JP2003306438A - Chemokine expression inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chemokine expression inhibitor which can inhibit the expression of a chemokine gene and especially inhibit the expression of interleukin-8, eotaxin, and RANTES genes. <P>SOLUTION: This chemokine expression inhibitor is characterized by containing the extract of one or more plants selected from Zingiber cassumunar Roxb. or Zingiber purpureum Rosc., Achillea millefolium L., Artemisia princeps Pampanini, Zanthoxylum piperitum Do Candolle, Thymus serpyllum L., and Phellodendron amurens Ruprecht as an active ingredient. At least one of four compounds as ingredients in the extract of the Zingiber cassumunar Roxb. or Zingiber purpureum Rosc. is contained as an active ingredient. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a chemokine (Chemot).
Actic Cytokine, Chemokine) expression inhibitors, especially interleukin-8 (IL-8), eotaxin,
RANTES (Regulated upon Activation, Normal T
Expressed and Secreted) gene expression to prevent rough skin, inflammation, and allergic symptoms due to various skin diseases such as atopic dermatitis, contact dermatitis, psoriasis, etc., as well as rough skin, sensitive skin, and acne skin of healthy people. The present invention relates to a chemokine expression inhibitor effective for prevention and improvement.

[0002]

2. Description of the Related Art Conventionally, it has an improving / preventing effect on rough skin and inflammation caused by skin diseases such as atopic dermatitis, contact dermatitis and psoriasis, allergic symptoms, and rough skin, sensitive skin and acne skin of healthy persons. As an active ingredient of the external preparation for skin, a steroid agent having an anti-inflammatory / anti-allergic effect and vaseline, urea, heparin, various amino acids and lipids having a moisturizing effect have been used. However, steroids have strong side effects, and moisturizers do not always have sufficient effects, and it has been desired to develop excellent active ingredients with higher safety.

Therefore, a highly safe crude drug with few side effects on the human body has been attracting attention, among which a platelet activating factor antagonist containing an extract of Zingiber cassumunar Roxb. Has been proposed (for example, refer to Patent Document 1). This therapeutic agent is a platelet activator (platelet activat) that is involved in the development of various inflammatory and allergic skin diseases and rough skin.
ing factor (PAF), it has an excellent PAF antagonistic effect and is effective in improving skin roughness and the like.

On the other hand, it is becoming clear that chemokines that control migration and activation of inflammatory cells are strongly involved in the development of various inflammatory and allergic skin diseases.
For example, chemokines are involved in allergic skin diseases such as atopic dermatitis and contact allergic dermatitis, and in psoriasis which is an inflammatory hyperkeratosis disease, IL, which is a kind of chemokine, is present in the affected skin area. It is known that -8 is present in a large amount. Similarly, in various inflammatory reactions, it has been found that a group of physiologically active proteins collectively called chemokines act mainly on the migration and activation of various cells involved in inflammation to the inflammatory site,
It is known that inflammatory cells such as neutrophils and eosinophils migrate and are activated at the site of inflammation in contact dermatitis of the skin, atopic dermatitis, etc., and are involved in the occurrence and exacerbation of inflammation,
In this case, it is known that cells such as skin keratinocytes and fibroblasts produce interleukin-8, eotaxin, RANTES, etc. as chemokines involved in migration and activation of inflammatory cells. There is. Interleukin-8, Eotaxin, RANTE
Chemokine expression inhibitors such as S are effective in ameliorating or preventing inflammatory or allergic disease symptoms involving chemokines such as interleukin-8, eotaxin, and RANTES.

As described above, interleukin-8, eotaxin, RANTES, etc. are known as chemokines, but there are diseases in which each substance is specifically or preferentially involved. For example, eotaxin is involved in atopic skin diseases, and lantes is involved in allergic skin diseases. Therefore, if information on the inhibition of expression of each substance is obtained, it is possible to more effectively improve the target disease and contribute to reduction of side effects.

[0006]

[Patent Document 1] Japanese Patent Laid-Open No. 2001-192339

[0007]

Therefore, an object of the present invention is to exert an excellent effect of suppressing the gene expression of chemokines and to improve and prevent inflammatory / allergic diseases based on the physiological actions of chemokines. Chemokine gene expression inhibitor having excellent effect on improving rough skin and various skin diseases, rough skin, etc. and prevention of rough skin containing it as an active ingredient.
It is intended to provide an improving skin external preparation composition.

[0008]

[Means for Solving the Problems] In view of such a current situation,
The present inventors believe that a chemokine gene expression-suppressing substance is effective in improving various inflammatory / allergic skin diseases and rough skin of healthy people, roughness, etc., and extensively investigate chemokine gene expression-suppressing action for various substances. As a result, it was found that the extract of Pontus ginger, which is a genus of Zingiberaceae, Zingiber, and the extracts of Achillea millefolium, Mugwort, Oataku, Sansho, and Thyme have a strong inhibitory effect on the expression of chemokine genes. . Further, the inventors have found that each plant extract selectively suppresses the expression of interleukin-8, eotaxin, and RANTES among chemokines, and completed the present invention.

That is, the present invention is a chemokine expression inhibitor characterized in that it contains one or more plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, thyme, and oat as an active ingredient. is there. Further, the present invention is an interleukin-8 expression inhibitor characterized in that one or two or more kinds of plant extracts selected from Pontsuku ginger, Achillea millefolium, and mugwort are added as an active ingredient.

Further, the present invention is an eotaxin expression inhibitor characterized in that it contains one or more plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, thyme and oat as an active ingredient. is there. Since a substance having an eotaxin expression inhibitory action is known to be effective for atopic skin diseases, the present invention provides an external preparation for atopic dermatitis based on the inhibition of eotaxin expression, which comprises the plant extract as an active ingredient. Can be used as

The present invention further comprises one or more kinds of plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, and rye as an active ingredient. RANTES.
It is an expression inhibitor. Since it is known that a substance having an inhibitory effect on the expression of RANTES is effective as an antiallergic agent, the present invention provides an antiallergic substance based on the inhibition of the expression of RANTES, which contains the plant extract as an active ingredient. It can be used as an agent.

