JPS60984B2 - 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, the present invention relates to a method for efficiently producing a large amount of naphthoquinone compounds and other organic components by tissue culturing plants of the family Prunusaceae using a liquid medium with a specific composition. The roots of the purple violet plant contain naphthoquinone-based compounds such as shikonin (R = 1OH) shown by the formula below, and have traditionally been called ``purple roots'' and used in Chinese medicine. .

In other words, the ointment obtained by extracting shikonin and other substances from the purple plate with oils such as sesame oil is called shiyun bone 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 are
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 the stable supply is at risk.

On the other hand, Mamoru Tabata, Hajime Mizukami, and others have proposed propagating plants of the PhyKaceae family using tissue culture methods as ``phytochemistry'' (phyK hemistry).
Volume 13, page 927, “Pharmaceutical Journal” No. 93, No. 13
Page 76, “Phytochemistry” (phybch
This is reported in Volume 16, page 1183 of Emjst, Vol. 17, page 95.

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 beach soil in the form of solid agar, and are unsuitable for mass production. Therefore, the present inventors investigated a method of growing callus in a similar manner using a liquid soil 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 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 widely, making it impossible to secure a stable yield.

The present inventors have conducted further studies on a liquid medium that is passed through tissue culture of plants of the family Murasaceae and produces large amounts of naphthoquinone compounds such as shikonin, and as a result, the inventors have determined that the concentration of specific components in the medium can be controlled. The inventors have discovered that, by doing so, the naphthoquinone compound can be rapidly propagated, a large amount of naphthoquinone compounds can be produced, and the amount of the naphthoquinone compound can be produced with little variation, ensuring stable production, leading to the completion of this invention.

That is, the present invention relates to a method for culturing the tissue of a Japanese purple plant, which is characterized by using a liquid medium having a copper ion concentration of 0.2 m or more.

In the present invention, the copper ion concentration in the liquid tower base is 0.2F
As mentioned above, other medium components can be changed over a wide range, especially as long as they are adjusted within the range of 0.2 French M to 25 AM, and the medium conventionally used for plant tissue culture can be modified in various ways. Can be used.

When the steel ion concentration is less than 0.2 France M, the amount of naphthoquinone compounds produced decreases, and even when it exceeds 25 France M, no major change is observed, but a slight decrease in the production amount is observed.

Conventional media used for plant tissue culture include those that contain inorganic components and carbon sources as essential components, and to which are added at least one component selected from plant hormones, vitamins, and amino acids. , and other ingredients may be used in combination as necessary.

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

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, and 2,4-dichlorophenoxyacetic acid (
Examples include auxins such as 2.41D), and cytokinins such as kinetin, zeatin, and dihydrozeatin.

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

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

The concentration of components other than copper ions in the liquid column base can be varied within a wide range.

Usually, the inorganic ingredients are about 0.11M to about 100M,
The carbon source is about 1 to 30 m/kg, the plant hormones are about 0.01 fm to about 10 fm, and the vitamins and amino acids are about 0.1 to 9 m/kg each. Approximately 1
The value is set to approximately 9' of 00. In the present invention, it is also possible to further increase the amount of naphthoquinone compounds produced by adjusting other components in the medium.

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 Prunusaceae, such as roots, growing points, leaves, stems, seeds, etc., the tissue pieces are solidified with agar or placed on a solid medium of Nsmeyer-Skoog for 10 to 30 minutes. 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. Normally, it is desirable to add about 1 to 200 hours of callus (fresh weight) to 1 volume of liquid medium. In the tissue culture of the present invention, light is not necessarily necessary, and it is preferable to culture in a stairwell. is desirable for the growth of pigments such as shikonin, and the culture temperature is about 10q○ to about 35°C, especially about 2yC to about 280°C.
0 is preferred.

Below about 10 o'clock C, the callus growth rate is low and about 35 o'clock
Even if qo is exceeded, the callus growth rate similarly decreases.
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 "Shikon" can be employed. According to the present invention, since a liquid medium is used, large-scale culture using a tank is possible, and simple operations such as decantation and furnace filtration can be adopted as a method for separating callus from the medium, which is industrially advantageous. be.

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

Comparative example Lithospermum ehithr
orhSeonSemountaetZucc. ) root tissue piece,
Purple callus was obtained by layering it on a Linsmayer-Skoog agar solid medium and using the Seiseki culture method.

The callus growth rate was increased by culturing this callus in a Linsmayer-Skoog liquid medium. On the other hand, in an Erlenmeyer flask above 100 ml, a white liquid solution having the composition shown in Table 1 was added (with the exception of 100 ml of indole acetic acid, 10 ml of kinetin as plant hormones, and 20 ml of sucrose as a carbon source). /Including) 30 pieces and sterilized for 10 minutes at 120 o'clock.

Add 0.5 liters of the above-mentioned fresh purple callus with increased growth rate to this white liquid tower, and add 25 ounces of fresh callus.
The callus was cultured with rotation (amplitude 25 mm, 10 pm) on a rotary shaker for 14 days at The dry weight of cultured cells per 1 volume of liquid medium was determined. Shikonin was separated from the obtained callus by extraction, its weight was measured, and the total shikonin per 1 volume of liquid medium was determined. The amount of produced was determined.

The results are shown in Table 2.

Examples 1 to 3 Comparative Examples were conducted in the same manner as in Comparative Examples except that among the medium components of the liquid medium, the copper ion concentration was set to the value shown in Table 2.

Table 1 Table 2

Claims (1)

[Scope of Claims] 1. A method for culturing the tissue of a plant of the family Murasaceae, characterized by using a liquid medium having a copper ion concentration of 0.2 μM or more. 2. Claim 1, characterized in that a liquid medium having a copper ion concentration of 0.2 μM to 25 μM is used.
The method described in section. 3 A plant of the family Lithospermum is Lithospermum erythrorhiz.
on Sieb. et Zucc. ) The method according to claim 1, characterized in that: