JP2010120946A - Antiinflammatory composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide antiinflammatory compositions (pharmaceutical compositions, food compositions, fodder compositions and cosmetic compositions) containing highly safe plant extracts as active ingredients. <P>SOLUTION: Ampelopsis extracts, preferably ampelopsis extracts provided by extraction treatment using a water-soluble organic solvent, a hydrate water-soluble organic solvent or water as the extraction solvent, are added to antiinflammatory compositions. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an anti-inflammatory composition containing a highly safe plant extract as an active ingredient.

Grapevine is a grapevine plant with the scientific name Ampelopsis brevipedunculata Trautv. Also known as gabion, it is a deciduous vine plant that grows naturally in Hokkaido, Okinawa, China, and the Korean Peninsula.
In China, the leaves and vines of vine grapes are said to be effective for diuresis and gastric fever, and have been decocted for a long time.

In addition, a preventive and therapeutic agent for cirrhosis consisting mainly of a leachate obtained by leaching no grapefruit with alcohol (see Patent Document 1), a methanol-cold extract of no grape root with an ethyl acetate-water mixed solvent, and ethyl acetate A therapeutic agent for liver disease comprising a vine grape root extract obtained by concentrating a layer (see Patent Document 2), a fibrosis inhibitor (see Patent Document 3) containing an effective amount of a gallic acid ester contained in vine grape, and an extract of vine grape There are known immunomodulators (see Patent Document 4) and the like mainly composed of.
However, the antioxidant effect, anti-skin aging effect, anti-inflammatory effect and lipid metabolism improving effect of no grape extract are not known.

Japanese Patent Publication No. 60-7974 Japanese Patent Laid-Open No. 2-48533 Japanese Patent Laid-Open No. 5-271667 JP-A-8-3052

  It is an object of the present invention to find a substance having a hyaluronidase inhibitory action and / or a cyclic AMP phosphodiesterase inhibitory action from highly safe natural products, and to provide an anti-inflammatory composition containing the substance as an active ingredient. .

  In order to achieve the above-mentioned object, the present invention provides an anti-inflammatory composition containing no grape extract as an active ingredient.

  In the anti-inflammatory composition of the present invention, it is preferable that the grapevine extract has a hyaluronidase inhibitory action and / or a cyclic AMP phosphodiesterase inhibitory action. In the anti-inflammatory composition of the present invention, it is preferable that the grape vine extract is obtained by an extraction treatment using a water-soluble organic solvent, a water-containing water-soluble organic solvent, or water as an extraction solvent. Moreover, it is preferable that the anti-inflammatory composition of this invention is a pharmaceutical composition, food-drinks composition, a feed composition, or a cosmetic composition.

  ADVANTAGE OF THE INVENTION By this invention, the anti-inflammatory composition which contains a highly safe no grape extract as an active ingredient is provided.

Hereinafter, the present invention will be described in detail.
In the present invention, the “no grape extract” refers to an extract obtained using no grape as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crudely purified product thereof. Alternatively, any purified product is included.

  The part of wild grape used as the extraction raw material is not particularly limited. For example, the above-ground part can be used, and among them, one or more of stem part, vine part and leaf part are used. It is preferable.

  The grapes used as the raw material for extraction are suitably dried and pulverized immediately after collection. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pre-processing, such as degreasing, with nonpolar solvents, such as hexane and benzene, you may use as an extraction raw material. By performing a pretreatment such as degreasing, the extraction process with the polar solvent of No grape can be efficiently performed.

  In the extraction process, it is preferable to use a polar solvent as the extraction solvent. Ingredients showing antioxidative action, skin aging preventive action, anti-inflammatory action or lipid metabolism improving action contained in grapevine can be easily extracted by extraction treatment using a polar solvent as an extraction solvent.

  Specific examples of the polar solvent include water and water-soluble organic solvents, and these can be used alone or in combination of two or more. Of these polar solvents, it is preferable to use a water-soluble organic solvent or a water-containing water-soluble organic solvent.

