JPH06321791A - Skin inflammation therapeutic agent - Google Patents

Abstract

PURPOSE:To obtain an acidic heteropolysaccharide not only excellent in a skin inflammation-inhibiting action but also little in side effects and extremely effective for the therapy of various skin inflammation diseases. CONSTITUTION:This skin inflammation therapeutic agent contains as an active ingredient an acidic heteropolysaccharide which is secreted from a callus derived from a plant belonging to the genus Polianthes L. outside the cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a therapeutic agent for skin inflammation having an excellent anti-skin inflammation effect and few side effects.

[0002]

2. Description of the Related Art Substances causing skin inflammation, especially hand dermatitis, are plants, metals (including cement), beauty products, detergents,
Paints, oils, inks, adhesives, rubbers, chemicals, external preparations, foods, clothing, etc. Occupational dermatitis is mainly caused by beauty products, paints, oils and metals, and non-occupational dermatitis is mainly caused by metals, detergents and plants, and in some cases, it may interfere with social life. In addition, cosmetics and quasi-drug components sometimes cause allergic dermatitis, pigmented contact dermatitis, contact urticaria and the like. The components of cosmetics and quasi drugs are oils and fats, surfactants, powders, pigments, fragrances, antiseptic / antifungal agents, antioxidants, humectants, astringents, polymer compounds, etc., all of which are Has the potential to develop allergic dermatitis. Currently, weak steroids or non-steroidal anti-inflammatory agents are used as therapeutic agents for skin inflammation. However, if steroids are continuously applied to the face, etc., it may cause steroid dermatosis, so an effective therapeutic agent with few side effects. Is required.

[0003]

Accordingly, it is an object of the present invention to provide a drug which has few side effects and is effective against various skin inflammations.

[0004]

[Means for Solving the Problems] Therefore, as a result of intensive investigations by the present inventors to obtain a new therapeutic agent for skin inflammation from the standpoints of both safety and efficacy, as a result, a specific acidic heteropolysaccharide has remarkable anti-skin The inventors have found that they have inflammatory activity and completed the present invention.

That is, the present invention relates to the genus Polyantes ( Poli
The present invention provides a therapeutic agent for skin inflammation containing an acidic heteropolysaccharide secreted extracellularly by callus derived from a plant belonging to anthes L. ).

The acidic heteropolysaccharide, which is the active ingredient of the present invention, is produced, for example, by culturing callus derived from a plant belonging to the genus Polyanthes in a plant hormone-containing medium and collecting from the culture (Japanese Patent Application Laid-Open No. 2000-242242). 64-10997 bulletin).

Examples of plants belonging to the genus Polyantes include tuberose ( Polianthes tuberosa L. ). The site that can be used as an explant includes a part of organs or tissues such as flowers, stems, leaves, bulbs, roots, etc., but a part of flowers is particularly preferable. As a basal medium for callus induction, Murasige which is usually used for plant tissue culture
-Skoog medium, Linsmaier-Skoog medium, Gamborg medium, White medium, Tuleeke medium, Nitsch & Nitsch
The medium or the like is used. It is necessary to add a plant hormone to this basal medium, and as a plant hormone, 2,
Auxins such as 4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), indoleacetic acid (IAA) and indolebutyric acid (IBA); furfurylaminopurine (kinetin), benzyladenine (BA) , Cytokinins such as dimethylaminopurine (2iP) and the like. Among them, 2,4-D alone, a combination of NAA and BA, or a combination of NAA and kinetin gives good results. The plant hormone concentration required for callus induction is 2,4
-D alone 5 × 10 ^-4 M to 1 × 10 ^-7 M, NAA and BA
Alternatively, in the case of the combination of NAA and kinetin, the concentration of NAA is 5 × 10 ⁻⁴ M to 1 × 10 ⁻⁷ M, and the concentration of BA or kinetin is 1 × 10 ⁻⁷ M. In addition to the above basic medium and plant hormones, sugar is added to the callus induction medium as a carbon source. Examples of the sugar include glucose, fructose, mannose, xylose, saccharose, rhamnose, fucose, starch and the like, but sucrose is usually used. Callus induction can be performed with a solid medium or a liquid medium, but a solid medium is usually used. The induced callus can be subcultured for 10 or more generations while maintaining the same morphology in the above callus induction medium.
The medium for subculture is usually Linsma
ier-Skoog's medium, Murasige-Skoog's medium is used,
1 × 10 ^-4 to 1 × 10 ^-7 M 2,4-D as a plant hormone
Or 1 × 10 ^-4 〜 1 × 10 ^-7 M NAA and 1 × 10 ^-4 〜 1 × 10 ^-7
BA of M, carbon sources such as glucose, fructose,
Mannose, xylose, sucrose, rhamnose,
Although fucose, starch and the like are used, sucrose is preferable, and the addition amount thereof is preferably 1 to 6% by weight (hereinafter simply referred to as%).

