JPS60986B2 - Tissue culture method for plants of the family Murasakiceae - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing the tissue of a plant of the family Murasaceae containing a naphthoquinone pigment such as shikonin.

More specifically, by controlling the concentration of specific components in the liquid medium and tissue culturing the purple plant,
The present invention relates to a method for efficiently producing large amounts of naphthoquinone compounds and other useful ingredients. The root of the purple plant, a plant belonging to the family Purple, has the following formula, (R=-OH,-OCOCH3
It contains naphthoquinone-based compounds such as shikonin (R=-OH), which is represented by the following formula, and has traditionally been called "purple root" and used in Chinese herbal medicine.

In other words, the ointment obtained by extracting shikonin and other substances from Shikonin using oils such as sesame oil is called Shiuncan and is used to treat various skin diseases, cuts, burns, hemorrhoids, etc., and has anti-inflammatory and granulation-forming effects. It is known that there is
However, the medicinal ingredients such as shikonin that can be extracted from the purple root are limited, and cultivating purple root takes time.
There are problems such as dependence on the natural environment and weather, and its stable supply is at risk.

In contrast, the use of tissue culture to propagate plants of the Phytochemistry family was proposed by Mamoru Tabata, Hajime Mizukami, and others as the first step in phytochemistry.
Kagami et al. page 927, “Pharmaceutical Journal” No. 93 No. 1376
Page, “Phytochemistry”
ist) Vol. 16, page 1183, Vol. 17, No. 9
It is reported on page 5.

According to this method, regardless of the season or weather,
It is very advantageous because it allows the cultivation of purple plants. However, the methods disclosed in these publications all use agar as a solid medium, and are unsuitable for mass production. Therefore, the present inventors investigated a method of growing callus in the same machine using a liquid tower suitable for mass production, and first added agar to the medium used by Tabata et al. (Linsmeyer-Skoog medium). It was used for tissue culture of purple violet in the form of a liquid base, but
Although the callus proliferated to some extent, the amount of pigments such as shikonin produced was small, and the amount produced also varied widely, making it impossible to secure a stable yield.

On the other hand, liquid media used for plant tissue culture include
Culture media containing inorganic salts, carbon sources, plant hormones, vitamins, amino acids, etc. are known.

The inventors further investigated these components and found that by controlling the concentration of specific components such as vitamins and amino acids, the amount of naphthoquinone compounds produced increased and the amount of production varied. There are few
We discovered that stable production can be ensured,
This invention was completed. That is, the present invention provides the following concentration conditions;
Below the tide {c- Pyridoxine concentration 0.25
Bench/below nicotinic acid concentration 0.
5/9/or less'e} Ascorbic acid concentration
1.0 female/less than 'f} Glycine concentration
p' limit of 1.0 or less (g) L cysteine concentration
5.0 p/or less (h) L-glutamine concentration
The present invention relates to a method for cultivating tissues of purple plants, characterized by using a liquid medium that satisfies at least one condition of 5.0 female or less.

The liquid medium used in the present invention can be used in a manner similar to that of a medium used in plant tissue culture, as long as the concentration of the above components is controlled, and other components can be varied widely within the range normally used. can be modified and used.

In addition, the liquid medium used for plant tissue culture is a liquid medium containing inorganic components and carbon sources as essential components, and at least one component selected from plant hormones, vitamins, amino acids, etc. Other ingredients may also be used as needed.

Inorganic components include nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, nitric acid Calcium, monopotassium phosphate, disodium phosphate, potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, Examples include molybdenum trioxide, potassium chloride, and cobalt chloride.

Examples of carbon sources include hydrocarbons such as sucrose, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol. Plant hormones include indoleacetic acid (IAA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (
Examples include auxins such as 2.4-D), and cytokinins such as kinetin, zeatin, and dihydrozeatin.

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

Amino acids include glycine, alanine, glutamine, and cysteine.

In the present invention, among these ingredients, vitamins {
a] Inositol, {b}thiamine, (c'pyridoxine, {d-nicotinic acid, (dascorbic acid and amino acids [f}glycine, (g}L-cysteine, (h
) It is necessary to control the concentration of at least one component selected from L-glutamine within the above range, and it is particularly desirable to control the concentration of not only one component but two or more components. It is most desirable to control the concentrations of all components of [h].

Furthermore, it is desirable that the concentrations of these components {a) to (h) be lower than the above-mentioned concentrations, and it is particularly desirable to use a liquid medium to which these components are not added.

