JPH07177893A - Production of tropolones - Google Patents

Abstract

PURPOSE:To obtain a tropolone compound useful as a raw material for antimicrobial agent, etc., on an industrial scale at a low cost by culturing a cell induced from a tissue piece of a plant of the genus Thujopsis in a medium containing carbon source, inorganic component, phytohormone and chitin as essential components. CONSTITUTION:This tropolone compound useful as a raw material for antimicrobial agent, etc., such as alpha-thujaplicin and beta-thujaplicin (hinokitiol) is produced by culturing a culture cell induced from a tissue piece of a plant of the genus Thujopsis (e.g. hiba arbor-vitae) for 4 weeks in a medium containing glucose as a carbon source, an inorganic component such as ammonium nitrate, potassium nitrate, sodium nitrate and dihydrogen sodium phosphate and a phytohormone such as auxin as essential components and further containing chitin dispersed in the medium as an essential component to accumulate the tropolone compound in the cultured cells and/or the medium, recovering and freeze-drying the cells, extracting with ethyl acetate at 60 deg.C for 2 hr and distilling out the ethyl acetate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing tropolones, which is obtained by extracting tropolones, which are raw materials for antimicrobial agents, from tissue culture of Asunaro plants.

[0002]

2. Description of the Related Art Plants of the genus Asunaro of the cypress family of evergreen Takagi conifers contain tropolones such as .alpha.-tsuyapricin and .beta.-tsuyapricin (hinokitiol) having antimicrobial activity. Therefore, tropolones useful as an antimicrobial agent are usually produced from sawdust of Asuna plants.

On the other hand, the inventors of the present invention thought that if the callus tissue of the plant of the genus Asunaro could be cultured, the callus tissue could be used as a raw material for antimicrobial agents such as tropolones, and as a result of repeated studies, It has been found that callus tissue of Asunaro plant can be efficiently cultured or subcultured in a medium containing a carbon source, an inorganic component and a plant hormone as essential components, and a patent application has already been filed (Japanese Patent Application No. 63- 327087, Japanese Patent Application No. 1-236360, Japanese Patent Application No. 2-79801, etc.).

Further, the present inventors have recently been using a culture medium derived from callus tissue of a plant of the genus Asunaro by the above method in a subculture medium containing a carbon source, an inorganic component and a plant hormone as essential components. Delay the culture as it is,
Alternatively, it was found that tropolones can be produced in the cultured cells and / or the culture medium by transferring to a production medium containing a carbon source, an inorganic component and a plant hormone as essential components and culturing, and already applied for a patent. (See Japanese Patent Application No. 2-226868).

[0005]

However, the above-mentioned conventional method has a problem that the production efficiency of tropolones is not good and that a sufficient amount of tropolones as a raw material of an antimicrobial agent cannot be obtained. In view of such circumstances, it is an object of the present invention to provide a method capable of efficiently obtaining a sufficient amount of tropolones as a raw material of an antimicrobial agent as compared with the conventional method.

[0006]

In order to solve the above problems, the inventors have made various studies. As a result, it was confirmed by experiments that the production efficiency of tropolones is improved by culturing the cultured cells of Asunaro plant in a medium containing chitin as an essential component together with a carbon source, an inorganic component, and a plant hormone. And completed this invention.

Therefore, the method for producing tropolones according to the present invention comprises culturing cultured cells derived from a tissue fragment of a plant of the genus Asunaro by culturing in a medium containing a carbon source, an inorganic component, and a plant hormone as essential components. A method for producing tropolones in cultured cells and / or a medium, characterized in that the medium also contains chitin as an essential component.

Examples of the plant of the genus Asunaro that can be used in the present invention include, but are not limited to, Hinoki asunaro and the like. Also, the tissue piece may be one of an appropriate site. A tissue piece collected from a plant of the genus Asuna is cultivated by an appropriate method and, if necessary, subcultured. Then, the induced cultured cells such as callus tissue are directed to the substance production.

