JPS60989B2 - 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 tissues of purple plants containing naphthoquinone pigments 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 roots of the purple root, which is a plant used as a purple root, contain naphthoquinone-based compounds such as shikonin (R=OH), which is represented by the following formula -(R=-OH, -OCOCH3, etc.). ” and is 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 Shiunsuga and is used for various skin diseases, cuts, burns, hemorrhoids, etc. It is known to have a forming effect. However, the medicinal ingredients such as shikonin that can be extracted from the purple root are only available in small quantities, and there are other problems such as the time it takes to process purple roots and the dependence on the natural environment and weather, so its stable supply is at risk. .

On the other hand, propagation of plants of the Phytochemistry family using tissue culture methods was proposed by Mamoru Tabata and Hajime Mizukami et al.
No. 927 page, “Pharmaceutical Magazine” No. 99 page 1376, “Phytochemistry”
st) Volume 16, page 1183, Volume 17, page 95
Reported on the page.

According to this method, regardless of the season or weather,
It is very advantageous because it can propagate plants of the family Murasakiceae. 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. (Rinsmeyer Skoog's medium). It was used for tissue culture of purple violet in the form of a liquid medium, but although the callus proliferated to some extent, the amount of pigments such as shikonin produced was small, and the amount produced also varied greatly, making it difficult to ensure a stable yield. I couldn't do it.

As proposed in Tokuiso Sho No. 55-115903, the present inventors have conducted repeated studies on a liquid tower suitable for tissue culture of purple plants and which produces a large amount of naphthoquinone compounds such as shikonin. , by reducing ammonium ions to 10 mol% or less of the nitrogen source in the medium,
It has been found that the growth is rapid, a large amount of naphthoquinone compounds are produced, there is little variation in the amount produced, and stable production can be ensured.

However, as a result of further investigation, it was found that even if ammonium ions exceed 10 mol% of the nitrogen source, succinic acid,
The present inventors have discovered that naphthoquinone compounds can be produced in the same manner as in the above case by adding at least one component selected from fumaric acid and malic acid, and have completed the present 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 present in a molar ratio of 1/20 to 5 times the molar ratio of ammonium ions. The present invention relates to a method for culturing the tissue of plants belonging to the family Murasaceae, which is characterized by using a liquid base containing . The liquid tower used in this invention contains ammonium ions and is not limited to other culture medium components as long as the concentration of the organic brewing components is within the above range. The nitrogen source of the beach composition used for tissue culture or other compositions can be appropriately modified and used.

In other words, normal beach land contains carbon sources or energy sources, inorganic salts, nitrogen sources, growth factors such as vitamins, etc. Carbon sources or energy sources include carbohydrates such as rice bran and their derivatives; Examples include primary alcohols such as ethanol, amino acids such as aspartic acid, and examples of inorganic salts include calcium chloride, magnesium sulfate, iron sulfate, potassium trihydrogen phosphate, etc.Nitrogen sources are usually ammonium ions, Examples include nitrogen-containing compounds such as nitrate ions, amino acids or decomposition products of complex proteins such as bebutone.

Specifically, the liquid substrates used in this invention include Linsmeyer Skoog's medium, Pleydes' medium, Gamborg's medium, Nitsuchi &Nitsuchi's medium, and their modified materials. The proportion of ammonium ions in 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 1'5 to
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.
When the organic brewing component is less than 1/2 molar amount of ammonium ion, the amount of naphthoquinone compounds produced decreases, and even when the amount exceeds 5 times the molar amount, no major change is observed, but the amount produced is slightly reduced. Can be seen. In the present invention, the yield of naphthoquinone compounds can be further improved by particularly adjusting the concentration of inorganic ions in the liquid column to a specific range. For example, if the copper concentration is set to 0.2 M or more, the amount of naphthoquinone compounds produced will further increase. In addition, auxins, such as 2
Compounds such as 04-dichlorophenoxyacetic acid (214-D), naphthaleneacetic acid, and indoleacetic acid have a concentration of 0.1 to 100
It is preferable that it is contained in a concentration of 0.5 to 1 OAM, and in this case, the content of cytokinins such as kinetin and zeatin is 0.

If it is allowed to coexist in the liquid column at a concentration of 1 to 100 rM, especially 1 to 15 rM, it is good 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, after sterilizing tissue pieces collected from the plant body of a plant belonging to the purple plant, such as roots, growing points, leaves, stems, seeds, etc., it is hardened with agar or layered on the solid surface of Smjerskoog. After about 7 to 30 days at 10 to 35 points, a part of the tissue piece is turned 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 necessary, and culturing in a cage is preferable for the growth of callus.

The culture temperature is preferably 10 to 3,500 degrees, particularly preferably 23 to 28 degrees. If it is less than 1000, the callus growth rate is low, and if it exceeds 35o0, the callus growth rate is also low.

The plants of the family Prunusaceae to which this invention is applied are not particularly limited, but among them, the plants of the family Prunusaceae (L.
perm skin m ery ratio rorhizen Sieb.
etZucc. ) is desirable, and as mentioned above, it is possible to obtain callus containing naphthoquinone compounds such as sinicone and other medicinal ingredients.

In order to extract active ingredients such as shikonin from callus, methods conventionally used for extracting purple lines can be employed. According to this invention, since a liquid tower is used, mass culture and tank culture are possible, and simple operations such as decantation and furnace filtration can be used as a method for separating the generated callus from the culture medium. Therefore, it is industrially advantageous.

In addition, callus multiplies quickly, 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 In a 100 m' Erlenmeyer flask, 30 g of Linsmeyer Skoog's liquid medium shown in Table 1 (containing 1 M of indole acetic acid, 10 M of kinetin, and 30 M of sucrose) was added. '' and sterilized for 120q010 minutes.

After cooling, 0.5 night-old moist callus (obtained in advance by the static culture method or liquid culture method) was placed and cultured with rotation on a rotary shaker at 2500 °C for 14 days (amplitude 2).
5 days, 10 days pm). The cultured purple callus was collected by filtering, dried at 35 qo for 2 hours, and then its weight (dry weight) was measured.

In addition, the content of naphthoquinone pigments such as shikonin in the dried callus was measured. The measurement method followed the method described in "PhytochemStry" by Hajime Mizukami et al., Vol. 16, pages 1185 to 186.

The results are shown in Table 2 (each expressed in terms of yield per night of liquid tower). Examples 1 to 9 Comparative Examples were conducted in the same manner as in Comparative Examples except that the concentration of organic acid among the medium components of the liquid medium was set to the values shown in Table 2.

The results are also shown in Table 2. Table 1 Table 2

Claims (1)

[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 added in a molar amount 1/20 to 5 times the amount of ammonium ions. 1. A method for culturing tissues of plants of the family Murasaceae, characterized by using a liquid medium containing a proportion of . 2 The plant of the family Lithospermum is Lithospermum erythrorhiz.
on Sieb. et Zucc. ) The tissue culture method according to claim 1, characterized in that: