JPS6112680B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for efficiently producing naphthoquinone compounds, which are secondary metabolites thereof, by utilizing uncomposed cell groups (callus) obtained by tissue culture of plants of the family Prunusaceae. More specifically, the production of naphthoquinone compounds can be achieved by culturing a cell group preliminarily grown in a liquid medium suitable for the growth of undifferentiated cell groups in a modified liquid medium suitable for producing naphthoquinone compounds. Concerning how to do it efficiently. Several attempts have been made to obtain secondary metabolites by tissue culturing undifferentiated plant cell groups (callus). For example, the roots of the purple plant, a plant belonging to the family Purple, contain naphthoquinone compounds such as shikonin, and are known to have various medical uses, so tissue culture is used to grow undifferentiated cells of the purple plant. Attempts have been made to produce naphthoquinone compounds. However, the conventional method uses an agar solid medium, which requires complicated operation and operation during production, making it unsuitable for mass production, and the amount of naphthoquinone compounds produced is also not good. . Therefore, we considered a method of using a liquid medium, and used the components of the solid medium that had been conventionally used for tissue culture of purple thorns in the form of a liquid medium without adding agar, but although the cell population grew, The amount of naphthoquinone compounds such as , shikonin, etc. produced was small, and the amount produced also varied widely and was not stable. The present inventors further investigated and found that by stepwise tissue culture in two types of liquid media with a specific relationship, the amount of naphthoquinone compounds obtained from undifferentiated cell groups of plants of the family Prunusaceae was significantly increased. This discovery led to the present invention. That is, the present invention uses an inorganic component and a carbon source as essential components, and is cultured in a liquid medium containing at least one component selected from plant hormones, vitamins, and amino acids (first step). of the naphthoquinone-based compound of the Murasaceae plant, which is characterized by culturing the cells of the Murasaceae plant in a modified liquid medium in which the concentration of at least one of the components of the liquid medium is reduced (second step). Regarding the manufacturing method. The liquid medium A used in the first step of the present invention is
It is a liquid medium that is commonly used for plant tissue culture, and contains inorganic components and carbon sources as essential components, and one or more of plant hormones, vitamins, and amino acids added thereto. 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,
Examples include manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, and cobalt chloride. Examples of carbon sources include hydrocarbons such as diosaccharide, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol. Plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, and 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and zeatin. Examples include cytokinins such as , dihydrozeatin and the like. Examples of vitamins include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid, ascorbic acid (vitamin C), inositol, and nicotinic acid. Examples of amino acids include glycine, alanine, glutamine, and cysteine. The concentration of each component in liquid medium A can vary within a wide range. Usually the inorganic component is about 0.1 μM ~
Approximately 100mM, carbon source approximately 1g/l to 30g/l, and plant hormones approximately 0.01μM to approximately 10μ
It is prepared by adding about 0.1 mg/l to about 100 mg/l of vitamins and amino acids, respectively. The liquid medium A is preferably one suitable for promoting the growth and proliferation of undifferentiated cell groups of plants belonging to the family Murasakiceae. Specific examples include Linsmeyer-Skoog's medium, Gamborg's B-5 medium, and modified media thereof. The undifferentiated cell group of the Murasaceae plant used in the present invention is the plant body of the Murasaceae plant, for example, the roots,
Tissue pieces collected from growing points, leaves, stems, seeds, etc. are sterilized and then added to a solid medium to allow some of the tissue pieces to grow into undifferentiated cell groups. In the case of plants of the family Murasakiceae, suitable examples include the following. In other words, after sterilizing a piece of tissue from a plant belonging to the family Purple, it was placed on a Linsmeyer-Skoog solid medium solidified with agar and incubated for about 10 minutes.
After about 7 to 30 days at ~35°C, a part of the tissue piece is turned into a callus. When the cell population thus obtained is subcultured, the growth rate gradually increases and a stabilized cell population is obtained. This cell group is added to the liquid medium A and cultured. The initial concentration of the cell population in liquid medium A can be varied within a wide range. Usually, it is desirable to add about 1 g to about 200 g (fresh weight) of the cell group to 1 liter of liquid medium. In the first stage, it is desirable that the liquid medium A is suitable for the proliferation of the cell group, and furthermore, it is desirable that the cell group be subcultured in the liquid medium A to gradually increase the growth rate. In the case of plants of the family Prunusaceae, a certain amount of secondary metabolites consisting of naphthoquinone compounds may be produced even in the first stage, but the amount is small and the conditions suitable for the growth of cell groups are not necessarily the same as those of naphthoquinone compounds. This does not provide good conditions for formation. In the present invention, it is preferable to allow the cell group to proliferate particularly vigorously in the first stage of tissue culture, and if a cell group with such a history is used, naphthoquinone in the modified liquid medium B of the second stage can be used. The amount of secondary metabolites produced, such as system compounds, can be further increased at any time. In the case of purple violet, it is particularly preferable to allow the cell group to proliferate without producing naphthoquinone compounds in the first stage, and then proceed to the second stage. The cell population cultured in the liquid medium A in the first stage is separated from the liquid medium A and added to the modified liquid medium B in the second stage to continue the tissue culture. The composition of the modified liquid medium B is such that the concentration of at least one of the medium components of the liquid medium A is reduced. In liquid medium B, the component that lowers the concentration compared to liquid medium A is preferably at least one component selected from inorganic components, plant hormones, vitamins, and amino acids. Among these, inorganic components such as ammonium ions, nitrate ions, phosphate ions, potassium ions, calcium ions, iron ions, manganese ions, cobalt ions, iodine ions, sodium ions, and chloride ions are particularly important as components that lower the concentration. At least one component selected from , cytokinins, vitamins, and amino acids is suitable. As long as the concentration of the above-mentioned preferred components is reduced, the production of naphthoquinone compounds is often not significantly affected by changes in the concentrations of other components. In the case of plants belonging to the family Murasakiceae, it may be preferable to increase the concentration of components other than those listed above. Such components include copper ions and sulfate ions. It is particularly desirable that the concentration of the component that lowers the concentration in liquid medium B be about 1/2 or less of the concentration in liquid medium A. In addition, the concentration of the component that lowers the concentration is further reduced to about 1/10 or less, or liquid medium B in which the component is not substantially added.
