to dermatitis.

Atopic disorders generally involve allergic reactions, atopic dermatitis, chronic or recurrent dermatitis, with a genetic predisposition with the characteristic shape and distribution of skin lesions and a personal or family history of atopic dermatitis. In other words, atopic dermatitis is a very common skin disease that occurs when born. Most infants or infants begin in the form of a stool, especially around 2 months of age, with about 50% occurring before 2 years of age and showing symptoms before 5 years of age. It is known that the first symptoms of adulthood are rare, and in most cases, symptoms are alleviated or disappeared with growth, and half of the infants who develop infants improve before 2 years of age.

This atopic dermatitis begins with erythematous papules and scales with severe itching and progresses to acute symptoms with blisters and serous extracts and thick crusts. The most characteristic symptom is pruritus, which is so severe that it scratches the affected area to bleed. Symptoms worsen by repeated itching and scratching, and if sustained, lichenifi cation may occur, resulting in thickening of the skin and wrinkling. Most clinical manifestations are the result of scratching or rubbing, and at night, pruritus worsens, causing sleep disorders.

Currently, the pathophysiology of atopic dermatitis is not fully understood. So far, it has been confirmed that chemical media such as histamine, lukotrien, and prostaglandin, which induce inflammation in atopic patients, have been increased in the blood. An increase in globulin E (IgE) is also observed. Furthermore, in the distribution of immune cells, it can be found that the balance between T _H 1 and T _H 2, which are subtypes of T cells, is inclined toward T _H 2. Another characteristic of patients with atopic dermatitis is that the content of γ-linolenic acid due to functional defects of delta- ^6- desaturase found in the fatty acid synthesis pathway is lower than that of normal people. Appears.

Gammalinolenic acid (chemical name; cis, cis, cis-6,9,12-octadecatrienoic acid, molecular formula; C ₁₈ H ₃₀ O ₂ ) is a metabolite of linoleic acid, an essential fatty acid in vivo, in the delta-6- It is formed from linoleic acid by disetase. The gamma-linolenic acid thus formed is converted to dihomo-γ-linolenic acid by elongase and to arachidonic acid by delta- ^5- desaturase. Is switched. Converted arachidonic acid is converted to 15-HETrE and PGE1 by lipoxygenase and cyclooxygenase. Converted 15-HETrE and PGE1 have been shown to have anti-inflammatory effects (Ziboh et al., Am. J. Clin. Nutr. 71: 361S-366S, 2000). Thus, deficiency of gamma linolenic acid reduces the production of substances with anti-inflammatory effects such as 15-HETrE and PGE1, resulting in increased production of inflammatory mediators, resulting in abnormalities in the skin lipid membrane structure. From this, it has been found that the proper supply of gamma linolenic acid in vivo is very important for the improvement and treatment of atopic diseases, especially atopic dermatitis diseases.

As the significance of the gamma linolenic acid in the prevention and treatment of atopic dermatitis diseases is revealed, a clinical study of atopic dermatitis diseases using gamma linolenic acid has been conducted. As a result, the diet of gamma linolenic acid in children with atopic dermatitis did not show a cure for atopic dermatitis, but a gradual improvement was observed in erythema, tachycardia and flaky skin, and the effect of itching was similar to that of antihistamines. The level was improved. On the other hand, no adverse effects on gamma linolenic acid were observed (Fiocchi et al., J. Int. Med. Res. 22: 24-32, 1994).

Another clinical study showed that dietary gamma-linolenic acid in patients with atopic dermatitis improved significantly from pruritus, erythema, vesicle formation, eczema, etc. (Andreassi et al., J. Int. Med). Res. 25: 266-274, 1997).

This improvement was also observed in children with dietary borage oil containing gamma linolenic acid. Although it does not prevent the development of atopic dermatitis itself, it has been reported that it is effective to alleviate the symptoms of atopic dermatitis after a little more growth of children fed borage oil (van Gool et al., Am. J. Clin). Nutr. 77: 943-951, 2003).

