JP2005213203A - Antioxidant and antiinflammatory/antiallergic agent, and skin cosmetic - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an antioxidant or an antiinflammatory/antiallergic agent, each containing at least one of an extract of Enterospermum madagascariensis, an extract of Incaina stellifera and an extract of Siegesbeckia abyssinica; and to prepare a skin cosmetic containing the agent as an active ingredient. <P>SOLUTION: The antioxidant or the antiinflammatory/antiallergic agent contains at least one kind of the extract of the Enterospermum madagascariensis, the extract of the Incaina stellifera and the extract of the Siegesbeckia abyssinica. The antioxidant or the antiinflammatory/antiallergic agent preferably has at least one selected from an active oxygen-scavenging activity, a radical-scavenging activity, a hyaluronidase-inhibiting activity, a hexosaminidase release-inhibiting activity, a platelet aggregation-inhibiting activity, a prostaglandin E2 production-inhibiting activity and a cyclic AMP (adenosine monophosphate) phosphodiesterase-inhibiting activity. The skin cosmetic is obtained by formulating the antioxidant, the anti-aging agent or the antiinflammatory/antiallergic agent. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an antioxidant and an anti-inflammatory / antiallergic agent comprising at least one of Enterospermum madagascariensis extract, Incaina sterifera extract and Siegesbecia abyssinica extract, and these as active ingredients It relates to a blended skin cosmetic.

  Active oxygen is necessary for the sterilization mechanism of phagocytic cells and plays an important role in removing viruses and cancer cells. However, excessive production of the active oxygen attacks in vivo molecules constituting membranes and tissues in the living body, decomposes, denatures or crosslinks biological tissues such as collagen, or oxidizes fats and oils and damages cells. It is thought to produce lipid peroxide to give and cause aging such as skin wrinkle formation and skin elasticity reduction. Therefore, it is considered that skin aging such as wrinkle formation and elasticity reduction can be prevented and treated by inhibiting and suppressing the generation of active oxygen and in vivo radicals. Examples of herbal medicines having such an active oxygen scavenging action or radical scavenging action include extracts of plants belonging to the genus Osbeckia (see Patent Document 1), extracts of plants belonging to the genus Plumeria (see Patent Document 2), canalium, and the like. The extract of the plant which belongs to the genus (refer patent document 3), the lantana extract (refer patent document 4), etc. are reported.

  Hyaluronic acid exists as an intercellular tissue and is also involved in vascular permeability. Furthermore, hyaluronidase is said to be involved in degranulation from mast cells when activated in mast cells. Therefore, by inhibiting the activity of hyaluronidase, which is a hydrolase of hyaluronic acid, it is possible to stabilize hyaluronic acid, prevent the release of various chemical mediators from mast cells, and to enhance moisturization or anti-inflammation . Examples of herbal medicines having such a hyaluronidase inhibitory activity include, for example, extracts of plants belonging to the genus Osbeckia (see Patent Document 1), Fuji tea extract (see Patent Document 5), rosemary extract, thyme extract, And Melissa extract (see Patent Document 6) have been reported.

Platelet aggregation leads to activation of the arachidonic acid cascade phospholipase A ₂ , whereby leukotriene B, prostaglandin E _{2 and the} like are released and become flame-inducing substances. For this reason, attempts have been made to prevent / treat allergic diseases and inflammatory diseases with substances that inhibit / suppress platelet aggregation. Examples of herbal medicines having an inhibitory action on platelet aggregation include, for example, an extract of a plant belonging to the genus Canalium (see Patent Document 3), an extract of Kusasanfu (see Patent Document 7), and a Fuji tea extract (see Patent Document 8). , Etc. have been reported.

  In addition, platelet aggregation is related to the concentration of cyclic AMP in the platelet. When cyclic AMP is decomposed by cyclic AMP phosphodiesterase and the concentration of cyclic AMP decreases, platelets tend to aggregate. Therefore, it is considered that platelet aggregation can be prevented by suppressing the action of cyclic AMP phosphodiesterase to prevent the decrease of cyclic AMP. Cyclic AMP phosphodiesterase is also involved in promoting fat metabolism. By inhibiting the action of cyclic AMP phosphodiesterase, the concentration of intracellular cyclic AMP increases, fat metabolism becomes active, and obesity is reduced. It is known to be resolved. As herbal medicines having such a cyclic AMP phosphodiesterase inhibitory action, for example, extracts of plants belonging to the genus Osbeckia (see Patent Document 1), Fuji tea extract (see Patent Document 5), and the like have been reported.

In this way, it is excellent in safety and productivity, and can be ingested on a daily basis, and is inexpensive, yet has high active oxygen scavenging action, radical scavenging action, hyaluronidase inhibitory action, hexosaminidase release inhibitory action, platelet aggregation inhibition of prostaglandin E ₂ production inhibitory action, and extremely strong demand for consumer for various formulations of the natural system having at least one of cyclic AMP phosphodiesterase inhibiting action, not provided what may yet satisfactory is the current situation.