  Water that can be used as the extraction solvent includes pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Examples of water-soluble organic solvents that can be used as the extraction solvent include lower aliphatic alcohols, acetone, tetrahydrofuran, and the like. Specific examples of lower aliphatic alcohols include monohydric alcohols such as methanol, ethanol, propanol, and butanol; 1 , 3-butylene glycol, glycerin, propylene glycol, isopropylene glycol, and other polyhydric alcohols.
As the extraction solvent, an intermediate polar solvent such as ethyl acetate, butyl acetate or methyl cellosolve can be used.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when a water-containing water-soluble organic solvent is used, the content of monohydric alcohol, polyhydric alcohol, acetone, tetrahydrofuran or the like is preferably 10% by weight or more.

  The extraction treatment is not particularly limited as long as the soluble component contained in wild grape can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, vine grapes are immersed in an extraction solvent 5 to 15 times the weight of the extraction raw material (weight ratio), and soluble components are eluted at room temperature or under reflux heating, followed by filtration to remove the extraction residue to obtain an extract. be able to. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed. Purification can be performed by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, liquid-liquid countercurrent distribution treatment, or the like. The obtained extract can be used as it is as an active ingredient of an antioxidant composition, a composition for preventing skin aging, an anti-inflammatory composition or a composition for improving lipid metabolism. It is easier to use.

  The grapevine extract can exert an antioxidant action based on the active oxygen scavenging action and / or the radical scavenging action. Here, “active oxygen” includes superoxide, hydrogen peroxide, hydroxy radical, singlet oxygen and the like. “Radical” means a molecule or atom having one or more unpaired electrons, and includes superoxide, hydroxy radical, DPPH and the like.

  Since the grape extract can exert an antioxidant action, it can be used as an active ingredient of an antioxidant composition. The grapevine extract has an active oxygen scavenging action or a radical scavenging action, and can therefore be used as an active ingredient of the active oxygen scavenging composition or radical scavenging composition.

  The antioxidant composition of the present invention has increased active oxygen concentration in the body (for example, increased active oxygen concentration due to excessive production of active oxygen in the body, increased active oxygen concentration due to decreased superoxide dismutase (SOD) action in the elderly). It is possible to effectively prevent and / or ameliorate diseases caused by such as rheumatoid arthritis, myocardial infarction, diabetes, nephritis, stiff shoulders, coldness and the like. Moreover, the antioxidant composition of the present invention can effectively prevent alteration of components that are oxidized or deteriorated by the action of active oxygen, such as oils and fats blended in drugs, foods and drinks, cosmetics and the like.

  Since the skin is directly affected by environmental factors such as ultraviolet rays, it is an organ that is prone to generate active oxygen. Therefore, for some reason, the active oxygen concentration rises if the body's antioxidant action does not work sufficiently, resulting in melanin. Skin aging phenomena such as abnormal production, wrinkles, sagging, and rough skin occur. In addition, when the cell growth ability of the skin becomes slow due to aging, the renewal of cells becomes difficult, and the supply of intercellular components such as collagen produced by the cells becomes insufficient, resulting in wrinkles, sagging, dullness. The skin aging phenomenon occurs. Furthermore, when elastin involved in skin elasticity is decomposed by elastase, which is a hydrolase, the elasticity of the skin decreases, and skin aging phenomena such as wrinkles and sagging occur. Therefore, the grapevine extract exhibits a skin aging preventing action based on one or more actions selected from the group consisting of an active oxygen scavenging action, a radical scavenging action, a fibroblast proliferation action and an elastase inhibitory action. Can do.

Since the grape extract can exert an anti-skin aging effect, it can be used as an active ingredient in a composition for preventing skin aging. In addition, since a grapevine extract has a fibroblast proliferation action or an elastase inhibitory action, it can also be utilized as an active ingredient of the composition for fibroblast proliferation or the composition for elastase inhibition.
The composition for preventing skin aging according to the present invention can effectively prevent and / or improve skin aging phenomena such as reduced skin elasticity, wrinkles, sagging and rough skin.

  Hyaluronidase is normally inactive in various tissues in the body, but when activated, it increases the permeability of capillaries, increases hyaluronidase activity in various inflammations, and is experimentally acute with hyaluronidase. It has been confirmed that hyaluronidase is involved in the inflammatory reaction due to the fact that an inflammation model can be prepared, and hyaluronidase is also called a pro-inflammatory enzyme. In addition, cyclic AMP is degraded by cyclic AMP phosphodiesterase, and when the cyclic AMP concentration is lowered, platelet aggregation occurs, and platelet aggregation leads to activation of phospholipase A2 of the arachidonic acid cascade, and thus released leukotriene B4 and prostas. Grungein E2 etc. cause an inflammatory reaction. Therefore, the grapevine extract can exert an anti-inflammatory action based on a hyaluronidase inhibitory action and / or a cyclic AMP phosphodiesterase inhibitory action.