[0009] Examples of the method for directing the cultured cells such as the callus tissue of the genus Asunaro to the substance production include a method of postponing the culture in the medium for passage and a method of subculturing in the medium for production. is there. The subculture medium contains a carbon source, an inorganic component and a plant hormone as essential components, and a growth medium in which 10% by weight or more of the carbon source contained in the medium is glucose, or a carbon source, an inorganic component and a plant. The basal medium is a medium for growth in which a hormone is an essential component, and the molar ratio of ammoniacal nitrogen and nitrate nitrogen contained as inorganic components is 1 to ammonia nitrogen and 4 to 8 nitrate nitrogen. A medium prepared by adding chitin is suitable. As the production medium, a medium prepared by adding B5 medium (however, auxins are added as plant hormones) as a basic medium and adding chitin thereto is suitable.
However, the medium is not limited to these, and any medium containing a carbon source, an inorganic component, a plant hormone, and chitin as essential components may be used. Further, it may be a liquid medium or a solid medium containing a polysaccharide such as agar and gelated.

Here, chitin is a polysaccharide having N-acetyl-D-glucosamine as a constitutional unit, and is widely used in the epidermis (shell) of arthropods such as crab and shrimp, and cell walls of fungi and bacteria. It is a substance that exists. The amount used is preferably 0.1 g or more per liter of medium, and 1 g
The above is more preferable. Further, chitin does not necessarily have to be used by dissolving it in a medium,
Since it usually does not dissolve in the medium, it may be simply dispersed in the medium before use.

The carbon source in the medium used in the present invention (hereinafter, simply referred to as "medium") is not particularly limited, but for example, carbohydrates such as glucose and sucrose and their derivatives, fatty acids and the like. Examples thereof include organic acids and primary alcohols such as ethanol. The inorganic component in the medium is not particularly limited, for example, nitrogen,
Phosphorus, potassium, sodium, calcium, magnesium, iron, manganese, zinc, molybdenum, copper, cobalt,
Inorganic salts containing elements such as sulfur, boron, chlorine and iodine can be mentioned. Specific examples thereof include, but are not limited to, potassium nitrate, sodium nitrate, ammonium nitrate, potassium chloride, sodium chloride, calcium chloride, cobalt chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate. ,
Examples thereof include ammonium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, zinc sulfate, sodium molybdate, potassium iodide, boric acid and the like.

In the present invention, it is necessary that the medium contains at least auxins of auxins and cytokinins as plant hormones. And, the concentration of auxins is in the range of 10 ^-6 to 10 ^-4 mol per liter of the medium, and if necessary, the concentration of cytokinins is 10 ^-5 mol or less per liter of the medium,
Preferably, it is contained in the medium in a range of 10 ^-6 mol or less.

The auxins are not particularly limited, but include, for example, α-naphthalene acetic acid (NAA),
Examples thereof include indole acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole butyric acid (IBA), and derivatives thereof. The cytokinins are not particularly limited, and examples thereof include kinetin, benzyladenine (BA), zeatin and the like. Of these, when the auxin is α-naphthalene acetic acid, the tissue culture of Asunaro plants can be performed particularly efficiently.

In addition to the above-mentioned essential components consisting of carbon source, inorganic components, plant hormones and chitin, the medium contains vitamins, amino acids, etc., if necessary. Good. The vitamins are not particularly limited, but include, for example, biotin, thiamine,
Pyridoxine, pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid, inositol,
Examples thereof include nicotinic acid and nicotinic acid amide.

The amino acids are not particularly limited, and examples thereof include glycine, alanine, glutamic acid, cysteine, and phenylalanine. The concentration of each of the above-mentioned essential components and optional components contained in the medium is usually 10 carbon sources per liter of the medium.
To 50 g, an inorganic component of 10 ⁻⁶ to 0.1 mol, plant hormones and chitin at the above predetermined concentrations, vitamins of 0.1 to 150 mg, and amino acids of about 0 to 1 g are preferable, but a carbon source, The concentrations of components other than plant hormones and chitin are not particularly limited to these.

