WO2015129525A1 - HYALURONIC ACID SYNTHESIS PROMOTER, HAS2 mRNA EXPRESSION PROMOTER, PHARMACEUTICAL, FOOD, OR COSMETIC HAVING HYALURONIC ACID SYNTHESIS-PROMOTING ACTION, AND PHARMACEUTICAL, FOOD, OR COSMETIC HAVING HAS2 mRNA EXPRESSION-PROMOTING ACTION - Google Patents

Abstract

　A hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter containing, as an active ingredient, at least a first fraction containing chrysin that is a fraction extracted from seeds of Oroxylum indicum by an aqueous organic solvent, another fraction obtained by purifying the first fraction, or chrysin. Also, a pharmaceutical, food, or cosmetic containing this hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter. This hyaluronic acid synthesis promoter, HAS2 mRNA expression promoter, pharmaceutical, food, or cosmetic serves as a useful item using a fraction or component obtained from highly safe Oroxylum indicum.

Description

Hyaluronic acid synthesis promoter, HAS2 mRNA expression promoter, pharmaceuticals, foods or cosmetics having hyaluronic acid synthesis promoting action, and pharmaceuticals, foods or cosmetics having HAS2 mRNA expression promoting action

The present invention relates to a hyaluronic acid synthesis promoter, a HAS2 mRNA expression promoter, a pharmaceutical product, food or cosmetic having a hyaluronic acid synthesis promoting action, and a pharmaceutical, food or cosmetic having a HAS2 mRNA expression promoting action.

Hyaluronic acid (also referred to as hyaluronan) is a polymer (glycosaminoglycan) composed of N-acetylglucosamine and glucuronic acid and has a high water retention capacity. Hyaluronic acid is widely used in extracellular matrix as a gel-like substance containing water, and is involved in maintaining viscoelasticity of living tissues (especially skin tissues), moisturizing living tissues, and maintaining the function of articular cartilage. It is known to do. Hyaluronic acid also has functions such as blocking the entry of macromolecular substances from outside the cell, regulating cell migration and proliferation, and serving as a signal transmission site.

By the way, the amount of hyaluronic acid in a living body decreases due to various stresses, ultraviolet rays, aging and the like. It is known that when the amount of hyaluronic acid in a living body decreases, symptoms such as dry skin (wrinkles, sagging and roughness) and joint pain occur. Direct causes for the decrease in the amount of hyaluronic acid in the body include the degradation of hyaluronic acid by hyaluronidase (hyaluronic acid-degrading enzyme) and ultraviolet rays, and the decrease in the production of hyaluronic acid in the living body. it can.

For this reason, if the amount of hyaluronic acid in the living body can be supplemented or increased, prevention, suppression, improvement or normalization of symptoms such as skin malfunction and joint pain can be expected.

Conventionally, retinoic acid in the epidermis and TGF-β1 in the dermis are widely known as components having an action of promoting the synthesis of hyaluronic acid in vivo. Moreover, various natural products and fractions thereof (for example, extracts from Agaricus) are known as those having an action of promoting the synthesis of hyaluronic acid in vivo (see, for example, cited document 1).

On the other hand, an antioxidant containing an extract from Oryzylum indicum (sometimes referred to as jade butterfly or tree butterfly, details will be described later) as an active ingredient is known (see, for example, Reference 2). . Soriyayanoki is a safe natural product that is sometimes used as food in its origin.

However, an extract from Sorilla japonica and a purified product thereof, in particular, a fraction extracted from the seeds of Sorusayanoki with an aqueous organic solvent, a predetermined fraction fractionated from the fraction, or a chrysin contained in Soralia A hyaluronic acid synthesis promoter containing (chrysin) as an active ingredient is not known.

In addition, HAS2 (hyaluronic acid synthase 2), which is an enzyme that synthesizes hyaluronic acid in vivo (particularly on the cell surface), is known. HAS2 is synthesized by the expression of HAS2 mRNA. That is, in order to supplement or increase the amount of hyaluronic acid in vivo, it is effective to promote the expression of HAS2 mRNA.

As in the case of the hyaluronic acid synthesis accelerator, the fraction extracted from the seeds of Soritsa japonica using an aqueous organic solvent, the predetermined fractions fractionated from the fractions, or the chrysin contained in Sorusayanoki as an active ingredient HAS2 mRNA expression promoter contained as is also not known.

JP 2013-035835 A JP 2006-321730 A

The object of the present invention is to provide “a hyaluronic acid synthesis accelerator using a fraction or a component obtained from Soriyayanoki having high safety”. Another object of the present invention is to provide a “HAS2 mRNA expression promoter using a fraction or a component obtained from Sorizayanoki that has high safety”. Another object of the present invention is to provide a “pharmaceutical, food or cosmetic containing the hyaluronic acid synthesis promoter of the present invention and having a hyaluronic acid synthesis promoting action”. A further object is to provide a “pharmaceutical, food or cosmetic comprising the HAS2 mRNA expression promoter of the present invention and having an HAS2 mRNA expression promoting action”.

As a result of diligent research by the inventors of the present invention, among the extracts from Sorusayanoki and purified products thereof, "fractions extracted from Sorusayanoki seeds with an aqueous organic solvent", "fractionated from the fractions It was found that “predetermined fractions” and “chrysin” have a hyaluronic acid synthesis promoting action. Moreover, it became clear from the research about the mechanism that each said fraction and component have a HAS2 mRNA expression promotion effect.
The inventors of this invention came to complete this invention from the above knowledge. The present invention includes the following items.

[1] A hyaluronic acid synthesis promoter containing, as an active ingredient, a first fraction containing at least chrysin, which is a fraction extracted from a solitary tree seed using an aqueous organic solvent.

[2] When liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, the fraction is distributed to the ethyl acetate side and contains at least chrysin A hyaluronic acid synthesis accelerator containing the second fraction as an active ingredient.

[3] Liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side with 90% methanol and hexane The hyaluronic acid synthesis promoter comprising, as an active ingredient, the third fraction that is distributed to the 90% methanol side and contains at least chrysin when further liquid-liquid partitioning is performed.

[4] Liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side with 90% methanol and hexane When the third fraction distributed to the 90% methanol side is fractionated by silica gel chromatography, the fraction eluted from silica gel with a 9: 1 mixed solvent of chloroform: methanol. A hyaluronic acid synthesis promoter comprising a fourth fraction containing at least chrysin as an active ingredient.

[5] A hyaluronic acid synthesis promoter containing chrysin as an active ingredient.

[6] A HAS2 mRNA expression promoter containing, as an active ingredient, a first fraction containing at least chrysin, which is a fraction extracted from the seeds of Sorisaya tree using an aqueous organic solvent.

[7] When liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, the fraction is distributed to the ethyl acetate side and contains at least chrysin A HAS2 mRNA expression promoter containing the second fraction as an active ingredient.

[8] Liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side with 90% methanol and hexane The HAS2 mRNA expression promoter comprising, as an active ingredient, the third fraction containing at least chrysin, which is a fraction distributed to the 90% methanol side when further liquid-liquid partitioning is performed.

[9] Liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side with 90% methanol and hexane When the third fraction distributed to the 90% methanol side is fractionated by silica gel chromatography, the fraction eluted from silica gel with a 9: 1 mixed solvent of chloroform: methanol. HAS2 mRNA expression promoter containing a fourth fraction containing at least chrysin as an active ingredient.

[10] A HAS2 mRNA expression promoter containing chrysin as an active ingredient.

[11] A pharmaceutical, food or cosmetic having hyaluronic acid synthesis promoting action, comprising the hyaluronic acid synthesis promoting agent according to any one of [1] to [5] above.

[12] A pharmaceutical, food or cosmetic having a HAS2 mRNA expression promoting action, comprising the HAS2 mRNA expression promoting agent according to any one of [6] to [10].

According to the present invention, as can be seen from the test examples described later, “a hyaluronic acid synthesis accelerator using a fraction or component obtained from a highly safe soybean tree”, “a fraction obtained from a highly safe soybean tree” HAS2 mRNA expression promoter using painting or ingredient ”,“ pharmaceutical, food or cosmetic containing hyaluronic acid synthesis promoter of the present invention and having hyaluronic acid synthesis promoter ”and“ HAS2 mRNA expression promoter of the present invention It is possible to provide a “pharmaceutical, food or cosmetic that contains HAS2 mRNA expression promoting action”.

In this specification and each drawing, when the concentration is simply described as “%”, it represents a percent concentration calculated by volume / volume (V / V).
Moreover, in this specification and each drawing, the component ratio (for example, “9: 1” above) of the mixed solvent represents the volume ratio of the solvent.

Chrysin (chrysin. 5,7-dihydroxy-2-phenyl-4H-chromen-4-one.) In the present invention is a compound described in the following formula (1).

FIG. 6 is a flowchart for explaining the first to fourth fractions and chrysin in Embodiments 1 to 5. FIG. 6 is a flowchart shown for explaining samples a to e in Test Examples 1 to 5.

Hereinafter, the hyaluronic acid synthesis promoter, HAS2 mRNA expression promoter, pharmaceuticals, foods or cosmetics having hyaluronic acid synthesis promoting action, and pharmaceuticals, foods or cosmetics having HAS2 mRNA expression promoting action of the present invention are based on the embodiments. I will explain.

[Embodiment]
FIG. 1 is a flowchart for explaining the first to fourth fractions and chrysin according to the first to fifth embodiments. In FIG. 1, the reason why the fourth fraction and chrysin are connected by a broken line is that the chrysin in Embodiment 5 is isolated from the fourth fraction, as will be described later. This is because there is not.

The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter of the present invention include the first fraction, the second fraction, the third fraction, the fourth fraction, or the like described in [1] to [10] above. Contains chrysin as an active ingredient.
Hereinafter, each of the above active ingredients will be described with reference to FIG. 1 and Embodiments 1 to 5. Moreover, Embodiment 6 demonstrates the pharmaceutical, foodstuff, or cosmetics containing said hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter.

[Embodiment 1]
The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to Embodiment 1 contain, as an active ingredient, a first fraction containing at least chrysin, which is a fraction extracted from the seeds of Sorisaya tree using an aqueous organic solvent. .

Oroxylum indicum is a deciduous tree plant with a height of 7 to 12 m. The place of origin is Southeast Asia, India and China. In these regions, they grow naturally along mountain slopes and mountain streams. The flowering period of Soriyayanoki is from July to August, and seeds can be collected from October to December. Soriyayanoki seeds are a general distribution that can be obtained in these regions.

In Southeast Asia etc., the fruit, young leaves, buds, etc. of Soriyayanoki are used as food products. Soriyayanoki is said to have medicinal properties and is used for treatment of voice and speech disorder, wound treatment (blood stop), healthy stomach, cough stop, and the like.

In addition to germ and endosperm, the seeds of Soriyayanoki have a wing-like thin film (site for scattering the seeds far away) and form a thin piece as a whole.
The seeds of Sorusayanoki used for obtaining the first fraction may be those with the above-mentioned thin film, or those from which the above-mentioned thin film has been removed. In the present specification and each drawing, “soraya tree seed” includes both those with the thin film and those with the thin film removed.

In order to obtain the hyaluronic acid synthesis promoter and the HAS2 mRNA expression promoter according to Embodiment 1, for example, a dried product obtained by drying and naturally drying seeds collected from Sorisayaki can be used. Further, unprocessed, heat-dried, freeze-dried, and frozen products of Sorusayanoki seed can be used.

In the extraction, for example, the seeds of Sorusayanoki may be used as they are, or may be used after being cut finely or in powder form.

In the present specification, the “aqueous organic solvent” refers to a solvent containing a hydrophilic organic solvent as a main component. “Hydrophilic organic solvent” refers to an organic solvent that mixes with water without being separated. Examples of hydrophilic organic solvents include lower aliphatic alcohols and acetone. Examples of lower aliphatic alcohols include monohydric alcohols such as methanol, ethanol, propanol, and butanol, and polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, 1,3-propanediol, pentylene glycol, and isoprene glycol. It can be illustrated.
The aqueous organic solvent may contain two or more types of organic solvents.

The aqueous organic solvent may contain water. In the case where the aqueous organic solvent contains water, whether or not the hydrophilic organic solvent is a main component is considered by removing water from the constituent components. That is, even when there is more water than the hydrophilic organic solvent, if the hydrophilic organic solvent is the main component in the organic solvent, it is an “aqueous organic solvent” as used herein.
When the aqueous organic solvent contains water, the aqueous organic solvent preferably contains 30% or more of the hydrophilic organic solvent, and more preferably contains 50% or more.

Specific examples of the first fraction include an extract obtained by extraction using sodayanoki seeds using an aqueous organic solvent, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, And these crudely purified products or purified products can be mentioned.

In addition, as shown in the test examples described later, the fraction extracted from the seeds of Sorisayaki using an aqueous organic solvent contains chrysin. Therefore, the condition “including chrysin” is satisfied unless a treatment for removing chrysin from the extract, concentrated liquid, diluted liquid, dried product, crude purified product, purified product or the like is performed. The same applies to the second to fourth fractions described later.