Japanese Patent Laid-Open No. 2003-55242 JP 2002-97151 A JP 2002-53478 A Japanese Patent Laid-Open No. 2002-179583 JP 2003-12532 A JP-A-8-333267 JP 2002-53477 A JP 2001-97873 A

This invention is made | formed in view of this present condition, 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 firstly finds a substance capable of scavenging active oxygen and in vivo radicals through at least one of active oxygen scavenging action and radical scavenging action among highly safe natural products. It aims at providing the antioxidant containing this.
The present invention, secondly, in a highly safe natural products, hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, prostaglandin E ₂ production inhibitory action, and cyclic AMP phosphodiesterase inhibitors An object of the present invention is to find a substance that can prevent or ameliorate antiallergic / inflammatory diseases through at least one selected from the actions, and to provide an antiinflammatory / antiallergic agent containing the substance as an active ingredient.
The third object of the present invention is to provide a highly safe skin cosmetic containing at least one of the antioxidant and the anti-inflammatory / anti-allergic agent of the present invention.

In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following knowledge. That is, at least one selected from an extract of Enterosperum madagascariensis, an extract of Incaina sterifera and an extract of Siegesbecia abyssinica is an active oxygen scavenging action, a radical scavenging action, a hyaluronidase inhibitory action, a hexosa Minidaze release inhibiting action, platelet aggregation inhibitory action, prostaglandin E ₂ production inhibitory action, and at least one selected from cyclic AMP phosphodiesterase inhibitory effect, anti-oxidants, or as an anti-inflammatory, anti-allergic agents This is a new finding that it can be suitably used.

The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> An extract of Enterospermum madagascariensis, an extract of Incaina stellifera, and an extract of at least one selected from Siegesbeckia abyssinica It is an antioxidant characterized by.
<2> The antioxidant according to <1>, which has at least one of active oxygen scavenging action and radical scavenging action.
<3> An extract of Enterosperum madagascariensis, an extract of Incaina stellifera, and an extract of at least one selected from Siegesbeckia abyssinica It is an anti-inflammatory / anti-allergic agent characterized by
<4> hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, according to the platelet aggregation inhibitory action, wherein the at least one selected from prostaglandin E ₂ production inhibitory action and cyclic AMP phosphodiesterase inhibitory effect <3> It is an anti-inflammatory and anti-allergic agent.
<5> containing at least one of the antioxidant according to any one of <1> to <2> and the anti-inflammatory / antiallergic agent according to any one of <3> to <4>. It is a featured skin cosmetic.

According to the present invention, an antioxidant and an anti-inflammatory / anti-allergic agent capable of solving various problems in the prior art are provided. According to the antioxidant of the present invention, aging phenomenon such as skin wrinkle formation and reduced elasticity is effectively prevented and treated through prevention of oxidation of biological components by active oxygen scavenging action and in vivo radical scavenging action. be able to.
According to anti-inflammatory, anti-allergic agent of the present invention, it is selected hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, prostaglandin E ₂ production inhibitory action, and the cyclic AMP phosphodiesterase inhibitory activity Through at least one, these involved inflammations can be effectively prevented or ameliorated. The antioxidant and the anti-inflammatory / anti-allergic agent of the present invention are suitable for blending into skin cosmetics because they are excellent in usability and safety when applied to the skin.
The skin cosmetic of the present invention is useful for preventing and / or improving inflammatory diseases and skin aging.

(Antioxidants and anti-inflammatory / anti-allergic agents)
The antioxidant and anti-inflammatory / anti-allergic agent of the present invention are all at least one selected from an extract of Enterosperum madagascariensis, an extract of Incaina sterifera, and an extract of Siagesbecia abyssinica And further contains other components as required.
The extract of Enterosperum / Madagascariensis, the extract of Incaina sterifera, and the extract of Siegesbekia / Abyssinica can be used alone, but they are used in combination from the viewpoint of efficiently exerting their effects. Can also be used.

The Enterosperum madagascariensis (scientific name: Enterospermum madagascariensis) is a protected tree of 10 to 15 meters, and is a plant of the Rubiaceae found in the semi-arid savanna zone.
The Incaina Stellifera (scientific name: Incaina stellifera) is a plant in which yellow flowers bloom and is a plant found in arid regions.
The said Siegesbekia Abyssinica (scientific name: Siegesbeckia abyssinica) is 40-50 cm, the stem is reddish and stiff, and the leaves are triangular.

  The use site of the enterospermum madagascariensis, incaina sterifera, or siegesbekia abyssinica is not particularly limited, and examples thereof include constituent parts such as leaves, branches, roots, bark parts, and fruit parts. Among them, enterosperum, madagascariensis and siagebeckia abyssinica are particularly preferably used as an extraction raw material. In addition, it is particularly preferable to use leaf parts as extraction raw materials for Incaina sterifera.