The method for preparing the medium is not particularly limited. For example, mediums conventionally used for tissue culture of plants, such as Murashige-Skoog medium, Rinsmeier-Skoog medium, White medium and Gamborg B.
5 medium, niche-niche medium, etc. as a basic medium, a carbon source, a plant hormone, and chitin, and if necessary, the above-mentioned components such as vitamins and amino acids may be added to prepare a method. . The basal medium originally contains the inorganic component, but the inorganic component may be appropriately added if necessary.

Culturing with the above-mentioned medium is not particularly limited, but is carried out, for example, at a temperature of 20 to 30 ° C., if necessary with stirring at 50 to 150 rpm, for 7 to 30 days. Tropolone produced by cultured cells such as callus tissue of Asunaro plant by culturing in the medium as described above, cells and /
Alternatively, it can be obtained from the medium. In the extraction method, organic solvents such as alcohols such as methanol, esters such as ethyl acetate, ethers, benzenes, halogenated hydrocarbons such as methylene chloride can be used. Depending on the type of solvent,
The efficiency of extraction can be improved by heating or performing reflux extraction. As a method for extracting from the medium, extraction is carried out using an organic solvent having a property of being immiscible with water, such as ethyl acetate, methylene chloride, n-hexane, ethyl ether, and toluene, and tropolones are easily extracted from these organic solvents. Can be blended in and acquired. In addition,
Besides the extraction method, tropolones can be efficiently obtained by a steam distillation method or the like.

[0018]

[Function] When the cultured cells of the genus Asunaro are cultured in a medium containing chitin as an essential component together with a carbon source, an inorganic component, and a plant hormone, it is more sufficient as a raw material for an antimicrobial agent than the conventional method. It becomes possible to efficiently obtain the amount of tropolones.

[0019]

EXAMPLES Specific examples and comparative examples of the present invention will be shown below, but the present invention is not limited to the following examples. Further, the culture in the following Examples and Comparative Examples,
All were carried out at 25 ° C. in the dark with rotary shaking at 100 rpm. Quantification of α-tsuyapurisin and β-tsuyapurisin (hinokitiol) is performed by gas chromatography,
The quantification of hinokitin, which is such an iron complex, was performed by liquid chromatography. The measurement conditions of gas chromatography and liquid chromatography are shown below. Gas chromatography device: Shimadzu GC-9A Column: CBP1-W12-100 (Shimadzu) Injection temperature: 250 ° C Column temperature: 100 to 275 ° C (heating rate 10 ° C / min) Liquid chromatography device: Hewlett Packard 1090 Column: Hypercil ODS 100 × 4.6 mm Liquid phase: 0.01M formic acid-triethylamine buffer (pH
5) / THF (1/1) In the following examples, chitin was obtained from the shell of a crab (Nacalai Tesque, Chitin from Crab).
Shells) was used. The compositions of the growth medium (liquid) and the production medium (B5 medium; liquid) used in the following Examples and Comparative Examples were as shown in Table 1 below.

[0020]

[Table 1]

Example 1 200 mg of chitin (10 g per 1 liter of medium) dispersed in 20 ml of a growth medium was placed in an Erlenmeyer flask having a volume of 100 ml, and further induced from callus tissue pieces of Hinoki Asunaro. Add 1.3g of cultured cells
Cultured for a week. Then, the cells were collected, freeze-dried, and then extracted with 50 ml of ethyl acetate at 60 ° C. for 2 hours, and the ethyl acetate was distilled off to obtain an extract.