From the above results, it was confirmed that the diet of gamma linolenic acid is effective in alleviating atopic dermatitis diseases, and increased the applicability of gamma linolenic acid to atopic diseases.

However, despite the excellent efficacy of gamma linolenic acid, various applications of gamma linolenic acid have been accompanied by certain limitations due to the physicochemical properties of gamma linolenic acid itself. Since gamma linolenic acid is present in the form of fat-soluble lipids (eg, fatty acids, triglycerides, fatty acid esters, etc.) that are not soluble in water, there is a problem that direct injection into blood is difficult. For this reason, it has been mainly absorbed or drinking in the body in the form of soft capsules.

In order to solve the above problems, many studies on the water-soluble lipidation of fatty acids have been conducted. Here are some of them.

First, the chloride method using an alkali metal salt (Na, K, Ca, etc.) is applied. However, since the method exists in the form of a strong basic aqueous solution, there is a problem in applying to the human body due to its toxicity and irritation.

Next, the microencapsulation method using an emulsifier and dextrin, milk protein. However, this method is accompanied by the limitation that the emulsifier should be bio-friendly and excellent solubility, and at the same time there is a problem that high stability to the environment such as heat and light is required.

In addition, Korean Patent No. 34895 discloses a method of increasing the oxidation stability of unsaturated fatty acids by mixing saturated hydrocarbon oil, glycerides of saturated fatty acids, esters of lower alcohols and saturated fatty acids, and the like. In addition, Korean Patent Laid-Open Publication No. 2001-0044622 discloses a method for repairing a skin barrier by applying a lipid complex of ceramide, essential fatty acids and cholesterol in connection with treating atopic skin diseases using lipids, and in 2000-0046633 The administration or application of gamma linolenic acid is disclosed by a composition containing a fatty acid, gamma linolenic acid and one or more of its precursors as an active ingredient. However, these technologies have problems such as increased peroxides due to automatic oxidation of lipids, instability of water-soluble state, etc. when applied to foods, cosmetics and pharmaceuticals.

Korean Patent Application No. 10-2003-30853 discloses a water-soluble lipid complex containing a basic amino acid and a preparation method thereof. However, this method is a reaction between unsaturated lipids and basic amino acids, which is limited in that all amino acids existing in nature such as acid, neutral, aromatic except basic amino acids cannot directly react with unsaturated lipids (particularly fatty acids). have. In addition, even if the lipid complex prepared by the above method has excellent water absorption, since the basic amino acid is used, the problem of human irritation as in the above-mentioned chloride method using alkali metals is raised.

Accordingly, the present inventors have completed the present invention by preparing a gamma linolenic acid complex with improved physicochemical properties in view of the above problems, and providing a composition having anti-inflammatory, anti-allergic and anti-atopic efficacy containing it as an active ingredient. .

The present invention is to provide a composition for the prevention and treatment of inflammatory, allergic and atopic diseases containing gamma linolenic acid complex as an active ingredient.

The present invention provides a composition for the prevention and treatment of inflammatory, allergic and atopic diseases containing gamma linolenic acid complex as an active ingredient.

Compositions of the present invention include pharmaceutical compositions and food compositions.

Hereinafter, the present invention will be described in detail.

The gamma linolenic acid complex of the present invention can be usefully used in medicine, cosmetic foods, etc. as a therapeutic agent or therapeutic adjuvant for atopic dermatitis by restoring the skin protective barrier and suppressing the inflammatory response by supplying gamma linolenic acid, which is insufficient for atopic skin diseases. Can be. Examples of such diseases include atopic dermatitis, allergic dermatitis, eczema, and dryness.

As a raw material of the gamma linolenic acid complex of the present invention, borage oil, evening primrose seed oil, black currant flower seed oil and the like may be used. In the present invention, it is separated and purified so that gamma linolenic acid is contained in high purity. Thus separated and purified gamma-linolenic acid exists in the form of fat-soluble unsaturated lipids, it is easy to take and absorb in the body through hydrogen bonds and ester exchange reactions with substances such as amino acids, sugars, and synergistic gamma-linolenic acid complex Switch. Specifically, the method is converted to the gamma linolenic acid complex of the present invention using a method using a catalyst and a method using an emulsifier.