Active oxygen scavenging action, radical scavenging action, hyaluronidase inhibitory action, hexosaminidase release inhibitory action, prostastasis, prosta, which is contained in the extract of Enterosperum madagascariensis, Incaina sterifera or Schiagebeckia abyssinica prostaglandin E ₂ production inhibitory action, and details of the material having at least one selected from cyclic AMP phosphodiesterase inhibitory action is unknown, can be obtained by extraction methods commonly used in the extraction of plant.

  In addition, any of the said extract, the dried material obtained by drying the diluted solution of this extract, or these rough refined | purified products or purified products is contained. For example, it can be obtained by drying the stem of Enterosperum madagascariensis, the stem of Incaina sterifera, or the leaf of Schiagebeckia abyssinica and then subjecting it to solvent extraction by pulverization as it is or using a crusher. . Drying may be performed in the sun or using a commonly used dryer. In addition, the stem of Enterosperum madagascariensis, the trunk of Incaina Sterifera, or the leaves of Siegesbecia abyssinica are used as raw materials for extraction after pretreatment such as defatting with a non-polar solvent such as hexane or benzene. May be. By performing pretreatment such as degreasing, extraction with a polar solvent can be performed efficiently.

As the solvent used for the extraction, it is preferable to use water, a hydrophilic organic solvent, or a mixture thereof at room temperature or a temperature not higher than the boiling point of the solvent.
Examples of the water include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, and fresh water, and those obtained by performing various treatments on these. Examples of the treatment applied to water include purification, heating, 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 the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used. In addition, when using the mixed solvent of water and a hydrophilic organic solvent, in the case of the said lower alcohol, 1-90 mass parts of lower alcohol is preferable with respect to 10 mass parts of water. In the case of the lower aliphatic ketone, 1 to 40 parts by mass of the lower aliphatic ketone is preferable with respect to 10 parts by mass of water. In the case of the said polyhydric alcohol, 1-90 mass parts of polyhydric alcohol is preferable with respect to 10 mass parts of water.

In the present invention, it is selected active oxygen scavenging action, radical scavenging effect, hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, prostaglandin E ₂ production inhibitory action, and the cyclic AMP phosphodiesterase inhibitory activity In order to obtain an extract having at least one, it is not necessary to adopt a special extraction method, and the extraction can be performed using any apparatus at room temperature or under reflux heating.
Specifically, enter at least one of Enterosperum madagascariensis executives, Siegesbecia abyssinica executives, and Incaina Sterifera leaves into a treatment tank filled with the extraction solvent, as needed. The mixture is allowed to stand for 30 minutes to 2 hours with occasional stirring, and the soluble components are eluted. Then, the solid is removed by filtration, the extraction solvent is distilled off from the obtained extract, and the extract is obtained by drying. It is done. The amount of the extraction solvent is usually 5 to 15 times the mass of the extraction raw material (mass ratio), and the extraction conditions are usually 50 to 95 ° C. and about 1 to 4 hours when water is used as the extraction solvent. Moreover, when using the mixed solvent of water and ethanol as an extraction solvent, it is about 30 minutes-4 hours at 40-80 degreeC normally.

In addition, the extract obtained by extracting with a solvent can be used as an antioxidant of the present invention or as an anti-inflammatory / anti-allergic agent as long as the extraction solvent is highly safe, A concentrated solution or a dried product thereof is easier to use. In obtaining a dried extract, a carrier such as dextrin or cyclodextrin may be added to improve hygroscopicity.
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.
The extract of Enterosperum madagascariensis, the extract of Incaina sterifera, or the extract of Siegesbekia abyssinica has a characteristic odor, but is not unpleasant, and is used as it is for skin cosmetics. Available as raw material. However, if necessary, purification for the purpose of improving the activity and decoloring / deodorizing may be performed, or it may be formulated by mixing with any auxiliary agent.

The extract obtained as described above, the active oxygen scavenging action, radical scavenging effect, hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, prostaglandin E ₂ production inhibitory action, and cyclic AMP Since it has at least one selected from phosphodiesterase inhibitory action, the extract can be used as an antioxidant, an anti-inflammatory / anti-allergic agent, an active oxygen scavenger, a radical scavenger, and a hexosaminidase release inhibitor , platelet aggregation inhibitors, prostaglandin E ₂ production inhibitor, or can be used as cyclic AMP phosphodiesterase inhibitors. When the various drugs of the present invention are applied to the skin, they may be applied directly to the skin, or may be blended in a skin cosmetic and applied.

The antioxidant of the present invention eliminates active oxygen and in vivo radicals through at least one of active oxygen scavenging action and radical scavenging action to prevent and / or ameliorate skin aging and various skin diseases caused thereby. can do. Here, the “active oxygen” includes superoxide, hydrogen peroxide, hydroxy radical, singlet oxygen and the like. The “radical” means a molecule or atom having one or more unpaired electrons, and includes superoxide, hydroxy radical, DPPH (diphenylpicrylhydrazide) and the like.
Antiinflammatory-antiallergic agent of the present invention, hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, at least one selected from prostaglandin E ₂ production inhibitory action, and cyclic AMP phosphodiesterase inhibitory activity Thus, various inflammatory diseases including skin diseases such as contact dermatitis (rash), psoriasis and pemphigus vulgaris can be prevented and / or improved.

