JPS63133995A - Production of naphthoquinone compound - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a remedy for skin diseases efficiently, by transforming a cell of a plant of the family Boraginaceae with plasmid of Agrobacterium rhizogenes, cultivating a formed hairy root in a medium containing nitrate ion, etc., and taking out a culture product after the cultivation. CONSTITUTION:A seed of a plant of the family Boraginaceae is sterilized with a fungicide such as a solution of sodium hypochlorite, etc., the seed is put on a solid medium, a cell of stem, leaf, etc., of germinated sterilized plant is inoculated with Agrobacterium rhizogenes containing Ri plasmid and transformed. Then after two-five weeks, a hairy root growing from the inoculated site is cut out, cultivated in a liquid medium containing 50-80mM nitrate ion and/or 0.00015-0.0003mM copper ion in 1l liquid medium and a substance produced from the hairy root is taken out to give a naphthoquinone compound such as shikonin shown by the formula (R is OH or OCOCH3), etc.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention is an efficient method for culturing the hairy roots of plants of the family Prunusaceae to produce naphthoquinone compounds, which are physiologically active substances and medicinal ingredients that are biosynthesized by plants of the family Prunusaceae. The present invention relates to a method for manufacturing the same.

[Conventional technology]

The roots of Murasaki, a plant belonging to the Purple family, contain naphthoquinone-based compounds such as shikonin (R=-OH) represented by the following formula (wherein, R is -OH, -○COCH3, etc.). , has traditionally been used in Chinese medicine called "purple root". Specifically, with oils and fats such as sesame oil,
The ointment obtained by extracting shikonin and other substances from Shikonin is called Shiun-yang, and is used to treat various skin diseases, cuts, burns, hemorrhoids, etc., and is known to increase vascular permeability and cause granulation formation. It is being

However, the amount of medicinal ingredients such as shikonin that can be extracted from purple roots is small, and cultivating purple roots takes time.
There are problems such as dependence on the natural environment and weather, and its stable supply is at risk.

On the other hand, there is a method for industrially producing naphthoquinone compounds using cell/tissue culture methods of plants of the family Murasakiceae.
Various types are known.

One of them is a method of culturing callus.

Tabata, Hara et al. have developed a medium suitable for this method, and a patent application has been filed regarding its components. For example, a method using a liquid medium containing cellulose fibers (Special Publication No. 6 O
-34), a method using a liquid medium in which the proportion of ammonium ions in the nitrogen source is 10 mol% or less (Special Publication No. 6
O-35), a method using a liquid medium containing at least one additive selected from cellulose acetate, chitin, activated carbon, and pectic acid (Special Publication No. 6 O-36), with a copper ion concentration of 0.2 μM or more. A method using a certain liquid medium (Special Publication No. 6O-984), a method using a liquid medium in which the concentration of cytokinins is 5 μM or less (Special Publication No. 6O-984)
985), a method using a specific amount of a specific amino acid (Japanese Patent Publication No. 6O-986), a method using a liquid medium with a sulfate ion concentration of 0.1 mM or more (Japanese Patent Publication No. 6O-987),
A method using a liquid medium containing a specific amount of manganese, molybdenum or iodine ions (Japanese Patent Publication No. 6O-988).

On the other hand, as a method other than callus culture, plants are grown with the hairy root disease fungus Agrobacterium lithogenes (Agrobacterium lysogenes).
A method is known in which the hairy roots that have grown are inoculated with Bacterium rhizogenes and cultured. This method is based on the following principle. In other words, when Agrobacterium rhizogenes is inoculated into the stems, leaves, roots, etc. of a plant, roots called hairy roots develop from the infected area. It is known that roots are generated when some of them are incorporated into the plant's genes, and that they grow much faster than normal roots or produce the same or higher amount of secondary metabolites than normal roots.

Furthermore, at this year's meeting of the Botanical Society of Japan, Kamata et al.
By culturing the hairy roots of the purple grass, we announced that naphthoquinone compounds were produced by the hairy roots and were also secreted into the medium.

However, the hairy roots of plants of the family Prunusaceae are grown on regular media (
When cultured in MS medium, etc.), problems arose in that the growth rate of the hairy roots, the content of naphthoquinone compounds in the hairy roots, and the amount secreted into the medium were still insufficient.

[Problem that the invention seeks to solve]

Therefore, the present invention develops a medium formulation suitable for the growth of hairy roots produced by transformation with an Ri plasmid, accelerates the growth rate of hairy roots, and provides a highly efficient method for producing naphthoquinone compounds. The purpose is to

[Means for solving problems]

The present invention was made based on the knowledge that the above problems can be effectively solved by using a liquid medium containing a specific amount of a specific element.