Since grapevine extract can exert an anti-inflammatory action, it can be used as an active ingredient of an anti-inflammatory composition. In addition, since the grapevine extract has a hyaluronidase inhibitory action or a cyclic AMP phosphodiesterase inhibitory action, it can also be used as an active ingredient of a hyaluronidase inhibitory composition or a cyclic AMP phosphodiesterase inhibitory composition.
The anti-inflammatory composition of the present invention can effectively prevent and / or improve inflammatory diseases such as contact dermatitis (rash), psoriasis, pemphigus vulgaris and the like.

When cyclic AMP involved in the promotion of lipid metabolism is degraded by cyclic AMP phosphodiesterase, lipid metabolism is suppressed, resulting in excessive accumulation of body fat. Therefore, the grape extract can exert a lipid metabolism improving action based on the cyclic AMP phosphodiesterase inhibitory action. That is, the grapevine extract can exert a lipid metabolism improving action by inhibiting cyclic AMP phosphodiesterase activity, increasing intracellular cyclic AMP concentration, and activating lipid metabolism.
Since the grape extract can exert an action of improving lipid metabolism, it can be used as an active ingredient of a composition for improving lipid metabolism.
The composition for improving lipid metabolism of the present invention can prevent and / or improve obesity by improving lipid metabolism.

  In each composition of the present invention, the “active ingredient” means a component having an antioxidant action, an anti-skin aging action, an anti-inflammatory action or a lipid metabolism improving action, and the grape extract is used in each composition of the present invention. It is not necessary to be a main component, and each composition of the present invention may contain an ingredient having an antioxidative action, an anti-skin aging action, an anti-inflammatory action or a lipid metabolism improving action in addition to the grape extract. .

  The form of each composition of the present invention is not particularly limited as long as the intended effect can be exhibited. Specific examples thereof include a pharmaceutical composition, a food and drink composition, a feed composition, and a cosmetic composition. Thing etc. are mentioned. Components other than the grape extract in each composition of the present invention can be appropriately selected according to the form of the composition.

  Examples of the pharmaceutical composition include an internal or external pharmaceutical product or quasi-drug (ie, internal preparation or external preparation), an additive added to a food or drink, a cosmetic, and the like. In the pharmaceutical composition, for example, vinegar extract is mixed with a pharmaceutically acceptable excipient or any other auxiliary agent (for example, binder, disintegrant, lubricant, stabilizer, flavoring / flavoring agent, etc.). Can be manufactured. Examples of the dosage form of the pharmaceutical composition include powders, granules, tablets, solutions, sprays, capsules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. The route of administration of the pharmaceutical composition can be appropriately selected based on the dosage form, etc., and the dosage and the number of administrations are appropriately adjusted according to the intended effect, administration method, treatment period, age, weight, etc. be able to.

  Food / beverage composition and feed composition can be obtained from, for example, sugars such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; cellulose and gum arabic and the like It can be produced by blending oils and fats such as soybean oil and medium chain fatty acid triglycerides.

  The food / beverage product composition can also be produced by blending the grape extract in any food / beverage product that does not interfere with its activity. The food and drink that can be mixed with the grape extract is not particularly limited, but specific examples thereof include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks (concentrated concentrates and adjustment powders of these drinks). Ice cream, ice sherbet, shaved ice, etc .; noodles such as buckwheat, udon, harusame, gyoza skin, shumai skin, chinese noodles, instant noodles; rice cake, chewing gum, candy, gum, chocolate, tablet confectionery, Sweets such as snacks, biscuits, jelly, jam, cream, baked goods; processed fishery and livestock products such as kamaboko, ham, sausage; dairy products such as processed milk and fermented milk; salad oil, tempura oil, margarine, mayonnaise, shortening , Whipped cream, oil and fat processed food such as dressing; seasonings such as sauce and sauce; soup, Chu, salad, prepared foods, pickles, bread and the like. The food / beverage composition of the present invention can be easily eaten or consumed when processed into a form such as a beverage, a tablet, a hard capsule, a soft capsule, or a granule. The grape extract may be blended in foods and drinks that do not contain lipids, but is preferably blended in foods and drinks that contain lipids. The food / beverage product composition produced in this way can be used not only as a food / beverage product for anti-obesity but also as a food / beverage product for beauty such as beautiful skin and slimming, that is, a food / beverage product for beauty.