According to the present invention, there is also provided an anti-inflammatory agent based on chemokine expression inhibition and a skin external preparation for improving rough skin, which comprises the above plant extract as an active ingredient.

There has been no report that the extract of Pontsuku ginger has a chemokine gene expression-suppressing action,
The application to inflammatory / allergic skin disease therapeutic agents and skin cosmetics for skin roughness prevention / improvement based on the chemokine gene expression inhibitory effect is not known at all. In addition, there has been no report that other plants belonging to the genus Zingiber (Zingiberaceae) have a chemokine gene expression-suppressing action. In addition, it is completely known that the plant extracts of Yarrow, Artemisia, Pleurotus cornucopiae, Pleurotus cornucopiae, and Thyme are applied to the therapeutic drug for inflammatory and allergic skin diseases and the skin cosmetics to prevent and improve skin roughness based on the chemokine gene expression inhibitory effect. Has not been done.

Among the plant extracts used in the present invention, Ponttsuku ginger is a plant belonging to the genus Zingiber of the Zingiberaceae family, and its scientific name is Zi.
ngiber cassumunar Roxb. or Zingiber purpureum
Known as Rosc., Zingiber cassumunar Roxb. And Zi
It has a synonym relationship with ngiber purpureum Rosc. In addition, pontsuku ginger is a plant that grows in tropical and subtropical regions such as Okinawa, southern China, and Southeast Asia.

The plant extract used in the present invention is obtained by immersing or heating and refluxing whole plants such as leaves, stems including rhizomes, fruits, etc. with an extraction solvent, filtering and concentrating. The extraction solvent used in the present invention may be any solvent that is usually used for extraction, particularly alcohols such as methanol and ethanol, hydrous alcohols, organic solvents such as acetone and ethyl acetate, or 1,3-butylene glycol. Etc. can be used alone or in combination.

The chemokine expression inhibitor of the present invention is preferably used as an external preparation, and the compounding amount of the plant extract at that time is 0.00 in terms of dry solid content in the total amount of the external preparation.
It is 1 to 20.0 mass%, preferably 0.01 to 10.0 mass%. If it is less than 0.001% by mass, the effect of the present invention is not sufficiently exerted, and even if it exceeds 20.0% by mass, further increase in the effect cannot be expected substantially, and it is compounded in each agent. Also tends to be difficult.

Furthermore, the inventors of the present invention particularly identified the components in the ponts ginger extract and investigated the inhibitory effect on gene expression of chemokines. As a result, the compounds (1) to (4) shown below showed strong chemokine gene expression. It was found that it has a suppressive effect. Further, they have found that each compound selectively suppresses the expression of interleukin-8, eotaxin and RANTES among chemokines, and completed the present invention.

That is, the present invention provides the following compounds (1) to
A chemokine expression inhibitor comprising at least one of (4) as an active ingredient.

[0019]

[Chemical 33]

[0020]

[Chemical 34]

[0021]

[Chemical 35]

[0022]

[Chemical 36]

The present invention also provides the above compounds (1) to (4).
It is an interleukin-8 expression inhibitor characterized by containing at least 1 sort (s) of these as an active ingredient.

The present invention also provides the above compounds (1) to (4).
An eotaxin expression inhibitor comprising at least one of the above as an active ingredient. Since a substance having an eotaxin expression inhibitory action is known to be effective for atopic skin diseases, the present invention uses the above compound as an external preparation for atopic dermatitis based on the inhibition of eotaxin expression containing the compound as an active ingredient. be able to.

The present invention also provides the compounds (1) to
A RANTES expression inhibitor comprising at least one of (4) as an active ingredient. Since a substance having a RANTES expression inhibitory action is known to be effective as an antiallergic agent, the present invention provides an antiallergic agent containing the above compound as an active ingredient based on RANTES expression inhibition. Can be used.

Further, according to the present invention, the above compound (1)
There is provided an anti-inflammatory agent based on chemokine expression inhibition and a skin external preparation for improving rough skin, which comprises at least one of (4) to (4) as an active ingredient.

The compounds (1) to (4) according to the present invention can be isolated from plants using known methods. For example, compounds (1) and (2) are available in Phytochemistry, vol.3
2, No.2, p357-363, 1993 (Author: Akiko Jitoe, Toshiya
Masuda, and Nobuji Nakatani), and the compounds (3) and (4) are described in JAOCS, vol.72, No.
The method described in 9, p.1053, 1995 (author: Toshiya Masuda, et.al.) can be used.

When the compounds (1) to (4) are prepared from an extract of ponts ginger, the site to be used is not particularly limited as long as the compounds (1) to (4) can be obtained. For example, roots, rhizomes, Examples thereof include stems, leaves, flowers, fruits, and whole plants, but roots or rhizomes are preferable. To give a specific example,
Compounds (1) to (4) are obtained by immersing the crushed product of roots or rhizomes of Pontsuku ginger in an extraction solvent at room temperature to heating for extraction, and purifying the extract by column separation and purification, recrystallization and the like. be able to.

The extraction solvent is not particularly limited, and examples thereof include water, primary alcohols such as methanol and ethanol,
Examples thereof include polyhydric alcohols such as 1,3-butylene glycol and propylene glycol, lower alkyl esters such as ethyl acetate, hydrocarbons such as benzene and hexane, ethyl ether, acetone and the like, and these may be used in combination of two or more kinds. it can. As a preferred extraction solvent,
Water, methanol, ethanol, 1,3-butylene glycol, or a mixed solvent thereof may be used.

In the present invention, the compounds (1) to (4)
Besides, it is also possible to use an extract containing these as an active ingredient. Examples of such an extract include a primary extract extracted with the above extraction solvent, and a crude fraction obtained by roughly purifying the primary extract by silica gel chromatography or the like. As the compounds (1) to (4) of the present invention, it is possible to use not only compounds extracted from natural products but also synthetic products produced by chemical synthesis or the like.