  The Enterosperum madagascariensis extract, Incaina sterifera extract, or Siegesbecia abyssinica extract has at least one of an antioxidant action and an antiallergic / anti-inflammatory action, and when applied to the skin It is suitable for blending into skin cosmetics because of its excellent usability and safety. By blending the above-described Enterosperum madagascariensis extract, Incaina sterifera extract, or Schiagebeckia abyssinica extract into skin cosmetics, the skin cosmetics have an antioxidative action, and an antiallergic and antiinflammatory action. At least one of them can be given.

(Skin cosmetic)
The skin cosmetic of the present invention is not particularly limited except that it contains at least one of the antioxidant and the anti-inflammatory / anti-allergic agent of the present invention, and other components appropriately selected according to the purpose. May be included.

The other components, active oxygen scavenging action, radical scavenging effect, hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, prostaglandin E ₂ production inhibitory action, and cyclic AMP phosphodiesterase inhibitory action As long as it does not hinder, there are no particular restrictions, and examples include ingredients appropriately selected according to the purpose. For example, whitening agents, astringents, bactericides / antibacterial agents, ultraviolet absorbers, moisturizers, cell activators, fats and oils, waxes , Hydrocarbons, fatty acids, alcohols, esters, surfactants, fragrances, and the like. These components, when used in combination with the Enterosperum / Madagascariensis extract, Incaina sterifera extract or Schiagezbekia abyssinica extract, act synergistically and have superior use effects that are normally expected. May bring.

The skin cosmetic containing at least one of the enterospermum madagascariensis extract, the incaina sterifera extract, and the siegesbekia abyssinica extract is an optional main agent or assistant that does not interfere with the physiological activity of the extract. What was mix | blended with the agent may be used, and the thing which has the said extract as a main component may be used.
The type of skin cosmetic that can be blended with at least one of the Enterosperum / Madagascariensis extract, Incaina sterifera extract and Siegesbekia abyssinica extract is not particularly limited, and specific examples thereof include Ointments, creams, emulsions, lotions, packs, foundations and the like can be mentioned.

The preferred blending ratio of at least one of the Enterosperum madagascariensis extract, Incaina sterifera extract and Schiagebeckia abyssinica extract is preferably 0.0001% to 10% by mass of the total amount of skin cosmetics, 0.001-1 mass% is more preferable.
The skin cosmetic may be applied directly to the skin, or may be used by impregnating it with a film, sheet, nonwoven fabric, etc. and applying it to the skin. Moreover, the skin cosmetics can be applied not only to the human body but also to animals.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Production Example 1)
-Water extract of Enterosperum madagascariensis-
3 L of water was added to 300 g of a dried product of the trunk of Enterosperum madagascariensis, and the mixture was heated and extracted at 80 ° C. for 2 hours with a reflux extractor and filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and then dried to obtain a solvent-extracted extract. The yield of the extract was as shown in Table 1.

(Production Example 2)
-80% ethanol extract of Enterosperum madagascariensis-
3 L of 80% by mass ethanol was added to 300 g of the dried product of the trunk of Enterosperum madagascariensis, heated and extracted at 80 ° C. for 2 hours with a reflux extractor, and filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and then dried to obtain a solvent-extracted extract. The yield of the extract was as shown in Table 1.

(Production Example 3)
-Water extract of Incaina Sterifera-
3 L of water was added to 300 g of the dried product of the leaves of Incaina sterifera, and the mixture was heated and extracted at 80 ° C. for 2 hours with a reflux extractor and filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and then dried to obtain a solvent-extracted extract. The yield of the extract was as shown in Table 1.

(Production Example 4)
-80% ethanol extract of Incaina Sterifera-
80 L% of ethanol 3 L was added to 300 g of dried leaves of Incaina sterifera, heated and extracted at 80 ° C. for 2 hours with a reflux extractor, and filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and then dried to obtain a solvent-extracted extract. The yield of the extract was as shown in Table 1.

(Production Example 5)
-Aqueous extract of Siegesbecia abyssinica-
3 L of water was added to 300 g of dried dried stem of Schiagebeckia abyssinica, heated and extracted at 80 ° C. for 2 hours with a reflux extractor, and filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and then dried to obtain a solvent-extracted extract. The yield of the extract was as shown in Table 1.

(Production Example 6)
-80% by weight ethanol extract of Siegesbecia abyssinica-
3 g of 80% by weight ethanol was added to 300 g of the dried portion of the trunk of Schiagebeckia abyssinica, heated and extracted at 80 ° C. for 2 hours with a reflux extractor, and filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and then dried to obtain a solvent-extracted extract. The yield of the extract was as shown in Table 1.