  A feed composition can also be manufactured by mix | blending a grape extract with arbitrary feed which does not prevent the activity. The feed to which the grape extract can be blended is not particularly limited, and specific examples thereof include pet food, livestock feed, fish feed and the like.

Cosmetic compositions can be obtained from grape extract, such as astringents, bactericides / antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-inflammatory / antiallergic agents, antioxidant / active oxygen scavengers, , Waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, and the like.
The cosmetic composition can also be produced by blending the grape extract with any cosmetic that does not interfere with its activity. Cosmetics to which the grape extract can be blended are not particularly limited, and specific examples thereof include skin cosmetics such as ointments, creams, emulsions, lotions, packs, bathing agents, lip balms, and lipsticks.

  The compounding amount of the grape extract in each composition of the present invention can be appropriately adjusted according to the form of the composition, the activity of the extract and the like. For example, when a standard grape extract is blended with the pharmaceutical composition, the preferred blending rate is 1 to 100% by weight, and the particularly preferred blending rate is 10 to 50% by weight. When a standard grape extract is blended in the composition, the preferred blending ratio is 0.01 to 50% by weight, and the particularly preferred blending ratio is 0.1 to 10% by weight. When blending a standard grape extract, a suitable blending ratio is 0.001 to 1% by weight, a particularly preferable blending ratio is 0.01 to 0.1% by weight,

Hereinafter, although this invention is demonstrated further in detail based on a manufacture example, a test example, and a compounding example, this invention is not limited to these content.
[Production Example 1]
100 g of dried pulverized leaves and vines were immersed in 1 L of 30% ethanol, 50% ethanol, and 80% ethanol and heated under reflux for 2 hours. Thereafter, the residue was filtered and the residue was again treated with 1 L of ethanol in the same manner. The extracts obtained by the above two treatments were combined and concentrated under reduced pressure to obtain 14 g, 9.9 g, and 7.8 g of a water-containing ethanol extract.

[Production Example 2]
100 g of dried pulverized product of leaves and vines were extracted with 1 L of hot water for 2 hours. Then, it filtered and the residue was similarly processed with 1 L hot water again. The extracts obtained by the above two treatments were combined and concentrated under reduced pressure to obtain 10 g of a hot water extract.

[Test Example 1] Superoxide scavenging action test The extracts obtained in Production Examples 1 and 2 were examined for superoxide scavenging action. The test method (NBT method) is as follows.
1. Reagent (1) 0.05 M Na ₂ CO ₃ buffer (pH 10.2)
(2) 3 mM xanthine solution: 45.64 mg of xanthine was dissolved in the buffer solution of the above (1) to make 100 mL.
(3) 3 mM EDTA solution: EDTA · 2Na 111.7 mg was dissolved in distilled water to make 100 mL.
(4) BSA solution: Bovine serum albumin (Sigma) 15 mg was dissolved in distilled water to make 100 mL.
(5) 0.75 mM NBT solution: 61.32 mg of NBT (nitroblue tetrazolium) was dissolved in distilled water to make 100 mL.
(6) Xanthine oxidase solution: Xanthine oxidase was diluted with distilled water. The concentration was such that the absorbance in the blank test of “2. Operation” below was in the range of 0.20 to 0.23.
(7) 6 mM CuCl ₂ solution: 102.29 mg of CuCl ₂ .2H ₂ O was dissolved in distilled water to make 100 mL.

2. Operation 2.4 mL of Na ₂ CO ₃ buffer solution was placed in a test tube, and xanthine solution, EDTA solution, BSA solution, NBT solution and 0.1 mL each were added thereto. 0.1 mL of the sample solution (80% ethanol solution) was added and allowed to stand at 25 ° C. for 10 minutes, and then 0.1 mL of the xanthine oxidase solution was added, stirred rapidly, and incubated at 25 ° C. After 20 minutes, 0.1 mL of CuCl ₂ solution was added to stop the reaction, and the absorbance at 560 nm was measured. Separately, a blank test was performed using distilled water instead of the sample solution. IC ₅₀ (superoxide 50% erase concentration) (μg / mL) is shown in Table 1.

