KR20070122211A - Anti-inflammatory agent - Google Patents

Abstract

An anti-inflammatory agent which comprises an oil-soluble licorice extract obtained by extraction treatment of at least either a leguminous plant of the genus Glycyrrhiza or a water extraction residue of a leguminous plant of the genus Glycyrrhiza with an organic solvent, and has at least one effect selected from an inhibitory effect on hyaluronidase activity, an inhibitory effect on hexosaminidase release (i.e., an inhibitory effect on histamine release), an inhibitory effect on platelet aggregation and an inhibitory effect on phospholipase A2 activity.

Description

Anti-inflammatory agent

The present invention contains an oil-soluble licorice extract (hereinafter sometimes referred to as a "soluble licorice extract"), inhibits hyaluronidase activity, inhibits hexosaminidase release, and inhibits platelet aggregation. operation and phospholipase a ₂ (phospholipase a ₂₎ has at least one action selected from the active inhibitory action, the present invention relates to anti-inflammatory agents that are particularly preferably used as a skin external preparation.

Licorice is known as a herbal medicine since ancient times, and is currently mainly used as a raw material for food sweeteners, pharmaceuticals, quasi-drugs and the like. Among them, glycyrrhizin and glycyrrhetic acid, which are water extracts of licorice, have excellent pharmacological effects such as anti-inflammatory, anti-ulcer and anti-allergic effects. It is widely used for such purposes.

On the other hand, it is known that oil-soluble licorice extract (oil-soluble licorice extract) which extracted licorice with organic solvents, such as ethanol and ethyl acetate, contains many flavonoids other than the said glycyrrhizin and glycyrrytic acid. This oil-soluble licorice extract has, for example, an antioxidant action (see Patent Document 1), an antioxidant action (see Patent Document 2), a whitening action (see Patent Document 3 and Patent Document 4), and an ultraviolet absorbing action (see Patent Document 5). It is disclosed to have a useful action such as.

However, the oil-soluble licorice extract has at least one action selected from inhibiting hyaluronidase activity, hexosaminidase free inhibitory action, platelet aggregation inhibitory action and phospholipase A ₂ activity inhibitory action, for example, contact At present, there is no known usefulness for sexual dermatitis (rash), psoriasis, vulgaris, and other skin diseases associated with roughening of the skin.

Patent Document 1: Japanese Patent Application Laid-Open No. 58-217583

Patent Document 2: Japanese Patent Publication No. 59-46210

Patent Document 3: Japanese Unexamined Patent Publication No. 1-49706

Patent Document 4: Japanese Patent Application Laid-Open No. 1-311011

Patent Document 5: Japanese Unexamined Patent Publication No. 1-57909

Disclosure of the Invention

This invention is made | formed in view of the present situation concerned, and makes it a subject to solve the said various problems in the past, and to achieve the following objectives. That is, the present invention prevents and ameliorates inflammatory diseases through at least one action selected from inhibiting hyaluronidase activity, inhibiting hexosaminidase release, inhibiting platelet aggregation, and inhibiting phospholipase A ₂ activity. It is an object of the present invention to provide an anti-inflammatory agent.

Means for solving the above problems are as follows.

<1> An anti-inflammatory agent characterized by containing oil-soluble licorice extract

In <2> <1>, the oil-soluble licorice extract is an anti-inflammatory agent obtained by extracting at least one of water extract residues of the soybean Glycyrrhiza plant and the soybean Glycyrrhiza plant.

In <3> <1> or <2>, at least any one of roots, root stems, leaves, stems, and water extract residues of soybean and Glycyrrhiza plants is obtained by extracting and treating with organic solvents. It is an anti-inflammatory.

In <4> <3>, the organic solvent is at least one anti-inflammatory agent selected from ethanol, hydrous ethanol and ethyl acetate.

<5> In any one of <2>-<4>, the plant of the genus Soybean Glycyrrhiza is Glycyrrhiza glabra, Glycyrrhiza inflata, Glycyrrhiza At least one anti-inflammatory agent selected from Glycyrrhiza araleasis, Glycyrrhiza uralensis, and Glycyrrhiza echinata.

<6> In any one of <1>-<5>, an oil-soluble licorice extract is an anti-inflammatory agent containing the flavonoid of at least any one of glabridine and ricochalcon A.