Extraction is not particularly limited as long as the soluble fraction contained in the seeds of Sorisaya tree can be eluted in an aqueous organic solvent, and can be performed according to a conventional method. In the extraction, it is not necessary to employ a special method or apparatus, and any method or apparatus can be employed.

Examples of extraction methods include extraction at room temperature, extraction by heating to a temperature below the boiling point of the aqueous organic solvent, and extraction under heating and reflux. At this time, the seeds of Sorusayanoki may be simply immersed, or may be stirred with a solvent. The amount of the aqueous organic solvent can be, for example, 5 to 50 times the weight (weight ratio) of the extraction raw material. The extraction time can be, for example, 1 to 72 hours. The extraction temperature can be, for example, room temperature to 95 ° C.

Before performing extraction with an aqueous organic solvent, degreasing treatment using a nonpolar solvent such as pentane, hexane, heptane, octane, petroleum ether or the like may be performed.

An eluate can be obtained by eluting the soluble fraction and then subjecting it to filtration, centrifugation, etc. to remove the extraction residue. In order to obtain a diluted solution, concentrated solution, dried product, crude purified product, purified product, etc. of the extracted solution from the obtained extracted solution, treatments such as dilution, concentration, drying, purification and the like may be further performed according to conventional methods. .

As the purification of the first fraction, purification for the purpose of decolorization, deodorization and the like may be performed. For the purification, for example, various adsorption treatments and various separation treatments (for example, treatment using activated carbon, adsorption resin, ion exchange resin, silica gel, gel filtration agent) can be used. The same applies to the second to fourth fractions described later.

[Embodiment 2]
The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to Embodiment 2 are used for liquid-liquid partitioning of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate. A fraction to be distributed and containing a second fraction containing at least chrysin as an active ingredient.

The first fraction can be obtained by the method described in Embodiment 1 above.

The liquid-liquid distribution is not particularly limited as long as the second fraction can be separated from the first fraction, and can be performed according to a conventional method. It is not necessary to employ a special method or apparatus for performing the liquid-liquid distribution, and any method or apparatus can be employed.

[Embodiment 3]
The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to Embodiment 3 are obtained by liquid-liquid partitioning the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and partitioning to the ethyl acetate side When the obtained second fraction is further subjected to liquid-liquid partition with 90% methanol and hexane, the third fraction containing at least chrysin as a fraction distributed to the 90% methanol side is used as an active ingredient. contains.

The first fraction can be obtained by the method described in Embodiment 1 above. The second fraction can be obtained by the method described in Embodiment 2 above.

Liquid-liquid distribution is not particularly limited as long as the third fraction can be separated from the second fraction, and can be performed according to a conventional method. It is not necessary to employ a special method or apparatus for performing the liquid-liquid distribution, and any method or apparatus can be employed.

[Embodiment 4]
The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to Embodiment 4 are obtained by liquid-liquid partitioning of the first fraction extracted from the seeds of Sorisaya tree with an aqueous organic solvent with water and ethyl acetate, and partitioning to the ethyl acetate side The second fraction thus obtained was further subjected to liquid-liquid partition with 90% methanol and hexane, and when the third fraction distributed to the 90% methanol side was fractionated by silica gel chromatography, chloroform: methanol 9: A fraction eluted from silica gel with one mixed solvent and containing at least a chrysin as an active ingredient.

The first fraction can be obtained by the method described in Embodiment 1 above. The second fraction can be obtained by the method described in Embodiment 2 above. The third fraction can be obtained by the method described in Embodiment 3 above.

Silica gel chromatography is not particularly limited as long as the fourth fraction can be separated from the third fraction, and can be performed according to a conventional method. In silica gel chromatography, it is not necessary to employ a special method or apparatus, and any method or apparatus can be employed.

[Embodiment 5]
The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to Embodiment 5 contain chrysin as an active ingredient.

Chrysin is a compound described in the following formula (1).

Chrysin is contained in the first to fourth fractions as shown in the test examples described later. The chrysin used in the hyaluronic acid synthesis promoter and the HAS2 mRNA expression promoter may be one obtained from the seeds of Sorilla japonica (see the test examples described later), or a natural product other than the seeds of Sorilla japonica, It may be obtained from a natural product containing, or may be a commercial product or a synthetic product.

In the case of obtaining chrysin from the seed of Soriyayanoki, for example, it can be isolated from the fourth fraction described in Embodiment 4. For the isolation and purification of chrysin, silica gel chromatography and gel filtration chromatography can be suitably used, but the present invention is not limited thereto.

Silica gel chromatography and gel filtration chromatography are not particularly limited as long as chrysin can be isolated and purified, and can be performed according to a conventional method. In silica gel chromatography and gel filtration chromatography, it is not necessary to employ a special method or apparatus, and any method or apparatus can be employed.

The hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to the above embodiments 1 to 5 can supplement or increase the amount of hyaluronic acid in vivo through the hyaluronic acid synthesis promoting action and HAS2 mRNA expression promoting action, As a result, prevention, suppression and improvement or normalization of symptoms such as skin malfunction and joint pain can be expected. However, the hyaluronic acid synthesis promoter and HAS2 mRNA expression promoter according to the present invention should be used for all purposes that are meaningful for exerting the hyaluronic acid synthesis promoter and HAS2 mRNA expression promoting action in addition to these uses. Can do.

[Embodiment 6]
The pharmaceutical product, food or cosmetic according to Embodiment 6 contains the hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter described in any of Embodiments 1 to 5.

When the drug according to Embodiment 6 is an internal drug, the administration method is not particularly limited. For example, enteral administration such as oral administration and rectal administration, mucosal administration such as nasal administration, intravenous administration and subcutaneous administration Or the like. The dosage form of the internal medicine of the present invention can be in a form suitable for the administration method, for example, solid preparations such as tablets, powders, fine granules, granules, capsules, powders, pills, troches and the like. , Solutions, suspensions, emulsions, syrups, liquids such as injections, and gel preparations. As a pharmaceutical product according to Embodiment 6, the hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter described in any of Embodiments 1 to 5 may be administered as it is, such as a pure product, a purified product, or a crude product. It may be administered with a pharmaceutically acceptable excipient. As the excipient, those generally usable as preparations such as monosaccharides, second class, polysaccharides, inorganic salts, fats and oils, and distilled water can be used. When formulating an internal medicine according to Embodiment 6, additives such as a binder, a lubricant, a dispersant, a suspending agent, an emulsifier, a diluent, a buffering agent, an antioxidant, and a bacterial inhibitor are added. It may be used.

