JPS58101687A - Tissue culture of plant belonging to boraginaceae family - Google Patents

Abstract

PURPOSE:To produce a naphthoquinone compound and other useful components, efficiently, in bulk, by the tissue culture of a plant belonging to Boraginaceae family in a liquid medium containing ammonium ion as a nitrogen source and an organic acid at a specific ratio. CONSTITUTION:Tissue culture of a plant belonging to Boraginaceae family, e.g. Lipthospermum erythrorhizon Sieb. et Zucc. is carried out in a liquid medium obtained by adjusting the amount of ammonium ion in the medium for the conventional plant tissue culture to 0.1-80mol%, preferably 5-50mol% of the nitrogen source, and adding 1/20-5mol, preferably 1/5-2mol of one or more organic acid components selected from succinic acid, fumaric acid and malic acid per 1mol of the ammonium ion.

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, the present invention relates to a method for efficiently producing a large amount of naphthoquinone compounds and other useful components by tissue culturing plants of the family Prunusaceae using a liquid medium with a specific composition.

The root of Murasaki, a plant belonging to the Purple family, contains naphthoquinone-based compounds such as shikonin (R=OH) shown by the following formula, and has traditionally been called "purple root" and used in Chinese medicine. In other words, an ointment obtained by extracting shikonin and other substances from Shikonin using oils such as sesame oil is called Shiun-yang and is used for various skin diseases, cuts, burns, hemorrhoids, etc., and is used to treat symptoms such as increased vascular permeability and granulation formation. It is known to have certain effects.

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, using tissue culture method to propagate plants of the family Murasakiceae was proposed by Mamoru Debata, Hikaru Mizukami et al. as ``Phytochemistry'' Vol. 13, No. 927.
Page, “Pharmaceutical Journal” Volume 95, Page 1376, “Phytochemistry J”
y) Reported in Volume 16, Page 1183 and Volume 17, Page 95. This method is very advantageous because it allows plants of the family Prunusaceae to be propagated without being affected by the season or weather. 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 for growing callus in a similar manner using a liquid medium suitable for mass production, and first added agar to the medium used by Debata et al. (Rinsmeyer Skoog's medium). The callus was used in a liquid medium for tissue culture of purple violet, 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 difficult to secure a stable yield. could not.

As proposed in Japanese Patent Application No. 55-115903, the present inventors have repeatedly studied a liquid culture medium suitable for tissue culture of plants of the family Murasaceae and which produces a large amount of naphthoquinone compounds such as shikonin. By reducing ammonium ions to 10 mol% or less among the nitrogen sources in the culture medium,
It has been found that the growth is rapid, a large amount of naphthoquinone compounds are produced, and there is little variation in the amount produced, ensuring stable production. However, as a result of further investigation, we found that even if the ammonium ion content in the nitrogen source exceeds 10 mol%, if at least one component selected from conotic acid fumaric acid and malic acid is added, the above case can be solved. The inventors discovered that naphthoquinone compounds can be produced in a similar manner, leading to the completion of this invention. That is, in this invention, ammonium ions are present as a nitrogen source, and at least one organic acid component selected from the group consisting of succinic acid, fumaric acid, and malic acid is added to 1/20 of the ammonium ions.
The present invention relates to a method for culturing the tissue of a plant belonging to the family Murasaceae, which is characterized by using a liquid medium containing a liquid medium at a molar ratio of ~5 times.

The liquid medium used in this invention is not limited to other medium components as long as ammonium ions are present and the concentration of the organic acid component is within the above range.
The nitrogen source of the culture medium composition used for normal plant tissue culture or other compositions can be appropriately modified and used.

That is, a normal culture medium contains a carbon source or an energy source, inorganic salts, a nitrogen source, growth factors such as vitamins, and the like. Examples of carbon sources or energy sources include carbohydrates such as sucrose and their derivatives, primary alcohols such as ethanol, and amino acids such as aspartic acid. Examples of inorganic salts include calcium chloride, magnesium sulfate, iron sulfate, and phosphoric acid. Examples include potassium dihydrogen.

Examples of the nitrogen source include nitrogen-containing compounds such as ammonium ions, nitrate ions, amino acids, and decomposition products of complex proteins such as peptone.

Specifically, the liquid medium used in this invention includes Linsmeyer-Skoog's medium, Preydes' medium, Gamborg's medium, Etch &Etch's medium, and modified media thereof, and the ammonium ion in these medium The proportion of the nitrogen source is 0.1 mol% or more, preferably 80 mol% or less, particularly preferably 5 to 50 mol%.
Furthermore, at least one organic acid component selected from succinic acid, fumaric acid, and malic acid is added in a molar amount of 1/20 to 5 times the ammonium ion, preferably 115 to 5 times the mole of ammonium ion.
It is used by adding twice the molar amount. The organic acid may be added in free form or may be used in the form of a salt such as a sodium or potassium salt.