In <7> <6>, the total content in the oil-soluble licorice extract of at least any one of glabridine and ricochalcone A is an anti-inflammatory agent that is 1 to 80 mass% in dry solid content.

The glabridine content in oil-soluble licorice extract of Glycyrrhiza glabra is an anti-inflammatory agent in any one of <8> <5>-<7> which is 1 mass% or more in dry solid content.

In any one of <5>-<8>, content of the lyocacalcon A in the oil-soluble licorice extract of Glycyrrhiza echinata is an anti-inflammatory agent which is 1 mass% or more in dry solid content.

In any one of <5>-<9>, content of the ricokalcon A in the oil-soluble licorice extract of Glycyrrhiza inflata is 1 mass% or more in dry solid content.

The oil-soluble licorice extract according to any one of <1> to <10>, wherein the oil-soluble licorice extract is inhibited from hyaluronidase activity inhibition, hexosaminidase free inhibition, platelet aggregation inhibition and phospholipase A ₂ activity inhibition. It is an anti-inflammatory agent having at least one action selected.

It is an anti-inflammatory agent in any one of <12> <1>-<11> used as an external preparation for skin.

The anti-inflammatory agent of the present invention contains an oil-soluble licorice extract, and in particular, by extracting and treating at least one of water extract residues of the soybean Glycyrrhiza plant and the soybean Glycyrrhiza plant with an organic solvent Effectively inhibit the inflammation associated with them through inhibition of hyaluronidase activity, hexosaminidase free inhibition (ie, histamine free inhibition), platelet aggregation inhibition and phospholipase A ₂ activity. And improvement can be achieved.

In addition, since the anti-inflammatory agent of the present invention is excellent in feeling and safety, it is particularly suitably used as an external preparation for skin. Here, in the present invention, "skin external preparation" means various drugs applied to the skin, for example, it is a concept including cosmetics, quasi-drugs, medicines and the like.

Best Mode for Carrying Out the Invention

The anti-inflammatory agent of the present invention contains an oil-soluble licorice extract, and further contains other components as necessary.

The oil-soluble licorice extract is not particularly limited and may be appropriately selected according to the purpose, but at least one of the water extract residues of the soybean Glycyrrhiza plant and the soybean Glycyrrhiza plant It is suitable to obtain by extraction with an organic solvent.

There is no restriction | limiting in particular as said soybean and Glycyrrhiza genus plant, Although it is possible to select suitably according to the target active ingredient, For example, Glycyrrhiza glabra (Glycyrrhiza glabra), Glycyrriza inplata ( Glycyrrhiza inflata), Glycyrrhiza araleasis, Glycyrrhiza uralensis, Glycyrrhiza echinata, and among them, Glycyrrhiza glabra), Glycyrrhiza echinata, Glycyrrhiza inflata are particularly preferred.

In addition, licorice is often called by the name of the place of production. , Afghanistan Licorice and others.

As an extracting material of the oil-soluble licorice extract, any part of the root, root stem, leaf and stem of the licorice can be used, but from the side with high content of flavonoids such as glabridine and ricokalcon A, the root and root stem At least one is particularly preferred. The raw material for extraction may be either raw or dried, but dried roots and dried roots are particularly preferred.

In addition, it is also possible to use the water extraction residue of the extract raw material of the licorice as the extraction raw material of the oil-soluble licorice extract.

The water extraction residue of licorice means a solid residue after extraction with water, hot water and hot water, and at least one of neutral to non-alkaline cold water, hot water and hot water in order to obtain glycyrrhizin and the like from the licorice. In addition, the residue after the said water extraction may be any of a water | moisture content and a dry state.

By using the water extraction residue of licorice as an extraction raw material in this way, the effective use of the water extraction residue of licorice can be aimed at, and there exists an advantage that productivity improves.

There is no restriction | limiting in particular as said organic solvent, It can select according to the objective, For example, benzene, toluene, xylene, ethyl ether, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, dichloroethane, chloroform, ethyl acetate, Propyl acetate, butyl acetate, acetone, methanol, ethanol, propanol, hydrous methanol, hydrous ethanol, hydrous propanol and the like. In addition, it is also possible to use carbon dioxide as a supercritical fluid. Among these organic solvents, ethanol, hydrous ethanol and ethyl acetate are preferably used from the viewpoint of safety. As said hydrous ethanol, the thing of ethanol concentration 30-99 mass% is suitable.