First, a method of converting gamma linolenic acid to gamma linolenic acid complex using a catalyst,

1-1) adding gamma-linolenic acid to pyridine to dissolve completely, and thionyl chloride was injected into the mixture to prepare gamma-linolenic acid-chloride derivative,

1-2) adding the gamma linolenic acid-chloride derivative prepared in 1-1) to a solution in which anhydrous ethanol and amino acid are dissolved, and reacting in the presence of a catalyst to form a gamma linolenic acid complex, and

1-3) The solution prepared in 1-2) is vacuum-dried to remove ethanol, and put in water to solubilize, followed by recovering the gamma linolenic acid complex using a lyophilizer.

Next, the method for converting gamma linolenic acid to gamma linolenic acid complex using the emulsifier according to the present invention,

2-1) maintaining gamma-linolenic acid and water at a temperature of 4 ° C ~ -1 ° C and emulsifying by adding an emulsifier while stirring at a stirring speed of 3,000 ~ 7,000 rpm,

2-2) adding an amino acid to the emulsion solution prepared in step 2-1) to form a gamma linolenic acid complex, and

2-3) recovering the gamma linolenic acid complex using the solution prepared in 2-2) using a lyophilizer.

In addition, the method of converting gamma linolenic acid to gamma linolenic acid complex according to the present invention includes both a method using a catalyst and a method using an emulsifier,

3-1) adding gamma linolenic acid, water and ethanol, sodium hydroxide, and phosphoric acid to form two phases,

3-2) removing the lower layer solution formed in step 3-1), adding pyridine to the upper layer solution to completely dissolve it, and dissolving thionyl chloride therein to prepare a gamma linolenic acid chloride derivative,

3-3) putting the gamma-linolenic acid-chloride derivative prepared in step 3-2) into anhydrous ethanol, maintaining the temperature at 4 ° C to -1 ° C, and emulsifying by adding an emulsifier while stirring at a stirring speed of 3,000 to 7,000 rpm. ,

3-4) adding an amino acid to the emulsion solution prepared in step 3-3) and reacting in the presence of a catalyst to form a gamma linolenic acid complex, and

3-5) The solution prepared in 3-4) is vacuum-dried to remove ethanol, and then put in water to solubilize, followed by recovering the gamma linolenic acid complex using a lyophilizer.

The above methods will be described in detail step by step.

In step 1-1), gamma linolenic acid is completely dissolved in pyridine, and partial injection of thionyl chloride is performed in small portions at a temperature of 4 ° C. If small amounts of infusion are not performed, the reaction proceeds violently and the rancidity of gamma-linolenic acid cannot be avoided. When the mixture is stirred while injecting thionyl chloride for about 40 minutes, crystals are formed. The mixture is allowed to stand at room temperature for about 2 hours, followed by filtration to obtain gammalinolenic acid-chloride derivatives as crystals. In the present invention, the activity of the reaction is increased by forming gamma linolenic acid-chloride derivatives without directly reacting gamma linolenic acid with amino acids.

In step 1-2), the gammalinolenic acid-chloride derivative prepared in step 1-1) is added to a solution in which anhydrous ethanol and amino acid are dissolved and reacted at 4 ° C. Pyridine and thionyl chloride (1: 1) are added as catalysts. When the catalyst is added, the solid particles present in the solution are dissolved to form an amino acid-gammarinolenic acid complex.

In the step 1-3), the reaction is terminated when the reaction product prepared in the 1-2 ) step is clear, and then the solution is ethanol removed using a vacuum rotary evaporator and water is solubilized after freezing. The gamma linolenic acid complex is recovered using a dryer.