When the pharmaceutical product according to Embodiment 6 is a topical pharmaceutical product, the form is not particularly limited, and for example, an ointment, a cream, a foaming agent, a tape, and an external preparation can be used. The external medicine according to Embodiment 6 contains, in addition to the hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter described in any of Embodiments 1 to 5, other pharmaceutical ingredients, excipients, and the like as necessary. May be. When manufacturing an external medicine according to Embodiment 6, additives such as a binder, a dispersant, a suspending agent, an emulsifier, a diluent, a buffer, an antioxidant, and a bacteria inhibitor may be used.

The amount of the hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter contained in the pharmaceutical product according to Embodiment 6 is various data (for example, administration methods, dosage forms, hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter for internal medicines) The optimal amount can be set based on the effective dose of itself and the effective dose as a preparation.

The form of the food according to Embodiment 6 is, for example, a pure product of the hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter described in any of Embodiments 1 to 5, a partially purified product thereof, a hyaluronic acid synthesis promoter or HAS2 mRNA. It can be set as the general food which mix | blended the paste containing an expression promoter, a hyaluronic acid synthesis promoter, or a HAS2 mRNA expression promoter. Specific examples include drinks, jellies, biscuits, teas, tablets, pills, soft capsules, hard capsules, powders, fine granules, and granules. Any form can be used. When the food according to Embodiment 6 is produced, as an auxiliary material, excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, diluents, buffers, antioxidants, bacteria inhibitors An additive such as may be used.

The form of the cosmetic according to Embodiment 6 can be, for example, lotion, cosmetic liquid, milky lotion, cream, gel, pack, cosmetic powder, facial cleansing foam, bath preparation, etc. Any form can be used. The cosmetic according to Embodiment 6 contains various blending ingredients that are blended into the cosmetic as necessary, in addition to the hyaluronic acid synthesis promoter or HAS2 mRNA expression promoter described in any of Embodiments 1 to 5. May be. Examples of the ingredients include solid oil, semi-solid oil, liquid oil, low molecular moisturizer, polymer moisturizer, fat-soluble moisturizer, emollient, surfactant, preservative, antioxidant, pH adjuster, Ethanol and water can be used.

The pharmaceutical, food and cosmetics according to Embodiment 6 described above have the effects of hyaluronic acid in vivo through the hyaluronic acid synthesis promoting action and the HAS2 mRNA expression promoting action possessed by the hyaluronic acid synthesis promoting agent and HAS2 mRNA expression promoting agent according to Embodiments 1 to 5. The amount can be supplemented or increased, and as a result, prevention, suppression and improvement or normalization of symptoms such as skin malfunction and joint pain can be expected. However, the pharmaceuticals, foods, and cosmetics according to Embodiment 6 can be used for all purposes that are meaningful in exerting the hyaluronic acid synthesis promoter and HAS2 mRNA expression promoting action, in addition to these uses.

Note that the hyaluronic acid synthesis promoter, HAS2 mRNA expression promoter, pharmaceuticals, foods and cosmetics described in the first to sixth embodiments are preferably applied to humans, but their respective effects are exhibited. As long as it can be applied to animals other than humans.

[Test example]
Hereinafter, as test examples, “1. Test example for fractionating and isolating first to fourth fractions and chrysin”, “2. Test example for confirming hyaluronic acid synthesis promoting action” and “ 3. “Test Example for Confirming HAS2 mRNA Expression Promoting Action” is given to describe the present invention in more detail. In addition, the following test examples show the result confirmed about the methods and actions, such as a fractionation, and this invention is not restrict | limited to the following test examples at all.

1. Test Example for Fractionation and Isolation of First Fraction to Fourth Fraction and Chrysin FIG. 2 is a flowchart shown for explaining samples a to e in Test Examples 1 to 5. The sample f in Test Example 6 is not shown in FIG. 2 because it is a sample of a different system from the samples a to e.
Hereinafter, fractionation / isolation of the first fraction to the fourth fraction and chrysin will be described with reference to Test Examples 1 to 6.
In the following test examples, distilled water was used as water, and Yamaichi Chemical Co., Ltd. domestic grade 1 was used as methanol, ethyl acetate and hexane. Moreover, the general goods (For example, a 100 mL Erlenmeyer flask and a 30 mL Erlenmeyer flask are those of Iwaki Glass Co., Ltd.) were used as the experimental instrument.

[Test Example 1]
Test Example 1 is a test example for obtaining a first fraction containing at least chrysin, which is a fraction extracted from the seed of Sorisayaki with an aqueous organic solvent. As shown in FIG. 2, the first fraction fractionated in Test Example 1 is designated as sample a.
In Test Example 1, methanol was used as the aqueous organic solvent.

First, 1 kg of seeds of Oroxylum indicum was prepared. As the seeds of Soriyayanoki, dried products (those with a winged thin film) were used.
Next, the seeds of Soriyayanoki were cut into fine pieces with scissors and extracted by immersing them in methanol. Extraction was performed in a stainless steel container. The extraction method was immersion (room temperature, methanol 20 L, extraction time 24 hours), and this was performed three times in total.

Thereafter, the mixture was naturally filtered at room temperature to obtain an extract (fluorescent yellow-green transparent liquid). The extract was further concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain 31.2 g of sample a (first fraction) as a black solid. The solid yield based on the weight of Sorisaya tree seeds (1 kg) was 3.12%.

In addition, although the test whether the sample a contains chrysin was not performed, as shown in Test Example 5 to be described later, the chrysin was obtained as a result of repeated fractionation and purification of the sample a. It is clear that it contains chrysin. The same applies to Samples b to d in Test Examples 2 to 4 described later.

[Test Example 2]
Test Example 2 is a fraction that is distributed to the ethyl acetate side when liquid-liquid partitioning of the first fraction extracted from the seeds of Sorisayaki with an aqueous organic solvent with water and ethyl acetate, and at least chrysin is present. It is a test example for obtaining the 2nd fraction containing. The second fraction fractionated in Test Example 2 is designated as sample b.

First, 800 mL of water was added to 31.2 g of sample a (first fraction) obtained in Test Example 1 to obtain a suspension, and the suspension was extracted with ethyl acetate (room temperature, ethyl acetate 1000 mL). Total 3 times. Extraction was performed using a separatory funnel.
Thereafter, ethyl acetate was collected and concentrated under reduced pressure (40 ° C., 75 mmHg) to obtain 11.8 g of sample b (second fraction) as a black solid. The solid yield based on the weight of Sorisayaki seed was 1.18%.

