JP2011231029A - Enzyme inhibitor and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme inhibitor using cholesterol-highly unsaturated fatty acid ester obtained by combining cholesterol with highly unsaturated fatty acid as an effective component, a method for producing the enzyme inhibitor, and skin care preparations, cosmetics, foods, medicines or the like containing the enzyme inhibitor.SOLUTION: The enzyme inhibitor includes cholesterol-highly unsaturated fatty acid ester obtained by combining cholesterol; and one or more kinds of highly unsaturated fatty acids selected from docosahexaenoic acid, eicosapentaenoic acid and docosapentaenoic acid as an effective component. The enzyme inhibitor is incorporated into skin care preparations, cosmetics, foods, medicines or the like.

Description

  The present invention relates to an enzyme inhibitor comprising a cholesterol highly unsaturated fatty acid ester obtained by synthesizing cholesterol and a highly unsaturated fatty acid as an active ingredient. The present invention also relates to a method for producing the enzyme inhibitor. Furthermore, this invention relates to the skin external preparation, cosmetics, foodstuffs, pharmaceuticals, etc. containing the said enzyme inhibitor.

  Hyaluronic acid is one of the matrix components widely present in mammalian connective tissues, and is distributed in human skin, joint fluid, vitreous body of eye, umbilical cord and the like. Hyaluronic acid functions include cell retention, skin retention, joint lubrication, and the like. In vivo, hyaluronic acid is turned over in a relatively short period of time, and the activity of hyaluronic acid synthase and degrading enzyme is normally balanced. However, in general, this balance is lost with aging, hyaluronidase activity, which is a hyaluronic acid-degrading enzyme, is increased with respect to the synthesis of hyaluronic acid, tissue flexibility and wettability are lost, skin wrinkles, tarmi, etc. Causes senile changes.

  Hyaluronidase itself has an action as a flame retardant, and its enzyme activity is inhibited by anti-inflammatory agents and anti-allergic agents. As for the hyaluronidase inhibitor, antiallergic agents have been developed by testing the hyaluronidase inhibitory activity. For example, reports on the hyaluronidase inhibitory effect of natural products and their antiallergic activity (see, for example, Non-Patent Document 1) There is.

  As a hyaluronidase inhibitor, a safe and effective substance is desired from the viewpoint of skin aging prevention, anti-inflammatory and anti-allergic activity.

Regarding the synthesis of esters of cholesterol and fatty acids, for example, a method for producing a sterol fatty acid ester, wherein a compound having a steroid skeleton and a hydroxyl group in a molecule is contacted with a fatty acid using lipase or cholesterol esterase ( For example, see Patent Document 1.
Also, for example, using a plant-derived sterol composition and oils and fats mainly composed of triacylglycerol as raw materials, an enzyme synthesis reaction of sterol fatty acid ester with lipase or cholesterol esterase as an enzyme having lipolytic activity is performed, and a series of purification steps There is a method for producing a sterol fatty acid ester excellent in safety and functional aspects (for example, see Patent Document 2).

Regarding the chemical synthesis of esters of cholesterol and fatty acids, for example, a method of reacting fatty acid chloride with cholesterol or a method of heating cholesterol with fatty acids, and a composition containing the ester for pharmaceutical, skin care or nutritional composition It is shown to be used as a product (see, for example, Patent Document 3).
For applications using cholesterol fatty acid esters, for example, there is an emulsion-type aqueous cosmetic in which solid fat containing cholesterol fatty acid esters is dispersed in an aqueous medium by an emulsifier and a method for producing the same (for example, see Patent Document 4). is there.

  However, there is no specific disclosure about synthesizing cholesterol and polyunsaturated fatty acids to make a hyaluronidase inhibitor, and there is no practical example.

JP-A-61-204197 JP 2002-171993 A JP-A-6-234644 Japanese Patent Laid-Open No. 4-112810

Hisao Kakegawa, et al, Chem. Pharm. Bull. 40, (6) 1439-1442 (1992)

  As described above, synthesis methods and uses of sterol fatty acid esters and cholesterol fatty acid esters have been disclosed, but hyaluronidase inhibitors containing cholesterol highly unsaturated fatty acid esters as active ingredients, cholesterol and highly unsaturated fatty acids are synthesized. There is no specific disclosure about the production method of hyaluronidase inhibitor to be purified and its application, and no practical examples are found.

  An object of the present invention is to provide a hyaluronidase inhibitor containing a cholesterol highly unsaturated fatty acid ester obtained by synthesizing cholesterol and a highly unsaturated fatty acid as an active ingredient. Moreover, an object of this invention is to provide the manufacturing method of the said enzyme inhibitor.

