JP6958344B2 - Elastase activity inhibitor - Google Patents

Description

The present invention relates to an elastase activity inhibitor containing a grape fruit extract and polymethoxyflavone as active ingredients.

Wrinkles and sagging of the skin are formed due to a decrease in the elasticity of the skin, and it is considered that the factors such as aging, dryness, oxidation, and ultraviolet rays are involved. Specifically, changes in the quality and quantity of extracellular matrix components such as elastin, collagen, and glycosaminoglycan in the dermis can be mentioned, and it has been clarified that changes in elastin are particularly important. Elastin fibers are highly elastic and give the skin moderate flexibility, but overexpression of elastase, an enzyme that decomposes elastin, due to aging and ultraviolet rays causes the decomposition of elastin and its amount decreases. It is thought to cause reduced elasticity, sagging and wrinkles of the skin. Therefore, it is considered that inhibiting elastase activity can prevent a decrease in skin elasticity, wrinkles and sagging.

As an elastase activity inhibitor, plant-derived components have been well studied. For example, as grape-derived components, resveratrol (Patent Document 1) obtained from grape skin and grape seed extract (Patent Document 2) have been studied. ), Red grape leaf extract (Patent Document 3) has been reported to have an elastase activity inhibitory effect.

However, the effects of the elastase activity inhibitor have not yet been satisfied, and a safe and excellent elastase activity inhibitor is desired.

Japanese Unexamined Patent Publication No. 2008-088123 Japanese Unexamined Patent Publication No. 2003-002820 Japanese Unexamined Patent Publication No. 2010-12586

An object of the present invention is to provide a safe and highly effective elastase activity inhibitor containing a plant-derived component.

As a result of diligent research to solve the above problems, the present inventors have found that the combination of grape fruit extract and polymethoxyflavon synergistically enhances the elastase activity inhibitory effect, and further research is conducted. Again, the present invention has been completed.

That is, the present invention includes the following aspects.
[1] An elastase activity inhibitor containing (a) an extract of grape fruits and (b) a polymethoxyflavone represented by the formula [I] as an active ingredient:

(In the formula, R ¹ , R ² , and R ³ independently represent a hydrogen atom or a methoxy group).
[2] (a) The inhibitor according to [1], wherein the grape fruit is a grape fruit containing seeds.
[3] (a) The inhibitor according to [1] or [2], wherein the grape fruit extract is an extract of water or a hydrophilic organic solvent.
[4] The polymethoxyflavones represented by the formula [I] in (b) are 3,5,6,7,8,3', 4'-heptamethoxyflavones, 5,6,7,8,3', The inhibitor according to any one of [1] to [3], which is at least one selected from the group consisting of 4'-hexamethoxyflavone and 5,6,7,8,4'-pentamethoxyflavone.
[5] The inhibitor according to any one of [1] to [4], wherein the mass ratio (a): (b) of (a) and (b) is 5: 1 to 1: 5.

Since the elastase activity inhibitor of the present invention suppresses the decomposition of elastin, it can be used for the prevention / improvement of sagging and wrinkles, which are skin aging phenomena.
Since the elastase activity inhibitor of the present invention is derived from a plant, it can be safely used for a long period of time.

The present invention relates to an elastase activity inhibitor containing (a) an extract of grape fruit and (b) a polymethoxyflavone represented by the following formula [I] as an active ingredient.

(In the formula, R ¹ , R ² , and R ³ independently represent a hydrogen atom or a methoxy group.)

(A) Extracts of grape fruits The grapes used in the present invention belong to the genus Grapevine, and examples thereof include European grapes (Vitis Vinifera) and American grapes (Vitis Labrusca). Among them, grapes containing seeds in fruits are preferable from the viewpoint of having an excellent elastase activity inhibitory effect, and examples thereof include varieties such as Niagara, Kyoho, Benifuji, Benizu, Rosario Bianco, and Rosario Rosso.