When the compounds (1) to (4) of the present invention are used as an external preparation, the compounding amount is 0.0001 in the total amount of the external preparation.
-20 mass%, preferably 0.001-10 mass%. If the amount is less than 0.0001% by mass, the effect of the present invention is not sufficiently exhibited, and even if it exceeds 20% by mass, further increase in the effect cannot be expected substantially, and it is difficult to compound each agent. Tends to become.

When the chemokine expression inhibitor of the present invention is used as an external preparation, in addition to the above-mentioned essential ingredients, ingredients usually used for external preparations for skin such as cosmetics and pharmaceuticals, such as whitening agents, moisturizers, antioxidants, oily ingredients. , UV absorbers, surfactants, thickeners, alcohols and the like, powder components, coloring agents, aqueous components, water, various skin nutrients and the like can be appropriately blended as necessary.

When the chemokine expression inhibitor of the present invention is used as an external preparation, it is further used as a sequestering agent such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate and gluconic acid, citric acid. , Glycolic acid, lactic acid, salicylic acid, sodium sulfite, caffeine, tannin, pantothenic acid and its derivatives, nicotinic acid and its derivatives, tranexamic acid and its derivatives, licorice extract, glabridin, horse chestnut, various plant extracts such as escin, Vitamin E and vitamin E derivatives such as tocopherol acetate, drugs such as glycyrrhizic acid and its derivatives or salts thereof, placenta extract, ascorbic acid and magnesium ascorbate phosphate, ascorbic acid derivatives such as glucoside ascorbate, al Whitening agents such as tin, kojic acid, ellagic acid, chamomile oil, 4-n-butylresorcinol, vitamin A derivatives such as vitamin A and retinol, sugars such as glucose, fructose, xylitol, mannose, sucrose, trehalose, vaseline, Moisturizers such as glycerin, urea and heparin, various amino acids such as serine and arginine, lipids such as ceramide and cholesterol, and the like can be appropriately added.

When the chemokine expression inhibitor of the present invention is used as an external preparation, it refers to a drug, a quasi drug, or a cosmetic product, for example, an ointment, a cream, an emulsion, a lotion, a pack, a bath preparation, etc. Any of the above may be used, and the dosage form is not particularly limited.

[0035]

The present invention will be described in more detail with reference to Examples. The present invention is not limited to this. The blending amount is% by mass. Prior to the Examples, the present invention relates to a test method for the chemokine gene expression inhibitory effect of the plant extract used in the present invention, the result (A), and the chemokine gene expression inhibitory effect of the compounds (1) to (4) used in the present invention. The test method and the result (B) will be described.

(A) Chemokine gene expression inhibitory effect of plant extract Preparation of Sample (1) Ponttsuku Ginger 50 g of the rhizome of the Pont Tsuku ginger was immersed in ethanol at room temperature for 1 week, and the extract was concentrated to obtain 67 mg of an ethanol extract. This extract was dissolved in DMSO at a concentration of 1%, the solution was diluted to adjust the concentration, and the following experiment was conducted using this.

(2) Achillea millefolium (Achilleamillefolium L.) 100 g of the whole plant was heated and extracted with 30% ethanol at 90 ° C. for 2 hours, and the obtained extract was distilled under reduced pressure to remove the solvent to obtain 13 g of an extract. Got This extract was dissolved in 50% ethanol to adjust the concentration, and the following experiment was conducted using this.

(3) Wormwood Wormwood (Artemisia princeps Pampanini) leaves 100 g
Was extracted with water at 90 ° C. for 2 hours while heating, and the solvent was distilled off from the obtained extract under reduced pressure to obtain 5 g of an extract. This extract was dissolved in 50% ethanol to adjust the concentration, and the following experiment was conducted using this.

(4) Pepper salmon (Zanthoxylum piperitum Do Candolle) 100 g of skin was extracted by heating with 70% ethanol at 90 ° C. for 2 hours, and the obtained extract was evaporated under reduced pressure to remove the solvent. 19 g were obtained. The extract was dissolved / suspended in 50% ethanol to adjust the concentration, and the following experiment was carried out using the extract.

(5) Time Thymus (Thymus serpyllum L.) 3 g of 100 g above ground
The extract was heated and extracted with 0% ethanol at 90 ° C. for 2 hours, and the solvent was distilled off from the obtained extract under reduced pressure to obtain 10 g of an extract. The extract was dissolved / suspended in 50% ethanol to adjust the concentration, and the following experiment was carried out using the extract.

(6) Tree bark 10 of Phellodendron amurens Ruprecht
0 g was extracted with water by heating at 90 ° C. for 2 hours, and the solvent was distilled off from the obtained extract under reduced pressure to obtain 20 g of an extract. The extract was dissolved / suspended in 50% ethanol to adjust the concentration, and the following experiment was carried out using the extract.

2. Test method for chemokine gene expression inhibitory action and its results Human skin fibroblasts were cultured in a culture dish with a diameter of 6 cm in DMEM medium (Dulbecco's modified Eagle medium) containing 10% fetal bovine serum until they became confluent and used for the experiment. did. Human dermal fibroblasts are commercially available and can be obtained from, for example, Kurabo (Kurashiki Spinning Co., Ltd.).

TN in the presence or absence of the above-prepared plant extracts of Pontsuku ginger, Achillea millefolium, Mugwort, Oat, Hawthorn and Thyme or the positive control hydrocortisone (10 ^-7 M).
F-α (1 ng / mL) was added, and the cells were cultured at 37 ° C for 6 hours (for interleukin-8) or 24 hours (for eotaxin and RANTES).

Next, RNA is isolated from the cells and cDNA is synthesized by a conventional method, and then the quantitative PCR method (Taq
The expression levels of interleukin-8, eotaxin, and RANTES genes were measured by the Man PCR method). The data obtained were corrected using GAPDH (glyceraldehyde 3 phosphate dehydrogenase) as an internal standard gene.