  From the results shown in Table 1, it was found that both water-containing ethanol extract of grapevine and hot water extract have superoxide scavenging action.

[Test Example 2] Singlet oxygen scavenging action test The extracts obtained in Production Examples 1 and 2 were examined for singlet oxygen scavenging action. The test method (erythrocyte hemolysis method) is as follows.
5 mL of 2% red blood cell suspension in a clear glass bottle (10 mL volume), 5 mL of isotonic phosphate buffer pH 7.4 containing a sample at a predetermined concentration, and a photosensitizer (10 mM hematoporphyrin-20 mM sodium hydroxide solution) ) 0.01 mL was mixed. The resulting solution was uniformly irradiated on a merry-go-round with a 7.5 W halogen lamp for 35 minutes to generate singlet oxygen ( ¹ O ₂ ), thereby causing red blood cell hemolysis. 1 mL of the reaction solution was collected, 2 mL of isotonic phosphate buffer was added and mixed, and then centrifuged at 4 ° C. and 3000 rpm for 5 minutes. Next, the supernatant was collected and the absorbance at a wavelength of 540 nm was measured. Separately, 1 mL of the reaction solution in which red blood cells were partially hemolyzed was taken, and 2 mL of distilled water was added thereto to completely hemolyze it, and the absorbance was measured in the same manner. The singlet oxygen scavenging rate was determined from the measured absorbance by the following formula.

Singlet oxygen elimination rate (%) = (1−B / A) × 100
In the formula, “A” is the absorbance of the control, and “B” is the absorbance of the reaction solution supernatant.
The above-mentioned erasure rate was measured by decreasing the sample concentration stepwise, and the sample concentration (μg / mL) at which the singlet oxygen erasure rate was 50% was determined by interpolation. IC ₅₀ (single oxygen 50% elimination concentration) (μg / mL) is shown in Table 2.

  From the results shown in Table 2, it was found that both the water-containing ethanol extract and the hot water extract of wild grapes have a singlet oxygen scavenging action.

[Test Example 3] Radical scavenging action test The extracts obtained in Production Examples 1 and 2 were examined for radical scavenging action. The test method (DPPH method) is as follows.
3 mL of the sample solution was added to 3 mL of 1.5 × 10 ⁴ M DPPH methanol solution, and the container was immediately sealed, shaken and allowed to stand for 30 minutes. Thereafter, the absorbance at 520 nm was measured. As a control test, the same operation was performed using the solvent instead of the sample solution, and the absorbance at 520 nm was measured. As a blank test, the absorbance at 520 nm was immediately measured after adding 3 mL of the methanol sample solution. Based on each measured absorbance, the radical scavenging rate was calculated by the following formula.

Radical scavenging rate (%) = [1- (BC) / A] × 100
In the formula, “A” represents the absorbance in the control test, “B” represents the absorbance when the sample solution was added, and “C” represents the absorbance in the blank test.
The above-mentioned erasure rate was measured while changing the sample concentration of the sample solution stepwise, and the concentration of the sample solution at which the DPPH radical erasure rate was 50% was determined by interpolation. IC ₅₀ (radical 50% elimination concentration) (μg / mL) is shown in Table 3.

  From the results shown in Table 3, it was found that both the water-containing ethanol extract of grapevine and the hot water extract have a radical scavenging action.

[Test Example 4] Elastase inhibitory action test The extract obtained in Production Example 1 was examined for elastase inhibitory action. The test method is as follows.
Prepare a 96-well microplate. 50 μL of sample solution (solvent: DMSO + water), 25 μL of substrate solution (2 mM N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide) and elastase solution (human preferred) Neutrophil-derived elastase; 6 μg / mL) was added. After reacting at 25 ° C. for 30 minutes, the reaction solution was collected and the absorbance at 415 nm was measured. Enzymatic reaction and absorbance measurement similar to the above were performed by adding only the solvent equivalent to the sample solution instead of the sample solution. Further, in each case, the same operation and measurement were performed without adding the elastase solution. From the measurement results, the elastase inhibition rate was calculated by the following formula.