There is no restriction | limiting in particular as extraction conditions for obtaining the licorice oily extract using the said organic solvent from the said licorice or licorice water extraction residue, According to the objective, it can select suitably, for example, it is 2-15 times with respect to the extraction raw material The method of extracting at normal temperature, adding an organic solvent, stirring, and the method of extracting by heating and drying are mentioned. In addition, it is more preferable to perform these methods individually or in combination, respectively, since extraction efficiency improves.

The obtained extract can be used as it is an oil-soluble licorice extract after removing the insoluble matter by centrifugation and filtration, and can be further concentrated and used according to a conventional method. Moreover, deodorization, decolorization, etc. may be performed suitably in the range in which the target physiological effect does not fall. For this deodorization and decolorization, it is common to use activated carbon, synthetic adsorbent resins, ion exchange resins, and the like. In addition, if the extract is dried by a suitable method, it is possible to obtain a yellowish brown extract powder as an oil-soluble licorice extract.

The obtained liquid extract is used as it is, or the liquid extract is concentrated, and further, powder or solid dried product of the liquid extract is used as the oil-soluble licorice extract.

There is no restriction | limiting in particular as a purification method of the flavonoid from the said oil-soluble licorice extract, It is possible to select suitably from a well-known organic compound purification method according to the objective, For example, normal phase silica gel chromatography and reverse phase It is possible to carry out by a method of crystallizing from acetone after treating by any one of chromatography. According to this method, it is possible to obtain the pure article of an active ingredient relatively easily. Moreover, as another purification method, column chromatography, liquid-liquid countercurrent extraction, etc. by synthetic adsorbents, such as Dion HP-20 (made by Mitsubishi Chemical Corporation), are mentioned.

The flavonoids contained in the oil-soluble licorice extract are different depending on the licorice species, which is an extract raw material, and cannot be uniformly defined. For example, glabridine, glabrene, ricochalcon A, ricokalcon B, glycicicoumarin, and glyco Lysoflavone, etc. are mentioned, Among these, from the side which has a high anti-inflammatory action, a globridine, a glablen, a ricokalcon A, a ricokalcon B is especially preferable, and a glabridine and a ricokalcon A are the most preferable.

1-80 mass% is preferable as dry solid content, and, as for the total content in the said oil-soluble licorice extract of at least any one of said glabridine and ricochalcone A, the 5 to 60 mass% is more preferable.

As for content of the glabridine in the oil-soluble licorice extract of the said glycyrrhiza glabra, 1 mass% or more is preferable as dry solid content, 10 mass% or more is more preferable, and 20-50 mass% is More preferred.

As for content of ricokalcon A in the oil-soluble licorice extract of the said glycyrrhiza echinata, 1 mass% or more is preferable as dry solid content, 5 mass% or more is more preferable, and 10-40 mass% is More preferred.

As for content of the ricokalcon A in the oil-soluble licorice extract of the said glycyrrhiza inflata, 1 mass% or more is preferable as dry solid content, 5 mass% or more is more preferable, and 10-40 mass% is More preferred.

The anti-inflammatory agent of the present invention exhibits at least one action selected from a hyaluronidase activity inhibitory effect, a hexosaminidase free inhibitory effect (ie, histamine free inhibitory effect), platelet aggregation inhibitory effect, and phospholipase A ₂ activity inhibitory effect. Through this, it is possible to prevent and improve various inflammatory diseases including skin diseases such as contact dermatitis (rash), psoriasis and vulgaris.

Here, the hyaluronidase is a hydrolase of hyaluronic acid, and is said to be involved in degranulation from mast cells by its activation in mast cells. Therefore, by inhibiting the activation of hyaluronidase, it is possible to stabilize the hyaluronic acid and to prevent the release of various chemical mediators from mast cells, thereby enhancing the moisturizing properties or anti-inflammatory action.

In addition, since the histamine in the cell is released and the hexosaminidase is also released, it is possible to evaluate the histamine release inhibitory activity using the hexosaminidase release as an index. The histamine is present in mast cells (mast cells), and when the mast cells are stimulated, they are released by degranulation reactions, acting as gastritis and allergens. In addition, histamine released from activated mast cells causes vascular permeability, smooth muscle contraction, mucus secretion, and the like, leading to allergic diseases such as bronchial asthma, allergic rhinitis and urticaria. Therefore, it is possible to prevent and treat allergic disease or inflammatory disease by free inhibition of hexosaminidase, that is, free inhibition of histamine.