(Example 1)
-Superoxide elimination test (NBT method)-
The extracts obtained in Production Examples 1 to 6 were tested for superoxide scavenging action by the following test method.
Take 0.1 mL each of 3 mM xanthine, 0.05 M Na ₂ CO ₃ buffer (pH 10.2), 3 mM EDTA, BSA solution, and 0.75 mM NBT (nitroblue tetrazolium) in a test tube. Was added with 0.1 mL of each sample solution of Production Examples 1 to 6 and allowed to stand at 25 ° C. for 10 minutes. Next, a xanthine oxidase solution was added and stirred rapidly, and allowed to stand at 25 ° C. for 20 minutes. Thereafter, 0.1 mL of 6 mM copper chloride was added to stop the reaction, and the absorbance at a wavelength of 560 nm was measured.
The same operation and measurement of absorbance were performed without adding the enzyme solution. Further, the same measurement was performed when distilled water was added without adding the sample solution.
From the above measurement results, the superoxide erasure rate was determined by the following formula 1.

<Formula 1>
Superoxide erasure rate (%) = {1- (AB) / (CD)} × 100
However, in the said Numerical formula 1, A represents the light absorbency when an enzyme solution and a sample solution are added. B represents the absorbance when the sample solution is added without adding the enzyme solution. C represents the absorbance when the enzyme solution is added and the sample solution is not added. D represents the absorbance when the enzyme solution and the sample solution are not added.

  Next, the concentration of the sample solution was decreased stepwise to measure the superoxide elimination rate, and the sample concentration (μg / mL) at which the superoxide elimination rate was 50% was determined by interpolation. The results are shown in Table 2.

表２の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物がスーパーオキサイド消去作用を有することが確認された。

From the results shown in Table 2, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Siagesbecia abyssinica have a superoxide scavenging action.

(Example 2)
-Radical scavenging test for DPPH (diphenylpicrylhydrazide)-
The extracts obtained in Production Examples 1 to 6 were tested for radical scavenging action on DPPH by the following test method.
Add 3 mL of each sample solution of Production Examples 1 to 6 to 3 mL of 1.5 × 10 ⁻⁴ M DPPH ethanol solution, immediately seal the container, shake and mix, let stand for 30 minutes, and then measure absorbance at 520 nm wavelength did.
As a control, the same operation was performed using a solvent in which the sample solution was dissolved instead of the sample solution, and the absorbance at a wavelength of 520 nm was measured. Further, as a blank, after adding 3 mL of the sample solution to ethanol, the absorbance at a wavelength of 520 nm was measured immediately.
From the above measurement results, the radical scavenging rate (%) was calculated by the following formula 2.

<Formula 2>
Radical scavenging rate (%) = {1− (BC) / A} × 100
However, in the said Numerical formula 2, A represents the light absorbency of control. B represents the absorbance when the sample solution is added. C represents the absorbance of the blank.

  Next, the concentration of the sample solution was decreased stepwise to measure the radical scavenging rate, and the sample concentration (μg / mL) at which the radical scavenging rate with respect to DPPH was 50% was determined by interpolation. The results are shown in Table 3.

表３の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物がＤＰＰＨに対するラジカル消去作用を有することが確認された。

From the results shown in Table 3, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Siagesbecia abyssinica have a radical scavenging action on DPPH.

(Example 3)
-Hydrogen peroxide elimination test-
The extracts obtained in Production Examples 1 to 6 were tested for hydrogen peroxide scavenging action by the following test method.
10 μL of each sample solution of Production Examples 1 to 6 was added to 10 μL of a standard solution of hydrogen peroxide (concentration: 1.5 mM), incubated at 37 ° C. for 20 minutes, and then a color reagent [DA-64 (Wako Pure Chemical Industries, Ltd.) 1 mL of peroxidase solution (100 units / mL, manufactured by Wako Pure Chemical Industries, Ltd.) was added to 0.1 M PIPES buffer (pH 7.0) containing 10 mM of Triton X-100 and 0.5% by mass, and the total amount was adjusted to 100 mL After 2.98 mL was added and incubated at 37 ° C. for 5 minutes, the absorbance at a wavelength of 727 nm was measured.
The same operation and absorbance measurement were performed without adding a standard solution of hydrogen peroxide. Further, the same measurement was performed when distilled water was added without adding the sample solution.
From the above measurement results, the hydrogen peroxide erasing rate was determined by the following mathematical formula 3.

<Formula 3>
Hydrogen peroxide elimination rate (%) = {1- (A−B) / (C−D)} × 100
In Equation 3, A represents the absorbance when the hydrogen peroxide standard solution and the sample solution are added. B represents the absorbance when the sample solution is added without adding the hydrogen peroxide standard solution. C represents the absorbance when the hydrogen peroxide standard solution is added and the sample solution is not added. D represents the absorbance when the hydrogen peroxide standard solution and the sample solution are not added.

  Next, the concentration of the sample solution was decreased stepwise to measure the hydrogen peroxide elimination rate, and the sample concentration (μg / mL) at which the hydrogen peroxide elimination rate was 50% was determined by interpolation. The results are shown in Table 4.