Elastase inhibition rate (%) = {1− (A−B) / (C−D)} × 100
In the formula, “A” is the absorbance when the enzyme is added and the sample solution is added, “B” is the absorbance when the enzyme is not added and the sample solution is added, “C” is the absorbance when the enzyme is added and the sample solution is not added, “ “D” represents the absorbance when no enzyme was added and no sample solution was added.
The inhibition rate was measured by gradually reducing the sample concentration, and the sample concentration IC ₅₀ (μg / mL) at which the inhibition rate was 50% was determined by interpolation. IC ₅₀ (elastase 50% inhibitory concentration) (μg / mL) is shown in Table 4.

  From the results shown in Table 4, it was found that the water-containing ethanol extract of grapevine has an elastase inhibitory action.

[Test Example 5] Fibroblast proliferation action test The extract obtained in Production Example 1 was examined for NB1RBG cell proliferation action. The test method was performed according to the MTT method (J. Immunol. Method, 93, 157, 1986).
Inoculating 1 × 10 ⁶ human normal neonatal dermal fibroblasts (NBIRGB) in a 10 v / v% FBS-containing medium (α-MEM medium: GIBCO BLR, pH 7.2) placed in a 25 cm ² culture flask, The cells were cultured for 4 days at 37 ° C. and 5% CO ₂ -95% air. Cells were then collected by trypsinization and centrifugation. Cells obtained as a precipitate were suspended in a medium containing 5 v / v% FBS (α-MEM medium: GIBCO BLR, pH 7.2), and dispensed at 7 × 10 ³ cells / 100 μL per well of a 96-well plate. did. After culturing for 24 hours, a medium containing 5 v / v% FBS in which the sample was dissolved was added at 100 μL per well, and cultured at 37 ° C. under 5% CO ₂ -95% air for 3 days. After the culture, the medium was removed by 100 μL per well, and MTT reagent (3- (4,5-dimethyl1-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide, 5 mg / mL PBS (−) solution) 20 μL Was added and incubated for 4.5 hours (reactive oxygen from mitochondria in the grown cells reacted with the MTT reagent, and the color of the reagent that was yellow turns blue-yellow with an absorption peak at 570 nm). Thereafter, 100 μL of 10 wt% sodium dodecyl sulfate-0.02 mol / L sulfuric acid solution was dispensed into each well and incubated for 18 hours. After completion of the incubation, the absorbance at 570 nm was measured using a microplate reader. Separately, the absorbance was measured by changing the presence / absence of the sample and the presence / absence of cells. In addition, each absorbance measurement value was corrected by subtracting the absorbance measured at 650 nm at the same time to correct the influence of turbidity caused by the grown cells. From each corrected absorbance, the cell growth promotion rate was determined by the following formula.

Cell growth promotion rate (%) = {(AC)-(BD)} / (BD) × 100
In the formula, “A” is the absorbance when the sample is added, “B” is the absorbance when the sample is not added, “C” is the absorbance when the sample is added and no cell is added, and “D” is the sample added and no cell is added. It represents the absorbance at the time of addition.
The elimination rate was measured by decreasing the sample concentration stepwise, and the sample concentration (μg / mL) at which the fibroblast proliferation rate was 50% was determined by interpolation. The results are shown in Table 5.

  From the results shown in Table 5, it was found that the water-containing ethanol extract of grapevine has a fibroblast proliferation action.

[Test Example 6] Hyaluronidase inhibitory action test The extract obtained in Production Example 1 was examined for hyaluronidase inhibitory action. The test method is as follows.
After mixing 0.1 mL of a hyaluronidase solution (400 units / mL, pH 3.5 acetate buffer) and 0.2 mL of a sample solution and incubating at 37 ° C. for 20 minutes, an activator solution (2.5 mM-CaCl ₂ ) 0.2 mL was added and incubated at 37 ° C. for 20 minutes to activate the enzyme. After adding 0.5 mL of potassium hyaluronate buffer and incubating at 37 ° C. for 40 minutes, 0.2 mL of 0.4N sodium hydroxide was added and ice-cooled to stop the reaction. Next, 0.2 mL of 0.8 M boric acid solution (pH 9.1) was added, heated in a boiling bath for 3 minutes, and immediately cooled on ice for 20 minutes. Add 6.0 mL of P-DABA reagent (10 g of p-dimethylaminobenzaldehyde dissolved in 12.5 mL of 10N hydrochloric acid and 87.5 mL of acetic acid and dilute 10-fold with acetic acid) and incubate at 37 ° C. for 20 minutes As a result, N-acetylglucosamine released by the enzyme reaction was colored and the absorbance at a wavelength of 585 nm was measured. The same operation and absorbance measurement were performed without adding the enzyme. Furthermore, the same measurement was performed when distilled water was added without adding the sample solution, and the hyaluronidase inhibition rate was determined by the following equation.