Table 1 shows that TNF-α (1 ng
/ ML) is added to induce chemokine expression, and the effect of the plant extract on chemokine gene expression is shown.
As the plant extract, ponts ginger, psyllium, salamander, yarrow, thyme and mugwort were applied, and a drug having a content concentration of each of them was 0.01% was added to the cells.

The relative value of the gene expression level of chemokine when the drug containing each of the above plant extracts was added was defined as the interleukin-8 expression level, with the expression level of the chemokine gene being 1 when cells without drug were used. (IL-8),
Each of RANTES and eotaxin is shown.

[0047]

[Table 1]

As can be seen from Table 1, when applied to Pontsuku ginger, all of IL-8, RANTES, and eotaxin had a gene expression suppressing effect of 20% or more. If you apply Oataku, RANT
ES and eotaxin had a gene expression suppressing effect of 20% or more. RA is applied when the sunshaw is applied
NTES and eotaxin had a gene expression suppressing effect of 20% or more. When the Yarrow was applied, all of IL-8, RANTES, and eotaxin had a gene expression suppressing effect of 20% or more. If thyme is applied, only eotaxin is 2
There was a gene expression suppressing effect of 0% or more. When mugwort was applied, IL-8, RANTES, and eotaxin all had a gene expression suppressing effect of 20% or more.

Next, ponts ginger extract was used as a plant extract, and the content concentration was 0.0005%,
R when 001%, 0.005% and 0.01% drugs were added to the cells and tested in the same manner as above
The test result of the expression level of the ANTES gene is shown in FIG. 1, and the test result of the expression level of the eotaxin gene is shown in FIG.

In FIGS. 1 and 2, the positive pair
Teru is hydrocortisone (10 ^-7When M) is added
RANTES and Eotaxin gene expression levels, drugs
When TNF-α (1 ng / mL) was added without any agent
RANTES and Eotaxin genes without addition
The expression level was simultaneously shown as vehicle. Figure 1 and
As shown in Fig. 2 and Fig. 2, the content of ponts ginger is high.
RANTES and eotaxin gene expression
It was found that the current suppression effect is high.

(B) Chemokine gene expression inhibitory effect of compounds (1) to (4) Production of Compounds (1) to (4) Production Example 1 Isolation of Compound (1) 500 g of rhizome of Zingiber purpureum Rosc. Was crushed, and immersed in 2.5 L of methanol at room temperature for 1 week for extraction.
The extract was filtered and concentrated, and then the concentrate was subjected to silica gel column chromatography (eluent: n-hexane / ethyl acetate mixed system) to obtain a crude fraction. Furthermore, reversed-phase HPLC preparative (mobile phase: 60% acetonitrile, column: CAPSELLPAK C18 UG120Å manufactured by Shiseido Co., Ltd., column size: 10
Compound (1) was isolated by mmφ × 250 mm, detection: UV210 nm). The yield was 410 mg and the yield was 0.082%. Table 2 shows the ¹³ C-NMR chemical shift values of the compound (1) together with literature values. Further, in the chemical formula (1A), carbon position No. The chemical structural formula corresponding to is shown.

[0052]

[Chemical 37]

[0053]

[Table 2] *: Jitoe, A., Masuda, T., Nakatani, N., Phytochemist
ry, vol.32 No.2 P357-363 (1993)

Production Example 2 Isolation of Compound (2) 500 g of a rhizome of Zingiber purpureum Rosc. Was crushed and immersed in 2.5 L of methanol at room temperature for 1 week for extraction.
The extract was filtered and concentrated, and then the concentrate was subjected to silica gel column chromatography (eluent: n-hexane / ethyl acetate mixed system) to obtain a crude fraction. Furthermore, reversed-phase HPLC preparative (mobile phase: 60% acetonitrile, column: CAPSELLPAK C18 UG120Å manufactured by Shiseido Co., Ltd., column size: 10
Compound (2) was isolated by mmφ × 250 mm, detection: UV 210 nm). The yield was 430 mg and the yield was 0.086%. Table 3 shows the ¹³ C-NMR chemical shift values of Compound 2 together with literature values. The carbon position numbers in Table 3 conform to the above chemical formula (1A).

[0055]

[Table 3] *: Jitoe, A., Masuda, T., Nakatani, N., Phytochemist
ry, vol.32 No.2 P357-363 (1993)

Production Example 3 Compound (3) (Cassumunarin
Isolation of B) 509.7 g of a rhizome of Zingiber purpureum Rosc. Was crushed, and allowed to stand at room temperature for 1 week in 2.5 L of methanol for extraction. The extract is filtered, concentrated, dried under reduced pressure,
50.6 g of extract solids was obtained. About 38 g of this was subjected to silica gel column chromatography (eluent: mixed system of n-hexane and ethyl acetate) to give a crude fraction of about 1.
2 g was obtained. Next, about 200 mg of this crude fraction was added to the reverse phase HP.
LC preparative (first / mobile phase: 50% acetonitrile, column: Shiseido CAPCELLPAK C18 UG120Å, column size: 20mmΦX250mm, detection: UV210nm second / mobile phase: 50
% Acetonitrile, column: SUPERIOREX ODS manufactured by Shiseido,
Column size: 10 mmΦ 250 mm, detection: UV210 nm) was performed twice, and finally 3.9 mg of compound (3) was isolated.

The ¹³ C-NMR chemical shift value and the ¹ H-NMR chemical shift value of the compound (3) are shown in Table 4 below. The measurement solvent is deuterated acetone, and the measurement temperature is 30 ° C. Chemical formula (3A) shows a chemical structural formula corresponding to the carbon position number in Table 4. Compound (3) is a specific component of curcuminoid, Cassumunarin B, in comparison with literature values.
Was identified.