If the organic acid component is less than 1/20 times the mole of ammonium ion, the amount of naphthoquinone compounds produced will decrease, and even if it exceeds 5 times the mole, no major change will be observed, but a slight decrease in the amount produced will be observed. It will be done.

In this invention, the yield of naphthoquinone compounds can be further improved by particularly adjusting the concentration of inorganic ions in the liquid medium within a specific range. For example, if the copper concentration is increased to 0.2 μM or more, the amount of naphthoquinone compounds produced will further increase.
-Compounds such as dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, indoleacetic acid, etc. are 0.1 to 100μ
M1 is preferably contained in the skin at a concentration of 5 to 10 μM, and in this case, cytokinins such as kinetin and zeatin are contained in the skin at a concentration of 0.1 to 100 μM1, particularly 1 to 15 μM.
When allowed to coexist in a liquid medium at a concentration of μM, it is favorable for callus growth and production of naphthoquinone compounds.

Further, nutrients such as yeast extract, malt extract, tomato juice, casamino acid, coconut milk, and vitamin mixture may be added to the liquid medium as necessary.

Preferred examples of the tissue culture method of the present invention include the following methods. That is, the plant body of a plant belonging to the family Murasakiceae,
For example, tissue pieces collected from roots, growing points, leaves, stems, seeds, etc. are sterilized and then placed on a solid Linsmeyer Skoog medium solidified with agar for about 7 to 30 days at 10 to 35°C. After a period of time, 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 added to the above-mentioned liquid medium and subjected to tissue culture by shaking.

In this invention, light is not necessarily required, and culturing in the dark is preferable for callus growth. The culture temperature is preferably 10 to 65°C, particularly 23 to 28°C. Below 10°C, the growth rate of callus is slow;
Even if the temperature exceeds C, the growth rate of callus similarly decreases.

The plants of the Lithoraceae family to which this invention is applied are not particularly limited, but among them, Litho
sperm ery-thrororhizon 5i
eb, etZucc, etc.), and as mentioned above, callus containing naphthoquinone compounds such as shikonin and other medicinal ingredients can be obtained.

In order to extract active ingredients such as shikonin from callus, a method conventionally used for extracting purple roots can be employed.

According to the present invention, since a liquid medium is used, mass culture and tank culture are possible, and simple operations such as decantation and p-filtration can be used to separate the generated callus from the medium. Industrially advantageous.

In addition, callus multiplies rapidly, and pigments such as shikonin are produced in large amounts, with little variation in the amount produced, and a large amount of pigments can be produced stably and reliably.

Hereinafter, preferred examples of the present invention will be explained in detail with reference to Examples. However, the present invention is not limited to these Examples in any way.

Comparative Example 30 ml of the Linsmeyer-Skoog liquid medium shown in Table 1 (containing 1 μM indole acetic acid, 10 μM kinetin, 30 g/l sucrose) was placed in a 100 m Erlenmeyer flask and sterilized at 120° C. for 10 minutes. After cooling, 0.5 g of wet callus (previously obtained by static culture or liquid culture) was added and cultured at 25°C for 14 days with rotation on a rotary shaker (amplitude 25 mm).
, 100 rpm) L/ta.

After culturing, the purple callus was collected by p-filtration and kept at 35°C.
After drying for 24 hours, the weight (transfer) was measured. In addition, the content of naphthoquinone pigments such as shikonin in the dried callus was measured.

The measurement method is based on Moto Mizukami et al.'s [Phytochemistry J (
Phytochemistry) Volume 16 No. 1185~
The method described on page 1186 was followed. The results are shown in Table 2 (each shown in terms of yield per 11 liquid medium)
.

Examples 1 to 9 Comparative Examples were carried out in the same manner as in Comparative Examples, except that among the medium components of the liquid medium, the concentration of organic acids was set to the values shown in Table 2. The results are also shown in Table 2.

Table 1

Claims (2)

[Claims] (1) Ammonium ions are present as a nitrogen source, and at least one organic acid component selected from the group consisting of succinic acid, fumaric acid, and malic acid is contained in a molar ratio of 1/20 to 5 times the amount of ammonium ions. A method for cultivating tissue of a plant of the family Murasaceae, characterized by using a diluted liquid medium. (2) Lithospermum erythrorhiz is a plant of the family Lithospermaceae.
on 5ieb. The method for culturing Mi tissue according to claim 1, wherein