This further increases the amount of naphthoquinone compounds produced in the callus. The concentrations of other components in the liquid medium can vary within wide limits.

Usually, the inorganic component is about 0.1 rM to about 100 mM.
The carbon source is about 1 night/even to 30 nights/so, the plant hormones are about 0.01 French M to about 10 French M, and the vitamins and amino acids are each about 0.1 female to about 100 French.
It is done to make it about female'Z. In the present invention,
It is also possible to further increase the amount of naphthoquinone compounds produced by adjusting other components in the soil.

For example, if the ratio of ammonium ions to the total nitrogen source is about 10 mol % or less, the amount of naphthoquinone compounds produced will further increase. A preferred example of this invention is the following method.

That is, after sterilizing tissue pieces collected from the plant body of a plant belonging to the family Murasakiceae, such as roots, growing points, leaves, stems, seeds, etc., it is hardened with agar or placed on a Smær-Skoog solid column. After about 7 to 30 days at ~35°C, a part of the tissue piece is formed into a callus. When the callus thus obtained is subcultured, the growth rate gradually increases and stable callus is obtained. This callus is transferred to a liquid medium suitable for growth, such as a Linsmeyer-Skoog liquid medium, and grown.

There is a method of culturing callus whose growth rate is further increased and stabilized in the liquid tower of the present invention by adding it to the liquid tower of the present invention.

In these methods, the initial concentration of callus in the liquid column can be varied within a wide range.

Usually, callus is added for about 1 hour to about 20 minutes per liquid tower.
It is desirable to add about 0.0 kg (fresh weight). In the tissue culture of the present invention, light is not necessarily necessary, and culturing in the dark is preferable for the growth of pigments such as shikonin, and the culture temperature is about 10 qo to about 35°C, especially about 23 oz.
~2800 is preferred.

Below about 10 o'clock, the callus growth rate is low, and about 35 o'clock
Similarly, the growth rate of callus decreases even if it exceeds MacC.
In order to separate and collect naphthoquinone compounds from callus and liquid medium, methods such as extraction that have been conventionally applied to the natural product "Shibaku" can be employed. According to the present invention, since a liquid tower is used, it is possible to perform mass culture using a tank, and furthermore, as a method for separating callus from a culture medium, simple operations such as decantation and furnace filtration can be adopted, which is industrially advantageous. It is.

Furthermore, the callus grows rapidly, and naphthoquinone compounds such as shikonin can be reliably produced in large quantities.

Comparative example: Lithospermum ehit
hrorhbonSecrying. etZMc. ) root tissue pieces were layered on a Linsmeier-Skoog agar solid medium and cultured in a static manner to obtain purple callus.

By culturing this callus in a Linsmeyer-Skoog liquid medium, the growth rate of the callus was increased. On the other hand 1
A liquid column having the composition shown in Table 1 was placed in a 0.00 Erlenmeyer flask (containing 1 AM of indole acetic acid, 1 OAM of kinetin as plant hormones, and 20 T of sucrose as a carbon source). [Put it in, 120o
o. Sterilized for 10 minutes.

To this liquid medium, 0.5 days of fresh callus of the above-mentioned purple callus with increased growth rate was added, and cultured with rotation (amplitude 25 ribs, 10
enemy pm). After culturing, the purple callus was collected in an oven, dried at 35o0 for 24 hours, and its weight (dry weight) was measured to determine the growth rate of cultured cells per liquid medium 1.

In addition, shikonin was separated from the obtained callus by extraction, its weight was measured, and the total amount of shikonin produced per liquid column was determined.

The results are shown in Table 2.

Examples 1 to 5 Comparative Examples were carried out in the same manner as in Comparative Examples except that the specific components of the medium components of the liquid medium were set to the values shown in Table 2.

Table 1 Table 2

Claims (1)

[Claims] 1 The following concentration conditions: (a) Inositol concentration 50 mg/l or less (b) Thiamine concentration 0.05 mg/l or less (c) Pyridoxine concentration 0
．． 25mg/l or less (d) Nicotinic acid concentration 0.5mg/l
l or less (e) Ascorbic acid concentration 1.0 mg/l or less (
Use a liquid medium that satisfies at least one of the following conditions: f) Glycine concentration 1.0 mg/l or less (g) L-cysteine concentration 5.0 mg/l or less (h) L-glutamine concentration 5.0 mg/l or less A method for culturing tissues of plants of the family Prunusaceae, characterized by the following.