[Test Example 3]
Test Example 3 is a liquid-liquid partition of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side is 90% methanol and This is a test example for obtaining a third fraction containing at least chrysin, which is a fraction distributed to the 90% methanol side when further liquid-liquid partition is performed with hexane. The third fraction fractionated in Test Example 3 is designated as sample c.

First, 300 mL of 90% methanol is added to 11.8 g of sample b (second fraction) obtained in Test Example 2 to obtain a suspension, and the suspension is extracted with hexane (room temperature, 300 mL of hexane). Total 3 times. Extraction was performed using a separatory funnel.
Thereafter, 90% methanol was recovered and concentrated under reduced pressure (40 ° C., 75 mmHg) to obtain 4.0 g of sample c (third fraction) as a black solid. The solid yield based on the weight of Sorisayaki seed was 0.40%.

[Test Example 4]
Test Example 4 is a liquid-liquid partition of the first fraction extracted from Sorisaya tree seeds with an aqueous organic solvent with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side was 90% methanol and Further liquid-liquid partition with hexane, and fractions eluted from silica gel with 9: 1 mixed solvent of chloroform: methanol when the third fraction distributed to 90% methanol side is fractionated by silica gel chromatography This is a test example for obtaining a fourth fraction containing at least chrysin. The fourth fraction fractionated in Test Example 4 is designated as sample d.

First, 4.0 g of sample c (third fraction) obtained in Test Example 3 was dissolved in a small amount (about several tens of mL) of a mixed solvent of chloroform and methanol to obtain a solution. Next, the solution was placed on a column (25 mmφ × 290 mm) packed with silica gel (Wakosil C-200 from Wako Pure Chemical Industries, Ltd.).
Subsequently, elution was performed with 300 mL of a mixed solvent of chloroform: methanol = 9: 1 to obtain a fraction A-1. Subsequently, elution was sequentially performed with 300 mL of a mixed solvent of chloroform: methanol = 4: 1, 300 mL of a mixed solvent of chloroform: methanol = 1: 1, and 300 mL of methanol, and in order, fraction A-2, fraction A-3, and fraction A-4 was obtained.
Among these, fraction A-1 was concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain 2.32 g of a sample d (fourth fraction) in the form of a brownish brown to black-green extract. The solid yield based on the weight of Sorisayaki seed was 0.232%.

[Test Example 5]
Test Example 5 is a test example for obtaining chrysin. Let chrysin isolated in Test Example 5 be sample e.
In silica gel chromatography and gel filtration chromatography in Test Example 5, a small amount (about 20 to 30 mL) of a sample was taken from each fraction, and spots were confirmed by TLC. Spots were confirmed by color or reaction to UV lamp. Fractions with the same spot appearance were grouped together and treated as one fraction. The amount of the solution per fraction was not always constant, and sometimes the fraction was divided while observing the color of the solution.

First, of the sample d (fourth fraction) obtained in Test Example 4, 2.27 g, which was obtained from the latter fraction, was dissolved in a small amount of a mixed solvent of chloroform and methanol to obtain a solution. . Next, the solution was placed on a column (25 mmφ × 340 mm) packed with silica gel (Wakosil C-200 from Wako Pure Chemical Industries, Ltd.).
Subsequently, elution was performed with 500 mL of a mixed solvent of chloroform: methanol = 9: 1. Fraction B-1 was obtained from the first three fractions, and fraction B-2 was obtained from the latter two fractions. Thereafter, elution was performed with 300 mL of methanol to obtain a fraction B-3.
Among these, the fraction B-1 was concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain 1.04 g of a fraction from the fraction B-1.

Further, 1.04 g of the fraction from fraction B-1 was dissolved in a small amount of a mixed solvent of chloroform and methanol to obtain a solution. Next, the solution was placed on a column (25 mmφ × 275 mm) packed with silica gel (Wakosil C-200 from Wako Pure Chemical Industries, Ltd.).
Subsequently, elution was performed with 500 mL of chloroform, and fraction C-1 was obtained from one fraction in the first half, and fraction C-2 was obtained from three fractions in the latter half. Thereafter, elution was successively carried out with 300 mL of a mixed solvent of chloroform: methanol = 19: 1 and 300 mL of methanol to obtain fraction C-3 and fraction C-4 in this order. Among these, the fraction C-2 was concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain 0.35 g of a fraction from the fraction C-2.

Further, 0.35 g of the fraction from fraction C-2 was dissolved in a small amount of a mixed solvent of chloroform and methanol to obtain a solution. Next, the solution was placed on a column (25 mmφ × 280 mm) packed with silica gel (Wakosil C-200 from Wako Pure Chemical Industries, Ltd.).
Subsequently, elution was performed with 500 mL of chloroform, and a fraction D-1 was obtained from one fraction in the first half, and a fraction D-2 was obtained from three fractions in the latter half. Thereafter, elution was performed with 300 mL of methanol to obtain a fraction D-3.
Among these, the fraction D-2 was concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain 0.31 g of a fraction from the fraction D-2.

Further, 0.31 g of the fraction from fraction D-2 was dissolved in a small amount of a mixed solvent of chloroform and methanol to obtain a solution. Next, the solution was placed on a column (25 mmφ × 250 mm) packed with a gel filtration agent for chromatography (Sephadex LH-20 from Sigma-Aldrich).
Subsequently, elution was carried out with 500 mL of a mixed solvent of chloroform: methanol = 1: 1, fraction E-1 was obtained from the first two fractions, fraction E-2 was obtained from the next one fraction, and the next one fraction was obtained. Fraction E-3 was obtained from
Among these, the fraction E-3 was concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain 0.07 g of a fraction from the fraction E-3.

Further, 0.07 g of the fraction from fraction E-3 was dissolved in a small amount of a mixed solvent of chloroform and methanol to obtain a solution. Next, the solution was placed on a column (25 mmφ × 220 mm) packed with a gel filtration agent for chromatography (Sephadex LH-20 from Sigma-Aldrich).
Subsequently, elution was carried out with 500 mL of a mixed solvent of chloroform: methanol = 1: 1. Fraction F-1 was obtained from the first fraction, fraction F-2 was obtained from the next three fractions, and the next one fraction was obtained. From this, fraction F-3 was obtained.
Among these, fraction F-2 was concentrated under reduced pressure (40 ° C., 60 mmHg) to obtain about 10 mg of sample e (chrysin). The solid yield based on the weight of Sorisayaki seed was about 0.001%.

With respect to the sample e (chrysin) isolated as described above, 1H-NMR spectrum data was obtained by nuclear magnetic resonance spectroscopy. For the acquisition of the data, JNM-ECA500 of JEOL RESONANCE Inc. was used.
As a result, the following peaks (only main peaks are described as approximate values) were observed, which substantially coincided with generally known 1H-NMR spectrum data of chrysin.