That is, the present invention transforms plant cells of the family Murasaceae with the R1 plasmid held by Agrobacterium rhizogenes, and transforms the resulting hairy roots with 50 to 80 mM nitrate ions and/or 0.00015 to 0.0003 mM.
Provided is a method for producing a naphthoquinone compound, which comprises culturing in a liquid medium containing copper ions per liter of the liquid medium, and extracting the naphthoquinone compound produced by the hairy roots.

In the present invention, any plant of the family Prunusaceae can be used, but it is preferable to use plants of the genus Prunus.

Specifically, Lithosperumum erythr.
orhizon) Firefly Kazura (L, zolling)
eri), L. canescens.

L, diffusum, L, graminifol
ium, L. petraeum.

L, purpure O-caeruleum, L,
Rosmorinifolium is exemplified, and Murasaki is particularly preferred.

Agrobacterium rhizogenes that can be used to make these plants produce hairy roots include Agrobacterium rhizogenes 25818 Agrobacterium rhizogenes 15834 Agrobacterium rhizogenes 8196 Agrobacterium rhizogenes
Examples include Rhizogenes A4. In addition, Ri plasmid or a part of it can be used in other bacteria such as E. coli.
Bacteria into which A gene has been introduced can also be used.

When a plant is treated with Agrobacterium rhizogenes according to the present invention, -R (T-DNA) of the R1 plasmid in the Rhizogenes bacterium is introduced (transformed) into the nuclear DNA of the plant cell.

Examples of methods for obtaining transformed hairy roots by introducing the R1 plasmid T-DNA into the stems, roots, leaves, etc. of the plants of the family Prunusaceae include the following method.

1. Direct inoculation method to individual plants 2. Leaf disc method using leaf discs (L, Comai
etal,, Nature, 317.741 (
1985)) 3. Co-culture method using plant protoplasts (2,!, 4. Wei et al., P
lant Ce1l Rep,, ir+press
(1986) ) 4. Plant protoplast and Agrobacterium rhizogenes spheroplast method (R9Hain et al.
al,, Plant Ce1l Rep,, 3
．． 605, Agrobacterium rhizogenes R
1 Method of directly injecting T-DNA of a plasmid or a part thereof into cells by a method such as microinjection Ri If the plasmid is introduced by methods 1 to 4 above, then sterilization treatment of Agrobacterium rhizogenes is performed. is required, and the methods for doing so are as follows.

O High temperature treatment (40°C) ○ Antibiotic treatment ○ Rapid cycle transplantation of hairy root tips The following methods are effective for culturing hairy roots obtained by methods other than those above.

In the present invention, the above-mentioned hairy roots are treated with a nitrate ion concentration of 50 to 8.
0mM preferably 60-70mM and/or copper ion concentration 0.0OO15-0.0003mM preferably 0.
It is characterized by culturing in a liquid medium containing 00017 to 0.00027 mM per lβ of liquid medium.

Here, as the nitrate ion source, ammonium nitrate, sodium nitrate, calcium nitrate, potassium nitrate, etc.
Examples of the copper ion source include copper sulfate.

In the present invention, for example, a medium conventionally used for plant tissue culture, that is, an inorganic component and a carbon source as essential components, and at least one component selected from plant hormones, vitamins, and amino acids. The amount of the above-mentioned specific element can be used as the specific value in a culture medium to which the above-mentioned specific element is added and other components are added as necessary.

Inorganic components in the above medium include, in addition to nitrogen, iron, zinc, and molybdenum, boron, phosphorus, cobalt, potassium, calcium, magnesium, sulfur, manganese, chlorine, sodium, and iodine. acid, phosphoric acid, monosodium phosphate, monopotassium phosphate, diphosphoric acid
Sodium, trisium phosphate, cobalt chloride,
Examples include potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, manganese sulfate, and potassium iodide.

Examples of carbon sources include sucrose, other hydrocarbons, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Plant hormones include indoleacetic acid (AA), naphthaleneacetic acid (NAA), I)-chlorophenoxyisoacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, zeatin, dihydrochloride, etc. Cytokinins such as zeatin are exemplified.

Vitamins include biotin and thiamin (vitamin B)
Examples include pyridoxine (vitamin B6), pantothenic acid, ascorbic acid (vitamin C), and inosytonobenicotinic acid.