表４の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物が過酸化水素消去作用を有することが確認された。

From the results shown in Table 4, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Siagesbecia abyssinica have a hydrogen peroxide scavenging action.

Example 4
-Hyaluronidase inhibition test-
The extracts obtained in Production Examples 1 to 6 were tested for hyaluronidase inhibitory action as follows.
After mixing 0.1 mL of a hyaluronidase solution (400 units / mL, pH 3.5 acetate buffer solution) and 0.2 mL of each sample solution of Production Examples 1 to 6, the mixture was heated at 37 ° C. for 20 minutes, and then activator 0.2 mL of a solution (2.5 mmol / L CaCl ₂ ) was added, and the enzyme was activated by heating at 37 ° C. for 20 minutes. After adding 0.5 mL of potassium hyaluronate buffer and reacting at 37 ° C. for 40 minutes, 0.2 mL of 0.4N sodium hydroxide was added and the reaction was stopped by cooling with ice.
Next, 0.2 mL of a 0.8 mol / L boric acid solution (pH 9.1) was added, and after heating in a boiling water bath for 3 minutes, the mixture was immediately cooled with ice for 20 minutes. 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 diluted 10-fold with acetic acid) was added to 6.0 mL and added at 37 ° C. for 20 minutes. Incubated. The 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. Further, as a control, the same operation and absorbance measurement were performed when distilled water was added instead of the sample solution.
From the above measurement results, the hyaluronidase activity inhibition rate (%) was calculated by the following mathematical formula 4.

<Formula 4>
Hyaluronidase activity inhibition rate (%) =
[1- (AB) / (CD)] × 100
However, in Formula 4, A means absorbance at the time of sample solution addition and enzyme addition. B means the absorbance when the sample solution is added and no enzyme is added. C means the absorbance when no sample is added and when the enzyme is added (control). D means the absorbance when no sample was added and no enzyme was added (control).

Next, the concentration of the sample solution was changed stepwise to measure the hyaluronidase activity inhibition rate, and the sample concentration IC ₅₀ (μg / mL) at which the inhibition rate was 50% was determined by interpolation (IC ₅₀ value). The smaller the value, the stronger the inhibitory action of hyaluronidase). The results are shown in Table 5.

表５の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物がヒアルロニダーゼ阻害作用を有することが確認された。

From the results shown in Table 5, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Siagesbecia abyssinica have a hyaluronidase inhibitory action.

(Example 5)
-Hexosaminidase release inhibition test-
The extracts obtained in Production Examples 1 to 6 were tested for hexosaminidase release inhibitory activity by the following test method. Since intracellular histamine is released and hexosaminidase is released at the same time, the histamine release inhibitory action can be evaluated using hexosaminidase release as an index.
Inoculate 1.0 × 10 ⁶ RBL-2H3 cells in a medium (15% FBS-added S-MEM medium; the same shall apply hereinafter) placed in a 25 mL culture flask at 37 ° C. under 5% CO ₂ -95% air. For 4 days. The cells were then collected by trypsinization and centrifugation (800 rpm, 4 minutes). The obtained cells were suspended in the medium at 4.0 × 10 ⁵ cells / mL, and mouse monoclonal anti-dinitrophenyl group IgE (DNP-Specific IgE) was added thereto at a concentration of 0.5 μg / mL. The cell suspension was seeded in 100 μL per well of a 96-well plate and cultured at 37 ° C. under 5% CO ₂ -95% air for 24 hours. After completion of the culture, the medium in each well was removed and washed twice with Silaganian buffer. Next, 30 μL of the buffer solution and 10 μL of each sample solution of Production Examples 1 to 6 were added and incubated at 37 ° C. for 10 minutes. Next, 10 μL of dinitrophenylated bovine serum albumin (DNP-BSA) was added, and further incubated at 37 ° C. for 15 minutes. Thereafter, 10 μL of the supernatant was transferred to a new 96-well plate under ice-cooling, 10 μL of 1 mmol / L p-nitrophenyl-N-acetyl-β-D-glucosamide solution was added thereto, and incubated at 37 ° C. for 1 hour. did. After completion of the reaction, 250 μL of a 0.1 mol / L NaCO—Na ₂ HCO ₃ solution was added, and the absorbance A at 415 nm was measured with a microplate reader at 650 nm as a control.
The same treatment and absorbance measurement were performed for the cell supernatant to which the Siraganian buffer was added instead of the sample solution (the absorbance measured at this time was designated as B). Absorbance measurement was also performed on the cell supernatant and 0.1 mol / L NaCO-Na ₂ HCO ₃ solution reacted in the same manner (the absorbance measured at this time was C). The same operation was performed for a sample in which a Siraganian buffer was added instead of DNP-BSA (at this time, the measured absorbance was D).
From the above measurement results, the hexosaminidase release inhibition rate was determined by the following formula 5.