The platelet aggregation causes activation of phospholipase A ₂ of the arachidonic acid cascade, whereby leukotriene B, prostaglandin E _2, and the like are released to become a base material. For this reason, it is possible to prevent and treat an allergic disease or an inflammatory disease by the substance which inhibits and suppresses aggregation of platelets.

The phospholipase A ₂ is an important enzyme of arachidonic acid cascade, which is an metabolic pathway of arachidonic acid. When phospholipase A ₂ is activated excessively, abnormality occurs in metabolism of arachidonic acid, inflammation, allergy, asthma, ischemia, Causes myocardial infarction. Therefore, by inhibiting the activation of phospholipase A ₂ , it is possible to prevent and treat an allergic disease or an inflammatory disease.

The anti-inflammatory agent of the present invention is not particularly limited and may be appropriately selected according to the purpose, but is suitably used as an external preparation for skin. The external preparation for skin refers to various drugs applied to the skin and includes, for example, cosmetics, quasi-drugs, and medicines. Examples of the external skin preparations include ointments, creams, emulsions, lotions, packs, jelly, lip creams, lipsticks, bath agents, tonics, rinses, shampoos, astringents, and the like.

The compounding amount of the anti-inflammatory agent in the external preparation for skin can be appropriately adjusted according to the type of external preparation for skin or the physiological activity of the extract, etc., but in terms of the oil-soluble licorice extract, preferably 0.001 to 10 mass%, and 0.01 to 5 mass % Is more preferable.

Although the anti-inflammatory agent of this invention demonstrated above is suitably applied to a human, it can also be applied to animals other than a human as long as each action effect is shown.

Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited only to a following example.

(Manufacture example 1)

Preparation of Soluble Licorice Extract

10 L of absolute ethanol was added to 1 kg of root of licorice (Glycyrrhiza glabra Linne var. Glandulifera Regel et Herder), and extraction was performed for 5 hours under reflux. The obtained extract was concentrated under reduced pressure. 1 L of ethyl acetate was added to the concentrated extract, extracted under reflux for 5 hours, dried under reduced pressure, and pulverized to obtain 10 g of an oil-soluble licorice extract.

As a result of quantitative determination of the flavonoid content in the oil-soluble licorice extract of Production Example 1 obtained by high performance liquid chromatography (manufactured by Japan Spectroscopic Co., Ltd.), 20% by mass of glabridine was contained.

(Manufacture example 2)

Preparation of Soluble Licorice Extract

10 L of absolute ethanol was added to 1 kg of root of licorice (Glycyrrhiza glabra Linne var. Glandulifera Regel et Herder), and extraction was performed for 5 hours under reflux. The obtained extract was concentrated under reduced pressure. 1 L of ethyl acetate was added to this concentrated extract, and extraction was performed under reflux for 5 hours. This was adjusted with a synthetic adsorbent (manufactured by Mitsubishi Chemical Corporation), dried under reduced pressure, and pulverized to obtain 5 g of an oil-soluble licorice extract.

As a result of quantifying the content of the flavonoids in the oil-soluble licorice extract of Production Example 2 obtained using high-performance liquid chromatography (manufactured by Japan Spectroscopic Co., Ltd.), 40% by mass of glybridine was contained.

(Manufacture example 3)

Preparation of Soluble Licorice Extract

10 L of anhydrous ethanol was added to 1 kg of roots of licorice (Glycyrrhiza inflata Batalin), and extraction was performed under reflux for 5 hours. The obtained extract was concentrated under reduced pressure. 1 L of ethyl acetate was added to this concentrated extract, and extraction was performed under reflux for 5 hours. The obtained extract was concentrated under reduced pressure. 1 L of ethyl acetate was added to the concentrate, and the mixture was extracted under reflux for 5 hours, dried under reduced pressure, and pulverized to obtain 30 g of an oil-soluble licorice extract.

As a result of quantitative determination of the content of flavonoids in the oil-soluble licorice extract of Production Example 3 obtained by high performance liquid chromatography (manufactured by Japan Spectroscopic Co., Ltd.), 20% by mass of lycochalcone A was contained.