In step 2-1), gamma linolenic acid and water is maintained at a temperature of 4 ℃ ~ -1 ℃, more preferably 4 ℃, while stirring at a stirring speed of 3,000 ~ 7,000 rpm, more preferably 5,000 rpm or more The emulsifier is emulsified to increase the complete dissolution and stability of the gamma linolenic acid. When the emulsifier is added, the particle size of the lipid is reduced to a very fine nanoparticle size (50 nm or less), thereby minimizing the interfacial tension and maximizing the surface area that can be reacted, resulting in a reaction conversion rate of 99% or more. The emulsifiers used here include 1-3 wt% lecithin, 1-3 wt% sucrose fatty acid ester, and 0.02-0.08 wt% ascorbic acid, preferably 2 wt% lecithin, 2 wt% of the total weight of the solution. Preference is given to using% sucrose fatty acid ester and 0.05% by weight of ascorbic acid.

In step 2-2), the amino acid is slowly added to the emulsion solution prepared in step 2-1) to prepare a gamma linolenic acid complex. The reason why the amino acid is injected slowly is that when a large amount is injected at once, the reaction product becomes viscous due to a sudden reaction and smooth stirring is impossible. If the reaction proceeds by injecting a large amount of amino acid at one time, the reaction proceeds to an instantaneous solidification state such as jelly, and thus it is not possible to induce a complete conversion to a water-soluble lipid state. Toxic problems in the body will be inevitable.

In step 2-3), the gamma linolenic acid complex is recovered using the solution prepared in 2-2) using a lyophilizer.

The amino acids used in the method of converting gamma linolenic acid to gamma linolenic acid complex according to the present invention is isoleucine (Isoleucine), leucine (Leucine), lysine (Lysine), phenylalanine (Phenylalanine), Methionine (Threonine), tryptophan Essential amino acids consisting of Trytophane, Valine, and Alanine, Arginine, Asparagine, Cysteine, Glutamine, Histidine, Proline, Serine And one or more selected from non-essential amino acids consisting of (Serine), tyrosine (Tyrosine), glycine (Glycine), but is not limited thereto.

The gamma linolenic acid complex according to the present invention exhibits superior antioxidant activity, inhibitory effect on tumor necrosis factor-α (TNF-α) production, inhibitory effect on PGE-2 (prostaglandin E-2) production, and inflammation compared to gamma linolenic acid oil. Thus, the gamma linolenic acid complex of the present invention can be used for the prevention and treatment of inflammatory, allergic or atopic diseases.

The content of gammalinolenic acid complex in the pharmaceutical composition according to the present invention is preferably 5 to 70%, more preferably 20 to 70% by weight of the total composition, depending on the dosage form. If it exceeds 70%, the improvement and treatment effect of atopic skin disease is no longer enhanced, while if it is less than 5%, it does not show an excellent effect compared to the use without converting to gammalinolenic acid complex.

The pharmaceutical composition containing the gamma linolenic acid complex according to the present invention as an active ingredient may be used in the form of a general pharmaceutical preparation. That is, the gamma linolenic acid complex of the present invention may be administered in various formulations, oral and parenteral, in actual clinical administration. In addition, when formulated, in addition to the gammalinolenic acid complex as an active ingredient, it may be prepared by including one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added.

Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, which may include at least one excipient such as starch, calcium carbonate, gamma linolenic acid complex, Sucrose (Lucose) or lactose (Lactose), gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

In addition, liquid preparations for oral administration include suspending agents, liquid solutions, emulsions, and syrups, and various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. This may include. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Furthermore, the pharmaceutical compositions of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. To formulate into a parenteral formulation, the gammalinolenic acid complex is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

In addition, the dosage or dosage of the pharmaceutical composition according to the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the patient, If the doctor decides to take 250-3000 mg based on the adult at regular intervals once a day or divided into several times.