1H-NMR (acetone-d ₆ ): δ (ppm) = 6.29 (1H, d, J = 1.7 Hz, H-6), 6.58 (1H, d, J = 1.7 Hz, H− 8), 6.79 (1H, s, H-3), 7.60 (2H, m, H-3 ′, H-5 ′), 7.61 (1H, m, H-4 ′), 8 .07 (2H, dd, J = 8.1, 1.7 Hz, H-2 ′, H-6 ′)

In addition, the sample e was analyzed by high performance liquid chromatography. For this analysis, a diode array detector SPD-M20A from Shimadzu Corporation, a liquid feeding unit LC-20AB, a column oven CTO-20A, and a HPLC column CAPCELL PAK MG II 4.6 mm ID X from Shiseido Co., Ltd. 250 mm was used. In the analysis, the solution A was phosphate buffer (pH 6.5), the solution B was 100% methanol, the linear gradient was A: B = 95: 5 to 20:80 (60 minutes), and the column temperature was 40. C. As a detection method, a diode array (190-800 nm) was used.

First, a chrysin standard product (purchased from Tokyo Chemical Industry Co., Ltd.) was analyzed under the above conditions, and it was confirmed that the largest peak occurred at 53.7 minutes. Next, the sample e was analyzed under the same conditions, and it was confirmed that the largest peak occurred at 52.6 minutes. Moreover, it confirmed that the spectrum of the largest peak of the chrysin standard goods and the sample e corresponded.

As described above, it was confirmed that the sample e was mainly composed of chrysin by nuclear magnetic resonance spectroscopy and high performance liquid chromatography.
In addition, since sample e was fractionated and isolated from samples a to d, it was confirmed that samples a to d also contained chrysin.

[Test Example 6]
Test Example 6 is another test example for obtaining a first fraction containing at least chrysin, which is a fraction extracted from the seed of Sorisayaki using an aqueous organic solvent. The first fraction fractionated in Test Example 6 is designated as sample f.
In Test Example 6, 50% ethanol was used as the aqueous organic solvent.

First, 10 kg of seeds of Oroxylum indicum were prepared. As the seeds of Soriyayanoki, we used a general-purpose dried product (with a wing-like thin film attached).
Next, Sorizayanoki seeds were chopped and extracted by soaking in 50% ethanol. The extraction method was extraction under heating and reflux (80 ° C., 150 L of 50% ethanol, extraction time 2 hours).
Thereafter, the mixture was naturally filtered at room temperature to obtain an extract, and the extract was further concentrated under reduced pressure (warming, but not higher than 60 ° C.) to obtain a sample f (first fraction).

2. Test example for confirming hyaluronic acid synthesis promoting effect [Test Example 7]
Test Example 7 is a test example for confirming the hyaluronic acid synthesis promoting action on the first fraction, the third fraction, the fourth fraction and chrysin in the present invention, and measuring the hyaluronic acid synthesis promoting action. Went.

First, the test method will be described.
No. 11900-024, a minimum essential medium α (MEMα, purchased from GIBCO) containing 10% (V / V) fetal calf serum (FBS, purchased from Nichirei Biosciences, CCB) )It was used. Using this medium, human neonatal dermal fibroblasts (NB1RGB, purchased from RIKEN BioResource Center. Resource. No. RBRC-RCB022) were seeded in a 96-well plate at 2 × 10 ⁴ cells / well. After 24 hours, it was confirmed that the cells had adhered, and in order to remove serum in the medium, the cells were washed twice with FBS-free minimum essential medium α. Thereafter, 0.5% (V / V) fetal calf serum-containing minimum essential medium α, which is an assay medium, was added to all wells so as to be 100 μL / well. Then, 100 μL / well of a medium containing various samples (prepared to be twice the final concentration in the assay medium) and a medium not containing the sample (for control) were added, and further cultured for 72 hours. Went.

After completion of the culture, the amount of hyaluronic acid in the culture supernatant is measured using an ELISA measurement kit such as Quantikine ELISA Hyaluronan Immunoassay (R & D Systems) and an absorption microplate reader Multiskan Spectrum (Thermo Fisher Scientific Co., Ltd.). Was measured. The cell viability was measured by the neutral red method. The cell viability was set to 80% as a standard, and the evaluation was performed in the range where the concentration of the sample did not affect the cell viability any more. Since the measurement of the amount of hyaluronic acid by the ELISA method and the measurement of the cell viability by the neutral red method were carried out by conventional methods in accordance with the method of using the measurement kit and instrument, detailed explanation is omitted.

As the sample, the sample f of Test Example 6 was used as the first fraction, the sample c of Test Example 3 was used as the third fraction, the sample d of Test Example 4 was used as the fourth fraction, and the test example was used as chrysin. 5 samples e were used.

Next, test results will be described.
Table 1 is a table showing the results of Test Example 7 (hyaluronic acid synthesis promoting effect by each sample). In Table 1, “concentration” represents the concentration of each sample during culture, and the unit is μg / mL. The “activity amount” is the hyaluronic acid production amount when the hyaluronic acid production amount in the control is 100, and the unit is%. Furthermore, “cell viability” is the cell viability when the cell viability in the control is 100, and the unit is%.

[Table 1]

As shown in Table 1, it was confirmed that the first fraction, the third fraction, the fourth fraction and chrysin according to the present invention show hyaluronic acid synthesis promoting action on human newborn skin fibroblasts. did it. In addition, among the samples used in Test Example 7, it was confirmed that sample c (third fraction) and sample e (chrysin) tend to exhibit high hyaluronic acid synthesis promoting action.
In Test Example 7, the second fraction was not tested, but hyaluronic acid synthesis was promoted for all of the other fractions and chrysin, so the second fraction was also promoted for hyaluronic acid synthesis. Naturally, it is considered to have an action.

3. Test example for confirming HAS2 mRNA expression promoting action [Test Example 8]
Test Example 8 is a test example for confirming the HAS2 mRNA expression promoting action on the first fraction and chrysin in the present invention, and the HAS2 mRNA expression promoting action was measured.

First, the test method will be described.
As the seeding / passaging medium and the assay medium, the same media as in Test Example 7 were used. Using a seeding / passaging medium, human neonatal skin fibroblasts (similar to Test Example 7 above) are seeded in a 12-well plate at 1 × 10 ⁵ cells / well, and after 24 hours, 1 × The plate was washed once with a PBS (−) solution. Thereafter, the assay medium was added to all wells at 700 μL / well. Thereafter, 700 μL / well of a medium containing various samples (prepared to be twice the final concentration in the assay medium) and a medium not containing the sample (for control) were added. The culture time was divided into 6 stages of 4, 8, 12, 16, 20, and 24 hours for the same sample. After completion of the culture, the supernatant of each well was removed and washed with 1 mL of 1 × PBS (−) solution.