(Manufacture example 4)

Purification of glybridine

20 g of the oil-soluble licorice extract obtained in Production Example 2 were dissolved in chloroform, applied to silica gel (silica gel 60, manufactured by Merck), and dried. The dried product was laminated and charged on a column packed with 1 kg of silica gel as described above, eluted with a chloroform / methanol mixture (30: 1), and a glabridine-containing fraction was collected. The solvent of this fraction was distilled off under reduced pressure to obtain 5.8 g of a solid. Subsequently, the obtained solid was dissolved in a small amount of methanol, applied to reversed phase silica gel (ODS DM1020T, manufactured by Fuji Silysia Co., Ltd.), dried, and laminated on a column packed with 800g of reverse phase silica gel in advance. A methanol / water mixture (60:40) was flowed into this column as an elution solvent, and the glabridine-containing fraction was collected. From this fraction, the solvent was distilled off under reduced pressure, the obtained solid (4.3 g) was dissolved in 40 ml of acetone, and left still at 5 ° C for 3 days to obtain 3.8 g of crystals of glabridine.

(Manufacture example 5)

Purification of Ricokalcon A

120 g of the oil-soluble licorice extract obtained in Production Example 3 was dissolved in chloroform, applied to silica gel (silica gel 60, manufactured by Merck), and dried. The dried product was laminated on a column packed with 3 kg of silica gel as described above, eluted with an n-hexane / ethyl acetate mixed solution (2: 1), and a fraction containing lyocacalcon A was collected. The solvent of this fraction was distilled off under reduced pressure to obtain 50 g of a solid, which was dissolved in a small amount of methanol, dried by applying to reversed phase silica gel (ODS DM1020T, manufactured by Fuji Silia Co., Ltd.) and laminated on a column previously filled with reversed phase silica gel. It was. Methanol / water mixture (60:40) was flowed into this column as an elution solvent, and the fraction containing ricokalcon A was collected. The solvent was distilled off from this fraction under reduced pressure, the obtained solid (25 g) was dissolved in a methanol / water mixture (70:30), and left at room temperature for 1 day to obtain 15.2 g of crystals of Ricokalcon A.

(Example 1)

Hyaluronidase activity inhibition test

The hyaluronidase activity inhibitory effect was tested as follows with respect to the extract prepared in the manufacture examples 1-5, and the purified matter (Hereinafter, it may be called a "sample.").

First, 0.1 mL of hyaluronidase solution (Type IV-S (from bovine testis; SIGMA 400 NF units / mL)) was added to 0.2 mL of 0.1 mol / L acetic acid buffer (pH 3.5) in which each sample was dissolved. The reaction was carried out for 20 minutes at.

Next, 0.2 mL of 2.5 mmol / L calcium chloride was added as an activator and reacted at 37 ° C for 20 minutes. 0.5 mL of 0.4 mg / mL potassium hyaluronic acid solution (from robster comb) was added thereto and reacted at 37 ° C for 40 minutes. Thereafter, 0.2 mL of 0.4 mol / L sodium hydroxide was added to stop the reaction, and after cooling, 0.2 mL of boric acid solution was added to each reaction solution, followed by boiling for 3 minutes. After ice-cooling, 6 mL of p-DABA (p-dimethylaminobenzaldehyde) reagents were added, and it reacted at 37 degreeC for 20 minutes. Thereafter, the absorbance at a wavelength of 585 nm was measured.

Next, the same operation and absorbance measurement were performed without adding an enzyme. In addition, the same operation and absorbance measurement were performed about the case where distilled water was added instead of the sample solution as a control.

From the above measurement result, the hyaluronidase activity inhibition rate (%) was computed by following formula (1).

However, in Equation 1, St represents the absorbance at a wavelength of 585 nm of the sample solution. Sb shows the absorbance at a wavelength of 585 nm of the sample solution blank. Ct shows the absorbance at a wavelength of 585 nm of the control solution. Cb shows the absorbance at a wavelength of 585 nm of the control solution blank.

Subsequently, the sample concentration was decreased in steps to measure the inhibition of the hyaluronidase activity, and the sample concentration for inhibiting the hyaluronidase activity by 50% was determined by the interpolation method. The results are shown in Table 1. The smaller this value, the stronger the inhibition of hyaluronidase activity.

From the results of Table 1, it was confirmed that the oil-soluble licorice extract of Preparation Examples 1 to 3, the glabridine of Preparation Example 4, and lycokalcon A of Preparation Example 5 had an inhibitory effect on hyaluronidase activity.