An object of the present invention is to provide an external skin preparation for preventing skin aging and / or for anti-allergy, which contains a hyaluronidase inhibitor containing a cholesterol highly unsaturated fatty acid ester as an active ingredient.
Furthermore, an object of the present invention is to provide cosmetics, foods, pharmaceuticals and the like containing a hyaluronidase inhibitor containing cholesterol highly unsaturated fatty acid ester as an active ingredient.

  As a result of earnest research focusing on substances having an enzyme inhibitory effect, the present inventors have found that cholesterol highly unsaturated fatty acid esters obtained by synthesizing cholesterol and highly unsaturated fatty acids have hyaluronidase inhibitory activity. Thus, the present invention has been completed in which the enzyme inhibitor can be effectively used in external preparations for skin, cosmetics, foods, pharmaceuticals and the like.

The present invention includes the following aspects.
Item (1)
A hyaluronidase inhibitor characterized by comprising a cholesterol highly unsaturated fatty acid ester obtained by synthesizing cholesterol and a highly unsaturated fatty acid as an active ingredient.
Item (2)
The hyaluronidase inhibitor according to item (1), wherein the highly unsaturated fatty acid is one or more of eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid.
Item (3)
The hyaluronidase inhibitor according to item (1) or item (2), wherein the content of the cholesterol highly unsaturated fatty acid ester in the hyaluronidase inhibitor is 60% or more.
Item (4)
A skin external preparation for skin aging prevention and / or antiallergy, comprising the hyaluronidase inhibitor according to any one of Items (1) to (3).
Item (5)
A food containing the hyaluronidase inhibitor according to any one of Items (1) to (3).
Item (6)
Using cholesterol and polyunsaturated fatty acids as raw materials, and performing a synthesis reaction in a temperature-controlled system;
A step of removing fats and oils mainly containing unreacted cholesterol and highly unsaturated fatty acids by molecular distillation as a first purification step;
A step of mainly removing pigment components and odor by adsorbent treatment with an adsorbent composed of activated carbon, activated clay or silica as the second purification step;
The manufacturing method of the hyaluronidase inhibitor which uses the cholesterol polyunsaturated fatty acid ester containing this as an active ingredient.
Item (7)
The method for producing a hyaluronidase inhibitor according to item (6), wherein the highly unsaturated fatty acid is one or more of eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid.
Item (8)
Item 8. The method for producing a hyaluronidase inhibitor according to item (6) or (7), wherein the content of the cholesterol highly unsaturated fatty acid ester in the hyaluronidase inhibitor is 60% or more.

  The present invention provides a hyaluronidase inhibitor comprising as an active ingredient a cholesterol highly unsaturated fatty acid ester obtained by synthesizing cholesterol and a highly unsaturated fatty acid, and a method for producing the same, and the hyaluronidase inhibitor includes: It has excellent hyaluronidase inhibitory activity, excellent oxidative stability, and can be effectively used in skin aging prevention and / or antiallergic skin preparations, cosmetics, foods, pharmaceuticals, etc. .

  In addition, the present invention effectively uses cholesterol polyunsaturated fatty acid esters obtained by synthesizing cholesterol and one or more polyunsaturated fatty acids of eicosapentaenoic acid, docosahexaenoic acid, or docosapentaenoic acid. The present invention provides a hyaluronidase inhibitor as a component and a method for producing the same, and the hyaluronidase inhibitor has excellent hyaluronidase inhibitory activity, excellent oxidation stability, and the hyaluronidase inhibitor is used for preventing skin aging and / or. It can be effectively used in anti-allergic skin external preparations, cosmetics, foods, pharmaceuticals and the like.

  As for polyunsaturated fatty acids, eicosapentaenoic acid and docosahexaenoic acid, etc., are related to prostaglandins and thromboxanes. It is being developed as a material for pharmaceuticals, feeds, cosmetics, etc.

  However, polyunsaturated fatty acids are easily deteriorated by oxidation and heating due to their structural characteristics. As a result, the acid value, peroxide value, anisidine value, carbonyl value are increased, and the appearance is not good. Deterioration of quality such as thickening and oxidative deterioration odor is observed. In the present invention, by using a highly unsaturated cholesterol fatty acid ester having excellent stability, the highly unsaturated fatty acid ester of cholesterol has characteristics such as excellent oxidative stability, which is easy to adjust to the skin, and is highly unsaturated. These problems of fatty acids can be solved and characteristics including the physiological activity of highly unsaturated fatty acids can be used.