The RNA was extracted using NucleoSpin RNA (Takara Bio Inc.), a total RNA purification kit.
After washing with 1 × PBS (−) solution, a mixed reagent of Buffer RA1 (cell lysis buffer of total RNA purification kit) and 2-mercaptoethanol was added to each well. The amounts of Buffer RA1 and 2-mercaptoethanol were 350 μL and 3.5 μL every 2 wells. The mixed reagent was collected from each well, transferred to a sample separation tube (sterilized 1.5 mL tube for RNA), and each tube was stirred on a vortex mixer. Total RNA was extracted from the contents of the tube using a purification kit and stored at −80 ° C. Since RNA was extracted by a conventional method according to the protocol of the purification kit, detailed description is omitted.

For the total RNA thus extracted, the RNA concentration was measured from the absorbance (260 nm) using a SmartSpec Plus spectrophotometer (Bio-Rad). Reverse transcription reaction was performed using PrimeScript RT Master Mix (Perfect Real Time) (Takara Bio Inc.) with 500 ng of total RNA, and incubated at 37 ° C. for 15 minutes at 85 ° C. for 5 seconds using a heat block to obtain 10 μL of cDNA. . Next, 1 μL each of sense and antisense primers (10 μM) of each gene, 12.5 μL of SYBR Premix EX Taq II (Takara Bio Inc.), and RNase Free H ₂ O as a balance are added to ₂ μL of the prepared cDNA. Was 25 μL. 5′-GACAGGCATCTCACGACCG-3 ′ was used as a sense primer for HAS2, and 5′-CAACGGGTCTGCTGGTTTAGC-3 ′ was used as an antisense primer. As an internal standard, glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used. Real-time PCR was performed using Thermal Cycler Dice Real Time System II (Takara Bio Inc.), and the expression level of HAS2 mRNA was measured.
Also in Test Example 8, the cell viability was measured by the same method as in Test Example 7.

As the samples, the sample f of Test Example 6 was used as the first fraction, and the sample e of Test Example 5 was used as chrysin.

Next, test results will be described.
Table 2 is a table showing the results of Test Example 8 (HAS2 mRNA expression promoting effect by each sample). In Table 2, “concentration” represents the concentration of each sample during culture, and the unit is μg / mL. The “promotion rate” is the magnification of the HAS2 mRNA expression level when the HAS2 mRNA expression level in the control is 1, and the unit is double. As described above, the culture time of the medium containing the sample is divided into 6 stages of 4 to 24 hours, but Table 2 shows the results for the culture time with the highest acceleration rate. The incubation time is shown in parentheses after the acceleration rate value. “Cell viability” is the cell viability when the cell viability in the control is 100, and the unit is%.

[Table 2]

As shown in Table 2, it was confirmed that the first fraction and chrysin according to the present invention exhibited HAS2 mRNA expression promoting action on human newborn skin fibroblasts.
In Test Example 7, the second fraction to the fourth fraction were not tested. However, since the HAS2 mRNA expression promoting action was confirmed for all of the first fraction and chrysin, the second fraction The fourth fraction is naturally considered to have a HAS2 mRNA expression promoting action.

Example 1. Preparation of ointment An ointment (100 g) is prepared by a conventional method using the first fraction prepared according to the method described in Test Example 1 above, with the following formulation.

(Oil phase component)
0.01 g of the first fraction
White petrolatum 20.00g
Mineral oil 20.00g
Stearyl alcohol 5.00g
Steareth-2 3.00g
Propylparaben 0.10g
Natural vitamin E 0.10g
(Aqueous phase component)
1,3-butylene glycol 5.00g
Phenoxyethanol 0.40g
Polysorbate 60 4.50g
Purified water Appropriate amount 100g

(Preparation method)
The oil phase component and the water phase component were each heated to 80 ° C. to be uniform, and the water phase was added to the oil phase with stirring. After emulsification, the mixture was cooled to prepare an ointment.

Example 2 Production of Tape Agent Using the second fraction prepared according to the method described in Test Example 2 above, a tape agent (100 g) is produced according to a conventional method with the following formulation.

(Adhesive solvent)
Styrene-isopropylene-styrene block copolymer 7.00 g
Picolite 25.00g
Isopropylene rubber 5.00g
Toluene 15.00g
14.20 g of ethyl acetate
Hexane 25.00g
(Medicinal ingredients)
0.01 g of second fraction
Ethanol 7.9g
(Transdermal absorption enhancer)
Oleyl alcohol 0.80g
Total amount 100g

(Preparation method)
The adhesive solvent and the medicinal component were made uniform, the medicinal component and the transdermal absorption accelerator were added to the adhesive solvent, and the mixture was stirred at room temperature to prepare a composition. This composition is spread on a silicone-treated polyester film, dried at 120 ° C. and cooled, and then the pressure-sensitive adhesive layer is transferred to a polyethylene film to produce a tape agent.

Example 3 FIG. Production of Lotion Using the third fraction prepared according to the method described in Test Example 3 above, a lotion (100 g) is produced according to a conventional method with the following formulation.

(Oil phase component)
Polyoxyethylene (60 mol) hydrogenated castor oil 1.0 g
Squalane 0.05g
(Aqueous phase component)
Third fraction 0.1g
1,3-butylene glycol 5.0 g
Glycerin 5.0g
Phenoxyethanol 0.3g
Citric acid 0.1g
Sodium citrate 0.2g
Ethanol 2.0g
Purified water Appropriate amount 100g

(Preparation method)
The oil phase component and the water phase component are each dissolved uniformly. The oil phase is heated and added to a part of the aqueous phase with stirring, and the prepared liquid is added to the remaining aqueous layer to prepare a lotion.

Example 4 Preparation of emulsion Using the fourth fraction prepared according to the method described in Test Example 4 above, an emulsion (100 g) is prepared in the usual manner with the following formulation.

Polyoxyethylene (40 mol) hydrogenated castor oil 3.0 g
Squalane 5.0g
Carboxyvinyl polymer 0.1g
Potassium hydroxide 0.05g
Behenyl alcohol 1.0g
Citric acid 0.1g
Sodium citrate 0.2g
4th fraction 1.5g
1,3-butylene glycol 4.0 g
Glycerin 5.0g
Paraoxybenzoic acid ester 0.1g
Edetic acid disodium 0.01g
Purified water Appropriate amount 100g

(Preparation method)
An emulsion is prepared by stirring and mixing the above materials.