The grape fruit extract, which is the active ingredient of the present invention, is an extract extracted from the fruit, but the extract extracted from the fruit residue (flesh fiber, pericarp and seed of the fruit) after squeezing the juice from the fruit is used. preferable. As the grapes, raw, dried and frozen grapes can be used, but dried grapes are preferable from the viewpoint of preservation.

The solvent used for extraction is a polar solvent, and examples thereof include water and a hydrophilic organic solvent.
Examples of the hydrophilic organic solvent include hydrocarbons such as hexane and benzene, esters such as ethyl acetate, ketones such as acetone, ethers such as diethyl ether and tetrahydrofuran, and halogenated hydrocarbons such as chloroform and methylene chloride. Examples thereof include alcohols such as lower alcohols and polyhydric alcohols. Among them, from the viewpoint of extraction efficiency, lower alcohols such as water, methanol, ethanol, propanol and isopropanol, and polyhydric alcohols such as propylene glycol, dipropylene glycol, 1,3-butylene glycol and glycerin are preferable, and water, ethanol, 1, 3-butylene glycol is more preferred.
In the present invention, one type of the extraction solvent may be selected and used alone, or two or more types may be used in combination.

The extraction treatment with a solvent can be carried out with respect to 100 parts by weight of the grape fruit residue, for example, when the extraction solvent is water, it can be extracted with usually 100 to 2000 parts by weight, preferably 200 to 1000 parts by weight. Similarly, when the extraction solvent is a lower alcohol, extraction can be usually carried out in an amount of 100 to 5000 parts by weight, preferably 500 to 2000 parts by weight.

The extraction temperature is appropriately determined according to the type of extraction solvent used. For example, when the extraction solvent is water, the extraction temperature is usually about 20 ° C. to 120 ° C., and when the extraction solvent is a lower alcohol, it is usually about 30 ° C. to 80 ° C.

As an extraction method, an extract can be obtained by adding an extraction solvent to a fruit residue containing grape seeds, immersing the fruit residue, and then filtering the residue. It may be immersed in an extraction solvent and stirred or the like.

The extraction time (immersion time) is usually 1 hour or more, preferably 3 hours or more, and more preferably 5 hours or more from the viewpoint of extraction efficiency. The upper limit of the extraction time is usually 10 hours or less, preferably 8 hours or less, from the viewpoint of extraction efficiency.

The number of extractions is not particularly limited, but it is preferable to extract once with the above amount of water or a hydrophilic organic solvent.

The obtained extract may be used as it is, or may be concentrated by distilling off a solvent, and if necessary, purified by treatment such as column chromatography or solvent fractionation, or may be dried and used. .. From the viewpoint of handling, it may be dissolved in a solvent such as water or alcohol. One type of the diluting solvent may be selected and used alone, or two or more types may be used in combination.

(B) Polymethoxyflavone The component (b) used in the present invention is a polymethoxyflavone represented by the formula [I]. In the formula, R ¹ , R ² , and R ³ independently represent a hydrogen atom or a methoxy group, respectively.

The component (b) of the present invention includes 3,5,6,7,8,3', 4'-heptamethoxyflavone (heptamethoxyflavone) represented by the following formulas [II] to [IV], 5 , 6,7,8,3', 4'-hexamethoxyflavone (nobiletin), 5,6,7,8,4'-pentamethoxyflavone (tangeletin) at least one poly selected from the group. Methoxyflavones are preferred.

The polymethoxyflavones represented by the formulas [II], [III] and [IV] can be obtained by various synthetic methods, and can also be obtained by solvent extraction as a natural product from plants.

Examples of the method of obtaining the component (b) from a plant by solvent extraction include a method of extracting from the peel of a plant belonging to the genus Citrus of the Rutaceae family.
Examples of Rutaceae Citrus plants include Satsuma mandarin, Shikuwasa, Ponkan, Hassaku, Lemon, Tachibana, Yuzu, Sudachi, Pomelo, Tangerine, Orange, Grapefruit, etc., which contain polymethoxyflavon. (Journal of Medicinal Plant Research Vol. 46, 162-166, (1982)). From the viewpoint of the content of polymethoxyflavone, the plants used for the solvent extraction are preferably unshu mikan, shikuwasa, tachibana, yuzu, sudachi and orange, and more preferably shikuwasa, orange and tachibana.