To produce polysaccharides from callus, callus
Is cultured in solid medium such as agar medium or liquid medium.
It is preferable to culture in a liquid medium. The basic medium is
The same as the Ruth induction medium is used. Plant hormone
The type and concentration of is related to the productivity of polysaccharides, for example
Auxins such as 2,4-D, NAA, IAA and IBA;
Cytokinins such as netin, BA, 2iP; Gibberellin A
₃(GA₃Gibberellins and the like are used. In this
, 2,4-D, NAA alone, or NAA and BA or
It is preferable to use kinetin in combination. Its rich
The degree is 5 × 10 when 2,4-D or NAA is used alone.
^-Four 1 x 10 from M^-7 M, especially 5 × 10^-Five 5 x 10 from M^-6 M
But; using NAA and BA or NAA and kinetin in combination
The concentration of NAA is 1 x 10^-Four 1 x 10 from M ^-7 M,
Especially 1 × 10^-Four 5 x 10 from M^-6 Concentration of M, BA or kinetin
The degree is 5 × 10^-Five 1 x 10 from M^-9 M, especially 1 × 10^-Five M to 1
× 10^-7 M is preferred. Carbon sources include glucose and fluorine
Lactose, mannose, xylose, sucrose,
Rhamnose, fucose, starch and the like are used. Many
Sugar production strongly influences the type of carbon source added
However, sucrose is usually used.
There is a deep relationship between the concentration of carbon source and the production of polysaccharides.
Although it does not matter, generally 1 to 6% is preferable. The culture method is special
Usually, but not limited to, at a temperature of 20-30 ° C for 15-30 days
Preferably, shaking culture is preferred.

Collection of the polysaccharide from the thus obtained culture is carried out by, for example, removing cells from the culture by centrifugation or filtration, and then concentrating the culture using a rotary evaporator or the like. Ethanol is added to the precipitate to precipitate, and the precipitate is freeze-dried. Purification of the above-mentioned polysaccharide can be carried out according to a usual method for purifying polysaccharides. For example, it is also possible to dissolve the above-mentioned roughly purified polysaccharide in water, centrifuge to completely remove the insoluble matter, and obtain a highly purified product by dialysis, ion exchange chromatography, or gel filtration chromatography.

Among the polysaccharides obtained by the above method,
The acidic heteropolysaccharide which is an active ingredient of the therapeutic agent for skin inflammation of the present invention is contained. This product was hydrolyzed with 2N H ₂ SO ₄ at 100 ° C. for 8 hours and then subjected to thin layer chromatography using a developing solvent having a mixing ratio of ethyl acetate: pyrimidine: acetic acid: water = 5: 5: 1: 3. When the product was chromatographed and colored with an aniline: diphenylamine: acetone: phosphate reagent, arabinose, mannose, galactose, glucuronic acid and xylose were detected. In addition, it was confirmed from the analysis results by gas chromatography that these were contained as constituent sugars. According to the gas chromatographic analysis (GC-MS method) after methylation by the Hakomori method,
The binding style and composition ratio are

[Chemical 2] Was found to be Further, 0 to 50% of the carboxyl group of glucuronic acid exists as a methyl ester form. Furthermore, since the polysaccharide, which is the active ingredient of the present invention, is adsorbed on an anion exchange resin or the like, it was judged to be acidic. Furthermore, high performance liquid chromatography (column: manufactured by Tosoh
According to TSK Gel 4000PW, 5000PW and 6000PW), the molecular weight was 1.0 × 10 ^{4 to} 2.0 × 10 ⁷ .