Embodiment 5 FIG. Preparation of cosmetic liquid Using the chrysin prepared according to the method described in Test Example 5 above, a cosmetic liquid (100 g) is prepared by a conventional method with the following prescription.

Chrysin 1.0g
Carboxyvinyl polymer 0.1g
2.0 g of polyglyceryl isostearate
Glycerin 6.5g
1,3-butylene glycol 7.5g
Phenoxyethanol 0.3g
Potassium hydroxide 0.05g
Sodium hyaluronate 0.1g
Squalane 1.0g
Vitamin E acetate 0.05g
Stearic acid 0.20g
Purified water Appropriate amount 100g

(Preparation method)
A cosmetic solution is prepared by stirring, mixing and dissolving the above materials.

Example 6 Preparation of tablets Using the first fraction prepared according to the method described in Test Example 6 above, tablets (500 mg per tablet) are prepared according to the following formulation.

1st fraction 10mg
Lactose 470mg
10mg dried corn starch
Talc 9mg
Calcium stearate 1mg

(Preparation method)
The first fraction (2 g), dried corn starch (2 g), talc (1.8 g) and calcium stearate (0.2 g) are added to and mixed with lactose (94 g). Subsequently, a tablet is produced by a conventional method using a single-shot tablet press.

Example 7 Production of Hard Capsule Using the first fraction prepared according to the method described in Test Example 6 above, a hard capsule (360 mg per capsule) is produced according to the following formulation.

1st fraction 5mg
Lactose 220mg
Corn starch 110mg
Hydroxypropylcellulose 25mg

(Preparation method)
Lactose (220 g) and corn starch (110 g) are added to and mixed with the first fraction (5 g), and an aqueous solution of hydroxypropylcellulose (25 g) is added thereto and kneaded. Next, granules are produced by an ordinary method using an extrusion granulator. A hard capsule is prepared by filling the granule into a gelatin hard capsule.

Example 8 FIG. Production of Soft Capsules Using the first fraction prepared according to the method described in Test Example 6 above, soft capsules (170 mg per capsule) are produced according to the following formulation.

1st fraction 5mg
Soybean oil 165mg

(Preparation method)
The first fraction (5 g) is added to soybean oil (165 g) and mixed. Next, soft capsules are prepared by filling the soft capsules using a rotary die type automatic molding machine according to a conventional method.

Example 9 Preparation of pills Using the first fraction prepared according to the method described in Test Example 6 above, pills (100 mg per tablet) are prepared according to the following formulation.

1st fraction 0.5mg
Morohaya powder 20.0mg
Starch 30.0mg
Molasses 20.0mg
Tea extract 15.0mg
Soy fiber 14.0mg
Shellac 0.5mg

(Preparation method)
After mixing the raw materials with the above blending, adding a suitable amount of water, producing a homogeneous kneaded product with a kneader, rolling the kneaded product, making a round shape using a round machine and drying it to produce a pill To do.

Example 10 Preparation of Jelly Using the first fraction prepared according to the method described in Test Example 6 above, a jelly (100 g) is prepared by a conventional method with the following formulation.

First fraction 0.02g
2.00 g of gelatin
Orange juice 20.00g
77.98 g of water

(Preparation method)
The above ingredients are mixed and heated to 90 ° C. After confirming the dissolution of gelatin, fill the container and cool. A jelly is prepared by solidifying gelatin.

Claims (12)

1. A hyaluronic acid synthesis promoter containing, as an active ingredient, a first fraction containing at least chrysin, which is a fraction extracted from the seeds of Sorisayanoki with an aqueous organic solvent.
2. When liquid-liquid distribution of the first fraction extracted from the seeds of Sorisayaki with aqueous organic solvent with water and ethyl acetate,
   A hyaluronic acid synthesis accelerator comprising, as an active ingredient, a fraction distributed to the ethyl acetate side and containing a second fraction containing at least chrysin.
3. The first fraction extracted from the seeds of Sorisaya tree with an aqueous organic solvent is subjected to liquid-liquid partition with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side is further liquidized with 90% methanol and hexane. -When dispensing liquid
   A hyaluronic acid synthesis accelerator containing, as an active ingredient, the third fraction that is distributed to the 90% methanol side and contains at least chrysin.
4. The first fraction extracted from the seeds of Sorisaya tree with an aqueous organic solvent is subjected to liquid-liquid partition with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side is further liquidized with 90% methanol and hexane. -Liquid partitioning, when the third fraction distributed to the 90% methanol side is fractionated by silica gel chromatography,
   A hyaluronic acid synthesis promoter containing, as an active ingredient, a fourth fraction that is eluted from silica gel with a 9: 1 mixed solvent of chloroform: methanol and contains at least chrysin.
5. Hyaluronic acid synthesis accelerator containing chrysin as an active ingredient.
6. HAS2 mRNA expression promoter containing, as an active ingredient, a first fraction containing at least chrysin, which is a fraction extracted from the seeds of Sorisaya tree using an aqueous organic solvent.
7. When liquid-liquid distribution of the first fraction extracted from the seeds of Sorisayaki with aqueous organic solvent with water and ethyl acetate,
   A HAS2 mRNA expression promoter containing, as an active ingredient, a second fraction that is distributed to the ethyl acetate side and contains at least chrysin.
8. The first fraction extracted from the seeds of Sorisaya tree with an aqueous organic solvent is subjected to liquid-liquid partition with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side is further liquidized with 90% methanol and hexane. -When dispensing liquid
   A HAS2 mRNA expression promoter containing, as an active ingredient, the third fraction that is distributed to the 90% methanol side and contains at least chrysin.
9. The first fraction extracted from the seeds of Sorisaya tree with an aqueous organic solvent is subjected to liquid-liquid partition with water and ethyl acetate, and the second fraction distributed on the ethyl acetate side is further liquidized with 90% methanol and hexane. -Liquid partitioning, when the third fraction distributed to the 90% methanol side is fractionated by silica gel chromatography,
   A HAS2 mRNA expression promoter containing, as an active ingredient, a fourth fraction that is eluted from silica gel with a 9: 1 mixed solvent of chloroform: methanol and contains at least chrysin.
10. HAS2 mRNA expression promoter containing chrysin as an active ingredient.
11. A pharmaceutical, food, or cosmetic having hyaluronic acid synthesis promoting action, comprising the hyaluronic acid synthesis promoting agent according to any one of claims 1 to 5.
12. A pharmaceutical, food, or cosmetic having a HAS2 mRNA expression promoting action, comprising the HAS2 mRNA expression promoting agent according to any one of claims 6 to 10.

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