Isolation and purification of polymethoxyflavones can be carried out by extracting the peels of these Rutaceae plants of the genus Citrus with a solvent and separating the extracts by column chromatography. Hereinafter, a specific description will be given.

First, the peel of a citrus plant is extracted using a solvent. Examples of such a solvent include water; a water-soluble alcohol such as methanol, ethanol, propanol, butanol, propylene glycol, and 1,3-butylene glycol; a solvent such as acetone, hexane, ethyl acetate, and chloroform; or a mixed solvent thereof. Can be mentioned. The preferred solvent is one or more solvents selected from the group consisting of methanol, ethanol, propanol, butanol, propylene glycol, 1,3-butylene glycol and ethyl acetate. More preferred solvents are one or more solvents selected from the group consisting of methanol, ethanol, propylene glycol, 1,3-butylene glycol and ethyl acetate.

The amount of the solvent used for extraction is 1 to 10 times by mass, preferably 2 to 6 times by mass, that of the peel (dried product) of a citrus plant. The temperature at the time of extraction is usually room temperature (1 to 30 ° C.), preferably room temperature (15 to 25 ° C.). The extraction period is, for example, 1 to 30 days, preferably 5 to 20 days.
Next, the extract is concentrated to obtain an extract containing about 1 to 15% by mass of the desired polymethoxyflavone. The extract may be further dried. This extract is dissolved in ethyl acetate having a mass of 2 times or more, preferably 3 to 10 times, water is added, the mixture is stirred and separated, the aqueous layer is removed, and ethyl acetate is distilled off to dryness. You can get things.

In the present invention, any one of the isolated heptamethoxyflavones, nobiletin, and tangeretin may be used alone, or two or more of these may be mixed and used. Further, it may be used as it is as a mixture at the time of synthesis or in a form containing a plurality of natural products such as plant extracts.

The obtained dry matter is usually redissolved in a suitable solvent and subjected to liquid column chromatography to obtain nobiletin, heptamethoxyflavone, and tangeretin. In liquid column chromatography, for example, a mixed solvent in which silica gel or alumina is used as a filler and hexane / ethanol is mixed at a volume ratio of 70/30 to 97/3 is used as an eluent. From the viewpoint of handling, it may be dissolved in a solvent such as water or alcohol. One type of the diluting solvent may be selected and used alone, or two or more types may be used in combination.

The elastase activity inhibitor of the present invention is a combination of the component (a) and the component (b) (that is, a concomitant agent).

In the elastase activity inhibitor of the present invention, the ratio of the combination of the component (a) and the component (b) is preferably 5: 1 to 1: 5 (mass ratio) in terms of dry weight. More preferably 1: 2 to 2: 1 (mass ratio).

In the elastase activity inhibitor of the present invention, the content of the component (a) is usually 10 to 80% by mass, preferably 15 to 75% by mass, based on the total amount of the inhibitor, by dry weight. It is preferably 30 to 60% by mass.
The content of the component (b) is usually 10 to 80% by mass, preferably 15 to 75% by mass, and more preferably 30 to 60% by mass with respect to the total amount of the inhibitor.

Since the elastase activity inhibitor of the present invention prevents or improves the decrease in elasticity of the skin, it is used for the prevention or improvement of wrinkles and sagging.

The elastase activity inhibitor of the present invention can be used as a pharmaceutical product, a quasi-drug, or a cosmetic product, but is preferably used as a quasi-drug product or a cosmetic product. In addition to the components (a) and (b), the elastase activity inhibitor of the present invention contains other components used in the fields of pharmaceuticals, quasi-drugs and cosmetics, such as solvents and excipients. can do.