Hyaluronidase inhibition rate (%) = {1− (A−B) / (C−D)} × 100
In the formula, “A” is the absorbance when the enzyme is added and the sample solution is added, “B” is the absorbance when the enzyme is not added and the sample solution is added, “C” is the absorbance when the enzyme is added and the sample solution is not added, “ “D” represents the absorbance when no enzyme was added and no sample solution was added.
The inhibition rate was measured by gradually reducing the sample concentration, and the sample concentration IC ₅₀ (μg / mL) at which the inhibition rate was 50% was determined by interpolation. The results are shown in Table 6.

  From the results shown in Table 6, it was found that the water-containing ethanol extract of grapevine has a hyaluronidase inhibitory action.

[Test Example 7] Cyclic AMP phosphodiesterase inhibitory action test The extract obtained in Production Example 1 was examined for a cyclic AMP phosphodiesterase inhibitory action. The test method is as follows.
0.2 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 1.5 mM magnesium chloride, 0.1 mL of fetal bovine serum albumin solution (2.5 mg / mL) and cyclic AMP phosphodiesterase solution (0.1 mg / mL) 0.1 mL was added, and 0.05 mL of a sample solution containing each sample was further added, and incubated at 37 ° C. for 5 minutes. Next, 0.05 mL of a cyclic AMP solution (0.5 mg / mL) was added and reacted at 37 ° C. for 60 minutes. After the reaction, the reaction was stopped by boiling for 3 minutes in boiling water, centrifuged at 4 ° C., and the reaction substrate (cyclic AMP) in the supernatant was quantified by high performance liquid chromatography.

The same enzyme reaction and reaction substrate were quantified without adding the sample solution, and the cyclic AMP phosphodiesterase inhibition rate (%) was determined from the ratio of the reaction substrate when the sample was added to the reaction group mass when no sample was added.
The cyclic AMP phosphodiesterase inhibition rate (%) is measured by changing the sample concentration of the sample solution stepwise, and the sample concentration IC ₅₀ (μg / mL) that inhibits cyclic AMP phosphodiesterase activity by 50% is determined by interpolation. It was. The results are shown in Table 7.

  From the results shown in Table 7, it was found that the water-containing ethanol extract of grapevine has a cyclic AMP phosphodiesterase inhibitory action.

[Formulation Example 1]
The following raw materials were uniformly mixed, granulated by a conventional method, and then tableted to produce a tablet-like health food.
Ground grapes 50% ethanol extract 75 parts by weight Turmeric extract 50 parts by weight Licorice extract 5 parts by weight Yeast extract (containing amino acids and vitamins) 25 parts by weight Powdered sugar 123 parts by weight Crystalline cellulose 10 parts by weight Sucrose fatty acid ester 12 parts by weight Part

[Formulation Example 2]
The following ingredients were mixed and a soft capsule-shaped health food was produced by a conventional punching method.
Ground grape water extract 50 parts by weight Vitamin E 25 parts by weight Silymarin 60 parts by weight Oyster meat extract 25 parts by weight Vegetable oil 113 parts by weight Glycerin fatty acid ester 12 parts by weight Beeswax 15 parts by weight

[Composition Example 3]
The following raw materials were uniformly mixed and granulated by a conventional method to produce a granular health food.
Grape vinegar ethanol extract 30 parts by weight Gymnema extract powder 25 parts by weight soybean peptide 65 parts by weight Maltitol 160 parts by weight Crystalline cellulose 20 parts by weight

Claims (4)

  An anti-inflammatory composition containing no grape extract as an active ingredient.   The anti-inflammatory composition according to claim 1, wherein the grapevine extract has a hyaluronidase inhibitory action and / or a cyclic AMP phosphodiesterase inhibitory action.   The anti-inflammatory composition according to claim 1 or 2, wherein the grape extract is obtained by an extraction treatment using a water-soluble organic solvent, a water-containing water-soluble organic solvent, or water as an extraction solvent.   The anti-inflammatory composition according to any one of claims 1 to 3, which is a pharmaceutical composition, a food / beverage product composition, a feed composition or a cosmetic composition.