The food composition of the present invention may be added to a health food for the purpose of preventing and treating inflammatory, allergic or atopic diseases. When the gamma linolenic acid complex of the present invention is used as an additive, the gamma linolenic acid complex may be added as it is, or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the gamma nolenic acid complex of the present invention is added in an amount of 1 to 20% by weight, preferably 5 to 10% by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition, the compositions of the present invention include various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Furthermore, the gamma linolenic acid complex of the present invention improves the safety and toxicity problems of gamma linolenic acid according to the acid and basicity, and increases the stability to environmental factors such as heat, air, and light, and at the same time absorbs water and water to the human body. By greatly increasing the solubility in the body, it is easy to take and absorb in the body. Therefore, the composition comprising the gamma linolenic acid complex of the present invention as an active ingredient can be widely applied as a constituent of functional cosmetics such as water-soluble creams and lotions, and bath products, soaps and the like.

Hereinafter, the present invention will be described in detail by Examples and Comparative Examples. However, the following Examples are merely to illustrate the invention, the present invention is not limited by the following Examples.

Example 1 Preparation of Gamma-linolenic Acid Complex

<Separation and purification of gamma linolenic acid>

Gamma-linolenic acid for preparing gamma-linolenic acid complex was isolated and purified from evening primrose or purple flower seed oil. For this purpose, the urea crystallization method was used in the present invention.

First, the seed oil was added to an aqueous 70% ethanol-urea solution, and then the temperature was adjusted to 25 to 30 ° C. to form urea crystals, thereby collecting gamma linolenic acid in the urea crystals. Water and hexane were added to the thus formed crystals in a weight ratio of 1: 1, and a small amount of hydrochloric acid was added to the resulting crystals to obtain a fatty acid including hexane in the upper portion. Hexane was removed from this to give gamma-linolenic acid concentrated at a yield of 60-99%.

<Production of Gammalinolenic Acid Complex>

The gamma linolenic acid complex of the present invention was prepared by a method using an emulsifier among the methods for converting gamma linolenic acid to gamma linolenic acid complex.

To 100 g of gamma linolenic acid (or Borage flower seed oil) obtained through the separation and purification process, 300 g of a mixed solvent of water and ethanol (equivalent to weight ratio) was added, and 40 g of sodium hydroxide was added while stirring at 3000 rpm. Thereafter, 60 g of phosphoric acid was added to form two phases and the lower layer liquid was removed. 1 of water was added to the supernatant liquid at a temperature of 4 ° C., and stirred at 5000 rpm to emulsify by adding 2% lecithin, 2% sucrose fatty acid ester, and 0.05% ascorbic acid. 50 g of glycine, phenylalanine, lysine and other amino acid mixture powder were slowly added, and then the reaction conditions were maintained at 4 ° C. and 5000 rpm until a uniform phase was obtained. The reaction was terminated when the reaction became clear, the solution was dried using a lyophilizer, and the gamma linolenic acid composite powder was recovered.

In order to confirm the recovered gamma linolenic acid complex, it was analyzed by infrared spectroscopy and the results are shown in FIG. 1. As shown in FIG. 1, the peaks of the carboxyl group ions at 1560 cm ⁻¹ and 1395 cm ⁻¹ of the recovered gamma linolenic acid complex were confirmed. This is a peak not observed when fatty acids are present alone, and it can be seen that the recovered gammalinolenic acid is in the form of a fatty acid-amino acid salt.

Experimental Example 1 Physicochemical Properties of Gammalinolenic Acid Complex

<Confirmation of water solubility>

In order to determine whether the gamma linolenic acid complex is water-soluble, the concentration of gamma linolenic acid complex powder prepared by the preparation method according to Example 1 was dissolved in water at 0.1, 1.0, and 2.0%, respectively, and gamma linolenic acid oil was used as a control. And the powder was dissolved. The turbidity change was observed at room temperature for two months, and the results are shown in FIG. 2.

As shown in Figure 2, unlike the gamma linolenic acid oil and powder used as a control, the turbidity change and the formation of precipitates were not observed in the aqueous solution in which the three concentrations of gamma linolenic acid complexes were dissolved. From this, it can be seen that the gamma linolenic acid complex of the present invention is water-soluble.