[0058]

[Chemical 38]

[0059]

[Table 4]

Production Example 4 Compound (4) (Cassumunarin
Isolation of C) 509.7 g of a rhizome of Zingiber purpureum Rosc. Was crushed and left to stand in 2.5 L of methanol at room temperature for 1 week,
Extraction was performed. The extract is filtered, concentrated, and dried under reduced pressure.
0.6 g of extract solids was obtained. About 38 g of this was subjected to silica gel column chromatography (eluent: a mixed system of n-hexane and ethyl acetate) to obtain about 1.2 g of a crude fraction.
Obtained. Then, about 200 mg of this crude fraction was subjected to reverse phase HPLC.
Preparative (1st time: mobile phase: 50% acetonitrile, column:
Shiseido CAPCELLPAK C18 UG120Å, column size: 20mm
ΦX250mm, detection: UV210nm 2nd time: mobile phase: 50% acetonitrile, column: SUPERIOREX ODS manufactured by Shiseido, column size: 10mm ΦX250mm, detection: UV210nm) was performed twice, and finally 4.1 mg of compound (4) was isolated. .

The ¹³ C-NMR chemical shift value and the ¹ H-NMR chemical shift value of the compound (4) are shown in Table 5 below. The measurement solvent is deuterated acetone, and the measurement temperature is 30 ° C. Chemical formula (4A) shows a chemical structural formula corresponding to the carbon position number in Table 5. Compound (4) was identified as Cascumunarin C, a specific component of curcuminoids, by comparison with literature values.
Was identified.

[0062]

[Chemical Formula 39]

[0063]

[Table 5]

Production Example 5 Mixture of Compound (1) and Compound (2) 500 g of Zingiber purpureum Rosc. Rhizome was ground,
Extraction was performed by immersing in ethanol for 1 week at room temperature. After the extract is filtered and concentrated, the concentrate is subjected to silica gel column chromatography (eluent: a mixture of n-hexane and ethyl acetate), and then reversed phase HPLC fractionation (mobile phase: 60% acetonitrile, column: Shiseido CAPCELLP
AK C18 UG120Å, column size: 10mmΦX250mm, detection:
UV210 nm) was performed to obtain 1.03 g of a mixture of the compound (1) and the compound (2) with a peak area ratio in HPLC analysis of about 1: 1.

Next, FAB-M of compounds (3) and (4)
S measurement was performed. The measurement conditions are as follows. The measurement results are shown in Table 6.

[0066] Measurement mass range: m / z 100 to 1000 (5sec / decade) Resolution: 1000 Ionization method: FAB-NEG Cs 20kV Accelerating voltage: 5kV Matrix: Thioglycerol

[0067]

[Table 6]

2. Test method of chemokine gene expression inhibitory effect of each compound and its results (1) Test method As a cell underlying chemokine gene expression, human skin fibroblasts similar to those used in the test on plant extract were used for the experiment. did. T for the expression of the chemokine gene
NF-α (1 ng / mL) was added, and at the same time, the following agents were added, and the mixture was cultured at 37 ° C. for 24 hours. Each sample was used as an ethanol solution. (A) Compound (1) and compound (2) obtained in Production Example 5
(B) The compound (3) obtained in Preparation Example 3 (content concentration: 0. 01%).
001%) (c) Compound (4) obtained in Preparation Example 4 (concentration of content: 0.
(001%)

Further, the following chemicals were added and the cells were similarly cultured. (D) Hydrocortisone (10 ^-8 M) (e) Pontuk ginger methanol extract (content concentration 0.001%) (f) Pontuk ginger methanol extract (content concentration 0.01%)

Then, RNA is isolated from the cells and cDNA is synthesized according to a conventional method, and then the quantitative PCR method (Taq
Man PCR method), eotaxin and RAN
The expression level of the TES gene was measured. The data obtained were corrected using GAPDH (glyceraldehyde 3 phosphate dehydrogenase) as an internal standard gene.

(2) Test Results The test results of the expression level of the RANTES gene when tested by the above test method are shown in FIG. 3, and the test results of the expression level of the eotaxin gene are shown in FIG. In addition, in FIG. 3 and FIG. 4, the gene expression levels of RANTES and eotaxin with and without addition of TNF-α (1 ng / mL) were simultaneously shown as vehicle without addition of a drug.

In FIG. 3, the RANTES gene expression inhibitory effect was observed in all of the mixture of compound (1) and compound (2), compound (3) and compound (4). Particularly, the mixture of the compound (1) and the compound (2) and the compound (3) were more excellent in the effect of suppressing the gene expression of RANTES than the ponts ginger extract having the same concentration.

In FIG. 4, the mixture of compound (1) and compound (2), compound (3) and compound (4) all showed the effect of suppressing gene expression of eotaxin. In particular, the compound (3) and the compound (4) were more excellent in the effect of suppressing the gene expression of eotaxin than the ponts ginger extract of the same concentration.

Thus, the compounds (1) to (1) of the present invention
By using (4), even with the same chemokine expression inhibitory action, it becomes possible to apply optimal compounds according to the suppression of RANTES gene expression suppression and the eotaxin gene expression suppression, respectively. Can be prevented and improved.

Hereinafter, prescription examples when the chemokine expression inhibitor of the present invention is applied to various dosage forms will be described as examples. In addition, in the examples, all plant extracts are masses converted to dry solids.

Example 1 Cream formulation mass% stearic acid 5.0 stearyl alcohol 4.0 isopropyl myristate 18.0 glycerin monostearate 3.0 propylene glycol 10.0 ponts ginger ethanol extract 0.01 caustic potash 0 .2 Sodium bisulfite 0.01 Preservative Suitable amount Perfume Suitable amount Ion-exchanged water Residual (production method) Propylene glycol, ethanol extract of ponts ginger and caustic potash were dissolved in ion-exchanged water and heated to 70 ° C (water phase) ). The oil phase is gradually added to the water phase, and after the addition is completed, the temperature is maintained for a while to cause the reaction. Then, it is uniformly emulsified with a homomixer and cooled to 30 ° C. with thorough stirring.