(Example 2)

Hexosaminidase free inhibitory test

The hexosaminidase free inhibitory effect was tested as follows with respect to the extract prepared in the manufacture examples 1-5, and the refined thing (Hereinafter, it may be called a "sample."). In addition, since the histamine in the cell is released and the hexosaminidase is also released, it is possible to evaluate the histamine release inhibitory activity using the hexosaminidase release as an index.

First, rat basophilic leukemia cells (RBL-2H3) were cultured using a medium (15% FBS-added S-MEM medium; the same below) in a 25 mL culture flask, and then cells were recovered by trypsin treatment. The recovered cells were diluted with S-MEM medium at a concentration of 4.0 × 10 ⁵ cells / mL, and the mouse monoclonal anti-dinitrophenyl group IgE (DNP-specific-IgE) was added so as to have a final concentration of 0.5 µg / mL, followed by 96 100 μL per well was sown in well plates and incubated overnight. After completion of the culture, the S-MEM medium was removed, and washed twice with 500 µL of Siraganian buffer.

Then 30 μL of Siraganian buffer and 10 μL of each sample prepared with Siraganian buffer were added, and left standing at 37 ° C. for 10 minutes. Then, 10 μL of a solution of dinitrophenylated bovine serum albumin (DNP-BSA) at 100 ng / mL was added, and the hexosaminidase was released by standing at 37 ° C. for 15 minutes.

The glass was then stopped by standing the 96 well plate on ice. Add 110 μL of the cell supernatant of each well and 10 μL of 1 mmol / L of p-nitrophenyl-N-acetyl-β-D-glycosamide (p-NAG) solution to a new 96 well plate and add 1 at 37 ° C. The reaction was time. After completion of the reaction, 250 μL of 0.1 mol / L Na ₂ CO ₃ / NaHCO ₃ was added to each well, and the absorbance at a wavelength of 415 nm was measured.

Was then ball (空) test as the absorbance of the cell supernatant was measured in 10 μL, and Na ₂ CO ₃ / NaHCO ₃ 415 ㎚ wavelength of 250 μL of a mixed solution of 0.1 ㏖ / L, and calibration.

From the above measurement result, hexosaminidase release inhibition was calculated | required by following formula (2).

However, in Formula (2), A represents the absorbance at a wavelength of 415 nm with no sample added. B shows absorbance at a wavelength of 415 nm in sample addition. C shows absorbance at wavelength 415 nm at sample addition and no p-NAG addition.

Subsequently, the hexosaminidase free inhibition rate was measured by decreasing the sample concentration stepwise, and the sample concentration which inhibits the glass of the hexosaminidase by 50% was determined by the interpolation method. The results are shown in Table 2. The smaller this value, the stronger the free inhibitory effect of hexosaminidase.

From the results of Table 2, it was confirmed that the oil-soluble licorice extract of Preparation Examples 1 to 3 and Ricokalcon A of Preparation Example 5 had a hexosaminidase free inhibitory action (that is, a histamine free inhibitory action).

(Example 3)

Platelet aggregation inhibitory effect

The platelet aggregation inhibitory effect was tested as follows with respect to the extract prepared in the manufacture examples 1-5, and the purified matter (Hereinafter, it may be called a "sample.").

First, 1/10 of 77 mmol / L EDTA (pH 7.4) was added, and the blood of the collected rabbit was centrifuged (180 x g, 10 minutes, room temperature) to obtain platelet suspension. Subsequently, it centrifuged (810xg, 10 minutes, 4 degreeC), supernatant was removed, and platelet was obtained. This was suspended in platelet washing solution (0.15 mol / L sodium chloride: 0.15 mol / L Tris-HCL buffer (pH 7.4): 77 mmol / L EDTA (pH 7.4) = 90: 8: 2) and centrifuged as above. The platelets obtained were suspended in platelet suspension (10 mmol / L HEPES buffer (pH 7.4) containing 145 mmol / L sodium chloride, 5 mmol / L potassium chloride and 5.5 mmol / L glucose), and the platelet count It was adjusted (3.0 × 10 ⁵ cells / μL) to prepare a platelet suspension after washing.

Next, 1 µL of 200 mmol / L calcium chloride solution was added to 222 µL of the obtained platelet suspension, which was then reacted at 37 ° C for 1 minute. 2 μL of each sample was added thereto, and the resultant was further reacted for 2 minutes, and stirred for 1 minute after adding a stirrer. Then, 25 μL of a 10 ppm collagen solution was added as a flocculation agent, and platelet aggregation rate for 10 minutes at 37 ° C. was measured. It measured by the aggregation measuring apparatus (PAM12CL, Mebanix Corporation make), and platelet aggregation inhibition rate A was obtained from following formula (3). Platelet aggregation rate B was measured in the same manner as above except that the solvent of the sample solution was not added instead of the sample solution.