  In the present invention, cholesterol polyunsaturated fatty acid ester is mainly produced by molecular distillation treatment as a first purification step, using cholesterol and polyunsaturated fatty acid as raw materials, and performing a synthesis reaction in a temperature-controlled system. Removal of fats and oils containing unreacted cholesterol and highly unsaturated fatty acids, and then removal of mainly pigment components and odors by adsorbent treatment with an adsorbent composed of activated carbon, activated clay or silica as the second purification step And performing the process.

In the present invention, cholesterol highly unsaturated fatty acid ester is obtained by synthesizing cholesterol and highly unsaturated fatty acid, and refers to an ester of cholesterol and highly unsaturated fatty acid.
In the present invention, examples of the synthesis method include enzyme synthesis, chemical synthesis, and combined use of enzyme synthesis and chemical synthesis.

  In the present invention, cholesterol includes cholesterol, isocholesterol, 7-dehydrocholesterol, and the like, preferably cholesterol.

In the present invention, the highly unsaturated fatty acid as a raw material refers to any of the highly unsaturated fatty acid itself, an oil or fat containing the highly unsaturated fatty acid, and a derivative of the highly unsaturated fatty acid, One or more of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA).
That is, eicosapentaenoic acid refers to any one of fatty acids, eicosapentaenoic acid, fats and oils containing eicosapentaenoic acid, and eicosapentaenoic acid derivatives. Oils and fats containing pentaenoic acid and docosapentaenoic acid derivatives It is.

In the present invention, the highly unsaturated fatty acid as a raw material is a fatty acid having three or more double bonds, and is an n-3 highly unsaturated group such as α-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, etc. Mention may be made of n-6 highly unsaturated fatty acids such as fatty acids, γ-linolenic acid, dihomo-γ-linolenic acid and arachidonic acid.
In the present invention, the highly unsaturated fatty acid as a raw material may be a fatty acid mixture containing a highly unsaturated fatty acid.

In the present invention, the fats and oils containing the highly unsaturated fatty acid include sardine oil, saury oil, bonito oil, fish oil such as tuna oil and cod liver oil, marine animal oil such as seal oil, and oils and fats obtained from plankton. it can.
In the present invention, the cholesterol highly unsaturated fatty acid ester purity reflects the highly unsaturated fatty acid content in the highly unsaturated fatty acid as a raw material, fats and oils containing the highly unsaturated fatty acid, and derivatives of the highly unsaturated fatty acid. Therefore, the higher the content of the highly unsaturated fatty acid that is the raw material, the higher the purity of the highly unsaturated fatty acid ester of cholesterol.

The fatty acid composition of the raw material and cholesterol fatty acid ester was determined by weight ratio by gas chromatography analysis (GC analysis) after adding Methyl heneicosanoate (manufactured by Sigma) as an internal standard substance to the sample and esterifying it. The esterification was carried out according to the standard oil analysis method defined by the Japan Oil Chemists' Society (Japan Oil Chemical Society established standard oil analysis method (1) 1996 version, fatty acid derivatization method, 2.4.1.1- 1996).
In the present invention, what is necessary is just to contain the highly unsaturated fatty acid in the highly unsaturated fatty acid as a raw material, and the higher the content, the more preferable, usually 40% or more, preferably 60% or more. .

  Examples of highly unsaturated fatty acid derivatives include derivatives of highly unsaturated fatty acids such as glycerides and alkyl esters. Examples of eicosapentaenoic acid derivatives include eicosapentaenoic acid methyl ester and eicosapentaenoic acid ethyl ester, and docosapentaene. Examples of the acid derivative include docosapentaenoic acid methyl ester and docosapentaenoic acid ethyl ester. Examples of the docosahexaenoic acid derivative include docosahexaenoic acid methyl ester and docosahexaenoic acid ethyl ester.

  In the present invention, cholesterol polyunsaturated fatty acid ester by enzymatic synthesis can be obtained by adding and reacting cholesterol and polyunsaturated fatty acid with an enzyme having a decomposition activity such as lipase and / or cholesterol esterase. Alternatively, an aqueous reaction can be selected as appropriate.

In the present invention, cholesterol and polyunsaturated fatty acid can be synthesized without using a solvent or using a solvent in the presence of a catalyst, and the reaction is performed stoichiometrically.
In the present invention, examples of the catalyst include alkoxide such as sodium ethoxide, sodium hydroxide, 4-dimethylaminopyridine, N, N′-dicyclohexylcarbodiimide, p-toluenesulfonic acid, sulfuric acid and the like.