Example 2 Cream formulation mass% stearic acid 2.0 stearyl alcohol 7.0 hydrogenated lanolin 2.0 squalane 5.0 2-octyldodecyl alcohol 6.0 polyoxyethylene (25 mol) cetyl alcohol ether 3. 0 Glycerin monostearate 2.0 Propylene glycol 5.0 1,3-butylene glycol extract of yarrow Astragalus 0.05 Sodium hydrogen sulfite 0.03 Ethylparaben 0.3 Perfume Ion-exchanged water Residual (production method) Ion-exchange Add propylene glycol to water,
Heat to maintain 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and maintained at 70 ° C. (oil phase). The oil phase is added to the aqueous phase to carry out preliminary emulsification, and the mixture is uniformly emulsified with a homomixer and then cooled to 30 ° C. with thorough stirring.

Example 3 Cream Formulation Mass% Solid Paraffin 5.0 Beeswax 10.0 Vaseline 15.0 Liquid Paraffin 41.0 Glycerin Monostearate 2.0 Polyoxyethylene (20 mol) Sorbitan Monolaurate 2.0 Soap powder 0.1 Borax 0.2 Oat water extract 0.05 Mugwort ethanol extract 0.05 Sodium bisulfite 0.03 Ethylparaben 0.3 Fragrance Ion exchange water Residual (production method) Ion exchange water soap The powder and borax are added, melted by heating and kept at 70 ° C. (aqueous phase). The other ingredients are mixed, heated and melted and maintained at 70 ° C. (oil phase). The oil phase is stirred into the water phase and gradually added to carry out the reaction. After the reaction is completed, the mixture is uniformly emulsified with a homomixer, and after emulsification, the mixture is cooled to 30 ° C. with thorough stirring.

Example 4 Emulsion formulation Mass% Stearic acid 2.5 Cetyl alcohol 1.5 Vaseline 5.0 Liquid paraffin 10.0 Polyoxyethylene (10 mol) Monooleate ester 2.0 Polyethylene glycol 1500 3.0 Tri Ethanolamine 1.0 Carboxyvinyl polymer 0.05 (trade name: Carbopol 941, BFGoodrich Chemical Company) 1,3-butylene glycol extract of salamander 0.01 Sodium bisulfite 0.01 Ethylparaben 0.3 Perfume Suitable amount Ion-exchanged water Residue (production method) A carboxyvinyl polymer is dissolved in a small amount of ion-exchanged water (phase A). Polyethylene glycol 1500 and triethanolamine were added to the remaining ion-exchanged water, and the mixture was heated and dissolved and maintained at 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and maintained at 70 ° C. (oil phase). The oil phase is added to the aqueous phase for preliminary emulsification, the phase A is added and the mixture is uniformly emulsified with a homomixer.

Example 5 Emulsion formulation Mass% Microcrystalline wax 1.0 Beeswax 2.0 Lanolin 20.0 Liquid paraffin 10.0 Squalane 5.0 Sorbitan sesquioleate 4.0 Polyoxyethylene (20 mol) Sorbitan mono Oleic acid ester 1.0 Propylene glycol 7.0 Water extract of thyme 10.0 Sodium bisulfite 0.01 Ethyl paraben 0.3 Perfume Ion-exchanged water Residual (production method) Add propylene glycol to ion-exchanged water,
Heat to maintain 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and kept at 70 ° C. (oil phase). While stirring the oil phase, the aqueous phase is gradually added to this and the mixture is uniformly emulsified with a homomixer. After emulsification, cool to 30 ° C while stirring well.

Example 6 Jelly Formulation Mass% 95% Ethyl alcohol 10.0 Dipropylene glycol 15.0 Polyoxyethylene (50 mol) Oleyl alcohol ether 2.0 Carboxyvinyl polymer 1.0 (trade name: Carbopol 940, BFGoodirch Chemical Company) Caustic soda 0.15 L-arginine 0.1 80% ethanol extract of Pontus ginger 7.0 Sodium 2-hydroxy-4-methoxy benzophenone sulfonate 0.05 Ethylenediaminetetraacetate ・ 3 sodium dihydrate 0.05 Methylparaben 0.2 Perfume A suitable amount of ion-exchanged water Residual (manufacturing method) Carbopol 940 is uniformly dissolved in ion-exchanged water, while 80% of ponts ginger in 95% ethanol.
% Ethanol extract, polyoxyethylene (50 mol)
The olein alcohol ether is dissolved and added to the aqueous phase. Next, after adding other ingredients, caustic soda, L
-Neutralize with arginine to thicken.

Example 7 Cosmetic Liquid Formulation Mass% (Phase A) Ethyl Alcohol (95%) 10.0 Polyoxyethylene (20 mol) Octyldodecanol 1.0 Pantothenyyl Ethyl Ether 0.1 0.13 of Achillea millefolium -Butylene glycol extract 1.5 Methylparaben 0.15 (Phase B) Potassium hydroxide 0.1 (Phase C) Glycerin 5.0 Dipropylene glycol 10.0 Sodium hydrogen sulfite 0.03 Carboxyvinyl polymer 0.2 (Product Name: Carbopol 940, BFGoodrich Chemical Company) Purified water Residue (production method) Phases A and C are uniformly dissolved, and phase A
Add phase and solubilize. Next, phase B is added and then filling is performed.

Example 8 Packing formulation Mass% (Phase A) Dipropylene glycol 5.0 Polyoxyethylene (60 mol) Hydrogenated castor oil 5.0 (Phase B) Water extract of Oat 0.01 Olive oil 5.0 Acetic acid Tocophenol 0.2 Ethylparaben 0.2 Perfume 0.2 (Phase C) Sodium hydrogen sulfite 0.03 Polyvinyl alcohol 13.0 (Saponification degree 90, degree of polymerization 2,000) Ethanol 7.0 Purified water Residual (manufacturing method ) A phase, B phase and C phase are each dissolved uniformly,
Add phase B to phase A to solubilize. Next, this is added to phase C and then filled.