<Confirmation of Oxidation Stability>

Peroxide value is generally useful as an indicator of fat stability and oxidative damage. From this, in order to confirm the oxidation stability of the gamma linolenic acid composite, the gamma linolenic acid composite powder prepared by the preparation method according to Example 1 was heat-treated at 100 ° C. for 96 hours. In order to measure the peroxide value, 1 g of gamma linolenic acid composite powder was dissolved in 10 ml of acetic acid: chloroform (3: 2), mixed in 250 µl of saturated potassium iodide solution, and left for 1 minute. Thereafter, 10 ml of water was added to the mixture, and then 500 µl of starch reagent was used as an indicator and titrated with 0.01 N sodium thiosulfate solution to measure peroxide value. Gammalinolenic acid oil was used as a control, and the measured peroxide value is shown in FIG. 3.

As shown in FIG. 3, the peroxide value of the gamma linolenic acid complex according to Example 1 was almost changed despite the heat treatment for 96 hours compared to the peak at 10 hours in the case of gamma linolenic acid oil, which increased with time. Turned out to be invisible. This shows that the oxidation stability of the gamma linolenic acid composite of the present invention is excellent.

Experimental Example 2 Physiological Characteristics of Gamma-linolenic Acid Complex

Antioxidant Effect

Skin cells are easily exposed to the attack of radicals produced by a variety of factors. Oxidative damage by these radicals has recently been recognized as a very important factor of the disease. Radical scavenging ability was measured by ABTS (2,2'-azinobis- (3-ethylbenzothiazoline-6-sulfonate) radical assay (Kim et al., J. Agric. Food Chem. 50: 3713-3717, 2002).

1 mM and 10 mM of gamma linolenic acid oil, 1 mM and 10 mM of gamma linolenic acid complex recovered from Example 1 were prepared, and a phosphate buffer solution was used as a control. About them, the amount of remaining ABTS was measured over time using the ABTS radical measurement method, and the results are shown in FIG. 4.

As shown in FIG. 4, the radical scavenging ability of the gammalinolenic acid oil and the gammalinolenic acid complex of Example 1 was both dependent on concentration. In particular, the gamma linolenic acid complex showed stronger antioxidant activity than gamma linolenic acid oil at each concentration. This shows that the oxidation stability of the gamma linolenic acid composite of the present invention is excellent.

<TNF-α (tumor necrosis factor-α) production inhibitory effect>

TNF-α inhibits bacterial invasion in the immune response by activating neutrophils, but in atopic diseases caused by excessive immune responses, excessive TNF-α production / secretion amplifies the inflammatory response. Control of production / secretion of TNF-α for the diseased is required. In order to investigate the inhibitory effect on the production / secretion of TNF-α of the gamma linolenic acid complex, the following experiment was performed.

Gamma linolenic acid oil and gamma linolenic acid complex recovered from Example 1 were treated with mouse macrophages with lipopolysaccharide (LPS, respectively) and incubated for 20 hours. LPS plays a role in activating macrophages. The amount of TNF-α secreted in the culture supernatant was measured using the mouse TNF-α ELISA kit (Amersham pharmacia, USA) according to the following method to determine the inhibitory effect of TNF-α production. That is, the culture supernatant was dispensed in a well-plate attached with an anti-mouse TNF-α antibody and reacted overnight at 4 ° C, and the unreacted antibody was washed with a phosphate buffer solution containing 0.2% Tween-20. After adding the biotinylated anti-TNF-α antibody solution, the mixture was reacted at 37 ° C. for 30 minutes and then washed as described above. Again, streptavidin-horseradish peroxidase antibody solution was added, followed by reaction at 37 ° C for 30 minutes. After the reaction was completed, the reaction was washed as described above and the color reaction was induced by adding TMB (tetramethylbenzidine) substrate, the reaction was stopped by adding 2N sulfuric acid solution, and the absorbance was measured and confirmed at 450 nm. Indicated. At this time, the concentration of TNF-α was calculated by creating a calibration curve using a standard product.