  In the present invention, the highly polyunsaturated fatty acid ester of cholesterol by chemical synthesis uses cholesterol and polyunsaturated fatty acid as raw materials, a step of performing a synthesis reaction in a system in which the temperature is controlled, and a molecular distillation as a first purification step. The process of removing fats and oils mainly containing unreacted cholesterol and polyunsaturated fatty acids by the treatment, and then the adsorbent treatment with an adsorbent composed of activated carbon, activated clay or silica as the second purification step And a step of performing the removal.

In the present invention, for the step of performing the synthesis reaction in a system in which the temperature is controlled, the temperature and time of the synthesis reaction may be any conditions as long as the synthesis reaction is completed, and usually 0.1 to 12 at 5 to 140 ° C. For example, when a highly unsaturated fatty acid derivative is used as a raw material, it may normally be 100 to 140 ° C. for 2 to 12 hours.
In the present invention, the pressure of the synthesis reaction may be a condition that allows the synthesis reaction to be completed, and when a highly unsaturated fatty acid derivative is used as a raw material, it may normally be 133 to 6,650 Pa.

  In the present invention, in order to increase the reaction efficiency, the reaction is usually carried out while stirring. Moreover, it can also be made to react by leaving still and in this case, an emulsifier etc. can be added to a reaction system. Moreover, in order to improve reaction efficiency, organic solvents, such as hexane and toluene, can also be used.

In the present invention, in order to obtain a high-quality cholesterol polyunsaturated fatty acid ester having high purity and excellent color, odor and other functional surfaces, purification after the synthesis reaction must be carried out carefully under certain conditions.
In the present invention, the cholesterol fatty acid ester can be increased in purity by undergoing a series of purification steps after the synthesis reaction.
Next, a series of purification steps will be described.

In the present invention, as the first purification step, a step of removing fats and oils mainly containing unreacted cholesterol and highly unsaturated fatty acid by molecular distillation is performed.
Examples of the apparatus for performing molecular distillation include a falling film type and a centrifugal type, and any of them may be used.
As molecular distillation conditions, it is usually 133 Pa or less and 100 to 300 ° C, and preferably 13.3 Pa or less and 100 to 250 ° C. The molecular distillation operation may be repeated a plurality of times.
Cholesterol highly unsaturated fatty acid ester is obtained as a distillation residue, and unreacted sterols, fatty acids and some pigments and odor components are obtained as a fraction.

In the present invention, next, as a second purification step, a step of mainly removing the pigment component and odor by the adsorbent treatment is performed. Cholesterol highly unsaturated fatty acid ester, which is a distillation residue after molecular distillation treatment, contains a pigment component derived from raw materials, a pigment component generated by heating during distillation, an odor component, and the like.
Examples of the adsorbent used include activated clay, acidic clay, activated carbon, silica, silica magnesia, and the like, and preferably activated carbon, activated clay, or silica. These adsorbents may be used alone or in combination of two or more.
In order to perform decolorization more efficiently, the activated carbon treatment may be performed in an organic solvent such as hexane, and the amount of the solvent to be used is usually 0.1 to 50 times the amount of the raw material to be treated. The amount is 5 to 20 times.
Moreover, when using activated clay, the usage-amount with respect to the process raw material weight of activated clay is 0.1-50 weight normally, Preferably, it is 1-20 weight%, When each processes in presence of hexane, Good.
When using silica, the usage-amount with respect to the process raw material weight of a silica is 0.1-50 weight normally, Preferably, it is 1-20 weight%. In each case, treatment with silica may be performed in the presence of hexane. Preferably, the treatment is performed by passing the solution through a silica gel column packed with silica.
When an organic solvent is used, it is necessary to remove the solvent after the adsorbent treatment. Note that the adsorbent treatment operation may be repeated a plurality of times.

Furthermore, in this invention, you may perform the process of removing an odor component mainly by a steam distillation process as a 3rd refinement | purification process.
Examples of the apparatus for performing the steam distillation include continuous, semi-continuous, and batch-type apparatuses, and any type of apparatus may be used.
As steam distillation conditions, it is 13.3 kPa or less and 50-250 degreeC normally, Preferably, it is 1,330 Pa or less, 50-150 degreeC. The steam distillation operation may be repeated a plurality of times.

  In the present invention, cholesterol highly unsaturated fatty acid ester obtained by synthesizing cholesterol and highly unsaturated fatty acid includes cholesterol linolenic acid ester, cholesterol arachidonic acid ester, cholesterol eicosapentaenoic acid ester, cholesterol docosapentaenoic acid ester. And cholesterol docosahexaenoic acid ester, etc., preferably cholesterol eicosapentaenoic acid ester, cholesterol docosapentaenoic acid ester, and cholesterol docosahexaenoic acid ester.