-Comparative Example 1-Culturing and extraction were carried out in the same manner as in Example 1 except that chitin was not used. -Example 2-Chitin 2 mg (0.1 g per liter of medium) dispersed in 20 ml of B5 medium was placed in an Erlenmeyer flask having a volume of 100 ml, and further cultured by induction from callus tissue pieces of Hinoki Asunaro. 1.8 g of cells were added and cultured for 2 weeks. Then, after collecting the cells and freeze-drying,
Extraction treatment was carried out with 50 ml of ethyl acetate at 60 ° C. for 2 hours, and ethyl acetate was distilled off to obtain an extract.

-Example 3-Culturing and extraction were carried out in the same manner as in Example 2 except that the amount of chitin used was changed to 20 mg (1 g per liter of medium). -Example 4- In Example 2, the amount of chitin used was 200 mg (medium 1
Cultivation and extraction were performed in the same manner as in Example 2 except that the amount was changed to 10 g per liter).

-Comparative Example 2-Culturing and extraction were carried out in the same manner as in Example 2 except that chitin was not used. Example 5-100 mg of chitin (1 g per 1 liter of medium) dispersed in 100 ml of B5 medium was placed in an Erlenmeyer flask having a volume of 300 ml, and the hinoki asunaro of a strain different from Examples 1 to 4 was further added. 3.7 g of cultured cells derived from callus tissue pieces were added and cultured for 17 days. Then, the cells were recovered, freeze-dried, and then subjected to extraction treatment with 70 ml of ethyl acetate at 60 ° C. for 2 hours, and the ethyl acetate was distilled off to obtain an extract.

-Comparative Example 3-Culturing and extraction were carried out in the same manner as in Example 5, except that chitin was not used. Dry weight of cells cultured in the above Examples 1 to 5 and Comparative Examples 1 to 3, the amount of extract obtained from the cultured cells, β-tsuyaprisin (hinokitiol) contained in the extract,
The amounts of hinokitin and α-tsuyapurisin are shown in Table 2 below in terms of 100 ml of the medium.

[0026]

[Table 2]

As shown in Table 2, the following were confirmed. Inclusion of chitin in the medium improves the production efficiency of tropolones. Among the tropolones, particularly, the production amount of α-tsuyaprisin increases.

[0028]

According to the method for producing tropolones according to the present invention, a large amount of tropolones useful as an antimicrobial agent can be efficiently obtained, as compared with the conventional method. Further, among the tropolones, it is possible to increase the production amount of α-tsuyaprisin, in particular.

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[Procedure amendment]

[Submission date] September 25, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction content]

Further, the present inventors have recently been using a culture medium derived from callus tissue of a plant of the genus Asunaro by the above method in a subculture medium containing a carbon source, an inorganic component and a plant hormone as essential components. Delay the culture as it is,
Alternatively, it was found that tropolones can be accumulated in the cultured cells and / or the medium by transferring to a production medium containing a carbon source, an inorganic component and a plant hormone as essential components and culturing, A patent application has already been filed (see Japanese Patent Application No. 2-226868).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

Accordingly, the production method of the tropolone compound according to the invention, the cultured cells derived from the tissue pieces dolabrata genus plant, culturing in a medium carbon source, an inorganic Ingredient Contact and plant hormones as essential components Is a method for accumulating tropolones in a cultured cell and / or a medium, characterized by including chitin as an essential component in the medium.

Claims (4)

[Claims] 1. A cultivated cell derived from a tissue fragment of a plant of the genus Asunaro is cultivated in a medium containing a carbon source, an inorganic component, and a plant hormone as essential components, to thereby obtain tropolone in the cultured cell and / or the medium. A method for producing tropolones, which comprises producing chitin as an essential component in the medium. 2. The method for producing tropolones according to claim 1, wherein the plant of the genus Asunaro is Hinoki Asunaro. 3. The method for producing tropolones according to claim 1, wherein the tropolones are α-tsuyapricin and β-tsuyapricin (hinokitiol). 4. The chitin content in the medium is 0.1 g or more per 1 liter of the medium.
A method for producing the described tropolones.