It is also possible to use Although the growth rate of the cell group in liquid medium B in the second stage is lower than that in liquid medium A, the amount of secondary metabolites such as naphthoquinone compounds is, on the contrary, significantly increased. The initial concentration of the cell population in liquid medium B can also be varied within a wide range, as in liquid medium A. According to the present invention, using a plant of the family Murasakiceae,
A naphthoquinone compound such as shikonin, which is a secondary metabolite, can be obtained preferably and efficiently. In the case of plants belonging to the Murasakiceae family, light is not necessarily necessary for tissue culture; on the contrary, culturing in the dark is preferable for the growth of secondary metabolites such as shikonin.
The culture temperature is about 10°C to about 35°C, especially about 23°C to about 28°C.
°C is preferred. Below about 10°C, the proliferation rate of the cell group is low, and even above about 35°C, the proliferation rate of the cell group is similarly low. In order to separate and collect the naphthoquinone compound from the cell group and liquid medium B, methods such as extraction that have been conventionally applied to the natural product "Shikon" can be employed. According to the present invention, a naphthoquinone compound can be efficiently produced from an undifferentiated cell group (callus) of a plant belonging to the family Murasaceae. Furthermore, since a liquid medium is used, scale-up and continuous operation are easy, and naphthoquinone compounds can be produced in large quantities with simple operations. The method of the present invention is applicable not only to cases where two types of liquid media are used to perform two-stage culture, but also to cases where two types of liquid media are used.
It is also possible to culture tissue in three or more stages using three or more types of liquid media that satisfy the conditions of the present invention. Examples 1 to 3 and Comparative Examples [First stage culture] A modified Linsmeyer-Skoog liquid medium having the composition shown in Table 1 was placed in a 100 ml Erlenmeyer flask (with the exception of 1 μM indoleacetic acid, 10 μM kinetin, and sucrose). (including 30g/l)30
ml and sterilized at 120°C for 10 minutes. After cooling, 0.5 g of fresh purple callus (previously obtained by the static culture method) was added and cultured at 25°C for 14 days on a rotary shaker with an amplitude of 25 mm and rotation at 100 rpm. Repeat the above operation (usually twice) using 0.5 g of callus obtained in the above culture. This stabilizes cell growth in the modified liquid medium. [Second stage culture] A culture medium (B ₁ , B ₂ or B _{3 )} shown in Table 1 in which the concentration of at least one component of the modified liquid medium A used in the first stage has been reduced is used. I used it. That is, 0.5 g of the wet callus obtained in the first step was added to 30 ml of the medium (B ₁ , B ₂ or B ₃ ), and cultured by rotation in the same manner as in the first step. After culturing, the callus of the purple callus was collected by filtration, dried at 35°C for 24 hours, and its weight (dry weight) was measured to determine the growth dry weight of the cultured cells per liter of liquid medium. In addition, shikonin was extracted from the obtained callus, its weight was measured, and the total amount of silicon produced per liter of liquid medium was determined. The results are shown in Table 2.

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Claims (1)

[Scope of Claims] 1 Cultivation in a liquid medium containing inorganic components and carbon sources as essential components and at least one component selected from plant hormones, vitamins, and amino acids (first step) ) The production of a naphthoquinone compound characterized by culturing the cells of a plant belonging to the family Murasaceae obtained by (second step) in a modified liquid medium in which the concentration of at least one of the components of the liquid medium is reduced. Method. 2 The components that reduce the concentration in the second stage are ammonium ions, nitrate ions, phosphate ions, potassium ions, calcium ions, iron ions, manganese ions, cobalt ions, iodine ions, sodium ions, chloride ions, cytokinins, and vitamins. The method according to claim 1, characterized in that the component is at least one type selected from amino acids and amino acids. 3. The method according to claim 1 or 2, characterized in that in the second step, the concentration of the component is reduced to at most one-half or less of the concentration used in the first step.