Examples of amino acids include glycine, alanine, glutamine, and cysteine.

The concentration of components other than those defined by the present invention in the liquid medium can vary within a wide range. Usually, the inorganic component is about 0.1μM to about 100mM, and the carbon source is about 1g/β.
~120g/f! , degree, and even plant hormones about 0
．． 01 μM to about 10 μM, vitamins and amino acids, respectively about 0.1 mg/β to about 100 mg/j
! It can be done to a certain extent.

In the present invention, the initial concentration of hairy roots in the liquid medium can be varied within a wide range. Usually, it is desirable to add about 1 g (fresh weight) of hairy roots to 50 ml of liquid medium.

In culturing the hairy roots of the present invention, light is not necessarily necessary, and culturing in the dark is preferable for the biosynthesis of naphthoquinone compounds, and the culture temperature is about 0°C to about 35°C, especially about 3°C. ~28°C is preferred. That is, about 10℃
This is because if the temperature is less than 35°C, the growth rate of hairy roots will be low, and if it exceeds about 35°C, the growth rate of hairy roots will be similarly low.

In the present invention, naphthoquinone compounds can be extracted from the hairy roots cultured as described above by various methods, but the method is carried out by extracting naphthoquinone compounds secreted by the hairy roots into a liquid medium. good.

〔Effect of the invention〕

According to the present invention, naphthoquinone compounds can be efficiently produced from cells of plants belonging to the family Murasaceae, and therefore the method of the present invention is extremely suitable as an industrial method for producing naphthoquinone compounds.

Next, the present invention will be explained by examples.

〔Example〕

Example 1 Lithosperumum erythr
orhizon) seeds were sterilized with a disinfectant such as sodium hypochlorite solution, and then sown on Murashige-Skoog (abbreviated as MS) solid medium, and the stems and leaves of the germinated sterile plants were Agrobacterium rhizogenes (15834) carrying the R1 plasmid was inoculated into the cells.

After 2 to 5 weeks, the hairy roots developed from the inoculation site were cut out and transplanted onto MS solid medium containing carbenin phosphorus Ig/β, and after 1 to 2 weeks, they were transplanted to a new medium with the same composition. 2
This operation was repeated ~3 times to obtain sterilized hairy roots.

50 ml of a liquid medium having the composition shown in Table 1 was placed in a 100 ml Erlenmeyer flask and sterilized at 120°C for 15 minutes. 100 mg of the above hairy roots were added to this liquid medium, and cultured with shaking (swivel rotation rate: 100 times/min, amplitude: 30 mm) at 25° C. for 30 days.

After culturing, the hairy roots of the purple grass were collected by filtration, weighed, and freeze-dried. After drying, it was weighed and ground into powder in a mortar. Next, the powder was extracted with chloroform to obtain a naphthoquinone compound. After measuring the weight of the culture medium, it was extracted with chloroform.

The naphthoquinone compound was quantified by measuring the absorbance value at 520 nm.

The amounts of both naphthoquinone compounds were added to determine the total amount produced per flask.

A table summarizing the nitrate ion concentration in the medium and the results obtained.
Shown in 2.

Table 1 Medium 1. l! As is clear from Hit Table-2, the conventionally used M
In a comparative example in which culture was performed in S liquid medium (containing 30 g/β of sucrose as a carbon source), the amount of total naphthoquinone compounds produced was extremely small, but according to the present invention, a large amount of naphthoquinone compounds was produced. I know what I can do.

Example 2 Cultivation and extraction were carried out in the same manner as in Example 1, except that the concentration of nitrate ions was 39.4 mmol and the concentration of copper ions in the table was changed.

The results are summarized in Table 3.

As is clear from Table 3, the conventionally used M
In a comparative example in which culture was performed in S liquid medium (containing 30 g/β of sucrose as a carbon source), the amount of total naphthoquinone compounds produced was extremely small, but according to the present invention, a large amount of naphthoquinone compounds was produced. I know what I can do.

Claims (2)

[Claims] (1) Transforming a plant cell of the Murasaceae family with an Ri plasmid carried by Agrobacterium rhizogenes,
The resulting hairy roots are cultured in a liquid medium containing 50-80mM nitrate ions and/or 0.00015-0.0003mM copper ions per liter of liquid medium, and the naphthoquinone system produced by the hairy roots is cultured. A method for producing a naphthoquinone compound, the method comprising extracting the compound. (2) The production method according to claim 1, wherein the plant of the family Purple is selected from plants of the genus Purple.