This acidic heteropolysaccharide has the following physicochemical properties. Solubility in solvent Soluble in water, insoluble in ethanol, ether, acetone. Color reaction Anthrone reaction: Positive Carbazole reaction: Positive Elson-Morgan reaction: Negative color and shape Ethanol-precipitated product is white to off-white powder. The product purified by ion exchange through dialysis and freeze-dried is white cotton-like or fibrous. Specific rotation [α] _D ²⁵ : 0 to +20 (c = 1.0, aqueous solution) infrared absorption spectrum The infrared absorption spectrum is as shown in FIG. Nuclear magnetic resonance spectrum The ¹³ C-nuclear magnetic resonance spectrum is as shown in FIG. 2 (solvent: D ₂ O, tube 5 mm, internal standard dioxane). The acidic heteropolysaccharide used in the present invention has the following repeating structure.

[Chemical 3]

The fact that the acidic heteropolysaccharide, which is the active ingredient of the present invention, is novel is apparent from comparison with other polysaccharides as already described in JP-A-64-10997. That is,
Glucuronomannan structure contained in the acidic heteropolysaccharide of the present invention [→ 2) α-D-Man- (1 → 4) -β-D-GlcU
A known polysaccharide having A- (1 →) as a partial structure is Dr.
Polysaccharide obtained from osera capensis (CHANNE et al., Carboh
ydr. Res. , 113, 113-124, 1983), Droser
Polysaccharide obtained from a binat a (CHANNE et al., Phytochemistr
y , Vol. 21, No. 9, pp. 2297-2300, 1982), Nicotiana
Polysaccharides obtained from cultured cells of tabacum (MORI et al., Carbo
hydr. Res. , 91, 49-58, 1981; AKIYAMA et al., Ag.
ric. Biol. Chem. , 48, No. 2, pp. 403-407, 1984
Years) are known. However, Drosera capens
The polysaccharides obtained from is and Drosera binate clearly differ from the acidic heteropolysaccharides of the present invention in that they have -2Man1- and -4GlcUA1- as the main binding modes and lack -3Ara1-. Regarding the polysaccharide obtained from Nicotianatabacum , MORI et al. Reported that -3Ara1- was not present in the main binding mode, and AKIYAMA et al. Reported that the main binding mode was not.
-4GlcUA1-, -2Man1- and -5Ara1- are present, and -3Ara1- is absent, which is clearly different from the acidic heteropolysaccharide used in the present invention. Therefore, it can be said that the acidic heteropolysaccharide which is the active ingredient of the present invention is a novel acidic heteropolysaccharide different from the conventionally obtained ones.

The above-mentioned acidic heteropolysaccharide is water-soluble, and is dissolved in physiological saline or the like to be used as an injection or mixed with a carrier such as kaolin, talc, lactose, starch or crystalline cellulose to prepare tablets, powders or granules. , Orally administered as capsules. It can also be administered as an external preparation such as an ointment, liniment or poultice.

The dose is preferably 1 to 500 mg / day for an adult male in the case of an injection, and 100 to 2000 mg / day for an adult male in the case of oral administration.

[0015]

INDUSTRIAL APPLICABILITY The therapeutic agent for skin inflammation of the present invention not only has an excellent anti-skin inflammation effect, but also has few side effects, and is extremely useful for treating various skin inflammations. Moreover, the polysaccharide, which is an essential component, can be uniformly produced by applying the plant tissue culture method.

[0016]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Production of Polysaccharides: As a plant belonging to the genus Polyanthes, tuberose ( Polianthes t.
uberosa L. ) was used. Callus is a sterilized tuberose 2 to 7 days before flowering, using buds as a plant hormone of 1 × 10 ^-5.
Induction was performed using a Linsmaier-Skoog medium (agar medium) containing M NAA and 1 × 10 ⁻⁶ M BA and containing 3% sucrose as a carbon source. The induced callus was subcultured in the same medium. Callus that has been passaged for several generations or more and stabilized and then contained in a Linsmaier-Skoog medium (liquid medium) containing 1 × 10 ^-5 M 2,4-D as a plant hormone and 5% sucrose as a carbon source. It was inoculated so that the concentration would be%. Culture using a rotary shaker in the dark at a shaking frequency of 120 r.
pm, 27 ± 1 ° C. for 30 days. After this, the cells were removed from the culture solution by filtration and centrifugation, and this was concentrated using a rotary evaporator. About 3 times the amount of ethanol was added to this concentrated solution, and the mixture was left standing at 5 ° C for 24 hours to obtain a precipitate. The precipitate was recovered by centrifugation, washed with 70% ethanol three times, and then lyophilized to remove water, thereby obtaining the desired polysaccharide. Do the above operation 5 times,
Changes in lots of polysaccharide yield, total sugar amount, uronic acid amount, protein amount, water amount, and neutral sugar composition ratio were compared. The results are shown in Table 1.