As shown in FIG. 5, both gamma linolenic acid oil and gamma linolenic acid complex recovered from Example 1 resulted in inhibiting the production of TNF-α. In particular, the gamma linolenic acid complex of Example 1 showed a stronger TNF-α production inhibitory effect as compared to gamma linolenic acid oil. From this, it can be seen that the gamma linolenic acid complex of the present invention has an excellent inhibitory effect on TNF-α production.

<PGE-2 (prostaglandin E-2) production inhibitory effect>

PGE-2 is a lipid inflammatory mediator that is biosynthesized from arachidonic acid, a lipid that makes up cells, catalyzed by a prostaglandin synthase or an enzyme called cyclooxygenase-2 (COX-2). Therefore, the following experiment was conducted to investigate the inhibitory effect of the gamma linolenic acid complex on the production of these inflammatory mediators PGE-2.

Gamma linolenic acid oil and gamma linolenic acid complex recovered from Example 1 were treated with mouse macrophages with lipopolysaccharide (LPS, respectively) and incubated for 20 hours. The amount of PGE-2 secreted in the cultured supernatant was measured at 450 nm using a mouse PGEE-2 ELISA kit (Amersham pharmacia, USA) and the results are shown in FIG. 6. At this time, the concentration of PGE-2 was calculated by preparing a calibration curve using a standard product.

As shown in FIG. 6, both gamma linolenic acid oil and gamma linolenic acid complex recovered from Example 1 resulted in inhibiting the production of PGE-2. In particular, it was confirmed that the gamma linolenic acid complex of Example 1 exhibited a stronger inhibitory effect on PGE-2 production than gamma linolenic acid oil, indicating that the PGE-2 production inhibitory effect of the gamma linolenic acid complex of the present invention was superior to gamma linolenic acid oil. Can be.

Inflammatory inhibitory effect

The following experiment was performed to investigate the inhibitory effect of the gamma linolenic acid complex using BALB / C mice 6-8 weeks old.

Ten BALB / C mice were prepared per experimental group and were acclimated in the kennel for 1 week before the experiment. Mice were fasted for 12 hours before the experiment, excluding the experimental interference factor, and orally administered gammalinolenic acid complexes at 0.5 and 1.0 g / kg per mouse weight. The control group was administered the same amount of saline. Thirty minutes after oral administration, anesthesia was performed with ketamine. After 5 minutes of anesthesia, intraperitoneal injection of 40 mg / kg of LPS induced inflammation, and the survival rate was measured at regular intervals up to 48 hours to confirm the difference in survival rate for the control group as an anti-inflammatory effect.

As shown in Figure 7, the group administered 0.5 and 1.0 g / kg of gamma linolenic acid complexes showed an inhibitory effect of 20% and 30%, respectively, compared to the control group administered only saline. From this, it can be seen that the inflammatory inhibitory effect of the gamma linolenic acid complex of the present invention is excellent.

Single oral dose toxicity test

In order to confirm the safety of the gamma linolenic acid complex of the present invention, a single oral dose toxicity test was performed. The gamma-linolenic acid complex was dissolved in physiological saline at different concentrations of 0.1, 0.5, 1.0, and 2.0 g / kg, respectively, and orally administered to mice (10 mice per group), and then observed for one week. Shown in

As shown in Table 1, no mice died in all experimental groups administered gamma-linolenic acid. From this it can be seen that the gamma linolenic acid complex of the present invention does not cause toxicity.

On the other hand, the compound according to the invention can be formulated in various forms depending on the purpose. The following are some examples of formulation methods containing the compound according to the present invention as an active ingredient, but the present invention is not limited thereto.