  In the present invention, the content of cholesterol polyunsaturated fatty acid ester in the hyaluronidase inhibitor comprising as an active ingredient cholesterol polyunsaturated fatty acid ester obtained by synthesizing cholesterol and polyunsaturated fatty acid is the hyaluronidase inhibitor. In order to effectively exhibit the above effect, a higher value is preferable, preferably 60% or more, more preferably 70% or more, and particularly preferably 80% or more.

In a normal state, hyaluronic acid in the living body maintains the equilibrium of the activities of hyaluronic acid synthase and degrading enzyme. This balance is lost with aging, hyaluronidase activity, which is a hyaluronic acid-degrading enzyme, is enhanced with respect to the synthesis of hyaluronic acid, tissue flexibility and wettability are also lost, and skin aging such as skin wrinkles and tarmi is caused .
Hyaluronidase is activated during inflammation in the body, induces allergies and inflammation, destroys the connective tissue matrix by the decomposition of hyaluronic acid, and facilitates the entry of inflammatory cells.
Therefore, inhibition of hyaluronidase activity can be expected to prevent skin aging, anti-allergy and anti-inflammation.

  The hyaluronidase inhibitor comprising the cholesterol highly unsaturated fatty acid ester of the present invention as an active ingredient may contain at least a cholesterol highly unsaturated fatty acid ester, has a hyaluronidase inhibitory effect, and utilizes skin aging prevention, antiallergy, and anti-inflammation. Thus, when used in a skin external preparation, its commercial value is improved. Further, the hyaluronidase inhibitor of the present invention may be in a mixed form with other components in the external preparation for skin. Furthermore, the hyaluronidase inhibitory effect can be effectively used in cosmetics, foods, pharmaceuticals and the like.

  The form of the external preparation for skin containing the hyaluronidase inhibitor containing the highly polyunsaturated fatty acid ester of the present invention as an active ingredient is arbitrary, for example, emulsion, cream, lotion, cosmetic liquid, pack, cleaning agent, makeup makeup. It may be any form of cosmetics, quasi-drugs, external medicines, etc. such as cosmetics, dispersions, ointments, liquids, aerosols, patches, poultices, liniments and the like.

  The skin external preparation containing the hyaluronidase inhibitor containing the highly polyunsaturated fatty acid ester of cholesterol of the present invention as an active ingredient includes, in addition to the enzyme inhibitor, oils and fats such as vegetable oil, waxes such as lanolin and beeswax, kidachi aloe and the like Plant extracts, hydrocarbons, fatty acids, higher alcohols, esters, various surfactants, pigments, fragrances, vitamins, animal extracts, ultraviolet absorbers, antioxidants, antiseptics, antiseptics, etc. Those used as cosmetic raw materials can be appropriately blended and used. Furthermore, an effect can also be heightened by using together with the other component and antiallergic component known as what has a whitening effect.

  In the present invention, the amount of hyaluronidase inhibitor containing a cholesterol highly unsaturated fatty acid ester as an active ingredient in cosmetics, foods, pharmaceuticals, etc. may be determined in consideration of its purity, dosage form, etc. It is 0.001 to 50 weight% with respect to it, Preferably, it is 0.01 to 20 weight%, Most preferably, it is 0.1 to 10 weight%.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited by the following examples. In the present invention, the blending percentage of each raw material and raw material is all by weight.

A raw material consisting of 10.0 g of cholesterol (purity: 95% or more) and 10.0 g of docosahexaenoic acid ethyl ester (DHA ethyl ester) (ethyl ester having a fatty acid composition with a DHA content of 72% and an EPA content of 5%) Sodium ethoxide (1.0 ml of 10% ethanolic solution) was mixed as a catalyst, and the mixture was stirred and reacted at 120 ° C. under a vacuum of 1,330 Pa for 8 hours. The purity of cholesterol fatty acid ester in the mixture immediately after the reaction was analyzed by TLC-FID method (manufactured by Yatron, thin-layer automatic detection device Iatroscan MK-5), and the purity was 91%. After the reaction, the mixture was cooled to 60 ° C., and the catalyst was inactivated with dilute acid. The mixture was washed until the aqueous phase became neutral, and the oil phase was dehydrated under reduced pressure. Subsequently, the obtained mixture was filtered, and molecular distillation treatment was performed as a first purification treatment to obtain 18 g of a crude product. The molecular distillation treatment was performed at 0.7 Pa and 170 ° C. Next, as a second purification treatment, 18 g of crude product was added with 18 ml of hexane and 0.9 g of activated carbon, and after stirring for 30 minutes at room temperature, filtration was performed, activated carbon was removed, hexane was distilled off, and cholesterol docosahexaenoic acid was obtained. 17 g of ester (cholesterol DHA ester) was obtained. The purity of the cholesterol fatty acid ester of the product was 99%. Moreover, according to the GC analysis result, the cholesterol DHA ester content was 72%.
As described above, regarding the purity of the cholesterol fatty acid ester, the purity of 91% after the synthesis reaction could be made 99% through the purification step. Moreover, the color was white, there was no off-flavor, and the quality was excellent in terms of functionality.