<Formula 5>
Release inhibition rate (%) = [1-{(ACD) / (BD)}] × 100
Subsequently, the above-mentioned inhibition rate was measured by gradually reducing the sample concentration, and the sample concentration (μg / mL) that inhibits hexosaminidase release by 50% was determined by interpolation. The results are shown in Table 6.

表６の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物がヘキソサミニダーゼ遊離抑制作用を有することが確認された。

From the results shown in Table 6, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Schiagebeckia abyssinica have a hexosaminidase release inhibitory action.

(Example 6)
-Platelet aggregation inhibition test-
The extracts obtained in Production Examples 1 to 6 were tested for platelet aggregation inhibitory action as follows.
1/10 volume of 77 mM EDTA was added to Japanese white rabbit blood and centrifuged at 1000 rpm for 10 minutes to remove precipitates. The supernatant was centrifuged at 2100 rpm for 10 minutes, and the precipitated platelets were collected. The obtained platelets were suspended in a platelet washing solution and centrifuged at 2100 rpm for 10 minutes. The precipitated platelets were collected and suspended in a platelet suspension so that the platelet count was 300,000 / μL.
Next, 1 μL of a calcium chloride solution was added to 223 μL of the prepared washed platelet suspension and held at 37 ° C. for 1 minute. 1 μL of each sample solution of Production Examples 1 to 6 was added to the solution, and the mixture was kept at the same temperature for 2 minutes, followed by stirring for 1 minute.
Next, 25 μL of a 10 ppm collagen solution was added as an aggregation inducer, and the mixture was incubated at 37 ° C. for 10 minutes. Separately, the 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.
From the above measurement results, the platelet aggregation inhibition rate (%) was determined by the following formula 6.

<Formula 6>
Platelet aggregation inhibition rate (%) = [(B−A) / B] × 100
However, in Formula 6, A means the platelet aggregation rate at the time of adding the aggregation initiator and the sample solution. B means the platelet aggregation rate when the aggregation inducer is added and the sample solution is not added.

Then, the concentration of the sample solution was decreased stepwise to measure the platelet aggregation inhibition rate, and the concentration IC ₅₀ (μg / mL) at which the inhibition rate was 50% was determined by interpolation (this IC ₅₀ value is The smaller the value, the stronger the platelet aggregation inhibitory action). The results are shown in Table 7.

表７の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物が血小板凝集抑制作用を有することが確認された。

From the results shown in Table 7, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Siagesbecia abyssinica have a platelet aggregation inhibitory action.

(Example 7)
- prostaglandin E ₂ production inhibitory test -
The extract obtained from preparation 1 in 6, were tested prostaglandin _{E 2} production inhibitory action mediated Cyclooxygenase-2 by the following test methods.
Hwang, B.M. -Y. The method (Planta Medica 67 (2001), 406-410) was modified with some modifications. That is, after pre-culturing mouse-derived macrophage-like cells (RAW264.7), the cells were collected, prepared to 1 × 10 ⁵ cells / mL, seeded in 100 μL each in a 96-well plate, and 5% CO ₂ at 37 ° C. Cultured for 18 hours. After culturing, in order to acetylate and inactivate COX-1 already present and COX-2 expressed in a small amount, the medium was replaced with a 500 μmol / L aspirin-containing medium and cultured for 4 hours. The cells were washed 3 times with PBS (−), and 200 μL of the sample of Production Examples 1 to 6 dissolved in a medium containing 1 μg / mL LPS was added and cultured for 18 hours. After culturing, prostaglandin E ₂ in the supernatant was quantified using PGE ₂ EIA Kit (Cayman Chemical, Ann Arbor, MI, USA), and the inhibition rate of prostaglandin E ₂ production was calculated by comparing with the control. did.
Then, the concentration of the sample solution was decreased stepwise to measure the prostaglandin E ₂ production inhibition rate, and the concentration IC ₅₀ (μg / mL) at which the inhibition rate was 50% was determined by interpolation. The results are shown in Table 8.

表８の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物がプロスタグランジンＥ_２産生抑制作用を有することが確認された。

From the results in Table 8, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Siegesbecia abyssinica have a prostaglandin E ₂ production inhibitory action.

(Example 8)
-Cyclic AMP phosphodiesterase inhibitory action test-
The extracts obtained in Production Examples 1 to 6 were tested for cyclic AMP phosphodiesterase inhibitory action as follows.
To 0.2 mL of Tris-HCl buffer (pH 7.5) containing 5 mL of magnesium chloride, 0.1 mL of fetal serum albumin solution and 0.1 mL of cyclic AMP phosphodiesterase solution were added, and then each sample solution of Production Examples 1 to 6 0.05 mL was added and incubated at 37 ° C. for 5 minutes.
Next, 0.05 mL of a cyclic AMP phosphodiesterase solution was added and incubated at 37 ° C. for 60 minutes. The reaction was stopped by boiling for 3 minutes in a boiling bath, centrifuged at 3500 rpm at 4 ° C., and 5′-AMP as a reaction substrate in the supernatant was quantified by high performance liquid chromatography.
In addition, the same enzyme reaction and reaction substrate were analyzed without adding the sample solution, and the cyclic AMP phosphodiesterase inhibition rate of the sample (from the ratio of the reactive group mass when the sample was added to the reactive group mass when no sample was added ( %).