[0018]

[Table 1]

As is clear from Table 1, since the polysaccharide obtained by the above-mentioned production method is obtained by culturing under complete artificial control, the polysaccharide yield, the total sugar amount, the uronic acid amount, and the protein amount depending on the lot. The water content and the neutral sugar composition ratio do not fluctuate, and the uniformity is high.

Example 2 Mouse hypersensitivity reaction test: 1
The abdomen of 10 BALB / c female mice (7 weeks old) in the group was shaved, and 0.1 ml of a 7% picryl chloride solution was applied to the villi. After 6 days, add 1% picryl chloride solution to both sides of both ears 20μ
l was applied to each to induce inflammation. The acidic heteropolysaccharide obtained in Example 1 was dissolved in a physiological saline solution for injection to prepare a test solution. The test solution was intraperitoneally administered at 1, 30, and 100 mg / kg 24 hours before induction. 24 hours after induction, both ears were punched out with a punch (φ7 mm) and the weight of the auricle was measured. The edema inhibitory rate was calculated from the following formula as an index of the anti-inflammatory effect.

[0021]

[Equation 1] Inhibition rate of edema (%) = (1-A / B) x 100 A: Difference between the weight of auricles inoculated with edema-inducing test solution and the weight of auricles in which edema non-induced diet was administered B: Edema-induced dietary administration Difference between mouse ear weight and ear weight without edema-induced saline

The results are shown in Table 2. As a result, as shown in Table 2, this polysaccharide had an excellent contact skin inflammation inhibitory action.

[0023]

[Table 2]

Example 3 Rat ear edema reaction test: 5
A croton oil containing% croton oil (croton oil: ether: pyridine: water = 1: 14: 4: 1) was infiltrated into a circular felt fixed to characteristic tweezers (0.4 ml / felt, 0.8 ml /
Ear) A group of 6 Wister male rats (5 weeks old) was pinched on both sides of the right auricle with tweezers for 15 seconds and applied at a constant pressure to induce edema. After 6 hours, punch both auricles (φ9m
It was punched out at m) and the weight of the auricle was measured. The acidic heteropolysaccharide obtained in Example 1 was dissolved in a physiological saline solution for injection to prepare a test solution. The test solution was 30 mg / kg and caused edema 7, 3, 1
It was intraperitoneally administered one day before. The edema inhibitory rate was calculated from the following formula as an index of the anti-inflammatory effect.

[0025]

[Equation 2] Inhibition rate of edema (%) = (1-A / B) x 100 A: Weight difference between the right auricle and the untreated left auricle of the test solution-administered rat B: Control applied only with the base Weight difference between right and untreated left auricles in group

As a result, as shown in Table 3, this polysaccharide had an excellent edema inhibitory action.

[0027]

[Table 3]

Example 4 Acute toxicity test: IC of 5 animals per group
An acute toxicity test was carried out using R type male mice (7 weeks old). The acidic heteropolysaccharide 20 mg / ml solution obtained in Example 1 was added to 0.2
4 ml was taken, and the mice were intraperitoneally administered 4 times at 2 hour intervals so that the total dose would be 1000 mg / kg. As a result, all mice survived on the 7th day, and no change was observed in the systemic symptoms after administration.

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Claims (2)

[Claims] 1. A therapeutic agent for skin inflammation comprising an acidic heteropolysaccharide secreted extracellularly by callus derived from a plant belonging to the genus Polianthes L. 2. It contains arabinose, mannose, galactose, glucuronic acid and xylose as constituent sugars, and their binding mode and constituent ratio are as follows: And a therapeutic agent for skin inflammation containing an acidic heteropolysaccharide having a molecular weight of 1 × 10 ⁴ to 2 × 10 ⁷ as an active ingredient.