In 30 ml of anhydrous toluene, 10 g of cholesterol (purity: 95% or more), 10 g of eicosapentaenoic acid free fatty acid (EPA free fatty acid) (free fatty acid having a fatty acid composition with an EPA content of 70% and a DHA content of 7%) To the starting material, 0.15 g of 4-dimethylaminopyridine as a catalyst and 5 g of N, N′-dicyclohexylcarbodiimide were mixed and stirred at 25 ° C. for 2 hours for reaction. The cholesterol fatty acid ester purity in the mixture immediately after the reaction was analyzed by the TLC-FID method as in Example 1. As a result, the purity was 82%. Next, 100 ml of hexane was added to the reaction mixture, followed by centrifugation (3,000 rpm, 5 minutes), and the supernatant was collected. The solvent of the collected supernatant was removed, and then the resulting mixture was filtered and subjected to molecular distillation treatment as a first purification treatment to obtain 17 g of a crude product. The molecular distillation treatment was performed at 0.7 Pa and 170 ° C. Next, as a second purification treatment, 34 ml of hexane was added to 17 g of the crude product and passed through a silica gel column to obtain 16 g of cholesterol eicosapentaenoic acid ester (cholesterol EPA ester). The purity of the cholesterol fatty acid ester of the product was 99%. Moreover, according to the GC analysis result, the cholesterol EPA ester content was 70%.
As described above, regarding the purity of cholesterol fatty acid ester, the purity after synthesis reaction of 82% could be 99% through the purification step. Moreover, the color was white, there was no off-flavor, and the quality was excellent in terms of functionality.

Free fatty acid with a fatty acid composition of 10 g of cholesterol (purity: 95% or more) and 10 g of docosapentaenoic acid free fatty acid (DPA free fatty acid) (70% DPA content, 30% oleic acid content) in 30 ml of anhydrous toluene ) 0.15 g of 4-dimethylaminopyridine and 5 g of N, N′-dicyclohexylcarbodiimide were mixed as a catalyst and reacted by stirring at 25 ° C. for 2 hours. The cholesterol fatty acid ester purity in the mixture immediately after the reaction was analyzed by the TLC-FID method as in Example 1. As a result, the purity was 82%. Next, 100 ml of hexane was added to the reaction mixture, followed by centrifugation (3,000 rpm, 5 minutes), and the supernatant was collected. The solvent of the collected supernatant was removed, and then the resulting mixture was filtered and subjected to molecular distillation treatment as a first purification treatment to obtain 17 g of a crude product. The molecular distillation treatment was performed at 0.7 Pa and 170 ° C. Next, as a second purification treatment, 51 g of hexane and 1.7 g of silica were added to 17 g of the crude product, stirred for 30 minutes at room temperature, filtered, silica removed, hexane distilled off, and cholesterol docosapenta. 16 g of enoic acid ester (cholesterol DPA ester) was obtained. The purity of the cholesterol fatty acid ester of the product was 99%. Moreover, according to the GC analysis result, the cholesterol DPA ester content was 70%.
As described above, regarding the purity of cholesterol fatty acid ester, the purity after synthesis reaction of 82% could be 99% through the purification step. Moreover, the color was white, there was no off-flavor, and the quality was excellent in terms of functionality.

[Measurement of hyaluronidase inhibitory activity]
In order to investigate the anti-skin aging effect of the cholesterol highly unsaturated fatty acid ester of the present invention, the hyaluronidase inhibitor which is the product of the present invention obtained in Examples 1 to 3 and the raw material used in Example 1 as a comparative control Hyaluronidase inhibition rate was measured for DHA ethyl ester (ethyl ester having a fatty acid composition of 72% DHA content and 5% EPA content).