The sample concentration of the sample solution was decreased stepwise and the above measurement was repeated, and the concentration IC ₅₀ (μg / mL) at which the inhibition rate of cyclic AMP phosphodiesterase activity was 50% was determined by interpolation (this) The smaller the IC ₅₀ value, the stronger the cyclic AMP phosphodiesterase inhibitory action). The results are shown in Table 9.

表９の結果から、エンテロスペルムム・マダガスカリエンシスの抽出物、インカイナ・ステリフェラの抽出物、及びシアゲスベキア・アビスシニカの抽出物がサイクリックＡＭＰホスホジエステラーゼ阻害作用を有することが確認された。

From the results shown in Table 9, it was confirmed that the extract of Enterosperum madagascariensis, the extract of Incaina sterifera, and the extract of Schiagebeckia abyssinica have a cyclic AMP phosphodiesterase inhibitory action.

(Formulation Example 1)
An emulsion having the following composition was produced by a conventional method.
Jojoba oil 4.0g
Olive oil 2.0g
Squalane 2.0g
Cetanol 2.0g
Glyceryl monostearate 2.0g
2.5 g of polyoxyethylene cetyl ether (20E.O)
Oleic acid polyoxyethylene sorbitan (20E.O) 2.0g
1,3-butylene glycol 3.0 g
Hinokitiol 0.15g
Fragrance 0.05g
Enterosperum madagascariensis water extract (Production Example 1) 0.01 g
INKAINA STELLIFERA 80% ethanol extract (Production Example 4) 0.01g
Sieges Beckia abyssinica water extract (Production Example 5) 0.01 g
Purified water balance
Total 100g

(Formulation Example 2)
A cream having the following composition was produced by a conventional method.
Liquid paraffin 5.0g
Salami beeswax 4.0g
Cetanol 3.0g
Squalane 10.0g
Lanolin 2.0g
Stearic acid 1.0g
Oleic acid polyoxyethylene sorbitan (20E.O) 1.5g
3.0 g glyceryl monostearate
1,3-butylene glycol 6.0 g
1.5 g of methyl paraoxybenzoate
Fragrance 0.1g
Enterosperum madagascariensis 80% ethanol extract (Production Example 2) 0.05 g
Incaina Sterifera water extract (Production Example 3) 0.05g
Siagesbecia abyssinica 80% ethanol extract (Production Example 6) 0.05 g
Purified water balance
Total 100g

(Formulation Example 3)
A pack having the following composition was produced by a conventional method.
Polyvinyl alcohol 15g
Polyethylene glycol 3g
7g of propylene glycol
Ethanol 10g
0.05 g ethyl paraoxybenzoate
Fragrance 0.05g
Enterosperum madagascariensis water extract (Production Example 1) 0.01 g
INKAINA STELLIFERA 80% ethanol extract (Production Example 4) 0.01g
Sieges bequia abyssinica 80% ethanol extract (Production Example 6) 0.01 g
Purified water balance
Total 100g

The antioxidant of the present invention can effectively prevent and treat aging phenomena such as skin wrinkle formation and reduced elasticity through prevention of oxidation of biological components by active oxygen scavenging action and in vivo radical scavenging action. it can.
Antiinflammatory-antiallergic agent of the present invention, hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory action, at least one selected from prostaglandin E ₂ production inhibitory action, and cyclic AMP phosphodiesterase inhibitory activity Through these, these involved inflammations can be effectively prevented or ameliorated.
Further, the skin cosmetic of the present invention is excellent in the feeling of use and safety when applied to the skin, and contains the antioxidant and the anti-inflammatory / anti-allergic agent of the present invention, so that inflammatory diseases and Useful for preventing and / or improving skin aging.

Claims (5)

  An extract of Enterosperumum madagascariensis, an extract of Incaina sterifera and an extract of at least one species selected from a species of Siegesbecia abyssinica Antioxidants.   The antioxidant according to claim 1, which has at least one of an active oxygen scavenging action and a radical scavenging action.   An extract of Enterosperumum madagascariensis, an extract of Incaina sterifera and an extract of at least one species selected from a species of Siegesbecia abyssinica Anti-inflammatory and anti-allergic agents. Hyaluronidase inhibitory effect, hexosaminidase release inhibitory action, platelet aggregation inhibitory effect, anti-inflammatory of claim 3 having at least one selected from prostaglandin E ₂ production inhibitory action and cyclic AMP phosphodiesterase inhibitory effect, Antiallergic agent. A skin cosmetic comprising at least one of the antioxidant according to any one of claims 1 to 2 and the anti-inflammatory / antiallergic agent according to any one of claims 3 to 4.