Example 9 Solid Foundation Formulation Mass% Talc 43.1 Kaolin 15.0 Sericite 10.0 Zinc white 7.0 Titanium dioxide 3.8 Yellow iron oxide 2.9 Black iron oxide 0.2 Squalane 8.0 Isostearic acid 4.0 POE sorbitan monooleate 3.0 Isocetyl octoate 2.0 Ethanol extract of mugwort 0.5 Preservative proper amount perfume appropriate amount (manufacturing method) Talc-black iron oxide powder components are thoroughly mixed with a blender, Squalane-isocetyl octoate oil component, mugwort ethanol extract, preservative, and fragrance are added and kneaded well, then filled into a container and molded.

Example 10 Emulsion type foundation (cream type) Prescription mass% (powder part) Titanium dioxide 10.3 Sericite 5.4 Kaolin 3.0 Yellow iron oxide 0.8 Bengala 0.3 Black iron oxide 0. 2 (Oil phase) Decamethylcinclopentasiloxane 11.5 Liquid paraffin 4.5 Polyoxyethylene modified dimethylpolysiloxane 4.0 (Aqueous phase) Purified water 50.0 1,3-Butylene glycol 4.5 Sansho 1,3-butylene glycol extract of 1.5 1.5 sorbitan sesquioleate 3.0 preservative appropriate amount perfume appropriate amount (production method) After stirring the aqueous phase with heating, thoroughly mix and pulverize the powder portion to add homogenate. Process with a mixer. Further, the heated and mixed oil phase is added and subjected to a homomixer treatment, and then a fragrance is added with stirring and cooled to room temperature.

Example 11 Cream Formulation Mass% stearic acid 5.0 stearyl alcohol 4.0 isopropyl myristate 18.0 glycerin monostearate 3.0 propylene glycol 10.0 thyme water extract 0.01 caustic potash 0.1. 2 Sodium bisulfite 0.01 Preservative Appropriate amount Perfume Appropriate amount Ion-exchanged water Residue (production method) Dissolve propylene glycol, thyme water extract and caustic potash in ion-exchanged water, heat and keep at 70 ° C phase). The oil phase is gradually added to the water phase, and after the addition is completed, the temperature is maintained for a while to cause the reaction. Then, it is uniformly emulsified with a homomixer and cooled to 30 ° C. with thorough stirring.

Example 12 Cream formulation mass% stearic acid 5.0 stearyl alcohol 4.0 isopropyl myristate 18.0 glycerin monostearate 3.0 propylene glycol 10.0 the above compound (1) 0.001 caustic potash 0.1. 2 Sodium bisulfite 0.01 Preservative Suitable amount Perfume Suitable amount Ion-exchanged water Residual (production method) Propylene glycol, the compound (1) and caustic potash are added to ion-exchanged water and dissolved, and heated to 70 ° C (water phase). The oil phase is gradually added to the water phase, and after the addition is completed, the temperature is maintained for a while to cause the reaction. Then, it is uniformly emulsified with a homomixer and cooled to 30 ° C. with thorough stirring.

Example 13 Cream formulation mass% stearic acid 2.0 stearyl alcohol 7.0 hydrogenated lanolin 2.0 squalane 5.0 2-octyldodecyl alcohol 6.0 polyoxyethylene (25 mol) cetyl alcohol ether 3. 0 Glycerin monostearate 2.0 Propylene glycol 5.0 Compound (2) 0.1 Sodium bisulfite 0.03 Ethylparaben 0.3 Perfume Ion-exchanged water Residual (production method) Propylene glycol is added to ion-exchanged water ,
Heat to maintain 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and maintained at 70 ° C. (oil phase). The oil phase is added to the aqueous phase to carry out preliminary emulsification, and the mixture is uniformly emulsified with a homomixer and then cooled to 30 ° C. with thorough stirring.

Example 14 Cream formulation mass% solid paraffin 5.0 beeswax 10.0 vaseline 15.0 liquid paraffin 41.0 glycerin monostearate 2.0 polyoxyethylene (20 mol) sorbitan monolaurate 2.0 Soap powder 0.1 Borax 0.2 Compound (3) 0.01 Sodium bisulfite 0.03 Ethylparaben 0.3 Perfume Ion-exchanged water Residual (production method) Soap powder and borax are added to ion-exchanged water and dissolved by heating And maintain at 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and maintained at 70 ° C. (oil phase). The oil phase is stirred into the water phase and gradually added to carry out the reaction. After the reaction is completed, the mixture is uniformly emulsified with a homomixer, and after emulsification, the mixture is cooled to 30 ° C. with thorough stirring.

Example 15 Emulsion formulation Mass% Stearic acid 2.5 Cetyl alcohol 1.5 Vaseline 5.0 Liquid paraffin 10.0 Polyoxyethylene (10 mol) Monooleate ester 2.0 Polyethylene glycol 1500 3.0 Tri Ethanolamine 1.0 Carboxyvinyl polymer 0.05 (Brand name: Carbopol 941, BFGoodrich Chemical Company) The compound (4) 0.005 Sodium hydrogen sulfite 0.01 Ethylparaben 0.3 Perfume Ion-exchanged water Residual (manufacturing method) ) Dissolve carboxyvinyl polymer in a small amount of ion-exchanged water (Phase A). Polyethylene glycol 1500 and triethanolamine were added to the remaining ion-exchanged water, and the mixture was heated and dissolved and maintained at 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and maintained at 70 ° C. (oil phase). The oil phase is added to the aqueous phase for preliminary emulsification, the phase A is added and the mixture is uniformly emulsified with a homomixer.

Example 16 Emulsion formulation Mass% Microcrystalline wax 1.0 Beeswax 2.0 Lanolin 20.0 Liquid paraffin 10.0 Squalane 5.0 Sorbitan sesquioleate 4.0 Polyoxyethylene (20 mol) Sorbitan mono Oleic acid ester 1.0 Propylene glycol 7.0 Mixture of the compound (1) and the compound (2) 1.0 Sodium bisulfite 0.01 Ethylparaben 0.3 Perfume Ion-exchanged water Residue (production method) To ion-exchanged water Add propylene glycol,
Heat to maintain 70 ° C (aqueous phase). The other ingredients are mixed, heated and melted and kept at 70 ° C. (oil phase). While stirring the oil phase, the aqueous phase is gradually added to this and the mixture is uniformly emulsified with a homomixer. After emulsification, cool to 30 ° C while stirring well.