The measurement of hyaluronidase inhibitory activity was performed by the following method.
Add 200 μl of sample to 100 μl of hyaluronidase (type IV-S from Bovine testis, Sigma) solution (4,000 units / ml; dissolved in 0.1 M acetate buffer (pH 4.9)) and leave at 37 ° C. for 20 minutes. did. Next, 200 μl of enzyme activator (Compound 48/80, Sigma) solution (0.5 mg / ml; dissolved in 0.1 M acetate buffer (pH 4.9)) was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes. 500 μl of a substrate potassium hyaluronate (from rooster comb, manufactured by Wako Pure Chemical Industries, Ltd.) solution (1.0 mg / ml; dissolved in 0.1 M acetate buffer (pH 4.9)) was added, and the mixture was stirred at 37 ° C. Left for a minute. Next, 200 μl of 0.4N sodium hydroxide solution was added to stop the reaction, and then 0.2 ml of 0.8M boric acid solution was added and heated in boiling water for 3 minutes. After cooling to room temperature, 6 ml of 1% p-dimethylaminobenzaldehyde acetic acid solution was added, and the mixture was allowed to stand at 37 ° C. for 20 minutes, and then the absorbance (A) at 585 nm was measured. The sample was mixed with 0.5 ml of a solution dissolved in chloroform to be 20 mg / ml, 50 mg / ml, and 70 mg / ml, and 2 ml of a 20 wt% Tween 40 (Polyoxyethylene Sorbitan Monopalmitate) chloroform solution. Chloroform was distilled off with nitrogen, and 50% ethanol was prepared to 2 mg / ml, 5 mg / ml, and 7 mg / ml. The enzyme reaction and absorbance (C) measurement similar to the above were performed by adding a 10 wt% Tween 40-50% ethanol solution instead of the sample solution. Further, in each case, absorbance (B) (D) was measured in the same manner by adding distilled water without adding enzyme. The inhibition rate was calculated by the following formula, and the results are shown in Table 1.

Hyaluronidase inhibition rate (%) = 100-100 × [(AB) / (CD)]
A: Absorbance when sample solution is added and enzyme solution is added B: Absorbance when sample solution is added and enzyme solution is not added C: Absorbance when sample solution is not added and enzyme solution is added D: When sample solution is not added and enzyme solution is not added Absorbance of

  As shown in Table 1, for the cholesterol highly unsaturated fatty acid ester of the present invention, cholesterol DHA ester, cholesterol EPA ester, and cholesterol DPA ester, they have concentration dependency and are significantly higher at a concentration of 7 mg / ml. Hyaluronidase inhibition rate was shown. Moreover, all this invention showed the higher inhibition rate than DHA ethyl ester of a comparison control. Therefore, the hyaluronidase inhibitor which is the product of the present invention can be effectively used in skin external preparations for skin aging prevention and / or antiallergy, cosmetics, foods, pharmaceuticals and the like.

  The example which used the hyaluronidase inhibitor of this invention for skin antiaging and / or antiallergic skin external preparation, cosmetics, and a foodstuff is shown.

[Formulation Example 1]
In the following formulation example, lotion was produced by a conventional method.
(Ingredients and weight%)
Product of the present invention (Example 1: Cholesterol DHA ester) 0.2
Glycerin 5.0
Oleyl alcohol 0.1
Polyethylene (20)
Sorbitan monolaurate ester 0.5
Ethyl alcohol 10.0
Perfume appropriate amount purified water 84.2

[Formulation Example 2]
In the following formulation example, an O / W cream was produced by a conventional method.
(Ingredients and weight%)
Product of the present invention (Example 2: Cholesterol EPA ester) 1.0
Stearyl alcohol 6.0
Stearic acid 2.0
Hydrogenated lanolin 4.0
Squalane 9.0
Octyldodecanol 10.0
1,3-butylene glycol 6.0
PEG1500 4.0
Polyethylene (25)
Cetyl alcohol ether 3.0
Glycerol monostearate 2.0
Preservative Appropriate amount Antioxidant Appropriate amount Fragrance Appropriate amount Purified water 53.0

[Composition Example 3]
In the following formulation example, an O / W cream was produced by a conventional method.
(Ingredients and weight%)
Product of the present invention (Example 3: Cholesterol DPA ester) 1.0
Stearyl alcohol 6.0
Stearic acid 2.0
Hydrogenated lanolin 4.0
Squalane 9.0
Octyldodecanol 10.0
1,3-butylene glycol 6.0
PEG1500 4.0
Polyethylene (25)
Cetyl alcohol ether 3.0
Glycerol monostearate 2.0
Preservative Appropriate amount Antioxidant Appropriate amount Fragrance Appropriate amount Purified water 53.0

[Formulation Example 4]
In the following formulation example, an O / W cream was produced by a conventional method.
(Ingredients and weight%)
Product of the present invention (Example 1: Cholesterol DHA ester) 1.0
Stearyl alcohol 6.0
Stearic acid 2.0
Hydrogenated lanolin 4.0
Squalane 9.0
Octyldodecanol 10.0
1,3-butylene glycol 6.0
PEG1500 4.0
Polyethylene (25)
Cetyl alcohol ether 3.0
Glycerol monostearate 2.0
Preservative Appropriate amount Antioxidant Appropriate amount Fragrance Appropriate amount Purified water 53.0