However, in the above Equation 3, A means platelet aggregation rate when the aggregation causing agent is added and the sample solution is added. B means the platelet aggregation rate at the time of addition of a flocculant and no sample solution.

Subsequently, the sample concentration was decreased in steps to measure the platelet aggregation inhibition rate, and the sample concentration for inhibiting platelet aggregation by 50% was determined by the interpolation method. The results are shown in Table 3. Smaller values indicate stronger platelet aggregation inhibition.

From the results of Table 3, it was confirmed that the oil-soluble licorice extract of Preparation Examples 1 to 3, the glabridine of Preparation Example 4, and lycokalcon A of Preparation Example 5 had a platelet aggregation inhibitory effect.

(Example 4)

Phospholipase A ₂ activity inhibitory activity

The phospholipase A ₂ activity inhibitory effect was tested as follows with respect to the extract prepared in the manufacture examples 1-5, and the refined thing (Hereinafter, it may be called a "sample.").

First, RBL-2H3 cells of rat leukemia cells were cultured in a 75 cm 2 flask in MEM medium containing 15 v / v% FBS under 37 ° C / CO ₂ -95% air, and the cells were collected by a conventional method. The obtained cells were adjusted to 5 × 10 ⁵ cells / mL in 15 v / v% FBS containing MEM medium. Further [ ³ H] arachidonic acid (50 μCi / 500 μL) was added at a rate of 3 μL / 10 mL. The resulting preparation was seeded in 1 mL of a 24 well plate, and incubated overnight at 37 ° C. and 5% CO ₂ 95% air. The medium in each well was discarded, washed with PBS (-), and then serum-free MEM medium was added, and incubated at 37 ° C for 30 minutes.

The solution in which the sample was dissolved was then added to each well and incubated for 10 minutes as well. Further, 10 mmol of 1 mmol / L of A23187 (manufactured by Sigma Co., Ltd.) was added, and incubated at 37 ° C for 5 minutes. After the reaction, 500 µL of the supernatant was taken under water cooling, and 6 mL of scintillation cocktail was added, and radioactivity was measured by a liquid scintillation counter.

Subsequently, the blank test (no A23187 irritation) and the control (solvent of sample solution) were measured by the same method.

To, from the obtained measurement result was obtained Phospholipase A ₂ activity inhibition rate by the equation (4).

However, in Formula 4, A represents the radioactivity at the time of sample addition. B represents the radioactivity of the control. C represents the radioactivity of the blank test.

Subsequently, the concentration of the phospholipase A ₂ activity was measured by decreasing the sample concentration stepwise, and the sample concentration for inhibiting the phospholipase A ₂ activity by 50% was determined by interpolation. The results are shown in Table 4. Smaller values indicate stronger phospholipase A ₂ activity inhibition.

From the results of Table 4, it was confirmed that the oil-soluble licorice extract of Preparation Examples 1-3 had a phospholipase A ₂ activity inhibitory effect.

(Formulation Example 1)

Creams having the anti-inflammatory action of the following composition were prepared by conventional methods.

5.0 g of liquid paraffin

4.0 g of refined beeswax

Cetanol 3.0 g

10.0 g of squalene

2.0 g of lanolin

1.0 g of stearic acid

1.5 g of oleic acid polyoxyethylene sorbitan (20E.O)

3.0 g glyceryl monostearate

6.0 g of 1,3-butylene glycol

1.5 g of paraoxy benzoate

0.1 g of spices

0.01 g of oil-soluble licorice extract of Preparation Example 1

Purified water balance

100 g in total

(Formulation Example 2)

An emulsion having an anti-inflammatory action of the following composition was prepared by a conventional method.

4.0 g of jojoba oil

0.1 g of placenta extract

2.0 g olive oil

2.0 g of squalene

Cetanol 2.0 g

2.0 g of glyceryl monostearate

2.5 g of polyoxyethylene cetyl ether (20E.O)

2.0 g of oleic acid polyoxyethylene sorbitan (20E.O)

1, 3-butylene glycol 3.0 g

0.15 g of hinokithiol

0.05 g of spices

0.01 g of oil-soluble licorice extract of Preparation Example 2

Purified water balance

100 g in total

(Compound Example 3)

Packs having the anti-inflammatory action of the following composition were prepared by conventional methods.