[0092]

INDUSTRIAL APPLICABILITY As described above, the chemokine expression inhibitor of the present invention suppresses the expression of chemokine gene, and particularly interleukin-8, eotaxin, RANTES.
Can inhibit the gene expression of. Therefore, it becomes possible to appropriately prevent and ameliorate various skin diseases specifically caused by the expression of each chemokine gene. Moreover, side effects caused by improper application can be avoided, and the target skin disease can be prevented and ameliorated with a smaller amount. Further, as the chemokine expression inhibitor of the present invention, the compounds most suitable for each disease can be applied by appropriately selecting compounds (1) to (4), and the occurrence of side effects can be reduced. Thus, the chemokine expression inhibitor of the present invention, atopic dermatitis, contact dermatitis, psoriasis, etc., skin roughening symptoms and inflammation due to various skin diseases, allergic symptoms, rough skin of healthy people,
For prevention and improvement of sensitive skin and acne skin, it becomes possible to apply optimal ingredients according to each disease.

[Brief description of drawings]

FIG. 1 is a diagram showing the test results of the expression level of RANTES gene in a plant extract according to the present invention.

FIG. 2 is a diagram showing the test results of the expression level of the eotaxin gene in the plant extract according to the present invention.

FIG. 3 is a RANT of compounds (1) to (4) according to the present invention.
It is a figure which shows the test result of the expression level of ES gene.

FIG. 4 is a diagram showing the test results of the expression level of the eotaxin gene of compounds (1) to (4) according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 7/00 A61K 7/00 K 7/48 7/48 31/09 31/09 31/12 31/12 A61P 17/00 A61P 17/00 29/00 29/00 37/08 37/08 43/00 111 43/00 111 (72) Inventor Masahiro Ohta 2-2-1 Hayabuchi, Tsuzuki Ward, Yokohama City, Kanagawa Prefecture Stock Company Shiseido Research Center (Shin-Yokohama) (72) Inventor Tomoko Kimura 2-2-1-1 Hayabuchi, Tsuzuki-ku, Yokohama-shi, Kanagawa Shiseido Research Center (Shin-Yokohama) (72) Inventor Hideyuki Ichikawa Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 2-12-1 F-term in Shiseido Research Center (Hakkei Kanazawa), a stock company (reference) 4C083 AA082 AA111 AA112 AA122 AB032 AB152 AB212 AB232 AB242 AB352 AB432 AB442 AC012 AC022 AC072 AC092 AC102 AC122 AC171 AC182 AC242 AC262 AC352 AC402 AC422 AC432 AC442 AC471 AC482 AC532 AC542 AC582 AC642 AC792 AD042 AD092 AD112 AD162 AD172 AD512 AD662 CC05 CC07 CC12 DD21 DD31 DD41 EE12 EE13 4C088 AB26 AB29 AB38 AB62 AB81 AC01 AC02 AC04 AC05 AC06 AC12 BA11 CA05 CA06 CA08 CA14 BAZ CA14 CA06 CA13 ZC01 ZC41 4C206 AA01 AA02 CA34 CB15 MA01 MA04 NA14 ZA89 ZB11 ZB13 ZC02 ZC41

Claims (16)

[Claims] 1. A chemokine expression inhibitor, which comprises, as an active ingredient, one or more kinds of plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, thyme and oat. 2. An interleukin-8 expression inhibitor, which comprises, as an active ingredient, one or two or more kinds of plant extracts selected from pontsuku ginger, yarrow, and mugwort. 3. An eotaxin expression inhibitor comprising one or two or more plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, thyme and oat as an active ingredient. 4. Atopic skin based on the inhibition of eotaxin expression, which comprises one or more kinds of plant extracts selected from ponts ginger, yarrow millet, mugwort, salamander, thyme and oat as an active ingredient. External preparation for flames. 5. A RANTES expression inhibitor, which comprises, as an active ingredient, one or two or more kinds of plant extracts selected from pontsuku ginger, yarrow plant, mugwort, salamander and psyllium. 6. An antiallergic agent based on inhibition of RANTES expression, which comprises one or more plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, and oat as an active ingredient. Agent. 7. An anti-inflammatory agent based on chemokine expression inhibition, comprising one or two or more kinds of plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, thyme and oat as an active ingredient. . 8. For improving skin roughness based on chemokine expression inhibition, comprising one or more kinds of plant extracts selected from pontsuku ginger, yarrow millet, mugwort, salamander, thyme and oat as an active ingredient. External skin preparation. 9. A chemokine expression inhibitor comprising at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 1] [Chemical 2] [Chemical 3] [Chemical 4] 10. An interleukin-8 expression inhibitor comprising at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 5] [Chemical 6] [Chemical 7] [Chemical 8] 11. An eotaxin expression inhibitor comprising at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 9] [Chemical 10] [Chemical 11] [Chemical 12] 12. An external preparation for atopic dermatitis based on the inhibition of eotaxin expression, comprising at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 13] [Chemical 14] [Chemical 15] [Chemical 16] 13. A RANTES expression inhibitor comprising at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 17] [Chemical 18] [Chemical 19] [Chemical 20] 14. An antiallergic agent based on RANTES expression inhibition, which comprises at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 21] [Chemical formula 22] [Chemical formula 23] [Chemical formula 24] 15. An anti-inflammatory agent based on chemokine expression inhibition, which comprises at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 25] [Chemical formula 26] [Chemical 27] [Chemical 28] 16. A skin external preparation for improving skin roughness based on chemokine expression inhibition, which comprises at least one of the following compounds (1) to (4) as an active ingredient. [Chemical 29] [Chemical 30] [Chemical 31] [Chemical 32]