In addition to the components (a) and (b), the external preparations include water-soluble components, oil-based components, powder components, surfactants, polymer components, thickeners, adhesive improvers, film-forming agents, and pH. Regulators, antioxidants, preservatives, preservatives, excipients, moisturizers, skin protectants, refreshers, fragrances, colorants, chelating agents, abundant agents, antipruritic agents, blood circulation promoters, astringents, tissues Restoration accelerators, antiseptic agents, plant extract components, animal extract components, additives necessary for cosmetics, non-pharmaceutical products, etc. can be blended as needed.

Examples of the present invention will be described below, but the present invention is not limited thereto.

(A) Ingredients Grape fruit (with seeds) extract The fruits of grapes with seeds (variety name: Niagara) were harvested, washed with water, and then squeezed to obtain fruit juice to obtain fruit residues. 50 g of the dried product of the fruit residue is immersed in 500 g of a 50 mass% 1,3-butylene glycol (BG) aqueous solution as an extraction solvent at 60 ° C. for 5 hours, and then filtered using a filter paper (5C manufactured by Advantech Toyo Co., Ltd.). And obtained a filtrate. This filtrate was allowed to stand at 5 ° C. for 7 days and then filtered using a filter paper to obtain a fruit extract containing grape seeds. The content of the solid content in this grape fruit extract excluding the extraction solvent is 1.9% by mass, and this is diluted with a 50% by mass BG aqueous solution to adjust the solid content to 1.0% by mass. Was used in the test as a fruit extract containing grape seeds.

(A') Ingredients Grape fruit (seedless) extract Seedless grapes (variety name: Pione) obtained by the same method as in Example 1 50 g of dried fruit residue was extracted as an extraction solvent for BG of 50% by mass. After immersing in 500 g of an aqueous solution at 60 ° C. for 5 hours, the mixture was filtered using a filter paper (5C manufactured by Advantech Toyo Co., Ltd.) to obtain a filtrate. This filtrate was allowed to stand at 5 ° C. for 7 days and then filtered using a filter paper to obtain a grape fruit (seedless) extract. The content of the solid content in this grape fruit (without seeds) extract excluding the extraction solvent is 1.8% by mass, and this is diluted with a 50% by mass BG aqueous solution to reduce the solid content to 1.0% by mass. Was used in the test as a grape fruit (seedless) extract.

(B) Isolation and purification of the component (polymethoxyflavone) will be described.
100 kg of dried peel of Rutaceae orange was crushed with a blender and immersed in 5 times the mass of 95% ethanol for 5 days for extraction. The extract was filtered and concentrated under reduced pressure to obtain 450 g of orange peel extract. This orange peel extract was dissolved in 5 times by mass of ethyl acetate, an equal amount of water was added to ethyl acetate, and the mixture was stirred and allowed to stand. After separation, the aqueous layer was removed. After repeating this operation three times, ethyl acetate was distilled off to obtain 280 g of a dry product.
Further, this dry matter was dissolved in a double mass hexane / ethanol mixed solvent (volume ratio 85/15) to obtain a solution. A column (diameter 30 cm, length 1 m) filled with silica gel was fully charged with this solution. After flowing hexane having a volume twice the column volume through this column, a hexane / ethanol mixed solvent (volume ratio 90/10) was used as an eluent, and the eluate was fractionated by 1 L.
Each of these fractions was subjected to silica gel thin layer chromatography using a hexane / ethanol mixed solvent (volume ratio 90/10) as a developing solvent and analyzed. As a result, they are represented by the above formulas [II] to [IV] 3 The presence of the species compound was confirmed. Then, the fractions containing the respective compounds are combined, and then the solvent is distilled off, and the compound 1 represented by the formula [II], the compound 2 represented by the formula [III], and the compound represented by the formula [IV] are represented. Compound 3 was obtained. The yields of compounds 1 to 3 were 13 g for compound 1 (heptamethoxyflavone), 9.2 g for compound 2 (nobiletin), and 2.2 g for compound 3 (tangeretin). In this way, three types of polymethoxyflavones were isolated and purified.