15 g of polyvinyl alcohol

3 g of polyethylene glycol

7 g of propylene glycol

10 g of ethanol

0.05 g of paraoxybenzoic acid

0.05 g of spices

Oil-soluble Licorice Extract of Preparation Example 3 0.05 g

Purified water balance

100 g in total

(Combination Example 4)

Creams having the anti-inflammatory action of the following composition were prepared according to conventional methods.

5.0 g of liquid paraffin

4.0 g of refined beeswax

Cetanol 3.0 g

10.0 g of squalene

2.0 g of lanolin

1.0 g of stearic acid

1.5 g of oleic acid polyoxyethylene sorbitan (20E.O)

3.0 g glyceryl monostearate

6.0 g of 1,3-butylene glycol

1.5 g of paraoxy benzoate

0.1 g of spices

0.01 g of glabridine of Preparation Example 4

Purified water balance

100 g in total

(Compound Example 5)

Packs having the anti-inflammatory action of the following composition were prepared by conventional methods.

15 g of polyvinyl alcohol

3 g of polyethylene glycol

7 g of propylene glycol

10 g of ethanol

0.05 g of paraoxybenzoic acid

0.05 g of spices

0.05 g of ricokalcon A of Preparation Example 5

Purified water balance

100 g in total

The anti-inflammatory agent of the present invention is obtained by extracting at least one of the water extract residues of the soybean Glycyrrhiza genus and the soybean Glycyrrhiza genus plant with an organic solvent and inhibiting hyaluronidase activity. It has at least one action selected from action, hexosaminidase free inhibitory action, platelet aggregation inhibitory action and phospholipase A ₂ activity inhibitory action, and is particularly suitable for skin cosmetics such as lotion, cream, emulsion, lotion, pack and the like. Is used.

Claims (12)

An anti-inflammatory agent comprising oil-soluble licorice extract. The anti-inflammatory agent according to claim 1, wherein the oil-soluble licorice extract is obtained by extracting at least one of a soybean Glycyrrhiza plant and a water extract residue of the soybean Glycyrrhiza plant. The anti-inflammatory agent according to claim 1 or 2, which is obtained by extracting at least one of roots, rhizomes, leaves, stems, and water extract residues of soybean and Glycyrrhiza genus plants with an organic solvent. The anti-inflammatory agent according to claim 3, wherein the organic solvent is at least one selected from ethanol, hydrous ethanol and ethyl acetate. The plant according to any one of claims 2 to 4, wherein the legume Glycyrrhiza genus plants are Glycyrrhiza glabra, Glycyrrhiza inflata, Glycyrrhiza inflata. At least one anti-inflammatory agent selected from Glycyrrhiza araleasis, Glycyrrhiza uralensis and Glycyrrhiza echinata. The anti-inflammatory agent according to any one of claims 1 to 5, wherein the oil-soluble licorice extract comprises at least one of the flavonoids of glabridine and ricokalcon A. The anti-inflammatory agent according to claim 6, wherein the total content of the oil-soluble licorice extract of at least one of the flavonoids of glabridine and ricochalcone A is 1 to 80 mass% in dry solid content. The anti-inflammatory agent according to any one of claims 5 to 7, wherein the content of glabridine in the oil-soluble licorice extract of Glycyrrhiza glabra is 1% by mass or more in dry solid content. The anti-inflammatory agent according to any one of claims 5 to 8, wherein the content of ricokalcon A in the oil-soluble licorice extract of Glycyrrhiza echinata is 1% by mass or more in dry solids. The anti-inflammatory agent according to any one of claims 5 to 9, wherein the content of ricokalcon A in the oil-soluble licorice extract of Glycyrrhiza inflata is 1% by mass or more in dry solid content. 11. The oil-soluble licorice extract according to any one of claims 1 to 10, wherein the oil-soluble licorice extract is selected from hyaluronidase activity inhibitory, hexosaminidase free inhibitory activity, platelet aggregation inhibitory activity, and phospholipase A ₂ activity inhibitory activity. Anti-inflammatory agent with one or more actions. The anti-inflammatory agent according to any one of claims 1 to 11, which is used as an external preparation for skin.