Preparation Example 1 Preparation of Pharmaceutical Formulation

1. Preparation of powder

2 g of gamma linolenic acid complex of the present invention

1g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

100 mg of gamma linolenic acid complex of the present invention

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

100 mg of gamma linolenic acid complex of the present invention

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Preparation of Injection Solution

1 mg / ml gamma linolenic acid complex of the present invention

Injectable sodium chloride BP up to 4 ml

The gammalinolenic acid complex of the present invention was dissolved in an appropriate volume of sodium chloride BP for injection, and the volume was adjusted using a sodium chloride BP for injection and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Preparation Example 2 Preparation of Food

Food comprising the gamma linolenic acid complex of the present invention was prepared as follows.

1. Preparation of Cooking Seasonings

Gamma-linolenic acid complex 0.2 ~ 10% by weight of the present invention was prepared for cooking spices for health promotion.

2. Preparation of Tomato Ketchup and Sauce

The gamma linolenic acid complex of the present invention was added to 0.2 ~ 1.0% by weight tomato ketchup or sauce to prepare a health promotion tomato ketchup or sauce.

3. Manufacturing of Flour Foods

0.1 to 5.0% by weight of the gamma linolenic acid complex of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare foods for health promotion.

4. Preparation of soups and gravy

Gamma-linolenic acid complex of the present invention was added to 0.1 ~ 1.0% by weight to soup and broth to prepare a health-promoting meat products, noodles soup and gravy.

5. Preparation of Ground Beef

10% by weight of the gamma linolenic acid complex of the present invention was added to ground beef to prepare a ground beef for health promotion.

6. Manufacture of Dairy Products

0.1 to 1.0% by weight of the gamma linolenic acid complex of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The gamma linolenic acid composite of the present invention was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

Cereals, seeds and the dry powder of the gamma linolenic acid composite of the present invention prepared in the above ratio was prepared.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder (1% by weight) of the gamma linolenic acid complex of the present invention,

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Preparation Example 3 Preparation of Beverage

1. Preparation of Carbonated Drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed, and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilizing at 20 ℃ for 180 seconds, mixed with a cooling water in a ratio of 1: 4, and then injected with 0.5 to 0.82% carbon dioxide gas to prepare a carbonated beverage containing the gamma linolenic acid complex of the present invention.

2. Manufacture of health drinks

After sterilization by homogeneously mixing the component materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and the gamma linolenic acid complex of the present invention This was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.

3. Preparation of Vegetable Juice

0.5 g of the gamma linolenic acid complex of the present invention was added to 1,000 ml of tomato or carrot juice to prepare a vegetable juice for health promotion.

4. Preparation of Fruit Juice

0.1 g of the gamma linolenic acid complex of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.

Since the gamma linolenic acid complex of the present invention is excellent in water solubility, and particularly excellent in antioxidant activity and anti-inflammatory effect, it is possible to improve and restore skin barrier by supplementing deficiency of gamma linolenic acid commonly found in atopic patients, and to mediate inflammation parameters. By inhibiting the production and secretion of these to exhibit an anti-inflammatory, anti-allergic effect, etc. can be useful as a pharmaceutical composition as a preventive and therapeutic agent for atopic dermatitis and skin diseases associated with atopic dermatitis.

Claims (4)

To gamma linolenic acid, a mixed solvent of water and ethanol, sodium hydroxide and phosphoric acid are sequentially added to form two phases, Removing the lower layer formed in the above step, completely dissolving pyridine in the upper layer, and dissolving thionyl chloride thereinto prepare a gammalinolenic acid-chloride derivative, The gamma linolenic acid-chloride derivative prepared in the above step was put in anhydrous ethanol, and emulsified by adding 1 to 3% by weight of lecithin, 1 to 3% by weight of sucrose fatty acid ester, and 0.02 to 0.08% by weight of ascorbic acid as an emulsifier. Steps, Gradually adding amino acids to the emulsion solution prepared in the step to form a gamma linolenic acid complex, and Vacuum drying the solution prepared in the above step to remove ethanol, water-solubilized, and then recovering the gamma linolenic acid complex powder using a lyophilizer. Method of producing a gamma linolenic acid complex comprising a. delete delete delete

Images