[Elastase activity inhibition test]
For the elastase substrate (N-Suc-Ala-Ala-Ala-p-nitronilide, manufactured by ELASTIN PRODUCTS COMPANY), DMSO was added to prepare a 0.1 M elastase substrate stock solution, and 0.1 M Tris was added to 100 μL of the elastase substrate stock solution. -HCl buffer (pH 8.3) 9900 μL was added to prepare a 1 mM elastase substrate solution. Elastase derived from porcine pancreas (ELASTIN PRODUCTS COMPANY) was prepared by adding 0.05 M sodium acetate buffer (pH 5.0) to make a 10 units / mL elastase stock solution, and adding 0.1 M Tris-HCl buffer (0.1 M Tris-HCl buffer) to 50 μL of the elastase stock solution. pH 8.3) 4950 uL was added to prepare a 0.1 units / mL elastase solution. Samples were prepared on a 96-well plate for dilution using 0.1 M Tris-HCl buffer (pH 8.3) in a 1/2 serial dilution series. 100 μL of 1 mM elastase substrate solution was added to all holes of the test 96-well plate. Next, 50 μL of the serial dilution series of the sample prepared on the 96-well plate for dilution was added to the 96-well plate for test. However, 50 μL of 0.1 M Tris-HCl buffer (pH 8.3) was added to the control. After incubating at 37 ° C. for 10 minutes, 50 μL of 0.1 units / mL elastase solution was added to the test 96-well plate. Immediately, the starting 405 nm absorbance was measured with a microplate reader. After incubating at 37 ° C. for 30 minutes, the absorbance at 405 nm was measured after 30 minutes. The elastase activity inhibition rate (%) was calculated by the following formula. The results are shown in Tables 1 and 2.

Elastase activity inhibition rate (%) = {1- (AB) / (CD)} × 100
A: Absorbance 30 minutes after the sample is added B: Absorbance immediately after the sample is added C: Absorbance 30 minutes after the control D: Absorbance at the start of the control

In Examples 1 to 3 in which a fruit extract containing grape seeds and 3,5,6,7,8,3', 4'-heptamethoxyflavone, which is a kind of polymethoxyflavone, were combined, an elastase activity inhibitory effect was obtained. It was confirmed that the effect of inhibiting elastase activity was dramatically improved as compared with the case of the fruit extract containing grape seeds known to be used alone (Comparative Examples 1 to 3). As for polymethoxyflavones, 5,6,7,8,3', 4'-hexamethoxyflavones and 5,6,7,8,4'-pentamethoxyflavones are also fruit extracts containing grape seeds. It was also confirmed that the elastase activity inhibitory effect was dramatically improved when combined (Examples 4 to 5).

On the other hand, polymethoxyflavone alone (Comparative Examples 4 to 6) did not confirm the elastase activity inhibitory effect. In Comparative Example 7 in which a seedless grape fruit extract and polymethoxyflavone were combined, and in Comparative Example 8 in which resveratrol and polymethoxyflavone contained in grape skin, which is known to have an elastase activity inhibitory effect, were combined. No excellent elastase activity inhibitory effect was confirmed as compared with Example 2 in which a fruit extract containing seeds and polymethoxyflavone were combined.

The elastase activity inhibitor of the present invention can be used as a skin external preparation or cosmetic for the purpose of preventing wrinkles and sagging.

Claims (4)

An elastase activity inhibitor containing (a) an extract of grape fruit and (b) a polymethoxyflavone represented by the formula [I] as an active ingredient:

(In the formula, R ¹ , R ² , and R ³ independently represent a hydrogen atom or a methoxy group). (A) The inhibitor according to claim 1, wherein the grape fruit is a grape fruit containing seeds. The polymethoxyflavone represented by the formula [I] is 3,5,6,7,8,3', 4'-heptamethoxyflavone, 5,6,7,8,3', 4'-. The inhibitor according to claim 1 or 2 , which is at least one selected from the group consisting of hexamethoxyflavone and 5,6,7,8,4'-pentamethoxyflavone. The inhibitor according to any one of claims 1 to 3 , wherein the mass ratio (a): (b) of (a) and (b) is 5: 1 to 1: 5.