[Formulation Example 5]
The lotion was manufactured by the conventional method in the following formulation example.
(Ingredients and weight%)
Product of the present invention (Example 1: Cholesterol DHA ester) 1.0
Stearic acid 2.0
Cetyl alcohol 1.0
Vaseline 4.0
Glycerol tri-2-
Ethylhexanoic acid ester 2.0
Sorbitan monooleate 2.0
Dipropylene glycol 5.0
PEG 1500 3.0
Triethanolamine 1.0
Perfume Appropriate amount Preservative Appropriate amount of purified water 79.0

[Composition Example 6]
In the following formulation example, a hair liquid was produced by a conventional method.
(Ingredients and weight%)
Product of the present invention (Example 1: Cholesterol DHA ester) 0.2
Polyethylene glycol 2.0
Polyoxyethylene (5)
Decyl tetradecyl ether 2.0
Polyoxyethylene Polyoxypropylene Oligosuccinate (3.E.O) (20.P.O) 2.0
Ethyl alcohol 65.2
Fragrance Appropriate amount of purified water 28.6

[Formulation Example 7]
A gelatin capsule was filled with 200 mg of the product of the present invention (Example 1: cholesterol DHA ester), and the cap part was bound to prepare a capsule food to be an antiallergic food.

[Formulation Example 8]
A gelatin capsule was filled with 200 mg of the product of the present invention (Example 2: cholesterol EPA ester), and the cap part was bound to prepare a capsule food to be an antiallergic food.

[Formulation Example 9]
A gelatin capsule was filled with 200 mg of the product of the present invention (Example 3: Cholesterol DPA ester), and a cap part was bound to prepare a capsule food to be an antiallergic food.

[Monitor test]
A monitor test was conducted on the creams of Formulation Example 2, Formulation Example 3 and Formulation Example 4 in Example 5 and the cream of Comparative Example in which the product of the present invention was not blended to examine the effect on the skin.
[Test method]
Randomly extracted 40 women aged 18-50 were divided into 4 groups of 10 subjects as subjects, and each group was assigned one of the creams of Example 5 and Comparative Example, respectively on the left and right cheeks Once applied twice a day (morning and evening) for one month, the effect of improving fine wrinkles and the improvement of skin tension and gloss is determined based on the following criteria based on the subject's own judgment. And evaluated.
[Evaluation criteria]
A: Improvement effect, B: Slight improvement effect, C: No change [Result]
The results are shown in Table 2. In addition, the number of each evaluation column of AC of Table 2 shows the number of the test subjects who performed the said evaluation among 10 test subjects.

  As is clear from the results in Table 2, the skin external preparation using the highly polyunsaturated fatty acid ester of the cholesterol product of the present invention showed a remarkable skin improvement effect and an effect of preventing skin aging.

Claims (8)

  A hyaluronidase inhibitor characterized by comprising a cholesterol highly unsaturated fatty acid ester obtained by synthesizing cholesterol and a highly unsaturated fatty acid as an active ingredient.   The hyaluronidase inhibitor according to claim 1, wherein the polyunsaturated fatty acid is one or more of eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid.   The hyaluronidase inhibitor according to claim 1 or 2, wherein the content of cholesterol polyunsaturated fatty acid ester in the hyaluronidase inhibitor is 60% or more.   The skin external preparation for skin aging prevention and / or antiallergy containing the hyaluronidase inhibitor of any one of Claims 1-3.   A food containing the hyaluronidase inhibitor according to any one of claims 1 to 3. Using cholesterol and polyunsaturated fatty acids as raw materials, and performing a synthesis reaction in a temperature-controlled system;
A step of removing fats and oils mainly containing unreacted cholesterol and highly unsaturated fatty acids by molecular distillation as a first purification step;
A step of mainly removing pigment components and odor by adsorbent treatment with an adsorbent composed of activated carbon, activated clay or silica as the second purification step;
The manufacturing method of the hyaluronidase inhibitor which uses the cholesterol polyunsaturated fatty acid ester containing this as an active ingredient.   The method for producing a hyaluronidase inhibitor according to claim 6, wherein the highly unsaturated fatty acid is one or more of eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid.   The method for producing a hyaluronidase inhibitor according to claim 6 or 7, wherein the content of the cholesterol polyunsaturated fatty acid ester in the hyaluronidase inhibitor is 60% or more.